c Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)-the major component of new-generation anthrax vaccines-affects vaccine immunogenicity, we created a "genetically deamidated" form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

Anthrax vaccines containing recombinant PA (rPA) as the only antigen face a stability issue: rPA forms aggregates in solution after exposure to temperatures ⩾40°C, thus losing its ability to form lethal toxin (LeTx) with Lethal Factor. To study rPA aggregation's impact on immune response, we subjected rPA to several time and temperature combinations. rPA treated at 50°C for 30min formed high mass aggregates when analyzed by gel electrophoresis and failed to form LeTx as measured by a macrophage lysis assay (MLA). Aggregated rPA-formed LeTx was about 30 times less active than LeTx containing native rPA. Mice immunized with heat-treated rPA combined with Al(OH)3 developed antibody titers about 49 times lower than mice immunized with native rPA, as measured by a Toxicity Neutralization Assay (TNA). Enzyme Linked Immunosorbent Assay (ELISA) of the same immune sera showed anti-rPA titers only 2-7 times lower than titers elicited by native rPA. Thus, rPA's ability to form LeTx correlates with its production of neutralizing antibodies, and aggregation significantly impairs the protein's antibody response. However, while these findings suggest MLA has some value as an in-process quality test for rPA in new anthrax vaccines, they also confirm the superiority of TNA for use in vaccine potency.

Leprosy is an infectious disease caused by Mycobacterium leprae, which is a noncultivable bacterium. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. M. habana is a cultivable nonpathogenic mycobacterium and candidate vaccine for leprosy, and several antigens that cross-react between M. leprae and M. habana have been discovered. The aim of the present study was to extend the identification of cross-reactive antigens by identifying M. habana proteins that reacted by immunoblotting with antibodies in serum samples from leprosy patients but not with antibodies in sera from tuberculosis (TB) patients or healthy donors (HDs). A 28-kDa antigen that specifically reacted with sera from leprosy patients was identified. To further characterize this antigen, protein spots were aligned in two-dimensional polyacrylamide gels and Western blots. Spots cut out from the gels were then analyzed by mass spectrometry. Two proteins were identified: enoyl-coenzyme A hydratase (lipid metabolism; ML2498) and antigen 85B (Ag85B; mycolyltransferase; ML2028). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of lepromatous leprosy, which is the most frequent form in Mexico.Intradermal immunization with killed Mycobacterium leprae renders mice immune to infection with viable M. leprae (28). This protection is long lasting and systemic. However, when other mycobacteria are used to immunize mice against infection with viable M. leprae bacilli, they have been shown to be either ineffective (i.e., Mycobacterium duvalii) or to confer only partial protection (i.e., M. bovis BCG) (29). In 1985 and then 1989, Mycobacterium habana TMC 5135 was found to be as effective as M. leprae in protecting mice against footpad infection (32, 33). This was surprising, since Shepard et al. found that among a large panel of mycobacteria tested, only M. leprae and BCG were able to confer protection (30).M. habana was first described following its isolation from 35 cases of pulmonary tuberculosis (TB) (40); subsequently, it was found to be closely related to the species Mycobacterium simiae serovar 1 and is now known as M. simiae serotype 1 (20). In 1996, Khoo et al. demonstrated that M. habana TMC 5135 and several M. simiae strains differed in their polar glycopeptidolipid (GPL) compositions, conferring sufficient specificity for identification of M. habana as a distinct serotype of M. simiae (14).M. habana is a cultivable organism, protects mice against Mycobacterium ulcerans challenge, and offers consistent protection against infection with Mycobacterium tuberculosis H37Rv and other strains of M. tuberculosis (11,12). Some studies suggest that the secretory antigens released by actively growing M. habana bacilli are protective against M. tuberculosis infections (6,8). Recent experimental data indicate that M. habana TMC 5135 and M. habana IPK-220 strains, which differ in the fine structure of their mycolates, a...

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