Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates.

BACKGROUND: Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. METHODS: In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. FINDINGS: Macaques that developed LTBI (n = 36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n = 21; p < 0.01, q < 0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p < 0.05) and increasing airway IgG responses to some proteins over time. INTERPRETATION: Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. FUNDING: NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Features and protective efficacy of human mAbs targeting Mycobacterium tuberculosis arabinomannan.

A better understanding of the epitopes most relevant for antibody-mediated protection against tuberculosis (TB) remains a major knowledge gap. We have shown that human polyclonal IgG against the Mycobacterium tuberculosis (M. tuberculosis) surface glycan arabinomannan (AM) and related lipoarabinomannan (LAM) is protective against TB. To investigate the impact of AM epitope recognition and Fcγ receptor (FcγR) binding on antibody functions against M. tuberculosis, we isolated a high-affinity human monoclonal antibody (mAb; P1AM25) against AM and showed its binding to oligosaccharide (OS) motifs we previously found to be associated with in vitro functions of human polyclonal anti-AM IgG. Human IgG1 P1AM25, but not 2 other high-affinity human IgG1 anti-AM mAbs reactive with different AM OS motifs, enhanced M. tuberculosis phagocytosis by macrophages and reduced intracellular growth in an FcγR-dependent manner. P1AM25 in murine IgG2a, but neither murine IgG1 nor a non-FcγR-binding IgG, given intraperitoneally prior to and after aerosolized M. tuberculosis infection, was protective in C57BL/6 mice. Moreover, we demonstrated the protective efficacy of human IgG1 P1AM25 in passive transfer with M. tuberculosis-infected FcγR-humanized mice. These data enhance our knowledge of the important interplay between both antibody epitope specificity and Fc effector functions in the defense against M. tuberculosis and could inform development of vaccines against TB.

Impact of Mycobacterium tuberculosis Glycolipids on the CD4+ T Cell-Macrophage Immunological Synapse.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

A non-neutralizing glycoprotein B monoclonal antibody protects against herpes simplex virus disease in mice.

There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2-vaccinated mice and evaluated these mAbs for binding, neutralizing, and FcγR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcγRIV, a biomarker of ADCC. The cryo-electron microscopic structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcγRIII when engineered as a human IgG1. These results highlight the importance of FcR-activating antibodies in protecting against HSV.

Monoclonal antibodies to lipoarabinomannan/arabinomannan - characteristics and implications for tuberculosis research and diagnostics.

Antibodies to the mycobacterial surface lipoglycan lipoarabinomannan (LAM) and its related capsular polysaccharide arabinomannan (AM) are increasingly important for investigations focused on both understanding mechanisms of protection against Mycobacterium tuberculosis (Mtb) and developing next-generation point-of-care tuberculosis (TB) diagnostics. We provide here an overview of the growing pipeline of monoclonal antibodies (mAbs) to LAM/AM. Old and new methodologies for their generation are reviewed and we outline and discuss their glycan epitope specificity and other features with implications for the TB field.

Synthetic nanobodies as tools to distinguish IgG Fc glycoforms.

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

Feasibility of novel approaches to detect viable Mycobacterium tuberculosis within the spectrum of the tuberculosis disease.

Tuberculosis (TB) is a global disease caused by Mycobacterium tuberculosis (Mtb) and is manifested as a continuum spectrum of infectious states. Both, the most common and clinically asymptomatic latent tuberculosis infection (LTBI), and the symptomatic disease, active tuberculosis (TB), are at opposite ends of the spectrum. Such binary classification is insufficient to describe the existing clinical heterogeneity, which includes incipient and subclinical TB. The absence of clinically TB-related symptoms and the extremely low bacterial burden are features shared by LTBI, incipient and subclinical TB states. In addition, diagnosis relies on cytokine release after antigenic T cell stimulation, yet several studies have shown that a high proportion of individuals with immunoreactivity never developed disease, suggesting that they were no longer infected. LTBI is estimated to affect to approximately one fourth of the human population and, according to WHO data, reactivation of LTBI is the main responsible of TB cases in developed countries. Assuming the drawbacks associated to the current diagnostic tests at this part of the disease spectrum, properly assessing individuals at real risk of developing TB is a major need. Further, it would help to efficiently design preventive treatment. This quest would be achievable if information about bacterial viability during human silent Mtb infection could be determined. Here, we have evaluated the feasibility of new approaches to detect viable bacilli across the full spectrum of TB disease. We focused on methods that specifically can measure host-independent parameters relying on the viability of Mtb either by its direct or indirect detection.

Tailor made: New insights into lipoarabinomannan structure may improve TB diagnosis.

Detecting the mycobacterial glycolipid lipoarabinomannan (LAM) in urine by anti-LAM antibodies fills a gap in the diagnostic armamentarium of much needed simple rapid tests for tuberculosis, but lacks high sensitivity in all patient groups. A better understanding of LAM structure from clinically relevant strains may allow improvements in diagnostic performance. De et al. have recently determined the structures of LAM from three epidemiologically important lineages of Mycobacterium tuberculosis and probed their interaction with an anti-LAM monoclonal antibody. Their results not only identify a series of tailoring modifications that impact antibody binding but also provide a roadmap for improving U-LAM-based diagnostics.

Plasma host protein biomarkers correlating with increasing Mycobacterium tuberculosis infection activity prior to tuberculosis diagnosis in people living with HIV.

BACKGROUND: Biomarkers correlating with Mycobacterium tuberculosis infection activity/burden in asymptomatic individuals are urgently needed to identify and treat those at highest risk for developing active tuberculosis (TB). Our main objective was to identify plasma host protein biomarkers that change over time prior to developing TB in people living with HIV (PLHIV). METHODS: Using multiplex MRM-MS, we investigated host protein expressions from 2 years before until time of TB diagnosis in longitudinally collected (every 3-6 months) and stored plasma from PLHIV with incident TB, identified within a South African (SA) and US cohort. We performed temporal trend and discriminant analyses for proteins, and, to assure clinical relevance, we further compared protein levels at TB diagnosis to interferon-gamma release assay (IGRA; SA) or tuberculin-skin test (TST; US) positive and negative cohort subjects without TB. SA and US exploratory data were analyzed separately. FINDINGS: We identified 15 proteins in the SA (n=30) and 10 in the US (n=24) incident TB subjects which both changed from 2 years prior until time of TB diagnosis after controlling for 10% false discovery rate, and were significantly different at time of TB diagnosis compared to non-TB subjects (p<0.01). Five proteins, CD14, A2GL, NID1, SCTM1, and A1AG1, overlapped between both cohorts. Furthermore, after cross-validation, panels of 5 - 12 proteins were able to predict TB up to two years before diagnosis. INTERPRETATION: Host proteins can be biomarkers for increasing Mycobacterium tuberculosis infection activity/burden, incipient TB, and predict TB development in PLHIV. FUNDING: NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Monoclonal antibodies from humans with Mycobacterium tuberculosis exposure or latent infection recognize distinct arabinomannan epitopes.

The surface polysacharide arabinomannan (AM) and related glycolipid lipoarabinomannan (LAM) play critical roles in tuberculosis pathogenesis. Human antibody responses to AM/LAM are heterogenous and knowledge of reactivity to specific glycan epitopes at the monoclonal level is limited, especially in individuals who can control M. tuberculosis infection. We generated human IgG mAbs to AM/LAM from B cells of two asymptomatic individuals exposed to or latently infected with M. tuberculosis. Here, we show that two of these mAbs have high affinity to AM/LAM, are non-competing, and recognize different glycan epitopes distinct from other anti-AM/LAM mAbs reported. Both mAbs recognize virulent M. tuberculosis and nontuberculous mycobacteria with marked differences, can be used for the detection of urinary LAM, and can detect M. tuberculosis and LAM in infected lungs. These mAbs enhance our understanding of the spectrum of antibodies to AM/LAM epitopes in humans and are valuable for tuberculosis diagnostic and research applications.

The knowns and unknowns of latent Mycobacterium tuberculosis infection.

Humans have been infected with Mycobacterium tuberculosis (Mtb) for thousands of years. While tuberculosis (TB), one of the deadliest infectious diseases, is caused by uncontrolled Mtb infection, over 90% of presumed infected individuals remain asymptomatic and contain Mtb in a latent TB infection (LTBI) without ever developing disease, and some may clear the infection. A small number of heavily Mtb-exposed individuals appear to resist developing traditional LTBI. Because Mtb has mechanisms for intracellular survival and immune evasion, successful control involves all of the arms of the immune system. Here, we focus on immune responses to Mtb in humans and nonhuman primates and discuss new concepts and outline major knowledge gaps in our understanding of LTBI, ranging from the earliest events of exposure and infection to success or failure of Mtb control.

The Many Hosts of Mycobacteria 8 (MHM8): A conference report.

Mycobacteria are important causes of disease in human and animal hosts. Diseases caused by mycobacteria include leprosy, tuberculosis (TB), nontuberculous mycobacteria (NTM) infections and Buruli Ulcer. To better understand and treat mycobacterial disease, clinicians, veterinarians and scientists use a range of discipline-specific approaches to conduct basic and applied research, including conducting epidemiological surveys, patient studies, wildlife sampling, animal models, genetic studies and computational simulations. To foster the exchange of knowledge and collaboration across disciplines, the Many Hosts of Mycobacteria (MHM) conference series brings together clinical, veterinary and basic scientists who are dedicated to advancing mycobacterial disease research. Started in 2007, the MHM series recently held its 8th conference at the Albert Einstein College of Medicine (Bronx, NY). Here, we review the diseases discussed at MHM8 and summarize the presentations on research advances in leprosy, NTM and Buruli Ulcer, human and animal TB, mycobacterial disease comorbidities, mycobacterial genetics and 'omics, and animal models. A mouse models workshop, which was held immediately after MHM8, is also summarized. In addition to being a resource for those who were unable to attend MHM8, we anticipate this review will provide a benchmark to gauge the progress of future research concerning mycobacteria and their many hosts.

Effects of anticoagulants and Ficoll on human serum antibody reactivities and functions against Mycobacterium tuberculosis.

The ability to utilize leftover samples containing anticoagulants or Ficoll would provide substantial opportunities for future antibody and biomarker studies. Some anticoagulants might influence antibody reactivity against pathogens, but comprehensive studies investigating effects in the context of TB are lacking. We enrolled 24 individuals with and without history of M. tuberculosis and/or HIV-infection and investigated TB antibody reactivities, function, and other host protein biomarkers in simultaneously obtained serum and plasma from serum separation, EDTA, heparin, acid citrate dextrose (ACD), or mononuclear cell preparation (CPT™) tubes which contain heparin and Ficoll. Antibody isotype reactivities to two mycobacterial antigens, as well as phagocytosis of M. tuberculosis, correlated strongly and significantly between serum and plasma, irrespective of type of anticoagulant or Ficoll present (r ≥ 0.85, p < 0.0001). However, the presence of ACD resulted in slightly lower values than those obtained with serum in both indirect (antibody reactivities to mycobacterial antigens) and Sandwich ELISAs (soluble CD14 measurements). Our data demonstrate that leftover plasma, regardless of containing anticoagulants or Ficoll, can be used in TB antibody or other host protein biomarker studies but suggest the value of a correction factor when using ACD plasma interchangeably with serum in antibody binding studies.

Capsular glycan recognition provides antibody-mediated immunity against tuberculosis.

A better understanding of all immune components involved in protecting against Mycobacterium tuberculosis infection is urgently needed to inform strategies for novel immunotherapy and tuberculosis (TB) vaccine development. Although cell-mediated immunity is critical, increasing evidence supports that antibodies also have a protective role against TB. Yet knowledge of protective antigens is limited. Analyzing sera from 97 US immigrants at various stages of M. tuberculosis infection, we showed protective in vitro and in vivo efficacy of polyclonal IgG against the M. tuberculosis capsular polysaccharide arabinomannan (AM). Using recently developed glycan arrays, we established that anti-AM IgG induced in natural infection is highly heterogeneous in its binding specificity and differs in both its reactivity to oligosaccharide motifs within AM and its functions in bacillus Calmette-Guérin vaccination and/or in controlled (latent) versus uncontrolled (TB) M. tuberculosis infection. We showed that anti-AM IgG from asymptomatic but not from diseased individuals was protective and provided data suggesting a potential role of IgG2 and specific AM oligosaccharides. Filling a gap in the current knowledge of protective antigens in humans, our data support the key role of the M. tuberculosis surface glycan AM and suggest the importance of targeting specific glycan epitopes within AM in antibody-mediated immunity against TB.

Combining urine lipoarabinomannan with antibody detection as a simple non-sputum-based screening method for HIV-associated tuberculosis.

BACKGROUND: Simple methods for the accurate triaging and screening of HIV-associated tuberculosis (TB) are urgently needed. We hypothesized that combining serum antibody with urine lipoarabinomannan (U-LAM) detection can improve the detection of HIV-associated TB. METHODS: We performed a case-control study with sampling from a prospective study of South African HIV-infected subjects who were screened for TB prior to initiating antiretroviral therapy. Sera from all available TB cases (n = 74) and randomly selected non-TB controls (n = 30), all tested for U-LAM, sputum microscopy, GeneXpert, and cultures, were evaluated for antibodies to LAM and arabinomannan (AM). Diagnostic logistic regression models for TB were developed based on the primary test results and the additive effect of antibodies with leave-one-out cross-validation. RESULTS: Antibody responses to LAM and AM correlated strongly (p<0.0001), and IgG and IgM reactivities were significantly higher in TB than non-TB patients (p<0.0001). At 80% specificity, the target specificity for a non-sputum-based simple triage/screening test determined by major TB stakeholders, combining U-LAM with IgG detection significantly increased the sensitivity for HIV-associated TB to 92% compared to 30% for U-LAM alone (p<0.001). Sputum microscopy combined with IgG detection increased sensitivity to 88% compared to 31% for microscopy alone, and Xpert with IgG increased sensitivity to 96% and 99% compared to 57% for testing one, and 70% for testing two sputa with Xpert alone, respectively. CONCLUSION: Combining U-LAM with serum antibody detection could provide a simple low-cost method that meets the requirements for a non-sputum-based test for the screening of HIV-associated TB.

Factors Associated With Sputum Culture-Negative vs Culture-Positive Diagnosis of Pulmonary Tuberculosis.

Importance: Recognition of active tuberculosis (TB) in its earliest stages could reduce morbidity and prevent advancement to transmissible disease. Little is published about the occurrence and presentation of sputum culture-negative pulmonary TB (PTB), an early paucibacillary but often underrecognized disease state. Objective: To assess differences between culture-negative and culture-positive PTB regarding occurrence, clinical presentation, radiographic findings, demographics, and comorbidities. Design, Setting, and Participants: Cross-sectional study in which surveillance data of adult patients with PTB reported to the New York City Department of Health in New York, New York, from 2011 through 2013, ie, years for which demographic, clinical, and radiographic data were collected. Patients were aged 18 years or older, had signs of pulmonary disease, and had mycobacterial sputum culture results; those with HIV coinfection or a TB diagnosis within 2 years prior to presentation were excluded. Culture-negative PTB was defined as clinical and radiographic presentation consistent with TB, 3 negative results on sputum culture, and improvement with antituberculous treatment. The analyses were performed between 2015 and 2016; notably, the proportion of reported patients with culture-negative PTB has remained consistent during the past 2 decades. Main Outcomes and Measures: The occurrence of culture-negative PTB among all patients with PTB was calculated, and demographics, comorbidities, symptoms, and radiographic findings were compared between culture-negative and culture-positive PTB. Results: Of the 796 patients with PTB (median [interquartile range] age, 41 [29-54] years; 499 [63%] men) who met criteria for analysis, 116 (15%) had negative results on sputum culture. Patients with culture-negative PTB compared with culture-positive PTB were less frequently male (53% vs 64%; P = .03) and presented with a significantly lower frequency of cough (68% vs 89%; P < .001), weight loss (39% vs 51%; P = .03), and cavitation on both chest radiograph (7% vs 28%; P < .001) and chest computed tomographic scan (26% vs 59%; P < .001). Conclusions and Relevance: Given the lack of criterion-standard test confirmation and the relative paucity of symptoms and radiological abnormalities, culture-negative PTB is likely underdiagnosed and its occurrence underestimated globally. Awareness of these findings, enhanced diagnostic approaches, and, ideally, better biomarkers could improve detection and treatment of this early disease and reduce the development of transmissible TB.

Utilization and Clinical Value of Diagnostic Modalities for Tuberculosis in a High HIV Prevalence Setting.

Human immunodeficiency virus (HIV) infection is a major risk factor for the development of active tuberculosis (TB), one of the deadliest infectious diseases globally. The high mortality associated with the disease can be reduced by early diagnosis and prompt antituberculous treatment initiation. Facilities in TB-endemic regions are increasing the use of nucleic acid amplification (e.g., GeneXpert), which provides rapid results but may have suboptimal sensitivity in HIV-associated TB. Our objective was to evaluate the current practices for TB diagnosis at Edendale Hospital, a large regional hospital in KwaZulu-Natal, South Africa-a TB-endemic region with high HIV prevalence. In this cross-sectional study, all adult inpatients newly started on TB treatment at Edendale were identified over a 6-week period. Demographics, clinical information, diagnostic test results, and outcomes were documented. Pulmonary TB (PTB), extrapulmonary TB (EXTB), and PTB + EXTB were defined as disease evidence in the lungs, other organs, or both, respectively. Ninety-four cases were identified, of which 83% were HIV-associated. Only 30% of all TB patients were microbiologically confirmed, consisting of 7/16 (44%) HIV-uninfected and 21/78 (27%) HIV-infected TB patients. Smear microscopy and mycobacterial culture were seldom ordered. Ultrasound was performed in about one-third of suspected EXTB cases and was valuable in identifying abdominal TB. In this clinical setting with a high incidence of HIV-associated TB, TB diagnosis was more commonly based on clinical assessment and imaging results than on mycobacterial gold standard test confirmation.

Serum-Mediated Cleavage of Bacillus anthracis Protective Antigen Is a Two-Step Process That Involves a Serum Carboxypeptidase.

Much of our understanding of the activity of anthrax toxin is based on in vitro systems, which delineate the interaction between Bacillus anthracis toxins and the cell surface. However, these systems fail to account for the intimate association of B. anthracis with the circulatory system, including the contribution of serum proteins to the host response and processing of anthrax toxins. Using a variety of immunological techniques to inhibit serum processing of B. anthracis protective antigen (PA) along with mass spectrometry analysis, we demonstrate that serum digests PA via 2 distinct reactions. In the first reaction, serum cleaves PA83 into 2 fragments to produce PA63 and PA20 fragments, similarly to that observed following furin digestion. This is followed by carboxypeptidase-mediated removal of the carboxy-terminal arginine and lysines from PA20IMPORTANCE Our findings identify a serum-mediated modification of PA20 that has not been previously described. These observations further imply that the processing of PA is more complex than currently thought. Additional study is needed to define the contribution of serum processing of PA to the host response and individual susceptibility to anthrax.

Updates on antibody functions in Mycobacterium tuberculosis infection and their relevance for developing a vaccine against tuberculosis.

A more effective vaccine to control tuberculosis (TB), a major global public health problem, is urgently needed. Current vaccine candidates focus predominantly on eliciting cell-mediated immunity but other arms of the immune system also contribute to protection against TB. We review here recent studies that enhance our current knowledge of antibody-mediated functions against Mycobacterium tuberculosis. These findings, which contribute to the increasing evidence that antibodies have a protective role against TB, include demonstrations that firstly distinct human antibody Fc glycosylation patterns, found in latent M. tuberculosis infection but not in active TB, influence the efficacy of the host to control M. tuberculosis infection, secondly antibody isotype influences human antibody functions, and thirdly that antibodies targeting M. tuberculosis surface antigens are protective. We discuss these findings in the context of TB vaccine development and highlight the need for further research on antibody-mediated immunity in M. tuberculosis infection.