1. No category

HIV Infection and Human Blood Cell
Development
Lessons Learned from the Humanized Mouse
Use of humanized mouse model as a translational medicine tool
Past successes:
1. Pathogenesis studies
2. Drug screening
3. Gene therapy
Future aims:
1. Gene therapy
2. Vaccine development
3. Immune reconstitution/enhancement
4. Viral transmission
5. Viral latency/ eradication
6. Organ regeneration/replacement
7. Novel therapeutic development
Immunodeficient Mice
SCID, NSG
Rag2-/-
Black 6 SCID
Use of Humanized mice
in HIV-related studies
Model #1:
The hu-PBL SCID Mouse Model
(the Mosier Model)
• First humanized mouse used in HIV studies.
• Involves injecting human Peripheral Blood
Lymphocytes into the peritoneum of SCID mice. Cells
are typically removed from mice by intraperitoneal
lavage.
• Key Strength: can assess effects rapidly and directly on
mature human blood cells in vivo.
• Key Weaknesses: limited number of time points, short
experimental duration (<3-4 weeks).
7 -14 days
Drug
HIV
Cells
Typical Experiment: hu-PBL SCID Mouse Model
7 -21 days
Typically there are 6-30 mice per experiment. The limiting factor in
these studies is the number of human cells. You need >2 x 107
human cells/mouse.
Lavage and
Assessment:
Real Time
Quantitative PCR
Analysis of radio-labeled HIV specific antibody
213Bismuth
HIV Infected
213Bismuth
HIV Infected
213Bismuth
213Bismuth
Experimental Results
HIV DNA Viral Load
120
Copies HIV/10000
100
80
60
40
20
0
Saline
n = 5 mice/group
Cold antigp41
non-specific
50 uCi
anti-gp41
50 uCi
non-specific
100 uCi
anti-gp41
100 uCi
non-specific
200 uCi
anti-gp41
200 uCi
Treatment
Dadachova et al. PLoS One 2012
Conclusions:
anti gp41 antibody in hu-PBL SCID mice
Bismuth213- labeled neutralizing anti-gp41 monoclonal
antibody targets and eliminates HIV infected cells in vivo.
Platelet counts and Pathology similar in all
experimental conditions:
Low toxicity of radioactive compound
Eliminates productively infected cells.
Decreases Viral DNA load.
Model #2:
The SCID-hu thy/liv mouse model
• Developed at Stanford, the SCID-human fetal
thymus and liver model.
• Involves transplanting human fetal thymus and
liver in SCID mice. Cells or HIV is then injected
into tissue. Tissue is obtained by biopsy.
• Key Strengths: can assess viral infection of
human T cells in human tissue in vivo, system to
study T cell development.
• Key Weaknesses: surgery required, technically
complex.
The SCID-hu Mouse Model
• Established in the late 1980’s/early 1990’s as a
model system to study HIV pathogenesis in
vivo.
• Played a key role in studies on:
–
–
–
–
–
HIV Pathogenesis
Gene Therapy
HIV Latency
Embryonic Stem Cell Development
Engineering T cell Immunity
The thymus is the organ that generates T cells
How does HIV infection perturb T cell
development?
Thymopoiesis
Peripheral
Circulation
Thymus
CD4 SP
Quiescent
-
CD4 / CD8
-
CD4+ / CD8+
Transcriptionally Active
CD4+ /
CD45RA+
CD8+ /
CD45RA+
CD8 SP
Quiescent
The SCID-hu mouse model
Human fetal
thymus
Human fetal
liver
Thy/Liv implant
3-4 months
SCID-hu mouse
CD8
HIV
Typical Experiment: hu-PBL SCID Mouse Model
Biopsy and
Assessment
SCID-hu
SCID-hu
3-22 weeks
Tissue
processing
Assay
1. PCR
2. Flow
cytometry
Experiments typically consist of 6-30 mice, and have 3 time points. The
limiting factor is the amount of fetal tissue.
Uninfected
HIV infected
HIV infection causes loss of immature thymocytes
What viral factors are involved in this process?
HIV Reporter Virus
muHSA (CD24)
HSA Expression in Thymocytes
Jamieson et al.
What is the effect of HIV infection on
the thymic microenvironment?
Do high levels of HIV destroy the ability of
thymic stroma to direct T cell differentiation?
Why is reconstitution of thymocytes transient?
Viral / Thymocyte Dynamics Following Antiretroviral Therapy
Conclusions
• Reconstitution of thymopoiesis is transient
following HAART.
• The transiency is caused by breakthrough in viral
replication to antiretroviral treatment.
• The SCID-hu thy/liv model is highly useful in
examining HIV infection in the context of
developing T cells.
Modeling HIV Latency
HIV-1
CD4 SP /HSA 4-6 weeks
Biopsy
+ Protease Inhibitor
CD8 SP /HSA Thy/Liv
Implant
Latent HIV in Thymocytes from SCID-hu Mice
Day 3
Day 0
CD24
99%
21%
<1%
MFI: 510
CD45
+ Protease Inhibitor
CD24
99%
<1%
5%
CD24
CD4
79%
MFI: 225
95%
CD8
CD45
Targeting The Latent Reservoir
The Search for Agents That Activate Latent HIV
Prostratin
Phorbol ester
Used in tea in Samoa to treat various illnesses
Activates latent virus without inducing T cell replication
Further testing is required to define effects on immune system
IL-7
Naturally occuring cytokine
Induces some cell proliferation, but phenotype is maintained
Potently induces expression of latent HIV
Further development is required
Anti-HIV Immunotoxin
Infected Cell
gp120
Anti-gp120
3B3:N31H/Q100eY(dsFv)-PE
McHugh et al.; 2002
Pseudomonas
Exotoxin
Elimination of Latent HIV
Conclusions
I.
Immunotoxins can be used to kill cells induced to express previously latent virus
II.
Pre-treatment with IL-7 or with prostratin plus immunotoxin results in a
decrease in rescuable latent virus upon subsequent co-stimulation.
III. These agents may prove useful as adjunctive therapeutics to purge the latent
HIV reservoir
Model #3:
The Non-obese diabetic (NOD), SCID, IL-2
receptor γ knockout (NSG), humanized bone
marrow, fetal liver and thymus (BLT) mouse model
The NSG-BLT model
• Recently developed model, pioneered by J. Victor Garcia in Texas.
• Involves transplanting human fetal thymus and liver in NSG mice,
the irradiating them and the injecting human stem cells
intraveneously, which allow them to become engrafted in mouse
bone marrow. Mice become engrafted with multiple human cell
types that arise from stem cells within 6-8 weeks.
• HIV is then injected. HIV replication and peripheral blood cells
are monitored following bleeding of the mice. Tissue is obtained
by biopsy and/ or sacrificing mice.
Model #3:
The Non-obese diabetic, SCID, IL-2 receptor γ
knockout (NSG), humanized bone marrow, fetal
liver and thymus (BLT) mouse model
• Key Strengths: can assess viral infection of multiple
types of human cells in vivo, slow and steady rate of T
cell depletion and viral replication (mimics natural
history in humans), easy to manipulate, develop immune
responses.
• Key Weaknesses: surgery required, technically complex,
immuodeficient status of mice make them highly
susceptible to graft versus host disease, lower
experimental numbers.
Humanized Mouse Model of HIV Infection:
The NSG-BLT Model
Infect with
HIV-1
1. Implant fetal thymus and
liver tissue.
liv
thy
Irradiate
6-12
weeks
3 weeks
NSG
2. Sort CD34+ Stem Cells
CD34+
NSG
4. Analyze
human cell
reconstitution
3. i.v. Inject
CD34+
CD34+
CD34+
CD34+
5. Analyze
effects of infection
Each experiment typically contains 6-15 mice. Human cell reconstitution frequency
is far lower than SCID –hu.
Multlineage Hematopoesis in
Humanized mice
CD34+
Myeloid stem cell
Erythroid
progenitor
Megakaryoblast
Eosinophil
progenitor
Lymphoid stem cell
BM Stem
cell
Basophil
progenitor
Myelomonocytic
progenitor
B progenitor
T progenitor
DP Thymocyte
Monocyte
NK Cell
Megakaryocyte
CD8+
T cell
Red blood cells
Platelets
Eosinophil
Basophil
Neutrophil
Macrophage
B cell
CD4+
T cell
Multilineage Hematopoiesis in NSG-LTL mice
%CD45+
mean=53% ± 29%
range 19%-80%
n=12
HIV Infection of NSG-BLT mice
Mice were either
1. Untreated
2. HIV infected
3. HIV infected but treated
with pre-exposure prophylaxis
of emtricitabine (FTC)/
tenofovir disoproxil
fumarate (TDF)
Conclusion: Pre-exposure prophylaxis
Prevents infection in this model
Denton et al.,PLoS Med. 2008 Jan 15;5(1)
Engineering HIV-Specific T-Cell Immunity
Targeting immune responses could augment rejection of
Infectious agents (chronic viruses) or tumors in certain
individuals or disease states
HIV disease: weakened immune system, viral drift
Cancer: Escape of tumor cells from immune surveillance
Transgenic mouse models suggest that introduction of antigen
receptors into stem cells can result in functional effector cells
targeting the antigen
Can this type of approach be done in humans?
Can we enhance immune capabilities in humans?
HIV Specific T Cell Responses
Killing of HIV
Infected Cells
HIV Infected
Cells
HIV-Specific
T Cell
Mature
T-Cells
Incomplete
Clearance of
HIV Infected
Cells
Stem Cell
Expansion of
HIV-Specific
Cells
Thymus
Periphery
T Cell Recognition Of Virally-Infected Cell
Step 1:
Strategy for Cloning HIV-Specific T Cell Receptors
T cell
T cell
T cell
HIV Infected
Individual
T cell
T cell
T cell
T cell
1) Purify T Cells
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
2) Culture in Presence of
Known HIV peptide
TCR 
TCR  IRES
eGFP
3) Molecularly Clone
HIV-Specific
TCR
Stem Cell
Gene Therapy
ES or
iPS Cells
Class I Restricted
TCR Gene
Myeloid stem cell
Erythroid
progenitor
Megakaryoblast
Eosinophil
progenitor
Lymphoid stem cell
BM Stem
cell
Basophil
progenitor
Myelomonocytic
progenitor
B progenitor
T progenitor
DP Thymocyte
Monocyte
NK Cell
Megakaryocyte
CD8+
T cell
Red blood cells
Platelets
Eosinophil
Basophil
Neutrophil
Macrophage
B cell
CD4+
T cell
HIV Gag SL9-Specific T Cell Receptor
Restricted to HLA-A2.01
2. Transduce with
Anti-HIV TCR
(SL9 Peptide Specific)
Fetal Liver
Irradiate
CD34+
CD34+
CD34+
CD34+
CD34+
ESC ESC
ESC ESC
ESC
SCID-hu
HLA-A2.1+
3-12 weeks
3. Analyze
TCR
Expression
1. Sort CD34+
CD8
Kitchen et al, PLoS One, 2009
HIV-TCR Transduced Thymocytes
Week 4
MHC Tetramers
Antigen Responsiveness of
Transgenic T Cells
HLA-A*0201+
Tissue
Humanized Mouse Model of HIV Infection:
The NSG-CTL Model
4. Thaw and Transduce with
Anti-HIV TCR
Or Control TCR
Fetal Liver
1. Sort CD34+
CD34+
CD34+
CD34+
CD34+
CD34+
CD34+
Infect with
HIV-1NL4-3HSA-HA
2a. Viably freeze fraction
Irradiate
CD34+
CD34+
NSG
2. Transduce with
Anti-HIV TCR or
Control TCR
3. Combine with fetal
thymus tissue and liver
stroma, implant under
kidney capsule
NSG
3 weeks
5. Tail Vein Inject
6-12
weeks
6. Analyze
TCR
Expression/Fu
nction
HIV-Specific TCR expressing cells are found in
multiple organs in NSG-LTL humanized mice
CD3
10
10
4
10
10
10
5
10
4
3
10
3
2
10
2
10
5
10
4
3
10
2
10
0.75%
0
1.26%
0
10
10
3
SL9 Tetramer
10
4
10
5
1.47%
0
0
2
0
10
2
10
3
10
4
10
5
Peripheral Blood
Liver
Spleen
Thymus
Bone Marrow
5
10
5
10
4
10
10
10
5
10
4
3
10
3
2
10
2
1.31%
0
0
10
2
10
3
10
4
10
5
1.28%
0
0
10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
Suppression of HIV Replication by HIV-specific TCR
35
%CD45+HIV(HSA-HA)+ Cells
p=0.004
30
25
20
p=0.43
15
10
5
0
SL9-TCR
Control Uninfected
TCR
Week 2
SL9-TCR
Control Uninfected
TCR
Week 6
Suppression of CD4 Depletion by HIV-specific TCR
100
% Cells CD45+CD4+
90
p=0.20
p=0.88
p=0.19
p=0.29
p=0.05
p=0.01
80
70
60
50
40
30
20
10
0
SL9-TCR
Control Uninfected
TCR
SL9-TCR
Control Uninfected
TCR
HIV-Specific TCR suppression of plasma vRNA in vivo
p=0.05
Copies vRNA/ml
1000000
100000
p=0.02
10000
1000
100
10
1
SL9-TCR
Control
TCR
Week 2
SL9-TCR
Control
TCR
Week 6
HIV-specific TCR does not drive short-term viral evolution
AMINO ACID ALIGNMENT
INPUT VIRUS: SLYNTVATL
Control TCR-CONT AINING MICE: SLYNTVATL
HIV SL-9 TCR-CON TAINING MICE: SLYNTVATL
Immune Correlates of Anti-Viral Efficacy
A.
Uninfected
C.
Infected
% SL-9 Tetramer+ CD8+
T Cells
16
Week 6
14
4.04
p=0.044
12
7.91
CD8
10
SL-9 Tetramer
57.2 16.8
12.7
25.8
8
6
4
2
0
6
D.
Week -2
5
p=0.0292
4
3
2
1
0
0
50
100
150
200
50
100
150
200
250
300
350
Copies vRNA /ml at Week 6
400
450
3.57
10.7 53.8
CCR7
% SL-9 Tetramer+ CD8+
T Cells
B.
19.3
% SL-9 Tetramer+ CD8+
T Cells
CD45RA
0
250
300
350
Copies vRNA/ml at Week 6
400
450
18
16
14
12
10
8
6
4
2
0
Week -2
Week 4
Week 6
“Genetic Vaccination” to HIV
Stem cell
TCR 
TCR 
TCR 
TCR 
TCR 
TCR 
TCR 
TCR 
Viral Vectors
Containing Cloned
TCRs
T cell
T cell
Virus
Infected cells
T cell
T cell
T cell
Conclusions
Human blood-forming stem cells can be
genetically modified with an HIV- specific T cell
receptor and mature into functional CD8+ T cells
in vivo in humanized mice.
HIV specific TCR lowers viral replication in vivo
in humanized mice.
Conclusions
• There are a variety of different type of mutations
that produce immunodeficient mice.
• Immunodeficient mice allow human cell
engraftment.
• The type of mouse and the system of
humanization has to be carefully considered
depending on the study.
• The interest in these models has significantly
expanded with the development of new strains
and better human cell engraftment and function.