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We report the safety and immunogenicity of a double lysine and pantothenate auxotroph of Mycobacterium tuberculosis in mice. The ⌬lysA ⌬panCD mutant is completely attenuated in immunocompromised SCID and gamma interferon knockout mice yet induces short-term and long-term protection in immunocompetent and CD4-deficient mice following single-dose subcutaneous vaccination.Tuberculosis (TB) remains a major global health problem, and the increased incidence of TB cases in human immunodeficiency virus (HIV)-infected and AIDS patient populations underlines the need for an improved and safe vaccine (12). The currently used Mycobacterium bovis BCG vaccine has several limitations, including variable protective efficacy in adults (1a, 16), waning of protective immunity over time (4,15), and safety concerns in immunocompromised individuals (1,6,25,38). In addition, comparative genomic analyses have identified more than 100 coding sequences that are missing from BCG but present in M. tuberculosis (5,8,26), including the secreted protein ESAT-6, which has been shown to be a protective antigen in several animal studies (7,31). These missing regions may encode potential antigenic determinants that could increase the immunogenicity of a vaccine if it were derived from an attenuated strain of M. tuberculosis.Live, attenuated vaccines offer a powerful means to prevent several important human diseases; a number of these live preparations are in routine use, including attenuated vaccines for polio, measles, mumps, and rubella. The advantages of live vaccines include the ability to replicate within host tissues, thereby facilitating the generation of prolonged memory T-cell responses. Several attenuated M. tuberculosis vaccine candidates that were constructed by deleting genes required for growth in mice (20,21,35) have been shown to confer protection against infection with virulent M. tuberculosis. We have previously reported the safety and immunogenicity of the single M. tuberculosis ⌬lysA (30) and ⌬panCD (32) auxotrophs in mice. In an attempt to further enhance the safety of a live, attenuated M. tuberculosis vaccine strain, we introduced the panCD deletion into an unmarked ⌬lysA mutant of M. tuberculosis by specialized transduction (2). The deletions of the lysA and panCD genes were confirmed by Southern blotting (Fig. 1A). Strain mc 2 6020 (⌬lysA ⌬panCD mutant) is strictly auxotrophic for lysine and pantothenate, and no growth of the mutant was observed in the absence of lysine and pantothenate supplementation. No revertants were recovered when 10 11 CFU of strain mc 2 6020 were plated on minimal medium or on medium containing either pantothenate or lysine, demonstrating the mutations to be highly stable and nonrevertible. Similarly, no revertants were observed following serial passage of the mutant or when it was cultured from infected mice.To evaluate the synergistic effect of the ⌬lysA and ⌬panCD mutations on bacterial virulence, immunocompetent BALB/c mice (6 to 8 weeks old, purchased from Jackson Laboratories, Bar Harbor, M...

Tuberculous pleuritis (TB) provides a good model to study the correlates of protective immune response at the site of infection. To study the in vivo correlates of immunity, cell subset profile and cytokine assay in plasma (BL) and pleural fluid (PF) of 82 patients were done. Lymphocyte proliferation and cytokine response to mycobacterial antigens were measured in 32 subjects to understand the in vitro correlates. Increase in CD4(+) cells and CD4(+)/CD8(+) ratio with selective concentration of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12 in PF suggests that the CD4(+) population may be of TH1 type. We observed an accelerated lymphoproliferative response to purified protein derivative (PPD) and heat killed Mycobacterium tuberculosis (MTB) in PF cells of both TB and non-TB (NTB) subjects. Interestingly, in in vitro studies, IL-4 levels together with IFN-gamma were significantly increased in the supernatants of PF mononuclear cells (PFMC) of TB patients and showed a shift in immune response towards TH0/TH2 type. PPD and MTB antigens induced an enhanced proliferation of PFMC and also increased in vitro IL-4 response together with apoptosis, thus eliciting a dual response.

Summary The global epidemic of tuberculosis, fuelled by acquired immune‐deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a ΔRD1ΔpanCD mutant of Mycobacterium tuberculosis (mc26030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild‐type mice and even in mice that completely lack CD4+ T cells as a result of targeted disruption of their CD4 genes (CD4–/– mice). Ex vivo studies of T cells from mc26030‐immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II‐restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8+, T‐cell receptor γδ (TCR‐γδ+) and NK1.1+ T cells from vaccinated CD4–/– mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4–/– mice with mc26030 were attributable to a population of CD4– CD8– (double‐negative) TCR‐αβ+, TCR‐γδ–, NK1.1– T cells. Transfer of highly enriched double‐negative TCR‐αβ+ T cells from mc26030‐immunized CD4–/– mice into naive CD4–/– mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double‐negative T cells transcribed significantly more interferon‐γ and interleukin‐2 mRNA than double‐negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II‐restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus‐infected humans who have chronic depletion of their CD4+ T cells.

Tuberculous pleuritis is a good human model to understand the local and protective immune response against tuberculosis, due to the self-limitedness of the disease. Although the cellular immune response has been well characterised in tuberculous pleurisy, much less is known about the humoral immune response operating at the site of infection. To understand the humoral immune response, B cells were enumerated in peripheral blood mononuclear cells (PBMC) and pleural fluid mononuclear cells (PFMC) of tuberculous (TP) and non-tuberculous pleuritis patients (NTP). The levels of IgG, IgA and IgM antibodies for PPD, culture filtrate (CF) and sonicate antigens (Son Ag) were assessed in plasma (BL) and pleural fluid (PF) and a western blot was carried out with the CF antigen. The percentage of CD19 + B-cells was similar in PBMC and PFMC of TP patients but was significantly lower in PFMCs of NTP patients. The IgG levels for PPD and CF antigens were higher in PF of TP than NTP patients. The antigen recognition patterns did not differ in plasma and pleural fluid of the same patient in both groups pointing out the passive diffusion of the plasma to the pleura. The antigens 25, 31, 33, 70, 110, 124 and 132 kDa were recognized exclusively by the TP patients. Thus our study showed that the local humoral response in TP did not differ from the systemic response. However, the humoral response differed in TP patients when compared to NTP patients.