Abstract
This article reviews briefly the making of an immunoprophylactic-cum-immunotherapeutic vaccine against leprosy. The vaccine is based on cultivable, heat-killed atypical mycobacteria, whose gene sequence is now known. It has been named Mycobacterium indicus pranii. It has received the approval of the Drug Controller General of India and the US Food and Drug Administration. Besides leprosy, M. indicus pranii has found utility in the treatment of category II (“difficult to treat”) tuberculosis. It also heals ugly anogenital warts. It has preventive and therapeutic action against SP2/O myelomas. It is proving to be a potent adjuvant for enhancing antibody titers of a recombinant vaccine against human chorionic gonadotropin, with the potential of preventing pregnancy without derangement of ovulation and menstrual regularity in sexually active women.
Introduction
The immune system has the task of defending the body from infections caused by a variety of microorganisms foreign to the body. Vaccines were and are made to boost the immune response against a given bacteria, virus, or harmful toxins generated by microorganisms. While the body has an armamentarium in terms of cells and molecules endowed with the property of responding and reacting with various “invaders”, the process to build up adequate immunity takes time. Vaccination with a killed or attenuated microbe performs this function and builds up adequate immunity against an eventual infection. Interestingly, some vaccines also have therapeutic properties.
This communication describes briefly the making of an immunoprophylactic-cum-immunotherapeutic vaccine initially developed against leprosy. The vaccine has received the approval of the Drugs Controller General of India (DCGI) and also of the US Food and Drug Administration (FDA). It is at present the only vaccine of its type in the world. It has been taken up by the industry and is available to the public. Besides leprosy, it has found therapeutic utility in the treatment of category II (“difficult to treat”) tuberculosis. Astonishingly, it cures ugly anogenital warts, presumably by potentiating both cellular and humoral immune responses. Its efficacy in prevention and treatment of SP2/O myeloma in mice has also been reported. It is employed as an adjuvant in a potential birth control vaccine against human chorionic gonadotropin (hCG) for preventing pregnancy.
Leprosy
Leprosy is caused by Mycobacterium leprae, a mycobacteria isolated by Armauer Hansen in Norway in 1873. He was unable to grow it in any of the many media that he tried. His thesis is one of the shortest on record. The majority (~99%) of humans can resist infection of M. leprae successfully, and do not become leprosy patients. The few who become victim to the disease manifest a spectrum ranging from a single lesion with few if any bacteria, classified as tuberculoid (TT) to multibacillary lepromatous leprosy (LL) with multiple lesions loaded with M. leprae. This spectrum is a reflection of the existence of complete or variable degrees of immune response against M. leprae in patients manifesting the different forms of leprosy. Our first task was to learn of what goes wrong or is deficient in humans who become victims of the disease. After learning this, the next task obviously was to see whether anything can be done to prevent humans contracting the disease.
Nature of immune deficit in leprosy
Those who contract leprosy have T cells that are unable to react against some key antigen(s) of M. leprae. Their immune system is otherwise fairly normal, and they respond normally to cholera or typhoid vaccines.Citation1 T lymphocytes generate the signal for macrophages to prevent the proliferation of phagocytosed M. leprae. gives data clearly supporting the role of T cells in this process. In this experiment, monocyte-derived macrophages from either TT leprosy patients or those suffering from the LL form of the disease were infected with M. leprae derived from patients. Radioactive thymidine (3H-thymidine) was used as a precursor in the medium. It was incorporated into DNA by M. leprae engulfed in macrophages derived from either LL or TT leprosy patients. However, if T cells derived from TT patients were also included in culture, the incorporation of 3H-thymidine by M. leprae was restricted, whereas T lymphocytes derived from LL patients lacked this property.Citation2
Can anything be done to restore this deficiency?
This was the basic requirement of an eventual vaccine against leprosy. Vaccines are usually made using the killed or attenuated forms of infecting microorganisms. Such a homologous approach was illogical for leprosy, as the basic defect in LL patients is their inability to respond to key antigen(s) of M. leprae. Therefore, a heterologous approach was adopted.
Search for an atypical Mycobacterium sharing antigens with M. leprae
Whereas M. leprae is noncultivable, a candidate for a vaccine against leprosy has to be cultivable in one medium or another to enable its production on a large scale for public use. We collected 16 cultivable, atypical mycobacteria from various sources, some already named and classified, others lying in various atypical collections. Each of them was coded and investigated for its ability to cause blast transformation of T cells from not only TT but also LL patients.Citation3 Their ability to generate cytokines influencing macrophage function was investigated.Citation4 The ensemble of these investigations led to the shortening of the list of 16 to 5 mycobacteria. These were M. vaccae, M. phlei, M. gordonae, ICRC bacillus, and Mycobacterium w (Mw).
To confirm their closeness to M. leprae, lepromin-like preparations were made of these five atypical mycobacteria and evaluated alongside lepromin prepared from M. leprae in TT and LL patients to evoke M. leprae lepromin-like responses in leprosy patients. An atypical mycobacteria (Mw) appeared to possess these molecular traits.Citation5
Could Mw serve as a vaccine? Can it convert lepromin-negative leprosy patients to lepromin-positive status? This investigation was carried by Chaudhary et al at the School of Tropical Medicine, Kolkata. Twenty of 32 leprosy patients who were consistently lepromin negative were converted from lepromin-negative to lepromin-positive status after a single intradermal injection of autoclaved Mw.Citation6 This was confirmed by Kar et alCitation7 in lepromin-negative family members and contacts of leprosy patients in Delhi. A total of 67 of 68 (98.5%) converted to lepromin-positive status after two immunizations with Mw. These observations pointed out the potential of Mw to serve as an immunoprophylactic vaccine to protect susceptible family members from contracting leprosy on continuous exposure to M. leprae from a multibacillary leprosy patient.
Therapeutic action of Mw
Zaheer et al administered autoclaved Mw as adjunct to standard multidrug therapy (MDT) to treat multibacillary leprosy patients. A parallel group received a tenth of the dose of tetanus toxoid as adjunct. It was observed that the inclusion of Mw with the drugs expedited bacterial clearance and shortened the period of complete recovery.Citation8 illustrates the remarkable property of Mw in not only expediting recovery but also clearing granulomas in patients.Citation9
Abbreviations: MDT, multidrug therapy; LL, lepromatous leprosy; BL, borderline leprosy.
Efficacy in slow responders
Fourteen patients who were not responding adequately to standard treatment with MDT were referred to our treatment center. They were randomly distributed into two groups: one group continued to receive MDT, and the second group was also given MDT with Mw vaccine as an adjunct. shows the dynamics of bacterial index values in two of these patients. Soon after immunization with Mw, the bacterial index started declining noticeably, followed by full recovery of the patients.Citation10 This was not the case in the group receiving drugs alone, in whom the decline continued to be extremely slow ().
Conversion of borderline leprosy/LL patients to lepromin-positive status by Mw
LL patients are all lepromin negative, one of the criteria employed for classifying the patient in this category, and they continue to be lepromin negative even after bacterial clearance is achieved with drugs. Drugs kill the bacteria but do not improve the immune response. It was pertinent to examine whether the inclusion of Mw for treatment had any effect on the delayed skin-hypersensitivity response to M. leprae, manifested as a positive response to lepromin. shows the dynamics of the conversion of patients from lepromin-negative to lepromin-positive status.
Clearance of bacilli from peripheral nerves
M. leprae infects the peripheral nerve cells, causing loss of sensitivity. Zaheer et alCitation10 had the good idea to investigate whether inclusive treatment with Mw in patients eliminated fully or partially the bacilli infecting the nerve cells. gives the observations. In contrast to drugs alone, where bacilli persisted in peripheral nerves of some patients, in the group receiving Mw, bacilli were eliminated in the peripheral nerves of all patients investigated.
Approval by drug regulatory authorities
Phase-wise clinical trials were conducted with due approval of ethics committees and drug regulatory authorities in recognized centers, followed by field trials in Kanpur Dehat in 272 villages inhabited by 420,823 people. Kanpur Dehat used to be one of the endemic districts for leprosy. Patients suffering from leprosy were treated with drugs and the Mw vaccine. Their household contacts who had been exposed received the vaccine based on Mw in the hope that they would be protected from becoming patients. Observations made during these trials are reported elsewhere.Citation11,Citation12 Vaccination with Mw shortened treatment time of patients. In addition, no side effects of any significance were seen by vaccination of healthy contacts with Mw. Our vaccine has received the approval of the DCGI and was licensed to a company to enable its availability to the public. In due course, it also received the approval of the FDA.
Move for eradication of leprosy
Eradication of small pox was achieved by planned use of a vaccine against that infectious disease. An Indian government committee led by Soumya Swaminathan, Director-general of the Indian Council of Medical Research, has decided to evaluate the use of M. indicus pranii (MIP) to eliminate if not eradicate leprosy in India. To begin with, a field project will be launched in five districts of high endemism in the country. The index patient will receive the vaccine in addition to MDT. Their family members and contacts will be immunized with the MIP vaccine twice at 6-month intervals with the hope that they will not become patients.
Tuberculosis
The company licensed to manufacture Mw vaccine asked from us a biological test to confirm that they were growing the right bacteria. As Mw shares antigens with M. tuberculosis besides M. leprae, we thought of testing whether Mw could prevent tuberculosis in guinea pigs following infection with the pathogenic strain M. tuberculosis H37RV. This was indeed the case. As shown in , after 3 weeks the lungs had nodules full of M. tuberculosis and the spleen was enlarged. Immunization with Mw prevented these from happening.
Similarities and differential properties of Mw with respect to BCG
Bacillus Calmette–Guérin (BCG) is effective only in live form and loses immunizing capability in a dead state, whereas Mw is effective in both live and in autoclaved form. Furthermore, in contrast to BCG, Mw seems to have no genetic restrictions. As shown in prevented the growth of pulmonary lesions in all four genetic strains (C3H, CBA, BALB/c, C57BL) of mice investigated, whereas BCG was effective in BALB/c and C57BL but not in CBA or C3H mice.Citation13
Abbreviations: Mw, Mycobacterium w; BCG, bacillus Calmette–Guérin.
Treatment of category II tuberculosis patients
Mw was employed as adjunct for treatment of category II (difficult to treat) tuberculosis patients in an exploratory trial conducted in Ahmedabad. gives the results. Inclusion of Mw with drugs improved recovery considerably. What is further significant is that patients receiving Mw with drugs had much lower relapse rates ().
Gene sequencing of Mw
Reddi et al carried out a molecular analysis of Mw, which indicated it as a unique species.Citation14 Three recognized centers were given a grant by the Department of Biotechnology, Government of India to determine the genome sequence and carry out molecular definition of Mw, an atypical cultivable mycobacterium with remarkable therapeutic traits of relieving leprosy and tuberculosis. Their findings are reported elsewhere.Citation15 Mw was considered an ancestor of both M. leprae and M. tuberculosis. No bacteria of this definition had heretofore existed in the International Depository. It was named Mycobacterium indicus pranii (MIP).Citation16
Unexpected astonishing properties of MIP
Every now and then, SG at the All India Institute of Medical Sciences receives patients harboring ugly anogenital warts. He found that administration of MIP caused remarkable recovery of these warts. shows the clearance of the warts. shows an ugly lesion on the anus. Among the first nine patients suffering from such anogenital warts, three were positive for HIV. In spite of that, they benefited from this treatment. These observations have been published elsewhere.Citation17 Similar warts occurring elsewhere in the body were also healed ().Citation18
Abbreviation: MIP, Mycobacterium indicus pranii.
Prevention and therapy of SP2/O myelomas in mice
Rakshit et al showed the prevention and anticancerous action of MIP on development of SP2/O myelomas in BALB/c mice.Citation19 gives a summary of their findings. They have also reported the generation by MIP of cytokines IL-2 and IFNγ, which may be involved in this action.
Abbreviations: MIP, Mycobacterium indicus pranii; Th, T-helper; PBS, phosphate-buffered saline.
Potent adjuvant properties of Mycobacterium indicus pranii
Our laboratory is involved in making a recombinant vaccine against hCG, which has the potential of preventing pregnancy in sexually active women without derangement of ovulation and menstrual regularity. Inclusion of MIP as an adjuvant in the vaccine enhances antibody titers remarkably (). MIP is a potent invigorator of immune response.
Abbreviations: hCGβ, human chorionic gonadotropin beta; MIP, Mycobacterium indicus pranii.
Summary
This review recapitulates briefly the making of an immunotherapeutic-cum-immunoprophylactic vaccine against leprosy. The vaccine is based on an atypical, cultivable mycobacteria, originally coded Mw. The gene sequence analysis of Mw and its molecular definition have been done. It is an ancestor of both M. leprae and M. tuberculosis. No bacteria of this definition had heretofore existed in the International Depository. It has been named Mycobacterium indicuss pranii (MIP).
MIP given as an adjunct to standard MDT expedites bacterial clearance and shortens the period of complete recovery of leprosy patients. It also upgrades the immune response of patients to M. leprae, which is not achieved by drugs. MIP has received the approval of the DCGI and the FDA. Besides leprosy, it has been used successfully as an adjunct in the treatment of category II (difficult to treat) tuberculosis patients. MIP appears to be a potent invigorator of both humoral and cellular immune responses. Used as an adjuvant, it enhances considerably the antibody response to the hCGβ-LTB vaccine, under development for control of fertility. Given intralesionally, it cures ugly anogenital warts. It suppresses growth of SP2/O myeloma tumors in BALB/c mice, presumably by inducing T-helper 1 cytokines.
Acknowledgments
The work reviewed in this article received research grants from the Indian Council of Medical Research and the Department of Biotechnology, Government of India. The authors acknowledge SA Zaheer, S Choudhary, VM Katoch, Dipankar Nandi, Shilpi P Vyas, and Kripa N Nand for their valuable contributions to the work.
Disclosure
The authors report no conflicts of interest in this work.
References
- TalwarGPAn immunotherapeutic vaccine for multibacillary leprosyInt Rev Immunol19991822924910614726
- TalwarGPKrishnanADJhaPMehraVIntracellular growth of an obligatory parasite Mycobacterium leprae: host bacterial interactionsBiochimie1974562312374135832
- TalwarGPKrishnanADMehraVLBlumEAPearsonJMEvaluation of cell mediated immune responses in untreated cases of leprosyClin Exp Immunol1972121952034567646
- MustafaASTalwarGPFive cultivable mycobacterial strains giving blast transformation and leukocyte migration inhibition of leukocytes analogous to Mycobacterium lepraeLepr India197850498508374869
- MustafaASTalwarGPEarly and late reactions in tuberculoid and lepromatous leprosy patients with lepromins from Mycobacterium leprae and five selected cultivable mycobacteriaLepr India197850566571374875
- ChaudhuriSFotedarATalwarGPLepromin conversion in repeatedly lepromin negative BL/LL patients after immunization with autoclaved Mycobacterium wInt J Lepr Other Mycobact Dis198351159686684642
- KarHKSharmaAKMishraRSInduction of lepromin positivity by a candidate anti-leprosy vaccine Mycobacterium w in lepromin negative healthy contacts of multibacillary leprosy patientsIndian J Lepr1992644955001308525
- ZaheerSAMukherjeeRRamkumarBCombined multidrug and Mycobacterium w vaccine therapy in patients with multibacillary leprosyJ Infect Dis19931674014108421173
- TalwarGPLeprosy is in principle eradicable: a possible approachCurr Sci201410613441345
- ZaheerSAMukherjeeARameshVImmunotherapy benefits multibacillary patients with persistently high bacteriological index despite long term multidrug therapyImmunol Infect Dis19955115122
- WaliaRSarathchandraKGPandeyRMField trials on the use of Mycobacterium w vaccine in conjunction with multidrug therapy in leprosy patients for immunotherapeutic and immunoprophylactic purposesLepr Rev1993643023118127216
- SharmaPMukherjeeRTalwarGPImmunoprophylactic effects of the anti-leprosy Mw vaccine in household contacts of leprosy patients: clinical field trials with a follow up of 8–10 yearsLepr Rev20057612714316038246
- SinghIGMukherjeeRTalwarGPResistance to intravenous inoculation of Mycobacterium tuberculosis H37Rv in mice of different inbred strains following immunization with a leprosy vaccine based on Myco-bacterium wVaccine1991910141901186
- ReddiPPAminAGKhandekarPSTalwarGPMolecular definition of unique species status of Mycobacterium w, a candidate leprosy vaccine strainInt J Lepr Other Mycobact Dis1994622292368046263
- SainiVRaghuvanshiSTalwarGPPolyphasic taxonomic analysis establishes Mycobacterium indicus pranii as a distinct speciesPLoS One20094e626319606228
- TalwarGPAhmedNSainiVThe use of the name Mycobacterium w for the leprosy immunotherapeutic bacillus creates confusion with M. tuberculosis-W (Beijing strain): a suggestionInfect Genet Evol2008810010117766188
- GuptaSMalhotraAKVermaKKSharmaVKIntralesional immunotherapy with killed Mycobacterium w vaccine for the treatment of anogenital warts: an open-label pilot studyJ Eur Acad Dermatol Venereol2008221089109318484970
- SinghSChouhanKGuptaSIntralesional immunotherapy with killed Mycobacterium indicus pranii vaccine for the treatment of extensive cutaneous wartsIndian J Dermatol Venereol Leprol20148050951425382507
- RakshitSPonnusamyMPapannaSSahaBAhmedANandiDImmunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: critical roles of IFNγInt J Cancer201213086587521455983
- PurswaniSTalwarGPDevelopment of a highly immunogenic recombinant candidate vaccine against human chorionic gonadotropinVaccine2011292341234821272600