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The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: We report a first-in-human trial evaluating safety and immunogenicity of a recombinant BCG, AERAS-422, over-expressing TB antigens Ag85A, Ag85B, and Rv3407 and expressing mutant perfringolysin., Methods: This was a randomized, double-blind, dose-escalation trial in HIV-negative, healthy adult, BCG-naïve volunteers, negative for prior exposure to Mtb, at one US clinical site. Volunteers were randomized 2:1 at each dose level to receive a single intradermal dose of AERAS-422 (>10(5)-<10(6)CFU=low dose, ≥10(6)-<10(7)CFU=high dose) or non-recombinant Tice BCG (1-8×10(5)CFU). Randomization used an independently prepared randomly generated sequence of treatment assignments. The primary and secondary outcomes were safety and immunogenicity, respectively, assessed in all participants through 182days post-vaccination. ClinicalTrials.gov registration number: NCT01340820., Findings: Between Nov 2010 and Aug 2011, 24 volunteers were enrolled (AERAS-422 high dose, n=8; AERAS-422 low dose, n=8; Tice BCG, n=8); all were included in the safety and immunogenicity analyses. All 24 subjects had at least one adverse event, primarily expected local reactions. High dose AERAS-422 vaccination induced Ag85A- and Ag85B-specific lymphoproliferative responses and marked anti-mycobacterial activity in a whole blood bactericidal activity culture assay (WBA), but was associated with varicella zoster virus (VZV) reactivation in two vaccinees. These volunteers displayed high BCG-specific IFN-γ responses pre- and post-vaccination possibly predisposing them to autocrine/paracrine negative regulation of immune control of latent VZV. A systems biology transcriptomal approach identified positive correlations between post-vaccination T cell expression modules and WBA, and negative correlations between post-vaccination monocyte expression modules and WBA. The expression of one key macrophage marker (F4/80) was constitutively elevated in the two volunteers with zoster., Interpretation: The unexpected development of VZV in two of eight healthy adult vaccine recipients resulted in discontinuation of AERAS-422 vaccine development. Immunological and transcriptomal data identified correlations with the development of TB immunity and VZV that require further investigation., Funding: Aeras, FDA, Bill and Melinda Gates Foundation., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Vaccines to protect against tuberculosis (TB) are urgently needed. We performed a case-control analysis to identify immune correlates of TB disease risk in Bacille Calmette-Guerin (BCG) immunized infants from the MVA85A efficacy trial. Among 53 TB case infants and 205 matched controls, the frequency of activated HLA-DR(+) CD4(+) T cells associates with increased TB disease risk (OR=1.828, 95% CI=1.25-2.68, P=0.002, FDR=0.04, conditional logistic regression). In an independent study of Mycobacterium tuberculosis-infected adolescents, activated HLA-DR(+) CD4(+) T cells also associate with increased TB disease risk (OR=1.387, 95% CI=1.068-1.801, P=0.014, conditional logistic regression). In infants, BCG-specific T cells secreting IFN-γ associate with reduced risk of TB (OR=0.502, 95% CI=0.29-0.86, P=0.013, FDR=0.14). The causes and impact of T-cell activation on disease risk should be considered when designing and testing TB vaccine candidates for these populations.

Background: MVA85A and AERAS-402 are two clinically advanced viral vectored TB vaccine candidates expressing Mycobacterium tuberculosis antigens designed to boost BCG-induced immunity. Clinical trials with candidate malaria vaccines have demonstrated that adenoviral vector based priming immunisation, followed by MVA vector boost, induced high levels of immunity. We present the safety and immunogenicity results of the first clinical trial to evaluate this immunisation strategy in TB., Methods: In this phase 1, open-label trial, 40 healthy previously BCG-vaccinated participants were enrolled into three treatment groups and vaccinated with 1 or 2 doses of AERAS-402 followed by MVA85A; or 3 doses of AERAS-402., Results: Most related adverse events (AEs) were mild and there were no vaccine related serious AEs. Boosting AERAS-402 with MVA85A significantly increased Ag85A-specific T-cell responses from day of vaccination. Two priming doses of AERAS-402 followed by MVA85A boost, resulted in a significantly higher AUC post-peak Ag85A response compared to three doses of AERAS-402 and historical data with MVA85A vaccination alone. The frequency of CD8+ T-cells producing IFN-γ, TNF-α and IL-2 was highest in the group receiving two priming doses of AERAS-402 followed by MVA85A., Conclusions: Vaccination with AERAS-402 followed by MVA85A was safe and increased the durability of antigen specific T-cell responses and the frequency and polyfunctionality of CD8+ T-cells, which may be important in protection against TB. Further clinical trials with adenoviral prime-MVA85A boost regimens are merited to optimise vaccination intervals, dose and route of immunisation and to evaluate this strategy in the target population in TB high burden countries., Trial Registration: ClinicalTrials.gov NCT01683773.

Background: Several novel tuberculosis vaccines are currently in clinical trials, including AERAS-402, an adenovector encoding a fusion protein of Mycobacterium tuberculosis antigens 85A, 85B, and TB10.4. A multicentred trial of AERAS-402 safety and immunogenicity in healthy infants was conducted in three countries in sub-Saharan Africa, using an adaptive design., Methods: In a double-blind, randomised, placebo-controlled, dose-finding trial, we enrolled BCG-vaccinated, HIV-uninfected infants aged 16-26 weeks. Infants in the safety/dose-finding phase received two doses of AERAS-402 across three dose levels, or placebo, intramuscularly on days 0 and 28. Infants in the expanded safety phase received three doses of the highest dose level, with the 3rd dose at day 280. Follow up for safety and immunogenicity was for up to two years., Results: We enrolled 206 infants (52 placebo and 154 AERAS-402 recipients) into the dose-finding phase and 281 (141 placebo and 140 AERAS-402 recipients) into the expanded safety phase. Safety data were acceptable across all dose levels. No vaccine-related deaths were recorded. A single serious adverse event of tachypnoea was deemed related to study vaccine. Antibodies directed largely against Ag85A and Ag85B were detected. Low magnitude CD4+ and CD8+ polyfunctional T cell responses were observed at all dose levels. The addition of a third dose of AERAS-402 at the highest dose level did not increase frequency or magnitude of antibody or CD8+ T cell responses., Conclusions: AERAS-402 has an acceptable safety profile in infants and was well tolerated at all dose levels. Response rate was lower than previously seen in BCG vaccinated adults, and frequency and magnitude of antigen-specific T cells were not increased by a third dose of vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The safety and immunogenicity of a replication deficient adenovirus serotype 35 tuberculosis (TB) vaccine containing gene inserts for Antigens (Ag) 85A, Ag85B and TB10.4 (AERAS-402/AD35.TB-S) was evaluated in previously BCG vaccinated, HIV-infected South African adults with baseline CD4 counts >350 cells/mm(3)., Methods: Subjects were randomized (1:1) to receive two doses of either intramuscular AERAS-402/AD35.TB-S or placebo at month 0 and at month 1. Participants were monitored for adverse events 28 days after each vaccination and for serious adverse events over 12 months. CD4(+) and CD8(+) T-cell and antibody responses to vaccine antigens were evaluated post first and second vaccination., Results: 26 subjects were randomly assigned to receive AERAS-402/AD35.TB-S (N=13) or placebo (N=13). The mean age was 29.0 years, all were Black-African, 88.5% were female, 46.2% were QuantiFERON Test (QFT) positive at baseline, and the median CD4 count was 559.5 cells/mm(3), all similar by treatment group. All subjects received their first vaccination and 24 subjects received their second vaccination. Injection site reactions and some systemic reactions were reported more commonly in the AERAS-402/AD35.TB-S versus placebo recipients. AERAS-402/AD35.TB-S did not appear to influence CD4 counts and HIV-1 viral load over the course of study follow-up. AERAS-402/AD35.TB-S induced a mixed CD4(+) T-cell and CD8(+) T-cell responses to Ag85B. The CD4(+) T-cell responses peaked to Ag85A and Ag85B 14 days after the second vaccination and had declined by Day 182. AERAS-402/AD35.TB-S predominantly induced CD4(+) T-cells expressing three (IFN-γ, TNF, IL-2) or two (IL-2 and TNF) cytokines, two weeks after the last vaccination, which did not differ by baseline Quantiferon test status. AERAS-402/AD35.TB-S induced strong Ag85A and Ag85B specific antibody responses, particularly after the second vaccination., Conclusion: AERAS-402/AD35.TB-S was well tolerated, safe and induced predominantly polyfunctional CD4(+) and CD8(+) T-cell responses to vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bacille Calmette-Guérin (BCG), the only licensed vaccine for the prevention of tuberculosis (TB), provides only limited protection against certain forms of Mycobacterium tuberculosis (Mtb) infection. While infection with Mtb can be treated with antibiotics, the therapy is expensive, toxic, and requires several months for treatment. In addition, the emergence of drug resistant strains limits the impact of antibiotics and underlines the importance of developing a more effective vaccine to control this disease. Given that pulmonary TB is the most common form of the disease, a vaccine capable of inducing lung-resident immunity may be advantageous for combating this infection. New advances in pulmonary delivery make this route of vaccination feasible and affordable. Here, we evaluate the safety and immunogenicity of an aerosolized Ad35-based vaccine, AERAS-402, delivered to the lungs in nonhuman primates as part of a GLP acute and chronic toxicology and safety study. In this study, animals received three high doses (1 x 10(11) vp) of AERAS-402 by inhalation via a nebulizer at 1-week intervals. Aerosol delivery of AERAS-402 resulted in an increase in relative lung weights as well as microscopic findings in the lungs, mediastinal lymph nodes, bronchus-associated lymphatic tissue, and the naso-oropharynx that were consistent with the induction of an immune response during the acute phase. These findings resolved by the chronic phase and were considered to be non-adverse. Furthermore, we observed transient vaccine-specific immune responses in the peripheral blood as well as sustained high-level polyfunctional CD4(+) and CD8(+) T cell responses in the bronchoalveolar lavage fluid of vaccinated nonhuman primates. The data suggest that pulmonary delivery of Ad35-based vaccines can be safe and can induce potent lung-resident immunity.

A powder vaccine intended for aerosol delivery was formulated by spray drying the Ad35-vectored tuberculosis (TB) AERAS-402 vaccine with mannitol-based stabilizers. Thermodynamic properties, water absorption, particle size distribution and morphology of the powders were evaluated. Virus survival during spray drying and storage was determined by medium Tissue Culture Infectious Dose (TCID(50)). A mannitol-based powder (mannitol-cyclodextrin-trehalose-dextran, MCTD) had a narrow size distribution with a median volume diameter around 3.2-3.5microm (suitable for pulmonary vaccination of humans) and good aerosolization characteristics. The spray dry powders generated from mannitol-based formulations were hydrophobic, which benefits virus survival during both production and storage at 4 degrees C, 25 degrees C and 37 degrees C as compared to the hygroscopic formulations (trehalose, sucrose, dextran, PVP, leucine). In conclusion, this study demonstrates that it is possible to produce in a one-step spray drying process a stable dry powder formulation of a TB vaccine suitable for mass vaccination.

Despite the extensive success with the introduction of M. bovis Bacille Calmette-Guérin (BCG), tuberculosis (TB) remains a major global epidemic infecting between 8 and 9 million people annually with an estimated 1.7 million deaths each year. However, because of its demonstrated effectiveness against some of the most severe forms of childhood TB, it is now realized that BCG vaccination of newborns is unlikely to be replaced. Therefore, BCG or an improved BCG will continue to be used as a prime TB vaccine and there is a need to develop effective boost vaccines that would enhance and prolong the protective immunity induced by BCG prime immunization. We report on a heterologous booster approach using two highly immunogenic TB antigens comprising Ag85B and TB10.4 (HyVac4) delivered as a fusion molecule and formulated in the proprietary adjuvant IC31. This vaccine was found to be immunogenic and demonstrated greater protection in the more stringent guinea pig model of pulmonary tuberculosis than BCG alone when used in a prime/boost regimen. Significant difference in lung involvement was observed for all animals in the HyVac4 boosted group compared to BCG alone regardless of time to death or sacrifice. A vaccine toxicology study of the HyVac4:IC31 regimen was performed and it was judged safe to advance the vaccine into clinical trials. Therefore, all non-clinical data supports the suitability of HyVac4 as a safe, immunogenic, and effective vaccination in a prime-boost regimen with BCG.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.

New post-exposure tuberculosis vaccination strategies are being developed to prevent disease in individuals latently infected with Mycobacterium tuberculosis. However, concerns about the potential induction of deleterious Koch-like reactions after immunization of persons with latent tuberculosis has limited progress in assessing the effectiveness of post-exposure vaccination. To evaluate the safety of immunization after M. tuberculosis infection, two mouse models were established, a drug treatment low bacterial burden model and an active disease model. Twelve different M. tuberculosis antigen preparations and vaccines (including DNA, subunit, viral vectored, and live, attenuated vaccines) were evaluated using these mouse models. In the low bacterial burden model, post-exposure vaccination did not induce significant reactivational disease and only injection of BCG evoked increases in lung inflammatory responses at 1 month after the immunizations. Additionally, although significant increases in lung inflammation were seen for animals injected with the hps65 DNA vaccine or a M. tuberculosis culture supernatant preparation, no differences in the survival periods were detected between vaccinated and non-vaccinated mice at 10 months post-immunization using the low bacterial burden model. For the active disease model, significantly more lung inflammation was observed at 1 month after administration of the hsp65 DNA vaccine but none of the antigen preparations tested increased the lung bacterial burdens at this early time point. Furthermore, vaccination of diseased mice with BCG or TB DNA vaccines did not significantly affect mortality rates compared to non-vaccinated controls at 10 months post-immunization. Overall, these data suggest that while the potential risk of inducing Koch-like reactions is low after immunization of persons with latent tuberculosis, extreme caution is still needed as post-exposure vaccines progress from pre-clinical experiments into the initial phases of clinical testing.

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.

Evaluation of expression of 16 PE&#95;PGRS genes present in Mycobacterium tuberculosis under various growth conditions demonstrated constitutive expression of 7 genes, variable expression of 7 genes, and no expression of 2 genes. An inverse expression profile for genes PE&#95;PGRS16 and PE&#95;PGRS26 was observed to occur in macrophages and in mice infected with M. tuberculosis. Variable expression of PE&#95;PGRS proteins could have implications for their role in the immunopathogenesis of tuberculosis.

Infection of mice with Mycobacterium avium or immunization with a novel PE gene expressed by M. avium (MaPE) showed that a dominant T-cell immune response was elicited. Immunization with an MaPE DNA vaccine protected mice against an aerosol challenge with Mycobacterium tuberculosis, suggesting that mycobacteria express PE antigens with cross-protective T-cell epitopes.

Research on mycobacteria-specific PE&#95;PGRS genes indicates that they code for cell surface proteins that may influence virulence and the infection of host cells by mycobacteria. In the studies presented here, we have expressed the PE&#95;PGRS 33 gene in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that it survives better in macrophage cultures, in vitro as well as in mice after intraperitoneal administration, than the parental strain containing the vector only or a strain expressing only the PE domain of PE&#95;PGRS 33. In macrophages, enhanced colonization by the M. smegmatis expressing PE&#95;PGRS 33 was associated with macrophage aggregation and clearance of macrophage monolayers, visible cell necrosis and significantly greater levels of TNF (TNF-alpha) in the cultures compared with controls. The presence of macrophage cell necrosis was confirmed by measurement of significantly greater levels of lactate dehydrogenase and nucleosomes in the supernatants of the macrophage cultures infected with M. smegmatis expressing PE&#95;PGRS 33. Antibodies directed against TNF partially reduced cytolysis, suggesting that this cytokine is critical but not sufficient for the observed macrophage necrosis and enhanced mycobacterial survival. These results extend earlier observations, which suggested that PE&#95;PGRS proteins may have a role in the pathogenesis of mycobacterial disease and that there may be a specific role for these proteins in influencing host cell responses to infection.

The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a approximately 0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) V beta expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR V beta families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR V beta families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR V beta families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR V beta families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR V beta family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) and Mycobacterium bovis BCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR V beta families readily recalled by PPD, while the disease status of the patients was attributed to TCR V beta downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR V beta usage.

Three genes encoding the secreted proteins (antigen 85-A, B, and C) of Mycobacterium avium subsp. paratuberculosis were cloned, sequenced and studied. The complete sequences of these three 85-complex proteins revealed their similarity with 85-complex proteins of other mycobacterial species. Specifically, these sequences showed 99% homology with M. avium 85-complex protein sequences. The multiple homology analysis of these sequences revealed that variations occur at only certain amino acid positions and this is true with all other 85-complex protein sequences of mycobacteria. However, the proposed three conserved regions involved in fibronectin binding in other mycobacteria were observed in N-terminal regions 85A, B and C of M. avium subsp. paratuberculosis.

HLA DRB1*02 and its subtypes predispose individuals for a far-advanced sputum-positive pulmonary tuberculosis transcending ethnic boundaries. Mycobacterium bovis BCG does not afford the desired protection against adult pulmonary tuberculosis, and a spectrum of immune reactivity exists in controls and hospital contacts. All of these findings have been identified and demonstrated in areas of endemicity. Skewing of immunity from protective to pathogenic may involve a shift in the Th1-Th2 paradigm. To elaborate these ideas, we studied gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-10 cytokine expression in 71 adult pulmonary tuberculosis patients and 74 controls from areas of endemicity in south India by 48-h microculture and reverse transcription-PCR. Most of the patients and controls expressed IFN-gamma de novo, and in the presence of purified protein derivative (PPD), all of them expressed significantly higher levels of IFN-gamma, suggesting a PPD-specific recall memory. HLA DRB1* allele-dependent IFN-gamma expression was identified only in controls, suggesting a skewing of the immune response in patients. In contrast to the case for IFN-gamma, only some patients and controls expressed IL-4 or IL-10 (Th2 profile); thus, the Th1 profile was identifiable only by a nonexpression of IL-4 or IL-10 in this area of endemicity. The Th2 profile was associated with HLA non-DRB1*02 and BCG scar-negative status in patients, attributing a significant risk (odds ratio = 2.074; 95% confidence interval = 0.612 to 7.07). It is possible that Mycobacterium tuberculosis (PPD)-specific IL-10 is expressed preemptively in unvaccinated (BCG scar-negative) individuals with a non-DR2 genetic background by chronic exposure in this area of endemicity and leads to pulmonary tuberculosis of adults.

Setting: Tuberculosis is endemic in south India: sputum positive pulmonary tuberculosis is predisposed by HLA-DR2 in south India and few other populations of the world., Objective: To study HLA-DRB1, DQB1, DQA1 and DPB1 allelic polymorphism in pulmonary tuberculosis patients and endemic controls from south India., Design: One hundred and twenty-six, sputum positive pulmonary tuberculosis patients and 87, endemic controls, from Madurai were studied for MHC class II allelic polymorphism by PCR-SSOP method. XI IHWC primers and probes and non-radioactive probing methods were employed., Results: HLA DRB1*1501 and DQB1*0601 predisposed for pulmonary tuberculosis (DRB1*1501: odds ratio (OR) = 2.68, 95% confidence interval (CI) = 1.30-5.89, P value (P) = 0.013, aetiological fraction (EF) = 0.17; DQB1*0601: OR = 2.32, CI = 1.29-4.27, P = 0.008, EF = 0.26). Haplotype DRB1*1501-DQB1*0601 was higher in patients (1324 per 10,000, X2 = 27.07) than controls (F = 404/10,000, X2 = 8.84). In a subset of 63 caste matched samples, DPB1*04 was preventive (OR = 0.45, CI = 0.21-0.95, P = 0.036, PF = 0.26): the distributions of DRB1*1501-DQB1*0601-DPB1*04 phenotypes were different between patients and controls (P = 0.0092). These alleles were predominant in patients and controls of T5SU caste., Conclusion: HLA-DRB1*1501 and DQB1*0601 predisposed to sputum positive pulmonary tuberculosis, and DPB1*04 was preventive and epistatic to this risk. Caste T5SU is an ideal model to study immunology of tuberculosis.