Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy.

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.

Immunologically reactive M. leprae antigens with relevance to diagnosis and vaccine development.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae that can manifest a wide variety of immunological and clinical outcomes ranging from potent humoral responses among borderline lepromatous (BL) and lepromatous (LL) patients to strong cellular responses among tuberculoid (TT) and borderline tuberculoid (BT) patients. Until recently, relatively little has been known about the immune responses to individual proteins of M. leprae recognized during leprosy. METHODS: The immune reactivity to a panel of 33 M. leprae recombinant proteins was evaluated among leprosy patients and controls from a high endemic area for leprosy (Goiania/GO, Central Brazil). Serum IgG responses were measured by ELISA (45 participants/group) and T cell responses (20 participants/group) were evaluated by IFN-gamma production in 24 hours whole blood cultures with antigen (whole blood assay-WBA). Study groups were newly diagnosed, untreated TT/BT and BL/LL leprosy patients classified by Ridley Jopling criteria and household contacts of BL/LL patients (HHC). Control groups were HIV-1 negative pulmonary tuberculosis patients (TB) and healthy individuals from the same endemic area (EC). In silico predictions indicated the level of identity of M. leprae proteins with homologues in other mycobacteria and the presence of T cell and B cell epitopes. RESULTS: Despite the prediction that all proteins would be reactive, 16 of 33 (48%) of the single proteins tested were immunogenic (recognized in WBA or ELISA) and seventeen were non-immunogenic (not recognized in either assay). Among the 16 immunogenic proteins, 9 were considered leprosy specific in WBA inducing cell-mediated IFN-gamma secretion from TT/BT patients and HHC. Three of these proteins were also leprosy specific in serology being recognized by serum IgG from LL/BL patients. Seven of the immunogenic proteins were not leprosy specific. CONCLUSIONS: New M. leprae antigens recognized by antibody responses of BL/LL patients and cellular responses of TT/BT leprosy patients were identified. An improved serological diagnostic test for leprosy could be developed by incorporating these IgG-reactive antigens to the current PGL-I based tests. Moreover our data indicate that the WBA is a robust, relatively simple and user friendly format for a T cell based diagnostic test. The field use of these test formats in leprosy endemic countries could contribute to early leprosy diagnosis before the development of deformities and disabilities.

Inhibition of the multiplication of Mycobacterium leprae by vaccination with a recombinant M. bovis BCG strain that secretes major membrane protein II in mice.

The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-gamma) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8(+) T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-gamma on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.

[The estimation of protective efficacy of the fusion gene vaccine encoding tubercle antigen 85B and MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To evaluate the protective efficacy of the fusion DNA vaccine (AM) encoding tubercle Ag85B and MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: C57BL/6 mice were intramuscularly immunized with the DNA vaccines. The mice were challenged with 10(6) CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B, pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). No IL-4 was found in all groups. The number of bacterial colonies in the lungs and spleens was significantly decreased at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). The pulmonary histopathological changes were observed 6 weeks later following challenge with M. tuberculosis H37Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by seroplastic inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B groups was characterized by seroplastic inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in lung. The survival time of BCG vaccinated mice after challenge with M. tuberculosis H37Rv was longer than that of other groups. The survival time of AM group was longer than that of other DNA vaccine groups. CONCLUSION: The pcDNA/AM can improve the protective efficacy in immunized mice against M. tuberculosis.

Anti-mycobacterial immunity induced by a single injection of M. leprae Hsp65-encoding plasmid DNA in biodegradable microparticles.

A single sub-cutaneous injection of a plasmid DNA encoding a mycobacterial heat shock protein 65 (Hsp65) entrapped in biodegradable poly(lactic-co-glycolic acid) microspheres produced high titers of antibodies, measured 5 months after the injection in BALB/c mice. Splenocytes secreted IFN-gamma and exerted an anti-bacterial effect on macrophages infected in vitro with Mycobacterium tuberculosis. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.

DNA encoding a single mycobacterial antigen protects against leprosy infection.

The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guérin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed. The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine. Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production. DNA-35 immunization induced significant levels of protection against M. leprae footpad infection, comparable to that produced by BCG. Therefore, DNA immunization with the 35 kDa antigen is effective against M. leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.

Towards a DNA vaccine against tuberculosis.

Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp65) in adult Balb/c mice resulted in substantial cell-mediated protection against challenge with M. tuberculosis. CD4 and CD8 T cells cloned from spleens of such immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. Three modes of expressing the gene have been tested: (1) expression from a retroviral vector (pZIPNeoSV) in implanted J774 tumour cells, (2) expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and (3) in a preliminary experiment, from CMV immediate-early and hydroxymethylglutaryl Co-A reductase promoters injected as plasmid DNA into muscle.

Protein antigens of Mycobacterium leprae.

Protein antigens of Mycobacterium leprae have been identified by screening the lambda gt11, pYA626 and pHC79::M. leprae genomic libraries with pooled sera from leprosy patients and with antiserum to M. leprae cell wall protein (CWP) aggregate. Immunological screening of the lambda gt11 library with pooled sera from 21 lepromatous (LL) leprosy patients resulted in the identification of 19 antigens that are apparently different from previously identified M. leprae antigens. Five additional antigens were identified by screening the lambda gt11 library with pooled sera from 30 borderline tuberculoid or tuberculoid patients. Four other antigens were identified by screening the lambda gt11 library with anti-CWP. Two groups of recombinant cosmids were identified by screening the pHC79 library with LL patients' sera: one group specified proteins that reacted with monoclonal antibodies (mAb) against the 65-kDa protein and against the 18-kDa protein; the other group specified a 15-kDa protein that did not react with any of the mAb that were tested. One pYA626 clone also specified a 15-kDa protein that reacted with LL patients' sera, but did not react with any mAb. Genes specifying several of these antigens have been subcloned into the Asd+ plasmid vector pYA292 and have been introduced into a delta cya delta crp delta asd Salmonella typhimurium strain to evaluate the ability of individual M. leprae proteins to elicit immune responses against M. leprae infection.

The etiologic agents of leprosy and tuberculosis share an immunoreactive protein antigen with the vaccine strain Mycobacterium bovis BCG.

The amino acid sequences of the 65-kilodalton antigens of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis BCG display greater than 95% homology.