Mycobacterium indicus pranii is a potent immunomodulator for a recombinant vaccine against human chorionic gonadotropin

Journal of Reproductive Immunology 91 (2011) 24–30

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Mycobacterium indicus pranii is a potent immunomodulator for a recombinant vaccine against human chorionic gonadotropin Shilpi Purswani a , G.P. Talwar a,∗ , Richa Vohra a , Rahul Pal b , Amulya K. Panda b , Nirmal K. Lohiya c , Jagdish C. Gupta a a b c

Talwar Research Foundation, E-8 Neb valley, Neb Sarai, New Delhi-110 068, India National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur-302 004, India

a r t i c l e

i n f o

Article history: Received 28 April 2011 Received in revised form 2 June 2011 Accepted 16 June 2011

Keywords: Genetic strains of mice Antibody sub classes Adjuvant

a b s t r a c t The objective of this work was to identify a human use-permissible adjuvant to enhance significantly the antibody response to a recombinant anti-hCG vaccine. Previous Phase II efficacy trials in sexually active women have demonstrated the prevention of pregnancy at hCG bioneutralization titers of 50 ng/ml or more. Mycobacterium indicus pranii (MIP), a non-pathogenic Mycobacterium employed as an autoclaved suspension in aqueous buffer, significantly increased antibody titers in the FVB strain of mice. Three other genetic strains of mice: SJL, C3H, and C57Bl/6 responded with antibody titers several-fold higher than 50 ng/ml, which is the protective threshold in women, although there were differences in the peak titers attained. In addition, the duration of the antibody response was lengthened. The vaccine hCG␤-LTB, given together with MIP, induces both a Th1 and Th2 response, which is reflected in the production of not only IgG1, but also a high proportion of IgG2a and IgG2b antibodies. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Human chorionic gonadotropin (hCG) is produced by the early embryo as observed and reported for the first time by Robert Edwards and colleagues in their seminal work on assisted reproductive technology, which led to the production of the first test tube baby in the world (Fishel et al., 1984). It plays a critical role in the implantation of the embryo and thereby in the onset and establishment of pregnancy. Based on this history, a vaccine was developed against hCG that could act by interception of implantation without interfering with the normal hypothalamus–pituitary–gonad axis of the woman. Indeed, pregnancy was prevented in sexually active women

∗ Corresponding author at: Talwar Research Foundation, E-8, Neb Valley, Neb Sarai, New Delhi-110 068, India. E-mail address: [email protected] (G.P. Talwar). 0165-0378/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2011.06.099

without impairment of ovulation and disturbance of menstrual regularity and normal bleeding profiles. Phase II efficacy trials, the first ever carried out with an anti-hCG vaccine, the HSD-TT, provided proof of concept and also indicated a requirement for bioneutralization titers of at least 50 ng/ml to achieve efficacy (Talwar et al., 1994). The vaccine employed in these trials generated titers above the protective threshold in only 60% of women, which was not adequate for a practical method of fertility regulation, given that alternatives available are effective in more than 90% of users. Hence there is a need to make a more immunogenic vaccine. We recently developed a recombinant anti-hCG vaccine that generated an antibody response in BALB/c mice (Talwar et al., 2009). Although the titers with the vaccine adsorbed on alum were above 50 ng/ml in every mouse, the duration of the antibody titers above 50 ng/ml was only 30 days. This indicated the need to employ an adjuvant in addition to alum. A non-pathogenic mycobacteria

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coded as Mw, now named Mycobacterium indicus pranii (MIP) (Saini et al., 2009) was developed as an immunomodulator for treatment of multibacillary lepromatous leprosy patients (reviewed by Talwar et al., 1999). It has undergone large-scale field trials in patients and healthy members of their household, and is approved for human use by the Drugs Controller General of India and by the US FDA in the orphan vaccine category. MIP was evaluated for its ability to enhance antibody titers with the recombinant vaccine. In BALB/c mice, the peak titers and duration of antibody response was significantly increased by inclusion of MIP (Purswani and Talwar, 2011). As the immune response varies with the genetic make-up of the recipient, it was appropriate to investigate whether this vaccine would also be equally immunogenic in mice of other genetic strains. Also, the earlier study did not provide formal evidence on its adjuvant property of enhancing anti-hCG titers. In this study we report on the response in FVB mice immunized with the vaccine, with and without addition of MIP. Data on the isotypes of antibodies generated by the vaccine + MIP are given, which are of interest in regard to potential use of the vaccine for treatment of hCG-expressing tumors. A number of papers have appeared reporting the ectopic expression of hCG or its subunits by a variety of cancers at advanced stages (Iles, 2007; Talwar et al., 2011). Such cancers are invariably refractory to available drugs and have poor prognosis and adverse survival (Iles et al., 1996; Hotakainen et al., 2002).

light/12 h dark cycle in the NII animal facility with free access to normal rodent chow and water. All procedures were conducted according to protocols and guidelines approved by the Institutional Ethics Committee. All animals were cared for by dedicated trained animal technicians. Animals were monitored daily for health and welfare.

2. Materials and methods

2.6. Bioneutralization capacity

2.1. Vaccine hCGˇ-LTB

The hCG bioneutralization capacity of antibodies was estimated as a function of the ability of sera to inhibit the binding of 125 I-hCG to the testicular receptors as described (Pal et al., 1990). Briefly, rat testicular homogenate was incubated with different dilutions of antiserum and 125 IhCG for 2 h at 37 ◦ C. The reaction was terminated by addition of cold Tris–HCl buffer pH 7.4 (50 mM Tris, 5 mM magnesium chloride, 0.1% BSA, 0.1% sodium azide). Tubes were centrifuged at 2000 × g and the pellets counted for radioactivity. Neutralization capacity was calculated by regression analysis at the 50% inhibition point and expressed as ng/ml of serum.

The vaccine was made and purified as described elsewhere (Purswani and Talwar, 2011). Its purity was checked by SDS–PAGE and Western blot analysis where it migrated as a single band of 43 kDa. On the mass spectrometer, it gave a main peak at 42,652 Da and a minor at 21,321 Da. The vaccine was adsorbed on Alhydrogel as described previously. 2.2. Mycobacterium indicus pranii (MIP, formerly Mw) The MIP was maintained on Lowenstein–Jensen medium (LJ) slants (BD Difco) and kept at −70 ◦ C. It was grown in Middlebrook 7H9 medium (BD Difco) with 0.02% glycerol, 0.05% Tween 80, and 10% albumin–dextrose complex enrichment (BD Difco) as a shake flask culture. Bacterial cells were harvested in the log growth phase by centrifugation at 840 × g for 15 min, washed twice by the centrifugal washing method, and suspended in saline at the desired concentration for immunization. The bacterial cells were inactivated by autoclaving for 20 min at a pressure of 15 lb/in.2 . 2.3. Inbred mice of different genetic background Four defined strains of mice FVB, C57Bl/6, SJL and C3H, were bred at the Animal House Facility of the National Institute of Immunology (NII), New Delhi, India. Mice were housed under controlled temperature conditions and a 12 h

2.4. Immunization The antigen employed was hCG␤ subunit fused with B subunit of heat labile enterotoxin of Escherichia coli (hCG␤-LTB). The cloning, purification, and characterization of this antigen is described elsewhere (Purswani and Talwar, 2011). hCG␤-LTB (2 ␮g) adsorbed on Alhydrogel was injected intramuscularly to mice of different strains. Autoclaved MIP (Fig. 2) suspended in saline at 0.5 × 107 cells, was given intramuscularly at the contralateral site. Primary immunization of each mouse consisted of three injections given at fortnightly intervals. Boosters were given as per need on the days indicated in Section 3. 2.5. Sera collection Blood was collected by retro-orbital puncture, at various times. The serum separated was diluted 10 times in phosphate buffered saline (100 mM PBS, pH 7.4) and stored at −70 ◦ C.

2.7. Antibody isotypes Antibody isotypes were determined by using isotyping kit (BD Biosciences, USA) as per the procedure described by the manufacturer. Briefly, 100 ␮l of antibody was coated onto wells in a 96-well, flat bottomed microtitration plate and 100 ␮l of pooled sera at dilutions of 1:103 , 1:104 , 1:105 , and 1:106 was then added, along with the provided positive control. After incubation for 1 h at 4 ◦ C, the diluted sera were removed and wells were washed three times with 100 mM PBS containing 0.5% Tween-20 (PBST). 100 ␮l of horse radish peroxidase (HRP)-labeled antibodies, provided in the kit, were added to each well. After washing three times, 100 ␮l of substrate was added to each well and the plate was incubated at room temperature for 3–10 min. The reaction was stopped by adding 50 ␮l of 5 N H2 SO4 solution to each well, and the OD was

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Table 1 Antibody response to hCG␤-LTB adsorbed on alum in FVB mice. Time (days)

15 30 37 52 67 82

Mice number

Geometric mean

1

2

3

4

5

6

7

8

0 0 34 96 322 77

0 0 23 123 151 105

0 0 50 134 366 131

0 0 0 197 276 106

0 0 0 134 380 61

0 0 0 105 376 97

0 0 54 125 299 43

0 0 69 215 256 85

measured at 450 nm. Antibody concentrations were chosen that gave the most sensitivity and the greatest signal difference between pre-immune and vaccinated sera. The ratios of various subclasses in the sera were calculated from the ratios of the OD values.

0 0 12 124 244 84

protective threshold for preventing pregnancy in women (Talwar et al., 1994), whereas those receiving the vaccine without MIP not only have lower titers, but three of them have no measurable antibodies and only two out of eight have titers above 50 ng/ml (Table 1). The antibody titers in the two groups keep rising until day 67, and start declining thereafter. On day 97 of the initiation of immunization, only two mice in the vaccine minus MIP group have titers above 50 ng/ml, whereas eight out of eight mice in the vaccine plus MIP group have titers well over 50 ng/ml (Table 2). Furthermore, the peak titers attained in FVB mice with vaccine plus MIP were 3952 ng/ml in one mouse and 2080 ng/ml in two mice, whereas without MIP, the vaccine generated up to 380, 376, 366, and 322 ng/ml as peak titers in four of the eight mice. There was thus a clear advantage of the inclusion of MIP as an adjuvant in the vaccine, even though the vaccine given on alum alone was immunogenic in all FVB strains of mice. An electron micrograph of MIP employed in these studies is given in Fig. 2.

3. Results In an earlier communication (Purswani and Talwar, 2011), the immunogenicity of the hCG␤-LTB vaccine was reported in BALB/c mice. Realizing that the immune response varies with the genetic constitution, studies were extended to four other genetic strains of mice, namely FVB, SJL, C3H and C57Bl/6. Experiments were also performed to determine the adjuvant activity of MIP. 3.1. Potentiation of antibody response by MIP Tables 1 and 2 give the bioneutralization titers of the antibodies generated in FVB mice immunized with hCG␤LTB with and without MIP. The hCG␤-LTB vaccine was a purified preparation meeting quality control criteria described previously, namely a single band of ∼43 kDa in SDS–PAGE reacting with a conformation-reading monoclonal antibody in Western blot analysis, a peak of 42,652 Da on mass spectrometry and the presence of ␤sheets as a single negative band around 205–207 nm as seen by circular dichroism. It was adsorbed on Alhydrogel. The hCG␤-LTB vaccine (2 ␮g) with or without MIP was given intramuscularly on days 0, 15, and 30. The antibody titers in bleeds of the eight mice used in each group are given on various days following primary immunization. These are expressed on the basis of their capacity for inactivation of hCG bioactivity. It is evident that on day 37, a week after completion of the primary immunization, all mice receiving the vaccine plus MIP have antibody titers well over 50 ng/ml, which was established earlier as the

3.2. Isotypes of antibodies induced by hCGˇ-LTB plus MIP Immunization with the vaccine adsorbed on Alhydrogel generated primarily an IgG1 response, the IgG1 to IgG2a and IgG2b ratios were 1.5 and 1.75. On the other hand immunization with the same vaccine plus MIP led to a higher proportion of IgG2a and IgG2b isotype antibodies than of IgG1; the IgG1/IgG2a and IgG1/IgG2b ratios being 0.89 and 0.98 respectively (Fig. 1). Therefore while MIP sustained IgG1 production, it increased the IgG2 response. IgG3 antibodies, which in humans have the highest affinity for complement activation and for binding to Fc receptors on phagocytic cells, were present in both cases in nearly equivalent proportions. In both cases, IgA antibodies were present in the serum, although the proportion of IgA to IgG was greater in mice receiving MIP (Fig. 1E).

Table 2 Effect of inclusion of MIP with the vaccine adsorbed on alum. Time (days)

Mice number 1

15 30 37 52 67 82 97

0 0 624 3536 3952 2328 2134

Geometric mean 2 0 0 468 1664 2080 1552 582

3 0 0 1456 2080 832 873 427

4 0 0 520 218 260 272 78

5

6

7

8

0 0 260 437 312 194 204

0 0 437 146 104 213 136

0 0 208 291 270 194 650

0 0 270 333 416 340 136

0 0 440 599 533 469 317

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Fig. 1. Isotypes of immunoglobulins generated in FVB mice by the vaccine ± MIP.

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Fig. 2. Electron micrograph of autoclaved Mycobacterium indicus pranii (MIP).

3.3. Safety studies The site at which intramuscular immunization was done with the vaccine with or without MIP in FVB mice showed no visible induration or erythema. It caused no apparent discomfort to the immunized mice, who remained alert. Their food intake as reflected by the body weight gain was nearly equivalent to that of the control animals and those immunized with Alhydrogel alone. 3.4. Immune response in mice of various other genetic strains The antibody response in BALB/c mice has been reported previously (Purswani and Talwar, 2011). In FVB mice, a very similar pattern of high antibody response was obtained with vaccine plus MIP, with titers ranging up to nearly 2000–3950 ng/ml (Table 2). In three other strains, namely SJL, C3H, and C57Bl/6, immunization with hCG␤-LTB plus MIP generated anti-hCG bio-neutralizing antibodies. Fig. 3 summarizes these results. All C3H mice made antibodies well above 50 ng/ml in bleeds on days 67, 82, and 97 after primary immunization. However, some but not all mice of SJL and C57Bl/6 had a response above 50 ng/ml following primary immunization. After a single booster, on day 127 all strains of mice namely SJL, C3H, and C57Bl/6, developed high titers of hCG bio-neutralizing antibodies and these were manifest in every mouse of each strain. Antibody titers during the six-month observation period were well above 50 ng/ml in every mouse except one C3H mouse in which the titers were marginal. These studies confirm the notion of variability of immune response in mice of different haplotypes. However three of the five strains generated 50 ng/ml titers after primary immunization and all mice of all five strains investigated, developed antibodies well above 50 ng/ml, after a booster following primary immunization. These findings

Fig. 3. hCG bioneutralization capacity (ng/ml) of antibodies generated in mice of haplotypes SJL, C3H, and C57Bl/6. All mice were immunized with hCG␤-LTB adsorbed on Alhydrogel + MIP as an adjuvant. Geometric mean of hCG binding capacity is represented by the bars and the symbols represent the individual animal titers.

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suggest that for any clinical trial to evaluate hCG␤-LTB plus MIP, it would be desirable to include a booster injection after the first month of three injections for primary immunization. 4. Discussion Initial studies (Purswani and Talwar, 2011) conducted with a recombinant vaccine hCG␤-LTB in BALB/c mice were encouraging and showed that all mice respond to this vaccine adsorbed on alum and given with M. indicus pranii (MIP) as an immuno-modulator. High titers of hCGbioneutralizing antibodies were present in every mouse and the antibody response was of respectable duration. During the eight months of the observation period, the antibody bioneutralization titers ranged up to 6600 ng/ml in the immunized mice with one booster after the first month of three primary immunizations. It was imperative to enquire whether the vaccine would be equally immunogenic in recipients of different genetic make-up. Studies were therefore carried out in mice of four other haplotypes namely FVB, SJL, C3H, and C57Bl/6. FVB mice show that high titers up to 3952 ng were elicited by the vaccine given along with MIP after the primary immunization of three injections. All C3H mice had titers well above 50 ng/ml after primary immunization with titers ranging from 125 to 1150 ng/ml. The response in SJL and C57Bl/6 mice was comparatively low. However, one booster injection after primary immunization caused every mouse of these strains to generate bioeffective titers ranging up to 1500 ng/ml. Extrapolation of these findings to eventual clinical trials would imply that primary immunization within the first month might generate protective threshold titers in some recipients, but a booster injection would be advisable for the low responders. The vaccine adsorbed on alum alone without MIP would not be optimally immunogenic (Talwar et al., 2009). An aqueous suspension of autoclaved M. indicus pranii (MIP) needs to be co-administered for evoking high antibody titers. Our data provide substantial evidence in support of the potency of MIP in enhancing the antibody titers. M. indicus pranii was discovered by Talwar et al. (1978, 1999) as an immunomodulator to evoke immune responsiveness in multibacillary lepromatous leprosy patients who are non-responsive to M. leprae. It is approved for human use by the Drugs Controller General of India and also by the USFDA. Additionally it is now being used as an adjunct to the standard multidrug regime in the treatment of category II tuberculosis (Patel and Trapathi, 2003; Nyasulu, 2010). Its inclusion expedites recovery and diminishes relapses. It is effective in the dead as well as live state and can evoke protective anti-tuberculosis response in experimental animals (Gupta et al., 2009). Injections of MIP (formerly Mw) into lesions caused by ugly ano-genital warts resulted in dramatic healing of the lesions (Gupta et al., 2008). Another interesting effect of MIP is in prevention and therapy of SP2 O myelomas in mice (Rakshit et al., 2011). The manner in which MIP exercises its various actions is only partially understood at present. It enhances IL12 and interferon-␥ synthesis as described by Rakshit

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et al. (2011). It has a stimulatory effect on both Th1 and Th2 responses. hCG␤-LTB was developed as a recombinant vaccine (Talwar et al., 2009) for more than one reason. It ensures a consistent covalent linkage of the carrier LTB to the ␤ subunit of hCG at the carboxy terminal amino acid, in contrast to the inherent variations of the amino/carboxylic groups to which carrier is linked chemically by bifunctional compounds in the vaccines made by us previously (Talwar et al., 1976, 1988). It is amenable to large-scale industrial production and the cost is expected to be substantially lower than the existing anti-hCG vaccines. The decision to retain hCG␤ as an antigen linked to the carrier LTB in the present vaccine instead of the carboxy terminal peptide (CTP) of hCG␤ was taken on the grounds that the CTP is a relatively poor antigen and elicits low affinity antibodies for binding with hCG. No efficacy of CTP-induced antibodies to prevent pregnancy has yet been demonstrated in spite of using strong oily adjuvants. hCG␤ no doubt elicits antibodies that are partially cross-reactive with hLH, but the degree of crossreactivity still leaves enough hLH in the mid-cycle surge to sustain ovulation, as repeatedly observed not only in Phase I clinical trials conducted in India (Kharat et al., 1990; Talwar et al., 1990) but also by a trial conducted by the Population Council in Finland, Sweden, Chile, and Brazil (Nash et al., 1980). Data from Phase II efficacy trials again confirms the maintenance of ovulation with regular cycles in sexually active women (Talwar et al., 1994). The ability of the circulating anti-hCG antibodies to prevent pregnancy in sexually active women was established in previous Phase II trials (Talwar et al., 1994). Also established is the antibody titer (50 ng/ml) at which protection is achieved. The short-coming of the vaccine used earlier was that it generated above 50 ng/ml titers in only 60% of women. The present vaccine along with MIP as an adjuvant engenders several-fold higher antibody titers in all five genetic strains of mice investigated, which indicates its likely suitability for clinical trial as a vaccine for control of fertility. A distinct advantage of preventing pregnancy by intercepting implantation with anti-hCG antibodies is the non-impairment of ovulation and non-disturbance of menstrual regularity. This vaccine is also likely to be useful for therapy of advanced-stage cancers with ectopic expression of hCG/subunits. Three vaccines, two in the USA (He et al., 2004; http://www.cancerbacteria.com/trial.html) and one in UK (http://www.dailymail.co.uk/health/article1293927/jab-halt-deadly-forms-cancer.html), are already used in trials for such cancers, with apparently encouraging results (Talwar et al., 2011). The high immunogenicity of the hCG␤-LTB vaccine, along with the extra properties of the exceptional adjuvant MIP, should add to the armamentarium of treatments for these ‘difficult to treat’ terminal cancers. Acknowledgements The development of this anti-hCG vaccine was supported by a DBT grant under Indo-US co-operation

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program. The Indian Council of Medical Research is currently supporting the production of this vaccine under GMP/GLP conditions and toxicology studies leading to the resumption of clinical trials. References Fishel, S.B., Edwards, R.G., Evans, C.J., 1984. Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science 223, 816–818. Gupta, S., Malhotra, A.K., Verma, K.K., Sharma, V.K., 2008. Intralesional immunotherapy with killed Mycobacterium w vaccine for the treatment of ano-genital warts: an open label pilot study. J. Eur. Acad. Dermatol. Venereol. 22, 1089–1093. Gupta, A., Geetha, N., Mani, J., Upadhyay, P., Katoch, V.M., Natrajan, M., Gupta, U.D., Bhaskar, S., 2009. Immunogenicity and protective efficacy of M.w against M.tb in mice immunized with live versus heat-killed Mw by the aerosol or parenteral route. Infect. Immun. 77, 223– 231. He, L.Z., Ramakrishna, V., Connolly, J.E., Wang, X.T., Smith, P.A., Jones, C.L., Valkova-Valchanova, M., Arunakumari, A., Treml, J.F., Goldstein, J., Wallace, P.K., Keler, T., Endres, M.J., 2004. A novel human cancer vaccine elicits cellular responses to the tumor-associated antigen human chorionic gonadotropin ␤. Clin. Cancer Res. 10, 1920–1927. Hotakainen, K., Ljungberg, B., Paju, A., Rasmuson, T., Alfthan, H., Stenman, U.H., 2002. The free beta-subunit of human chorionic gonadotropin as a prognostic factor in renal cell carcinoma. Br. J. Cancer 86, 185–189. Iles, R.K., 2007. Ectopic hCGbeta expression by epithelial cancer: malignant behaviour, metastasis and inhibition of tumor cell apoptosis. Mol. Cell. Endocrinol., 260–262, 264–270. Iles, R.K., Persad, R., Trivedi, M., Sharma, K.B., Dickinson, A., Smith, P., et al., 1996. Urinary concentration of human chorionic gonadotropin and its fragments as a prognostic marker in bladder cancer. Br. J. Urol. 77, 61–69. Kharat, I., Nair, N.S., Dhall, K., Sawhney, H., Krishna, U., Shahani, S.M., Banerjee, A.K., Roy, S., Hingorani, V., Singh, O., Talwar, G.P., 1990. Analysis of menstrual records of women immunized with anti-hCG vaccine inducing antibodies partly cross-reactive with hLH. Contraception 41, 293–299. Nash, H., Talwar, G.P., Segal, S., Luukkainen, T., Johannson, E.D.B., Vasquez, J., Coutinho, E., Sundaram, K., 1980. Observation on the antigenicity and clinical effects of a candidate antipregnancy vaccine: B-subunit of human chorionic gonadotropin linked to tetanus toxoid. Fertil. Steril. 34, 328–335. Nyasulu, P.S., 2010. The role of adjunctive Mycobacterium w immunotherapy for tuberculosis. J. Exp. Clin. Med. 2 (3), 124–129.

Pal, R., Singh, O., Rao, L.V., Talwar, G.P., 1990. Bioneutralization capacity of the antibodies generated in women by the beta subunit of human chorionic gonadotropin (beta hCG) and beta hCG associated with the alpha subunit of ovine luteinizing hormone linked to carriers. Am. J. Reprod. Immunol. 22, 124–126. Patel, N., Trapathi, S.B., 2003. Improved cure rates in pulmonary tuberculosis category II (retreatment) with Mycobacterium w. J. Indian Med. Assoc. 101, 680–682. Purswani, S., Talwar, G.P., 2011. Development of a highly immunogenic recombinant candidate vaccine against human chorionic gonadotropin. Vaccine 29, 2341–2348. Rakshit, S., Ponnusamy, M., Papanna, S., Saha, B., Ahmed, A., Nandi, D., 2011. Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: critical roles of IFN␥. Int. J. Cancer, doi:10.1002/ijc.26099. Saini, V., Raghuvanshi, S., Talwar, G.P., Ahmed, N., Khurana, J.P., Hasnain, S.E., Tyagi, A.K., Tyagi, A.K., 2009. Polyphasic taxonomic analysis establishes Mycobacterium indicus pranii as a distinct species. PLoS One 4 (7), e6263. Talwar, G.P., 1999. An immunotherapeutic vaccine for multibacillary leprosy. Int. Rev. Immunol. 18, 229–249. Talwar, G.P., Sharma, N.C., Dubey, S.K., Salahuddin, M., Das, C., Ramakrishnan, S., Kumar, S., Hingorani, V., 1976. Isoimmunisation against human chorionic gonadotropin with conjugates of processed beta subunit of the hormone and tetanus toxoid. Proc. Natl. Acad. Sci. U.S.A. 73, 218–222. Talwar, G.P., et al., 1978. 14 Papers in Golden Jubilee Issue Leprosy in India, vol. 50 , pp. 492–597. Talwar, G.P., Singh, O., Rao, L.V., 1988. An improved immunogen for anti hCG vaccine eliciting antibodies reactive with a conformation native to the hormone without cross reaction with human follicle stimulating hormone. J. Reprod. Immunol. 14, 203–212. Talwar, G.P., Hingorani, V., Kumar, S., Roy, S., Banerjee, A.K., Shahani, S.M., Krishna, U., Dhall, K., Sawhney, H., Sharma, N.C., Singh, O., Gaur, A., Rao, L.V., Aruman, K., 1990. Phase I clinical trials with three formulations of anti-human chorionic gonadotropin vaccine. Contraception 41, 301–316. Talwar, G.P., Singh, O., Pal, R., Chatterjee, N., Sahai, P., Dhall, K., et al., 1994. A vaccine that prevents pregnancy in women. Proc. Natl. Acad. Sci. U.S.A. 91, 8532–8536. Talwar, G.P., Vyas, H.K., Purswani, S., Gupta, J.C., 2009. Gonadotropinreleasing hormone/human chorionic gonadotropin ␤ based recombinant antibodies and vaccines. J. Reprod. Immunol. 83, 158–163. Talwar, G.P., Gupta, J.C., Shankar, N.V., 2011. Immunological approaches against human chorionic gonadotropin for preventing unwanted pregnancy and therapy of advanced stage cancers expressing hCG/subunits. Am. J. Reprod. Immunol. 66, 26–39.