Prologue

Any worthwhile advance in Biotechnology has to reach people via industry so as to benefit the users. Described below are a few products which have resulted from our work.

A Unique Immunoprophylactic Cum Immune-Therapeutic Vaccine Against Leprosy Endowed with Multiple Additional Applications of High Utility

We discovered a saprophytic, cultivable mycobacteria, which shares antigens with both M. leprae and M.tuberculosis. This mycobacterium coded as Mw is now defined by its gene sequence [1-3]. Being a hitherto new micro-organism, previously unregistered in International Depository, it has been named as Mycobacterium indicuspranii (MIP). Pran is my familiar name and NII, the National Institute of Immunology where bulk of research was done on it under my leadership. (Figure 1) gives the electron micrograph and atomic force microscopic vision of MIP.

MIP as adjunct to standard multi-drug regime expedites bacterial clearance and shortens the period of recovery of leprosy patients [4]. It converts upto 68% of Lepromatous Leprosy (LL) patients from lepromin negativity to lepromin positivity status. LL patients lack immunity to respond to M. leprae antigens and remain lepromin negative even after clearance of bacilli by drugs. Drugs kill bacteria but do not improve the immuno-reactivity of LL patients to M. leprae antigens. MIP has thus this unique ability to not only clear bacilli, but also rectify to a large extent the immune deficit in persons lacking it completely.

Another notable feature of using MIP along with drugs is to attain complete recovery without residual blemishes. (Figure 2) shows few patients treated with MIP in addition to drugs. 

MIP has received the approval of the Drugs Controller General of India (DCGI) and also of USFDA. It is at present the only vaccine of its type in the world. It has been passed on to industry. M/s Cadilla Pharma Ahmedabad, manufacture and make it available to public.

Additional Applications of MIP

Tuberculosis

Guinea pigs immunized with MIP do not fall victim to H₃₇R_V virulent strain of tuberculosis. MIP has been employed with good results for treatment of category II “Difficult to treat” tuberculosis patients. (Table 1) gives the results of one of these trials.

Furthermore, the relapse rate of patients treated with MIP, in addition to standard drugs, is far lower than those treated with drugs alone (Figure 3). 

MIP has several advantages over BCG for prophylactic immunization. It is active in both live and killed form, whereas BCG is inactive in non-live state.

BCG is protective in some countries, such as Uganda, but not in others, such as India. There appears to be genetic restriction on its immunization capability. (Figure 4) shows that BALB/c and C57BL/6 strains of mice are protected by both BCG and MIP, whereas C3H and CBA strains of mice immunized with BCG do not resist challenge with H₃₇R_V,but all mice immunized with MIP are protected [5].

MIP: A Potent Adjuvant

We are in process to develop a Birth Control Vaccine, directed against Human Chorionic Gonadotropin (hCG). Human Chorionic Gonadotropin (hCG) is not made by any organ of non-pregnant non-cancerous woman. That is why its appearance in urine /blood is a reliable test for diagnosis of pregnancy. It is the early embryo following fertilization of the egg that starts making HCG as observed by Bob Edwards [6]. Human Chorionic Gonadotropin (hCG) plays an important role in implantation of the embryo onto the endometrium resulting in onset of pregnancy. Antibodies binding with hCG prevent this process, hence prevent pregnancy. These do not interfere with normal reproductive functions of the woman. She ovulates normally and makes her sex hormones normally. Regularity of menstrual cycles is maintained with normal bleeding profiles. Vaccines against hCG made by us earlier have undergone Phase I/Phase II clinical trials in India and also in Finland, Sweden, Chile and Brazil, which have shown the safety and reversibility of the vaccine [7]. Antibodies above 50 ng/ml titre prevent pregnancy without derangement of menstrual regularity [8]. 

We have now developed a recombinant vaccine against hCG, which is amenable to industrial production. This vaccine, hCGβ-LTB, is fairly immunogenic in mice. MIP has been used as adjuvant in this vaccine. As shown in (Figure 5), inclusion of MIP as adjuvant enhances by several folds antibody titres against hCG [9].

Preventive and Therapeutic Action of MIP on Myelomas

Prof. Dipankar Nandi at the Indian Institute of Science, Bangalore has published highly interesting properties of MIP in curtailing the development of Myelomas caused by SP2/o cells in BALB/c mice. (Figure 6) is a summary of his findings reported elsewhere [10].

Healing of Ugly Warts on Ano-Genital Region and Feet

Prof. Somesh Gupta at the All India Institute of Medical Sciences, New Delhi employs MIP intra-lesionally to cure warts on sperm and rectum (Figure 7-8) as well as on feet (Figure 9). These findings are reported in 2 publications [11,12]. 

Probiotics

A healthy human vagina has an acidic pH of about 4, whereas the pH of the rest of body of normal individuals is rigorously maintained at 7.4. The local acidic pH of vagina is caused and maintained by Lactobacilli resident in harmony with endometrial cells of the vagina. Lactobacilli make and secrete D- and L-Lactic acid in addition to Bacteriocins, and H₂O₂. Good strains of Lactobacilli have also the enzyme Arginine deiminase, which prevents the formation of foul odor derivatives.

We isolated 80 Probiotic strains from women with healthy vagina. These were characterized on the basis of Genus, Group and Species by specific PCRs along with Random Amplified Polymorphic DNA (RAPD) and 16s rDNAs. The findings are reported elsewhere [13]. The predominant species isolated were L. gasseri, L. fermentum, L. salivarius, L. plantarum). All bacilli were gram positive and catalase negative. They all made and secreted lactic acid. Many made also H₂O₂. It was observed that strains of even the same species differed in their capacity to make and secrete lactic acid [14]. Strains isolated from women with healthy vagina made twice as much D-lactic acid as those isolated from women suffering from Vaginosis (Figure 10).

On basis of their property of making and secreting high amounts of D-lactic acid, Bacteriocins, H₂O₂ and positive for Arginine deiminase, we selected 3 meritorius strains: L. salivarius TRF # 30,L. fermentum TRF # 36, and L. gasseri TRF # 8. These have been passed on to M/s Microbax Hyderabad for large-scale culture, lyophilisation along with stabilizers and packaging in easily dispersible cellulose capsules, which can be inserted in vagina. Their high acceptability by women and their ability to restore healthy vaginal pH has been established by appropriate clinical trial.

Polyherbal Formulation BASANT

Vaginosis is manifested as abnormal vaginal discharge, vaginal pH>5 and presence of aerobic, anaerobic microorganisms. It is a commonly occurring syndrome. Its incidence is 30% in women in urban localities [15], 50% in rural Maharastra [16] and 80% in slums of India [17]. It is not confined to India, 29.2 % of women are reported to suffer from it in USA [18].

We developed a polyherbal formulation, named as BASANT, containing 95% pure Curcumin, purified extracts of Amla (Emblica officinalis), Neem (Azadirachtaindica) leaves, and Aloe vera (Aloe barbadensis) together with pharmacopoeially approved excipients. BASANT has wide spectrum action on sexually transmitted pathogens. It inhibits Neisseria gonorrhoeae, including strains resistant to Penicillin, Tetracycline, Nalidixic acid and Ciprofloxacin [19]. It has pronounced inhibitory action against Candida glabrata, Candida albicans and Candida tropicalis isolated from women with vulvovaginal candidiasis, including isolates resistant to azole drugs and amphotericin. It has inhibitory action on free as well as cell infected Chlamydia trachomatis [20]. BASANT was found to be totally safe as per USFDA recommendations on rabbit vagina after application for 7 consecutive days and twice daily for 3 weeks.

BASANT inhibits HIV. It prevents the entry of CCR5 and CXCR4 tropic HIV 1 lab-adapted strains and primary isolates belonging to different clades [21]. BASANT inhibits the entry of HPV16 in Hela cells [19]. Furthermore, it has the unique property of eliminating HPV16 from the infected cervical cells on path of progression to Carcinoma of Cervix at early stages when the virus is not yet integrated in host genome. In a trial conducted in Aligarh, Uttar Pradesh, in 11/11 women whose cervical cells were infected with HPV16, the virus was eliminated from infected cells, restoring normal Pap smear [22].

BASANT is highly effective in providing relief to women suffering from recurring episodes of vaginosis. (Figure 11) gives typical results obtained by using BASANT.

(c)  Healing of fishy odour:

Fishy Odour No Fishy Odour                

(After KOH addition on slide)   (After KOH addition on slide) 

ON ENROLLMENTAFTER TREATMENT

(d) pH of vagina restored to acidic range:

 pH > 5     pH < 4.5

ON ENROLLMENTAFTER TREATMENT

Figure 11: An Illustrative Representation of Typical Woman Receiving the Treatment with Combination of BASANT plus Probiotics.

A combination of BASANT along with the 3 selected meritorious Probiotics, cures almost 95% of women suffering from Vaginosis (Table 2). This is the highest efficacy achieved by any treatment in the world literature. Antibiotics cure only up to 65% of such patients.

Summary

Recapitulated above is our work on Problems of relevance. In each case, the work was followed up by developing potential solutions. After defining the nature of immune deficit in leprosy patients, a vaccine was developed based on a cultivable non-pathogenic atypical mycobacterium whose gene sequence has been determined. It has been named as Mycobacterium indicuspranii (MIP). MIP along with standard drugs expedites bacterial clearance and shortens the period of blemish free recovery. This vaccine is approved by the DCGI (India) and USFDA. It is passed on to Industry and is available to public.

MIP prevents H37RV induced tuberculosis in guinea pigs. Unlike BCG, it has no genetic restriction for response. It is active in both live and killed form. Used as adjunct to standard drugs, it improves significantly the cure rate of Category II ‘Difficult to treat’ tuberculosis patients.

MIP heals dramatically ugly ano-genital warts and prevents SP2/o myelomas in mice.

Also, passed on to Industry are 3 selected meritorious Probiotics strains for vaginal use, which are high secretors of lactic acid and prevent foul odor derivatives. At final stage of going to Industry is a Polyherbal microbicide BASANT, which inhibits N.gonorrhoeae,C. trachomatis, HIV, HPV, Candida’sincluding drugs resistant strains. It eliminates HPV-16 from infected cervical cells restoring normal Pap smear.

Acknowledgements

Research reported here was supported by the Indian Council of Medical Research, Department of Biotechnology, and Department of Science and Technology, Govt. of India.

Figure 1: Mycobacterium indicuspranii (MIP). [A] Electron Micrograph [B] Atomic Force Microscopy Perspective.

Figure 2: Some Representative Cases of LL/BL Multibacillary Patients Treated with MDT plus MIP (Mycobacterium indicuspranii).

Figure 3: Relapse Rate of Category II Tuberculosis Patients After Treatment with MDT Alone or MDT+MIP.

Figure 4: Effect of Immunization with killed MIP or live BCG on development of pulmonary lesions in C3H, CBA (Panel a) and in Balb/c, C57BL/6 (Panel b) strains of mice. Animals were immunized either with 10⁷ heat-killed MIP s.c. or 10⁴ live BCG i.v. Four weeks later, the animals were challenged with 10⁷ M. tuberculosis H₃₇R_vi.v. Four weeks’ post challenge, the animals were killed and visible lesions in the lungs were recorded. The data plotted are the mean of results obtained from three sets of experiments with MIP and two sets of experiments with BCG, with n=7-10 animals per group. The data for the non-immunized group of animals are also presented (Blank bars).

Figure 5:Antibody titres against Human Chorionic Gonadotropin (hCG). Symbols represent titres in individual mice, Bars give the geometrical mean titres [9].

Figure 6:  Preventive and Therapeutic Action of MIP Against SP2/O Myelomas in Mice.

Figure 8. (A) Before treatment (B) After treatment with MIP.

Figure 10: D-lactic acid Production by (A) 80 Lactobacillus Strains from Women with Healthy Vagina (B)124 Strains from Women Suffering from Bacterial Vaginosis [14].

                                      ON ENROLLMENT                                               AFTER TREATMENT

                                         ON ENROLLMENT                                          AFTER TREATMENT

                                                                                                                         (Lactobacilli colonised)

1. Saini V, Raghuvanshi S, Talwar GP, Ahmed N, Khurana JP, et al. (2009) Polyphasic taxonomic analysis establishes Mycobacterium indicuspranii as a distinct species. PLoS One4: e6263.
2. Talwar GP, Ahmed N, Saini V (2008) The use of the name Mycobacterium w for the leprosy immunotherapeutic bacillus creates confusion with M. tuberculosis-W (Beijing strain): a suggestion. Infect Genet Evol8: 100-101.
3. Saini V, Raghuvanshi S, Khurana JP, Ahmed N, Hasnain SE,et al. (2012)Massive gene acquisitions in Mycobacterium indicuspranii provide a perspective on mycobacterial evolution. Nucleic Acids Res 40: 10832-10850.
4. Zaheer SA, Mukherjee R, Ramkumar B, Mishra RS, Sharma AK, et al. (1993) Combined multidrug and Mycobacterium w vaccine therapy in patients with multibacillary leprosy. J Infect Dis 167:401-410.
5. Singh IG, Mukherjee R, Talwar GP (1991) Resistance to intravenous inoculation of Mycobacterium tuberculosis H₃₇R_v in mice of different inbred strains following immunization with a leprosy vaccine based on Mycobacterium. Vaccine9: 10-14.
6. Fishel SB, Edwards RG, Evans CJ (1984) Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science223:816-818.
7. Nash H, Johansson ED, Talwar GP, Vasquez J, Segal S,et al. (1980) Observation on the antigenicity and clinical effects of a candidate antipregnancy vaccine: β-subunit of human chorionic gonadotropin linked to tetanus toxoid. FertilSteril 34:328-335.
8. Talwar GP, Singh O, Pal R, Chatterjee N, Sahai P, et al. (1994) A vaccine that prevents pregnancy in women. Proc Natl Acad Sci USA 91: 8532-8536.
9. Purswani S, Talwar GP (2011) Development of a highly immunogenic recombinant candidate vaccine against human chorionic gonadotropin. Vaccine 29: 2341-2348.
10. Rakshit S, Ponnusamy M, Papanna S, Saha B, Ahmed A, et al. (2012) Immunotherapeutic efficacy of Mycobacterium indicuspranii in eliciting anti-tumor T cell responses: critical roles of IFNÉ£. Int J Cancer 130: 865-875.
11. Gupta S, Malhotra AK, Verma KK, Sharma VK (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.
12. Singh S, Chouhan K, Gupta S (2014) Intralesional immunotherapy with killed Mycobacterium indicuspranii vaccine for the treatment of extensive cutaneous warts. Indian J DermatolVenereolLeprol80:509-514.
13. Garg KB, Ganguli I, Das R, Talwar GP (2009) Spectrum of Lactobacillus species present in healthy vagina of Indian women. Indian J Med Res 129: 652-657.
14. Garg KB, Ganguli I, Ram Das, Kriplani A, Lohiya NK, et al. (2009) Metabolic properties of lactobacilli in women experiencing recurring episodes of bacterial vaginosis with vaginal pH≥5.Eur J clinMicrobiol Infect Dis 29: 123-125.
15. Bhujwala RA, Buckshee K, Shriniwas (1985) Gardnerellavaginitis and associated aerobic bacterial in nonspecific Vaginitis. Ind J Med Res81: 251-256.
16. Bang RA, Bang AT, Baitule M, Sarmukaddam S, Choudhary Y, et al. (1989) High prevalence of gynaecological diseases in rural Indian women. The Lancet333: 85-88.
17. Salhan S, Tripathi V, Sehgal R, Kumar G, Talwar GP, et al. (2009) A Phase II Randomized Controlled Trial to Evaluate the Safety and Efficacy of Praneem Polyherbal Vaginal Tablets Compared to Betadine Vaginal Pessary in Women with Symptoms of Abnormal Vaginal Discharge. Asia Pac J Public Health 21: 461-468.
18. Cudmore SL, Delgaty KL, Hayward-McClelland SF, Petrin DP, Garber GE (2004) Treatment of infections caused by Metronidazole-resistant Trichomonas vaginalis. ClinMicrobiol Rev 17: 783-793.
19. Talwar GP, Dar SA, Rai MK, Reddy KV, Mitra D, et al. (2008) A novel Polyherbal microbicide with inhibitory effect on bacterial, fungal and viral genital pathogens. Int J Antimicrob Agents 32: 180-185.
20. Bhengraj AR, Dar SA, Talwar GP, Mittal A (2008) Potential of a novel polyherbal formulation BASANT for prevention of Chlamydia trachomatis infection. Int J Antimicrob Agents 32: 84-88.
21. Maselko MB, Joshic RS, Prescott M, Talwar GP, Kulkarni S, et al. (2014)Basant, a Polyherbal Topical Microbicide Candidate Inhibits Different Clades of Both CCR5 and CXCR4 Tropic, Lab-Adapted and Primary Isolates of Human Immunodeficiency Virus-1 in Vitro Infection. J VirolAntivir Res3:3.
22. Talwar GP, Sharma R, Singh S, Das BC, Bharti AC, et al. (2015) BASANT, a Polyherbal Safe Microbicide Eliminates HPV-16 in Women with Early Cervical Intraepithelial Lesions. Journal of Cancer Therapy 6: 1163-1166.
23. Talwar GP, Kavita Garg, Atrey N, Singh P, Gaur J, et al. (2015) A Safe Wide Spectrum Polyherbal Microbicide and Three Meritorious Strains of Probiotics for Regressing Infections and Restoration of Vaginal Health -Regression of Vaginosis with BASANT and Probiotics. J Women’s Health Care 4: 256.