1. health and fitness
2. disease
3. aids and hiv

FACULTE DE BIOLOGIE ET MEDECINE
Immunology of Viral Disease, How to Curb
Persistent Infection
Thèse de doctorat ès sciences de la vie (PhD)
présentée à la
Faculté de biologie et médecine de
l'Université de Lausanne
par
Simone C. Zimmerli
Licenciée en Biologie
Université de Bâle
Jury
Prof. Pavel Kucera, Président
Prof. Philippe Moreillon, Directeur de thèse
Prof. Peter Erb, Expert
Prof. Ed Palmer, Expert
LAUSANNE
2005
Acknowledgments
First of all, I would like to thank Prof. Giuseppe Pantaleo for giving me the opportunity to
perform my PhD-thesis in his laboratory and for introducing me to the fascinating world of
Immunology.
I am deeply indebted to Prof. Philippe Moreillon for accepting to be the director of my thesis
during its final phase, for hosting me in his Laboratory during the last months, for his
admirable patience, his valuable advice and his time and effort which he invested in the
manuscript of my thesis.
I am grateful to Alexandre Harari and Florence Vallelian for many helpful discussions and
technical help during my thesis. My thanks go also to the other collaborators of the
Laboratory of AIDS Immunopathogenesis, especially to Samantha for her support and
Patricia for her help at the last minute.
I would like to thank Professors Peter Erb, Ed Palmer and Pavel Kucera for offering their time
by accepting to be in the jury of my PhD thesis.
I acknowledge Prof. Stéphanie Clarke and Prof. Philippe Moreillon for helpful discussions
and their effort to find a solution in a difficult period.
Last but not least, I would like to thank my parents. Without your encouragement, the
thousands of discussions and of course your financial support during the last months, I would
never have been able to be where I am now. Thank you very much!
Simone C. Zimmerli
Table of Contents
Table of Contents
1. Summaries ...................................................................................................................... 1
1.1 Preamble and extended abstract ............................................................................. 1
1.2. Préamblule et résumé élargi ................................................................................... 4
1.3. Résume pour un large public.................................................................................. 7
2. Abbreviations ................................................................................................................. 8
3. Introduction.................................................................................................................... 11
3.1. General principles of immunology ......................................................................... 11
3.1.1 The principle of innate and adaptive immunity............................................ 11
3.1.2 The origin of the cellular partners of innate and adaptive immunity .......... 11
Innate immunity cells..................................................................... 11
Adaptive immunity cells ................................................................. 12
The lymphoid organs ..................................................................... 12
3.1.3 Innate immunity ............................................................................................. 14
3.1.4 Adaptive immunity......................................................................................... 14
3.1.5 The innate and adaptive immunity cooperation ........................................... 16
B cells and T cells.......................................................................... 16
Humoral immune response ............................................................ 17
Cell-mediated immune respone ...................................................... 19
3.2. The immune response to viral infection ................................................................. 20
3.2.1 The surface T cell receptors (CD4 and CD8) ................................................ 20
3.2.2 The TH1-TH2 decision of CD4 T cells............................................................. 24
3.2.3 The special case of CD8 T cells ...................................................................... 24
3.2.3.1 CD8+ cytolytic T lymphocytes............................................................... 25
Simone C. Zimmerli
Table of Contents
3.2.3.2 CD8 memory cells ................................................................................ 25
3.2.3.3 Additional cluster of differentiation (CD) markers................................ 26
3.2.3.4 Cytokine secretion and T cell proliferation........................................... 27
Interferon-γ.................................................................................... 27
Interleukin-2 .................................................................................. 28
3.3. General characteristics of viruses........................................................................... 30
3.3.1 Morphology .................................................................................................... 30
3.4. Viruses used in these studies................................................................................... 32
3.4.1 Human Immunodeficiency Virus (HIV-1)..................................................... 32
3.4.1.1 HIV genes and lifecycle ........................................................................ 32
3.4.1.2 HIV-1 and the immune system ............................................................. 34
Humoral immunity............................................................................... 34
Cell-mediated immunity...................................................................... 35
3.4.1.3 Clinical manifestations......................................................................... 36
3.4.1.4 Treatment and prevention..................................................................... 40
Treatment ........................................................................................... 40
Post exposure prophylaxis .................................................................. 40
The perspective of anti-HIV vaccines .................................................. 41
3.4.2 Cytomegalovirus (CMV)................................................................................ 43
3.4.2.1 Viral structure, genes and lifecycle....................................................... 43
3.4.2.2 CMV and the immune system................................................................ 45
3.4.2.3 CMV pathogenesis and clinical manifestations..................................... 45
3.4.2.4 Treatment and prevention..................................................................... 46
Therapy.............................................................................................. 46
Prevention.......................................................................................... 47
Simone C. Zimmerli
Table of Contents
3.4.3 Epstein-Barr virus (EBV) .............................................................................. 47
3.4.3.1 Viral structure, genes and lifecycle....................................................... 47
3.4.3.2 EBV and the immune system................................................................. 49
Viral escape strategies ....................................................................... 49
3.4.3.3 EBV pathogenesis, clinical manifestations and complications .............. 50
3.4.3.4 Treatment ............................................................................................ 51
3.4.4 Influenzavirus (Flu)........................................................................................ 51
3.4.4.1 Viral structure, genes and lifecycle....................................................... 51
3.4.4.2 Influenza and the immune system.......................................................... 54
Humoral immunity .............................................................................. 54
Cell-mediated immunity ...................................................................... 54
Viral escape strategies ........................................................................ 54
Antigenic shift................................................................................ 55
Antigenic drift................................................................................ 55
3.4.4.3 Pathogenesis, clinical manifestations and complications ...................... 55
3.4.4.4 Treatment and prevention..................................................................... 56
Therapy .............................................................................................. 56
Prevention .......................................................................................... 56
4. Results ............................................................................................................................ 58
4.1. Publication #1.......................................................................................................... 58
Summary ................................................................................................................. 58
HIV-1-specific IFN-γ/IL-2 secreting CD8 T cells support CD4-independent
proliferation of HIV-1-specific CD8 T cells (pdf) .................................................. 60
4.2. Publication #2......................................................................................................... 66
Summary ................................................................................................................ 66
Simone C. Zimmerli
Table of Contents
Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2restricted cytotoxic T-cell responses (pdf) ............................................................ 68
4.3. Publication #3 ........................................................................................................ 77
Cytomegalovirus (CMV)-specific cellular immune responses (pdf) .................... 78
5. Conclusions and Outlook .............................................................................................. 85
6. References...................................................................................................................... 89
7. Glossary ........................................................................................................................ 103
Simone C. Zimmerli
1. Summaries
1. Summaries
1.1. Preamble and extended abstract
The present thesis dissertation addresses the question of antiviral immunity from the
particular standpoint of the adaptive T cell-mediated immune response. The experimental
work is presented in the form of three published articles (two experimental articles and one
review article, see sections 4.1, 4.2 and 4.3 on pages 73, 81 and 91, respectively), describing
advances both in our understanding of viral control by CD8 T lymphocytes, and in vaccine
development against the Human Immunodeficiency Virus Type 1 (HIV-1).
Because the articles focus on rather specialized areas of antiviral immunity, the article
sections are preceded by a general introduction (section 3) on the immune system in general,
and on four viruses that were addressed in the experimental work, namely HIV-1,
Cytomegalovirus (CMV), Epstein Barr Virus (EBV) and Influenzavirus (Flu). This
introduction section is aimed at providing a glimpse on viral molecular biology and immunity,
to help the hypothetical non-expert reader proceeding into the experimental part. For this
reason, each section is presented as individual entity and can be consulted separately.
The four viruses described are of peculiar relevance to immunity because they induce
an array of opposite host responses. Flu causes a self limiting disease after which the virus is
eradicated. CMV and EBV cause pauci-symptomatic or asymptomatic diseases after which
the viruses establish lifelong latency in the host cells, but are kept in check by immunity.
Eventually, HIV-1 establishes both latency – by inserting its genome into the host cell
chromosome – and proceeds in destroying the immune system in a poorly controlled fashion.
Hence, understanding the fundamental differences between these kinds of viral host
interactions might help develop new strategies to curb progressive diseases caused by viruses
such as HIV-1.
Publication #1: The first article (section 4.1, page 73) represents the main frame of my
laboratory work. It analyses the ability of CD8 T lymphocytes recovered from viral-infected
patients to secrete interferon γ (IFN-γ) alone or in conjunction with interleukin 2 (IL-2) when
exposed in vitro to their cognate viral antigens. CD8 T cells are instrumental in controlling
viral infection. They can identify infected cells by detecting viral antigens presented at the
surface of the infected cells, and eliminate both the cell and its infecting virus by triggering
apoptosis and/or lysis of the infected cell. Recognition of these antigens triggers the cognate
1
Simone C. Zimmerli
1. Summaries
CD8 cells to produce cytokines, including IFN-γ and IL-2, which in turn attract and activate
other pro-inflammatory cells. IFN-γ triggers both intrinsic antiviral activity of the infected
cells and distant activation of pro-inflammatory cells, which are important for the eradication
of infection. IL-2 is essential for clonal expansion of the antigen (Ag)-specific CD8 T cell.
Hence the existence of Ag-specific CD8 cells secreting both IFN-γ and IL-2 should be
beneficial for controlling infection.
In this first work we determined the percentage of IFN-γ/IL-2 double positive and
single IFN-γ secreting CD8 T cells against antigens HIV-1, CMV, EBV and Flu in three
groups of subjects: (i) HIV-1 infected patients progressing to disease (progressors), (ii) HIV1-infected subjects not progressing to disease (long-term non progressors or LTNP), and (iii)
HIV negative blood donors. The results disclosed a specific IFN-γ/IL-2 double positive CD8
response in all subjects able to control infection. In other words, IFN-γ/IL-2 double positive
CD8 cells were present in virus-specific CD8 T cells against Flu, CMV and EBV as well
against HIV-1 in LTNP. In contrast, progressors only had single IFN-γ secreting CD8 T cells.
Hence, the ability to develop an IFN-γ/IL-2 double positive response might be critical to
control infection, independently of the nature of the virus.
Additional experiments helped identify the developmental stage of the missing cells
(using different markers such as CD45RA and CCR7) and showed a correlation between the
absence of IL-2 secreting CD8 T cells and a failure in the proliferation capacity of virusspecific CD8 T cells. Addition of exogenous IL-2 could restore clonal expansion of HIV-1
specific CD8 T cells, at least in vitro. It could further been shown, that IL-2 secreting CD8 T
cells are sufficient to support proliferation even in absence of CD4 help. However, the reason
for the missing IFN-γ/IL-2 double positive CD8 T cell response in HIV-1 progessors has yet
to be determined.
Publication #2: The second article (section 4.2, page 81) explores new strategies to trigger
CD8 T cell immunity against specific HIV-1 proteins believed to be processed and exposed as
"infection signal" at the surface of infected cells. Such signals consist of peptide fragments (813 amino acids) originating from viral proteins and presented to CD8 T cells in the frame of
particular cell surface molecules of the major histocompatibility complex class I∗ (MHC I). To
mimic "natural" viral infection, the HIV-1 polyprotein Gagpolnef was inserted and expressed
in either of two attenuated viruses i.e. vaccinia virus (MVA) or poxvirus (NYVAC). Mice
∗
words in italic (except for names of pathogens) are explained in the glossary section 7 p. 103
2
Simone C. Zimmerli
1. Summaries
were infected with these recombinant viruses and specific CD8 T cell response to Gagpolnef
peptides was sought.
Mice could indeed mount a CD8 T cell response against the HIV-1 antigens,
indicating that the system worked, at least in this animal model. To further test whether
peptides from Gagpolnef could also be presented in the frame of the human MHC class I
proteins, a second round of experiments was performed in "humanized" transgenic mice
expressing human MHC molecules. The transgenic mice were also able to load Gagpolnef
peptides on their human MHC molecule, and these cells could be detected and destroyed by
Ag-specific CD8 T cells isolated from HIV-1-infected patients. Therefore, expressing
Gagpolnef on attenuated recombinant viruses might represent a valid strategy for anti-HIV-1
immunization in human.
Publication #3: This is a review paper (section 4.3, page 91) describing the immune response
to CMV and newly developed methods to detect this cellular immune response. Some of it
focuses on the detection of T cells by using in vitro manufactured tetramers. These consist of
four MHC class I molecules linked together and loaded with the appropriate antigenic
peptide. The tetramer can be labeled with a fluorochrome and analyzed with a fluorescenceactivated cell sorter.
Taken together, the work presented indicates that (i) an appropriate CD8 T cell response
consisting of IFN-γ/IL-2 double positive effectors, can potentially control viral infection,
including HIV-1 infection, (ii) such a response might be triggered by recombinant viral
vaccines, and (iii) CD8 T cell response can be monitored by a variety of techniques, including
recently-developed MHC class I tetramers.
3
Simone C. Zimmerli
1. Summaries
1. 2. Préambule et résumé élargi
Le présent travail de thèse s'intéresse à l'immunité antivirale du point de vue particulier de la
réponse adaptative des cellules T. Le travail expérimental est présenté sous la forme de trois
articles publiés (2 articles expérimentaux et 1 article de revue, voir sections 4.1, 4.2 et 4.3,
pages 58, 66 et 77, respectivement), décrivant des progrès dans la compréhension du contrôle
de l'infection virale par les lymphocytes T CD8, ainsi que dans le développement de
nouveaux vaccins contre le Virus d'Immunodéficience de Humaine de type 1 (VIH-1).
En raison du caractère spécialisé de l'immunité antivirale de type cellulaire, les
articles sont précédés par une introduction générale (section 3), dont le but est de pourvoir le
lecteur non avisé avec des bases nécessaire à une meilleure appréhension du travail
expérimental. Cette introduction présente les grandes lignes du systèmes immunitaire, et
décrit de façon générale les 4 virus utilisés dans le travail expérimental: à savoir le virus VIH1, le Cytomégalovirus (CMV), le virus Epstein Barr (EBV) et le virus Influenza A (Flu).
Toutes les sections sont présentées de façon individuelle et peuvent être consultées
séparément.
La description des 4 virus a une pertinence particulière quant à leur interaction avec le
système immun. En effet, ils induisent une panoplie de réponses immunitaires s'étendant aux
extrêmes de la réaction de l'hôte. Influenza A est à l'origine d'une maladie cytopathique aiguë,
au décours de laquelle le virus est éradiqué par l'hôte. CMV et EBV sont classiquement à
l'origine d'infections pauci-symptomatiques, voire asymptomatiques, après lesquelles les virus
persistent de façon latente dans la cellule hôte. Cependant, ils restent sous le contrôle du
système immun, qui peut prévenir une éventuelle réactivation. Enfin, VIH-1 s'établit à la fois
en infection latente – par l'insertion de son génome dans le chromosome des cellules hôtes –
et en infection productive et cytopathique, échappant au contrôle immunitaire et détruisant ses
cellules cibles. La compréhension des différences fondamentales entre ces différents types
d'interactions virus-hôte devraient faciliter le développement de nouvelles stratégies
antivirales.
Article 1 : Le premier article (section 4.1 Page 58) représente l'objet principal de mon travail
de laboratoire. Il analyse la capacité des lymphocytes T CD8 spécifiques de différent virus à
sécréter de l’interféron gamma (IFN-γ) et/ou de l’interleukine 2 (IL-2) après stimulation par
leur antigène spécifique. Les cellules T CD8 jouent un rôle crucial dans le contrôle des
infections virales. Elles identifient les cellules infectées en détectant des antigènes viraux
4
Simone C. Zimmerli
1. Summaries
présentés à la surface de ces mêmes cellules, et éliminent à la fois les cellules infectées et les
virus qu'elles contiennent en induisant l'apoptose et/ou la lyse des cellules cibles.
Parallèlement, l’identification de l'antigène par la cellule T CD8 la stimule à sécréter des
cytokines. L'IFN-γ en est un exemple. L'IFN-γ stimule les cellules infectées à développer une
activé antivirale intrinsèque. De plus, il attire sur place d'autres cellules de l'inflammation, et
active leur fonction d'éradication des pathogènes. L'IL-2 est un autre exemple. L'IL-2 est
essentielle à l'expansion clonale des cellules T CD8 spécifiques à un virus donné. Elle est
donc essentielle à augmenter le pool de lymphocytes antiviraux. En conséquence, la double
capacité de sécréter de l’IFN-γ et de IL-2 pourrait être un avantage pour le contrôle antiviral
par les cellules T CD8.
Dans ce travail nous avons comparé les proportions de lymphocytes T CD8 doubles
positifs (IFN-γ/IL-2) et simples positifs (IFN-γ) chez trois groupes de sujets: (i) des patients
infectés par VIH-1 qui ne contrôlent pas l’infection (progresseurs), (ii) des patients infectés
par VIH-1, mais contrôlant l’infection malgré l'absence de traitement (“long term non
progressors“ [LTNP]) et (iii) des donneurs de sang négatifs pour l'infection à VIH-1. Les
résultats ont montré que les individus capables de contrôler une infection possédaient des
cellules T CD8 doubles positifs (IFN-γ/IL-2), alors que les patients ne contrôlant pas
l'infection procédaient prioritairement des CD8 simples positifs (IFN-γ). Spécifiquement, les
lymphocytes T spécifiques pour Flu, CMV, EBV, et VIH-1 chez les LTNP étaient tous IFNγ/IL-2 doubles positifs. Au contraire, les lymphocytes T CD8 spécifique à VIH-1 étaient IFNγ simples positifs chez les progresseurs.
La capacité de développer une réponse IFN-γ/IL-2 pourraient être primordiale pour le
contrôle de l'infection, indépendamment de la nature du virus. En effet, il a été montré que
l’absence de sécrétion d'IL2 par les lymphocytes T CD8 corrélait avec leur incapacité de
proliférer. Dans nos mains, cette prolifération a pu être restaurée in vitro par l'adjonction
exogène d’IL-2. Toutefois, la faisabilité de ce type de complémentation in vivo n'est pas clair.
Des expériences additionnelles ont permis de préciser de stade de développement des
lymphocytes doubles positifs et simples positifs par le biais des marqueurs CD45RA et
CCR7. Il reste maintenant à comprendre pourquoi certains lymphocytes T CD8 spécifiques
sont incapables à sécréter de l’IL-2.
Article 2: Le deuxième article explore des nouvelles stratégies pour induire une immunité T
CD8 spécifique aux protéines du VIH-1, qui sont édités et exposés à la surface des cellules
5
Simone C. Zimmerli
1. Summaries
infectées. Ces signaux consistent en fragments de peptide de 8-13 acide aminés provenant de
protéines virales, et exposées à la surface des cellules infectées dans le cadre des molécules
spécialisées d'histocompatibilité de classe I (en anglais "major histocompatibility class I" ou
MHC I). Pour mimer une infection virale, la polyprotéine Gagpolnef du VIH-1 a été insérée et
exprimée dans deux vecteurs viraux atténués, soit MVA (provenant de vaccinia virus) ou
NYVAC (provenant d'un poxvirus). Ensuite des souris ont été infectées avec ces virus
recombinants et la réponse T CD8 aux peptides issus de Gagpolnef a été étudiée. Les souris
ont été capables de développer une réponse de type CD8 T contre ces antigènes du VIH-1.
Pour tester si ces antigènes pouvaient aussi êtres présentés par dans le cadre de molécules
MHC humaines, des expériences supplémentaires ont été faites avec des souris exprimant un
MHC humain. Les résultats de ces manipulations ont montré que des cellules T CD8
spécifique aux protéines du VIH pouvaient êtres détectées. Ce travail ouvre de nouvelles
options quant à l'utilisation des virus recombinants exprimant Gagpolnef comme stratégie
vaccinale contre le virus VIH-1 chez l'homme.
Article 3: Ces revues décrivent la réponse immunitaire à CMV ainsi que des nouvelles
méthodes pouvant servir à sa détection. Une partie du manuscrit décrit la détection de cellule
T à l'aide de tétramères. Il s'agit de protéines chimériques composées de 4 quatre molécules
MHC liées entre elles. Elles sont ensuite "chargées" avec le peptide antigénique approprié, et
utilisée pour détecter les cellules T CD8 spécifiques à ce montage. Elles sont aussi marquées
par un fluorochrome, qui permet une analyse avec un cytomètre de flux, et l'isolement ultime
des CD8 d'intérêt.
En résumé, le travail présenté dans cette thèse indique que (i) une réponse T CD8 appropriée
– définie par la présence des cellules effectrices doublement positives pour l’IFN-γ et l'IL-2 –
semble indispensable pour le contrôle des infections virales, y compris par le VIH-1, (ii) une
telle réponse peut être induite par des vaccin viral recombinant, et (iii) la réponse T CD8 peut
être analysée et suivie grâce à plusieurs techniques, incluant celle des tétramères de MHC
class I.
6
Simone C. Zimmerli
1. Summaries
1.3. Résume pour un large public
Le système immunitaire humain est composé de différents éléments (cellules, tissus et
organes) qui participent aux défenses de l’organisme contre les pathogènes (bactéries, virus).
Parmi ces cellules, les lymphocytes T CD8, également appelés cellules tueuses, jouent un rôle
important dans la réponse immunitaire et le contrôle des infections virales. Les cellules T
CD8 reconnaissent de manière spécifique des fragments de protéines virales qui sont exposés
à la surface des cellules infectées par le virus. Suite à cette reconnaissance, les cellules T CD8
sont capables de détruire et d’éliminer ces cellules infectées, ainsi que les virus qu’elles
contiennent.
Dans le contexte d’une infection par le virus de l’immunodéficience humaine (VIH),
le virus responsable du SIDA, il a pu être montré que la présence des cellules T CD8 est
primordiale. En effet, en l’absence de ces cellules, les individus infectés par le VIH
progressent plus rapidement vers le SIDA.
Au cours de la vie, l’Homme est exposé à plusieurs virus. Mais à l’opposé du VIH,
certains d’entre eux ne causent pas des maladies graves : par exemple le virus de la grippe
(Influenza), le cytomégalovirus ou encore le virus d’Epstein-Barr. Certains de ces virus
peuvent être contrôlés et éliminés de l’organisme (p. ex. le virus de la grippe), alors que
d’autres ne sont que contrôlés par notre système immunitaire et restent présents en petite
quantité dans le corps sans avoir d’effet sur notre santé.
Le sujet de mon travail de thèse porte sur la compréhension du mécanisme de contrôle
des infections virales par le système immunitaire : pourquoi certains virus peuvent être
contrôlés ou même éliminés de l’organisme alors que d’autres, et notamment le VIH, ne le
sont pas.
Ce travail a permis de démontrer que les cellules T CD8 spécifiques du VIH ne
sécrètent pas les mêmes substances, nécessaires au développement d’une réponse antivirale
efficace, que les cellules T CD8 spécifiques des virus contrôlés (le virus de la grippe, le
cytomégalovirus et le virus d’Epstein-Barr). Parallèlement nous avons également observé que
les lymphocytes T CD8 spécifiques du VIH ne possèdent pas la capacité de se diviser. Ils sont
ainsi incapables d’êtres présents en quantité suffisante pour assurer un combat efficace contre
le virus du SIDA. La (les) différence(s) entre les cellules T CD8 spécifiques aux virus
contrôlés (grippe, cytomégalovirus et Epstein-Barr) et au VIH pourront peut-être nous amener
à comprendre comment restaurer une immunité efficace contre ce dernier.
7
Simone C. Zimmerli
2. Abbreviations
2. Abbreviations
Ab
antibody
Ag
antigen
APC
antigen-presenting cells
BCR
B cell receptor
BMT
bone marrow transplanted
C
constant region
CA
capsid
CC
cystein-cystein
CCR7
chemokine receptor 7
CD
Cluster of differentiation
CFSE
5- (and 6-) carboxyfluorescein diacetate succinimidyl ester
CMV
Cytomegalovirus
CTL
cytolytic/cytotoxic T lymphocytes
CXC
cystein-X-cystein
DN
double negative
DNA
desoxyribonucleic acid
DP
double positive
ds
double stranded
EA
early antigen (EBV)
EBERs
Epstein-Barr encoded small RNAs
EBNA
EBV nuclear antigens
EBV
Epstein Barr virus
ER
endoplasmatic reticulum
FACS
fluorescence activated cell sorter
Flu
Influenza
H
hinge region
HA
heamagglutinine
HIV-1
Human immunodeficiency virus 1
IFN-γ
Interferon γ
Ig
immunoglobuline
IL-2
Interleukin 2
IM
Infectious mononucleosis
IN
integrase
ITAMs
immunoreceptor tyrosine-based activation motifs
8
Simone C. Zimmerli
2. Abbreviations
JNK
c-Jun N-terminal kinase
LMP
latent membrane protein
LTNP
long term non progressor
LTR
long terminal repeat (sequence)
M
matrix (Influenza)
MA
matrix (HIV-1)
MHC
major histocompatibility complex
MIP-1α/β
macrophage inflammatory protein 1α/β
m.o.i.
multiplicity of infection
mRNA
messenger RNA
NA
neuraminidase
NC
nucleocapsid
NEP
nuclear export protein
NFκB
nuclear factor κB
NK
natural killer cell
NP
nucleoprotein
NS1/2
nonstructural 1/2
PA
acidic polymerase (Flu)
PAMPs
Pathogen-associated molecular patterns
PB
basic polymerase (Flu)
PB1/2
basic polymerase 1/2
PBMC
peripheral blood mononuclear cells
PR
protease
pTα:β
pre TCR α:β
RANTES
regulated upon activation normal T cell expressed and secreted
RNA
ribonucleic acid
RT
reverse transcriptase
SP
single positive
ss
single stranded
STAT
signal transducers and activators of transcription
SU
surface or gp120
TCM
central memory T cell
TCR
T cell receptor
TEM
effector memory T cell
TH1/TH2
T helper cell 1/2
TM
transmembrane or gp41
9
Simone C. Zimmerli
TNF
tumor necrosis factor
TNFR
tumor necrosis factor receptor
TT
tetanus toxoid
V
variable region
VCA
virus capsid antigen (EBV)
wt
wild type
2. Abbreviations
10
Simone C. Zimmerli
3. Introduction
3. Introduction
3.1. General principles of immunology
3.1.1 The principle of innate and adaptive immunity
Microbes (viruses, prokaryotes, fungi and parasites) represent the vast majority of the earth's
biomass (1). Most of them are indifferent to higher eukaryotes because they never engage
contact with them. In contrast, higher eukaryotes live in the middle of microbes and
constantly interact with them. Therefore they have developed specialized molecules that can
recognize conserved bacterial components and activate intracellular cascades that kill and
digest the microbes. This is a feature of the so-called innate immunity. However, recognition
of conserved microbial molecules by the host has pushed microorganisms to surround
themselves with anti-phagocytic structures, including surface polysaccharids and proteins.
These structures hide the microbial sensitive structures from detection by the host. To cope
with this evolution, higher eukaryotes have developed an additional layer of defense, called
adaptive immunity. Adaptive immunity allows detecting specific molecules of the non-self,
including microbial molecules that are used to trick the first line innate immunity. Typical
examples of such molecules are the pneumococcal antiphagocytic capsule, and the
antiphagocytic M protein on the surface of Streptococcus pyogenes.
3.1.2 The origin of the cellular partners of innate and adaptive immunity
The cells of the immune system are generated in the bone marrow (BM). Most of these cells
mature in the BM before they circulate in the blood and lymphatic system to guard peripheral
tissues. All the cellular elements from the blood arise from the same pluripotent
hematopoietic stem cell in the bone marrow (Fig. 1). Each of them carries specific surface
molecules, called cluster differentiation (CD) markers, which define special functions and
help classify them in different "CD" types.
Innate immunity cells: The myeloid progenitor is the precursor cells of the innate immune
system such as granulocytes, macrophages, dendritic cells and mast cells. Macrophages, the
mature form of monocytes, are distributed in all the tissues. Dendritic cells (DC) are
specialized in uptaking and displaying Ag to adaptive immunity T cells. Before encountering
a pathogen DC are "immature" and continuously traffic from the blood to tissues. They are
both phagocytic and macropinocytic. After engulfing an intruder they mature, express
cytokines and chemokines, and migrate to lymph nodes.
11
Simone C. Zimmerli
3. Introduction
Adaptive immunity cells: The common lymphoid progenitor gives rise to lymphocytes, natural
killer cells and some dendritic cells, although most of dendritic cells arise from the myeloid
progenitors. Most lymphocytes are small, resting cells with few cytoplasmic organelles and
most of their chromatin is inactive. They have no functional activity until they encounter their
specific antigen in combination with co-stimulatory molecules.
Lymphocytes can be divided into B and T lymphocytes. B lymphocytes mature in the bone
marrow. Once they are activated, they differentiate into plasma cells and produce antibodies.
In contrast, T cell precursors are produced in the bone marrow and migrate then to the thymus
for their differentiation into mature T cells (see section 3.2.1, p. 25).
The lymphoid organs: Lymphocytes are generated in the bone marrow and the thymus, which
are referred to as the primary lymphoid organs. They then migrate to the secondary lymphoid
organs, which include the lymph nodes, spleen and lymphoid tissues associated with mucosa
(e.g. gut-associated tonsil, Peyer patches and appendix), where they mature, and initiate and
maintain the adaptive immunity. Lymphoid organs are composed of large numbers of
lymphocytes gathered in a framework of nonlypmphoid cells (e.g. macrophages, granulocytes
and mature dendritic cells displaying the Ag to the lymphocytes).
12
Simone C. Zimmerli
3. Introduction
Figure 1. All the cellular elements of the blood arise from a pluripotent hematopoietic stem cell. Figure from
Immunobiology 6th edition chapter 1.
13
Simone C. Zimmerli
3. Introduction
3.1.3 Innate immunity
Innate immunity is present in newborns, and does not need pre-exposure to foreign microbial
materials to react. It has an intrinsic ability to recognize conserved microbial molecules, also
referred to as PAMPs (pathogen-associated molecular patterns). Hence, it can directly react
against a large number of viruses, bacteria, fungi and parasites (for review see (2-5)).
Innate immunity is always involved in the primary detection of invading microbes, and in the
inflammatory response that aims at calling in other players of the host defense system. It can
kill microbes by itself. Moreover, after having digested a pathogen, professional phagocytes
(which are part of innate immunity) present small fragments of the microbe to cognate Tlymphocytes from adaptive immunity. This is achieved by a sophisticated intercellular
signaling implying the surface receptors of the major MHC class II family on the side of the
innate immunity partner cells, and the T-cell receptor on the side of adaptive immunity
partner cell. The cognate T-lymphocytes then expand and can promote the production of
either cytotoxic cells (TH1 pathway) or antibody (Ab) production (TH2 pathway) to get rid of
the invader. This sophisticated defense network allows both responding to unknown intruders,
and preventing re-infection with a previously known one (see also section 3.1.4., page 17).
The cellular and humoral components in innate immunity have been extensively reviewed (for
instance, see (6)). Cellular factors primarily include macrophages and dendritic cells (Fig. 1),
which are abundant in tissues, and polymorphonuclear neutrophils and monocytes (the
precursors of macrophages) that are abundant in the blood. Soluble factors comprise all the
cellular mediators (cytokines and chemokines) that amplify the inflammatory response to
infection, the complement cascade, as well as alternative soluble components such as the Creactive protein, mannan-binding lectin proteins, platelet-activating factor, and
lipopolysaccharide-binding protein.
Thank to their array of PAMPs-recognizing receptors, the cellular components of innate
immunity can recognize a vast number of microbes, including viruses, bacteria, fungi and
parasite (6). One important antiviral innate protein is INF-γ, which acts mainly as a cytokine,
but can also modify transcription and translation viral polynucleotides by the host cell.
3.1.4 Adaptive immunity
Adaptive immunity results from an adaptation to infection with a specific pathogen. Recall
that the cells of innate immunity recognize only a limited number of microbial molecules, the
repertoire of which is constrained by germ line-encoded receptors. To ensure coverage of the
14
Simone C. Zimmerli
3. Introduction
whole palette of pathogens, including viruses, lymphocytes of adaptive immunity have
evolved Ag receptors specific for infinity of molecules, excluding only those carried by the
host itself. These receptors are determined by a unique genetic mechanism that operates
during lymphocyte development and not described in details here (for review see (6)).
Lymphocyte maturation and survival are regulated by signals received through their very Ag
receptors. A strong signal causes immature lymphocytes to die or to undergo further receptor
rearrangement. The complete absence of a signal can also lead to cell death. This process is
called clonal selection and is a central principle of the adaptive immune system (Fig. 2).
If the Ag is recognized as foreign to the host, then lymphocytes undergo clonal expansion. On
the other hand, if the Ag is recognized as belonging to the self, the particular clone is
eliminated.
A.
C.
foreign antigen
B.
self antigens
D.
self antigens
Effector cells eliminate antigen
Figure 2. Clonal selection. (A) A single progenitor cell gives rise to a large number of lymphocytes each with a
different specificity. (B) Lymphocytes with specificity to ubiquitous self antigens are eliminated before the finish
maturation. This ensures tolerance to self antigens. (C) The remaining mature naïve lymphocytes can recognize
foreign antigens. When an antigen is recognized and bound to the T cell receptor on a naïve lymphocyte, the cell
becomes activated and (D) starts to proliferate. The resulting clone of progeny has all the same receptor binding
the same antigen. Activated lymhocytes differentiate into effector cells and eliminate the antigen. Figure adapted
from Immunobiology 6th edition chapter 1.
15
Simone C. Zimmerli
3. Introduction
3.1.5 The innate and adaptive immunity cooperation
The innate immune response is important for the initiation and subsequent direction of the
adaptive immune response, but also to remove pathogens that have been targeted by the
adaptive immune system. Adaptive immunity is delayed by 4-7 days. In this period the innate
immunity is crucial in controlling infections.
Phagocytic cells may eliminate a number of microbes thank to their PAMPs-recognizing
surface receptors. After microbial engulfment macrophages and dendritic cells mature into
antigen-presenting cells (APC), so called because they can present pieces of the microbial
proteins on their surface, for recognition by T-lymphocytes (see details in section 3.2.3, page
24).
T cells receive survival signals from self-molecules on specialized epithelial cells in the
thymus during development (see Fig. 2) and from the same or similar molecules expressed by
APC in peripheral lymphoid tissues. Lymphocytes must become activated and proliferate to
generate sufficient Ag-specific effector cells to overcome an infection (Fig. 2C and D). When
a lymphocyte recognizes its specific Ag on an APC it stops migrating and starts to enlarge.
The chromatin becomes less dense and the volume of the cytoplasm and nucleus increases.
mRNA and proteins are synthesized. The lymphocyte starts to divide two to four times every
24 hours for three to five days and then differentiate into effector cells. After four to five days,
the clonal expansion is completed. After eradication of infection, most of the Ag-specific cells
undergo apoptosis. Yet, enough of them persist even after clearance of the pathogen to build a
memory cell pool. These cells are then ready to rapidly react and eliminate the pathogen in
case of reinfection with or reactivation of the same pathogen.
B cells and T cells: The adaptive immune response has developed two types of lymphocytes
responding to the different lifestyles of the different pathogens. B cells recognize Ag that are
present in the extra cellular milieu of the host, as it is the case for most bacteria, fungi,
parasites, and extra cellular forms of viruses. In contrast, T cells recognize Ag generated
inside of infected host cells, as in productive or persistent viral infections, as well as in some
infections due to intracellular bacteria (e.g. Listeria monocytogenes) and parasites (e.g.
Plasmodium spp.).
16
Simone C. Zimmerli
3. Introduction
Humoral immune response: B cells recognize and bind soluble Ag via their surface B cell
receptor (BCR). Binding of Ag to BCR transmits a signal that leads to B cell activation,
clonal expansion, and ultimately Ab production. Ab are identical to the B cell receptor (BCR)
except for a small portion of the C-terminus that allows them to be soluble instead of being
anchored to the membrane. They have different functions: they neutralize or opsonize
pathogens, or they activate the complement system, which then directly destroy bacteria, coat
surfaces of pathogens to target them for phagocytes or enhance phagocyte function (Fig. 3).
Ab secreted during infection are found in the plasma and interstitial fluids. Some subclasses
(called isotypes) are also secreted on mucosal surfaces. However, because Ab are soluble
they do not detect intracellular pathogens. Hence, another mechanism is required against such
type of microbes.
17
Simone C. Zimmerli
3. Introduction
Figure 3. Different functions of Ab. Antibodies can (a) neutralize toxins and target them for ingestion by
macrophages (left panel) (b) opsonize baceterias in the extracellular space or (middle panel) or (c) activate
complement system, which then activates phagocytes (right panel). Figure from Immunobiology 6th edition,
chapter 1.
18
Simone C. Zimmerli
3. Introduction
Cell-mediated immune response: This second system depends on a direct interaction between
T lymphocytes and infected host cells. Cells that are infected display an "infection signal" on
their surface. The infection signal is represented by pieces of the invaders' proteins, digested
by the cell and presented in the frame of their MHC class I or MHC class II surface receptors
(Fig. 4).
This response is ensured by two kinds of T cells bearing different tasks. CD8 T cells
recognize Ag displayed on the surface of infected cells and end up killing them (Fig 4A).
CD4 T cells, also called helper cells, do not kill the cognate cells, but activate them to kill
microbes, and produce additional soluble cytokines and chemokines to trigger local
inflammation. One subset of CD4 T cells (TH1 cells) is important in the control of infections
by activating macrophages. Another subset (TH2 cells) stimulates B cells to secrete Ab (Fig.
4B and C). Both CD4 and CD8 T cells recognize their targets by detecting peptide fragments
derived from foreign proteins.
CD8 T cells recognize MHC class I molecules that collect peptides from proteins of the
pathogens (i.e. viral proteins) processed in the cytosol of the infected cell. CD4 T cells
recognize MHC class II molecules binding to peptides derived from proteins in intracellular
vesicles (Fig. 4).
A.
B.
C.
Figure 4. Interaction between T cells and their target cells. (A) Cytotoxic CD8 T cells recognize viral
peptides displayed by MHC class I molecules and kill the infected cells. (B) CD4 TH1 cells recognize bacterial
peptides presented by MHC class II molecules at the surface of macrophages and subsequently activate the latter.
(C) CD4 helper cells recognize antigenic peptides displayed by B cells and activates them to produce Ab. Figure
adapted from Immunobiology 6th edition chapter 1.
19
Simone C. Zimmerli
3. Introduction
3.2. The immune response to viral infection
As mentioned, cell-mediated immunity is the adaptive immune response to infections by
intracellular microbes. Thus, it is important against viruses. While innate immunity and
humoral immunity are also implicated in antiviral defenses, the present dissertation principally
focuses on cell-mediated antiviral immunity.
Viruses bind to receptors on a wide range of cells, infect these cells and replicate in their
cytoplasm. Many viruses cause cytopathic infections, where they kill and sometimes lyse the
host cell after replication. On the other hand, some viruses cause latent infections, where the
viral DNA persists in the nucleus and may become integrated in the host genome. Viral
proteins, but not infectious viral particles, are produced in these infected cells. To handle such
infections, cell-mediated immunity must first activate naïve T cells to proliferate and
differentiate into effector cells, which in turn will eliminate both the host cell and its intruding
virus simultaneously (7).
3.2.1 The surface T cells receptors (CD4 and CD8)
Each T cell bears about 30’000 identical Ag-receptor molecules on its surface. This receptor
is referred to as the T-cell receptor (TCR), and is responsible of Ag recognition. It consists of
two different polypeptides called α and β chains, respectively, linked via a disulfide bond (left
panel of Fig. 5). Some T cells have an alternative γ:δ TCR, the function of which is not fully
understood.
Both chains of the TCR have an extracellular amino-terminal variable region (V) with
homology to the immunoglobulin (Ig) V region, a constant region (C) with homology to the
immunoglobulin C region, and a short hinge region (H) with a cystein residue that forms the
interchain disulfide bond. Each chain spans the plasma membrane by a hydrophobic
transmembrane domain, followed by a carboxy-terminal hydrophilic intracytoplasmic tail (6).
In addition to their TCR, functionally different CD8 and CD4 T lymphocytes express cluster
differentiation types 8 and 4 surface molecules. These CD molecules are co-receptors that
recognize the MHC class I and II molecules, respectively, on the surface of their cognate
target cells. Figure 5 exemplifies the situation with CD8 and MHC class I.
20
Simone C. Zimmerli
3. Introduction
T cell receptor
CD8 co-receptor
MHC class I molecule
carbohydrate
α chain β chain
α
β
peptide-binding
cleft
variable
region (V)
α2
α1
constant
region (C)
α3
β2-microglobulin
hinge (H)
transmembrane
region
+
+
+
cytoplasmic tail
Disulfide bond
Fig. 5 T cell receptor, CD8 co-receptor and MHC class I molecule. The T cell receptor heterodimer is
composed of two transmembrane glycoprotein chains α and β. The extracellular portion consists of a variable
and a constant region which both have carbohydrate side chains attached. The short hinge region contains a
cystein residue, which forms a disulfide bond to connect the two chains. The CD8 molecule is a heterodimer of
an α and a β chain linked by a disulfide bond. MHC class I molecules are expressed on cells of the lymphoid
tissue (T cells, B cells, macrophages, other Ag-presenting cells as for example Langerhans’ cells and weakly on
epithelial cells of the thymus) and other nucleated cells (neutrophils, weakly on hepatocytes, cells of the kidney
and brain). They are heterodimers of a membrane-spanning α chain noncovalently bound to a β 2-microglobulin.
The α chain folds into three domains: α1, α2 and α3. The folding of the α 1 and α2 creates a long cleft which is
the site at which peptide Ag bind to the MHC molecules. Figure modified according to chapter 3
Immunobiology 6th edition
T cell precursors are produced in the bone marrow, and migrate to the thymus where they
differentiate (for ca. one week) before entering a phase of intense proliferation. In young adult
mice, the thymus contains around 108 to 2 x 108 thymocytes. Every day about 5 x 107 new
cells are generated but only about 2-5% of these new cells leave the thymus as mature T cells
to colonize lymphoid organs. About 95% of the cells die within the thymus by apoptosis.
The development of mature T cells is characterized by changes in the status of T cell receptor
genes, in the expression of TCR and by changes in the expression of cell surface proteins such
as CD3 complex and the co-receptor proteins CD4 and CD8. The stages of maturation are
illustrated in Figure 6. In early development, two distinct lineages of T cells with different
TCR (α:β or γ:δ) are produced. Later, α:β T cells develop into the two functional subsets,
namely CD4 and CD8 T cells, as mentioned above. Development of α:β T cells proceeds
21
Simone C. Zimmerli
3. Introduction
through stages where both CD4 and CD8 are expressed by the same cell. These doublepositive cells first express a pre-TCR (pTα:β). They enlarge and divide. Later they become
small resting double-positive cells with low expression of the TCR (α:β). Among these cells,
those which carry a TCR able to interact with self-peptide:self-MHC molecular complexes (it
has to be mentioned that cells undergo apoptosis when the receptor strongly recognize the
MHC-peptide in the thymus a process called negative selection [for review see (8-10)]. This
happens when self-peptide is abundant in the thymus and therefore everywhere in the body
and with TCR with high affinities) lose either expression of CD4 or CD8 to become single
positive thymocytes.
At the same time, they increase their level of TCR expression. These small, resting singlepositive thymocytes are then exported from the thymus as mature single-positive CD4 or CD8
T cells.
22
Simone C. Zimmerli
3. Introduction
DN thymocyte
DN = double negative
DP = double positive
SP = single positive
pT = pre T cell receptor
CD3-4-8-
CD3+γ:δ4-8-
large active
DP thymocyte
apoptosis
CD3+pTα:β4+8+
> 95%
small resting DP
thymocyte
CD3+α:β+4+8+
< 5%
CD3+α:β+4+8-
small resting SP
thymocytes
THYMUS
CD3+α:β+4-8+
export to the periphery
BLOOD
Figure 6. T cell development. Double negative thymocyte precursors develop into CD3+ cells with different
TCR (either γ:δ or α:β). α:β T cells then develop into CD4 and CD8 double positive cells with a precursor TCR.
They enlarge and divide. Later they become small and resting again. Most cells die by apoptosis. The surviving
cells develop in either CD4 or CD8 single positive thymocytes, which are then exported via blood as mature
single positive CD4 or CD8 T cells. Figure modified according to chapter 7 Immunobiology 6th edition
23
Simone C. Zimmerli
3. Introduction
3.2.2 The TH1 - TH2 decision of CD4 T cells
Upon activation (see Fig. 4, page 19), naïve CD4 T cells can differentiate into either TH1 or
TH2 cells. These cells differ in their type of cytokine production and consequently in their
function. Their differentiation fate is decided after the first encounter with the Ag. Stimuli that
determine whether a proliferating CD4 T cell will differentiate into a TH1 or a TH2 cell are not
fully understood. Factors such as cytokines elicited in response to the specific infectious agent
(IFN-γ, IL-12 and IL-2), co-stimulators and the nature of peptide:MHC ligand all play a role.
Selective production of TH1 cells leads to the production of opsonizing Ab classes
(predominantly IgG) and thus is crucial for the activation of macrophages. TH1 cells also
boost cell-mediated immunity by producing IL-2 and IFN-γ. IL-2 is required for CD8 T cell
proliferation and IFN-γ stimulates amongst others CD8 T cell action by upregulating the
expression of the MHC class I molecules of their target cells. In the presence of IL-12
(produced by dendritic cells or macrophages) and IFN-γ (secreted by NK cells or CD8 T
cells), CD4 T cells generally develop into TH1. This is partly due to the inhibition of TH2 cell
proliferation by IFN-γ.
TH2 cells activate B cells, especially in primary responses. Thus, they favor humoral
immunity, and especially the secretion of IgM, IgA and IgE. The presence of IL-4 and
especially of IL-6 preferentially targets CD4 cells to the TH2 differentiation. The initial source
of IL-4 is not yet entirely clear, but it may be the mast cells. IL-4 or IL-10 either alone or in
combination inhibits the generation of TH1 cells.
Hence, although CD4 and CD8 cells apparently guarantee different functions, they are closely
linked by a subtle network of cytokines, chemokines and effector cells, the synchronization of
which is critical to control both infection, via an inflammatory response, and damper
exuberant inflammation, in order to preserve the host from uncontrolled tissue damages.
3.2.3 The special case of CD8 T cells
Because CD8 T cells are implicated in the control of viral infections, they are of special
interest in the present thesis. The CD8 co-receptor is a disulfide-linked heterodimer consisting
of an α and a β chain (different from the α and a β chain of TCR), each consisting of a single
immunoglobulin-like domain linked to the membrane by a segment of extended polypeptide
chain (see middle panel of Fig. 5, page 21). CD8 binds weakly to an invariant site in the α 3
domain of an MHC class I molecule (see right panel in Fig. 5, page 21) and also interacts with
24
Simone C. Zimmerli
3. Introduction
the α2 domain of this molecule. CD8 binds to the membrane-proximal domain of MHC class
I, leaving the rest of the receptor available for interaction with the TCR. CD8 also binds Lck,
a molecule of the signaling cascade, and brings it in close proximity to the TCR.
On their side, MHC class I molecules present short peptides of 8 to 10 amino acids derived
from the infecting microbes (MHC class II molecules bind peptides of at least 13 amino
acids) in a specific grove. These peptides, presented in the context of the rest of the MHC
molecule, drive recognition by specific cognate T-cells.
3.2.3.1 CD8+ cytolytic T lymphocytes (CTL)
CTL recognize MHC class I-associated peptides on infected cells and kill these cells to
eliminate the reservoir of infections. CTL adhere tightly to cells mainly through integrins (e.g.
lymphocyte-function-associated Ag-1 [LFA-1]) on their surface, which bind to their ligands
(e.g. intracellular adhesion molecule-1 [ICAM-1]) on the infected cell. Cell adherence results
in CTL activation, which in turn triggers a signal transduction pathway leading to the
exocytosis of the contents of their granules (i.e. granzymes which target cells through perforin
pores). Each CTL can kill a target cell, detach, and proceed to kill additional target cells.
CTL also secrete cytokines, such as tumor necrosis factor (TNF) and IFN-γ, which play a role
in the inflammatory and antiviral responses (11, 12).
3.2.3.2 CD8 memory T cells
CD8 T cells express numerous additional surface molecules that help identify both
differentiation and functional stages. For instance, expression of the chemokine receptor
CCR7 controls homing to secondary lymphoid organs. CCR7- memory cells mediate
immediate effector function and home preferentially to inflamed tissues. In contrast, CCR7+ T
cells lack immediate effector function and home to secondary lymphoid organs, such as the
lymph nodes.
At least four differentiation subsets of CD8 memory T cells were found based on the coexpression of CCR7 and supplementary CD45RA cluster differentiation molecules (Fig. 7)
(13, 14). First, CD45RA+CCR7+ cells, which are the precursors for the other subsets, and
expand upon Ag re-encountering to ensure continuous replenishment. Second, a subset of
CD45RA-CCR7+ CD8 T cells, also called central memory T cells (TCM). Third, a subset of
CD45RA-CCR7- cells, referred to as effector memory cells (TEM). Fourth, a subset of memory
25
Simone C. Zimmerli
3. Introduction
cells that re-express CD45RA after transient downregulation of it, representing the terminally
differentiated CD45RA+CCR7- cells. These cells are effectors cells that can rapidly intervene
following Ag re-encounter. They secrete IFN-γ and show high levels of perforin expression
(13).
Additional markers include the CD27 and CD28 co-stimulatory molecules, which also
provide information on the CD8 T cell developmental stage (15-18). These co-stimulatory
molecules are downregulated with progressive differentiation. CD45RA-CD27+CD28+ T cells
represent early memory cells. CD45RA-CD27+CD28- T cells are at an intermediate stage.
CD45RA-CD27-CD28- T cells are at a late differentiation stage. And CD45RA+CD27-CD28cells are effector cells with a potent ex vivo cytolytic activity (15-18).
3.2.3.3 Additional cluster differentiation (CD) markers
Brenchley et al. (19) reported that CD57 was a marker of replicative senescence. They
showed that CD8+CD57+ T cells are unable to proliferate, have shorter telomere length, and
have significantly lower numbers of T cell receptor excision circles (TREC) compared to
CD57- cells. This means that this subset of CD8 T cells has undergone more rounds of
proliferation and might be at the end of the differentiation process. In addition, they could
further show that the CD57+ subset is more susceptible to apoptosis, which strengthens the
hypothesis of being terminally differentiated.
Aandahl et al. (20) used the expression of CD7 as a new marker of T cell differentiation.
They found three subsets according to the level of expression of CD7. CD7high, CD7low and
CD7neg. The CD7high subset contained both naïve and memory CD8 T cells, whereas the other
two subsets were only composed of memory cells. The latter two subsets showed significantly
higher levels of cytokine or perforin levels than CD7high cells. Cells secreting cytokines
tended not to express perforin and vice versa. Therefore, the authors proposed that these cells
can be further divided into cytokine producing effector T cells and cytolytic effector T cells.
CD7high cells were able to proliferate in contrast to the other two subsets.
26
Simone C. Zimmerli
3. Introduction
Figure 7. Lineage differentiation pattern of memory CD8+ T lymphocytes. The model is based on the
identification of four subsets of memory CD8 T lymphocytes characterized by the expression of CD8, CD45RA
and CCR7. CD8+CD45RA+CCR7+ cells function as precursor cells for the other memory subsets.
CD45RA+CCR7- cells are the most differentiated cells and therefore referred to as terminally differentiated cells
whereas the CD45RA-CCR7+ are the central memory cells and the CD45RA-CCR7- effector memory cells. The
capacity of effector function increases with increasing degree of differentiation. Figure from Champagne et al.
(2001) Nature 410:106-11
3.2.3.4 Cytokine secretion and T cell proliferation
Cytokines are small soluble proteins secreted by one cell that can either alter its own behavior
and properties (autocrine effect), or alter the behavior of other cells (paracrine effect). Most
cytokines produced by T cells are called interleukin (IL) followed by a number (6). Most of
them demonstrate different biological effects.
Interferon-γ (IFN-γ): The main cytokine released by CD8 effector T cells is IFN-γ, which can
block viral replication or even lead to the elimination of virus from infected cells without
killing them. This includes lowering the intracellular concentration of tryptophan, and hence
decreasing synthesis of polypeptides from the pathogen.
Additional effects of IFN-γ are recruiting macrophages to the infection site and activating
them into APC, and increasing the expression of MHC class I and other molecules involved in
MHC peptide loading for improved recognition of target cells by CD8 effectors. IFN-γ further
27
Simone C. Zimmerli
3. Introduction
activates NK cells. The importance of IFN-γ for intracellular infections was demonstrated in
IFN-γ knock-out mice, which had an increased susceptibility to infections by intracellular
bacteria such as Mycobacterium spp., as well as some viruses (6).
Interleukin-2 (IL-2): Another important cytokine is IL-2. It is produced by an array of cells
including naïve T cells, CD4 TH1 cells and some CD8 T cells. In B cells, IL-2 stimulates both
cell division and J-chain synthesis. In T cells and NK cells, IL-2 stimulates principally cell
division.
Resting T cells constitutively express a moderate affinity IL-2 receptor, which allows them to
respond to very high concentrations of IL-2. Upon activation they resume rapid division, thus
providing the progeny that will differentiate into armed effector cells. The source of IL-2 is
multiple (see above) and includes activated T cells themselves. Activation of T cells by
specific Ag triggers the expression both of IL-2 and of the IL-2 receptor α chain. The α chain
then clusters with the β and γ chains to form a heterotrimer with high affinity for IL-2. This
high affinity allows the newly activated T cells to respond to low concentrations of IL-2. This
leads T cells to divide two or three times a day, allowing one single cell to give rise to a clone
of thousands in a few days, all carrying the same antigen receptor (Fig. 8). In addition of
being a stimulator, IL-2 is also a survival factor for these cells. Removal of IL-2 from
activated T cells results in their death (6).
28
Simone C. Zimmerli
moderate affinity IL-2 receptor
(only IL-2Rβ and γ chains)
3. Introduction
IL-2
high affinity IL-2 receptor
(IL-2Rα, β and γ chains)
IL-2
IL-2Rα
resting T cell
activated T cell
binding of IL-2 stimulates
the T cell to enter the cell
cycle and thus induces cell
proliferation
Figure 8. IL-2 secretion. IL-2 secreted by activated T cells induces T cell proliferation. Resting T cells express
moderate affinity IL-2 receptor consisting of only the β and γ chains. Once T cells become activated they start to
express IL-2 and the IL-2Rα chain. IL-2 binds then to this high affinity IL-2 receptor consisting of an α, a β and
a γ chain, and thus promotes T cell proliferation in an autocrine fashion. Figure adapted from chapter 8
Immunobiology 6th edition.
29
Simone C. Zimmerli
3. Introduction
3.3. General characteristics of viruses
Viruses are small obligate intracellular parasites. This means that they are strictly dependent
on the host cell’s machinery for their replication. The genome of viruses consists either of
DNA or RNA, but never both. The nucleic acids may be single stranded (ss) or double
stranded (ds) and linear or circular. The genome may be constituted of either one single
polynucleotide (in the majority of cases) or by several polynucleotides, as in the
Influenzaviruses (Flu) (Table 1). Viruses are classified by the "International Committee on
Taxonomy of Viruses" according to their type of nucleic acids, their mode of replication and
their shape.
3.3.1 Morphology
Four groups of viruses exist according to their shape: helical, polyhedral, enveloped and
complex viruses. Helical viruses have a helicoidal structure (e.g. viruses that cause rabies or
Ebola). Polyhedral viruses have complex symmetric shapes, e.g. icosahedron, or polyhedron
with 20 triangular faces and 12 corners (e.g. adenovirus, poliovirus). When viruses are
enclosed by an envelope, they are called enveloped viruses (e.g. Influenzavirus is a helical
enveloped virus; and CMV is a polyhedral enveloped virus). Complex viruses are viruses with
a more complicated structure as it is seen in mammalian poxviruses and bacterial
bacteriophages Lambda and T4.
30
Simone C. Zimmerli
Family
3. Introduction
Example
Type of nucleic
Genome
acid
size (kb
Envelope
Capsid
symmetry
or kbp)
RNA containing viruses
Picornaviridae
Poliovirus
SS (+) RNA
7-8
No
I
Astroviridae
Astrovirus
SS (+) RNA
7-8
No
I
Caliciviridae
Norwalk virus
SS (+) RNA
8
No
I
Togaviridae
Rubella virus
SS (+) RNA
10-12
Yes
I
Flaviviridae
Yellow fever virus
SS (+) RNA
10-12
Yes
P
Coronaviridae
Coronavirus
SS (+) RNA
20-33
Yes
H
Rhabdoviridae
Rabies virus
SS (-) RNA
13-16
Yes
H
Paramyxoviridae
Measles virus
SS (-) RNA
15-16
Yes
H
Filoviridae
Ebola virus
SS (-) RNA
19
Yes
H
Arenaviridae
Lymphocytic
2 circular SS RNA
5-7
Yes
H
choriomeningitis virus
segments
California encepahilitis
3 circular SS RNA
10-23
Yes
H
virus
segments
Influenza virus
8 SS (-) RNA
12-15
Yes
H
18-30
No
I
7-11
Yes
I-capsid
Bunyaviridae
Orthomyxoviridae
segments
Reoviridae
Rotavirus
10-12 DS RNA
segments
Retroviridae
HIV-1
2 identical SS (+)
segments
H-nucleocapsid
DNA containing viruses
Hepadnaviridae
Hepatitis B virus
Circular DS DNA
3
Yes
I
with SS portions
Parvoviridae
Human Parvovirus B19
SS (+) or (-) DNA
5
No
I
Polyomaviridae
JC virus
Circular DS DNA
5
No
I
Papillomaviridae
Human Papillomavirus
Circular DS DNA
8
No
I
Adenoviridae
Adenovirus
Linear DS DNA
30-42
No
I
Herpesviridae
Herpes simplex virus
Linear DS DNA
120-122
Yes
I
Poxviridae
Vaccinia virus
130-375
Yes
complex
Linar DS DNA with
covalently closed
ends
Table 1. Classification of Viruses. Kb, kilobase; kbp kilobase pairs; DS; double stranded; SS, single stranded;
(+), message sense; (-), complement of message sense; I, icosahedral; H, helical; P, polyhedral. Information from
Condit RC. Principle of virology. In: Knipe DM, Howley PM, eds. Fields Virology, 4th ed. Philadelphia:
Lippincott-Raven; 2001: 19-51. Table from Mandell 6th edition.
31
Simone C. Zimmerli
3. Introduction
3.4. Viruses used in these studies
3.4.1 Human Immunodeficiency Virus (HIV-1)
3.4.1.1 HIV genes and lifecycle
HIV-1 (Fig. 9) is a complex retrovirus encoding 15 distinct proteins (21). The genome of
HIV-1 encodes 9 open reading frames. Three of these reading frames encode the polyproteins
Gag, Pol and Env. These polyproteins are subsequently proteolyzed into individual proteins
that are common in all retroviruses.
Gag is cleaved into matrix (MA), capsid (CA), nucleocapsid (NC) and p6 proteins. Env gives
rise to surface (SU or gp120) and transmembrane (TM or gp41) proteins. These proteins are
the structural components of the retrovirus. They are found in the core and outer membrane.
The Pol polyprotein is proteolyzed into protease (PR), reverse transcriptase (RT) and
integrase (IN) which all provide essential enzymatic functions.
Six additional - so-called accessory proteins - are encoded by the HIV-1 genome. Vif, Vpr
and Nef are found in the viral particle, Tat and Rev provide essential gene regulatory
functions and Vpu indirectly assists in the assembly of the virion.
5 LTR
vpu
vif
gag
pol
env
vpr
5 LTR
rev
rev
tat
tat
nef
Uncertain function
gag: nuclear core protein
pol: reverse transcriptase protease, integrase
env: envelope glycoprotein
NC
vif: promotes infecitivity
CA
gp120
vpr: weak transcriptional acitvator
vpu: required for efficient virion budding
rev: regulator of structural gene expression
tat: potent transcriptional activator
gp41
p6
PR Vif
RT Vpr
IN Nef
MA
Figure 9. Genetic organization and structure of HIV-1 virus. The HIV-1 genome encodes 9 open reading
frames which give rise to the three polyproteins Gag, Pol and Env and six accessory proteins, Vif, Vpr, Vpu, Tat,
Rev and Nef. For more details see text. Figure adapted from Frankel et al. (1998) Annu. Rev. Biochem 67:1-25
32
Simone C. Zimmerli
3. Introduction
The HIV-1 replication cycle can be divided in 15 steps (Fig. 10)(21). It begins with the
transcription of the viral genes, which are expressed from the promoter located in the 5’LTR.
The rate of transcription is greatly enhanced by Tat (step 1). A set of spliced and genomic
length RNA is transported from the nucleus to the cytoplasm (step 2). This is regulated by
Rev. The viral mRNA is then translated (step 3). The Gag and Gag-Pol polyproteins are
targeted to the cell membrane, whereas Env is targeted to the endoplasmatic reticulum (ER),
where it tends to become associated with naturally synthesized CD4. Thereafter the core Gag
and Gag-Pol polyproteins (which are later processed into MA, CA, NC, p6, PR, RT and IN),
the Vif, Vpr and Nef proteins and the genomic RNA are assembled into an immature virion,
which buds at the cell surface (step 4).
To mature Env into plasma membrane protein SU and TM, the Env polyprotein must be
released from the complex with CD4. Vpu assists the degradation of CD4 (step 5). The Env
polyprotein is then transported to the cell surface (step 6). To prevent further binding of Env
to CD4 at the cell surface Nef promotes endocytosis and degradation of surface CD4 (step 7).
The particle then buds at the cell surface and is released from the host cell (step 8).
During the budding step, the particle is coated with SU and TM. Once released from the cell,
the virion undergoes maturation, which involves morphologic changes such as proteolytic
processing of the Gag and Gag-Pol polyproteins by PR and Vif (step 9). The mature virion is
now ready to infect the next cell by interaction of SU with CD4 and CC or CXC (step 10).
Binding of the virion to the new cell induces conformational changes in TM, which promotes
the fusion of the viral membrane and cell membrane, thus allowing entry of the viral core into
the cytoplasm (step 11). Once inside, the virion is uncoated and the viral core (MA, RT, IN,
Vpr and RNA) becomes exposed (step 12). The nucleoprotein complex is transported into the
nucleus (step 13), where the genomic RNA is reverse transcribed by RT into partially duplex
linear DNA (step 14). After replication, IN catalyzes the integration of viral DNA into the
host chromosome (step 15).
33
Simone C. Zimmerli
3. Introduction
LTR
LTR
LTR
LTR
1
15
2
14
13
3
5
3
6
7
4
12
8
9
10
11
Figure 10. HIV-1 replication cycle. The HIV-1 replication cycle can be divided in 15 steps: (1) viral
transcription, (2) RNA processing and transport, (3) translation and membrane localization, (4) assembly and
budding, (5) CD4 degradation in the ER, (6) Env transport, (7) CD4 endocytosis and degradation, (8) release, (9)
maturation, (10) receptor and co-receptor binding, (11) fusion, (12) uncoating, (13) nuclear transport, (14)
reverse transcription, (15) integration into the host chromosome. Figure adapted from Frankel et al. (1998)
Annu. Rev. Biochem 67:1-25
3.4.1.2 HIV-1 and the Immune System
Humoral immunity
Neutralizing antibodies arise against both Env and gp41 proteins, which are exposed at the
surface of the virus. The first anti-Env response targets the V3 loop of the protein. This
neutralizing Ab response is specific for different viral isolates (22). Because this is a variable
target, it is not appropriate vaccine development. A second anti-Env neutralizing Ab response
targets the Env binding site to the CD4 receptor on CD4 helper cells. This binding site is
highly conserved among viral isolates. However, Ab directed against this site are weakly
neutralizing. A third target of neutralizing Ab is the transmembrane gp41 (23), but its
protective efficacy is not demonstrated. Thus, although a neutralizing Ab-response does exist
against HIV-1, humoral immunity does not contribute much to blocking viral replication. On
34
Simone C. Zimmerli
3. Introduction
the contrary, it rather selects for viral variants that may pass undetected by existing Abs (23,
24).
Cell-mediated immunity
CD8 T cells: There are several arguments that point to the importance of specific CD8 T cell
response to control HIV-1 infection. First depletion of CD8 T cells by specific anti-CD8
monoclonal antibodies resulted in a failure to control the early peak of viremia in infected
animals (25). Second HLA types may significantly influence the rate of HIV-1 disease
progression. HLA-B27 and HLA-B57 haplotypes are associated with slow disease
progression, whereas HLA-B35 is associated with faster disease progression. This
phenomenon could be due to differences in the capacities of HLA molecules to present the Ag
(26). Third HIV-specific CD8 T cell responses have been observed in subjects and/or animals
that had been exposed to the virus but did not become infected (27-29).
In primary HIV-1 infection, a potent CTL response coincides with the peak in viremia and
preceeds the Ab response (30-32). HIV-1-specific CD8 T cells are found in large numbers in
a variety of anatomic compartments, such as peripheral blood, bronchoalveolar spaces, lymph
nodes, spleen, skin, cerebrospinal fluid, semen, and vaginal and gastrointestinal mucosal
tissues. These CD8 T cells were shown to inhibit viral replication in vitro (33). They secrete
MIP-1α, MIP-1β and RANTES to lyse HIV-1 infected cells and block viral propagation (34).
Moreover, there is an association between the appearance of CTL and a decline in the viral
RNA in primary infection (30, 31, 35).
Yet, despite the appearance of a vigorous CTL response during primary HIV-1 infection the
immune system is unable to sufficiently control the infection. CD8 T cells show abnormalities
at both functional and maturation levels. Appay et al. (36) showed a selective defect in the
levels of intracellular perforin, and two other studies demonstrated poor lytic activity in
freshly isolated HIV-1-specific T cells (37, 38). HIV-1-specific CD8 T cells show a skewed
maturation, the memory population of HIV-1-specific CD8 T cells being mainly composed of
preterminally differentiated cells. In comparison, CMV-specific CD8 T cells are
predominantly terminally differentiated cells (14). Eventually, certain clones of HIV-1specific CD8 T cells are rapidly deleted during primary infection (39). CD8 T cells expressing
Fas receptor – a receptor that renders cells susceptible to apoptosis - are significantly higher
in HIV-1 infected than in non-infected individuals and the difference is even more
pronounced in patients with a more advanced stage of infection (40). A decreased expression
of Bcl-2 – a repressor of apoptosis – was shown ex vivo as well in CD8 as CD4 T cells and
35
Simone C. Zimmerli
3. Introduction
there was an association with apoptosis of those cells. Interestingly antiretroviral therapy
increased the proportion of Bcl-2 expressing T cells (41).
CD4 T cells: Virus-specific CD4 T lymphocytes also have an important role in controlling
HIV-1 replication. Indeed, many HIV-1 infected individuals have virus-specific CD4 T cells
(42, 43). Rosenberg et al. (44) showed that the magnitude of the proliferation and cytokine
secretion in HIV-1-specific CD4 T cells correlated with the clinical status. Several studies
have reported a selective defect of HIV-1-specific CD4 helper T cells in progressive HIV-1
infection. In contrast, HIV-1-specific CD4 T cells with effector function (secreting IFN-γ and
TNF-α) persist (42, 44, 45).
The decrease of CD4 T cells with the progression of HIV could be shown to be independent
of Fas expression (40). As mentioned in the preceding paragraph CD4 T cells expressing low
levels of Bcl-2 can be detected. But in apoptotic cells not only Bcl-2 but also CD4 are
downregulated and therefore it is likely that the percentage of apoptotic CD4 T cells is
underestimated. The same is true for CD8 T cells even though in a smaller extend (41).
In long term non-progressors (LTNP, see next paragraph) the situation is different. Both,
HIV-1 specific CD4 helper and effector T cell responses can be found (46).
3.4.1.3 Clinical manifestations
The first cases of AIDS were described in the United States in 1981, in homosexual males
from New York and San Francisco. These patients presented with unexplained opportunistic
infections, such as Pneumocystis carinii (recently renamed Pneumocystis jirovecii)
pneumonia and Kaposi’s sarcoma (47-49).
Primary HIV infection is difficult to diagnose, because symptoms such as fever, lethargy,
malaise, sore throat, arthralgias, headaches, retroorbital pain, photophobia, lymphadenopathy
and maculopapular rash are non-specific and can precede the humoral immune response to
HIV (50-53). The duration of these symptoms can last from a few days up to more than 10
weeks. However, usually the duration does not go beyond 14 days (54).
Primary infection is followed by a long phase of clinical latency, which lasts in typical
progressors between 8-10 years in absence of treatment. A minority of patients, the so-called
rapid progressors, develop AIDS within 2-3 years after primary infection (55, 56). Figure 7
shows the natural history of HIV-1 infection in a typical untreated patient. By the end of the
disease, the count of CD4 T cells drops radically and is accompanied by sharp increase in
36
Simone C. Zimmerli
3. Introduction
viral load. With the introduction of highly active antiretroviral therapies (HAART), the
mortality rate dramatically decreased. In the United States death due to AIDS decreased by
23% in 1996 and by 44% in 1997 (57, 58). A study of the Swiss HIV cohort showed that
treatment with HAART lead to a mean reduction of viremia of 1.8 log10 copies per milliliter.
63% of the patients reached undetectable viremia. Regarding CD4 counts the course was
more variable. After a peak-increase of 40 cells per microliter during the first 6 months, a
gradual decrease was observed thereafter (59). Another study performed in Switzerland
revealed that the risk of death in patients treated with HAART is equal to that in patients with
cured cancer (60).
Figure 11. Natural history of HIV infection in absence of therapy (from Fauci AS et al. (1996) Ann Intern Med
124:654-663)
37
Simone C. Zimmerli
3. Introduction
HIV infection can be divided in different stages. Table 2 shows the 1993 revised classification
system. The last stage of HIV-infection is marked by a series of tumors, opportunistic
infections and HIV-related neurological diseases (Table 2).
Clinical
categories
CD4
T cell categories
1
≥ 500/µl
2
200-499/µl
3
≤ 200/µl
A
B
C
Asymptomatic,
acute (primary) HIV
Symptomatic not A
or C conditions
AIDS-Indicator
conditions*
A1
B1
C1
A2
B2
C2
A3
B3
C3
Table 2. Classification system for HIV-1 infection. * AIDS-indicator conditions are listed in table 3. From
CDC and Prevention 1993 (1992) MMWR 41:1-19
About 5% of the HIV-infected patients do not progress to HIV disease and have stable CD4 T
cell counts within the normal range for many years (61, 62). This group of patients is called
long term non-progressors (LTNP). Low degree of virus trapping is observed in lymph node
biopsies of LTNP, and virus-expressing cells are rarely seen (63). The virological parameters
(HIV-1 RNA and proviral DNA) are significantly lower (4-20 fold) compared to progressors
(64). CD4 T cell counts are constantly in the normal range (>500 cells/µl), whereas CD8 T
cell counts were found to be significantly increased (500-2500 cells/µl). The proliferative
response to mitogens and alloantigens is conserved and a vigorous HIV-1-specific humoral
and cellular immune response can be observed. HIV-1-specific cytotoxicity can be seen ex
vivo or in vitro. On histology, there is a lower degree of reactivity of lymphoid tissues, as
compared to progressors (50).
38
Simone C. Zimmerli
-
3. Introduction
multiple or recurrent bacterial infections
Candidiasis of bronchi, trachea or lungs
esophageal Candidiasis
invasive, cervical cancer
disseminated or extrapulmonary Coccidiodomycosis
extrapulmonary Cryptococcosis
chronic intestinal Cryptosporidiosis (>1 month duration)
CMV disease (other than liver, spleen, or nodes)
CMV retinitis (with loss of vision)
HIV related encephalopathy
Herpes simplex, chronic ulcer(s) (> 1 month duration); or bronchitis,
pneumonitis, or esophagitis
disseminated or extrapulmonary Histoplasmosis
chronic, intestinal Isosporiasis (> 1 month duration)
Kaposi’s sarcoma
Lymphoid interstitial pneumonia and/or pulmonary lymphoid
hyperplasia
Burkitt’s lymphoma
immunoblastic lymphoma
primary lymphoma of the brain
disseminated or extrapulmonary Mycobacterium avium-intracellulare complex or
Mycobacterium kansaii
pulmonary or extrapulmonary (any site) Mycobacterium tuberculosis
disseminated or extrapulmonary Mycobacterium (other or unidentified species)
Pneumocystis jirovecii pneumonia
recurrent pneumonia
progressive mulitfocal leukoencephalopathy
recurrent Salmonella septicemia
Toxoplasmosis of the brain
wasting syndrome of HIV infection
Table 3. AIDS indicator conditions (adapted from Mandell 6th edition)
39
Simone C. Zimmerli
3. Introduction
3.4.1.4 Treatment and prevention
Treatment
HIV-1 infection is treated with antiretroviral therapies (ART). In general a combination of
different drugs is indicated due to development of drug resistances. It is now common to
administer so-called highly active antiretroviral therapy (HAART). Three classes of
substances are used: nucleoside reverse transcriptase inhibitors (NRTI), non-NRTI (NNRTI)
and protease inhibitors (PI). NRTI are used in combination with PI and/or NNRI.
Combinations of two nucleoside analogs RT inhibitors with a potent protease inhibitor have
shown to be effective (65, 66). It is important to underscore that HAART cannot eradicate the
virus. AIDS is therefore still not curable.
NRTI target the enzyme reverse transcriptase. They are nucleoside analogues that lack 3’OH
groups. Hence, when mistakenly incorporated in polynucleotide strands during transcription
of replication, they irreversibly block strand elongation (67). Zidovudine (AZT) and D4T are
thymidine analogs, DCC and 3TC are cytosine analogs, DDI is an inosin analog, and abacavir
is a guanosin analog. Nucleoside analogs are activated inside the cell by phosphorylation.
They are mainly eliminated via the kidney (67).
NNRTIs bind directly to the reverse transcriptase, close to the binding site of nucleosides.
Examples for NNRTIs are neverapin, delavirdin and efavirenz. They do not require
phosphorylation or intracellular processing to become activated (67)
Protein inhibitors inhibit maturation of the gag-pol polyprotein. This results in non-infectious
particles (68). Protein inhibitors bind directly to the active site of the enzyme to inhibit its
action. They result in a clear improvement of therapy, but they are associated with several
problems, especially in long term treatment. They affect the lipid metabolism and can trigger
lipodystrophy and dyslipidemia. Furthermore, they have quite a short half-life and have to be
taken three times a day.
Post exposure prophylaxis
This intervention has been proposed since 1990 in case of inadvertent exposure to HIV. If
provided within the first 24 hours following exposure, post exposure prophylaxis can
attenuate viral replication and prevent systemic HIV infection. It is likely to act by blocking
the infection of T cells in the regional lymph nodes. Additionally it might allow the
development of a robust HIV-1-specific cellular response (67).
40
Simone C. Zimmerli
3. Introduction
The efficacy of post exposure prophylaxis was demonstrated in animal models. (1) It delayed
or completely suppressed viremia, (2) it inhibited viral replication and resulted in a longlasting, protective cellular immune response, and (3) it provided complete protection (69-74).
Importantly, protection appeared closely dependent on both the schedule of drug
administration and the window between exposure and treatment start. Tsai et al. (70) showed
in a macaque/SIV study that all the macaques treated for 28 days remained uninfected, while
50% of the animals treated only for 10 days and none of those treated for 3 days were
protected. Likewise, animals treated within 24 hours remained uninfected, whereas only half
of the animal treated within 48 hours and 25% of the animals that started treatment 72 hours
after exposure were protected (70). Thus, drug posology and timing of administration is
critical.
The perspective of anti-HIV vaccines
Twenty-four years after discovery of the first AIDS cases, HIV infection is still one of the
major health problems worldwide. In the year 2004, 39.4 million people living with HIV were
counted, and 4.9 million were newly infected with HIV, whereas 3.1 million people died of
AIDS (75). Especially in the Sub-Saharan Africa with 25.4 million HIV-infected individuals
and no accessibility of HAART AIDS is a major problem (Fig. 12). To break through this
pandemic a safe, effective and affordable vaccine would be the only solution. However,
developing a vaccine against HIV requires overcoming several challenges:
•
Natural infection with HIV does not result in protective immunity.
•
HIV is difficult to neutralize with Ab for several reasons, including carbohydrate
shielding of gp120 subunit, occlusion of envelope epitopes via oligomerization (76),
conformational masking of receptor binding sites (77) and mutational variation of
envelope variable loops (23, 24).
•
There is both a progressive destruction and impaired regeneration, of CD4 helper cells
(50).
•
HIV has a high mutation rate and is therefore able to escape from humoral (78) and
cellular response (35, 79, 80).
•
High levels of HIV genetic diversity might require more than one vaccine for global
prevention and prevent HIV superinfections in patients with broad CD8 T cell. (8183).
41
Simone C. Zimmerli
3. Introduction
Difficulties in generating neutralizing Ab is revealed by the failure of this approach in early
vaccination studies (84-87). The first Phase III trial with recombinant monomeric HIV-1
envelope gp 120 vaccine (AIDSVAX; VaxGen Inc, Brisbane, CA, USA) showed no efficacy
(84). Likewise, the second phase III trial of VaxGen’s AIDSVAX B/E composed of
monomeric recombinant gp120s representing HIV subtypes B and E, did not provide antiHIV protection either (86).
Because of these failures scientists now focus on eliciting a potent antiviral CD8 T cell
response. This type of vaccine will not be able to prevent HIV infection, but might control
viral expansion, thus slowing down the progression to AIDS and decreasing the risk of HIV
transmission (88-91). Moreover, inducing HIV-specific CD8 T cell response before HIV
infection might be favorable in preserving the function of CD4 helper cells, which in turn
promote a sustained CD8 T cell response (92).
There are now several candidate vaccines focusing on the CD8 T cells in clinical evaluation.
Most of them are still phase I trials, in order to assess safety and generate data to choose the
best HIV immunogens and vector systems (92).
North America
1.0 million
(540 000-1.6 million)
Western Europe
610 000
North Africa
and Middle East
25.4 million
(270 000-780 000)
(23.4-28.4 million)
(1.3-2.2 million)
(920 000-2.1 million)
(480 000-760 000)
Caribbean
440 000
Latin America
1.7 million
Eastern Europe
and Central Asia
1.4 million
Sub-Saharan Africa
25.4 million
(23.4-28.4 million)
East Asia
1.1 million
(560 000-1.8 million)
South and South-East Asia
7.1 million
(4.4-10.6 million)
Oceania
35 000
(25 000-48 000)
Figure 12. Geographic distribution of HIV-infections. Figure from UNAIDS: AIDS epidemics update
December 2004
42
Simone C. Zimmerli
3. Introduction
3.4.2 Cytomegalovirus (CMV)
3.4.2.1 Viral structure, genes and lifecycle
CMV is a member of the human herpes virus family (β-Herpes viridae; Table 1). The virion
of CMV consists of a 100 nm icosahedral nucleocapsid. It contains a 230 kb double stranded
linear DNA genome, surrounded by a protein aqueous layer called the tegument. The
tegument itself is enclosed by a lipid bilayer spun by several viral glycoproteins (Fig. 13). The
mature virion particle is about 150-200nm in size (93).
tegument
nucleocapsid
glycoprotein
dsDNA genome
membrane
Figure 13. Structure of herpes viruses. The tegument surrounds the icosahedral nucleocapsid containing the
double stranded DNA genome. A lipid bilayer membrane covered with glycoproteins envelops the tegument
itself.
The genome of CMV has a protein-coding capacity of 170-200 open reading frames (94).
Predominant structural proteins of the virion are the envelope proteins gB and gH and the
matrix proteins pp65/pp150/pp71 and pp28 (95). The life cycle of herpes viruses is depicted
in Figure 14. It starts with attachment and penetration of the virus into the cell via cell surface
proteoglycans (step 1). As in HIV, the membranes of the virus and the host cell fuse together,
leading to the release of the viral particle into the cytoplasm. Inside the cell the viral DNA is
uncoated and rapidly translocated to the nucleus (step 2). Sequential transcription (step 3A) of
immediate-early, early and late viral genes in the nucleus is followed by their translation in
43
Simone C. Zimmerli
3. Introduction
the cytosol (step 3B). DNA is replicated by the mechanism of rolling circle (step 4). The new
viral genome is cleaved, assembled with viral proteins translocated from the cytosol and
packed inside the nucleus (step 5). After viral maturation (step 6) the nucleocapsid buds out of
the nucleus and acquires thus a primary envelope (step 7). In the cytoplasm the virion further
matures through a de-envelopment/re-envelopment process thereby acquiring their tegument
(step 8). The tegument capsids then receive their definitive envelope by budding into vesicles
of the Golgi apparatus. Finally mature virions are released from the cell via an exocytosis-like
pathway (step 9) (96-98). The CMV genome can also be kept latently in the cell. Productive
infection is suppressed by histones binding to the genome establishing latency.
uncoating of viral DNA
attachment and
penetration
1
DNA replication
(rolling circle)
2
Sequential
transcription and
translation of
immediate-early,
early and late
genes
4
cleavage,
assembly and
packaging
5
3A
6
α mRNA
β mRNA
γ mRNA
3B
α, β and γ
proteins
7
release of virions
8
maturation
9
viral glycoproteins
Figure 14. Life cycle of herpes viruses. 1. Herpes entry by attachment and penetration. 2. Release of viral DNA
3. Sequential transcription (A) and translation (B) of viral immediate-early, early and late genes 4. DNA
replication by rolling circle mechanism 5. Assembly and packaging 6. Maturation and 7. Budding of the
nucleocapsid out of the nucleus 8. further maturation 9. release of the virion via exocytosis-like pathway.
44
Simone C. Zimmerli
3. Introduction
3.4.2.2 CMV and the Immune System
CMV is a very potent immunogen, which triggers all arms of the immune system. Humoral
immunity is established early in infection. IgG antibodies measurement is the standard assay
for determining infection history. However, the cellular immune response is thought to be the
major mechanism by which viral replication is controlled.
CD8 T cell response seems to be the most important component of it, even though CD4 T
cells and NK cells also play an important role in controlling CMV (99). CD8 T cell response
is focused on two CMV proteins, which are the pp65 tegument protein and IE-1 protein (100,
101). The use of a pp65-deleted viral mutant showed that 70-90% of all the CD8 T cell clones
isolated by limiting dilution analysis were specific for peptides derived from this protein.
Nevertheless, it was recently observed that many other proteins also carry peptides that are
epitopes for CD8 T cells (102). (101). Such alternative epitopes are responsible for 1-2% of
the total anti-CMV repertoire of CD8 T cells.
3.4.2.3 CMV pathogenesis and clinical manifestations
CMV initially enters the host via the epithelium layers of the upper alimentary tract,
respiratory tract, or genitourinary tract (103). Leukocytes and vascular endothelial cells help
in disseminating the virus. During acute infection, viral antigens are mainly found in
neutrophils and monocytes (103). Virus encoded chemokines can also support the spread of
the virus by attracting neutrophils and monocytes (104, 105). The hematogeneous spreading is
followed by infection of ductal epithelial cells. CMV-infected cells can be found in the
salivary gland, bile duct, bronchial and renal tubular epithelium, islet cells, epithelial cells of
the inner ear, the capillary endothelium, astrocytes and neurons (106, 107). CMV resides in
the host throughout life without causing any symptoms in healthy, immuocompetent
individuals. Depending on the residence country and social status, up to 50-90% of adults are
seropositive for the virus (108-110).
Primary infection is in most cases unapparent but can be associated with infectious
mononucleosis (IM). IM is caused by EBV, HIV and in 21% of the cases by CMV (111). This
can be associated with several complications including interstitial pneumonia specially in
bone marrow transplanted (BMT) patients but rarely in immunocompetent hosts (111),
hepatitis, mostly with mild or even without symptoms in immunocompetent individuals (67),
G u i l l a i n - B a r r é syndrome (67, 112), meningoencephalitis (113), m y o c a r d i t i s ,
thrombocytopenia and hemolytic anemia as well as skin eruptions (67).
45
Simone C. Zimmerli
3. Introduction
These symptoms are usually mild in immunocompetent patients, but can become dramatic in
the immunocompromised host (see below). Primary CMV infection is followed by a
prolonged, unapparent infection during which the virus remains alive but usually dormant and
resides in cells without causing detectable damage or clinical illness.
Either primary or reactivated infection is frequent in immunocompromised patients. In the
case of AIDS, CMV is the cause of the most common opportunistic viral infections (67). It
can cause retinitis (114, 115), neurological infections such as encephalitis, polyradiculopathy,
or peripheral neuropathy, and gastrointestinal infections such as esophageal ulcer, colitis,
acute pancreatitis or cholecystitis (67).
Transplant patients also have a special risk of CMV infection, since the virus is either
transmitted by the donor organ (primary infection) or reactivated due to immunosuppressive
drugs (reactivation). The most common complication is CMV pneumonia in BMT patients,
and a potentially fatal CMV hepatitis following transplantation of a liver, especially if the
organ comes from a CMV positive donor. Following kidney transplantation, the generalized
CMV syndrome is more frequent than CMV hepatitis or CMV pneumonia (67).
Eventually, a particular form of CMV disease is represented by intrauterine infection of the
fetus. Intrauterine infection may occur in 0.5-22% of pregnant women. In the non-immune
mothers it may be associated with serious disease of the fetus, including jaundice,
hepatosplenomegaly, petechial rush, and multiorgan involvement. Neurologic complications
include microcephaly, motor disability, chorioretinitis and cerebral calcifications (67).
3.4.2.4 Treatment and prevention
Therapy
Therapy includes 3 antiviral drugs inhibiting viral DNA polymerase for treatment of CMV
end-organ disease, antisense inibitor CMV directly injected into intravitreal fluid for
treatment of CMV retinitis and antiviral therapy following BMT or solid organ transplantation
(67).
In addition to classical antiviral drugs, several studies indicated that prophylactic treatment of
patients with CMV-specific CD8 T cells results in a CD8 T cell response that was equivalent
to the responses found in healthy seropositive subjects resisting CMV infection (95, 116-118).
This was useful in the early period following allogeneic stem cell transplantation. This kind of
therapy was also shown to be effective in a patient infected with a multidrug resistant CMV
46
Simone C. Zimmerli
3. Introduction
isolate (119). Thus, adoptive transfer of CMV-specific T cell clones represents a new strategy
for restoring protective host immunity and preventing CMV disease in these patients (116).
Prevention
CMV disease can be prevented after bone marrow or solid organ transplantation by antiviral
therapy. Four independent studies from 1991-95 showed that ganciclovir administrated orally
following transplantation was effective in preventing CMV pneumonia or other CMV
associated diseases (67).
As for HIV, an effective vaccine against CMV is not yet available. However, several vaccine
candidates have been or are being tested in humans. The first candidate is a live attenuated
CMV isolate. The second is a chimera between an attenuated CMV and wild-type virus. The
third is a non-replicating canarypox vector with either a pp65 core or a gB envelope Ag. The
fourth is composed of a recombinant envelope glycoprotein vaccine. The fifth is a mixture of
synthetic peptides including a CD4 T helper epitope, known CD8 epitopes and a lipid tail.
Other vaccines such as a DNA vaccine and a recombinant vaccine are also proposed (120).
All these candidates have proved safe. However, their efficacy in clinical trials has yet to be
demonstrated (120).
3.4.3. Epstein-Barr virus (EBV)
3.4.3.1 Viral structure, genes and lifecycle
EBV is a γ -herpes virus with a linear double-stranded DNA genome (Table 1). Viral
replication involves expression of over 60 proteins involved in the lytic cycle (immediateearly [IE], early [E] and late [L] proteins). BZLF1 and BRLF1 act as transactivators and are
IE genes, BMRF1, BALF2, BALF5, BBLF2/3, BBLF4, BSLF1 play a role as replication
factors and are E genes. Gp350/220, VCA, Gp35, Gp25, Gp42 are structural proteins and are
L genes (121). The BALF5 gene encodes the catalytic subunit of a DNA polymerase (122),
BMRF1 the DNA polymerase accessory subunit (123-125) and BALF2 a ssDNA binding
protein (126, 127). BBLF4, BSLF1 and BBLF2/3 are thought to form a tight complex, which
acts as helicase, primase and helicase-primase associated proteins (128). It is suggested that
all of them, with the exception of BZLF1, work at the replication fork to synthesize the
leading and lagging strands of the concatemeric EBV genome (129).
BZLF1 acts on viral replication and transcription as oriLyt binding protein and transactivator.
It can mediate the switch between latent and lytic forms of the EBV infection, and is
47
Simone C. Zimmerli
3. Introduction
sufficient for the activation of the lytic cascade (130). It further interacts with the CREBbinding protein to activate EBV early gene expression (131). BZLF1 can inhibit host cell
proliferation by causing a cell cycle arrest in the G0/G1 phase (132, 133) and it can interact
with the activated form of p53 inhibiting its transactivation function, which then prevents
downstream signal transduction (134).
Beside the lytic cycle proteins, the EBV genome encodes nine latency-associated proteins. Six
are localized in the nucleus and are called EBV nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C
and LP, whereas three are membrane proteins and referred to as latent membrane proteins
(LMPs) 1, 2A and 2B. EBNA1 links the viral genome to the cellular chromosome, which
enables viral replication in dividing host cells as if it was a part of the cellular genome.
EBNA2 controls the transcriptional machinery, which is required to block B-cell
differentiation at the proliferative stage. EBNA3C allows the activated cells to progress into
the G1/S boundary of the cell cycle.
LMP1 is a ligand-independent cell-surface signaling molecule providing a surrogate T-helper
cell signal. It interacts with the signaling system of the tumor necrosis factor receptor (TNRF)
family. It activates the nuclear factor κB (NFκB), c-Jun N-terminal kinase (JNK) and signal
transducers and activators of transcription (STAT) pathways in order to provide both survival
and growth signals. LMP2A is also a ligand-independent cell surface signaling molecule. It
interacts with members of the Src family of tyrosine kinases through immunoreceptor
tyrosine-based activation motifs (ITAMs), which are also found in the α- and β- chains of the
B cell receptor (BCR). The signal triggered by LMP2A resembles the signal provided by the
intact BCR for positive selection of B cells in the bone marrow and for survival of mature B
cells in the periphery in absence of cognate Ag (135).
EBV specifically infects resting B cells via CD21 and HLA class II molecules on the cell
surface (136). This infection first induces continuous proliferation, which results in
lymphoblastoid cell lines. These cells express a limited number of gene products, such as
EBNAs, LMPs, two EBV-encoded small RNAs (EBER1 and EBER2), and transcripts from
the BamHI region. This type of cell cycle is called the latent phase. Quiescent B cells are
activated to enter the cell cycle, to maintain continuous proliferation and are prevented from
apoptosis (121). During the latent cell cycle EBV genomic DNA, which is in form of a closed
circular plasmid, behaves like the host chromosomal DNA. It associates with cellular
histones, it replicates only once during the S phase and is then distributed to the daughter cells
(137-139). Only one viral cis element, the OriP and one viral protein, EBNA1 is required for
maintenance (140, 141).
48
Simone C. Zimmerli
3. Introduction
The latent cell cycle can change into a lytic cell cycle, where multiple rounds of replication
are initiated within the OriLyt (130). In the lytic cell cycle, the replication has a greater
dependence on EBV-encoded proteins (128). The IE lytic genes BZLF1 and BRLF1 are
expressed to activate viral and certain cellular promoters and to lead to a cascade of viral gene
expression. First early genes which play a role in DNA replication and metabolism are
expressed followed by late genes, which code for structural proteins. The EBV genome is
amplified 100- to 1000 fold.
3.4.3.2 EBV and the immune system
During primary infection, anti-virus capsid antigen (VCA) IgM and anti-early antigen (EA)
IgM are secreted followed by a transient development of anti-EA IgG. This is found in about
80% of the infected individuals. Anti-EBNA IgG are absent during acute infection (142).
Early in the acute phase of infection, NK cells limit the number of EBV-infected cells (143).
Later a large proportion of CTL that are directed towards EBV emerge. There are two
immunodominant EBV lytic cycle epitopes and one immunodominant EBV latent cycle
epitope. During primary infection, there is a massive expansion of activated Ag-specific CD8
T cells, which can persist at in relatively high proportion for at least three years after primary
infection (144).
During acute infection the cellular response to lytic cell cycle proteins is about ten times
stronger than to latent cycle proteins. In follow-up samples at 6 and 37 months after primary
infection, the response to lytic proteins decreases, but is still detectable. In contrast, the
frequencies of CD8 T cells specific to latent proteins is similar during acute and chronic
infection (144).
The EBV glycoprotein gp350 binds to B cells via CD21, whereas gp85 plays an important
role in the fusion of the virus to the host cell. Neutralizing Ab are directed against these two
glycoproteins, and a CTL response induced against these targets have shown to protect mice
against infection with recombinant vaccinia virus expressing those epitopes (145).
Viral escape strategies
EBV has developed several strategies to avoid host defense. It limits gene expression during
latency in B cells, produces proteins that inhibit apoptosis and Ag processing, and expresses a
cytokine and a soluble cytokine receptor.
49
Simone C. Zimmerli
3. Introduction
There are different viral proteins modulating the immune response. LMP-1 is the homolog of
cellular CD40. It binds TNF-receptor-associated factors, activates B cells, stimulates B cell
proliferation, up regulates NFκB and kinase and inhibits apoptosis (146-155). BHRF1 is a
homolog of bcl-2 and therefore inhibits apoptosis (156). BARF1 a homolog of Colony
stimulating factor 1 receptor inhibits proliferation and function of macrophages (157). BCRF1
an IL-10 homolog inhibits IFN-γ and IL-12, promotes B cell growth and inhibits dendritic
cells (158, 159) and EBNA-1 is able to inhibit antigen presentation (160).
3.4.3.3 EBV pathogenesis, clinical manifestation and complications
EBV preferentially infects B cells and epithelial cells but is also able to infect NK cells and
smooth muscle cells (161, 162). EBV infects B cells through binding of gp350 to CD21 and
gp42 to HLA II as co receptor (136). The incubation period lasts between 2 and 7 weeks
(163).
Primary infection is often unapparent. The classical clinical manifestation is a glandular fever
tonsillitis also called infectious mononucleosis (IM). Rarely a virus-associated
haemophagocytic syndrome (HPS) occurs (164, 165). During primary infection, a marked
lymphocytosis is common, due to an hyperexpansion of CTL reacting to lytic and latent viral
Ag (149). In rare cases, IM can trigger several complications including neutropenia,
thrombocytopenia, splenic rupture, airway obstruction by tonsillar hypertrophy, central
nervous system involvements and acute liver failure (166).
In the latent phase, several complications can occur such as different types of neoplasia and
hematological disorders including X-linked lymphoproliferative disorder (Duncan disease),
malignant lymphomas in patients with AIDS or immunosuppressive therapy, gastric cancer
and pleural lymphoma (167-170). In some geographical regions, hematological disorders
occurs in association with endemic EBV infection (Table 4) (171-177).
Africa
Burkitt’s lymphoma
Southern China
Nasopharyngeal carcinoma
Asian and central and south American countries
Nasal-type NK/T cell lymphomas
Japan
Chronic active EBV infection
EBV-associated haemophagocytical
lmphohistiocytosis (178)
Table 4. Endemic appearance of EBV associated hematological disorders
50
Simone C. Zimmerli
3. Introduction
3.4.3.4 Treatment
There is no established treatment against EBV infection. Thus supportive therapy is given.
More than 95% of the patients recover without specific therapy.
The treatment of lymphoproliferative secondary complications usually consists in decreasing
the immune suppression, in patients receiving active immnosuppressive therapy (regression in
up to 50% of the cases) or antiviral therapy generally combined with immunoglobulins.
However latently EBV infected cells do not express EBV DNA polymerase. Therefore,
polymerase inhibitors do not act against latent EBV. In addition, Ab against B cells or cellular
immunotherapy, which reconstitutes the cellular immune response, may be tried. The
disadvantage of this therapy is the induction graft versus host disease by the infused
alloreactive T cells in transplanted patients (67).
3.4.4 Influenzavirus (Flu)
3.4.4.1 Viral structure, genes and lifecycle
The Influenza virus belongs to the family of Orthomyxoviridae (Table 1 and Fig. 15)(179). Its
genome is constituted of 8 single-stranded RNA segments, referred to as negative strands
because the viral mRNAs are transcribed directly from them. The entire genome is embedded
in a helical-shaped nucleocapsid, further enveloped in a lipid bilayer acquired by budding
through the membrane of the host cell.
The whole virion has a diameter of about 80-120nm. The Influenza A virion has a layer of
about 500 spikes (haemagglutinine [HA] and neuraminidase [NA]) radiating through the outer
membrane (179).
The first RNA segment encodes for PB2, which is responsible for Cap binding and
endonucleolytic cleavage. Segment 2 encodes for a basic polymerase 1 (PB1), which is an
RNA transcriptase. Segment 3 encodes for an acidic polymerase (PA) of unknown function.
Segment 4 encodes for haemagglutinine (HA). HA is exposed at the surface of the virus and
mediates viral attachment to cellular receptors and subsequent membrane fusion. Segment 5
encodes for the nucleoprotein (NP). NP encapsulates the RNA and plays a role in the
regulation of transcription and translation. Segment 6 encodes for neuraminidase (NA). NA is
part of the envelope and cleaves sialic acid from the cell surface. It facilitates the release from
membranes and prevents aggregation. Segment 7 encodes for the matrix proteins M, which
surrounds the viral core and controls nuclear transport and M2. M2 is an ion channel required
for uncoating the virus after entry into the cell. Segment 8 encodes for nonstructural proteins
51
Simone C. Zimmerli
3. Introduction
NS1 and NS2. NS1 is an antagonist of type I interferons, probably involved in regulation of
the mRNA transport from the nucleus to the cytoplasm. NS2, also called nuclear export
protein (NEP), plays a role in the transport of newly assembled ribonucleoprotein from the
nucleus to the cytoplasm (180).
neuraminidase
haemagglutinine
protein layer
lipid bilayer
RNA
ribonuleoprotein
(nucleocapsid)
nucleoprotein
Figure 15. Structure of Influenzavirus. The ssRNA genome is associated with nucleoproteins, enveloped in
the nucleocapsid and surrounded by a matrix. The surface of the virion is covered with haemagglutinine and
neuraminidase molecules. Figure modified according to Fields Virology Chapter 45, Orthomyxoviridae: The
Viruses and Their Replication.
52
Simone C. Zimmerli
3. Introduction
The lifecycle of Influenzavirus (Fig. 16) starts with its entry into the host cell by receptormediated endocytosis (step 1). Once inside the cell, the virus is uncoated and disassembled in
the endosome (step 2). The nucelocapsid consisting of viral RNA and nucleoproteins
translocates to the nucleus, where the viral genome is replicated (step 3) and transcribed (step
4). Viral proteins are translated in the cytosol (step 5). Nucleoproteins re-enter the nucleus
where they are packed together with the RNA in the ribonucleoprotein core (step 6). Other
proteins are further modified in the ER and Golgi-apparatus (step 7) and some are transported
to the cell membrane of the host cell (step 8). The new virions leave the cell by budding (step
9). At the end the viruses are released and spread (step 10) (180).
Viral envelope
Viral genome (RNA)
endosome
1.
5.
3.
2.
ribosome
cell membrane
4.
nucleus
ribonucleoprotein core
viral proteins
6.
Via
endoplasmatic
reticulum
7.
9.
8.
10.
Golgi apparatus
Figure 16. Life cycle of Influenza virus. Step 1, entrance of virus into the cell by receptor mediated
endocytosis. Step 2, uncoating of the viral particle. Step 3, replication of viral RNA . Step 4, transcription of
viral genome. Step 5, translation of viral proteins. Step 6, packaging of the ribonucleoproteincore and
translocation out of the nucleus. Step 7, maturation of viral proteins in the endoplasmatic reticulum and Golgi
apparatus. Step 8, budding. Step 9,.release and spread of virus.
53
Simone C. Zimmerli
3. Introduction
3.4.4.2 Influenza and the immune system
Humoral immunity
Antibodies (IgM, IgA and IgG) generated during the primary response are completely
protective against a secondary challenge with the same virus (181-183). However, they are
generated late in primary response and do not play a critical role in clearance of primary
infection unless the viral load is very high (184-186). Moreover, they are not protective
against challenge with serologically different viruses (181-183).
Only antibodies to HA are neutralizing. Antibodies to NA are not neutralizing, but may
reduce efficient release from infected cells. Antibodies to M or NP do not play a role in
protective immunity (67).
Cell-mediated immunity
During primary infection, CD8 T cell response is essential to clear Influenzavirus from the
lungs of infected mice (184, 187-189). Dendritic cells and macrophages carry the antigen
from the lungs to the lymph nodes (190), where they prime naïve CD8 T cells. The activated
CD8 T cells then migrate to the lungs to clear the infection (191). Under normal
circumstances Flu-specific CD8 T cells appear in the lung at day7 and peak at day 9 to 10.
There is a correlation between the appearance of virus-specific CD8 T cells and the clearance
of primary infection, which is either performed via perforin or the Fas mechanism (192, 193).
In contrast to humoral immunity, cellular response to cross-reactive epitopes can provide
substantial protection against serologically different viruses. Even though they do not prevent
re-infection, they are able to reduce the viral load and clear the virus faster (194, 195).
Flu-specific CD8 T cells primed during primary infection persist and enable a rapid and
vigorous response to secondary challenges. Some of the virus-specific CD8 T cells persist in
the lungs. These cells were shown to be able to secrete IFN-γ directly ex vivo. Yet, they did
not exhibit cytolytic function directly ex vivo, although they could be induced to proliferate
and acquire cytolytic function after re-stimulation in vitro (196). CD4 T cells also contribute
to protection both by promoting the humoral antiviral response (188, 197), and by the
secretion of IFN-γ (184, 198, 199).
Viral escape strategies
Several Influenza epidemics have been recorded during the 19th and 20th century, but the first
pandemic was not accurately recorded until 1889-92. A second pandemic occurred in 191819, known as the Spanish Influenza, which was lethal for 20-25 Mio people, especially young
54
Simone C. Zimmerli
3. Introduction
adults. There were further pandemics in 1957 (Asian Flu) and 1968 (Hong Kong Flu). Every
year, almost identical epidemics occur in most of the countries. However extensive pandemics
occur only every 10 to 12 years.
Antigenic shift: Viruses recovered from different pandemics show a wide variation, which
means that pandemics occur because of appearance of new Influenza A subtypes, against
which the population has no immunity (antigenic shift). The appearance of new subtypes
parallels the disappearance of old subtypes. HA is always involved in the antigenic shift since
HA is a binding site for Ab.
There are three different theories for the appearance of antigenic shift. The first implies a
mechanism of reassortment. Reassorted viruses result from double infections with human and
animal or bird viruses, where the 8 RNA segments of each strain reassort to form a new virus.
This is possible because Influenza A is capable of crossing species barriers. The second
possibility is a gradual adaptation of animal viruses to human. The third possibility is the
recirculation of existing subtypes. This implicates that several Influenza A subtypes exist in
the human population and that they are recycled. However, the evidence supporting this
hypothesis is not very strong (180).
Antigenic drift: In addition to large pandemics smaller epidemics occur regularly. Viruses
causing these epidemics show some strain differences since they do not completely crossreact with preexisting antibodies (antigenic drift). This antigenic drift results from the
accumulation of spontaneous mutations, which are responsible for amino acid substitutions at
antigenic sites. This evolution is likely to be driven by the pressure of neutralizing Ab in the
population. In case of antigenic drift, prior immunization yields only partial immunity (180).
3.4.4.3 Pathogenesis, clinical manifestations and complications
Infection of cells with Influenzavirus causes cell death by necrosis and apoptosis. Especially
bronchiolar epithelial cells and alveolar cells are concerned. The virus is released from the
cell before the latter dies and infects nearby or adjacent cells. Infection of polymorphic
nuclear cells, lymphocytes and monocytes is nonproductive but causes dysfunctions of those
cells such as defective chemotaxis and phagocytosis (200) or decreased proliferation and costimulation (201, 202).
Influenzavirus is spread via respiratory droplets. The virus binds to cells of the respiratory
tract, which are rich in viral receptors. The neuraminidase in the envelope helps the virus to
55
Simone C. Zimmerli
3. Introduction
be spread by releasing virus particles, which have been bound by mucous present on the
surface of epithelial cells.
The incubation period lasts between 18 and 72 hours, before an abrupt onset of symptoms
such as fever, headache, photophobia, shivering, dry cough, malaise, myalgia and a dry
tickling throat occurs. Several complications can arise in association with Influenza infection.
Pulmonary complications include primary Influenza viral pneumonia, as well as secondary
bacterial pneumonia mostly caused by Streptococcus pneumoniae, Haemophilus influenzae or
Staphylococcus aureus. Further pulmonary complications are Croup and acute exacerbation
of chronic bronchitis.
Examples for nonpulmonary complications are myositis, cardiac complications such as
myocarditis and pericarditis (rarely) and central nervous complications such as Guillain-Barré
syndrome and Reye’s syndrome (67).
3.4.4.4 Treatment and prevention
Therapy
Flu normally is a self-limiting virus, which means that the virus is cleared without treatment.
Nevertheless in some cases treatment is indicated. There are two kinds of approaches. First
M2 inhibitors inhibiting the M2 ion channel activity and thus inhibiting virus uncoating (203,
204). Second, NA inhibitors, which prevent detachment of the virus from host cells and curb
the local and distant spread of the virus (67).
Prevention
Vaccines have been available for 50 years, but their efficacy is only partial, i.e. in
approximately 65% (205, 206). Moreover, their ability to prevent epidemics could not be
proven. Antibodies against HA reduce the severity of infection and decrease virus spreading
in infected persons. Antibodies against NA also contribute to protection.
There are different types of vaccines:
•
Whole virus vaccine, which was the first Influenza vaccine to be produced. The virus
was inoculated into embryonated eggs, harvested 2-3 days later and inactivated. This
confers 60-90% of protection during 1-5 years. The protection is less effective if the
infecting virus undergoes progressive Ag drift.
•
Split virus vaccines, where inactivated particles are disrupted with detergents. This
type of vaccine induces fewer side effects than whole virus vaccines, but has the same
immunogenicity.
56
Simone C. Zimmerli
•
3. Introduction
Subunit virus vaccines. These contain only HA and NA antigens. They are used in
aqueous suspension or absorbed to carriers such as alhydrogel. Aqueous suspensions
demonstrate fewer side effects than whole virus vaccines or vaccines absorbed to
carriers.
•
Life attenuated vaccines. These induce a stronger immunity than inactivated vaccines.
The problem is that normal methods for attenuation such as repeated passages and
temperature adaptation require long periods of development and manufacturing.
Hence an attenuated vaccine against a given strain may already be obsolete at the time
of application. One possibility is to mix already attenuated strains with wild type (wt)
virus to produce recombinants with appropriate RNA fragments. However, even with
modern technologies the purification and safety procedures require ca. 2 years for
production. Much too long considering the yearly turnover of new viruses.
57
Simone C. Zimmerli
4. Results
4. Results
4.1. Publication #1
HIV-1-Specific IFN-γ/IL-2 Secreting CD8 T Cells Support CD4Independent Proliferation of HIV-1-Specific CD8 T Cells
Simone C. Zimmerli, Alexandre Harari, Cristina Cellerai, Florence Vallelian, PierrreAlexandre Bart and Giuseppe Pantaleo
Proc Natl Acad Sci U S A (2005) 102: 7239-7244)
Summary
Previous studies in monkeys and humans have indicated the importance of CD8 T cells in the
initial control of HIV-1 infection. The absence of CD8 T cells was correlated with high viral
load and rapid progression to AIDS. However, the different conditions defining the type of
CD8 T cell response are not yet established. Important criteria could be the Ag load (dose) on
the one hand, or the duration of persistence (time) on the other hand.
In this work, functional and phenotypic characterization of virus-specific CD8 T cells against
Cytomegalovirus (CMV), Epstein Barr virus (EBV), Influenzavirus (Flu) and HIV-1 were
performed on the basis of the ability of CD8 T cells to secrete IFN-γ and IL-2, and their
capacity to proliferate and to express CD45RA and CCR7. The different viruses served as
models of distinct Ag persistence and dose. Flu reflects a situation of pathogen clearance,
CMV, EBV and HIV-1 in LTNP of repetitive Ag exposure at low Ag dose, whereas HIV-1 is
a model for Ag persistence at high Ag dose.
We identified two functional distinct populations of CD8 T cells, namely dual IFN-γ/IL-2 and
single IFN-γ secreting cells. Virus-specific IFN-γ/IL-2 secreting CD8 T cells were CD45RACCR7-. In contrast, single IFN-γ
CD8 T cells were either CD45RA
CCR7- or
CD45RA+CCR7-. We found a good correlation (p<0.02) between the proportion of virusspecific IFN-γ/IL-2 secreting CD8 T cells on the one hand, and the specific proliferation
capacity of CD8 T cells on the other hand. The loss of HIV-1-specific CD8 T cells, capable to
secrete IL-2, was associated with the failure of HIV-1-specific CD8 T cells to proliferate.
Even in CD4 T cell-depleted populations, or after stimulation with MHC class I tetramer-
58
Simone C. Zimmerli
4. Results
peptide complexes, a substantial proliferation of virus-specific CD8 T cells could be
observed. IL-2 was the factor responsible for the CD4-independent CD8 T cell proliferation.
Taken together, our results indicate: a) a wide heterogeneity of antiviral CD8 T cell immune
responses under different conditions of virus persistence, b) a combined loss of virus-specific
IFN-γ/IL-2 secreting and proliferating CD8 T cells in progressive HIV-1 infection, c) a typical
phenotype of effector cells, i.e. CD45RA-CCR7-, for the IFN-γ/IL-2 secreting CD8 T cells, d)
a correlation between the proportion of virus-specific IL-2 secreting and proliferating CD8 T
cells, and e) the occurrence of Ag-specific CD8 T cell proliferation also in experimental
conditions excluding the involvement of Ag-specific helper CD4 T cells.
59
Simone C. Zimmerli
4. Results
HIV-1-specific IFN-␥兾IL-2-secreting CD8 T cells
support CD4-independent proliferation
of HIV-1-specific CD8 T cells
Simone C. Zimmerli, Alexandre Harari, Cristina Cellerai, Florence Vallelian, Pierre-Alexandre Bart,
and Giuseppe Pantaleo*
Department of Medicine, Division of Immunology and Allergy, Laboratory of AIDS Immunopathogenesis, Centre Hospitalier Universitaire Vaudois,
University of Lausanne, 1011 Lausanne, Switzerland
helper CD4 T cells in sustaining Ag-specific CD8 T cell proliferation (13).
Recent studies (14–16) investigating antiviral memory CD4 T
cell responses have shown that the combined assessment of IL-2 and
IFN-␥ is instrumental to distinguish functionally distinct populations of memory CD4 T cells and patterns of antiviral immune
responses associated with different conditions of virus persistence
and control.
In the present study, we have performed functional and phenotypic characterization of antiviral CD8 T cell responses specific for
HIV-1, CMV, EBV and influenza (flu) on the basis of their ability
to proliferate, to secrete IL-2 and IFN-␥, and to express CD45RA
and CCR7. Our results indicate: (i) a wide heterogeneity of antiviral
CD8 T cell immune responses under different conditions of virus
persistence; (ii) a combined loss of virus-specific IFN-␥兾IL-2secreting and -proliferating CD8 T cells in progressive HIV-1
infection; (iii) a typical phenotype of effector cells, i.e.,
CD45RA⫺CCR7⫺, for the IFN-␥兾IL-2-secreting CD8 T cells; (iv)
a correlation between the proportion of virus-specific IL-2secreting and -proliferating CD8 T cells; and (v) the occurrence of
Ag-specific CD8 T cell proliferation also in experimental conditions, excluding the involvement of Ag-specific helper CD4 T cells.
Functional and phenotypic characterization of virus-specific CD8 T
cells against cytomegalovirus, Epstein–Barr virus, influenza (flu),
and HIV-1 were performed on the basis of the ability of CD8 T cells
to secrete IFN-␥ and IL-2, to proliferate, and to express CD45RA and
CCR7. Two functional distinct populations of CD8 T cells were
identified: (i) dual IFN-␥兾IL-2-secreting cells and (ii) single IFN-␥secreting cells. Virus-specific IFN-␥兾IL-2-secreting CD8 T cells were
CD45RAⴚCCR7ⴚ, whereas single IFN-␥ CD8 T cells were either
CD45RAⴚCCR7ⴚ or CD45RAⴙCCR7ⴚ. The proportion of virusspecific IFN-␥兾IL-2-secreting CD8 T cells correlated with that of
proliferating CD8 T cells, and the loss of HIV-1-specific IL-2-secreting CD8 T cells was associated with that of HIV-1-specific CD8 T cell
proliferation. Substantial proliferation of virus-specific CD8 T cells
(including HIV-1-specific CD8 T cells) was also observed in CD4 T
cell-depleted populations or after stimulation with MHC class I
tetramer–peptide complexes. IL-2 was the factor responsible for
the CD4-independent CD8 T cell proliferation. These results indicate that IFN-␥兾IL-2-secreting CD8 T cells may promote antigenspecific proliferation of CD8 T cells even in the absence of helper
CD4 T cells.
C
D8 T cells play a critical role in the control of viral infections
(reviewed in ref. 1). Several studies have shown a wide heterogeneity of memory CD8 and CD4 T cells with multiple phenotypes
and functions in response to virus infections (2–7). Functionally
distinct populations of CD8 T cells can be defined by the expression
of CD45RA and CCR7 (8) and are able to proliferate and兾or to
secrete cytokines such as IL-2, IFN-␥, and TNF-␣ after antigen
(Ag)-specific stimulation (9–11). The determination of quantitative
and qualitative changes of virus-specific CD8 T cells in rapidly
controlled acute, more slowly controlled or uncontrolled chronic
infections showed that high load of lymphocytic choriomeningitis
virus resulted in the progressive diminution of the ability of CD8 T
cells to produce IL-2, TNF-␣, and IFN-␥ (9). Of interest, the
capacity to secrete cytokines could be restored if the viral load was
brought under control (9).
IL-2 production from virus-specific CD8 T cells has been the
object of few studies in humans. Recent studies have shown that a
variable percentage of cytomegalovirus (CMV)- and Epstein–Barr
virus (EBV)-specific CD8 T cells were able to secrete IL-2 (10, 11),
whereas IL-2 was not produced by melanoma-1-specific CD8 T cells
obtained from patients with stage IV melanoma (10). With regard
to HIV-1 infection, no studies have investigated the ability of
HIV-1-specific CD8 T cells to secrete IL-2. However, it has been
shown that HIV-1-specific CD8 T cells of HIV-1-infected subjects
with nonprogressive disease, i.e., long-term nonprogressors
(LTNPs), had greater proliferation capacity as compared with
HIV-1-specific CD8 T cells from progressors (12), and this finding
was associated with a better ability to control virus replication (12).
A recent study has shown that the loss of HIV-1-specific CD8 T cell
proliferation was associated with the loss of HIV-1-specific helper
CD4 T cells and has proposed a critical role of HIV-1-specific
Materials and Methods
Study Groups. The 21 subjects with progressive chronic HIV-1
infection enrolled in this study were naı¨ve to antiviral therapy, with
CD4 T cell counts of ⬎250 cells per microliter (mean ⫾ SE: 810 ⫾
39) and plasma viremia counts of ⱖ5,000 HIV-1 RNA copies per
ml (mean ⫾ SE: 41,854 ⫾ 12,339). Five HIV-1-infected patients
with nonprogressive disease, i.e., LTNPs, as defined by documented
HIV-1 infection for ⬎14 years, stable CD4 T cell counts of ⬎500
cells per microliter (mean ⫾ SE: 912 ⫾ 125) and plasma viremia of
⬍1,000 HIV-1 RNA copies per ml (mean ⫾ SE: 97 ⫾ 38) were also
included. Patient 1010 has a documented HIV-1 infection since
March 1999. He was treated with antiviral therapy at the time of
primary infection and remained on antiviral therapy for 18 months.
He interrupted therapy spontaneously in December 2000. During
the last 4 years, he constantly had levels of viremia of ⬍50 HIV-1
RNA copies per ml and CD4 T cell count in the range of 1,400 cells
per microliter. In addition, blood from 28 HIV-negative subjects
was obtained from the local blood bank or from laboratory coworkers. The studies were approved by the Institutional Review
Board of the Centre Hospitalier Universitaire Vaudois.
Freely available online through the PNAS open access option.
Abbreviations: EBV, Epstein–Barr virus; CMV, cytomegalovirus; Ag, antigen; LTNP, longterm nonprogressor; CFSE, carboxyfluorescein succinimidyl ester; SEB, staphylococcal enterotoxin B.
*To whom correspondence should be addressed at: Laboratory of AIDS Immunopathogenesis, Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, Rue
Bugnon, 1011 Lausanne, Switzerland. E-mail: [email protected].
© 2005 by The National Academy of Sciences of the USA
PNAS 兩 May 17, 2005 兩 vol. 102 兩 no. 20 兩 7239 –7244
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0502393102
60
IMMUNOLOGY
Communicated by Anthony S. Fauci, National Institutes of Health, Bethesda, MD, March 23, 2005 (received for review December 18, 2004)
Simone C. Zimmerli
4. Results
lococcal enterotoxin B (SEB) stimulation (200 ng兾ml) served as
positive control. Where indicated, 10% exogenous IL-2 (Roche,
Basel) was added 48 h after peptide stimulation. For neutralization
experiments, anti-IL-2-neutralizing Ab or isotype control Ab (Becton Dickinson) were added at 10 ␮g兾ml. At day 5, cells were
harvested and stained with CD4-PE-Cy5 (Becton Dickinson) and
CD8-APC (Becton Dickinson). Cells were fixed with CellFix
(Becton Dickinson) and acquired (1–8 ⫻ 105 nongated events) on
a FACScalibur (Becton Dickinson).
Synthetic Peptides and Tetramers. The following individual peptides
were used: A2-restricted CMV pp65 (amino acids 495–503: NLVPMVATV) peptide (17), B7-restricted CMV pp65 (amino acids
415–429: TPRVTGGGAM) peptide (17), A2-restricted EBV
BMLF1 (amino acids 259–267: GLCTLVAML) peptide (18),
B8-restricted EBV EBNA3A (amino acids 325–333: FLRGRAYGL) peptide (18), B8-restricted EBV BZLF1 (amino acids
190–197: RAFKQLL) peptide (18), A2-restricted flu matrix 1
(amino acids 58–66: GILGFVFTL) peptide (19), A2-restricted
HIV-1 pol (amino acids 476–484: ILKEPVHGV) (20), A2restricted HIV-1 gag (amino acids 77–85: SLYNTVATL) (21),
B8-restricted HIV-1 gag (amino acids 259–267: GEIYKRWII),
(22) or B8-restricted HIV-1 nef (amino acids 89–97: FLKEKGGL)
(23) peptides. Cells were stimulated with HIV-1 (strain IIIB)
peptide pools. Each pool consisted of 50–62 15-mers peptides
overlapping by 11 amino acids (Synpep, Dublin, CA). Pools 1–6
spanned the gag, pol, and nef sequence; pool 1: amino acids 1–230;
pool 2: amino acids 220–432; pool 3: amino acids 421–655; pool 4:
amino acids 645–879; pool 5: amino acids 871-1103; and pool 6:
amino acids 1043–1326. CMV-, EBV-, or flu-derived peptides were
used either all in a pool or grouped as virus-specific pools (24).
For tetramer stimulations, A2- and B7-restricted class I peptide
tetramers were produced as described (25, 26).
CD4 T Cell Depletion. CFSE-labeled cells were stained with CD4-
APC and sorted by using a FACS Vantage (Becton Dickinson). The
purity of the CD4-depleted cell populations was 99%.
Statistical Analysis. Statistical significance (P values) of the results
was calculated by using a two-tailed Student t test. A two-tailed P
value of ⬍0.05 was considered significant. The correlations among
variables were tested by simple regression analysis.
Results
Distinct Cytokine Secreting Populations of Virus-Specific CD8 T Cells.
We used different models of virus-specific CD8 T cell responses,
including HIV-1-, CMV-, EBV-, and flu-specific CD8 T cell
responses. Based on the observation that functionally distinct
Ag-specific CD4 T cell populations are defined by the secretion of
IL-2 and IFN-␥ (14–16), we performed functional characterization
of virus-specific CD8 T cell responses by simultaneous assessment
of IFN-␥ and IL-2 secretion after Ag-specific stimulation. Representative examples obtained from the analysis of 21 HIV-1-infected
progressors and 28 HIV-negative blood donors in whom CMV-,
EBV-, or flu-specific CD8 responses were detected are shown in
Fig. 1A. The dual IFN-␥兾IL-2-secreting T cells were absent in
HIV-1-specific CD8 T cells, whereas they were found within CMV-,
EBV-, and flu-specific CD8 T cells (Fig. 1 A). These observations
were confirmed by the analysis of a larger number of subjects. A
significant difference was found between the percentage of HIV1-specific IFN-␥兾IL-2-secreting cells in progressive HIV-1 infection
and that found in the virus-specific IFN-␥兾IL-2-secreting CD8 T
cells (P ⬍ 0.05) of the other virus infections (Fig. 1B). We also
evaluated the proportion of IL-2-secreting cells within IFN-␥secreting CD8 T cells. Cumulative data of this analysis are shown
in Fig. 1C. The proportion of CMV-specific (12.7 ⫾ 1.8%, n ⫽ 11)
and EBV-specific (19.2 ⫾ 3.2%, n ⫽ 10) IL-2-secreting CD8 T cells
was significantly higher (P ⬍ 0.05) compared with that of HIV-1specific IL-2-secreting CD8 T cells (2.3 ⫾ 0.6%, n ⫽ 21) (Fig. 1C).
The proportion (25.6 ⫾ 3.6%, n ⫽ 7) of flu-specific IL-2-secreting
CD8 T cells was significantly higher (P ⬍ 0.05) compared with that
Detection of IFN-␥ and IL-2 Secretion. Cell stimulations were performed as described (14). For stimulation of CD8 T cells, individual
peptides (5 ␮g兾ml) or peptide pools (1 ␮g兾ml for each peptide)
were used. Cells were then stained with CD8-PerCP-Cy5.5, CD69FITC, IFN-␥-APC and IL-2-PE (Becton Dickinson, Franklin, NJ).
For phenotypic analysis, the following Abs were used in combination: Rat anti-human CCR7 (Becton Dickinson) followed by goat
anti-rat IgG(H⫹L)-APC (Caltag, Burlingame, CA), CD8-Pacific
blue (DAKO, Glostrup, Denmark), CD45RA-Biotin followed by
anti-Streptavidin-PercP, anti-CD69-APC-Cy7, anti-IL-2-PE, and
anti-IFN-␥-FITC (Becton Dickinson). Data were acquired on a
FACScalibur or an LSR II and analyzed by using CELLQUEST and
DIVA software (Becton Dickinson). The number of nongated events
ranged between 105 and 106 events.
Ex Vivo Proliferation Assay. After an overnight rest, cells were
washed twice, resuspended at 1 ⫻ 106 ml in PBS, and incubated for
7 min at 37°C with 0.25 ␮M carboxyfluorescein succinimidyl ester
(CFSE; Molecular Probes). The reaction was quenched with 1
volume of FCS, and cells were washed and cultured in the presence
of anti-CD28 Ab (0.5 ␮g兾ml) (Becton Dickinson). Cells were either
stimulated with HIV-1 peptide pools (1 ␮g兾ml of each peptide),
individual peptides (5 ␮g兾ml), or tetramers (0.31 ␮g兾ml). Staphy-
Fig. 1. Analysis of different virus-specific IFN-␥- and IL-2-secreting CD8 T cells after stimulation with single peptides. (A) Distribution of IFN-␥- and IL-2-secreting
virus-specific CD8 T cells. Cells were stimulated with single peptides. One representative profile is shown for HIV-1-, CMV-, EBV-, or flu-specific CD8 T cell responses.
The cluster of events shown in red corresponds to the responder CD8 T cells, i.e., secreting IFN-␥ or IL-2, and the blue clusters correspond to the nonresponder
cells. (B) Cumulative data on the percentage (mean ⫾ SE) of IFN-␥兾IL-2-secreting cells within the different virus-specific CD8 T cell responses. (C) Cumulative data
on the proportion (mean ⫾ SE) of IL-2-secreting cells within IFN-␥-secreting CD8 T cells. *, P ⬍ 0.05.
7240 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0502393102
Zimmerli et al.
61
Simone C. Zimmerli
4. Results
IMMUNOLOGY
of HIV-1- and CMV-specific but not with that of EBV-specific
IL-2-secreting CD8 T cells (Fig. 1C). Finally, the proportion of
EBV-specific IL-2-secreting cells was also significantly higher compared with that of CMV-specific IL-2-secreting CD8 T cells (P ⬍
0.05) (Fig. 1C). CMV-, EBV-, and flu-specific CD8 T cell responses
were also studied in HIV-1-infected individuals either by using
peptides specific to CMV and EBV (n ⫽ 7) and flu (n ⫽ 6) or a pool
of 21 CMV-, EBV-, and flu-derived peptides in 30 HIV-1-infected
subjects. The proportion of CMV-, EBV-, or flu-specific IL-2secreting CD8 T cells in HIV-1-infected subjects was not significantly different from that observed in HIV-negative subjects
(P ⬎ 0.05).
To exclude the possibility that the lack of detection of HIV-1specific IFN-␥兾IL-2-secreting CD8 T cells was specific of the
response to certain peptides, we performed stimulation with peptide pools spanning gag, pol, and nef proteins of HIV-1. A
representative flow cytometry profile of one (of 21) HIV-1-infected
subjects with progressive disease (progressors) is shown in Fig. 2A.
Despite the presence of HIV-1-specific IFN-␥-secreting CD8 T
cells after stimulation with different HIV-1 peptide pools, IL-2secreting CD8 T cells were not detected (Fig. 2 A).
Previous studies (12) have shown that HIV-1-specific CD8 T cells
of LTNPs, but not of progressors, proliferated in response to
Ag-specific stimulation (12). The evaluation of the presence of
HIV-1-specific IFN-␥兾IL-2-secreting CD8 T cells in three of five
representative LTNPs showed variable intensities of the response to
the different peptide pools (Fig. 2B). HIV-1-specific IFN-␥secreting CD8 T cells were detected consistently after stimulation
with different peptide pools (Fig. 2B), and a substantial percentage
of dual IFN-␥兾IL-2-secreting cells was also found after stimulation
with peptide pools 1 and 2 (Fig. 2B). The percentage (0.13 ⫾ 0.04,
n ⫽ 5) of IFN-␥兾IL-2-secreting cells in LTNPs was significantly
different (P ⫽ 0.0003) compared with progressors (0.01 ⫾ 0.002,
n ⫽ 21).
Phenotypic Analysis of Cytokine-Secreting Virus-Specific CD8 T Cells.
Previous studies in humans and mice have shown that IL-2secreting CD8 T cells were contained within the CCR7⫹ central
memory CD8 T cell population, whereas the IFN-␥-secreting CD8
T cells were contained within the CCR7⫺ effector CD8 T cells (8,
27). Blood mononuclear cells of LTNPs and HIV-negative donors
with known HIV-1, flu, or CMV CD8 T cell responses were
stimulated with the appropriate virus-derived peptides, and cells
were stained with CD8, CD45RA, CCR7, IL-2, IFN-␥, and CD69
Abs. The results obtained indicated that the virus-specific IFN-␥兾
IL-2 CD8 T cells were contained within the CD45RA⫺CCR7⫺
effector cell population and the IFN-␥-secreting CD8 T cells within
the CD45RA⫺CCR7⫺ and CD45RA⫹CCR7⫺ effector cell populations (Fig. 3). These results were representative of the analysis of
two LTNPs and seven HIV-negative subjects.
Fig. 2. Analysis of HIV-1-specific IFN-␥- and IL-2-secreting CD8 T cells in
progressors and LTNPs after stimulation with peptide pools. Flow cytometry
profiles of IFN-␥- and IL-2-secreting HIV-1-specific CD8 T cells of progressor
2113 (A) and three different LTNPs (B) after stimulation of blood mononuclear
cells with different peptide pools spanning gag, pol, and nef proteins.
proliferation was measured by the loss of CFSE in the dividing CD8
T cells. A substantial proportion of CD8 T cells of subject 248
proliferated after stimulation with CMV- and Flu-derived peptides
(Fig. 4A). Similarly, CD8 T cells of subject 359 proliferated after
stimulation with two different EBV-derived peptides (Fig. 4A). We
then determined the proliferation of HIV-1-specific CD8 T cells
after stimulation with HIV-1-derived peptide pools in progressors
(n ⫽ 9) and LTNPs (n ⫽ 5). HIV-1-specific CD8 T cell proliferation
was barely detected or was absent in these two representative
progressors [two of nine patients each tested with one to three pools
(16 responses were tested in total)] (Fig. 4B). However, CD8 T cells
of progressors were able to proliferate after SEB stimulation (Fig.
4B), thus indicating a selective loss of HIV-1-specific proliferation.
Consistent with results previously shown by Migueles et al. (12),
vigorous HIV-1-specific CD8 T cell proliferation was observed in
two of five representative LTNPs (Fig. 4C). The mean ⫾ SE
percentage of HIV-1-specific CD8 T cell proliferation in progressors was 0.45 ⫾ 0.16 compared with 6.88 ⫾ 1.69 in LTNPs (P ⬍
0.00001).
We then determined the correlation between the proportion of
Ag-specific proliferating CD8 T cells and the proportion of IL-2secreting CD8 T cells within IFN-␥-secreting cells. This analysis was
performed by pooling together 32 individual determinations from
21 subjects of Ag-specific CD8 T cell-proliferating and IL-2secreting CD8 T cells. We found a significant correlation between
Proliferation Capacity of Virus-Specific CD8 T Cells. Recent studies
(12, 13) have shown the loss of proliferation capacity of HIV-1specific CD8 T cells of subjects with progressive disease, whereas
HIV-1-specific CD8 T cell proliferation was retained in CD8 T cells
of LTNPs. Based on these observations, it has been proposed that
Ag-specific CD8 T cell proliferation represents a characteristic of
effective and protective immune response (12). Furthermore, it has
been proposed that the loss of HIV-1-specific CD8 T cell proliferation depended on the loss of HIV-1-specific CD4 helper T cells
(13). In the present study, we decided to investigate (i) the correlation between the ability of virus-specific CD8 T cells to secrete
IL-2 and their proliferation capacity and (ii) the potential mechanism responsible for Ag-specific CD8 T cell proliferation. Representative examples of the proliferation capacity of CMV-, EBV-,
flu-, and HIV-1-specific CD8 T cells after virus-specific stimulation
are shown in Fig. 4 A–C. Cells were labeled with CFSE, stimulated
for 5 days with virus-derived peptides, and virus-specific CD8 T cell
PNAS 兩 May 17, 2005 兩 vol. 102 兩 no. 20 兩 7241
Zimmerli et al.
62
Simone C. Zimmerli
4. Results
Fig. 3. IFN-␥- and IL-2-secreting CD8 T cells in different populations defined by CD45RA and CCR7. Shown is the distribution of IFN-␥- and IL-2-secreting CD8
T cells in different populations defined by CD45RA and CCR7. (A) Cells of LTNP 2073 were stimulated with different peptide pools spanning gag, pol, and nef
proteins. (B) Cells of subjects 205 and 35 were stimulated with CMV or flu peptides, respectively.
and CMV-derived peptides induced vigorous Ag-specific proliferation of CD8 T cells of subjects 172 and 180 (Fig. 5A). It is important
to underscore that no CD4 T cell proliferation was observed (Fig.
5A), thus indicating that Ag-specific CD8 T cell proliferation was
not associated with the stimulation of Ag-specific helper CD4 T
cells. Consistent with the observations previously reported (12, 13),
HIV-1-specific CD8 T cell proliferation was barely detected in
progressors after stimulation with the HLA-A2 tetramer complexed with an HIV-1 pol ILKEPVHGV-derived peptide (20) (Fig.
5B). Of interest, in agreement with the work of Lichterfeld et al.
(13), HIV-1-specific CD8 T cell proliferation was recovered in the
presence of exogenous IL-2 (Fig. 5B). No proliferation was observed in CD4 T cells after MHC class I tetramer–peptide complex
the proportion of Ag-specific IL-2-secreting and -proliferating CD8
T cells (Fig. 4D). The correlation was even stronger when only
HIV-1-specific CD8 T cell responses were analyzed (R ⫽ 0.53, P ⬍
0.01, n ⫽ 24).
Having demonstrated a correlation between the ability to secrete
IL-2 and the proliferation capacity of CD8 T cells, we further
investigated the mechanism responsible for Ag-specific CD8 T cell
proliferation. Firstly, we assessed Ag-specific CD8 T cell proliferation under experimental conditions excluding the involvement of
CD4 T cells. For this purpose, Ag-specific CD8 T cell proliferation
was determined by using either MHC class I tetramer–peptide
complexes as stimuli or CD4 T cell-depleted populations in the
absence of exogenous IL-2. HLA-A2 tetramer complexed with flu-
Fig. 4. Virus-specific CD8 T cell proliferation after stimulation with single peptides or peptide pools. (A) CFSE-labeled cells of HIV-negative donors 248 and 359
were stimulated with CMV-, flu-, or EBV-derived peptides. Profiles of proliferating cells, i.e., CFSE low cells, are gated on CD8 T cells. (B) HIV-1-specific CD8 T cell
proliferation in HIV-1 progressors after stimulation with different HIV-1 peptide pools or SEB. (C) HIV-1-specific CD8 T cell proliferation in LTNPs after stimulation
with different HIV-1 peptide pools. (D) Correlation between the proportion of IL-2-secreting and -proliferating virus-specific CD8 T cells.
7242 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0502393102
Zimmerli et al.
63
Simone C. Zimmerli
4. Results
cells from subject 180 observed after stimulation with the CMV
tetramer NLVPMVATV was completely abolished (95% inhibition
of proliferation) in the presence of anti-IL-2 Ab (Fig. 6B). Therefore, virus-specific CD8 T cell proliferation, including HIV-1specific proliferation, depends on IL-2 and on the presence of the
IFN-␥兾IL-2 CD8 T cells, and may occur in the absence of helper
CD4 T cells. The finding that CD8 T cell proliferation was
independent of CD4 T cell help and dependent on the presence of
IFN-␥兾IL-2-secreting CD8 T cells was also confirmed for CMVand EBV-specific CD8 T cell-mediated proliferation in three
HIV-negative subjects (data not shown).
stimulation (Fig. 5B). To further confirm the hypothesis that
HIV-1-specific CD8 T cell proliferation was independent of CD4
helper T cells, we compared the HIV-1-specific CD8 T cell proliferation in response to the p24-derived GPGHKARVL peptide that
has been previously characterized as a CD8 epitope (17) restricted
by HLA-B7. Unfractionated blood mononuclear cells or CD4 T
cell-depleted populations of patient 1010 with chronic HIV-1
infection were stimulated with the peptide GPGHKARVL. As
reported in Materials and Methods, patient 1010 had constantly
controlled viremia since 4 years after interruption of antiviral
therapy. A large percentage (59%) of HIV-1-specific CD8 T cells
proliferated after stimulation of unfractionated cell populations
with the p24 peptide (Fig. 6A). Substantial HIV-1-specific CD8 T
cell proliferation (32.7%) occurred also in the CD4 T cell-depleted
populations although it was reduced (45% reduction) compared
with the cell cultures containing CD4 T cells (Fig. 6A). It is
important to underscore the fact that the CD8 T cell proliferation
in the CD4-depleted cell populations was not due to contaminating
CD4 T cells because CD4 T cells were almost absent (0.6%) in the
CD4-depleted cell populations at day 5 (Fig. 6A). The experiments
shown in Fig. 6A were performed in the absence of exogenous IL-2.
Secondly, Ag-specific CD8 T cell proliferation was assessed in the
presence of anti-IL-2 Ab. The substantial proliferation of CD8 T
Discussion
In the present study, we have investigated the function and phenotype of memory CD8 T cells in different models of virus-specific
T cell responses, including HIV-1, CMV, EBV, and flu. HIV-1specific CD8 T cell responses were studied in subjects with progressive and nonprogressive infection who were naı¨ve to therapy.
The other virus-specific CD8 T cell responses were analyzed in
HIV-negative donors. Functional characterization was performed
by the measurement of the ability of CD8 T cells to proliferate and
to secrete IFN-␥ and IL-2 after Ag-specific stimulation.
Fig. 6. Virus-specific CD8 T cell proliferation in CD4-depleted cells or after neutralization of IL-2. (A) CD8 T cell proliferation was evaluated in CD4 T cell-depleted
populations stimulated with HIV-1-derived peptide. The purity of the sorted CD4⫺ T cell populations was 99%. (B) Inhibition of virus-specific CD8 T cell proliferation
with anti-IL-2 Ab. Cells of subject 180 were stimulated with an A2-restricted CMV tetramer and cultured in the presence of anti-IL-2 or isotype control Abs.
PNAS 兩 May 17, 2005 兩 vol. 102 兩 no. 20 兩 7243
Zimmerli et al.
64
IMMUNOLOGY
Fig. 5. Virus-specific CD8 T cell proliferation after stimulation with HLA class I tetramers. (A) Blood mononuclear cells of
HIV-negative donors 172 and 180 were stimulated with A2-flu
or -CMV tetramers, respectively. Flow cytometry profiles of
proliferating CD8 (Left) and CD4 (Right) T cells are shown. (B)
Blood mononuclear cells of progressor 2056 were stimulated
with an A2-pol tetramer and cultured in the absence or presence of 10% of exogenous IL-2.
Simone C. Zimmerli
4. Results
Most studies performed on CD8 T cells in different models of
antiviral responses in both mice and humans were predominantly
focused on the characterization of effector functions such as
perforin and granzyme expression or secretion of IFN-␥ and TNF-␣
(9–11). Recently, a series of studies have shown the importance of
investigating other functions such as the ability to proliferate and to
secrete IL-2 (14–16) that have generally been the object of extensive
investigation in CD4 T cells. With regard to CD8 T cells, it has been
shown that the preservation of the proliferation capacity and the
ability to secrete IL-2 were generally associated with an apparently
effective immune response because virus replication was controlled
in both mouse and human models of virus infection (12, 28). In
addition, a recent study has shown a paralleled loss of HIV-1specific helper CD4 T cells and HIV-1-specific CD8 T cell proliferation, and concluded that HIV-1-specific helper CD4 T cells are
critical for the maintenance of HIV-1-specific proliferating CD8 T
cells (13).
This is the first study, to our knowledge, investigating IL-2
secretion in HIV-1-specific CD8 T cells. In addition, it compares
the function of HIV-1-specific CD8 T cells with that of CMV-,
EBV-, and flu-specific CD8 T cells that are able to keep either
on check (CMV and EBV) or clear (flu) the virus. The rationale
for studying antiviral CD8 T cell responses in different models
of virus persistence resides on recent studies (28) performed in
mice, demonstrating that the function of CD8 T cells was
modulated by different conditions of Ag levels and兾or persistence. HIV-1 infection in subjects with progressive disease
corresponded to the model of immune failure with Ag persistence and high Ag levels. CMV, EBV, and HIV-1 infection in
subjects with nonprogressive disease corresponded to the model
of immune control with protracted virus persistence and low Ag
levels and flu to the model of Ag clearance. Our results
demonstrated the presence of an Ag-specific IFN-␥兾IL-2secreting CD8 T cell population in the models of virus infections
associated with resolved virus infection or with virus control, i.e.,
CMV, EBV, and nonprogressive HIV-1 infection or virus clearance, i.e., flu. This cell population was absent in progressive
HIV-1 infection. Therefore, we provided evidence for (i) a loss
of IFN-␥兾IL-2-secreting CD8 T cells in progressive HIV-1
infection and (ii) a skewed representation of functionally distinct
memory HIV-1-specific CD8 T cells in progressive HIV-1
infection. The present results showed that the same pathogen,
i.e., HIV-1, can be associated with substantially different CD8 T
cell responses in progressive and nonprogressive infection where
the major difference between these two conditions was indeed
represented by Ag levels. Therefore, along with the observation
from the lymphocytic choriomeningitis virus model (28), our
results rather supported the hypothesis that also in humans the
functional heterogeneity of virus-specific CD8 T cell responses
was influenced by Ag persistence and Ag levels.
In agreement with previous studies (12, 13), HIV-1-specific
CD8 T cell proliferation was lost in progressive HIV-1 infection.
Of interest, we have provided evidence for the combined loss of
HIV-1-specific IFN-␥兾IL-2-secreting and -proliferating CD8 T
cells in progressive HIV-1 infection. This association raised the
issue on the role of IFN-␥兾IL-2-secreting CD8 T cells in
Ag-specific CD8 T cell proliferation. To address this issue, we
evaluated the virus-specific CD8 T cell proliferation under
experimental conditions excluding any involvement of helper
CD4 T cells. These latter have been proposed to be critical for
sustaining HIV-1-specific CD8 T cell proliferation (13). Virusspecific CD8 T cell proliferation, including HIV-1-specific,
occurred in CD4 T cell-depleted populations or after stimulation
with MHC class I tetramer–peptide complexes. Under these
experimental conditions, virus-specific CD8 T cell proliferation
was found in the HIV-1-, CMV-, EBV- and flu-specific immune
responses, and a significant correlation between the proportion
of IL-2-secreting and -proliferating CD8 T cells was observed.
These results demonstrated that the persistence of virusspecific IFN-␥兾IL-2-secreting CD8 T cells was associated with
the persistence of CD8 T cell proliferation. Virus-specific CD8
T cell proliferation was supported by IL-2 because it was
completely abolished in the presence of the anti-IL-2 Ab.
Therefore, taken together, they indicate that IFN-␥兾IL-2secreting CD8 T cells are able to promote CD8 T cell proliferation through the secretion of IL-2 even in the absence Agspecific helper CD4 T cells. Despite the demonstration in vitro
of a CD4-independent CD8 T cell proliferation, it is important
to underscore that Ag-specific helper CD4 T cells are crucial in
vivo for the maintenance and for preventing impairment of
optimal CD8 T cell function (29). Of interest, this CD4indepndent proliferation capacity was present in the effector,
i.e., CD45RA⫺CCR7⫺ cell population. The importance in vivo
of this CD4-independent proliferation capacity of effector CD8
T cells during the expansion phase of the immune response
remains to be determined.
These results represent a further step in the understanding of
the functional characterization of virus-specific CD8 T cell
responses and in the understanding of the impairment of CD8 T
cell functions in progressive HIV-1 infection.
Wong, P. & Pamer, E. G. (2003) Annu. Rev. Immunol. 21, 29–70.
Ahmed, R. & Gray, D. (1996) Science 272, 54–60.
Doherty, P. C. & Christensen, J. P. (2000) Annu. Rev. Immunol. 18, 561–592.
Kaech, S. M., Wherr y, E. J. & Ahmed, R. (2002) Nat. Rev. Immunol. 2,
251–262.
Kaech, S. M., Hemby, S., Kersh, E. & Ahmed, R. (2002) Cell 111, 837–851.
Seder, R. A. & Ahmed, R. (2003) Nat. Immunol. 4, 835–842.
Sprent, J. & Surh, C. D. (2002) Annu. Rev. Immunol. 20, 551–579.
Sallusto, F. & Lanzavecchia, A. (1999) Nature 401, 708–712.
Fuller, M. J., Khanolkar, A., Tebo, A. E. & Zajac, A. J. (2004) J. Immunol. 172,
4204 – 4214.
Mallard, E., Vernel-Pauillac, F., Velu, T., Lehmann, F., Abastado, J. P., Salcedo, M. &
Bercovici, N. (2004) J. Immunol. 172, 3963–3970.
Sandberg, J. K., Fast, N. M. & Nixon, D. F. (2001) J. Immunol. 167, 181–187.
Migueles, S. A., Laborico, A. C., Shupert, W. L., Sabbaghian, M. S., Rabin, R., Hallahan,
C. W., Van Baarle, D., Kostense, S., Miedema, F., McLaughlin, M., et al. (2002) Nat.
Immunol. 3, 1061–1068.
Lichterfeld, M., Kaufmann, D. E., Yu, X. G., Mui, S. K., Addo, M. M., Johnston, M. N.,
Cohen, D., Robbins, G. K., Pae, E., Alter, G., et al. (2004) J. Exp. Med. 200, 701–712.
Harari, A., Petitpierre, S., Vallelian, F. & Pantaleo, G. (2004) Blood 103, 966–972.
Younes, S. A., Yassine-Diab, B., Dumont, A. R., Boulassel, M. R., Grossman, Z., Routy, J. P.
& Sekaly, R. P. (2003) J. Exp. Med. 198, 1909–1922.
Iyasere, C., Tilton, J. C., Johnson, A. J., Younes, S., Yassine-Diab, B., Sekaly, R. P., Kwok,
W. W., Migueles, S. A., Laborico, A. C., Shupert, W. L., Hallahan, C. W., et al. (2003) J. Virol.
77, 10900–10909.
17. Wills, M. R., Carmichael, A. J., Mynard, K., Jin, X., Weekes, M. P., Plachter, B. & Sissons,
J. G. (1996) J. Virol. 70, 7569–7579.
18. Rickinson, A. B. & Moss, D. J. (1997) Annu. Rev. Immunol. 15, 405–431.
19. Lalvani, A., Brookes, R., Hambleton, S., Britton, W. J., Hill, A. V. & McMichael, A. J. (1997)
J. Exp. Med. 186, 859–865.
20. Walker, B. D., Flexner, C., Birch-Limberger, K., Fisher, L., Paradis, T. J., Aldovini, A.,
Young, R., Moss, B. & Schooley, R. T. (1989) Proc. Natl. Acad. Sci. USA 86, 9514 –
9518.
21. Johnson, R. P., Trocha, A., Yang, L., Mazzara, G. P., Panicali, D. L., Buchanan, T. M. &
Walker, B. D. (1991) J. Immunol. 147, 1512–1521.
22. Gotch, F. M., Nixon, D. F., Alp, N., McMichael, A. J. & Borysiewicz, L. K. (1990) Int.
Immunol. 2, 707–712.
23. Robertson, M. N., Buseyne, F., Schwartz, O. & Riviere, Y. (1993) AIDS Res. Hum.
Retroviruses 9, 1217–1223.
24. Currier, J. R., Kuta, E. G., Turk, E., Earhart, L. B., Loomis-Price, L., Janetzki, S., Ferrari,
G., Birx, D. L. & Cox, J. H. (2002) J. Immunol. Methods 260, 157–172.
25. Champagne, P., Ogg, G. S., King, A. S., Knabenhans, C., Ellefsen, K., Nobile, M., Appay,
V., Rizzardi, G. P., Fleury, S., Lipp, M., et al. (2001) Nature 410, 106–111.
26. Ellefsen, K., Harari, A., Champagne, P., Bart, P. A., Se´kaly, R. P. & Pantaleo, G. (2002) Eur.
J. Immunol. 32, 3756–3764.
27. Wherry, E. J., Teichgra¨ber, V., Becker, T. C., Masopust, D., Kaech, S. M., Antia, R.,
von Adrian, U. H. & Ahmed, R. (2003) Nat. Immunol. 4, 225–234.
28. Wherry, E. J., Blattmann, J. N., Murali-Krishna, K., van der Most, R. & Ahmed, R. (2003)
J. Virol. 77, 4911–4927.
29. Sun, J. C., Williams, M. A. & Bevan, M. J. (2004) Nat. Immunol. 5, 927–933.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
This work was supported by Swiss National Foundation Grant FN 3100058913兾2 and European Commission Grant QLK2-CT-1999-01321.
7244 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0502393102
Zimmerli et al.
65
Simone C. Zimmerli
4. Results
4.2. Publication #2
Attenuated poxviruses expressing a synthetic HIV protein stimulate HLAA2-restricted cytotoxic T cell responses
Arnaud Didierlaurent, Juan-Carlos Ramirez, Magdalena Gherardi, Simone C. Zimmerli,
Marcus Graf, Hans Acha Orbea, Giuseppe Pantaleo, Ralf Wagner, Mario Esteban, Jean-Pierre
Krähenbühl, Jean-Claude Sirard
Vaccine (2004) 22:3395-4034
Summary
The aim of this study was to test the immunogenicity of modified vaccinia ankara (MVA) and
poxvirus (NYVAC) containing a construct harboring a fusion of the HIV-1 IIIB gag, pol and
nef genes (gpn) in HLA-A2 transgenic mice.
Poxviruses are useful for vaccination since they can accommodate large amounts of foreign
genes, infect mammalian cells and express foreign proteins in the latter. MVA was developed
towards the end of the campaign for the eradication of smallpox. Passages on chicken embryo
for over 500 times lead to the loss of about 15% of the genetic material and the ability to grow
in human and most other mammalian cells. MVA shows similar levels of recombinant gene
expression as replication-competent vaccinia viruses in human cells. NYVAC is a highly
attenuated strain of vaccinia, in which 18 open reading frames from the viral genome have
been deleted.
The gagpolnef polygene was generated by back-translation of HIV-1 IIIB amino-acid
sequence and optimized by elimination of splice-donor/acceptor sites and RNA destabilizing
sequences. The resulting gagpolnef fusion protein is composed of Gag fused and in frame to
Pol and Nef, which replaces the active site of RT. The RT sequence overlapping the active
site was translocated to the 3’ end. The terminal Glycine was substituted by Alanine to
prevent myristoylation of the N-terminus and a point mutation was introduced to the active
site of the protease domain to damage its enzymatic activity. Gagpolnef was then cloned into
a vaccinia virus insertion vector and MVA or NYVAC were transfected with this construct.
To assess if Gpn is immunogenic mice were immunized intraperitoneally or subcutaneously
with peptides, recombinant MVA or NYVAC and splenocytes of immunized mice were tested
66
Simone C. Zimmerli
4. Results
on their ability to secrete IFN-γ and to perform cytotoxic activity. Next it was checked if Gpn
expressed by MVAgpn was recognized by human MHC class I molecules. Transgenic mice
exclusively displaying a chimeric human HLA-A2.1 MHC class I molecule were immunized
with six HLA-A2 restricted peptides, MVAgpn or MVA expressing luciferase (MVAluc) as
control. Again IFN-γ and CTL response was tested following immunization.
My task in this work was to investigate whether MVAgpn could stimulate human pol- or gagspecific CD8 T cells. For this purpose, human PBMC containing pol- and gag- specific CD8
T cells (as verified by tetramer staining at baseline) were stimulated with MVAgpn at
increasing multiplicity (m.o.i) of infection or MVAluc as negative control. At day 10
following stimulation, the frequency of pol- or gag-specific CD8 T cells, respectively, was
measured using HLA-A2 restricted pol- or gag- tetramers. To ensure that the stimulation is
specific PBMC of an HIV-negative subjects were stimulated in parallel.
We found that Gpn delivered by recombinant poxvirus vectors induced a robust HIV-specific
CTL response. The HIV-specific CD8 T cell epitopes were immunogenic in transgenic mice
displaying human MHC class I molecules. Furthermore MVAgpn was able to trigger
expansion of human pol- and gag-specific CD8 T cells proportional to the m.o.i.
67
Simone C. Zimmerli
4. Results
Vaccine 22 (2004) 3395–3403
Attenuated poxviruses expressing a synthetic HIV protein stimulate
HLA-A2-restricted cytotoxic T-cell responses
Arnaud Didierlaurent a,1 , Juan-Carlos Ramirez b,1 , Magdalena Gherardi b , Simone C. Zimmerli c ,
Marcus Graf d , Hans-Acha Orbea a , Giuseppe Pantaleo c , Ralf Wagner d , Mariano Esteban b ,
Jean-Pierre Kraehenbuhl a,∗ , Jean-Claude Sirard a,e
a
c
Swiss Institute for Experimental Cancer Research (ISREC), Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland
b Centro Nacional Biotecnolog´ıa, CSIC; Campus Universidad Autonoma de Madrid, 28049 Madrid, Spain
Division of Immunology and Allergy, Laboratory of AIDS Immunopathogenesis, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
d GENEART GmbH, 93053 Regensburg, Germany
e Equipe Avenir, Institut de Biologie de Lille, 59021 Lille, France
Received 25 July 2003; received in revised form 16 February 2004; accepted 26 February 2004
Available online 17 March 2004
Abstract
Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the
breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring
a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins.
The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice
characterised by stimulation of Gpn-specific IFN-␥-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA
elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA
vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity
as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic,
efficiently processed and presented by human MHC class I molecules.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: HIV vaccine; HLA-A2; Recombinant poxviruses
1. Introduction
though induction of neutralising antibodies at mucosal surfaces is probably a prerequisite to induce sterile protection,
a robust CTL response may lower viral load and, therefore,
limit transmission [7].
Among the vectors used to carry HIV antigens, Modified Vaccinia Ankara (MVA), an attenuated vaccinia virus,
was shown to induce HIV-specific CTL responses [3,8,9].
Upon MVA infection of mammalian cells, early and late protein synthesis is restricted to the first 24–48 h post-infection
[10,11]. MVA is safe for humans [12] and is currently used
in several HIV vaccine trials [13,14]. Within the family of
attenuated poxvirus, NYVAC carrying multiple SIV/HIV
antigens has also been used in monkeys [15,16]. NYVAC
is a highly attenuated strain of vaccinia virus generated by
precise deletion of 18 open reading frames from the viral
genome, which affects non essential genes for virus growth
in some cell lines but are important for virulence in animal models [17]. MVA has been generated by passage on
Many HIV candidate vaccines that target both cellular and
humoral immunity have been tested for a decade in mice
and non-human primates [1,2]. Successful HIV vaccination,
however, has been hampered by the complexity of HIV infection and the absence of a defined correlate of protection.
More recently, protective immunity against simian immunodeficiency (SIV) challenges in macaques has been reported
[3–5]. These data indicate that induction of cytotoxic T cells
(CTLs) prevents the spread of the viral infection [6]. Al∗ Corresponding author. Tel.: +41-21-692-58-56;
fax: +41-21-652-69-33.
E-mail address: [email protected]
(J.-P. Kraehenbuhl).
1 Contributed equally to this work. Present address: Laboratorio de
Reumatolog´ıa, Unidad de Investigaci´on, Hospital Universitario 12 de
Octubre, 28041 Madrid, Spain.
0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2004.02.025
68
Simone C. Zimmerli
3396
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
chicken embryo fibroblast cells more than 500 times, loosing about 15% of genetic information and ability to grow
in human and most other mammalian cells [18]. Both NYVAC and MVA share common deleted genes, like the serpins
(B13R and B14R) and host range (K1L), but most other of
the deleted genes are distinct between the two virus strains.
A careful evaluation of the differences in MVA and NYVAC
biology remains to be done [19].
Peptides corresponding to CTL epitopes administered
with adjuvant or via recombinant vectors are immunogenic
but their use is limited due to HLA variability and high
mutation rate of HIV. A strategy that targets as many viral
proteins as possible and that is not restricted to a few HLA
molecules is more likely to be successful. So far, few candidate vaccines have used fusion proteins that contain multiple HIV determinants and little is known about the breadth
and the magnitude of responses against such vaccines. This
information is critical as immunodominance may favour
the induction of restricted sets of specificities in vaccinated
individuals. In order to develop strategies overcoming immunodominance, it is important to validate mouse model,
such as transgenic mice carrying human MHC class I to
predict human CD8+ T cell responses [20]. In particular, the
HHD mouse, that is H2-Db−/− ␤2-microglobulin−/− and
transgenic for a chimeric HLA-A2 molecule, was shown to
be a good model for the prediction of HLA-A2-restricted
responses [21].
Within the frame of EuroVacc (http://www.eurovac.net),
a European effort to bring HIV vaccines into clinical trials, candidate vaccines harbouring a fusion of the HIV-1
IIIB gag, pol and nef genes (gpn) have been constructed.
Before entering clinical trials in humans, we sought to verify that immunisation with Gpn induced CTLs with similar
specificities than found in natural infections. We show here
that MVA and NYVAC expressing Gpn induce strong CTL
and IFN-␥-producing T cell responses in conventional mice.
HLA-A2 transgenic mice immunised with MVA expressing
Gpn developed HLA-A2-restricted responses against two
CTL epitopes known to be immunogenic in HIV infected individuals. In addition, the candidate vaccine specifically triggers the in vitro expansion of human HIV-1-specific CD8+
T cells. Therefore, we provide evidence that these vaccines
tested for safety in mice [22], are immunogenic and elicit
CTL responses against HLA-A2-restricted epitopes recognised by HIV-infected patients.
2. Material and methods
2.1. Construction of the chimeric gpn
The synthetic read-through gagpolnef polygene was synthesized and provided by GENEART GmbH (Regensburg,
Germany). Gagpolnef was generated by back-translating the
amino-acid sequence of the HIV-1IIIB (BH10, GenBank Accession no. M15654) using a matrix for the most frequently
69
occurring codons in mammalian cells. By optimising the
gagpolnef sequence, cryptic splice-donor/acceptor sites and
RNA destabilising sequence motifs were eliminated. The
synthetic gagpolnef DNA was designed in fragments of
300–800 nucleotides, assembled via unique restriction sites
and cloned into pCRscript giving rise to pCRscript/gpn.
The gagpolnef fusion protein comprises the group-specific
antigen Gag including p7 (aa 1–432; GI: 326388) fused
and in frame to Pol (aa 1–793; GI: 326385) lacking the
integrase domain. Furthermore, the active site of the reverse
transcriptase (RT) was replaced by a scrambled nef gene
(aa 1–74 fused to aa 75–123; GI: 326393) resulting in an
artificial budding defective 1326 aa read-through gagpolnef
fusion protein. The RT sequence overlapping the active site
was translocated in frame to the 3 end (aa 296–396; GI:
326385) of the gagpolnef polygene. Accordingly to prevent
myristoylation, the N-terminal glycine was substituted by
alanine (G to A). A point mutation was also introduced into
the active site of the protease domain (aa 93; GI: 326385)
to impair its enzymatic activity.
2.2. Construction of recombinant poxvirus vectors
After digestion with EcoRI and SacI, the DNA fragment containing gagpolnef sequence was isolated from
pCRscript/gpn and cloned into the Vaccinia virus (VV)
insertion vector pJR101. In the resulting plasmid, the expression of Gpn and the selection marker ␤-glucuronidase
is regulated by the VV synthetic early/late promoter e/l [23]
and promoter p7.5, respectively. The synthetic e/l promoter
contains 40 bp which largely overlap early and late regulatory elements [23]. This construct was further checked by
sequencing and inserted into the VV haemagglutinin gene.
The selection of recombinant MVA viruses (MVAgpn) was
performed as in [11]. The purity of the recombinant virus
was confirmed by PCR analysis. The construction of MVA
delivering the luciferase protein is described elsewhere
(MVAluc [11]). MVA recombinants were grown in CEF,
purified by sucrose-cushion and titrated by immunostaining in CEF [11]. Recombinant NYVAC expressing Gpn
(NYVACgpn) was provided by Dr. Frachette (Aventis Pasteur, Lyon). NYVACgpn was grown in CEF and titrated
by immunostaining in CEF or by plaque assay in BSC-40
cells. Both MVAgpn and NYVACgpn recombinants represent a homogeneous virus population, as established after
PCR analysis. In NYVACgpn, Gpn expression is regulated
by the same synthetic e/l promoter as in MVAgpn except
that the expression cassette is inserted into the thymine
kinase gene. To generate WRgp, the sequences encoding
natural HIV Gag and Pol proteins as well as the selection
marker ␤-galactosidase were inserted in the thymine kinase gene. The expression of HIV antigens was assessed in
BHK-21 cells infected at a multiplicity of infection (m.o.i.)
of five with either sucrose-purified MVAgpn, NYVACgpn
or WRgp. Cells were collected at various times after infection and extracts (12 ␮g) were run on 10% SDS-PAGE.
Simone C. Zimmerli
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
Gpn was visualized after Western blotting using rabbit
polyclonal anti-gag p24 serum (ARP 432, NIBSC, Centralised Facility for AIDS reagents, UK). Alternatively, the
analysis of Gpn intracellular location was performed at
24 hpi by immunofluorescence and confocal microscopy on
permeabilized BHK-21 cells that were incubated with the
antibody ARP 432 and the nuclei staining reagent To-Pro
(Molecular Probes).
3397
were revealed using biotinylated secondary anti-IFN-␥
antibodies (clone XMG1.2, Pharmingen) and extravidin
conjugated to the Alkaline Phosphatase (Sigma). The spots
were counted using a ProgRes microscope (Zeiss). For CTL
assay or expanded IFN-␥ ELISPOT assay, effector cells
(5 × 106 splenocytes) were stimulated in vitro in 10 ml
complete medium for 6 days once or twice. For C57BL/6
effectors, Gpn-specific stimulation was achieved by adding
5 × 105 EL4gpn and 5 × 106 irradiated na¨ıve C57BL/6
splenocytes. For HHD effectors, incubation was performed
with 5 × 105 LPS blasts derived from HHD mice and independently loaded with each peptide [26]. Target cells for
CTL assays were EL4gpn effector cells and RMA-S/HHD
loaded with individual HLA-A2-restricted peptide, respectively for C57BL/6- and HHD-stimulated effector cells
[24].
2.3. Mice immunisation
C57BL/6 mice were purchased from Harlan. Transgenic
HHD mice kindly provided by Dr. Lemonnier (Pasteur
Institute, France) are double-knockout for H2-Db and
␤2-microglobulin and transgenic for a chimeric HLA-A2
molecule, the only MHC class I, therefore, expressed [24].
For peptide immunisation, 25 ␮mol of each peptide were
mixed with 50 ␮g of tetanus toxin universal helper peptide
P30 in 200 ␮l IFA/PBS 1:1 emulsion and injected at days 0
and 15 at the base of the tail. Recombinant MVA and NYVAC were injected intraperitoneally (i.p.) or subcutaneously
(s.c.) at 5 × 107 p.f.u. in 200 ␮l PBS.
2.5. Stimulation of HIV-1-specific human CD8+ T cells
with MVAgpn
The expansion of Pol2- and Gag-specific CD8+ T cells
induced by MVAgpn was studied as described in [27] using
cryo-preserved peripheral blood mononuclear cells (PBMC)
from an HIV-1-infected patient. Patient CNA 2099 is part of
a cohort of HIV-1 infected patients with progressive disease
enrolled in therapeutic clinical trials with anti-retroviral
regimens [28,29]. These studies were approved by the local
Institutional Review Board and the subject gave written
informed consent. PBMCs from an HIV-seronegative subject (LDH 197) were included in the study as negative
control.
PBMCs were cultured at 1 × 106 cells per ml with a sonicated preparation of MVAgpn or MVAluc at the indicated
m.o.i. Fresh culture medium supplemented with recombinant human IL-2 (0.02 U/ml, Roche Diagnostics) was added
every 2–3 days to the PBMC cultures. On day 10, the expansion of Pol2- and Gag-specific CD8+ T cells was monitored by staining with APC-conjugated anti-CD8 antibodies (clone SK1) and PE-labelled tetramers. Class I-peptide
tetramers were produced as previously described [30]. Data
were acquired on a FACSCaliburTM system and analysed
using CellQuestTM software (Becton Dickinson).
2.4. IFN-γ ELISPOT and CTL assays
Six peptides chosen from the Los Alamos Database
(http://hiv-web.lanl.gov/immunology/index.html) were synthesised at the Biochemistry Institute facility: Gag (SLYNTVATL), Pol1 (VLDVGDAYFSV), Pol2 (ILKEPVHGV),
Pol3 (ALQDSGLEV), Pol4 (VIYQYMDDL) and Prot1
(VLVGPTPVNI). EL4 cells were a gift of Dr. Pedro
Romero (Ludwig Institute, Lausanne branch). RMAS-HHD
cells were obtained from Dr. Lemonnier. EL4gpn are
stable EL4 clones transfected with the synthetic gagpolnef coding region inserted into the in pCi/neo expression vector (Promega) under the transcriptional control of
the immediate-early promoter-enhancer of the human cytomegalovirus. EL4gpn cells thus constitutive express Gagpolnef. Cell cultures were performed in RPMI 1640 containing 2 mM l-glutamine, 20 mM HEPES, 1 mM sodium pyruvate, 1% penicillin-streptomycin and 2-␤-mercaptoethanol
(Gibco BRL), and 10% foetal calf serum (FCS, Myoclone
Superplus). Ten days after the last immunisation, spleens
from immunised animals were removed and pooled. Fresh
IFN-␥ ELISPOT analysis was performed as described [25].
Briefly, 105 –106 splenocytes were incubated with either
medium containing 10 ␮M specific peptide (for HHD animals), 105 MVA-infected splenocytes, EL4 or EL4gpn (for
C57BL/6) depending on the experiments. MVA-infected
splenocytes were prepared from na¨ıve splenocytes infected
at a m.o.i. of five in FCS-free medium for 1 h. The cells
were further incubated for 5 h in medium containing 2%
FCS to allow expression of the viral antigens and used in
the ELISPOT assay after extensive washing.
The cells were incubated 18 h in 96 well plates coated
with IFN-␥ antibodies (clone R4-6A2, Caltag). The spots
3. Results
3.1. Construction of the attenuated poxviruses candidate
vaccines
3.1.1. Characteristics of the chimeric Gagpolnef
Gagpolnef is a fusion protein of 1326 amino acids composed of Gag, Pol and Nef from the HIV-1 clade B clone
IIIB [31] that has been modified to enhance its immunogenicity and for safety reasons (Fig. 1). The gag sequence
was fused in frame to the polnef part by creating a 1 frame
shift in the natural slippery sequence. A glycine to alanine
70
Simone C. Zimmerli
3398
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
Fig. 1. Schematic drawing of the synthetic HIV IIIB fusion protein Gagpolnef. The Gag derived-domains are constituted of the matrix (p17MA),
the capsid (p24CA), p2 and the nucleocapsid (p7Nc). The Pol and Nef
derived domains, i.e. Protease (PR), N-terminal (N-RT) and C-terminal
(RT-C) portion of the reverse transcriptase (RT), the active site of the
RT (RT-A) and a scrambled Nef (SC-NEF) are also depicted. Modifications include the G to A amino acid substitution abolishing N-terminal
myristilation (Myr) of Gag, the in frame fusion (FS) of the Gag and
Polnef domains and the D to N amino acid substitution inactivating
the PR.
substitution in Gag was introduced to prevent formation of
virus-like particles [32]. The active sites of protease (PR)
and integrase were inactivated by mutagenesis and deletion,
respectively. The active site of the RT was translocated at
the C-terminus of the fusion protein. The nef gene was partially deleted, mixed up to generate a fusion between the
C-terminal and N-terminal parts of the protein, and inserted
into the fragment coding the RT. In addition, the codon usage
was adapted to highly expressed human genes (GENEART
GmbH, Germany) based on a previous study showing that
such optimisation of an HIV Gag DNA vaccine increases its
expression and its immunogenicity [33].
3.1.2. Expression of Gagpolnef
The synthetic gagpolnef gene was introduced into the
MVA or NYVAC genome, referred to as MVAgpn and NY-
VACgpn, respectively. After infection of permissive BHK21
cells with MVAgpn, a band corresponding to the full length
Gagpolnef protein (∼150 kDa) was revealed on immunoblot
with anti-Gag polyclonal antibodies (Fig. 2A). Pol- and
Nef-specific antibodies recognised the same band (data not
shown). BHK-21 infection by WR harbouring wild type HIV
IIIB gag and pol genes (WRgp) resulted in PR-mediated
Gag processing as shown by the presence of p55 and p24.
In contrast, native HIV proteins resulting from PR activity
were not observed in cells infected with MVAgpn. Therefore, according to the construct design, MVAgpn-infected
cells produce a Gagpolnef fusion protein that cannot be processed by PR into native and potentially harmful HIV proteins. As compared to MVAgpn, the full length Gpn fusion
protein was produced in NYVACgpn infected cells but at
different levels depending on the time point after infection
(Fig. 2B). In these cells, expression of Gpn was higher at
6 h post infection (hpi) in NYVAC-gpn when compared to
MVAgpn-infected cells. We have consistently found that in
permissive (CEF, BHK-21) and non-permissive (TK-143,
HeLa) cells infected with NYVACgpn, the levels of Gpn at
6 hpi are somewhat higher or similar to those found in cells
infected with MVAgpn depending on the cell lines (Fig. 2B
and data not shown). This observation rules out the possibility that putative mutations in the early/late promoter of NYVACgpn influences expression. With time (18 and 24 hpi),
the Gpn expression level decreases in NYVACgpn versus
MVAgpn-infected cells. This results from differences of cytopathic effect and inhibition of virus-induced protein synthesis between both poxviruses (Najera et al, manuscript in
Fig. 2. Gagpolnef expression in MVAgpn and NYVACgpn infected cells. BHK-21 cells were infected either with MVAgpn, MVAluc, NYVACgpn or
with recombinant Vaccinia virus WR expressing the HIV IIIB gag and pol (WRgp) as indicated. Cell extracts were analysed after 24 h (A) or various
hpi (B) by SDS-PAGE followed by immunoblotting using anti-Gag serum (A,B) or immunofluorescence (C). Fluorescence analysis was performed at
24 hpi on BHK-21 permeabilized cells for detection of Gagpolnef fusion protein (anti-Gag serum, white) and cell nuclei (grey).
71
Simone C. Zimmerli
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
3399
and presented in a MHC class I-restricted pathway to CD8+
T cells.
To allow a direct comparison between immunisation
protocols, Gpn-specific T-cell response was analysed by
measuring the frequency of IFN-␥-secreting cells using ELISPOT [25]. Gagpolnef-specific IFN-␥-producing
splenocytes were found directly ex-vivo after immunisation
with MVAgpn (Fig. 3B). This corresponds to a frequency
of 0.4% Gagpolnef-specific CD8+ T cells. This response
correlates with the CTL activity described in Fig. 3A, confirming that Gagpolnef is an immunogenic protein when
expressed in cells infected with MVA. NYVACgpn also
induced the development of Gpn-specific IFN-␥-producing
cells similar than the one observed with MVA. Specific
responses were also monitored after subcutaneous immunisation with both vectors. Altogether, these results indicate
that Gpn delivered by recombinant poxvirus vectors induce
a robust HIV-specific CTL response.
3.3. MVAgpn induces HIV-specific HLA-A2-restricted
CTL in HHD mice
3.3.1. Immunogenicity of selected HLA-A2-restricted
peptides
To test whether Gagpolnef expressed by MVAgpn was
recognised by human MHC class I molecules, we immunised transgenic HHD mice that exclusively display
a chimeric human HLA-A2.1 as MHC class I molecule
[24]. Six HLA-A2-restricted peptides recognised by CTLs
from HIV-infected individuals were selected throughout the
fusion protein (Fig. 4A) and tested for their intrinsic immunogenicity in HHD mice. Seven days after immunisation
with a mixture of the peptides, splenocytes were stimulated in vitro with LPS blasts loaded with each peptide.
All peptides except Pol4 induced an HLA-A2-restricted
IFN-␥-producing (Fig. 4B) and CTL (Fig. 4C) responses
against RMAS-HHD cells loaded with each peptide. The
magnitude of the response varied among the peptides, Gag,
Pol2 and Pol3 being more immunogenic than Prot and
Pol1. These results show that the selected HIV-specific
CD8 epitopes found to be immunodominant in humans are
immunogenic in HHD mice.
Fig. 3. Immunogenicity of Gagpolnef carried by MVAgpn and NYVACgpn. (A) C57BL/6 mice (n = 3) were injected i.p. with MVAgpn
or MVAluc as control. Ten days later, splenocytes from immunised animals were stimulated in vitro for 6 days and tested for their ability to
kill target cells in a chromium release assay. Target cells were EL4 for
non-specific response (dashed line and open symbols) and EL4gpn for
Gagpolnef-specific response (filed symbols). E:T stands for effector/target.
Data are representative of three experiments. (B) C57BL/6 mice (n = 3)
were injected i.p. or s.c. with MVAgpn or NYVACgpn. Ten days later,
splenocytes from immunised animals were tested in fresh IFN-␥ ELISPOT
assay. Target cells were EL4 for non-specific response (hatched bars) and
EL4gpn for Gagpolnef-specific response (black bars). SFC stands for spot
forming cells. Data are representative of two experiments.
preparation). Immunofluorescence analysis by confocal microscopy in infected BHK-21 cells at 24 hpi, revealed that
Gpn is localised in the cytoplasm of cells infected by both
recombinant viruses but not by a control virus delivering
luciferase (MVAluc, Fig. 2C) [11].
3.3.2. MVAgpn triggers CTLs specific for peptides
immunogenic in HLA-A2+ HIV-infected individuals
To assess whether HHD mice could respond to MVAgpn
immunisation, an IFN-␥ ELISPOT assay specific for MVA
antigens was first performed using na¨ıve splenocytes infected with MVAluc as MVA-specific antigen presenting
cells (Fig. 5A). The frequency of IFN-␥-producing cells was
lower in HHD mice than in C57BL/6 animals, which correlates with the lower proportion of total splenic CD8+ T
cells in HHD mice (on average 3% of total splenocytes).
The response against the different HLA-A2 restricted epitopes spanning Gpn was next investigated after i.p. injection of MVAgpn. After 6 days in vitro expansion with each
3.2. Recombinant MVA and NYVAC induce
Gagpolnef-specific CTL response
To assess the immunogenicity of Gpn, C57BL/6 mice
(H-2b ) were immunised with MVAgpn and NYVACgpn and
the response against the whole fusion protein was assessed
using EL4 cells (H-2b ) expressing Gpn (EL4gpn). As shown
in Fig. 3A, a significant CTL activity against EL4gpn was
detected in mice immunised with MVAgpn but not in animals treated with MVAluc. These results indicate that the
fusion protein delivered by MVAgpn is efficiently processed
72
Simone C. Zimmerli
3400
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
Fig. 4. HLA-A2-restricted peptides from Gagpolnef stimulate CTL responses in HHD mice. (A) The position on Gagpolnef of six HLA-A2-restricted
peptides (Gag, Prot, Pol1, Pol2, Pol3 and Pol4) recognised by HIV infected individuals is depicted. (B, C) HHD mice (n = 5) were immunised with a
mixture of the HLA-A2 peptides with helper peptide P30 in IFA. One week later, splenocytes of immunised animals were collected and stimulated in
vitro for 1 (B) or 2 (C) weeks with LPS blasts loaded independently with each peptide. An IFN-␥ ELISPOT assay (B) and a chromium release assay
(C) were then performed using RMAS-HHD loaded with each peptide as target cells. Values obtained with non-loaded RMAS-HHD as target cells were
subtracted from values obtained with peptide-loaded RMAS-HHD. This experiment was reproduced twice. SFC, asterisk and E:T mean spot forming
cells, no detectable response, and effector/target, respectively.
peptide, IFN-␥-producing cells specific for Prot (VLVGPTPVNI) and Pol2 (ILKEPVHGV) peptides were only detected
(Fig. 5B). These cells were shown to be CTLs since they
were able to kill RMAS-HHD cells loaded with Prot and
Pol2, respectively (Fig. 5C). No CTL nor IFN-␥-producing
cell responses were observed under similar conditions following immunisation with MVAluc. Moreover, two successive injections of MVAgpn did not alter the broadness of
Gpn-specific T cell responses (data not shown). Altogether,
these data show that MVAgpn induces HLA-A2-restricted
CTL responses directed against peptides generated during
natural HIV infection.
3.4. MVAgpn stimulates human Pol2-specific CD8+ T cells
To define the relevance of our observations in a human
model, we investigated whether MVAgpn could stimulate
Pol2-specific human CD8+ T cells. To this aim, we used
the in vitro assay developed by Dorrell and co-workers to
show that recombinant MVA efficiently stimulates human
CTLs [27]. PBMCs from an HLA-A2+ HIV-1-infected patient (CNA 2099) were found to contain Pol2-specific CD8+
T cells as shown by staining with Pol2 HLA-A2 tetramer
(Fig. 6). The cells were then stimulated with MVAgpn at
increasing m.o.i. or with MVAluc as control. Pol2-specific
CD8+ T cells were specifically expanded in the presence of
MVAgpn but not in the presence of MVAluc. Moreover, the
expansion of Pol2-specific CD8+ cells, ranging from a 5.2to 16.7-fold increase in cell number, was proportional to the
m.o.i. No Pol2-specific T cells were detected in PBMCs from
73
seronegative donors (LHD 197) stimulated with MVAgpn.
Thus, these data show that the Pol2 epitope is generated in
MVAgpn-infected human cells, loaded onto HLA-A2 and
presented to human CD8+ T cells, confirming the results
obtained in the HHD mice. Gag-specific CD8+ T cells were
also specifically expanded upon stimulation with MVAgpn
(13.5-fold increase compared to baseline, at a m.o.i. of 1).
This observation suggests that some epitopes found to be
cryptic upon immunisation of the HHD mice are however
generated in human cells.
4. Discussion
Here we report the construction of a novel antigen containing multiple HIV proteins and its immunogenicity when
expressed in poxvirus vectors. The antigen reported in this
study has several advantages. First, it is constituted of the
major proteins targeted by CTLs in a natural infection and
gathered in a single expression cassette. In particular, the
Pol protein is often seen by the immune system but its expression level is low during a natural infection. Here, the removal of the frameshift between gag and pol results in similar expression level of Gag and Pol proteins, i.e. Gag- and
Pol-specific epitopes. Second, it has been designed for optimal safety as it does not produce functional proteins and,
therefore, no potential virus-like particles (Fig. 1). Third,
it has been engineered for human codon usage, which is
known to increase both antigen expression and immunogenicity [34–36]. Gpn has been inserted into several vac-
Simone C. Zimmerli
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
3401
Recently, Belyakov and colleagues have reported that
MVA and NYVAC induce similar levels of protection against
virulent Vaccinia virus challenge in mice [37]. However, no
studies have thoroughly compared the immunogenicity of
both vectors when carrying an heterologous antigen. Our
study indicates that Gpn-specific immune responses are
similar when induced by NYVACgpn and MVAgpn using
two different routes of inoculation. This also indicates that
the different insertion sites for the heterologous antigen
(TK or HA) have no apparent effect on the strength of the
immune response.
Mice transgenic for human HLA molecules are important
models to study the immunogenicity of vaccines dedicated
to human trials. Our results support the use of HHD mice
to study HIV-specific HLA-A2-restricted responses since
selected HIV-specific CD8 epitopes found to be immunodominant in humans are immunogenic in HHD mice.
Moreover, we provide strong evidence that an immunodominant epitope found after immunisation in the HHD mice is
similarly processed in humans cells, thereby reinforcing the
relevance of this mouse model in the prediction of vaccine
trials.
When the peptides were tested individually for their
intrinsic immunogenicity, the magnitude of the response
was found to be different between peptides: Gag, Pol2 and
Pol3 were more immunogenic than Prot and Pol1. This
observation could reflect a variation in HLA-A2 binding
affinity within these epitopes. Pol4 for instance is not immunogenic in HHD mice and has indeed a poor binding
and stabilising capacity for HLA-A2 [38]. Gagpolnef fusion protein delivered upon infection of mammalian cells
with MVAgpn is processed into HLA-A2-restricted epitopes. Among the selected epitopes, Prot and Pol2 are
immunodominant. The response against Pol2, also called
I9V, is particularly interesting since this peptide has been
reported in most HLA-A2 infected patients and is well conserved among HIV isolates, in particular B and C clades
(http://hiv-web.lanl.gov/immunology/index.html). This indicates that the CTL response induced by MVAgpn could
be cross-reactive in humans. The magnitude of the CTL
response observed against Pol2 and Prot was weaker than
the level of the Gagpolnef-specific response in conventional
mice. The difference could be due to the immunological
limitation of the HHD mice, i.e. low CD8+ T cell number
and limited CD8+ T-cell repertoire [39]. In addition, the
response in conventional mice is assumed to be directed
against the three antigenic determinants including Gag, Pol
and Nef and to be polyclonal, i.e. specific for various epitopes on each determinant. The strong Gagpolnef-specific
response in C57BL/6 supports this hypothesis. Therefore,
many HLA-A2-restricted epitopes different from those selected in this study are likely to stimulate CTL responses in
the HHD mice.
The Gag peptide (known also as S9L) combined to an adjuvant elicit strong CD8 responses in the HHD mice whereas
it is cryptic in the context of the fusion protein delivered
Fig. 5. MVAgpn induces Gagpolnef-specific HLA-A2-restricted CTLs in
HHD mice. Mice were immunised i.p. with a single injection of MVAgpn.
Ten days later, spleens of treated animals were sampled and assayed
for CD8+ T cell responses. (A) An IFN-␥ ELISPOT assay specific for
MVA antigens (with MVAluc-infected irradiated splenocytes as antigen
presenting cells) was performed on pooled splenocytes from three mice.
Number of spots per 106 cells against non-infected APC is <20. (B, C)
Splenocytes were also stimulated in vitro for 6 days with LPS blasts loaded
independently with each peptide. An IFN-␥ ELISPOT assay (B) and a
chromium release assay (C) were then performed using RMAS-HHD as
target cells. In panel C, squares and triangles represent specific lysis of
Pol2- and Prot1-loaded RMAS-HHD cells by Pol2- and Prot1-stimulated
effectors, respectively. Dashed line is background lysis of non loaded
RMAS-HHD. The difference in the magnitude of the response against the
Pol2 and Prot1 epitopes was not significant. SFC, asterisks and E:T mean
spot forming cells, no detectable response, and effector/target, respectively.
Data are representative of three experiments.
cine candidates which will be tested in a near future in
phase I clinical trials within the EuroVacc program. We
show that Gpn induces T cell response with peptide specificity similar to that found in HLA-A2+ HIV-infected individuals. Importantly, the dominance towards the Pol2 epitope was further extended to humans since MVAgpn but
not a control MVA stimulated pre-existing Pol2-specific human CD8+ T cells in vitro. Moreover, Gpn was immunogenic when expressed either from NYVAC or MVA. Altogether, these results indicate that Gpn delivered by recombinant poxviruses is a suitable antigen to use in human
vaccination.
74
Simone C. Zimmerli
3402
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
Fig. 6. MVAgpn specifically stimulates Pol2-specific human CD8+ T cells. Diagrams represent flow cytometry analysis of PBMCs from HIV-seropositive
(CNA 2099) and seronegative (LHD 197) patients stimulated or not with recombinant MVA for 10 days at indicated m.o.i. The number of Pol2-specific
CD8+ T cells was evaluated by staining with anti-human CD8 antibodies and Pol2-specific tetramer before (baseline) and after stimulation. Diagrams
represent cells gated on lymphocytes and numbers indicate the percentage of Pol2-specific cells among total lymphocytes.
by MVA. Therefore, the lack of Gag-specific response upon
immunisation with MVAgpn can not be explained by the
absence of specific na¨ıve cells in the HHD mice. In humans, the Gag epitope emerges only after chronic HIV infection and has been, therefore, referred as subdominant
peptide [40]. However, we found that Gag-specific human
CD8+ T cells are stimulated by MVAgpn in vitro. The difference between human and mouse could mirror some differences in the respective processing machineries. Overlapping peptide pools spanning the entire Gpn molecule should
be useful to address this point. This observation also means
that human vaccination with MVAgpn could induce CTL
specificities even broader than anticipated using the HHD
model.
The induction of a restricted set of CTLs might favour
the emergence of viral escape mutants, accelerating disease
progression [41]. It is therefore crucial for a successful vaccine to induce the greatest breadth of CTL responses. As it
was also shown for hepatitis B DNA vaccination [42], we
demonstrate here that a novel complex multi-determinant
HIV antigen is efficiently processed and is likely to induce
multiepitopic T-cell response in humans. As antibodies may
also play an important role in neutralising free viruses, the
Gp120 membrane protein will be included in the vaccine
to be used in clinical trials. The use of mouse model could
be valuable in comparing various vaccines and vaccination
protocols. The clinical trials that will be conducted by the
EuroVacc consortium are going to establish whether CTL
responses in HHD mice are predictive of those that the vaccines will elicit in humans.
75
Acknowledgements
We thank Murielle Michetti, Hiltrud Stubbe and Delphine
Galaud for the excellent technical assistance. We are grateful to the Centralised Facility for AIDS reagents for the
gift of antibodies and purified proteins. This work was supported by Grants from the Swiss National Science Foundation to JPK (3100-067926.02), the Spanish Foundation
for AIDS Research (FIPSE-36344/02), the NCCR Molecular Oncology Program and by an EU Grant EUROVAC
QLRT-PL1999-01321 (OFES 99.041-1 & 2).
References
[1] Nabel GJ. Challenges and opportunities for development of an AIDS
vaccine. Nature 2001;410:1002–7.
[2] Letvin NL, Barouch DH, Montefiori DC. Prospects for vaccine
protection against HIV-1 infection and AIDS. Ann Rev Immunol
2002;20:73–99.
[3] Amara RR, Villinger F, Altman JD, Lydy SL, O’Neil SP, Staprans
SI, et al. Control of a mucosal challenge and prevention of AIDS
by a multiprotein DNA/MVA vaccine. Science 2001;292:69–74.
[4] Belyakov IM, Hel Z, Kelsall B, Kuznetsov VA, Ahlers JD, Nacsa J,
et al. Mucosal AIDS vaccine reduces disease and viral load in gut
reservoir and blood after mucosal infection of macaques. Nat Med
2001;7:1320–6.
[5] Shiver JW, Fu TM, Chen L, Casimiro DR, Davies ME, Evans RK, et
al. Replication-incompetent adenoviral vaccine vector elicits effective
anti-immunodeficiency-virus immunity. Nature 2002;415:331–5.
[6] Robinson HL, Montefiori DC, Johnson RP, Manson KH, Kalish
ML, Lifson JD, et al. Neutralizing antibody-independent containment
of immunodeficiency virus challenges by DNA priming and
Simone C. Zimmerli
4. Results
A. Didierlaurent et al. / Vaccine 22 (2004) 3395–3403
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
3403
[25] Miyahira Y, Murata K, Rodriguez D, Rodriguez JR, Esteban M,
Rodrigues MM, et al. Quantification of antigen-specific CD8(+)
T-cells using an Elispot assay. J Immunol Methods 1995;181:45–54.
[26] Vitiello A, Sette A, Yuan L, Farness P, Southwood S, Sidney J,
et al. Comparison of cytotoxic T lymphocyte responses induced by
peptide or DNA immunization: Implications on immunogenicity and
immunodominance. Eur J Immunol 1997;27:671–8.
[27] Dorrell L, O’Callaghan CA, Britton W, Hambleton S, McMichael
A, Smith GL, et al. Recombinant modified vaccinia virus Ankara
efficiently restimulates human cytotoxic T lymphocytes in vitro.
Vaccine 2000;19:327–36.
[28] Rizzardi GP, Tambussi G, Bart PA, Chapuis AG, Lazzarin A,
Pantaleo G. Virological and immunological responses to HAART
in asymptomatic therapy-naive HIV-1-infected subjects according to
CD4 cell count. AIDS 2000;14:2257–63.
[29] Bart PA, Rizzardi GP, Tambussi G, Chave JP, Chapuis AG, Graziosi
C, et al. Immunological and virological responses in HIV-1-infected
adults at early stage of established infection treated with highly active
antiretroviral therapy. AIDS 2000;14:1887–97.
[30] Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K,
Nobile M, et al. Skewed maturation of memory HIV-specific CD8
T lymphocytes. Nature 2001;410:106–11.
[31] Ratner L. Complete nucleotide sequence of the AIDS virus,
HTLV-III. Nature 1985;313:277–84.
[32] Göttlinger HG, Sodroski JG, Haseltine WA. Role of capsid precursor
processing and myristoylation in morphogenesis and infectivity of
human immunodeficiency virus type 1. Proc Natl Acad Sci USA
1989;86:5781–5.
[33] Deml L, Schirmbeck R, Reimann J, Wolf H, Wagner R. Recombinant
human immunodeficiency Pr55(Gag) virus-like particles presenting
chimeric envelope glycoproteins induce cytotoxic T-cells and
neutralizing antibodies. Virology 1997;235:26–39.
[34] zur Megede J, Chen MC, Doe B, Schaefer M, Greer CE,
Selby M, et al. Increased expression and immunogenicity of
sequence-modified human immunodeficiency virus type 1 gag gene.
J Virol 2000;74:2628–35.
[35] Bojak A, Wild J, Deml L, Wagner R. Impact of codon usage
modification on T-cell immunogenicity and longevity of HIV-1
gag-specific DNA vaccines. Intervirology 2002;45:275–86.
[36] Liu WJ, Gao F, Zhao KN, Zhao W, Fernando GJ, Thomas R, et
al. Codon modified human papillomavirus type 16 E7 DNA vaccine
enhances cytotoxic T-lymphocyte induction and anti-tumour activity.
Virology 2002;301:43–52.
[37] Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt
LS, et al. Shared modes of protection against poxvirus infection
by attenuated and conventional smallpox vaccine viruses. Proc Natl
Acad Sci USA 2003;100:9458–63.
[38] Firat H, Tourdot S, Ureta-Vidal A, Scardino A, Suhrbier A, Buseyne
F, et al. Design of a polyepitope construct for the induction of HLAA0201-restricted HIV 1-specific CTL responses using HLA-A*0201
transgenic, H-2 class IKO mice. Eur J Immunol 2001;31:3064–74.
[39] Ureta-Vidal A, Firat H, Perarnau B, Lemonnier FA. Phenotypical
and functional characterization of the CD8+ T-cell repertoire of
HLA-A2.1 transgenic* H-2KbnullDbnull double knockout mice. J
Immunol 1999;163:2555–60.
[40] Goulder PJR, Altfeld MA, Rosenberg ES, Nguyen T, Tang
YH, Eldridge RL, et al. Substantial differences in specificity of
HIV-specific cytotoxic T cells in acute and chronic HIV infection.
J Exp Med 2001;193:181–93.
[41] Koenig S, Conley AJ, Brewah YA, Jones GM, Leath S, Boots LJ, et
al. Transfer of Hiv-1-Specific Cytotoxic T-Lymphocytes to an Aids
Patient Leads to Selection for Mutant Hiv Variants and Subsequent
Disease Progression. Nat Med 1995;1:330–6.
[42] Loirat D, Lemonnier FA, Michel ML. Multiepitopic HLA-A*0201restricted immune response against hepatitis B surface antigen after
DNA-based immunization. J Immunol 2000;165:4748–55.
recombinant pox virus booster immunizations. Nat Med 1999;5:526–
34.
McMichael A, Hanke T. The quest for an AIDS vaccine: is the
CD8+ T-cell approach feasible? Nat Rev Immunol 2002;2:283–91.
Amara RR, Villinger F, Staprans SI, Altman JD, Montefiori DC,
Kozyr NL, et al. Different patterns of immune responses but similar
control of a simian-human immunodeficiency virus 89.6P mucosal
challenge by modified vaccinia virus Ankara (MVA) and DNA/MVA
vaccines. J Virol 2002;76:7625–31.
Ourmanov I, Brown CR, Moss B, Carroll M, Wyatt L, Pletneva L,
et al. Comparative efficacy of recombinant modified vaccinia virus
Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol
and/or Env in macaques challenged with pathogenic SIV. J Virol
2000;74:2740–51.
Sutter G, Moss B. Nonreplicating vaccinia vector efficiently expresses
recombinant genes. Proc Natl Acad Sci USA 1992;89:10847–51.
Ramirez JC, Gherardi MM, Esteban M. Biology of attenuated
modified vaccinia virus Ankara recombinant vector in mice: virus
fate and activation of B- and T-cell immune responses in comparison
with the Western Reserve strain and advantages as a vaccine. J Virol
2000;74:923–33.
Stickl H, Hochstein-Mintzel V, Mayr A, Huber H, Schäfer CH,
Holzner A. MVA vaccination against smallpox:clinical trials of an
attenuated live vaccinia virus strain (MVA). Dtsch Med Wochenschr
1974;99:2386–92.
Hanke T, McMichael AJ. Design and construction of an experimental
HIV-1 vaccine for a year-2000 clinical trial in Kenya. Nat Med
2000;6:951–5.
Robinson HL. Vaccines: new hope for an aids vaccine. Nat Rev
Immunol 2002;2:239–50.
Hel Z, Venzon D, Poudyal M, Tsai WP, Giuliani L, Woodward R, et
al. Viremia control following antiretroviral treatment and therapeutic
immunization during primary SIV251 infection of macaques. Nat
Med 2000;6:1140–6.
Hel Z, Nacsa J, Tryniszewska E, Tsai WP, Parks RW, Montefiori DC,
et al. Containment of simian immunodeficiency virus infection in
vaccinated macaques: correlation with the magnitude of virus-specific
pre- and postchallenge CD4+ and CD8+ T-cell responses. J
Immunol 2002;169:4778–87.
Tartaglia J, Perkus ME, Taylor J, Norton EK, Audonnet JC, Cox WI,
et al. NYVAC: a highly attenuated strain of vaccinia virus. Virology
1992;188:217–32.
Sutter G, Staib C. Vaccinia vectors as candidate vaccines: the
development of Modified vaccinia virus Ankara for antigen delivery.
Curr Drug Target-Infect Disord 2003;3:263–71.
Gallego-Gomez JC, Risco C, Rodriguez D, Cabezas P, Guerra S,
Carrascosa JL, et al. Differences in virus-induced cell morphology
and in virus maturation between MVA and other strains (WR, Ankara,
and NYCBH) of vaccinia virus in infected human cells. J Virol
2003;77:10606–22.
Faulkner L, Borysiewicz LK, Man S. The use of human leucocyte
antigen class I transgenic mice to investigate human immune function.
J Immunol Methods 1998;221:1–16.
Firat H, Garcia-Pons F, Tourdot S. HLA-A2.1-transgenic mice:
a versatile animal model for preclinical evaluation of antitumor
immunotherapeutic strategies. Eur J Immunol 1999;29:3112–21.
Ramirez JC, Finke D, Esteban M, Kraehenbuhl JP, Acha-Orbea H.
Nasal inoculation of Modified Vaccinia Virus Ankara (MVA) is safe
in mice. Arch Virol 2003;148:827–39.
Chakrabarti S, Sisler JR, Moss B. Compact, synthetic, vaccinia
virus early/late promoter for protein expression. Biotechniques
1997;23:1094–7.
Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Perarnau
B. HLA-A2.1-restricted education and cytolytic activity of CD8(+)
T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1
monochain transgenic H-2Db beta2m double knockout mice. J Exp
Med 1997;185:2043–51.
76
Simone C. Zimmerli
4. Results
4.3. Publication #3
Cytomegalovirus (CMV)-Specific Cellular Immune Responses
Alexandre Harari, Simone C. Zimmerli, and Giuseppe Pantaleo
Human Immunol (2004) 65: 500-506
77
Simone C. Zimmerli
4. Results
Cytomegalovirus (CMV)-Specific Cellular
Immune Responses
Alexandre Harari, Simone C. Zimmerli, and
Giuseppe Pantaleo
ABSTRACT: A large percentage of healthy individuals
(50 –90%) is chronically infected with Cytomegalovirus
(CMV). Over the past few years, several techniques were
developed in order to monitor CMV-specific T-cell responses. In addition to the identification of antigen-specific T cells with peptide-loaded MHC complexes, most of
the current strategies to identify CMV-specific T cells are
centered on the assessment of the functions of memory T
cells including their ability to mediate effector function,
to proliferate or to secrete cytokines following antigenspecific stimulation. The investigation of these functions
has allowed the characterization of the CMV-specific T-
cell responses that are present during different phases of
the infection. Furthermore, it has also been shown that the
combination of virus-specific CD4 and CD8 T-cell responses are critical components of the immune response in
the control of virus replication. Human Immunology 65,
500⫺506 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier
Inc.
KEYWORDS: CMV; immune response; T cells; HIV;
monitoring
ABBREVIATIONS
BMT
bone marrow transplantation
CMV
cytomegalovirus
CTL
cytotoxic T lymphocytes
ER
endoplasmatic reticulum
HIV
human immunodeficiency virus
HLA
human leukocyte antigen
IE
immediate early
IFN
IL
MHC
PBMC
TNF
US
INTRODUCTION
Cytomegalovirus (CMV) is a member of the human
herpesvirus family, which includes herpes simplex virus
types 1 and 2, Epstein-Barr virus, varicella-zoster virus,
and human herpesvirus 5– 8. It is a human double-strand
DNA 230kb ␤-herpesvirus. More than 200 proteins are
produced in three overlapping phases (immediate early
(IE), early, and late). The predominant proteins critical
for virion production are envelope proteins gB, gH, gM,
and gL and the matrix proteins pp65/pp150/pp71 and
pp28 [1].
CMV resides in the host throughout life without
interferon
interleukin
major histocompatibility complex
peripheral blood mononuclear cells
tumor necrosis factor
unique short
causing any symptoms in healthy, immunocompetent
individuals, and 50 –90% of the population have become
seropositive by adulthood [2].
Primary CMV infection is always followed by a prolonged, inapparent infection during which the virus remains alive but usually dormant and resides in cells
without causing detectable damage or clinical illness.
The occurrence of CMV diseases is almost exclusively
restricted to immunocompromised hosts. In acquired
immunodeficiency syndrome (AIDS) patients, CMV
causes retinitis or enteritis [2]. CMV syndrome is characterized by fever, leukopenia, hepatosplenomegaly, myalgias and occasionally pneumonitis in organ transplant
recipients. Interstitial pneumonitis and rarely retinitis
may complicate bone marrow transplantation (BMT) [3].
In some cases CMV is also a cause of mononucleosis-like
illness [1].
CMV cellular reservoirs are leukocytes, epithelial cells
of salivary glands, and cervix. Infectious CMV may be
From the Laboratory of AIDS Immunopathogenesis, Division of Immunology and Allergy, Department of Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Address reprint requests to: Giuseppe Pantaleo, M.D., Laboratory of
AIDS Immunopathogenesis, Division of Immunology and Allergy, Centre
Hospitalier Universitaire Vaudois, Rue Bugnon, 1011 Lausanne, Switzerland; Tel: 41-21-3141071; Fax: 41-21-3141070; E-mail:
[email protected].
Human Immunology 65, 500⫺506 (2004)
© American Society for Histocompatibility and Immunogenetics, 2004
Published by Elsevier Inc.
0198-8859/04/$–see front matter
doi:10.1016/j.humimm.2004.02.012
78
Simone C. Zimmerli
4. Results
CMV-Specific Cellular Immune Responses
501
experienced early or late CMV reactivation. CMV-specific cytotoxic T lymphocyte (CTL) responses are generally lost in subject undergoing allogeneic BMT and
restoration of those responses requires an extended period
[6].
A series of genes are directly involved in these mechanisms of immune evasion [7]. The primary target of the
proteins encoded by these genes is the class I antigen
processing pathway: the unique short (US) region 3,
which is expressed in the IE phase binds to and retains
major histocompatibility complex (MHC) class I molecules in the endoplasmatic reticulum (ER). US2 and
US11, both early gene products, cause the translocation
of MHC class I to the cytosol where it is degraded. US6
blocks the transport of antigen-peptide into the ER.
CMV protein US2 contributes to the degradation of
human leukocyte antigen (HLA)-Dr␣ and HLA-Dm␣
through the inhibition of class II transactivator. The
inhibitory effects on T-cell antigen recognition is active
when viral or self-antigens are synthesized within the
cells, but not if the specific epitope is given as a peptide
[7]. These strategies may explain the immunodominance
of CTL directed against viral antigens that can be presented before expression of the US genes. In CMVinfected cells, the expression of the viral phosphoprotein
pp65 inhibits the generation of CMV-specific T-cell
epitopes [7].
shed in body fluids of infected persons, and may be
detected in urine, saliva, blood, tears, semen, and breast
milk. Examination of organ tissues and of peripheral
blood obtained from patients with CMV disease has
suggested that peripheral blood mononuclear cells
(PBMC) are also a viral reservoir, and further analyses of
PBMC revealed monocytes as the predominant infected
cell type [2]. On allogeneic stimulation of PBMC, latent
CMV can be reactivated to produce infectious virus.
CMV is thought to be controlled by antigen-specific
antiviral CD8 T cells. However, reactivation of CMV
occurs often in certain high-risk groups such as immunocompromised subjects, but also in asymptomatically
healthy individuals. A series of mechanisms have been
proposed to be responsible for CMV reactivation. These
include: (1) stress (through catecholamine using the
cAMP system), (2) inflammation (through tumor necrosis factor [TNF]-␣ using nuclear factor ␬B or through
prostaglandins using the cAMP pathway), and (3) some
cAMP-elevating drugs (e.g., pentoxifylline). The ultimate result is the activation of the CMV immediate early
(IE) enhancer/promoter, which is responsible for initiation of virus replication [4]. The high frequency of
CMV-specific effector CD8 T cells found in healthy
individuals indicates that CMV is more frequently reactivated than previously expected [4] but reactivation
remains unnoticed and asymptomatic. In contrast, most
of the situations of reactivation associated with clinical
CMV diseases occur after transplantation or in immunocompromised subjects.
Reactivation of CMV from latency results in serious
morbidity and mortality in immunocompromised transplant recipients or immunodeficient individuals and has
both direct and indirect effects. Actually, CMV is the
leading cause of death in allogenic BMT recipients and
CMV-associated disease affects 40% of AIDS patients
[5]. In AIDS patients, CMV reactivation is associated
with the loss of CMV-specific proliferative responses.
Interestingly, CMV infection is reactivated during bacterial sepsis. Four days after the onset of sepsis 32.4% of
patients with sepsis show signs of active CMV infection
[5]. This percentage is equally high in renal transplantation patients [5].
The loss of immune control of CMV that is evidenced
by the detection of antigenemia is closely associated with
an impaired function of CMV-specific CD8 T cells. In
fact, it is the reduced cytokine production rather than a
lower frequency or absolute number of CMV-specific
CD4 or CD8 T cells that is thought to be responsible for
the loss of immune control. Reduced numbers of cytokine-producing CMV-specific CD8 T cells were found in
individuals with a higher risk of CMV reactivation. The
highest frequencies and absolute numbers of CMV-specific CD8 T cells were noted in those subjects who
Monitoring
The monitoring of CMV replication is critical after transplantation. Among the laboratory tests that have been
used, the CMV DNA amplification assay in plasma has
shown limited sensitivity as compared with the detection
of pp65 antigen in leukocytes. Recently, Kaiser et al. [8]
developed an ultrasensitive plasma DNA polymerase
chain reaction assay with a limit of detection of 20 CMV
DNA copies/ml of plasma. By using this latter technique, the occurrence of CMV reactivation in stem cell
transplantation patients and renal transplant recipients is
detected on average 4 and 12 days earlier, respectively,
compared with the pp65-positive test [8].
Until recently, chromium release assays and limitingdilution analyses were the most common techniques used
to measure specific T-cell responses for research purposes.
However, these techniques are time-consuming and not
very sensitive [9]. An additional strategy to evaluate
antigen-specific T-cell responses is the proliferation assay, which is based on the ability of cells (mostly CD4 T
cells) to proliferate after antigen stimulation. The major
advantage of this technique is that it requires only very
few cells, but this strategy does not allow precise quantification of the actual frequencies of helper CD4 T cells
and presents a series of technical constraints that have
made difficult its standardization.
79
Simone C. Zimmerli
4. Results
502
A. Harari et al.
effector and memory differentiation have been extensively described [10].
The major expansion of CMV-specific CD8 T cells
during primary CMV infection is well-documented but
until recently little was known about CD4 T-cell responses. In this regard, it has recently been shown, in a
study analyzing CD4 T-cell responses during primary
and chronic CMV and human immunodeficiency virus
(HIV)-1 infections, that a major expansion of virusspecific interferon (IFN)-␥ secreting CD4 T cells is associated with primary CMV but not with HIV-1 infection [11] (Figure 3). In contrast to HIV-1 infection, the
magnitude of primary CMV-specific CD4 T-cell response was indeed significantly different from that observed during chronic infection. It is also worth noting
that the large majority (⬎90%) of the expanded IFN-␥
secreting virus-specific CD4 T cells was contained within
the cell population lacking CCR7 and therefore belonged
to the effector memory cell population [11] (Figure 3).
Of interest, the kinetics and characteristics of CMVspecific CD4 and CD8 T cells were recently investigated
in the course of primary CMV infection in asymptomatic
and symptomatic recipients of renal transplants [12]. It
has been shown that in the case of an asymptomatic
primary CMV infection, the peak of IFN-␥ secreting
CD4 T cells appears 10 days after CMV DNA is detectable and is followed, 7 days later, by the appearance of
immunoglobulin M and immunoglobulin G and 14 days
later by the appearance of CMV-specific CD8 T cells.
Interestingly, in the few symptomatic patients, there
were no significant differences in the kinetic or magnitude of the antibody or CD8 T-cell responses but there
was a delay in the CD4 T-cell response [12].
In contrast to CD4 T-cell response during primary
infection that was only analyzed recently [10], the clonal
expansion of virus-specific CD8 T cells during primary
infection is a hallmark of the adaptive immune response.
Recently, Gamadia et al. [12] have performed an analysis
of the CMV-specific CD8 T-cell response during primary
infection. They have demonstrated that these cells had a
phenotype of typical effector cells (i.e., CCR7CD27⫹CD45RA-). They also demonstrated that, in contrast to the previous experiments performed in mice,
functional CD8 T cells were not sufficient to control viral
replication and that CMV-specific IFN-␥ secreting CD4
T cells were necessary for recovery from the infection
[12].
A novel methodology has been recently developed to
determine cell division and proliferation in mononuclear
cells after antigen-specific stimulation. This method is
based on the use of a dye, carboxyfluorescein succinimidyl ester (CFSE), which allows to visualize proliferating
cells by flow cytometry (Figure 1A). After cell division,
CFSE equally distributes within the two daughter cells
and, after each cell division, fluorescence intensity is
reduced of 50%. The advantage of this technique is the
possibility to combine multiple surface markers to identify and characterize the type of dividing cells.
The enzyme-linked immunospot assay (Elispot) allows
the delineation of the functional properties of T cells and,
particularly, their ability to secrete cytokines after antigen stimulation [9]. T cells (either PBMC or purified
CD4 or CD8 T cells) and antigens are mixed in anticytokine antibodies precoated plates (Figure 1B). A second
chromogenic substrate coupled to anticytokine antibodies is added and the secreted cytokine molecules form
spots. The advantage of this technique is the high degree
of sensitivity (i.e., at least 30 –100 times more sensitive
than the chromium release assay [9]). Furthermore, it is
the most efficient assay available for the quantitative
evaluation of antigen-specific immune responses [9].
Intracellular cytokine staining is another methodology available for the functional characterization and
quantification of antigen-specific T cells. The advantage
of using this technique resides in the possibility of assessing simultaneously the phenotype, the function, the
replicative history, and the number of antigen-specific T
cells. The limitations are mostly associated with the
technical difficulties in the standardization of flow cytometry procedures (Figure 1C).
The development of MHC class I tetramer complexes
has substantially advanced the characterization of antigen-specific CD8 T-cell responses. Along the same line,
MHC class II tetramers were developed to study CD4
T-cell responses. However, the development of MHC
class II tetramers is still in a preliminary phase. Fluorescent multimers of MHC-peptide complexes are used to
quantify, to perform functional and phenotypic characterization, and to isolate peptide-specific T cells by flow
cytometry (Figure 2).
Acute Infection
Primary CMV infection is usually asymptomatic but can
also cause a mononucleosis-like illness, with leukopenia,
fever, and hepatitis. Acute CMV infection is usually
localized within the salivary gland epithelium [1].
After antigenic stimulation, naive T cells follow a
program of proliferation and differentiation that leads to
the generation of effector cells and, ultimately, to the
generation of memory cells. The different models of
Chronic Infection
In the immunocompetent host, the virus remains efficiently controlled and several components of the immune
system are shown to play a role. In mice it has been
demonstrated that both T and B cells play a role in the
control of CMV [2, 13].
80
Simone C. Zimmerli
4. Results
CMV-Specific Cellular Immune Responses
503
FIGURE 1 (A) Representative
flow cytometry profiles of carboxyfluorescein succinimidyl ester
(CFSE) labeled CD4 T cells. After
6 days of culture, staphylococcal
enterotoxin B (SEB)–stimulated
cells (right panel) show three
peaks of cell division as compared
with the unstimulated cells (left
panel). (B) Representative enzyme-linked immunospot (Elispot) wells after different stimulations. Each dark spot correspond
to one cytokine-secreting cell. (C)
Flow cytometry profiles of cytomegalovirus (CMV)-specific interferon (IFN)-␥ secreting CD4 and
CD8 T cells of one representative
subject. CD4 T cells were stimulated with CMV lysate, whereas
CD8 T cells were stimulated with
a pool of pp65 peptides and then
stained with anti-CD4 or CD8
PerCp Cy5.5, anti-CD69 FITC
and anti–IFN-␥-APC, and analyzed by flow cytometry. The cluster of events shown in red corresponds to the responder T cells
(i.e., co-expressing CD69 and
IFN-␥), whereas the cluster of
events in blue correspond to the
nonresponder T cells. Data are expressed as the percentage of cells
coexpressing IFN-␥ and CD69
within CD4 or CD8 T cells.
81
Simone C. Zimmerli
4. Results
504
A. Harari et al.
FIGURE 2 (A) Major histocompatibility complex (MHC)
class I heavy chain molecules
lacking the transmembrane domain are refolded with ␤2-microglobulin together with a
peptide of interest. Bacterial
BirA enzyme is used to attach a
biotin molecule to the specific
BirA-recognition sequence that
has been incorporated into the
C-terminus of the MHC molecule. Tetramers are finally
formed by incubation with fluorescence labeled streptavidin
molecules. (B) Example of a tetramer staining of blood mononuclear cells (PSMC) from an
human
leukocyte
antigen
(HLA)–matched cytomegalovirus (CMV)-infected subject.
The comparison between HIV- and CMV- specific
CD8 T cells has also allowed the identification of a weak
level of perforin in HIV-specific CD8 T cells as compared
to CMV-specific CD8 T cells. This was related to a
significantly higher cell lysis capacity for a given effector/
target ratio [13]. However, conflicting results have been
reported regarding the ability of CMV-specific CD8 T
cells to express perforin.
Most of the CMV-specific CD8 T-cell responses are
specific to pp65 but, in a number of cases, the response
to the identified HLA-A2–restricted IE-1 peptides was
shown to exceed that of A2-pp65. IE-1–specific cells
have high CD57, low CD28 expression, absence of
CCR7, and high levels of intracellular perforin, which is
typical of CMV-specific memory T cells [17].
Even HIV-1–infected individuals can maintain high
and stable frequencies of CMV-specific CD4 and CD8 T
cells. In contrast, there is a decrease in the frequency of
CD4 T cells (as assessed by the proportion of CMVspecific TNF-␣ secreting CD4 T cells) that leads to the
inability to sustain CD8 T cells in patients with CMVassociated end-organ disease [2].
In HIV and CMV coinfected patients, elevated and very
stable CD4 and CD8 T-cell responses to CMV were observed [14, 15]. The majority of CMV-specific CD8 T cells
in peripheral blood were able to produce a range of antiviral factors after stimulation with specific antigens (IFN-␥,
macrophage inflammatory protein-1␤, TNF-␣) [13].
With regard to CD4 T cells, CMV-specific lymphoproliferation and interleukin (IL)-2 secreting CD4 T-cell
responses were positive in healthy subjects [14] but
deficient CD4 T-cell responses were reported in BMT
recipients [2]. The majority of CMV-specific IFN-␥ secreting CD4 T cells belonged to the CCR7- compartment whereas interleukin-2–secreting CD4 T cells represented the dominant virus-specific population within
CCR7⫹ cells [14].
Our group has previously reported that CMV-specific
memory CD8 T cells were composed by 50% of terminally differentiated cells (CD45RA⫹CCR7-), 40% of
preterminally differentiated cells (CD45RA–CCR7⫺),
and 10% of CCR7⫹ cells in both blood and lymph nodes
[15]. A phenotype of differentiated cells was also confirmed by others using different immunologic markers,
such as CD27 and CD28 or CD7 [13, 16]. In this regard,
Aandahl et al. [16] have also recently shown a model in
which CD7 identifies three populations of CD8 T cells.
CD7high CD8 T cells include naive and memory cells and
retained proliferative capacity and in contrast CD7low
and CD7neg CD8 T cells discriminate between two subsets of effector cells (i.e., cytokine-secreting cells and cells
with high lytic potential respectively [16]).
Tissue Distribution
As shown in mice, memory T cells with effector function
accumulate in the target organ of the pathogen away
from the lymphoid tissue [18]. The anatomic distribution (e.g., blood and lymph nodes) of CMV-specific as
compared with HIV-1–specific T cells was recently investigated. Although HIV-1–specific IFN-␥ CD4 T cells
82
Simone C. Zimmerli
4. Results
CMV-Specific Cellular Immune Responses
505
FIGURE 3 (A) Analysis of
human immunodeficiency virus (HIV)-1–specific and cytomegalovirus (CMV)-specific CD4 T cells within
different populations of
memory cells defined by the
expression of CCR7. Patients
3 and 4 had primary HIV-1
and CMV coinfection, and
patient 8 had primary HIV-1
infection and chronic CMV
infection. Blood mononuclear
cells were stimulated with
p55 gag (i.e., an HIV-1 protein) and CMV lysates and
analyzed for the expression of
CD4, CCR7, CD69, and interferon (IFN)-␥ (intracellular expression). The data show
the expression of CD69 and
IFN-␥ within CD4⫹CCR7and CD4⫹CCR7⫹ T-cell populations. Negative control: unstimulated blood mononuclear
cells. After in vitro antigenspecific stimulation, blood
mononuclear
cells
were
stained with anti–IFN-␥
APC, anti-CD69 FITC, antiCCR7 PE, and anti-CD4 Cychrome. Adapted from Harari
et al., Blood 2002 [11]. (B)
Summary of the magnitude of
the response of HIV-1–specific and CMV-specific primary and chronic infection.
MHC complex. CTL from different donors that recognize
the same peptide-MHC complex use only one or two V␤
gene segments. A given ␤-chain is always associated with
the same ␣-chain. Memory CTL response to individual
CMV pp65 epitopes contains individual clones that have
undergone extensive expansion in vivo [20]. In some individuals, IE-1 (72kDa major IE protein), which is a nonstructural protein found very early and throughout the
replicative cycle, may be of the same importance as pp65.
In healthy subjects, the percentage of individuals with
pp65-specific CD8 T-cell response seems clearly larger
than the percentage of those with IE-1–specific CD8
T-cell response. CD8 T cells specific to pp65 are found in
a similar proportion of healthy subjects and renal transplant patients (85% vs 83%), whereas CD8 T cells
reactive to IE-1 occurred in a higher percentage of renal
transplant patients than of healthy subjects (75% vs
48%) [21]. The ubiquity of pp65-specific CTL in natural
CMV infection suggests that pp65 epitopes can be presented by a wide variety of HLA alleles. Kondo et al. [17]
recently identified 14 novel CTL epitopes derived from
were equally distributed in the blood and lymph node
compartments, CMV-specific IFN-␥ CD4 T cells predominantly accumulated in the blood [14]. Similar observations were obtained for CD8 T cells where memory
CMV-specific CD8 T cells were accumulated mostly
(10-fold higher frequency) in the blood as compared with
the lymph nodes, whereas HIV-1–specific CD8 T cells
were equally distributed [15]. Therefore, the results obtained are compatible with the different target organs of
HIV-1 (i.e., the lymphoid tissue) and of CMV (i.e., lung,
cervix, salivary glands, the retina) [19].
Clonality, Repertoire, and Immunodominance
CMV matrix protein pp65 has been identified as a significant target antigen for CMV-specific CTL. CMVspecific CTL recognition of pp65 on target cells occurs
immediately after infection of a cell, before the onset of
viral gene expression, and persists throughout the duration of the replicative cycle [1].
There is a high degree of clonal focusing among
memory CTL–specific for a given CMV pp65 peptide-
83
Simone C. Zimmerli
4. Results
506
A. Harari et al.
CMV pp65 antigen, restricted to several HLA-A,
HLA-B, and HLA-C alleles.
For CMV-specific CD4 T cells, stable clonotypic hierarchy could be shown in which 1–3 clonotypes accounted for 10 –50% of the overall CMV response and
comprised 0.3– 4.0% of peripheral blood CD4 T cells
[22]. The CMV-specific CD4 T-cell memory repertoire
in normal subjects is characterized by striking clonotypic
dominance suggesting that primary responsibility for
immunosurveillance against CMV reactivation rests with
a handful of clones recognizing a limited array of CMV
determinants [22].
10. Kaech SM, Wherry EJ, Ahmed R: Effector and memory
T-cell differentiation: implications for vaccine development. Nat Rev Immunol 2:251, 2002.
11. Harari A, Rizzardi GP, Ellefsen K, Ciuffreda D, Champagne P, Bart PA, Kaufmann D, Telenti A, Sahli R,
Tambussi G, Kaiser L, Lazzarin A, Perrin L, Pantaleo G:
Analysis of HIV-1- and CMV-specific memory CD4 Tcell responses during primary and chronic infection. Blood
100:1381, 2002.
12. Gamadia LE, Remmerswaal EB, Weel JF, Bemelman F,
van Lier RA, Ten Berge IJ: Primary immune responses to
human CMV: a critical role for IFN-gamma-producing
CD4⫹ T cells in protection against CMV disease. Blood
101:2686, 2003.
13. Van Lier RA, Ten Berge IJ, Gamadia LE: Human CD8(⫹)
T-cell differentiation in response to viruses. Nat Rev
Immunol 3:931, 2003.
14. Harari A, Petitpierre S, Vallelian F, Pantaleo G: Skewed
representation of functionally distinct populations of virus-specific CD4 T cells in HIV-1-infected subjects with
progressive disease. Changes after antiretroviral therapy.
Blood 103:966, 2004.
15. Ellefsen K, Harari A, Champagne P, Bart PA, Sekaly RP,
Pantaleo G: Distribution and functional analysis of memory antiviral CD8 T cell responses in HIV-1 and cytomegalovirus infections. Eur J Immunol 32:3756, 2002.
16. Aandahl EM, Sandberg JK, Beckerman KP, Taske´ n K,
Moretto WJ, Nixon DF: CD7 is a differentiation marker
that identifies multiple CD8 T cell effector subsets. J Immunol 170:2349, 2003.
17. Kondo E, Akatsuka Y, Kuzushima K, Tsujimura K,
Asakura S, Tajima K, Kagami Y, Kodera Y, Tanimoto M,
Morishima Y, Takahashi T: Identification of novel CTL
epitopes of CMV-pp65 presented by a variety of HLA
alleles. Blood 103:630, 2004.
18. Sprent J, Surh CD: T cell memory. Ann Rev Immunol
20:551, 2002.
19. Harari A, Ellefsen K, Champagne P, Nobile M, Pantaleo
G: Antiviral memory T cell responses: correlation with
protective immunity and implication for vaccine development. Adv Exp Med Biol 512:155, 2002.
20. Weekes MP, Wills MR, Mynard K, Carmichael AJ, Sissons JG: The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone
extensive expansion in vivo. J Virol 73:2099, 1999.
21. Kern F, Faulhaber N, Frommel C, Khatamzas E, Prosch S,
Schonemann C, Kretzschmar I, Volkmer-Engert R, Volk
HD, Reinke P: Analysis of CD8 T cell reactivity to
cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol 30:1676, 2000.
22. Bitmansour AD, Waldrop SL, Pitcher CJ, Khatamzas E,
Kern F, Maino VC, Picker LJ: Clonotypic structure of the
human CD4⫹ memory T cell response to cytomegalovirus. J Immunol 167:1151, 2001.
Conclusion
A large percentage (50 –90%) of healthy individuals is
chronically infected with CMV. Of interest, CMV infection is successfully controlled by the host immune system and the infection goes generally unnoticed with the
exception of immune-compromised hosts. The ability to
control CMV infection is mediated by the combination
of both virus-specific CD4 and CD8 T-cell responses.
REFERENCES
1. Landolfo S, Gariglio M, Gribaudo G, Lembo D: The
human cytomegalovirus. Pharmacol Therap 98:26, 2003.
2. Scholz M, Doerr HW, Cinatl J: Human cytomegalovirus
retinitis: pathogenicity, immune evasion and persistence.
Trends Microbiol 11:171, 2003.
3. Hummel M, Abecassisa MM: A model for reactivation of
CMV from latency. J Clin Virol 25(Suppl 2):S123, 2002.
4. Reinke P, Prosch S, Kern F, Volk HD: Mechanisms of
human cytomegalovirus (HCMV) (re)activation and its
impact on organ transplant patients. Transpl Infect Dis
1:157, 1999.
5. Kutzka AS, Muhl E, Hackstein H, Kirchner H, Bein G:
High incidence of active cytomegalovirus infection among
septic patients. Clin Infec Dis 26:1076, 1997.
6. Reusser P, Riddell SR, Meyers JD, Greenberg PD: Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of
recovery and correlation with cytomegalovirus infection
and disease. Blood 78:1373, 1991.
7. Ploegh HL: Viral strategies of immune evasion. Science
280:248, 1998.
8. Kaiser L, Perrin L, Chapuis B, Hadaya K, Kolarova L,
Deffernez C, Huguet S, Helg C, Wunderli W: Improved
monitoring of cytomegalovirus infection after allogeneic
hematopoietic stem cell transplantation by an ultrasensitive plasma DNA PCR assay. J Clin Microbiol 40:4251,
2002.
9. Romero P, Cerottini JC, Waanders GA: Novel methods to
monitor antigen-specific cytotoxic T-cell responses in cancer immunotherapy. Molec Med Today 4:305, 1998.
84
Simone C. Zimmerli
5. Conclusions and Outlook
5. Conclusions and Outlook
The aim of my thesis was to investigate memory CD8 T cells in different models of virusspecific T cell responses. In particular, CD8 T cell responses to HIV-1, CMV, EBV and
Influenza virus (Flu). HIV-1-specific CD8 T cell responses were studied in cohorts of subjects
with progressive infection and compared to individuals with non-progresssive infection who
where naïve to antiretroviral therapy. Knowledge about the keypoints of an effective CD8 T
cell response would be helpful developing efficient therapeutic vaccines against HIV-1
infection.
Several studies highlight the importance of CD8 T cells in the rhesus monkey model:
1. Depletion of CD8 T cells by monoclonal Ab results in enhanced viral replication (25,
207, 208).
2. CTL are able to exert selective pressure on the viral genome (209, 210).
3. Vaccine strategies that are able to elicit virus-specific CD8 T cell response are also
capable of controlling viral replication and prevent the onset of disease (211-213).
The importance of CD8 T cells has also been proven in human studies:
1. CTL are able to control viral replication in vitro (33).
2. The appearance of HIV-1-specific CTL occurs simultaneously with the control of a
burst of viral replication (31).
3. HIV-1 infected individuals with low viral loads show a potent virus-specific CTL
response (63, 214-217).
4. Loss of HIV-1-specific CD8 T cells is linked to a rapid progression to AIDS (217).
5. Similar as in monkeys, CTL exert a potent selective pressure on HIV, which can be
shown by the appearance of epitope escape mutants (35, 79, 80).
In preliminary experiments, we performed a phenotypic characterization of the virus-specific
CD8 T cell respsonses by staining PBMC with MHC class I tetramers loaded with virusspecific peptides and CD45RA and CCR7 markers. Confirming previous studies from our and
other groups (14, 36, 218), we found a phenotypic heterogeneity within the different models
of virus-specific CD8 T cell responses. HIV-1-specific CD8 T cells in subjects with
progressive disease were almost exclusively CD45RA-CCR7- TEM while CMV- and EBV85
Simone C. Zimmerli
5. Conclusions and Outlook
specific CD8 T cells were mostly CD45RA+CCR7- (14, 219, 220). The latter phenotype is
therefore typical of a CD8 T cell response associated with an immune control of the pathogen.
In support of this hypothesis, a recent study has shown a correlation between the proportion of
HIV-1-specific CD45RA+CCR7- CD8 T cells and control of viral replication (221).Thus, the
CD45RA+CCR7- phenotype seems to be a marker of immune control in virus infections,
where the pathogen is able to establish chronic infection. However, the same phenotype could
not been found in the pool of Flu-specific CD8 T cells, despite the fact that the virus is
efficiently cleared by the immune response. Thus, it is likely that the CD45RA+CCR7subpopulations are only generated under conditions of protracted Ag stimulation which is not
the case in Flu infection. The CD45RA-CCR7- phenotype which is typical for TEM CD8 T
cells, was found both under conditions of high Ag load (progressive HIV-1 infection) and of
Ag clearance (Flu infection), therefore reflecting situations of ongoing or pre-existing effector
responses. Finally, consistently with what has been shown in the LCMV model (222-224), the
highest proportion of CD45RA-CCR7+ TCM were found under conditions of complete Ag
clearance, i.e. Flu-specific CD8 T cell response.
The results of functional characterization of virus-specific CD8 T cell responses have
demonstrated a selective defect of IL-2 secreting cells in the settings of progressive HIV-1
infection accompanied by an inability of HIV-1-specific CD8 T cells to proliferate in
response of specific stimulation. Virus-specific IL-2 secreting cells were consistently detected
in CMV, EBV, non-progressive HIV-1 and Flu infections. In all these conditions, there is an
effective immune control, where the Ag level is either low or even cleared. Previous studies
have hypothesized that phenotypic and functional heterogeneity of virus-specific CD8 T cell
responses was pathogen-specific (36). However, the present results show that the same
pathogen (i.e. HIV-1) is associated with substantially different CD8 T cell responses in
progressive as compared to non-progressive HIV-1 infection. The major difference between
those two settings is the level of Ag. Therefore, consistent with the observations in the LCMV
model (223), our results rather support the hypothesis that the phenotypic and functional
heterogeneity of virus-specific CD8 T cells is influenced by Ag persistence and Ag load. The
fact that IFN-γ/IL-2-secreting cells are lacking in progressive HIV-infection, but not in
LTNPs indicate that IL-2 secretion by CD8 T cells might be associated with control of HIV-1
infection or viral infections in general. Interestingly therapies administrating exogenous IL-2
have only an effect on CD4 T cells but not CD8 T cells (225-228). Another study
demonstrated that the capacity of IL-2 to increase CD8 T cell counts and the expression of
perforin and granzyme B is limited to the time of cytokine administration. Prolonged
86
Simone C. Zimmerli
5. Conclusions and Outlook
observations indicated rather a tendency of CD8 T cells to decline over time (229). In view of
the results of our studies, this might suggest that autocrine secretion of IL-2 by virus-specific
CD8 T cells, but not exogenous IL-2 exposure is crucial for the maintenance of their own
proliferation.
The factors responsible for the capacity of a CD8 T cell to secrete IL-2 still remain to be
investigated. From our studies it is not clear whether the high viral load exhausts IL-2
secretion, or whether IL-2 secretion is responsible for the low viral load. Since our results
demonstrate that only the CD45RA-CCR7- memory subset secrets IL-2, the presence of
different memory subsets in the various virus infections might be the key. However, this
subset is also predominant in individuals with progressive HIV-1 infection. Thus, the
mechanism of total Ag clearance must have an alternative explanation. It may be interesting
to look at a molecular level. The strength of signal (peptide affinity, avidity), influenced by
the higher viral load might influence the decision if IL-2 or IFN-γ is secreted or which
receptor is expressed or downregulated.
To evaluate whether the Ag-load is responsible for the absence of IL-2 secreting CD8 T cells,
different manipulations of the in vivo Ag level and exposure might be performed. Therefore
the capacity of CD8 T cells to secrete IL-2 or to proliferate should be investigated in HIV-1
infected individuals under ART. This would be equal to the model of protracted viral
infection with controlled virus replication. Under these conditions, we would expect to find
IL-2 secreting HIV-specific CD8 T cells that are able to proliferate as it could be shown in
LTNP. To prove that the absence of IL-2 secreting cells is not restricted to HIV-infection,
cytokine secretion should be addressed in individuals with acute CMV or EBV infection.
Furthermore, it would be interesting to compare the viral immune response in the same
individual before ART, during ART and after treatment interruption. Last but not least,
individuals with cleared viral infections should be re-exposed to the same virus by reimmunization. We tried to perform this in individuals getting vaccinated against Influenza.
Unfortunately we were not able to detect Flu-specific CD8 T cells following immunization
possibly because the vaccine didn’t contain the same peptides as used for our experiments. To
avoid this, we should probably screen Flu-specific CD8 T cells by stimulating PBMCs with
the vaccine, but it is not sure whether a 12h-stimulation would be long enough for peptideprocessing and presentation to CD8 T cells.
Previous studies performed in our lab (46, 230, 231) have addressed these issues in Agspecific CD4 T cells. As a model of Ag clearance, tetanus toxoid (TT) was used, CMV, EBV
and HIV-1 from LTNP were used as models of Ag-persistence and protracted Ag exposure
87
Simone C. Zimmerli
5. Conclusions and Outlook
with controlled viral replication. Progressive HIV-1 infection represented a situation of Ag
persistence with uncontrolled virus replication. In adddition, primary CMV and HIV infection
was analyzed as fourth model of acute Ag exposure and high viral Ag laod. As shown for
CD8 T cells, different functional subsets could be defined by the secretion of IFN-γ and IL-2.
However, in CD4 T cells there is an additional subset i.e. single IL-2 secreting cells which is
the dominant response to TT, and which is also present for CMV, EBV and HIV in nonprogressive infection. The results are quite similar for CD4 and CD8 T cells. In both T cell
subsets, the absence of IL-2 secreting cells is associated with a situation of high Ag levels. In
vivo manipulations of Ag levels and exposure confirmed that the Ag level is critical for the
appearance of the different functional memory subsets. In the case of CD4 T cells, it was
possible to re-immunize subjects having exclusively TT-specific single IL-2 secreting cells. A
substantial increase in the percentage of IL-2 secreting cells could be observed with a peak at
day 11 but more interestingly, the appearance of dual IL-2/IFN-γ and single IFN-γ could be
demonstrated.
In contrast, manipulation of the conditions with virus persistence with controlled virus
replication and low Ag exposure (ART, HIV-treatment interruption) showed a change from a
polyfunctional profile (i.e. presence of single IL-2, dual IL-2/IFN-γ and single INF-γ subsets)
to a single IFN-γ secreting CD4 T cell response.
Since there seems to be an analogy of the CD4 and CD8 T cell response to virus infection, we
would expect similar results in CD8 T cells.
Along the same line, the phenotype with regard to CD45RA and CCR7 of Ag-specific T cells
is always similar between CD4 and CD8 T cells. Three different memory subsets can be
found in CD4 T cells as well as in CD8 T cells (14, 219, 231). In both cell types, the
CD45RA-CCR7- subset dominates in HIV-1 infection, whereas CMV- or EBV-specific T
cells are predominantly CD45RA+CCR7-.
While in CD4 T cells it could clearly be demonstrated that the Ag is dictating the functional
and phenotypic profile of the response, this remains to be done in CD8 T cells.
88
Simone C. Zimmerli
6. References
6. References
1.
Pace, N. R. (1997) Science 276, 734-40.
2.
Heine, H. & Lien, E. (2003) Int Arch Allergy Immunol 130, 180-92.
3.
Elward, K. & Gasque, P. (2003) Mol Immunol 40, 85-94.
4.
Takeda, K., Kaisho, T. & Akira, S. (2003) Annu Rev Immunol 21, 335-76.
5.
Beutler, B. & Rietschel, E. T. (2003) Nat Rev Immunol 3, 169-76.
6.
Janeway, C. A., Travers, P., Walport, M. & Shlomchik, M. J. (2005) Immunobiology
6, the immune system in health and disease (Garland Science Publishing Churchill
Livingstone, New York).
7.
Abbas, A. K. & Lichtman, A. H. (2001) Basic Immunology (W.B. Saunders
Compagny.
8.
Palmer, E. (2003) Nat Rev Immunol 3, 383-91.
9.
Werlen, G., Hausmann, B., Naeher, D. & Palmer, E. (2003) Science 299, 1859-63.
10.
Palmer, E. (2005) Nat Immunol 6, 9-10.
11.
Harty, J. T., Tvinnereim, A. R. & White, D. W. (2000) Annu Rev Immunol 18, 275308.
12.
Wong, P. & Pamer, E. G. (2003) Annu Rev Immunol 21, 29-70.
13.
Sallusto, F. & Lanzavecchia, A. (1999) Nature 401, 708-12.
14.
Champagne, P., Ogg, G. S., King, A. S., Knabenhans, C., Ellefsen, K., Nobile, M.,
Appay, V., Rizzardi, G. P., Fleury, S., Lipp, M., Förster, R., Rowland-Jones, S.,
Sekaly, R. P., McMichael, A. J. & Pantaleo, G. (2001) Nature 410, 106-11.
15.
Hamann, D., Baars, P. A., Rep, M. H., Hooibrink, B., Kerkhof-Garde, S. R., Klein, M.
R. & van Lier, R. A. (1997) Journal of Experimental Medicine 186, 1407-18.
16.
Hamann, D., Kostense, S., Wolthers, K. C., Otto, S. A., Baars, P. A., Miedema, F. &
van Lier, R. A. (1999) Int Immunol 11, 1027-33.
17.
Van Baarle, D., Kostense, S., van Oers, M. H., Hamann, D. & Miedema, F. (2002)
Trends in Immunology 23, 586-91.
18.
Appay, V., Dunbar, P. R., Callan, M., Klenerman, P., Gillespie, G. M., Papagno, L.,
Ogg, G. S., King, A., Lechner, F., Spina, C. A., Little, S., Havlir, D. V., Richman, D.
D., Gruener, N., Pape, G., Waters, A., Easterbrook, P., Salio, M., Cerundolo, V.,
McMichael, A. J. & Rowland-Jones, S. L. (2002) Nature Medicine 8, 379-85.
89
Simone C. Zimmerli
19.
6. References
Brenchley, J. M., Karandikar, N. J., Betts, M. R., Ambrozak, D. R., Hill, B. J., Crotty,
L. E., Casazza, J. P., Kuruppu, J., Migueles, S. A., Connors, M., Roederer, M., Douek,
D. C. & Koup, R. A. (2003) Blood 101, 2711-20.
20.
Aandahl, E. M., Sandberg, J. K., Beckerman, K. P., Taskén, K., Moretto, W. J. &
Nixon, D. F. (2003) Journal of Immunology 170, 2349-2355.
21.
Frankel, A. D. & Young, J. A. (1998) Annu Rev Biochem 67, 1-25.
22.
Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L.,
Profy, A. T., Rusche, J. R., Bolognesi, D. P. & Putney, S. D. (1989) Proc Natl Acad
Sci U S A 86, 6768-72.
23.
Wei, X., Decker, J. M., Wang, S., Hui, H., Kappes, J. C., Wu, X., Salazar-Gonzalez, J.
F., Salazar, M. G., Kilby, J. M., Saag, M. S., Komarova, N. L., Nowak, M. A., Hahn,
B. H., Kwong, P. D. & Shaw, G. M. (2003) Nature 422, 307-12.
24.
Richman, D. D., Wrin, T., Little, S. J. & Petropoulos, C. J. (2003) Proc Natl Acad Sci
U S A 100, 4144-9.
25.
Schmitz, J. E., Kuroda, M. J., Santra, S., Sasseville, V. G., Simon, M. A., Lifton, M.
A., Racz, P., Tenner-Racz, K., Dalesandro, M., Scallon, B. J., Ghrayeb, J., Forman, M.
A., Montefiori, D. C., Rieber, E. P., Letvin, N. L. & Reimann, K. A. (1999) Science
283, 857-60.
26.
Kaslow, R. A., Carrington, M., Apple, R., Park, L., Munoz, A., Saah, A. J., Goedert, J.
J., Winkler, C., O'Brien, S. J., Rinaldo, C., Detels, R., Blattner, W., Phair, J., Erlich, H.
& Mann, D. L. (1996) Nat Med 2, 405-11.
27.
Rowland-Jones, S. L., Dong, T., Fowke, K. R., Kimani, J., Krausa, P., Newell, H.,
Blanchard, T., Ariyoshi, K., Oyugi, J., Ngugi, E., Bwayo, J., MacDonald, K. S.,
McMichael, A. J. & Plummer, F. A. (1998) J Clin Invest 102, 1758-65.
28.
Putkonen, P., Makitalo, B., Bottiger, D., Biberfeld, G. & Thorstensson, R. (1997) J
Virol 71, 4981-4.
29.
Lifson, J. D., Rossio, J. L., Arnaout, R., Li, L., Parks, T. L., Schneider, D. K., Kiser,
R. F., Coalter, V. J., Walsh, G., Imming, R. J., Fisher, B., Flynn, B. M., Bischofberger,
N., Piatak, M., Jr., Hirsch, V. M., Nowak, M. A. & Wodarz, D. (2000) J Virol 74,
2584-93.
30.
Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M. & Oldstone, M. B. (1994) J Virol
68, 6103-10.
31.
Koup, R. A., Safrit, J. T., Cao, Y., Andrews, C. A., McLeod, G., Borkowsky, W.,
Farthing, C. & Ho, D. D. (1994) J Virol 68, 4650-5.
90
Simone C. Zimmerli
32.
6. References
Pantaleo, G., Demarest, J. F., Soudeyns, H., Graziosi, C., Denis, F., Adelsberger, J.
W., Borrow, P., Saag, M. S., Shaw, G. M. & Sekaly, R. P. (1994) Nature 370, 463-7.
33.
Walker, C. M., Moody, D. J., Stites, D. P. & Levy, J. A. (1986) Science 234, 1563-6.
34.
Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C. & Lusso, P.
(1995) Science 270, 1811-5.
35.
Borrow, P., Lewicki, H., Wei, X., Horwitz, M. S., Peffer, N., Meyers, H., Nelson, J.
A., Gairin, J. E., Hahn, B. H., Oldstone, M. B. & Shaw, G. M. (1997) Nat Med 3, 20511.
36.
Appay, V., Nixon, D. F., Donahoe, S. M., Gillespie, G. M., Dong, T., King, A., Ogg,
G. S., Spiegel, H. M., Conlon, C., Spina, C. A., Havlir, D. V., Richman, D. D.,
Waters, A., Easterbrook, P., McMichael, A. J. & Rowland-Jones, S. L. (2000) Journal
of Experimental Medicine 192, 63-75.
37.
Zajac, A. J., Blattman, J. N., Murali-Krishna, K., Sourdive, D. J., Suresh, M., Altman,
J. D. & Ahmed, R. (1998) J Exp Med 188, 2205-13.
38.
Kalams, S. A. (1998) Journal of Experimental Medicine 188, 2199-2204.
39.
Soudeyns, H. & Pantaleo, G. (1999) European Journal of Immunology 29, 3629-35.
40.
Gehri, R., Hahn, S., Rothen, M., Steuerwald, M., Nuesch, R. & Erb, P. (1996) Aids 10,
9-16.
41.
Regamey, N., Harr, T., Battegay, M. & Erb, P. (1999) AIDS Res Hum Retroviruses 15,
803-10.
42.
Pitcher, C. J., Quittner, C., D.M., P., M., C., R.A., K., V.C., M. & L.J., P. (1999)
Nature Medicine 5, 518-525.
43.
McNeil, A. C., Shupert, W. L., Iyasere, C. A., Hallahan, C. W., Mican, J., Davey, R.
T. J. & Connors, M. (2001) Proceedings of the National Academy of Sciences of the
United States of America 98, 13878-83.
44.
Rosenberg, E. S., Altfeld, M., Poon, S. H., Phillips, M. N., Wilkes, B. M., Eldridge, R.
L., Robbins, G. K., D'Aquila, R. T., Goulder, P. J. & Walker, B. D. (2000) Nature
407, 523-26.
45.
Rosenberg, E. S., Billingsley, J. M., Caliendo, A. M., Boswell, S. L., Sax, P. E.,
Kalams, S. A. & Walker, B. D. (1997) Science 278, 1447-1450.
46.
Harari, A., Petitpierre, S., Vallelian, F. & Pantaleo, G. (2004) Blood 103, 966-72.
47.
Gottlieb, M. S., Schroff, R., Schanker, H. M., Weisman, J. D., Fan, P. T., Wolf, R. A.
& Saxon, A. (1981) N Engl J Med 305, 1425-31.
91
Simone C. Zimmerli
48.
6. References
Masur, H., Michelis, M. A., Greene, J. B., Onorato, I., Stouwe, R. A., Holzman, R. S.,
Wormser, G., Brettman, L., Lange, M., Murray, H. W. & Cunningham-Rundles, S.
(1981) N Engl J Med 305, 1431-8.
49.
Durack, D. T. (1981) N Engl J Med 305, 1465-7.
50.
Pantaleo, G. & Fauci, A. S. (1995) Annu Rev Immunol 13, 487-512.
51.
Clark, S. J., Saag, M. S., Decker, W. D., Campbell-Hill, S., Roberson, J. L.,
Veldkamp, P. J., Kappes, J. C., Hahn, B. H. & Shaw, G. M. (1991) N Engl J Med 324,
954-60.
52.
Daar, E. S., Moudgil, T., Meyer, R. D. & Ho, D. D. (1991) N Engl J Med 324, 961-4.
53.
Tindall, B. & Cooper, D. A. (1991) Aids 5, 1-14.
54.
Schacker, T., Collier, A. C., Hughes, J., Shea, T. & Corey, L. (1996) Ann Intern Med
125, 257-64.
55.
Phair, J., Jacobson, L., Detels, R., Rinaldo, C., Saah, A., Schrager, L. & Munoz, A.
(1992) J Acquir Immune Defic Syndr 5, 490-6.
56.
Haynes, B. F., Pantaleo, G. & Fauci, A. S. (1996) Science 271, 324-8.
57.
Hogg, R. S., O'Shaughnessy, M. V., Gataric, N., Yip, B., Craib, K., Schechter, M. T.
& Montaner, J. S. (1997) Lancet 349, 1294.
58.
CDC & Prevention, C. o. D. C. a. (1997) in morbidity and mortality weekly report,
Vol. 46, pp. 861-867.
59.
Erb, P., Battegay, M., Zimmerli, W., Rickenbach, M. & Egger, M. (2000) Arch Intern
Med 160, 1134-40.
60.
Jaggy, C., von Overbeck, J., Ledergerber, B., Schwarz, C., Egger, M., Rickenbach,
M., Furrer, H. J., Telenti, A., Battegay, M., Flepp, M., Vernazza, P., Bernasconi, E. &
Hirschel, B. (2003) Lancet 362, 877-8.
61.
Sheppard, H. W., Lang, W., Ascher, M. S., Vittinghoff, E. & Winkelstein, W. (1993)
Aids 7, 1159-66.
62.
Levy, J. A. (1993) Aids 7, 1401-10.
63.
Pantaleo, G., Menzo, S., Vaccarezza, M., Graziosi, C., Cohen, O. J., Demarest, J. F.,
Montefiori, D., Orenstein, J. M., Fox, C., Schrager, L. K. & et al. (1995) N Engl J Med
332, 209-16.
64.
Cao, Y., Qin, L., Zhang, L., Safrit, J. & Ho, D. D. (1995) N Engl J Med 332, 201-8.
65.
Hammer, S. M., Squires, K. E., Hughes, M. D., Grimes, J. M., Demeter, L. M.,
Currier, J. S., Eron, J. J., Jr., Feinberg, J. E., Balfour, H. H., Jr., Deyton, L. R.,
Chodakewitz, J. A. & Fischl, M. A. (1997) N Engl J Med 337, 725-33.
92
Simone C. Zimmerli
66.
6. References
Saag, M. S., Tebas, P., Sension, M., Conant, M., Myers, R., Chapman, S. K.,
Anderson, R. & Clendeninn, N. (2001) Aids 15, 1971-8.
67.
Mandell, G. L., Bennet, J. E. & Dolin, R. (2004) Principles and practice of infectious
diseases (Churchill Livingstone.
68.
Greene, W. C. (1991) N Engl J Med 324, 308-17.
69.
Tsai, C. C., Emau, P., Sun, J. C., Beck, T. W., Tran, C. A., Follis, K. E.,
Bischofberger, N. & Morton, W. R. (2000) J Med Primatol 29, 248-58.
70.
Tsai, C. C., Emau, P., Follis, K. E., Beck, T. W., Benveniste, R. E., Bischofberger, N.,
Lifson, J. D. & Morton, W. R. (1998) J Virol 72, 4265-73.
71.
Tsai, C. C., Follis, K. E., Sabo, A., Beck, T. W., Grant, R. F., Bischofberger, N.,
Benveniste, R. E. & Black, R. (1995) Science 270, 1197-9.
72.
Van Rompay, K. K., Marthas, M. L., Ramos, R. A., Mandell, C. P., McGowan, E. K.,
Joye, S. M. & Pedersen, N. C. (1992) Antimicrob Agents Chemother 36, 2381-6.
73.
Bottiger, D., Johansson, N. G., Samuelsson, B., Zhang, H., Putkonen, P., Vrang, L. &
Oberg, B. (1997) Aids 11, 157-62.
74.
Bottiger, D., Putkonen, P. & Oberg, B. (1992) AIDS Res Hum Retroviruses 8, 1235-8.
75.
UNAIDS (2004), Geneva, Switzerland).
76.
Johnson, W. E. & Desrosiers, R. C. (2002) Annu Rev Med 53, 499-518.
77.
Kwong, P. D., Doyle, M. L., Casper, D. J., Cicala, C., Leavitt, S. A., Majeed, S.,
Steenbeke, T. D., Venturi, M., Chaiken, I., Fung, M., Katinger, H., Parren, P. W.,
Robinson, J., Van Ryk, D., Wang, L., Burton, D. R., Freire, E., Wyatt, R., Sodroski, J.,
Hendrickson, W. A. & Arthos, J. (2002) Nature 420, 678-82.
78.
Parren, P. W., Moore, J. P., Burton, D. R. & Sattentau, Q. J. (1999) Aids 13 Suppl A,
S137-62.
79.
Goulder, P. J., Phillips, R. E., Colbert, R. A., McAdam, S., Ogg, G., Nowak, M. A.,
Giangrande, P., Luzzi, G., Morgan, B., Edwards, A., McMichael, A. J. & RowlandJones, S. (1997) Nat Med 3, 212-7.
80.
Price, D. A., Goulder, P. J., Klenerman, P., Sewell, A. K., Easterbrook, P. J., Troop,
M., Bangham, C. R. & Phillips, R. E. (1997) Proc Natl Acad Sci U S A 94, 1890-5.
81.
Jost, S., Bernard, M. C., Kaiser, L., Yerly, S., Hirschel, B., Samri, A., Autran, B., Goh,
L. E. & Perrin, L. (2002) N Engl J Med 347, 731-6.
82.
Altfeld, M., Allen, T. M., Yu, X. G., Johnston, M. N., Agrawal, D., Korber, B. T.,
Montefiori, D. C., O'Connor, D. H., Davis, B. T., Lee, P. K., Maier, E. L., Harlow, J.,
93
Simone C. Zimmerli
6. References
Goulder, P. J., Brander, C., Rosenberg, E. S. & Walker, B. D. (2002) Nature 420, 4349.
83.
McMichael, A. J. & Rowland-Jones, S. L. (2002) Nature 420, 371, 373.
84.
Cohen, J. (2003) Science 299, 1290-1.
85.
Follmann, D., Gilbert, P. & Self, S. (2004) in 11th Conference on Retroviruses and
Opportunistic Infections, San Francisco, CA).
86.
Pitisutithum, P. (2004) in 11th Conference on Retroviruses and Opportunistic
Infections, San Francisco, CA).
87.
Anon (2003) (http://www.vaxgen.com/pressroom/index.html, Vol. 2004.
88.
Garber, D. A. & Feinberg, M. B. (2003) AIDS Rev 5, 131-9.
89.
Mellors, J. W., Rinaldo, C. R., Jr., Gupta, P., White, R. M., Todd, J. A. & Kingsley, L.
A. (1996) Science 272, 1167-70.
90.
Quinn, T. C. (2000) Hopkins HIV Rep 12, 1, 5, 11.
91.
Gray, R. H., Wawer, M. J., Brookmeyer, R., Sewankambo, N. K., Serwadda, D.,
Wabwire-Mangen, F., Lutalo, T., Li, X., vanCott, T. & Quinn, T. C. (2001) Lancet
357, 1149-53.
92.
Garber, D. A., Silvestri, G. & Feinberg, M. B. (2004) Lancet Infect Dis 4, 397-413.
93.
Landolfo, S., Gariglio, M., Gribaudo, G. & Lembo, D. (2003) Pharmacol Ther 98,
269-97.
94.
Reddehase, M. J. (2000) Curr Opin Immunol 12, 390-6.
95.
Riddell, S. R. & Greenberg, P. D. (1997) Rev Med Virol 7, 181-192.
96.
Mocarski, E. S. & Courcelle, C. T. (2001) Cytomegalovirus and thier replication
(Lippincott, Williams and Wilkins, Philadelphia).
97.
Sanchez, V., Greis, K. D., Sztul, E. & Britt, W. J. (2000) J Virol 74, 975-86.
98.
Mettenleiter, T. C. (2002) J Virol 76, 1537-47.
99.
Moss, P. & Khan, N. (2004) Hum Immunol 65, 456-64.
100.
Rodgers, B., Borysiewicz, L., Mundin, J., Graham, S. & Sissons, P. (1987) J Gen
Virol 68 ( Pt 9), 2371-8.
101.
Wills, M. R., Carmichael, A. J., Mynard, K., Jin, X., Weekes, M. P., Plachter, B. &
Sissons, J. G. (1996) J Virol 70, 7569-79.
102.
Elkington, R., Walker, S., Crough, T., Menzies, M., Tellam, J., Bharadwaj, M. &
Khanna, R. (2003) J Virol 77, 5226-40.
103.
Sinzger, C. & Jahn, G. (1996) Intervirology 39, 302-19.
94
Simone C. Zimmerli
104.
6. References
Saederup, N., Lin, Y. C., Dairaghi, D. J., Schall, T. J. & Mocarski, E. S. (1999) Proc
Natl Acad Sci U S A 96, 10881-6.
105.
Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble,
G. W. & Schall, T. J. (1999) Proc Natl Acad Sci U S A 96, 9839-44.
106.
Griffiths, P. D. (2000) in Principles and Practice of Clinical Virology, eds.
Zuckerman, A. J., Banatvala, J. E. & Pattison, J. R. (John Wiley and Sons, London),
pp. 79-116.
107.
Pass, R. F. (2001) in In Fields Virology, eds. Knipe, D. M. & Howley, P. M.
(Lippincott, Williams and Wilkins, Philadelphia), pp. 2675-2705.
108.
Britt, W. J. & Alford, C. A. (1996) Cytomegalovirus (Lippincott-Raven, New York).
109.
Goodrich, J. M., Bowden, R. A., Fisher, L., Keller, C., Schoch, G. & Meyers, J. D.
(1993) Ann Intern Med 118, 173-8.
110.
HO, M. (1991) Cytomegalovirus, biology and infection (Plenum Medical Book, New
York).
111.
Klemola, E., Von Essen, R., Henle, G. & Henle, W. (1970) J Infect Dis 121, 608-14.
112.
Klemola, E., Weckman, N., Haltia, K. & Kaariainen, L. (1967) Acta Med Scand 181,
603-7.
113.
Klemola, E., Kaariainen, L., von Essen, R., Haltia, K., Koivuniemi, A. & von
Bonsdorff, C. H. (1967) Acta Med Scand 182, 311-22.
114.
Scholz, M., Doerr, H. W. & Cinatl, J. (2003) Trends Microbiol 11, 171-8.
115.
McKenzie, R., Travis, W. D., Dolan, S. A., Pittaluga, S., Feuerstein, I. M., Shelhamer,
J., Yarchoan, R. & Masur, H. (1991) Medicine (Baltimore) 70, 326-43.
116.
Riddell, S. R. & Greenberg, P. D. (2000) J Antimicrob Chemother 45 Suppl T3, 3543.
117.
Riddell, S. R., Watanabe, K. S., Goodrich, J. M., Li, C. R., Agha, M. E. & Greenberg,
P. D. (1992) Science 257, 238-41.
118.
Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas,
E. D. & Riddell, S. R. (1995) N Engl J Med 333, 1038-44.
119.
Einsele, H., Roosnek, E., Rufer, N., Sinzger, C., Riegler, S., Loffler, J., Grigoleit, U.,
Moris, A., Rammensee, H. G., Kanz, L., Kleihauer, A., Frank, F., Jahn, G. & Hebart,
H. (2002) Blood 99, 3916-22.
120.
Arvin, A. M., Fast, P., Myers, M., Plotkin, S. & Rabinovich, R. (2004) Clin Infect Dis
39, 233-9.
121.
Tsurumi, T., Fujita, M. & Kudoh, A. (2005) Rev Med Virol 15, 3-15.
95
Simone C. Zimmerli
122.
6. References
Tsurumi, T., Kobayashi, A., Tamai, K., Daikoku, T., Kurachi, R. & Nishiyama, Y.
(1993) J Virol 67, 4651-8.
123.
Tsurumi, T. (1993) J Virol 67, 1681-7.
124.
Tsurumi, T., Daikoku, T., Kurachi, R. & Nishiyama, Y. (1993) J Virol 67, 7648-53.
125.
Tsurumi, T., Daikoku, T. & Nishiyama, Y. (1994) J Virol 68, 3354-63.
126.
Tsurumi, T., Kishore, J., Yokoyama, N., Fujita, M., Daikoku, T., Yamada, H.,
Yamashita, Y. & Nishiyama, Y. (1998) J Gen Virol 79 ( Pt 5), 1257-64.
127.
Tsurumi, T., Yamada, H., Daikoku, T., Yamashita, Y. & Nishiyama, Y. (1997)
Biochem Biophys Res Commun 238, 33-8.
128.
Fixman, E. D., Hayward, G. S. & Hayward, S. D. (1995) J Virol 69, 2998-3006.
129.
Tsurumi, T. (2001) Curr Top Microbiol Immunol 258, 65-87.
130.
Hammerschmidt, W. & Sugden, B. (1988) Cell 55, 427-33.
131.
Adamson, A. L. & Kenney, S. (1999) J Virol 73, 6551-8.
132.
Cayrol, C. & Flemington, E. K. (1996) Embo J 15, 2748-59.
133.
Rodriguez, A., Jung, E. J., Yin, Q., Cayrol, C. & Flemington, E. K. (2001) Virology
284, 159-69.
134.
Zhang, Q., Hong, Y., Dorsky, D., Holley-Guthrie, E., Zalani, S., Elshiekh, N. A.,
Kiehl, A., Le, T. & Kenney, S. (1996) J Virol 70, 5131-42.
135.
Thorley-Lawson, D. A. (2001) Nat Rev Immunol 1, 75-82.
136.
Borza, C. M. & Hutt-Fletcher, L. M. (2002) Nat Med 8, 594-9.
137.
Dyson, P. J. & Farrell, P. J. (1985) J Gen Virol 66 ( Pt 9), 1931-40.
138.
Adams, A. (1987) J Virol 61, 1743-6.
139.
Kirchmaier, A. L. & Sugden, B. (1995) J Virol 69, 1280-3.
140.
Yates, J., Warren, N., Reisman, D. & Sugden, B. (1984) Proc Natl Acad Sci U S A 81,
3806-10.
141.
Yates, J. L., Warren, N. & Sugden, B. (1985) Nature 313, 812-5.
142.
Okano, M., Thiele, G. M., Davis, J. R., Grierson, H. L. & Purtilo, D. T. (1988) Clin
Microbiol Rev 1, 300-12.
143.
Steven, N. M., Annels, N. E., Kumar, A., Leese, A. M., Kurilla, M. G. & Rickinson,
A. B. (1997) J Exp Med 185, 1605-17.
144.
Callan, M. F., Tan, L., Annels, N., Ogg, G. S., Wilson, J. D., O'Callaghan, C. A.,
Steven, N., McMichael, A. J. & Rickinson, A. B. (1998) J Exp Med 187, 1395-402.
145.
Khanna, R., Sherritt, M. & Burrows, S. R. (1999) J Immunol 162, 3063-9.
96
Simone C. Zimmerli
146.
6. References
Mosialos, G., Birkenbach, M., Yalamanchili, R., VanArsdale, T., Ware, C. & Kieff, E.
(1995) Cell 80, 389-99.
147.
Devergne, O., Cahir McFarland, E. D., Mosialos, G., Izumi, K. M., Ware, C. F. &
Kieff, E. (1998) J Virol 72, 7900-8.
148.
Devergne, O., Hatzivassiliou, E., Izumi, K. M., Kaye, K. M., Kleijnen, M. F., Kieff, E.
& Mosialos, G. (1996) Mol Cell Biol 16, 7098-108.
149.
Izumi, K. M. & Kieff, E. D. (1997) Proc Natl Acad Sci U S A 94, 12592-7.
150.
Miller, W. E., Cheshire, J. L. & Raab-Traub, N. (1998) Mol Cell Biol 18, 2835-44.
151.
Kilger, E., Kieser, A., Baumann, M. & Hammerschmidt, W. (1998) Embo J 17, 17009.
152.
Busch, L. K. & Bishop, G. A. (1999) J Immunol 162, 2555-61.
153.
Wang, S., Rowe, M. & Lundgren, E. (1996) Cancer Res 56, 4610-3.
154.
Fries, K. L., Miller, W. E. & Raab-Traub, N. (1996) J Virol 70, 8653-9.
155.
Kawanishi, M. (1997) Virology 228, 244-50.
156.
Fanidi, A., Hancock, D. C. & Littlewood, T. D. (1998) J Virol 72, 8392-5.
157.
Strockbine, L. D., Cohen, J. I., Farrah, T., Lyman, S. D., Wagener, F., DuBose, R. F.,
Armitage, R. J. & Spriggs, M. K. (1998) J Virol 72, 4015-21.
158.
Kanegane, H., Wakiguchi, H., Kanegane, C., Kurashige, T. & Tosato, G. (1997) J
Infect Dis 176, 254-7.
159.
Takayama, T., Nishioka, Y., Lu, L., Lotze, M. T., Tahara, H. & Thomson, A. W.
(1998) Transplantation 66, 1567-74.
160.
Levitskaya, J., Sharipo, A., Leonchiks, A., Ciechanover, A. & Masucci, M. G. (1997)
Proc Natl Acad Sci U S A 94, 12616-21.
161.
Straus, S. E., Cohen, J. I., Tosato, G. & Meier, J. (1993) Ann Intern Med 118, 45-58.
162.
Rickinson, A. B. & Kieff, E. (2001) Epstein-Barr virus (Lippincott Williams and
Wilkins, Philadelphia).
163.
Iwatsuki, K., Yamamoto, T., Tsuji, K., Suzuki, D., Fujii, K., Matsuura, H. & Oono, T.
(2004) Acta Med Okayama 58, 169-80.
164.
Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. L., Farrell, P. J., Gibson, T. J.,
Hatfull, G., Hudson, G. S., Satchwell, S. C., Seguin, C. & et al. (1984) Nature 310,
207-11.
165.
Rowe, M., Lear, A. L., Croom-Carter, D., Davies, A. H. & Rickinson, A. B. (1992) J
Virol 66, 122-31.
166.
Jenson, H. B. (2000) Curr Opin Pediatr 12, 263-8.
97
Simone C. Zimmerli
6. References
167.
Ambinder, R. F. & Mann, R. B. (1994) Am J Pathol 145, 239-52.
168.
Hanto, D. W., Sakamoto, K., Purtilo, D. T., Simmons, R. L. & Najarian, J. S. (1981)
Surgery 90, 204-13.
169.
Shibata, D., Tokunaga, M., Uemura, Y., Sato, E., Tanaka, S. & Weiss, L. M. (1991)
Am J Pathol 139, 469-74.
170.
Aozasa, K. (1996) Int J Hematol 65, 9-16.
171.
Kawa-Ha, K., Ishihara, S., Ninomiya, T., Yumura-Yagi, K., Hara, J., Murayama, F.,
Tawa, A. & Hirai, K. (1989) J Clin Invest 84, 51-5.
172.
Su, I. J., Wang, C. H., Cheng, A. L. & Chen, R. L. (1995) Leuk Lymphoma 19, 401-6.
173.
Harabuchi, Y., Yamanaka, N., Kataura, A., Imai, S., Kinoshita, T., Mizuno, F. &
Osato, T. (1990) Lancet 335, 128-30.
174.
Oono, T., Arata, J., Masuda, T. & Ohtsuki, Y. (1986) Arch Dermatol 122, 1306-9.
175.
Magana, M., Sangueza, P., Gil-Beristain, J., Sanchez-Sosa, S., Salgado, A., Ramon, G.
& Sangueza, O. P. (1998) J Am Acad Dermatol 38, 574-9.
176.
Ohtsuka, M., Iwatsuki, K., Kaneko, R., Akiba, H., Kikuchi, S., Harada, H. & Kaneko,
F. (1999) Br J Dermatol 140, 358-9.
177.
Iwatsuki, K., Ohtsuka, M., Harada, H., Han, G. & Kaneko, F. (1997) Arch Dermatol
133, 1081-6.
178.
Imashuku, S. (2002) Crit Rev Oncol Hematol 44, 259-72.
179.
Murphy, B. R. & Webster, R. G. (1996) Orthomyxoviruses (Lippincott-Raven
Publishers, Philadelphia).
180.
Lamb, R. A. & Krug, R. M. (1996) Orthomyxoviridae: The Viruses and Their
Replication (Lippincott-Raven Publishers, Philadelphia).
181.
Ada, G. L. & Jones, P. D. (1986) Curr Top Microbiol Immunol 128, 1-54.
182.
Gorman, O. T., Bean, W. J. & Webster, R. G. (1992) Curr Top Microbiol Immunol
176, 75-97.
183.
Couch, R. B. & Kasel, J. A. (1983) Annu Rev Microbiol 37, 529-49.
184.
Graham, M. B. & Braciale, T. J. (1997) J Exp Med 186, 2063-8.
185.
Zhong, W., Marshall, D., Coleclough, C. & Woodland, D. L. (2000) J Immunol 164,
3274-82.
186.
Murphy, B. R., Nelson, D. L., Wright, P. F., Tierney, E. L., Phelan, M. A. & Chanock,
R. M. (1982) Infect Immun 36, 1102-8.
187.
Epstein, S. L., Lo, C. Y., Misplon, J. A. & Bennink, J. R. (1998) J Immunol 160, 3227.
98
Simone C. Zimmerli
188.
6. References
Eichelberger, M., Allan, W., Zijlstra, M., Jaenisch, R. & Doherty, P. C. (1991) J Exp
Med 174, 875-80.
189.
Doherty, P. C., Allan, W., Eichelberger, M. & Carding, S. R. (1992) Annu Rev
Immunol 10, 123-51.
190.
Harmsen, A. G., Muggenburg, B. A., Snipes, M. B. & Bice, D. E. (1985) Science 230,
1277-80.
191.
Cerwenka, A., Morgan, T. M. & Dutton, R. W. (1999) J Immunol 163, 5535-43.
192.
Topham, D. J., Tripp, R. A. & Doherty, P. C. (1997) J Immunol 159, 5197-200.
193.
Kagi, D. & Hengartner, H. (1996) Curr Opin Immunol 8, 472-7.
194.
Yewdell, J. W., Bennink, J. R., Smith, G. L. & Moss, B. (1985) Proc Natl Acad Sci U
S A 82, 1785-9.
195.
Rimmelzwaan, G. F. & Osterhaus, A. D. (1995) Vaccine 13, 703-5.
196.
Garlepp, M. J., Rose, A. H., Bowman, R. V., Mavaddat, N., Dench, J., Holt, B. J.,
Baron-Hay, M., Holt, P. G. & Robinson, B. W. (1992) Immunology 77, 31-7.
197.
Hou, S., Doherty, P. C., Zijlstra, M., Jaenisch, R. & Katz, J. M. (1992) J Immunol 149,
1319-25.
198.
Graham, M. B., Braciale, V. L. & Braciale, T. J. (1994) J Exp Med 180, 1273-82.
199.
Kast, W. M., Bronkhorst, A. M., de Waal, L. P. & Melief, C. J. (1986) J Exp Med 164,
723-38.
200.
Larson, H. E., Parry, R. P. & Tyrrell, D. A. (1980) Br J Dis Chest 74, 56-62.
201.
Roberts, N. J., Jr. & Steigbigel, R. T. (1978) J Immunol 121, 1052-8.
202.
Roberts, N. J., Jr., Prill, A. H. & Mann, T. N. (1986) J Exp Med 163, 511-9.
203.
Richman, D. D., Yazaki, P. & Hostetler, K. Y. (1981) Virology 112, 81-90.
204.
Richman, D. D., Hostetler, K. Y., Yazaki, P. J. & Clark, S. (1986) Virology 151, 20010.
205.
Demicheli, V., Rivetti, D., Deeks, J. J. & Jefferson, T. O. (2004) Cochrane Database
Syst Rev, CD001269.
206.
Negri, E., Colombo, C., Giordano, L., Groth, N., Apolone, G. & La Vecchia, C.
(2005) Vaccine 23, 2851-61.
207.
Jin, X., Bauer, D. E., Tuttleton, S. E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C. E.,
Safrit, J. T., Mittler, J., Weinberger, L., Kostrikis, L. G., Zhang, L., Perelson, A. S. &
Ho, D. D. (1999) J Exp Med 189, 991-8.
99
Simone C. Zimmerli
208.
6. References
Metzner, K. J., Jin, X., Lee, F. V., Gettie, A., Bauer, D. E., Di Mascio, M., Perelson,
A. S., Marx, P. A., Ho, D. D., Kostrikis, L. G. & Connor, R. I. (2000) J Exp Med 191,
1921-31.
209.
Allen, T. M., O'Connor, D. H., Jing, P., Dzuris, J. L., Mothe, B. R., Vogel, T. U.,
Dunphy, E., Liebl, M. E., Emerson, C., Wilson, N., Kunstman, K. J., Wang, X.,
Allison, D. B., Hughes, A. L., Desrosiers, R. C., Altman, J. D., Wolinsky, S. M., Sette,
A. & Watkins, D. I. (2000) Nature 407, 386-90.
210.
Chen, Z. W., Craiu, A., Shen, L., Kuroda, M. J., Iroku, U. C., Watkins, D. I., Voss, G.
& Letvin, N. L. (2000) J Immunol 164, 6474-9.
211.
Amara, R. R., Villinger, F., Altman, J. D., Lydy, S. L., O'Neil, S. P., Staprans, S. I.,
Montefiori, D. C., Xu, Y., Herndon, J. G., Wyatt, L. S., Candido, M. A., Kozyr, N. L.,
Earl, P. L., Smith, J. M., Ma, H. L., Grimm, B. D., Hulsey, M. L., Miller, J., McClure,
H. M., McNicholl, J. M., Moss, B. & Robinson, H. L. (2001) Science 292, 69-74.
212.
Barouch, D. H., Santra, S., Schmitz, J. E., Kuroda, M. J., Fu, T. M., Wagner, W.,
Bilska, M., Craiu, A., Zheng, X. X., Krivulka, G. R., Beaudry, K., Lifton, M. A.,
Nickerson, C. E., Trigona, W. L., Punt, K., Freed, D. C., Guan, L., Dubey, S.,
Casimiro, D., Simon, A., Davies, M. E., Chastain, M., Strom, T. B., Gelman, R. S.,
Montefiori, D. C., Lewis, M. G., Emini, E. A., Shiver, J. W. & Letvin, N. L. (2000)
Science 290, 486-92.
213.
Seth, A., Ourmanov, I., Schmitz, J. E., Kuroda, M. J., Lifton, M. A., Nickerson, C. E.,
Wyatt, L., Carroll, M., Moss, B., Venzon, D., Letvin, N. L. & Hirsch, V. M. (2000) J
Virol 74, 2502-9.
214.
Rinaldo, C., Huang, X. L., Fan, Z. F., Ding, M., Beltz, L., Logar, A., Panicali, D.,
Mazzara, G., Liebmann, J., Cottrill, M. & et al. (1995) J Virol 69, 5838-42.
215.
Musey, L., Hughes, J., Schacker, T., Shea, T., Corey, L. & McElrath, M. J. (1997) N
Engl J Med 337, 1267-74.
216.
Ogg, G. S., Bonhoeffer, S., Dunbar, P. R., Nowak, M. A., Monard, S., Segal, J. P.,
Cao, Y., Rowland-Jones, S. L., Cerundolo, V., Hurley, A., Markowitz, M., Ho, D. D.,
Nixon, D. F. & McMichael, A. J. (1998) Science 279, 2103-6.
217.
Klein, M. R., van Baalen, C. A., Holwerda, A. M., Kerkhof Garde, S. R., Bende, R. J.,
Keet, I. P., Eeftinck-Schattenkerk, J. K., Osterhaus, A. D., Schuitemaker, H. &
Miedema, F. (1995) J Exp Med 181, 1365-72.
218.
Catalina, M. D., Sullivan, J. L., Brody, R. M. & Luzuriaga, K. (2002) J Immunol 168,
4184-91.
100
Simone C. Zimmerli
219.
6. References
Ellefsen, K., Harari, A., Champagne, P., Bart, P. A., Sékaly, R. P. & Pantaleo, G.
(2002) European Journal of Immunology 32, 3756-64.
220.
Hislop, A. D., Annels, N. E., Gudgeon, N. H., Leese, A. M. & Rickinson, A. B. (2002)
J Exp Med 195, 893-905.
221.
Hess, C., Altfeld, M., Thomas, S. Y., Addo, M. M., Rosenberg, E. S., Allen, T. M.,
Draenert, R., Eldrige, R. L., van Lunzen, J., Stellbrink, H. J., Walker, B. D. & Luster,
A. D. (2004) Lancet 363, 863-6.
222.
Fuller, M. J. & Zajac, A. J. (2003) Journal of Immunology 170, 477-86.
223.
Fuller, M. J., Khanolkar, A., Tebo, A. E. & Zajac, A. J. (2004) Journal of Immunology
172, 4204-4214.
224.
Wherry, E. J., Blattmann, J. N., Murali-Krishna, K., van der Most, R. & Ahmed, R.
(2003) Journal of Virology 77, 4911-4927.
225.
Kovacs, J. A., Baseler, M., Dewar, R. J., Vogel, S., Davey, R. T., Jr., Falloon, J., Polis,
M. A., Walker, R. E., Stevens, R., Salzman, N. P. & et al. (1995) N Engl J Med 332,
567-75.
226.
Kovacs, J. A., Vogel, S., Albert, J. M., Falloon, J., Davey, R. T., Jr., Walker, R. E.,
Polis, M. A., Spooner, K., Metcalf, J. A., Baseler, M., Fyfe, G. & Lane, H. C. (1996)
N Engl J Med 335, 1350-6.
227.
Losso, M. H., Belloso, W. H., Emery, S., Benetucci, J. A., Cahn, P. E., Lasala, M. C.,
Lopardo, G., Salomon, H., Saracco, M., Nelson, E., Law, M. G., Davey, R. T.,
Allende, M. C. & Lane, H. C. (2000) J Infect Dis 181, 1614-21.
228.
Marchetti, G., Meroni, L., Molteni, C., Bandera, A., Franzetti, F., Galli, M., Moroni,
M., Clerici, M. & Gori, A. (2004) Aids 18, 211-6.
229.
De Paoli, P. (2001) Clin Diagn Lab Immunol 8, 671-7.
230.
Harari, A., Vallelian, F., Meylan, P. R. & Pantaleo, G. (2005) J Immunol 174, 103745.
231.
Harari, A., Vallelian, F. & Pantaleo, G. (2004) Eur J Immunol 34, 3525-33.
232.
Kawa, K., Okamura, T., Yagi, K., Takeuchi, M., Nakayama, M. & Inoue, M. (2001)
Blood 98, 3173-4.
233.
Okano, M., Matsumoto, S., Osato, T., Sakiyama, Y., Thiele, G. M. & Purtilo, D. T.
(1991) Clin Microbiol Rev 4, 129-35.
234.
Kikuta, H., Taguchi, Y., Tomizawa, K., Kojima, K., Kawamura, N., Ishizaka, A.,
Sakiyama, Y., Matsumoto, S., Imai, S., Kinoshita, T. & et al. (1988) Nature 333, 4557.
101
Simone C. Zimmerli
235.
6. References
Iwatsuki, K., Xu, Z., Takata, M., Iguchi, M., Ohtsuka, M., Akiba, H., Mitsuhashi, Y.,
Takenoshita, H., Sugiuchi, R., Tagami, H. & Kaneko, F. (1999) Br J Dermatol 140,
715-21.
102
Simone C. Zimmerli
7. Glossary
7. Glossary
adaptive immunity
antigen-specific defense mechanisms that take several days to become
protective and are designed to remove a specific antigen. This immunity
persists throughout life. There are two major branches of the adaptive
immune response: humoral and cell-mediated immunity
antibodies
Y-shaped protein of the surface of B cells that is secreted into blood or
lymph in response to an antigenic stimulus, such as a bacterium, virus,
parasite, or transplanted organ, and that neutralizes the antigen by
binding specifically to it. Also called immunoglobulin.
antigen
any molecule that can bind specifically to an antibody. Their name
arises from their ability to generate antibodies. However some antigens
do not, by themselves, elicit antibody production; those antigens that
can induce antibody productions are called immunogens.
antigenic drift
refers to mutations in the Influenza virus over time. Such mutations
occur almost yearly in the Influenza virus. Mutations happen frequently
because the virus is highly unstable and has no way of proofreading.
antigenic shift
process by which two different strains of Influenza combine to form a
new subtype which is characterized by a mixture of the surface antigens
of the two original strains.
apoptosis
programmed cell death; form of cell death in which the cell activates an
internal death program. It is characterized by nuclear degradation,
nuclear degeneration and condensation, and the phagocytosis of cell
remains. Proliferating cells frequently undergo apoptosis, which is a
natural process in the development and during immune responses.
Apoptosis contrasts with necrosis, death caused by external factors,
which occurs in situations such as poisoning or anoxia.
arthralgias
Pain in the joint
attenuation
a dilution, thinning or weakening of a substance, especially a reduction
in the virulence of a pathogen through repeated inoculation, growth in a
103
different culture medium,
or exposure to heat, light, air or other
weakening agents
Simone C. Zimmerli
7. Glossary
in the virulence of a pathogen through repeated inoculation, growth in a
different culture medium, or exposure to heat, light, air or other
weakening agents
autocrine
mode of hormone action in which a hormone binds to receptors on and
effects the function of the cell type that produced it.
Burkitt lymphoma
form of undifferentiated malignant lymphoma usually found in central
Africa, but also in other parts of the world; commonly manifested as a
large osteolytic lesion in the jaw or as an abdominal mass. B cell Ag are
expressed on the immature cells that make up the tumor in virtually all
cases of Burkitt lymphoma.
Cap
7-methyl guanosin added to the 5’ end of a pre-mRNA molecules in
eukaryotes. This cap is necessary for recognition by the ribosom, for
export and also serves as protection against phosphatases and nucleases.
(capping is specific for transcripts produced by RNA polymerase II)
CD3 complex
complex of α:β or γ:δ T cell receptor chains with the invariant subunits
CD3γ, δ and ε, and the dimeric ζ chains.
CFSE
5- (and -6)-Carboxyfluorescein diacetate succinimidyl ester is a
membrane-permanent, fluorescein-based dye that can be used to follow
proliferation of cells. The two acetate side chains render the molecule
highly membrane permanent. Once inside the cells, the acetate groups
are removed by intracellular esterases and the carobxyfluorescein exits
from cells at much slower rate. CFSE couples covalently to intracellular
molecules by reaction with intracellular amine groups forming a highly
stable amide bond. When cells divide, CFSE labeling is equally
distributed between the daughter cells, which are therefore half as
fluorescent as the parent cells. As a result each successive generation in
a population of proliferating cells is marked by a halving of cellular
fluorescence intensity.
chemokines
small chemoattractant proteins that stimulate the migration and
activation of cells, especially phagocytic cells and lymphocytes. The
have a central role in inflammatory responses.
104
Simone C. Zimmerli
7. Glossary
have a central role in inflammatory responses.
chronic active EBV
lymphoproliferative disorder characterized by abnormally high titers of
infection
anti-EBV Ab and increased levels of EBV-DNA in PBMC and plasma;
50% of patients die of complications such as hepatic failure,
gastrointestinal bleeding, HLH and malignant lymphomas several years
after onset of the disease (232-234)
cis element
regulatory sequence in DNA that can control a gene only on the same
chromosome. In bacteria cis-acting elements are adjacent or proximal to
the gene(s) they control, whereas in eukaryotes they may also be far
away. (trans-acting elements → DNA sequences encoding diffusible
proteins [e.g. transcription activators or repressors] that control genes on
the same or different chromosome)
coccidioidomycosis
infection caused by inhalation the microscopic spores of the fungus
Coccidioides immitis; acute form produces flu-like symptoms whereas
the chronic form can develop as late as 20 years after initial infection
with purulent lung disease
concatemer
a DNA segment composed of repeated sequences linked end to end
croup
condition characterized by resonant barking cough, hoarseness and
persistent stridor and caused by allergy, foreign body or infection. It
mainly occurs in infants and children.
cytokines
proteins made by cells that affect the behavior of other cells. Cytokines
made by lymphocyte are often called lymphokines or interleukins (IL),
but the generic term cytokine is used in this book and in most of the
literature. Cytokines act via specific cytokine receptors on the cells that
they affect.
endemic infection
a disease that is constantly present to a greater or lesser degree in a
defined population.
endocytosis
process of cellular ingestion by which the plasma membrane folds
inward to bring substance into the cell.
105
Simone C. Zimmerli
7. Glossary
inward to bring substance into the cell.
epidemic infection
outbreak of a contagious disease that spreads rapidly and widely
epitope
site on an antigen recognized by an antibody or an antigen receptor;
epitopes are also called antigenic determinants. A T cell epitope is a
short peptide derived from a protein antigen. It binds to an MHC
molecule and is recognized by a particular T cell.
exocytosis
process of cellular secretion or excretion in which substances contained
in vesicles are discharged from the cell by fusion of the vesicular
membrane with the outer cell membrane.
FACS
fluorescence activated cell sorter. Measures cell size, granularity and
fluorescence due to bound fluorescent antibodies as single cells pass in a
stream past photodetectors. The analysis of single cells this way is
called flow cytometry and the instruments that carry out the
measurement and/or sort cells are called flow cytometers.
Gianotti-Crosti-syndrome
synonym: papular acrodermatitis; discrete papules on the cheeks, dorsal
surfaces of the hands and buttocks and the extensor aspects of the arms
and thighs of infants.
Guillain-Barré syndrome
temporary inflammation of the nerves, causing pain, weakness, and
paralysis in the extremities and often progressing to the chest and face.
Typically occurs after recovery from a viral infection or, in rare cases,
following immunization against influenza.
haemophagocytic
synonym: hemophagocytic lymphohistiocytosis (HLH); unusual
syndrome
syndrome characterized by fever, splenomegaly, jaundice, pancytopenia,
disseminated
intravascular
coagulation
and
features
of
haemophagocytosis of erythrocytes, leucocytes and platelets by
macrophages in the bone marrow and other tissues.
helicase
protein/enzyme that unwinds DNA molecules at the replication fork
106
Simone C. Zimmerli
histoplasmosis
7. Glossary
disease caused by the fungus Histoplasma capsulatum; infects lungs,
skin, mucous membranes, bone, skin and eyes
HIV encephalophathy
any disorder or disease of the brain caused by HIV
HIV wasting syndrome
involuntary weight loss > 10% associated with intermittent or constant
fever and chronic diarrhea or fatigue for more than 30 days in the
absence of a defined cause other than HIV infection. A constant feature
is major muscle wasting with scattered myofiber degeneration. A variety
of etiologies, which vary among patients, contributes to this syndrome.
Hodgkin’s lymphoma
synonym: Hodgkin’s disease; malignant progressive, sometimes fatal
disease, marked by enlargement of the lymph nodes, spleen and liver.
humoral immunity
the component of the immune system involving antibodies that are
secreted by B cells and circulate as soluble proteins in blood plasma and
lymph..
Hydroa vacciniforme
photosensitive dermatitis of childhood, mediated by the infiltration of
EBV-infected T cells and reactive cytotoxic T cells (235). Resolves in
most patients spontaneously with age.
Kaposi’s sarcoma
vascular proliferation characterized by the presence of spindle cells and
vascular channels mixed with cellular infiltrates.
lymphadenopathy
chronic, abnormal enlargement of the lymph nodes, usually associated
with disease.
Macropinocytic
Macropinocytosis is a form of regulated endocytosis that involves the
formation of large endocytic vesicles after the closure of cell-surface
membrane ruffles
maculopapular rash
maculopapular describes a rash that contains both macules (flat
discolored area of the skin) and papules (small raised bump). Usually
manifested as large red area with small confluent bumps.
107
Simone C. Zimmerli
meningoencephalitis
7. Glossary
Inflammatory process involving the brain and meninges, most often
produced by pathogenic organisms which invade the central nervous
system, and occasionally by toxins, autoimmune disorders and other
conditions
MHC
major histocompatibility complex; cluster of genes of human
chromosome 6 or mouse chromosome 17. It encodes a set of membrane
glycoproteins called the MHC molecules. The MHC class I molecules
present peptides generated in the cytosol to CD8 T cells, and the MHC
class II presents peptides degraded in intracellular vesicles to CD4 T
cells. The MHC also encodes proteins involved in antigen processing
and other aspects of host defense. The MHC is the most polymorphic
gene cluster in the human genome, having large numbers of alleles at
several different loci. Because this polymorphism is usually detected by
using antibodies or specific T cells, the MHC molecules are often called
major histocompatibility antigens.
mononeuritis multiplex
disorder characterized by simultaneous or sequential damage to more
than one nerve group.
multifocal
demyelinating disease that predominantly affects immunocompromised
leukoencephalopathy
hosts.
myalgia
muscular pain or tenderness, especially when diffuse and nonspecific.
myocarditis
inflammation of the heart muscle
myositis
inflammation of a muscle, especially a voluntary muscle, characterized
by pain, tenderness, and sometimes spasm in the affected area.
nasopharyngeal
malignant transformation of EBV-infected epithelial cells; subset of
carcinoma
gastric adenocarcinomas and certain saliverygland carcinomas
necrosis
death of cells due to chemical or physical injury, as opposed to
apoptosis; leaves extensive cellular debris that needs to be removed by
phagocytes.
108
Simone C. Zimmerli
neutralization
7. Glossary
inhibition of the infectivity of a virus or the toxicity of a toxin molecule
by antibodies (neutralizing antibodies)
opsonization
alteration of the surface of a pathogen or other particle so that it can be
ingested by phagocytes. Antibody and complement opsonize
extracellular bacteria for destruction by neutrophils and macrophages.
oral hairy leukoplakia
refers to a white patch – or white patches – that can develop in the
mouth; patches usually occur along the sides of the tongue, although
they can sometimes develop on the top and underside of the tongue or
along the inside of the cheek; it is caused by Epstein-Barr virus
pandemic
epidemic over a wide geographic area and affecting a large proportion
of the population.
pericarditis
inflammation of the lining sac (pericardium), which surrounds the heart.
Can be associated with a collection of fluid in the space between the
heart and the pericardium.
phagocytosis
Phagocytosis is the term describing the ingestion of micro-organisms,
cells, and foreign particles by phagocytes, eg phagocytic macrophages.
polyradiculopathy
inflammation of multiple spinal nerve roots
primase
enzyme that creates an RNA primer for the initiation of DNA
replication.
pyomyositits
primary acute bacterial infection of skeletal muscles, usually caused by
Staphylococcus aureus
RANTES
a chemokine that is a chemoattractant for eosinophils, monocytes, and
lymphocytes. It is a potent and selective eosinophil chemotaxin that is
stored in and released from platelets and activated T-cells. RANTES
affects HIV activity and is believed to act in conjunction with 2 other
chemokines, MIP-1α and MIP-1β.
109
Simone C. Zimmerli
7. Glossary
retroorbital
behind the eye (orbita)
Reye’s syndrome
acute encephalopathy characterized by fever, vomiting, fatty infiltration
of the liver, disorientation, and coma, occurring mainly in children and
usually following a viral infection, such as chicken pox or influenza.
rolling circle replication
a model of DNA replication that accounts for a circular DNA molecule
producing linear daughter double helices. A long dsDNA strand is built
consisting of many copies of the originally circular DNA. This copies
are joined, which is called concatemere DNA, and are later cut in single
copies. One of the DNA cleaved and nucleotides are joined to the 3’OH
end to push away the 5’ end. Subsequently okazaki fragments are bound
to the 5’ end. These are then polymerized to build a complementary
strand. The strang is continuously elongated and can possess many
copies.
tetramers
soluble versions of the MHC class I molecules are synthesized in E. coli
bacteria. The molecules achieve an appropriate conformation following
addition of β2 microglobulin and a synthetic peptide that represents the
epitope that is recognized by the TCR of interest. In addition the enzyme
BirA is used to attach a biotin molecule to the specific BirA-recognition
sequence, which has been incorporated into the C terminus of the MHC
molecule. Four MHC-biotin complexes are linked to a single
fluorochrome “tagged” streptavidin molecule. Tetramers can be used to
detect antigen-specific T cells by flow cytometry.
transcription
process whereby one strand of a DNA molecule is used as a template for
synthesis of a complementary RNA by RNA polymerase.
translation
the ribosome-mediated production of a polypeptide whose amino acid
sequence is specified by the nucleotide sequence in an mRNA.
TREC
T cell receptor excision circles, also called T cell receptor rearrangement
circles, are DNA circles resulting from genen rearrangement. They are
used as makers of T cells of thymic origin. TRECs are stable and cannot
replicate. They are maintained in T cells after exit from the thymus, but
are diluted out by cell division as the T cells get older.
110
Simone C. Zimmerli
7. Glossary
are diluted out by cell division as the T cells get older.
virion
a complete virus particle that exists outside of a host cell ; a
mature infectious virus particle existing outside a cell ; The
mature virus. The name of the virus once it is out of a cell ; a
complete viral particle; nucleic acid and capsid (and a lipid
envelope in some viruses)
virus
ultramicroscopic infectious agent that replicates itself only within cells
of living hosts; many are pathogenic; a piece of nucleic acid (DNA or
RNA) wrapped in a thin coat of protein
111