Towards designing an improved hCG vaccine

Project Associates / Assistants
Sarita Ahlawat (till Aug 2002)
Shaifuddin
Nimisha Suman (till Aug 2002)
Shailja Chhikara
Sarita Singh

Collaborators
Rahul Pal

Potentiation of the immune response by synthetic carriers and adjuvants is the theme of research. Clinical studies with a prototype hCG vaccine formulation demonstrated the general safety of the approach and feasibility of preventing pregnancy in women of proven fertility. Human chorionic gonadotropin (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. The objective of the study is to devise 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 antibody levels in most recipients.

We have previously reported that use of a combination(s) of pathogen derived broadly reactive Th peptides as carriers helped induce significantly elevated anti-hCG antibody responses in mice of different haplotypes as compared to those produced by protein carrier TT or DT. Initial results of the study extended in outbred Wistar rats confirmed the beneficial effects of the peptide based formulation; a mixture of Th peptides conjugates with bhCG enhanced anti-hCG response several-fold, clearly demonstrating the potential of Th peptides as alternate carriers for bhCG with permissible adjuvant. These animals (n=8) were followed up to study the duration of antibody responses. Rats were bled at regular intervals and anti-hCG antibody titers determined in individual sera. Kinetics of the antibody responses show that significantly elevated antibody titers were maintained for six months of immunization in the groups immunized with peptide based formulation. After six months, these animals were given a booster injection to test the immunological memory. The antibody responses were comparable to that obtained during primary immunization. Analysis of the anti-hCG antibody responses indicate that affinity of the antibodies for hCG remained of the same order (10¹⁰ M^-1) and bioneutarlization capacity was also a function of antibody titers. No anti-peptide reactivity was detectable in sera of animals immunised with the peptide-based formulations, confirming the non-B status of the peptides under the protocol employed.

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.

Preimmunization with a carrier protein may result in a diminished response to the ligand upon subsequent immunization with a ligand-carrier conjugate. This form of hyporesponsiveness has been attributed to the presence of anti-carrier antibodies, induction and activation of suppressor T cells, and antigenic competition between carrier and hapten specific B cells. We evaluated the effects of pre-sensitization with protein carrier on the anti-hCG immune response elicited by a cocktail of peptides. Groups of 8 mice of various haplotypes were immunized by giving three injections of alum adsorbed antigen at an interval of four weeks. Animals were bled at regular intervals and anti-hCG antibody titers determined in individual sera. An analysis of anti-hCG responses elicited revealed that prior immunity against TT/DT did not inhibit the subsequent response against bhCG-peptide conjugate, confirming our earlier observation in inbred mice that peptide-conjugate may not be susceptible to carrier induced immuno-suppression. Indeed, responses were higher when TT-sensitized animals were subsequently immunized with combination of TT peptide conjugates, indicating that prior natural exposure to the infectious agents from which peptides are derived may in fact prove be beneficial rather than detrimental. Based on these results, an advantage of choosing peptides from infectious agents for human immunizations can be envisaged; a significant percentage of the population may be naturally pre-sensitized to them.

The adjuvanticity of the compound AS2 was previously evaluated in inbred mice of different haplotypes and also in an outbred strain of mice. Having demonstrated consistently higher adjuvanticity of AS2 in inbred as well as outbred mice, the potential of the compound was evaluated in outbred Wistar rats. Groups of 8 female Wistar rats were given three injections at an interval of four weeks. Conjugate vaccine (ßhCG linked chemically with promiscuous helper T-cell peptides) was adsorbed on alum or mixed with a novel adjuvant AS2. Animals were bled at intervals and individual sera analyzed for hCG binding capacity. All the animals in the group immunized with AS2 responded with elevated anti-hCG antibody responses; mean titers were significantly higher than responses observed in the alum group. Results of the present experiment confirmed our earlier observation that inclusion of AS2 enhances the anti-hCG response significantly compared to that obtained with alum adsorbed immunogen. Antibodies were of high affinity for hCG (Ka»10 M^-1) and neutralized the hormonal bioactivity as function of the antibody titers. Overall, the enhancement is appears to be additive: antibody responses increase several-fold by carrier conjugation and inclusion of permissible adjuvants.

The immunization schedule followed in all experiments comprised of three injections. In order to investigate whether number of injection can be reduced with the improved formulation, groups of 8 rats each were given one, two or three injections at an interval of 4 weeks. Preliminary results indicate that while one injection schedule generated low response, 2-injection schedule may induce antibody responses comparable to 3-injection schedule.

In order to establish the long-term safety of the hCG vaccine approach, follow up of immunized women was continued. Sixty one women have completed more than ten years after immunization. Results obtained so far are not indicative 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.    Raghuvanshi RS, Singh O and Panda AK (2002) Correlation between in vitro release and in vivo immune response from biodegradable polymer particles entrapping tetanus toxoid. Drug Deliv 9:113-120.

2.    Raghuvanshi RS, Katare YK, Lalwani K, Ali MM, Singh O and Panda AK (2002) Improved immune response from biodegradable polymer particles entrapping tetanus toxoid by use of different immunization protocol and adjuvants. Int J Pharm 245:109-121.