Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol.

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn­glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Inflammatory responses in SARS-CoV-2 associated Multisystem Inflammatory Syndrome and Kawasaki Disease in children: An observational study.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe inflammatory disease in children related to SARS-CoV-2 with multisystem involvement including marked cardiac dysfunction and clinical symptoms that can resemble Kawasaki Disease (KD). We hypothesized that MIS-C and KD might have commonalities as well as unique inflammatory responses and studied these responses in both diseases. In total, fourteen children with MIS-C (n=8) and KD (n=6) were included in the period of March-June 2020. Clinical and routine blood parameters, cardiac follow-up, SARS-CoV-2-specific antibodies and CD4+ T-cell responses, and cytokine-profiles were determined in both groups. In contrast to KD patients, all MIS-C patients had positive Spike protein-specific CD3+CD4+ T-cell responses. MIS-C and KD patients displayed marked hyper-inflammation with high expression of serum cytokines, including the drug-targetable interleukin (IL)-6 and IFN-γ associated chemokines CXCL9, 10 and 11, which decreased at follow-up. No statistical differences were observed between groups. Clinical outcomes were all favourable without cardiac sequelae at 6 months follow-up. In conclusion, MIS-C and KD-patients both displayed cytokine-associated hyper-inflammation with several high levels of drug-targetable cytokines.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections.

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide’s and Fluspirilene’s efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

[Better vaccines against tuberculosis]. / Betere vaccins tegen tuberculose.

In the past 2 years, research on new tuberculosis vaccines and correlates of immunity against tuberculosis has led to significant breakthroughs. This gives real hope for the development and evaluation of effective, life-saving tuberculosis vaccines. These are urgently needed to control the tuberculosis pandemic and to fight increasing multidrug resistance of Mycobacterium tuberculosis. Effective tuberculosis vaccines, as well as more effective drugs and better diagnostics, are the cornerstone of the WHO End TB Strategy.

Use of resuscitation promoting factors to screen for tuberculosis infection in household-exposed children in The Gambia.

BACKGROUND: Interferon-γ release assays (IGRA) with Resuscitation promoting factor (Rpf) proteins enhanced tuberculosis (TB) screening and diagnosis in adults but have not been evaluated in children. Children often develop paucibacillary TB and their immune response differs from that of adults, which together affect TB disease diagnostics and immunodiagnostics. We assessed the ability of Rpf to identify infection among household TB-exposed children in The Gambia and investigated their ability to discriminate Mycobacterium tuberculosis complex (MTBC) infection from active TB disease in children. METHODS: Detailed clinical investigations were done on 93 household TB-exposed Gambian children and a tuberculin skin test (TST) was administered to asymptomatic children. Venous blood was collected for overnight stimulation with ESAT-6/CFP-10-fusion protein (EC), purified protein derivative and RpfA, B, C, D and E. Interferon gamma (IFN-γ) production was measured by ELISA in supernatants and corrected for the background level. Infection status was defined by IGRA with EC and TB disease by mycobacterial confirmation and/or clinical diagnosis. We compared IFN-γ levels between infected and uninfected children and between infected and TB diseased children using a binomial logistic regression model while correcting for age and sex. A Receiver Operating Characteristics analysis was done to find the best cut-off for IFN-γ level and calculate sensitivity and specificity. RESULTS: Interferon gamma production was significantly higher in infected (IGRA+, n = 45) than in uninfected (IGRA-, n = 20) children after stimulation with RpfA, B, C, and D (P = 0.03; 0.007; 0.03 and 0.003, respectively). Using RpfB and D-specific IFN-γ cut-offs (33.9 pg/mL and 67.0 pg/mL), infection was classified with a sensitivity-specificity combination of 73-92% and 77-72% respectively, which was similar to and better than 65-75% for TST. Moreover, IFN-γ production was higher in infected than in TB diseased children (n = 28, 5 bacteriologically confirmed, 23 clinically diagnosed), following RpfB and D stimulation (P = 0.02 and 0.03, respectively). CONCLUSION: RpfB and RpfD show promising results for childhood MTBC infection screening, and both performed similar to and better than the TST in our study population. Additionally, both antigens appear to discriminate between infection and disease in children and thus warrant further investigation as screening and diagnostic antigens for childhood TB.

Differential gene expression of activating Fcγ receptor classifies active tuberculosis regardless of human immunodeficiency virus status or ethnicity.

New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings.

IgA response to ESAT-6/CFP-10 and Rv2031 antigens varies in patients with culture-confirmed pulmonary tuberculosis, healthy Mycobacterium tuberculosis-infected and non-infected individuals in a tuberculosis endemic setting, Ethiopia.

Little attention has been given to the role of antibodies against Mycobacterium tuberculosis (Mtb) infection. We have compared the levels of IgA and IgG against ESAT-6/CFP-10 and Rv2031c antigens in sera of patients with culture-confirmed pulmonary tuberculosis (PTB), healthy Mtb-infected and non-infected individuals in endemic TB settings. Venous blood samples were collected from 166 study participants; sera were separated and assayed by an enzyme-linked immunosorbent assay (ELISA). QuantiFERON-TB Gold In-Tube (QFTGIT) assay was used for the screening of latent TB infection. The mean optical density (OD) values of IgA against ESAT-6/CFP-10 and Rv2031 were significantly higher in sera of patients with culture-confirmed PTB compared with healthy Mtb-infected and non-infected individuals (P < 0.001). The mean OD values of IgG against ESAT-6/CFP-10 and Rv2031 were also significantly higher in sera of patients with culture-confirmed PTB compared with healthy Mtb-infected and non-infected individuals (P < 0.05). The mean OD values of IgA against both antigens were also higher in sera of healthy Mtb-infected cases compared with non-infected individuals. There were positive correlations (P < 0.05) between the level of IFN-γ induced in QFTGIT assay and the OD values of serum IgA against both antigens in healthy Mtb-infected subjects. This study shows the potential of IgA response against ESAT-6/CFP-10 and Rv2031 antigens in discriminating clinical TB from healthy Mtb-infected and non-infected cases. Nevertheless, further well-designed cohort study is needed to fully realize the full potential of this diagnostic marker.

The knowns and unknowns of the immunopathogenesis of tuberculosis.

In this review, we discuss the 'knowns' and 'unknowns' of host defence towards Mycobacterium tuberculosis. With regard to the 'knowns', both protective and immunopathogenic mechanisms are discussed, including recently discovered new pathways of host defence and inflammatory immunopathology. With regard to the 'unknowns', there will be emphasis on unanswered key questions, accompanied by a perspective on needs and priorities for future research. The role of the inflammatory response and its dysregulation in tuberculosis-related immune reconstitution inflammatory syndrome will also be discussed.

Identification of biomarkers for tuberculosis disease using a novel dual-color RT-MLPA assay.

Owing to our lack of understanding of the factors that constitute protective immunity during natural infection with Mycobacterium tuberculosis (Mtb), there is an urgent need to identify host biomarkers that predict long-term outcome of infection in the absence of therapy. Moreover, the identification of host biomarkers that predict (in)adequate response to tuberculosis (TB) treatment would similarly be a major step forward. To identify/monitor multi-component host biomarker signatures at the transcriptomic level in large human cohort studies, we have developed and validated a dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) method, permitting rapid and accurate expression profiling of as many as 60-80 transcripts in a single reaction. dcRT-MLPA is sensitive, highly reproducible, high-throughput, has an extensive dynamic range and is as quantitative as QPCR. We have used dcRT-MLPA to characterize the human immune response to Mtb in several cohort studies in two genetically and geographically diverse populations. A biomarker signature was identified that is strongly associated with active TB disease, and was profoundly distinct from that associated with treated TB disease, latent infection or uninfected controls, demonstrating the discriminating power of our biomarker signature. Identified biomarkers included apoptosis-related genes and T-cell/B-cell markers, suggesting important contributions of adaptive immunity to TB pathogenesis.

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines.

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.

Dynamics of interferon-gamma release assay and cytokine profiles in blood and respiratory tract specimens from mice with tuberculosis and the effect of therapy.

There are limitations on diagnostic methods to differentiate between active and latent tuberculosis (TB), and the prediction of latent progression to TB disease is yet complex. Traditionally, tuberculosis-specific host immune response was visualized using the tuberculin skin test. Nowadays, IFN-γ release assays (IGRA) provide a more specific and sensitive tool, by which exposure to Mtb could be determined. However, the merit of IGRA aids in diagnosing active TB is yet unclear. We adapted IGRA for use in mice, and quantifying bead-based flow cytometry techniques were used to assess cytokine profiles during the course of untreated infection and to investigate the value of IGRA and cytokines as biomarkers for therapy response. High variability of IGRA results during progression of active TB infection related to various phases of infection was obtained. However, a significant decrease in IGRA results and in levels of IFN-γ, IL-17, IP-10 or MIG was observed and appeared to be associated with successful therapy. This outcome does not support the value of IGRA to accurately diagnose active TB or to monitor infection progression. However, IGRA proved to be a useful biomarker to monitor therapy success. In addition, different cytokines might serve as biomarkers.

Detection of Mycobacterium tuberculosis infection in dogs in a high-risk setting.

Dogs infected with Mycobacterium tuberculosis can develop clinical tuberculosis (TB) but there are currently no validated immunological assays for diagnosing this infection in this species. Using a post mortem survey we investigated the prevalence of non-clinical M. tuberculosis infection and clinical TB disease in a high-risk population of dogs and developed and utilised a novel interferon-gamma release assay to determine the risk of transmission of M. tuberculosis from TB patients to contact dogs. The prevalence of clinical TB in dogs from a high-risk setting was 1% (95% CI: 0-5%) while the prevalence of immunological sensitization to M. tuberculosis antigens in dogs living in contact with sputum smear-positive TB patients was 50%. The IGRA proved a useful test of M. tuberculosis infection in dogs and the high levels of transmission of this pathogen from humans to companion dogs should be considered when assessing the zoonotic risks associated with such animals.

Mycobacterium bovis BCG-itis and cervical lymphadenitis due to Salmonella enteritidis in a patient with complete interleukin-12/-23 receptor beta1 deficiency.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder with predisposition to severe, sometimes lethal, disease caused by otherwise poorly virulent, non-tuberculous environmental mycobacteria and poorly virulent salmonellae. In patients with MSMD, mutations have been identified in five genes that encode for the proteins IL-12/IL-23p40, IL-12/ IL-23Rbeta1, IFN-R1, IFN-gammaR2 and STAT1. These proteins play important roles in the type-1 cytokine pathway, which is crucial for human host defence against intracellular pathogens such as mycobacteria and salmonellae. We report a girl with mild Mycobacterium bovis Bacille Calmette-Guérin (BCG) disease and Salmonella enteritidis cervical lymphadenitis. Despite treatment, she has remained a fecal carrier of S. enteritidis for the past 14 years. She was found to have complete IL-12/IL-23Rbeta1 deficiency. A homozygous r.518G>C IL12RB1 mutation was identified, leading to a non-functional R173P substitution in the IL-12/IL-23Rbeta1 protein. This mutation abrogated IL-12/IL-23Rbeta1 cell-surface expression and resulted in complete lack of T cell responsiveness to both IL-12 and IL-23.

Response to Rv2628 latency antigen associates with cured tuberculosis and remote infection.

Interferon-gamma release assays based on region of difference 1 antigens have improved diagnosis of latent tuberculosis infection (LTBI). However, these tests cannot discriminate between recently acquired infection (higher risk of progression to active tuberculosis) and remote LTBI. The objective of the present study was to evaluate the T-cell interferon-gamma responses to Mycobacterium tuberculosis DosR-regulon-encoded antigens (latency antigens) compared with QuantiFERON TB-Gold In-Tube (QFT-GIT) in subjects at different stages of tuberculosis. A total of 16 individuals with remote LTBI and 23 with recent infection were studied; 15 controls unexposed to M. tuberculosis and 50 patients with active tuberculosis and 45 with cured tuberculosis were also analysed. The results indicated that subjects with remote LTBI showed significantly higher whole-blood interferon-gamma responses to M. tuberculosis latency antigen Rv2628 than did individuals with recent infection, active tuberculosis and controls (p<0.003), whereas no significant differences between these groups were found for other latency antigens tested (Rv2626c, Rv2627c, Rv2031c and Rv2032). The proportion of responders to Rv2628 was five-fold higher among QFT-GIT-positive-individuals with remote infection than among those with recently acquired infection. These data suggest that responses to M. tuberculosis latency antigen Rv2628 may associate with immune-mediated protection against tuberculosis. In contact-tracing investigations, these preliminary data may differentiate recent (positive QFT-GIT results without responses to Rv2628) from remote infection (positive to both tests).

The role of interferon-gamma in the increased tuberculosis risk in type 2 diabetes mellitus.

As patients with diabetes mellitus are at increased risk of developing tuberculosis, we hypothesized that this susceptibility to mycobacterial infection is due to a defective Th1-cytokine response. To explore this hypothesis, we examined four groups of subjects in Indonesia: 23 patients with tuberculosis, 34 patients with tuberculosis and diabetes, 32 patients with diabetes only and 36 healthy controls. Ex-vivo production of interferon (IFN)gamma, tumour necrosis factor-alpha and interleukin (IL)-1beta, 6, 10, -12 and -4 was measured following stimulation with Mycobacterium tuberculosis, Escherichia coli lipopolysaccharide and phytohaemagglutinin. Patients with active tuberculosis were found to have lower IFNgamma levels and a higher production of other pro-inflammatory cytokines and IL-4, both in the presence and absence of diabetes. Diabetes patients without tuberculosis, however, showed strongly reduced non-specific IFNgamma production, which is essential for inhibition of the initial growth of M. tuberculosis. Our data suggest that a defective non-specific immune response in diabetes may contribute to an increased susceptibility to develop tuberculosis.

Plasma granulysin levels and cellular interferon-gamma production correlate with curative host responses in tuberculosis, while plasma interferon-gamma levels correlate with tuberculosis disease activity in adults.

Granulysin is a recently identified cytolytic protein which is expressed by human cytotoxic T-lymphocytes and natural killer (NK)-cells, and has broad antimicrobial and tumoricidal activity. Circulating granulysin levels are associated with T- and NK-cell activity, and may thus reflect protection-associated cellular immune responses. In a case-control study in Indonesia, a highly tuberculosis (TB)-endemic country, we therefore determined plasma granulysin levels in adults with active pulmonary TB before, during, and after TB treatment, both in mild/moderate-TB and advanced-TB patients, and compared these to healthy neighbourhood controls. Adults with active pulmonary TB had significantly lower plasma granulysin levels compared to controls. After 2 months of anti-TB therapy, levels in TB patients had significantly increased, reaching values similar to those in controls. Plasma granulysin levels further increased after completion of TB therapy, being significantly higher than those in controls. Plasma granulysin levels correlated inversely with TB disease activity but not with TB disease severity. In contrast, plasma interferon-gamma (IFN-gamma) levels were significantly higher in active TB cases than in controls, normalised during treatment and correlated with both TB disease activity and TB disease severity. At the cellular level, granulysin and IFN-gamma expression both correlated inversely with disease activity. Interestingly, granulysin was predominantly expressed by IFN-gamma negative T-cells, suggesting that the cellular sources of IFN-gamma and granulysin in TB are partly non-overlapping. The observation that plasma granulysin levels and cellular IFN-gamma production correlate with curative host responses in pulmonary tuberculosis points to a potentially important role of granulysin, next to IFN-gamma, in host defence against M. tuberculosis.

Effect of tuberculin skin testing on a Mycobacterium tuberculosis-specific interferon-gamma assay.

Recently, interferon-gamma release assays (IGRA) for specific diagnosis of Mycobacterium tuberculosis infection have become available. In recent UK tuberculosis (TB) guidelines, it has been advised to screen for latent M. tuberculosis infection using the tuberculin skin test (TST), followed by IGRA if the TST is positive. Since TST can boost immune responses to tuberculin, the present authors evaluated whether TST administration affects the result of QuantiFERON-TB Gold in-tube (QFT-GIT), a whole blood-based IGRA. QFT-GIT was performed on the day of TST administration and the day of reading in 15 TST-negative subjects, 46 TST-positive subjects with recent or remote exposure to M. tuberculosis and five cured TB patients. No systematic boosting of QFT-GIT responses from negative to positive was observed. Only in a few TST-positive persons did TST enhance pre-existing QFT-GIT responses. Screening for latent Mycobacterium tuberculosis infection using tuberculin skin testing followed by interferon-gamma release assays on the day of reading is a reliable approach, as the specificity of QuantiFERON-TB Gold in-tube is not affected by prior tuberculin skin test administration.

Evaluation of recombinant serine-rich 45-kDa antigen (ML0411) for detection of antibodies in leprosy patients.

The potential of the recombinant serine-rich 45-kDa antigen (ML0411) of Mycobacterium leprae to aid in detecting M. leprae-specific serum antibodies was assessed by an enzyme-linked immunosorbent assay (ELISA) in leprosy patients and controls comprising of tuberculosis patients, other unrelated skin-diseased patients and healthy individuals from India. All 18 multibacillary (MB) and 18/38 (47.4%) of the paucibacillary (PB) leprosy patients were found positive. None of the controls was positive, yielding complete (0/49) specificity in the series tested here. On the other hand, an anti-phenolic glycolipid-1 (PGL-I) antibody-detecting assay yielded detectable responses in 94.4% (17/18) of MB and 36.8% (14/38) of PB leprosy patients. Only two of 49 (4.1%) controls were positive, giving a specificity of 95.9%. Further, there was a good concordance (agreement of 83.8%; chi(2) = 40.3, P < 0.001; kappa = 0.63) between the two assays. Thus, the 45-kDa-based assay was slightly better than anti-PGL-I antibody-detecting assay. Interestingly, when combining the results of both the assays together for all leprosy patients (MB + PB), the combined sensitivity was significantly higher than that of the anti-PGL-I antibody-detecting ELISA alone (73.2% versus 55.4%; P < 0.05), but not (P > 0.05) compared with the 45-kDa antigen-based assay alone. Similarly, in case of PB patients, using both assays in combination, the sensitivity was significantly higher compared with anti-PGL-I antibody-detecting assay alone (60.5% versus 36.8%; P < 0.05). While adopting the combinatorial approach, the specificity remained invariably high (>95%). In conclusion, the results of the present study indicate that the M. leprae 45-kDa protein is a potent B-cell antigen and may be a useful serodiagnostic reagent.