The HSD vaccine was given with alum as adjuvant. The dose and the adjuvant, at which it was given, generated above protective threshold of antibodies in 60–80 % of recipients. This order of efficacy may be acceptable for vaccines against infectious diseases but as other contraceptives protecting up to 98–99 % of recipients are available, this order of efficacy is not sufficient to make it eligible as an option in the family planning basket.

Research on hCG vaccine was revived under an Indo-US program in 2006. the previous HSD vaccine had hormonal subunits purified from natural sources (hence costly), linked chemically to carrier (DT/TT). The efficiency of conjugation, besides the uncertainties of the numbers and the position at which carrier was conjugated, lowered the yield and created nonhomogeneity of the product from batch to batch. To overcome these limitations, we decided to make a fully recombinant vaccine. hCGβ was cloned and expressed in Pichia pastoris to obtain a glycosylated hormonal subunit linked in a defined position to LTB, the B subunit of heat labile enterotoxin of E. coli (Fig. 19.3). The yield was good and the procedure amenable to industrial production. This recombinant conjugate has been tested for immunogenicity in Balb/C mice, and every mouse immunized so far with this vaccine given on alum with SPLPS (sodium pthylylated derivative of lipopolysaccharide of Salmonella typhi) in the first injection has generated far above 50 ng/ml antibody titers [16]. We also extended the studies to five inbred strains of mice of different genetic background, encompassing haplotypes H-2^d, H-2^k, H-2^b, H-2^s, H-2^q, in order to ascertain the genetic restriction, if any, of antibody response to this vaccine. Mice of all strains generated antibodies in response to this vaccine [17].

DNA vaccines are not only cheaper to make but also thermostable without requiring cold chain. Therefore, recombinant hCGb-LTB vaccine as DNA, in addition to proteinic form of the vaccine expressed in yeast Pichia pastoris, was made. Priming with the DNA form of the recombinant hCGβ-LTB vaccine twice at fortnightly interval followed by boosting with the proteinic form of the vaccine induced distinctly higher antibody response, showing synergy in the actions of DNA and protein versions of recombinant hCGβ-LTB vaccine [18].

All immunized and control animals were then cohabitated with adult fertile male marmosets for 2 weeks per cycle and their fertility was tested for 6 months. Except for one animal, in 80 μg dose group, none of the immunized animals became pregnant, whereas all animals in the control group conceived. After 6 months, with no booster injections given, all immunized animals regained fertility following the decline of antibody titers, indicating thereby that the circulating anti-hCG antibodies were indeed responsible for preventing them from becoming pregnant.

Preclinical toxicology studies were also conducted in rodents as per the regulatory requirements before heading for the clinical trials in humans. These studies were carried out by M/s Bioneeds at their GLP Facility in Bangalore, India, based on biosafety issues related to Genetically Modified Organisms, Schedule “Y” guidelines on Drugs and Cosmetics, and guidelines of Institutional Animal Ethics Committees (IAEC).

It was observed that both DNA and protein vaccines were devoid of sensitizing the skin of guinea pigs. The two forms of hCGβ-LTB vaccine were nonmutagenic at the highest concentration tested both in Bacterial Reverse Mutation and Mammalian Chromosome Aberration Tests. Similar nonmutagenicity observations were made in vivo Mammalian Erythrocyte Micronucleus Test conducted in mice. Single-dose acute toxicity study conducted in Sprague Dawley rats demonstrated the safety of the vaccine. Segment II studies conducted in rats showed that vaccines did not affect the embryo-fetal development, body weight, and food consumption.

Thus, extensive toxicology studies on the hCGβ-LTB vaccine in two species of rodents and a subhuman primate species the marmosets have shown the total safety of the recombinant hCGβ-LTB vaccine. These are now ready to go to clinical trials. In fact, the technology has been transferred to Bharat Biotech, a company in Hyderabad, which will make available the vaccine produced under GMP conditions for the clinical trial. A clinical trial protocol has been developed, and the Indian Council of Medical research is awaiting approval from the Regulatory Authorities to initiate the clinical trials.

Immunization against hCG has no doubt applications for preventing pregnancy. A number of recent papers report ectopic expression of hCG or subunits in a variety of cancers: lung [19], bladder [20], colon [21], gastric [22], pancreatic [23], breast [24], cervical [25], oral [26], head and neck [27], vulva/vaginal [28, 29], prostate [30], and renal cancers [31]. It has been further observed that patients with cancers expressing hCGβ ectopically have poor prognosis and adverse survival [20, 31]. It follows that a vaccine against hCG or recombinant antibodies against hCG may have additional applications for therapy of such cancers.

We engineered a recombinant chimeric antibody of high affinity and specificity [32, 33]. This antibody, cPiPP, bound to T-lymphoblastic leukemia MOLT-4 cells expressing hCG, whereas it had no binding with peripheral blood lymphocytes (PBLs) of normal healthy subjects [34]. Vyas et al. [35] observed that this antibody linked to curcumin, a safe anticancerous compound, killed 100 % of MOLT-4 cells as well as histocytic lymphomas U937 cells, both expressing ectopically hCG. On the other hand, the immuno-conjugate had no deleterious effect on PBLs of healthy subjects.

Besides hCG, the only other vaccine which has undergone clinical trial was directed against LHRH. It was a semisynthetic vaccine in which glycine at position 6 was replaced by D-lysine which created a functional NH₂ group for linking the carrier either TT or DT [36]. The vaccine was highly immunogenic and induced bioeffective response in rodents [37] and in monkeys [38] with alum alone as adjuvant. Immunization caused cessation of spermatogenesis in rodents along with testosterone declining to castration levels [39]. Spermatogenesis and fertility was regained on decline of antibodies. Immunization against LHRH could also block fertility of female rodents in a reversible manner [40].

In male rats and in monkeys, a drastic atrophy of prostate was observed. The vaccine inhibited the growth of Dunning R3327-PAP tumor implanted in rats by suppression of cell division [41]. Preclinical toxicology studies in India and Austria indicated the safety of semisynthetic vaccine LHRH vaccine. With permission of the Drugs Regulatory Authority in India and Austria and with the approval of the Institutional Ethics Committees, Phase I/II clinical trials were conducted with this vaccine in 28 patients of carcinoma of prostate, 12 patients each at the All India Institute of Medical Sciences (AIIMS) New Delhi, and Postgraduate Institute of Medical Education and Research (PGI) Chandigarh, and four patients at the Urology Department of Salzburg General Hospital. The vaccine was well tolerated in all subjects. No ill effect of immunization was seen. Subjects developing more than 200 pg/ml of antibodies experienced a decline of testosterone to castration levels with marked reduction of PSA. Patients receiving 400 μg LHRH equivalent dose showed better clinical improvement than those receiving 200 μg. Table 19.1 is a summary of observations on 12 patients of carcinoma of prostate on whom trial was conducted by Prof. S. Wadhwa, at AIIMS. Serial nephrostograms of a patient at PGIMER, Chandigarh, showed clearance of prostatic tissue mass [42] (Fig. 19.4).