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The impact of new technologies on vaccines
G. P. TALWAR, MANISH DIWAN, FAREHA RAZVI, RITU MALHOTRA
ABSTRACT
Vast changes are taking place in vaccinology consequent to the
introduction of new technologies. Amongst the vaccines included
in the Expanded Programme of Immunization (EPI), the pertussis
vaccine has been replaced by acellular purified fractions devoid
of side-effects. Non-pathogenic but immunogenic mutants of
tetanus and dlptheria toxins are likely to replace the toxoids. An
effective vaccine against hepatitis B prepared by recombinant
technology is in large-scale use. Conjugated vaccines against
Haemophilus influenzae b, S. pneumococcus and meningococcus
are now available, as also vaccines against mumps, rubella and
measles. Combination vaccines have been devised to limit the
number of injections. Vaccine delivery systems have been
developed to deliver multiple doses of the vaccine at a single
contact point. A genetically-engineered oral vaccine for typhoid
imparts better and longer duration of immunity. Oral vaccines for
cholera and other enteric infections are under clinical trials. The
nose as a route for immunization is showing promise for mucosal
immunity and for anti-inflammatory experimental vaccines against
multiple sclerosis and insulin-dependent diabetes mellitus. The
range of vaccines has expanded to include pathogens resident in
the body such as Helicobacter pylori (duodenal ulcer), S. mutans
(dental caries), and human papilloma virus (carcinoma of the
cervix). An important progress isthe recognition that DNA alone
can constitute the vaccines, inducing both humoral and cellmediated immune responses. A large number of DNA vaccines
have been made and shown interesting results in experimental
animals. Live recombinant vaccines against rabies and rinderpest
have proven to be highly effective for controlling these infections
Talwar Research Foundation, New Delhi, India
G.P.TALWAR,
MANISHDIWAN,
FAREHARAZVI,
RITU MALHOTRA
Correspondence
to G. P. TAL W AR
© The National Medical Journal of India 1999
in the field, and those for AIDS are under clinical trial. Potent
adjuvants have added to the efficacy of the vaccines.
New technologies have emerged to 'humanize' mouse
monoclonals by genetic engineering and express these efficiently
in plants. These recombinant antibodies are opening out an era
of highly specific and safe therapeutic interventions. Human
recombinant antibodies would be invaluable for treating patients
with terminal tetanus and rabies. Antibodies are already in use for
treatment of cancer, rheumatoid arthritis and allergies. An
advantage of preformed antibodies directed at a defined target
and given in adequate amounts is the certainty of efficacy in every
recipient, in contrast to vaccines, where the quality and quantum
of immune response varies from individual to individual.
Natl Med
J
India 1999;12:274-80
INTRODUCTION
Vaccines have had a profound effect on health care. The mere
introduction of a handful of vaccines in the Expanded Programme
of Immunization (EPI) drastically reduced infant mortality, espe- .
cially in economically-emerging countries. Thanks to a vaccine,
albeit of Jennerian descent, smallpox has been eradicated from the
surface of the earth. It is expected that with aggressive and
systematic use of two vaccines (the Salk killed vaccine and the
Sabin oral vaccine), poliomyelitis will be eradicated globally in
the future. Similar achievements can be expected for other infections such as leprosy, where the infecting microorganism breeds
primarily in humans and for which vaccines have now been
developed. Vaccines not only prevent disease but also by virtue of
their property of enhancing immunity in humans (and animals),
render them as inhospitable territory for propagation of pathogens, thereby curtailing the focus of infection and its transmission
to others.
_
With the input of new technologies, old vaccines are undergoing improvement. New vaccines are being made to replace those
conferring insufficient or regionally variable immunity, as is 'the
case with tuberculosis. The range of vaccines has expanded.
TALWAR
et al. :
NEW TECHNOLOGIES
AND VACCINES
Infections such as hepatitis B can now be prevented by a highly
effective recombinant vaccine. Vaccines are presently conceived
not only against external pathogens, but also against microorganisms resident within the body, as is the case with Helicobacter
pylori which causes duodenal ulcers or against S. mutans which
causes dental caries.
Besides infections, vaccines are under development against
cancers, control of fertility, autoimmune diseases and allergies.
Advances in technologies for purification and stabilization of
proteins have led to purer and safer vaccines. Genetic engineering
and recombinant technology have enabled the making of antigenic components of those viruses which are difficult to cultivate
in vitro, such as hepatitis B, hepatitis C, HIV, and human papilloma virus. Monoclonal antibodies generated by hybridoma technology are revolutionizing not only immunodiagnostics, but have
also provided precious reagents for delineation of the epitopes
involved in protective immunity. New methodologies for synthesis and sequencing of peptides and genes, and knowledge-based
computer graphics have laid the basis of 'designer' vaccines. Last
but not the least, has been the impact of better understanding of the
immune system, the Th. and Th2 types of immune responses and
their role in conferring immunity. Parameters of non-responsiveness or tolerance are now better understood, leading to approaches such as conjugation to carriers, to make better vaccines.
New adjuvants, which have converted a non-protective vaccine
against malaria to a highly protective entity are underproduction.'
Single contact-point delivery systems have been developed for
administering multiple doses of the vaccines by a single shot. Oral
and nasal vaccines have been shown to impart mucosal immunity,
which is critically needed for some infections. Edible vaccines are
on the horizon. This article aims to briefly review the fastchanging spectrum of vaccinology with the advent of new technologies. While notable trends are cited, no attempt has been
made to comprehensively review individual developments in
various fields.
IMPROVEMENT OF EPI VACCINES
The EPI proposed by World Health Organization for children has
been adopted by many countries. Several hundred millions of
children receive these vaccines. In the DPT vaccine, the diphtheria and tetanus toxoids are made from respective purified toxins,
whereas the pertussis vaccine hitherto used in many countries is
a crude entity and causes side-effects. A number of candidate
acellular vaccines are under clinical trial. It is expected that in the
near future, these will replace the previous pertussis vaccine.
Another change expected in course of time would be the replacement of the diphtheria and tetanus toxoids by recombinant
mutants of respective toxins, which retain the desirable immunogenicity but are not pathological.
Bacillus Calmette Guerin (BCG) is still used in India and in
some other countries as an EPI vaccine. Large-scale trials conducted in south India by the Indian Council of Medical Research
(ICMR) with the cooperation of WHO and Centers for Disease
Control of USA showed that this vaccine does not confer any
protection in children against pulmonary tuberculosis. Hence,
there is a pressing need to have a better vaccine for tuberculosis.
We observed that two genetic strains of mice non-immunizable by
BCG were protected by immunization with the Mycobacterium
W. vaccine developed originally for immunotherapy of leprosy.P
Barry Bloom and his colleagues are engineering BCG with genes
coding for protecti ve antigens. In collaboration with a biotechnology company, they have obtained a mutant of BCG with dimin-
275
ished reaction. Similar work is being carried out by Tyagi" at the
University of Delhi, who has engineered mycobacterial promoters
expressing homologous or heterologous genes in M. smegmatis
and BCG. A host of secretory proteins of M. tuberculosis which
are protective against tuberculosis in experimental animals have
been identified. Their relative merits are unknown, so is the utility
of employing a combination of these. What is apparent is that
future vaccines for tuberculosis would be recombinant engineered propositions.
Poliomyelitis is nearing eradication. However, to achieve this
goal, both the oral (Sabin) as well as the killed (Salk) intravenous
vaccine would have to be employed. Research supported by WHO
has indicated stabilizing modalities. The uptake of the oral vaccine is, however, not high and several repeat doses are required to
induce full immunity. Also, it requires a cold chain to retain
efficacy. On the other hand, the Salk killed vaccine, administered
systemically, assures high immunity and has in fact been responsible for elimination of poliomyelitis in many countries.
NEW TYPHOID AND CHOLERA VACCINES
The previous typhoid vaccine based on killed bacilli had sideeffects and imparted only short term immunity, mostly of the
humoral type with no cell-mediated component. It has been
replaced by two better vaccines. One of them developed by
Germanier and Cryz" is given orally and employs a self-limiting
metabolic mutant of S. typhi Ty21a. It conferred very high
efficacy in a trial conducted in Egypt and is commercially available. The other is an injectable vaccine developed at the National
Institutes of Health, in which the polysaccharide Vi antigen is
conjugated to tetanus or diphtheria toxoid to rope in the T-cell to
help and enhance immunogenicity.
Holmgren et al. 6 proposed the use of the B subunit of cholera
toxin (prepared by recombinant DNA technology) along with
inactivated whole cells of Vibrio or E. coli as a vaccine. Clinical
trials of the vaccine in Bangladesh showed 60%-80% efficacy."
Oral live vaccines for cholera are under development in USA and
India. The Indian vaccine" under development seeks to employ an
engineered mutant of V. cholerae isolated from a carrier, which is
totally non-toxigenic, in which genes from a cholera toxin A
chain-negative, B chain-positive Vibrio have been inserted. This
engineered attenuated V. cholerae is non-reactogenic and confers
high immunity against cholera in experimental models. Early
clinical trials have started.
THE FIRST RECOMBINANT VACCINE FOR
WORLDWIDE USE IN HUMANS
Hepatitis B is rampant in China and has millions of carriers all
over the world. The development and introduction of a vaccine
against hepatitis B was an important step forward. The vaccine is
based on the surface protein of this virus, which was made in both
yeast and mammalian cells by recombinant DNA techniques. The
vaccine protects not only against hepatitis B but also against
hepatocellular carcinoma which develops as a consequence of
this infection in a certain percentage of infected people. The initial
vaccines marketed by Merck USAlPasteur-Merieux-Connaught
and SmithKline Beecham were fairly expensive. Two Indian
companies (Shantha Biotech and Bharat Biologicals) have obtained highly effective, efficiently expressed hepatitis B vaccines
which have cut down the price substantially. More compa~ies
within India are indigenizing technology and it is expected that in
the near future, this vaccine will be abundantly and cheaply.
available, and could be included in the EPI programme of vacci-
276
THE NATIONAL MEDICAL JOURNAL OF INDIA
nation. Meanwhile, emanating from research, the second-generation hepatitis B vaccines incorporating pre S regions and naked
DNA vaccines have emerged. Michelet al. at the Institute Pasteur
have observed the safety of the hepatitis B DNA vaccine and its
ability to clear the antigen. The vaccine induces both humoral and
cytotoxic T-cell responses."
Thanks to new technologies, the hepatitis E virus, responsible
for large epidemics has been isolated, cloned and sequenced.'? It
is transmitted by the faecal-oral route through contaminated
drinking water. A monkey experimental model of the disease has
been developed. Fortunately, this virus causes a mild self-healing
disease in adults, except in pregnant women where it can be fatal.
Significant progress has been made in delineating the molecular
structure of hepatitis C, responsible for chronic liver infection and
cirrhosis.'! Molecular tools are now available for diagnosis and
cytokines such as interferons prepared by recombinant DNA
technology are commercially available for therapy, a non-existent
possibility in the past.
LIVE RECOMBINANT VACCINES
A number of vectors have been identified and engineered to
express a variety of antigens. Vaccinia virus was an early favourite
for many reasons. It induces strong humoral and cell-mediated
responses. It has a large DNA genome, in which 25 kb of foreign
DNA can be inserted at the TK locus." The virus does not get
integrated in the host genome and multiplies in the cytoplasm of
the host. The genes of hepatitis B,Herpes and many other viruses
have been engineered in vaccinia. However, those which have
reached field-use and commercialization stages are the vacciniabased live recombinant vaccines for animals. Vaccinia-rabies
glycoprotein imparts high efficacy against rabies in dogs and
other mammals tested. 13The vaccine can be used intradermally or
through a bait, which on chewing transfers the engineered vaccinia on to the oral mucosa. Large-scale use of this vaccine in
Europe and USA has enabled a drastic reduction of rabies in wild
foxes and raccoons. Another remarkable success has been in the
control of rinderpest in Africa by the use of a recombinant
vaccinia expressing two proteins of the rinderpest viruS.14,15
At times, it is important to anchor the vaccinia-expressed
protein on the membrane of the host cell to induce good immune
response. This was the case when the ~ subunit of hCG (human
chorionic gonadotrophin) was engineered in vaccinia. A low
antibody response was observed with ~-hCG alone. However, a
high antibody response was observed (every animal exhibiting
high titres of antibodies against hCG) when ~-hCG was coexpressed with a 48 amino acid-fused peptide anchored in the
membrane. 16The vaccine engendered a long-duration, very high
antibody response in monkeys during which they were protected
from pregnancy.'?
Canary pox virus has replaced vaccinia for human vaccines, as
the avian pox viruses express the engineered antigen but do not
replicate in humans, hence these are deemed to be safer vectors.
Clements-Mann et al. have made a vaccine against HIV18in this
vector, which has entered Phase II and Phase III trials. We have
expressed ~-hCG-rabies glycoprotein (as membrane anchor) in
fowl pox virus, which expresses ~-hCG on the surface of the host
cells.19
Two other interesting vectors being experimented with for live
recombinant vaccines are avirulentSalmonelia and adenoviruses.
The former has the advantage that it can be used orally, the latter
appears to be carried widely by humans without any apparent ill
effects. Curtiss et al.20have developed genetic strains of Salmo-
VOL.
12, No.6,
1999
nella, which are safe and in which the engineered genes can be
expressed to elicit high humoral and mucosal response. A spermspecific protein SP-lO expressed in a recombinant Salmonella is
undergoing investigation to determine its fertility-controlling
potential. The hepatitis B surface protein has also been expressed
in this avirulent vector.
NUCLEIC ACID VACCINES
The observation that naked DNA introduced in either skin or
muscles generates both cell-mediated immunity and antibody
response against the protein encoded by administered DNA was
a major advance. 21Plasmids with eukaryotic promoter, enhancing
oligonucleotide motifs, eukaryotic termination and poly A signals" are available commercially to enable engineering of the
desired DNA. Engineered plasmids can be replicated in a fast
growing bacteria, for instance E. coli, to obtain large amounts of
DNA vaccine at low cost.
DNA vaccines have been made against two hypervariable
viruses, HIV and influenza. The testing of one ofthe HIV-l DNA
vaccines was done in cynomolgous monkeys and the feedback of
immunization was favourable, as the vaccine could produce broad
immune reactivities against several regions of the envelope protein of the virus (e.g. gp120, gp41) and provided additive protection by stimulating both T-helper cell and cytotoxic T-lyrnphocyte
(CTL) response against HIV-l antigens." The antibodies which
were neutralizing the virus were measurable within 30 days of
inoculation.
Experimental studies on influenza DNA vaccine were carried
out in mice and immunization led to generation of specific CTL
response and protection from a subsequent challenge with a
heterologous strain of influenza A virus." HIV and influenza
virus mutate rapidly and have hypervariable regions. In both
cases, it was of great interest that the immune response was
generated against both variable and constant domains of the
antigen, with the result that the immune response would prevail
over minor variations.
DNA vaccines have now been made against a variety of other
infections. Of particular interest are those against hepatitis B,25
rabies," tuberculosis," and malaria. 28,29
Both malaria and tuberculosis would demand immunization against more than one antigen
for high protective efficacy. The malarial parasite passes through
multiple stages-sporozoite,
merozoite and gamete-each having vulnerable critical antigens. An advantage offered by DNA
vaccines is the possibility of combining genes corresponding to
several antigens in the same construct. Another benefit of nucleic
acid immunization is simultaneous induction of both cytotoxic Tcells and neutralizing antibodies. This happens by processing and
presentation of antigens coded by DNA of both class I and class
II MHC molecules.
A number of concerns were raised about the safety of DNA
vaccines. For some of these, salient observations have been made.
Unlike live viral vaccines, DNA vaccines have no potential of
causing infection. No case of integration of the DNA vaccine has
been noted in the chromosomes of the host cells, nor has any
malignant transformation or induction of autoimmune response
occurred. At present, these vaccines appear to be safe and devoid
of toxicity and side-effects. An in-built advantage of DNA vaccines is the possibility of testing them in various mammals and
experimental laboratory animals, as the plasmid ca~ing the
DNA infects all eukaryotic cells.
Currently, clinical trials using DNA vaccines are underway for
herpes, influenza, hepatitis B, HIV and malaria. A DNA vaccine
TALWAR
et al. :
NEW TECHNOLOGIES
AND VACCINES
for HIV -1 env and rev genes has been tested for safety and host
immune response in 15 HIV-infected asymptomatic patients
receiving no antiviral therapy." Patients received three doses of
the vaccine at 10-week intervals in a dose-escalation trial. Data
were analysed for CTL responses, T-lymphocyte proliferation and
~-chemokines. More changes were noted in these immunological
parameters in the high-dose groups, thus showing a stronger antiHIV immune response. The vaccine was well tolerated and no
side-effects ascribable to immunization were noted.
DNA vaccines exhibit few limitations and many positive
points. These can be used to raise immune responses against the
protein components of the pathogen. However, certain microbes
have outer capsular structures of polysaccharides, which carry the
protective antigens. DNA vaccines cannot substitute for the
polysaccharide-based vaccines.
MINIMIZATION OF VISITS FOR VACCINA TION
The full course of childhood immunizations currently requires up
to 18 visits to a clinic. This is not only costly, but failure of
compliance for repeat injections leaves a percentage of children
incompletely immunized. Attempts are being made to develop
combination vaccines. For instance, a quadruple vaccine combines polio with DPT. Similarly, hepatitis Band Haemophilus
influenzae vaccines are being tested as partners in combination
vaccines.
Two alternate strategies are also possible. In one, multivalent
DNA vaccines are being conceived to carry protective proteins
coding genes for more than one infection. The second strategy is
to develop biodegradable delivery systems, whereby multiple
doses of the vaccine can be given at a single contact point.
VACCINE DELIVERY SYSTEMS
Over 70 years ago, Raman discovered that the antitoxin response
to tetanus and diphtheria toxin was increased by injection of
vaccines together with other compounds such as starch, oil,
saponins or even breadcrumbs." Glenny et al. 32 reported the
immune stimulating properties of aluminum salts. Vaccine technology has come a long way since then and so has the technology
for the delivery of antigens.
New delivery systems and adjuvants are being investigated
that would potentiate the immunogenicity of antigens which
otherwise induce 'weak' immune response when given adsorbed
on alum. The inclusion of vaccines in liposomes or biodegradable
microspheres has been found to be useful for developing singlecontact point delivery systems.
Liposomes
Liposomes have been successfully used as drug carriers" and
tested as carriers for antigens. In 1974, Allison and Gregoriadis"
identified these as potent, non-toxic adjuvants that enhance
immune response. These are microspherical structures consisting
of vesicles that are concentric bilayers or multilayers of phospholipids and cholesterol in which antigens can be introduced in
aqueous or lipid phase." Liposomes have been used for a variety
of antigenic substances including proteins, carbohydrates, lipids
and glycolipids." In most cases, the association of the antigen
with the lipid vesicle has been by their internal entrapment in the
aqueous phase. The adjuvant property ofliposomes is due to their
depot effect and the ability to target the encapsulated antigens to
antigen-presenting cells (APCs) because of their greater susceptibility to phagocytosis. They induce both humoral and cellmediated immunity.
277
Additional adjuvants can also be incorporated into liposomes
together with the antigen. Incorporation of Lipid A has been
shown to markedly improve the immune enhancement capacity of
liposomes."
One non-phospholipid liposomal preparation (Novasome"
vesicle) available commercially for two poultry vaccines is approved by the US Department of Agriculture." Recently, another
liposome-based preparation, namely Epaxal Berna", against hepatitis A has also been licensed in Switzerland.
Biodegradable microspheres
The development of controlled release formulations for vaccine
delivery was a priority area of research for the WHO. 39 Microspheres
made of biodegradable polymers slowly release the encapsulated
antigen into the tissue of a vaccinated individual, while simultaneously serving as a repository for unreleased antigen, a phenomenon known as the depot adjuvant action." The subsequently
released antigen acts as a secondary stimulus to the sensitizing
action of the antigen released earlier, leading to sustained antibody production.
One of the first studies demonstrating the concept of a singleshot immunization was the work of Preis and Langer in 1979
using a non-degradable polymer, polyethylene-vinyl acetate." A
major disadvantage of non-biodegradable polymers is that these
must be retrieved surgically after the expiry of the delivered
antigen. The long term effects of these polymers have not been
determined and these can have potential adverse side-effects as
have been observed for silicone breast implants, previously considered safe and biocompatible."
Biodegradable polyesters based on lactic and glycolic acids are
approved for human use. They have been employed for the last 20
years in resorbable sutures and have a well-established safety and
toxicology record." Polycaprolactone, an inexpensive alternate
to PLGA polymers, is also approved for human use.
Preclinical investigations in experimental animals have yielded
promising results and the use of micro spheres as delivery systems
for vaccines appears possible in the near future.
A subunit vaccine for HIV -1 consisting of recombinant glycoprotein 120 (rgp 120) when given in micro spheres together with
QS21 adjuvant, elicited long term bioneutralizing antibody titres
in baboons." A single immunization with tetanus toxoid entrapped in microspheres generates protective antitoxin antibodies
in mice which are comparable to those elicited after immunization
with the conventional alum adsorbed vaccine." Our laboratory
has shown that luteinizing hormone releasing hormone (LHRH)
conjugate, an anti-prostatic cancer vaccine, encapsulated in PLGA
microspheres given along with alum and SPLPS adjuvant in
rodents, not only generates a sustained antibody response for 5 to
7 months but the bioeffective antibody response is initiated within
2 weeks post immunization." The induction of a similar response
takes 8 weeks when the vaccine is given adsorbed on alum after
three immunizations.
Microspheres are taken from the intestine by Peyer's patches,
hence have considerable potential as carriers for oral and nasal
immunization. Microencapsulated staphylococcal enterotoxin B47
or ricin toxoid" when given orally or nasally in mice have been
shown to induce not only mucosal IgA response but also systemic
immune response. Holmgren et al" observed the prevention gf
allergic encephalitis if myelin protein is given orally. The nasal
and oral routes are advocated for anti-inflammatory vaccines for
multiple sclerosis and insulin-dependent diabetes mellitus.
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THE NATIONAL MEDICAL JOURNAL OF INDIA
IMMUNE STIMULTATING COMPLEXES (lSCOMs)
ISCOMs have a micellar structure which consist of glycosides of
the adjuvant Quil A, cholesterol, the antigen, and, in most cases,
phospholipids." ISCOMs have been proven to be potent stimulators of both humoral and cell-mediated immune responses. It has
been suggested that these complexes act as stimulants by targeting
antigens to APCs. ISCOMs are used in veterinary vaccines" and
have not been approved so far for human vaccines. Attention is
being paid to the reduction of toxicity of the respective components ofISCOM preparations, especially Quil A. Ultimately, the
ISCOM approach may generate promising vaccines against viral
infections.
HUMANIZED AND HUMAN RECOMBINANT
THERAPEUTIC ANTIBODIES
With the emergence of new technologies, a new mode of immunotherapy has dawned, which offers the use of preformed human
or chimeric antibodies of desired specificity and characteristics.
These can be administered in the appropriate dose to achieve
efficacy in every recipient, doing away with the uncertainty and
quantum of antibody response to a vaccine, which varies from
individual to individual. Antibodies as 'drugs' would be highly
safe in situations where the target molecule is defined. The inbuilt specificity of the antibody for the given target molecule
would avoid side-effects invariably associated with pharmacological preparations.
Monoclonal antibodies (MoAb) have been used in therapeutic
clinical trials as: (i) mediators of immune effector function; (ii)
carriers of cytotoxic agents (magic bullets); (iii) agents to block
tumour growth factor; or (iv) anti-idiotype vaccines.
Anti-idiotypic MoAb can mimic both protein and non-protein
antigenic epitopes. In animal models, and now in humans, it is
possible to induce immune responses against tumour antigens
using anti-idiotypic MoAb vaccines. They have potential advantages when the antigen is not readily available in sufficient
quantities or purity, or when the antigen is a non-protein moiety.
Yao et al." used BEC2, an anti-idiotypic mouse MoAb that
mimics GD3 ganglioside for immunization of melanoma patients.
Clinical trials demonstrated that BEC2 with BCG can induce antiGD3 antibodies in patients. Zhang et at. 52 have developed an
MoAb against GD2 (mAb 3F8), richly expressed at the cell
surface of human neuroblastomas, sarcomas and melanomas. In a
syngeneic murine model, they demonstrated that passively
administered and vaccine-induced anti-ganglioside antibodies
prevent outgrowth of micrometastases. The level of protection
was proportional to the antibody titre. Also, protection was
demonstrated even when immunization was initiated after tumour
implantation.
Monoclonal antibodies against tumour-associated antigens
have also been tested with various imaging techniques to improve
detection and staging of lung cancer."
Genetically-engineered chimeric human-mouse MoAbs have
been developed by replacing the mouse Fe region with the human
constant region. The starting material is the mouse hybrid cell
clone, making MaAbs of high affinity and proven bioefficacy.
The RNA of these cells is reverse transcribed employing PCR and
primers coding for the variable heavy and light chains. These are
cloned, amplified and sequenced to confirm that the putative
CDRs have framework residues of immunoglobulins. The variable light and heavy chains are then linked into a single chain
variable fragment (ScFv) with a spacer in between, enabling the
VOL. 12, No.6,
1999
flexibility of conformation of the two variable chains to cooperate
for binding with the target antigen. The ScFv is then expressed in
a prokaryote host to determine whether the recombinant product
has the requisite specificity and affinity for binding. Chimeric
antibodies can be obtained by retaining the mouse antibodybinding regions and aligning these with constant regions of the
human immunoglobulin genes. Alternatively, ScFv is used as a
probe for sifting complementary human variable regions from a
library to eventually make a totally human antibody. Site-directed
mutagenesis may be necessary to achieve high-affinity human
antibodies. Antibody gene constructs can be expressed in yeast or
in plants. Gene expression in plants has several advantages. An
antibody expressed in plants would not demand exclusion of
oncogenic DNA, endotoxins and eukaryote pathogens during the
purification procedure. 54 High yields of antibodies are obtained
and they can be stored in a stable condition in seeds. Plants are able
to make antibodies efficiently. Even a complex antibody such as
IgA has been made successfully by Maet al.55 in plant cells. This
antibody directed at S. mutans protects against dental caries. The
antibody stays in the oral cavity for three days after a single rinse,
in contrast to IgG which stays only for 24 hours.
.
A humanized antibody against the FcERI binding regions on
IgE is undergoing clinical trials in Switzerland. 56 The antibody
prevents the binding of IgE to mast cells. The half-life of this
antibody is about two weeks. It is well tolerated and has given
beneficial results in both short term cure and chronic allergies.
Akatsu et al." in Japan are planning treatment with anti-IL-5
antibody. Ozakiet at. 58 have reported that humanized anti-HM 1.24
MoAb that detects human plasma cell-specific antigen has potential as a new therapeutic agent in multiple myeloma; and that cotreatment of effector cells with immunomodulating cytokines,
interleukin-2 (IL-2), IL-12 orIL-15, restores the effect of humanized anti-HM1.24 MoAb in patients with diminished antibodydependent cell-mediated cytotoxicity (ADCC) activity.
Another major trial with a chimeric anti-TNFa antibody has
been carried out by Feldmanet al. 59 in rheumatoid arthritis. A dose
of3 or 10 mg/kg body weight was given for 12 weeks. Seventy per
cent of patients experienced improvement and a few achieved
clinical remission.
Monoclonal antibodies of mouse origin have been used for
some years for therapy of tumours in humans. Their replacement
by human or chimeric antibodies would avoid anti-mouse sensitization on repeat administration. With human, mouse or chimeric
(human-mouse) MoAbs, beneficial results have been obtained
for treatment of colorectal cancers'? and refractory neuroblastoma
and osteosarcoma. 61 The antibodies exercise their effect by complement-mediated lysis and by ADCC. These effects are augmented
by co-therapy with recombinant cytokines IL-2, GM-CSF and
CSF. A single chain human ScFv co-expressed in alignment with
IgG, against the melanoma-associated chondroitin sulphate
proteoglycan causes specific lysis of human melanoma cells by
NK cells and complement mediation."
Antibodies with two binding ends-bispecific
antibodies
(BsAb) provide improvement for targeting cancer cells." These
are prepared by chemically linking two different MoAbs or by
fusing two hybridoma cell lines to produce a hybrid-hybridoma.
BsAbs have been used to demonstrate that specific surface molecules can trigger leucocytes to either phagocytose or kill tumour
cells, viruses, parasites and infected cells. They have-also been
used to direct toxins to tumour sites and fibrinolytic agents to
areas of thrombosis.
TALWAR etal.
NEW TECHNOLOGIES
279
AND VACCINES
IMMUNOTHERAPEUTIC VACCINES FOR CANCERS
Though immunotherapy of cancers is still largely experimental,
there have been marked innovations in this field of vaccine
development. There has been progress in identifying both viral
and non-viral tumour-associated antigens since it was known that
the immune system usually reacts against the most immunodominant antigenic determinants." Molecularly defined vaccines, containing the minimal essential components, can be used
to elicit specific T-cell responses against both dominant and
subdominant T-cell epitopes." The application of molecularly
defined vaccines composed of a limited number of tumour specific T-cell epitopes has resulted in protective antitumour T-cell
immunity in several mouse tumour models."
An important group of antigens encoded by the melanoma
antigen encoding gene (MAGE) family of genes is expressed in a
significant proportion of melanoma tumours of several histological types. These genes are not expressed in normal tissues except
the testis." Clinical trials are underway to immunize patients
suffering from metastatic melanoma using peptides coded by
MAGE. Regression has been observed in 5 of the 17 immunized
patients.
A novel way to enhance the immunogenicity of tumourassociated antigens is by transfection in dendritic cells, which
process and present the antigens to induce both cytotoxic and
humoral antibody responses. Fonget al. 68 have designed a vaccine
for prostate cancer whereby they primed the human dendritic cells
with xenogenic prostatic acid phosphatase (rat PAP) and administered it to patients with prostate cancer. The assessment of the
immune response of patients after 12 weeks of the initial dose of
mouse PAP-pulsed dendritic cells indicated a stability in the
levels of prostate specific antigen (PSA), a marker of prostate
tumour activity.
Besides immunization against tumour-associated antigens,
.another approach for vaccine development is to immunize against
a key growth factor supporting the proliferation of cancer cells.
Pilot clinical trials are underway to define the safety, toxicity and
immunogenicity of active immunotherapy with human epidermal
growth factor (huEGF) coupled to a carrier protein." Patients in
advanced clinical stages of malignant carcinomas (colon, lung,
stomach and prostate) were immunized with huEGF linked to
either tetanus toxoid (TT) or Neisseria meningitides recombinant
protein (P64k) inducing anti-EGF antibody titres without evidence of toxicity.
Prostatic hypertrophy and carcinoma may also be amenable to
control by administering vaccines and relevant MoAbs. Talwaret
al. 70 developed a semisynthetic vaccine against LHRH, in which
D-glycine at position 6 was substituted by D-lysine to freeze the
native conformation of the decapeptide," concomitantly creating
a functional group to link the decapeptide to a spacer and a carrier.
The vaccine causes marked atrophy of the rat" and monkey
prostate." The vaccine also inhibits the growth of Dunning
tumours in rats." After preclinical toxicology, drug regulatory
and ethical approvals, the vaccine has been clinically tested in 28
patients suffering from D2 ~dva~ed prostatic carcinoma in India
and Austria. In all patients, the vaccine was well tolerated, and in
those generating 400 pg/rnl or more of antibody titres, clinical
benefits were observed. The levels of PSA and PAP declined
sharply in these patients. Serial nephrostograms and ultrasound
scans indicated the regression of tumours." Development of a
recombinant vaccine and humanized anti-LHRH antibodies is
ongoing.
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