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Towards designing an improved hCG vaccine |
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Principal Investigator : Om Singh Co-investigators Project
Associates/Assistants Potentiation of the
immune response by synthetic carriers and adjuvants is the theme of research. Immunization with a
vaccine eliciting antibodies against human chorionic gonadotropin (hCG) can
prevent pregnancy in women without any notable side-effects. HCG is considered
essential for the establishment of early pregnancy, and is also a potential
target for tumor immunotherapy. While all immunized women respond by making
anti-hCG antibodies, protective levels were however, not attained in some
individuals. Ongoing research efforts are focussed on the new strategies such
as use of Th-cell peptides as alternate carriers and inclusion of additional
adjuvant with an aim to design a vaccine formulation that can elicit and
maintain effective levels of antibody response in most recipients. The adjuvanticity of
the compound SBAS2 was previously evaluated in mice of different haplotypes.
Subsequently, it was found that the batch of Balb/c mice used was
contaminated. The experiment was therefore repeated in this strain. Groups of
10 mice each were given three injections at an interval of four weeks.
Conjugate vaccine, bhCG
linked chemically with promiscuous helper T-cell peptides, was adsorbed on
alum or mixed with a novel adjuvant SBAS2. Animals were bled at intervals and
individual sera analyzed for hCG binding capacity and for determination of IgG
subclass distribution of anti-hCG antibodies. Results of the present
experiment confirmed our earlier observation that inclusion of SBAS2 enhances
the anti-hCG response significantly compared to that obtained with alum
adsorbed immunogen. Anti-hCG antibody response in the group immunized with
alum based formulation was predominantly of IgG1 subclass, with a peak titer
of 522±85
(mean±SE) antibody units (AU). The IgG2 subclass showed considerably lower
mean levels (IgG2a, < 2; IgG2b, 12±2 AU). Immunization
with SBAS2 significantly enhanced the antibody response; hCG-specific IgG1
levels were 2087±178 AU, IgG2a 1082±196
AU and IgG2b 258±55 AU. Antibodies were of high
affinity for hCG (Ka»10 M-1)
and neutralized the hormonal bioactivity as function of the antibody titers. Having demonstrated
consistently higher adjuvanticity of SBAS2 in inbred mice, potential of the
compound was evaluated in an out-bred strain. Groups of 10 female CD4 mice
each received the peptide-conjugates adsorbed on alum or mixed with SBAS2. All
the animals in the group immunized with SBAS2 responded with elevated anti-hCG
antibody responses; mean titers were significantly higher than responses
observed in the alum group. The results indicate that SBSA2 potentiate the
antibody response not only in inbred animals but outbred population as well. We have earlier
demonstrated the enhanced immunogenicity of Th peptide based formulation in
only inbred strains of mice. The study was extended in out bred rats in order
to confirm the beneficial effects of the peptide based formulation. Wistar
rats in a group of 8 animals each were immunized by giving three injections of
alum adsorbed bhCG, bhCG conjugates with diverse Th
peptides or bhCG-DT conjugate. Animals were bled at regular intervals and anti-hCG
antibody titers determined in individual sera. bhCG
being ‘foreign’, rats do respond by producing anti-hCG antibodies, albeit
of low order, when it is injected after adsorption on alum. If, however, the
molecule is linked to Th peptides, anti-hCG response increases several-fold,
clearly demonstrating the potential of Th peptide as carrier for bhCG,
employing only alum as an adjuvant. Anti-hCG antibody titers generated in
animals immunized with Th peptide were significantly higher at all time points
than those achieved in the group immunized with protein carrier. Antibodies
have high affinity (Ka »1010
M-1) for binding with hCG, and have better hCG
neutralization index (neutralization capacity/binding capacity) than that
observed with antibodies generated in DT conjugated group. No anti-peptide
antibodies were detected in animals immunised with the peptide-based
formulations, confirming the non-B status of the peptides under the protocol
employed. Our studies
demonstrate that anti-hCG antibody response is of low order even in mice and
rats, where bhCG is a ‘foreign’ molecule. The responses increase several-fold
by carrier conjugation and inclusion of permissible adjuvants. Our
well-documented data obtained from series of clinical studies have clearly
established that improvement observed in animals is valid for humans as well. Follow up of
immunized women was continued to establish the long-term safety of the hCG
vaccine. Seventy women have completed more than nine years after immunization.
There is no indication of any residual effects of vaccination. All biochemical
and hematological parameters are within normal ranges. Menstrual and bleeding
pattern is comparable to age matched controls, indicating the lack of any
long-term harmful consequences attributable to LH cross-reactive antibodies. Publications Original
peer-reviewed articles 1.
*Gupta A, Pal R, Ahlawat S, Bhatia P and Om Singh (2001) Enhanced
immunogenicity of a contraceptive vaccine using diverse synthetic carriers. Vaccine
19:3384-3389 (*in press last year, since published). 2.
*Pal R and Om Singh (2001) Absence of corpus luteum rescue by chorionic
gonadotropin in women immunized with a contraceptive vaccine. Fertil Steril
76:332-336 (*in press last year, since published). 3.
*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 26:281-287 (*in press
last year, since published). 4.
*Raghuvanshi RS, Om Singh and Panda AK (2001) Formulation and
characterization of immunoreactive TT biodegradable polymer particles. Drug
Delivery 8:99-106 (*in press last year, since published). Patent 1.
Panda AK, Pal, R, Venkatesan N and Om Singh (2001) A process for
improving the immunogenicity of a weak antigen. Indian patent application
No.794/Del/2001, filed in Jul 2001. |