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Towards designing an improved hCG vaccine |
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Principal Investigator : Om Singh Co-investigators Project Associates/Assistants Focus of this project is on potentiation of the
immune response by carriers, adjuvants and delivery systems. A vaccine based
on the entire b-subunit of human chorionic gonadotropin (hCG) is under development
as a contraceptive option. The vaccine aims at eliciting antibody response
that can neutralize hCG, a hormone considered essential for the establishment
of early pregnancy. It is also a potential target for tumor immunotherapy.
Clinical trials with a prototype vaccine demonstrated the lack of notable
adverse effects and feasibility of the approach preventing pregnancy in
fertile women. Protective levels of anti-hCG antibodies were however not
attained in all recipients although every single immunized woman responded by
making anti-hCG antibodies. Current research efforts are focussed on new
strategies to develop a vaccine formulation that can elicit and maintain
effective levels of antibody titers in recipients of diverse genetic
backgrounds. Studies on a novel adjuvant We had previously demonstrated significantly
enhanced immunogenicity of cocktail formulations, employing alum as the
adjuvant. The adjuvanticity of the compound SBAS2 (obtained from SmithKline
Beecham) was evaluated employing two cocktail peptide formulations. The first
comprised peptides derived from cirumsporozoite protein of P.falciparum,
1A protein of respiratory syncytial virus, and from haemaglutinin of influenza
virus, fusion protein of measles virus, reverse transcriptase of HIV. The
other formulation included four promiscuous TT derived peptides. For both
formulations, SBAS2 increased the anti-hCG response significantly in low
responder as well as high responder strains, compared to animals immunized
with alum-adsorbed immunogens. All animals immunized with SBAS2 responded with
high anti-hCG titers of long duration. Affinity of the antibody-hCG
interaction was high (1010 M-1). Antibodies were capable of neutralizing the
bioactivity of hCG, neutralization capacity being a function of antibody titre. T-cell helper peptides as alternate carriers In order to confirm that prior immunity against
TT would not have deleterious effects on the anti-hCG response elicited by the
Th epitope based formulations, groups of 8-10 mice were pre-sensitized with TT
adsorbed on alum, followed by immunization with bhCG
conjugated with non-TT Th peptides. Anti-hCG antibody responses were
comparable, irrespective of whether or not animals had received TT
pre-immunization. No anti-peptide antibodies were detected in animals
immunised with the peptide-based formulations. Thus, prior immunity to large
protein carriers may not have any suppressive effects upon Th peptide based
vaccines, indicating the importance of employing pathogen-derived peptides in
vaccine formulations. Studies on DNA vaccination Attempts are also being made to genetically
engineer and express bhCG, along with selected T helper
epitopes, in the form of well-defined fusion proteins. This method offers the
flexibility of altering the orientation of epitopes, of changing the position
of one determinant in relation to the other, and also of expressing bhCG
containing variable numbers of T cell epitope(s). In vitro expression
studies were conducted using the transformed primary human embryonal kidney
cell line HEK-293. Transfection of the bhCG construct
(pEGFP-N3) resulted in a protein expression of 5 ng/ml as measured by a
specific radioimmunoassay, whereas no expression was observed when a control
plasmid was employed. Balb/c mice received three intramuscular
injections (50 mg DNA/mouse) of the bhCG
construct, or a control plasmid, at fortnightly intervals. No anti-hCG
antibodies were evident in either group. When boosted with bhCG
adsorbed on alum, animals primed with the bhCG construct
responded by generating anti-hCG titres comparable to those produced by
conventional protein/alum only immunization. In contrast, animals primed with
the control plasmid did not respond to the protein booster. These observations
indicate that DNA vaccination, though not eliciting an overt antibody
response, induced immunological memory. Upon increasing the DNA dose to 100 mg/injection,
anti-hCG responses were observed after the second DNA injection. Long term follow-up Women immunized during clinical trials are being
followed to establish the long-term immunological safety of the approach.
Thirty eight subjects each have completed 9 and 10 years after vaccination.
Follow-up results available are not indicative of any residual effects of
immunization with the hCG vaccine. All biochemical and hematological
parameters are within normal ranges. Menstrual regularity and bleeding pattern
is comparable to matched controls or non-immunized women, indicating lack of
any long-term harmful consequences attributable to partial LH cross-reactivity
of antibodies. Publications Original peer-reviewed articles 1. Mandokhot A, Pal R, Nagpal S, Chauhan VS, Ahlawat S and Om Singh (2000) Humoral hyporesponsiveness to a conjugate contraceptive vaccine and its bypass by diverse carriers using permissible adjuvant. Clin Exp Immunol 122:101-108. 2. Gupta A, Pal R, Ahlawat S, Bhatia P and Om Singh (2001) Enhanced immunogenicity of a contraceptive vaccine using diverse synthetic carriers. Vaccine (in press). 3.
Pal R and Om Singh (2001) Absence of corpus luteum rescue by chorionic
gonadotropin in women immunized with a contraceptive vaccine. Fertil Steril
(in press). 4.
Gupta A, Chandrasekhar S, Pal R, Ahlawat S and Om Singh (2001) High expression
of human chorionic gonadotropin b-subunit using a synthetic vaccinia virus
promoter. J Mol Endocrinol (in press). 5.
Raghuvanshi RS, Om Singh and Panda AK (2001) Formulation and characterization
of immunoreactive TT biodegradable polymer particles. Drug Delivery (in
press). |