Jacqueline M. Achkar

B cells and antibodies in the defense against Mycobacterium tuberculosis infection

Better understanding of the immunological components and their interactions necessary to prevent or control Mycobacterium tuberculosis (Mtb) infection in humans is critical for tuberculosis (TB) vaccine development strategies. Although the contributory role of humoral immunity in the protection against Mtb infection and disease is less defined than the role of T cells, it has been well‐established for many other intracellular pathogens. Here we update and discuss the increasing evidence and the mechanisms of B cells and antibodies in the defense against Mtb infection. We posit that B cells and antibodies have a variety of potential protective roles at each stage of Mtb infection and postulate that such roles should be considered in the development strategies for TB vaccines and other immune‐based interventions.

The role of B cells and humoral immunity in Mycobacterium tuberculosis infection

Mycobacterium tuberculosis remains a major public health burden. It is generally thought that while B cell- and antibody-mediated immunity plays an important role in host defense against extracellular pathogens, the primary control of intracellular microbes derives from cellular immune mechanisms. Studies on the immune regulatory mechanisms during infection with M. tuberculosis, a facultative intracellular organism, has established the importance of cell-mediated immunity in host defense during tuberculous infection. Emerging evidence suggest a role for B cell and humoral immunity in the control of intracellular pathogens, including obligatory species, through interactions with the cell-mediated immune compartment. Recent studies have shown that B cells and antibodies can significantly impact on the development of immune responses to the tubercle bacillus. In this review, we present experimental evidence supporting the notion that the importance of humoral and cellular immunity in host defense may not be entirely determined by the niche of the pathogen. A comprehensive approach that examines both humoral and cellular immunity could lead to better understanding of the immune response to M. tuberculosis.

Protease Mutations in HIV-1 Non-B Strains Infecting Drug-Naive Villagers of Cameroon

To describe the presence of protease inhibitor (PI) resistance-associated mutations and subtype distribution in drug-naive villagers of six provinces of Cameroon, we sequenced the protease (PR) gene (297 bp) of 128 viruses. Secondary PI resistance-associated mutations were identified at five sites: L10I/V (16%), K20R (8%), M36I (98%), L63P (13%), and V77I (6%). No primary mutation in the PR was identified. Of the 128 specimens analyzed, subtypes A (11%), C(2%), D (6%), F2 (3%), G (6%), H (0.8%), J (6%), and CRF02_AG (60%) were identified. The mutations identified were not characteristic to any particular subtype. The absence of primary mutations, in addition to the few secondary mutations, gives good perspectives for PI treatment interventions in these rural areas.

Adjunctive tests for diagnosis of tuberculosis: Serology, ELISPOT for site-specific lymphocytes, urinary lipoarabinomannan, string test, and fine needle aspiration

The diagnostic gold standard for active tuberculosis (TB) is the detection of Mycobacterium tuberculosis (MTB) by culture or molecular methods. However, despite its limited sensitivity, sputum smear microscopy is still the mainstay of TB diagnosis in resource-limited settings. Consequently, diagnosis of smear-negative pulmonary and extrapulmonary TB remains challenging in such settings. A number of novel or alternative techniques could provide adjunctive diagnostic use in the context of difficult-to-diagnose TB. These may be especially useful in certain patient groups such as persons infected with human immunodeficiency virus (HIV) and children, who are disproportionably affected by smear-negative and extrapulmonary disease and who are also most adversely affected by delays in TB diagnosis and treatment. We review a selection of these methods that are independent of nucleic acid amplification techniques and could largely be implemented in resource-limited settings in current or adapted versions. Specifically, we discuss the diagnostic use and potential of serologic tests based on detection of antibodies to MTB antigens; interferon gamma release assays using site-specific lymphocytes; detection of lipoarabinomannan, a glycolipid of MTB, in urine; the string test, a novel technique to retrieve lower respiratory tract samples; and fine needle aspiration biopsy of lymph nodes.

Differences in Clinical Presentation among Persons with Pulmonary Tuberculosis: A Comparison of Documented and Undocumented Foreign-Born versus US-Born Persons

BACKGROUND Most cases of tuberculosis (TB) in the United States are diagnosed in foreign-born persons, and undocumented foreign-born persons may face particular barriers to timely access to health care services. This study investigates whether differences in clinical presentations among persons with pulmonary TB are associated with foreign birth or documentation status. METHODS In this cross-sectional study, we reviewed the medical records of patients who had received a diagnosis of microbiologically proven pulmonary TB at a New York City public hospital during the period April 1999 through March 2005. Three groups of patients with pulmonary TB (US-born persons, foreign-born persons with documents, and undocumented, foreign-born persons) were defined and compared at presentation. Odds ratios (ORs) for a symptom duration >or=8 weeks before hospital admission for each group were estimated using logistic regression. RESULTS Among 194 subjects with newly diagnosed pulmonary TB, 61 (31%) were US born, 62 (32%) were documented foreign-born persons, and 71 (37%) were undocumented foreign-born persons. Undocumented foreign-born persons presented with significantly higher frequencies of cough (P = .020) and hemoptysis P = .012 and had a significantly longer median duration of symptoms, compared with US-born persons (8 vs. 4 weeks; P = .023). No statistically significant differences between documented foreign-born and US-born persons were observed. Multivariate analysis revealed that undocumented status (compared with being US born; adjusted OR, 4.1; 95% confidence interval, 1.7-10.2; P = .0002) and being unemployed (adjusted OR, 2.2; 95% CI, 1.1-4.5; P = .023) were independently associated with a prolonged symptom duration (i.e., >or=8 weeks). CONCLUSIONS Undocumented status was associated with an increased frequency of cough and hemoptysis and a longer duration of symptoms before medical evaluation for pulmonary TB. Whether reducing barriers to health services for undocumented foreign-born persons could enhance TB control deserves additional study.

Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

ABSTRACT Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. IMPORTANCE This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation. This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

Mycobacterium tuberculosis Malate Synthase- and MPT51-Based Serodiagnostic Assay as an Adjunct to Rapid Identification of Pulmonary Tuberculosis

ABSTRACT The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from ∼80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.

Combined Use of Serum and Urinary Antibody for Diagnosis of Tuberculosis

Efforts to devise immunoassays for tuberculosis (TB) that can be adapted to rapid formats are ongoing. The present study was aimed at determining whether urinary anti-Mycobacterium tuberculosis antibodies are present in patients with TB, to evaluate the feasibility of developing a urine antibody-based diagnostic test. Urinary antibodies directed against the culture filtrate proteins of M. tuberculosis, MPT 32, and the 81-kDa GlcB protein were detectable in patients with TB, although the sensitivity of antibody detection was lower (53%-64%), compared with serum antibodies (68%-77%). Surprisingly, with all 3 antigens, the use of paired serum and urine samples provided higher sensitivities of antibody detection than either single specimen, and anti-GlcB antibodies were present in the serum and/or urine of 39 (90%) of 43 smear-positive patients with TB. Although, with the current methods and antigens, the level of sensitivity is insufficient to design a urinary antibody diagnostic test, these studies provide the foundation for further studies on the development of a urine antibody-based immunoassay for TB.

Antibodies against immunodominant antigens of Mycobacterium tuberculosis in subjects with suspected tuberculosis in the United States compared by HIV status.

ABSTRACT The immunodominance of Mycobacterium tuberculosis proteins malate synthase (MS) and MPT51 has been demonstrated in case-control studies with patients from countries in which tuberculosis (TB) is endemic. The value of these antigens for the serodiagnosis of TB now is evaluated in a cross-sectional study of pulmonary TB suspects in the United States diagnosed to have TB, HIV-associated TB, or other respiratory diseases (ORD). Serum antibody reactivity to recombinant purified MS and MPT51 was determined by enzyme-linked immunosorbent assays (ELISAs) of samples from TB suspects and well-characterized control groups. TB suspects were diagnosed with TB (n = 87; 49% sputum microscopy negative, 20% HIV+) or ORD (n = 63; 58% HIV+). Antibody reactivity to MS and MPT51 was significantly higher in U.S. HIV+/TB samples than in HIV−/TB samples (P < 0.001), and it was significantly higher in both TB groups than in control groups with latent TB infection (P < 0.001). Antibody reactivity to both antigens was higher in U.S. HIV+/TB samples than in HIV+/ORD samples (P = 0.052 for MS, P = 0.001 for MPT51) but not significantly different between HIV−/TB and HIV−/ORD. Among U.S. HIV+ TB suspects, a positive anti-MPT51 antibody response was strongly and significantly associated with TB (odds ratio, 11.0; 95% confidence interval, 2.3 to 51.2; P = 0.002). These findings have implications for the adjunctive use of TB serodiagnosis with these antigens in HIV+ subjects.

Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction

Background. The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. Methods. Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. Results. Immunoglobulin G (IgG) responses to AM increased significantly 4–8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. Conclusions. Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.