Lipids, glycolipids and lipopeptides derived from Mycobacterium tuberculosis (Mtb) are presented to T cells by monomorphic molecules known as CD1. This is the case of the Mtb-specific sulfoglycolipid Ac2SGL, which is presented by CD1b molecules and is recognized by T cells found in tuberculosis (TB) patients and in individuals with latent infections. Our group, using filamentous phage display technology, obtained two specific ligands against the CD1b-Ac2SGL complex: (i) a single chain T cell receptor (scTCR) from a human T cell clone recognizing the CD1b-AcSGL complex; and (ii) a light chain domain antibody (dAbκ11). Both ligands showed lower reactivity to a synthetic analog of Ac2SGL (SGL12), having a shorter acyl chain as compared to the natural antigen. Here we put forward the hypothesis that the CD1b endogenous spacer lipid (EnSpacer) plays an important role in the recognition of the CD1b-Ac2SGL complex by specific T cells. To support this hypothesis we combined: (a) molecular binding assays for both the scTCR and the dAbκ11 antibody domain against a small panel of synthetic Ac2SGL analogs having different acyl chains, (b) molecular modeling of the CD1b-Ac2SGL/EnSpacer complex, and (c) modeling of the interactions of this complex with the scTCR. Our results contribute to understand the mechanisms of lipid presentation by CD1b molecules and their interactions with T-cell receptors and other specific ligands, which may help to develop specific tools targeting Mtb infected cells for therapeutic and diagnostic applications.
The search for new vaccines more efficacious than bacille Calmette-Guérin for tuberculosis prevention is of paramount importance for the control of the disease. The expression of Mycobacterium tuberculosis antigens in Mycobacterium smegmatis is one of the current strategies for the development of new-generation vaccines against tuberculosis. The objective of this study was to evaluate the immunogenicity in mice of M. smegmatis expressing epitopes from Ag85B antigen.
Globally, it is estimated that one-quarter of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), also known as latent tuberculosis infection (LTBI). Recently, this condition has been referred to as tuberculosis infection (TBI), considering the dynamic spectrum of the infection, as 5-10% of the latently infected population will develop active TB (ATB). The chances of TBI development increase due to close contact with index TB patients. The emergence of multidrug-resistant TB (MDR-TB) and the risk of development of latent MDR-TB has further complicated the situation. Detection of TBI is challenging as the infected individual does not present symptoms. Currently, there is no gold standard for TBI diagnosis, and the only screening tests are tuberculin skin test (TST) and interferon gamma release assays (IGRAs). However, these tests have several limitations, including the inability to differentiate between ATB and TBI, false-positive results in BCG-vaccinated individuals (only for TST), false-negative results in children, elderly, and immunocompromised patients, and the inability to predict the progression to ATB, among others. Thus, new host markers and Mtb-specific antigens are being tested to develop new diagnostic methods. Besides screening, TBI therapy is a key intervention for TB control. However, the long-course treatment and associated side effects result in non-adherence to the treatment. Additionally, the latent MDR strains are not susceptible to the current TBI treatments, which add an additional challenge. This review discusses the current situation of TBI, as well as the challenges and efforts involved in its control.
The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST(+) ) and uninfected TST(-) ) with M. tuberculosis. From a total of 22 V genes analysed, the TST(-) population preferred the VH 3-23 and Vκ1 genes. The VH 3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST(-) population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST(+) population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST(-) population. The antibodyome difference between both populations suggests a preference for antibodies with VH 3-23, D3-3, JH 4 gene usage by the TST(-) population that could be associated with resistance to infection with M. tuberculosis.
Horseshoe crabs (HSCs) are living fossil species of marine arthropods with a long evolutionary history spanning approximately 500 million years. Their survival is helped by their innate immune system that comprises cellular and humoral immune components to protect them against invading pathogens. To help understand the genetic mechanisms involved, the present study utilised the Illumina HiSeq platform to perform transcriptomic analysis of hemocytes from the HSC, Tachypleus gigas, that were challenged with lipopolysaccharides (LPS). The high-throughput sequencing resulted in 352,077,208 and 386,749,136 raw reads corresponding to 282,490,910 and 305,709,830 high-quality mappable reads for the control and LPS-treated hemocyte samples, respectively. Based on the log-fold change of > 0.3 or
Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is an infectious disease with more than 10.4 million cases and 1.7 million deaths reported worldwide in 2016. The classical methods for detection and differentiation of mycobacteria are: acid-fast microscopy (Ziehl-Neelsen staining), culture, and biochemical methods. However, the microbial phenotypic characterization is time-consuming and laborious. Thus, fast, easy, and sensitive nucleic acid amplification tests (NAATs) have been developed based on specific DNA markers, which are commercially available for TB diagnosis. Despite these developments, the disease remains uncontrollable. The identification and differentiation among MTBC members with the use of NAATs remains challenging due, among other factors, to the high degree of homology within the members and mutations, which hinders the identification of specific target sequences in the genome with potential impact in the diagnosis and treatment outcomes. In silico methods provide predictive identification of many new target genes/fragments/regions that can specifically be used to identify species/strains, which have not been fully explored. This review focused on DNA markers useful for MTBC detection, species identification and antibiotic resistance determination. The use of DNA targets with new technological approaches will help to develop NAATs applicable to all levels of the health system, mainly in low resource areas, which urgently need customized methods to their specific conditions.
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis (TB). This contagious disease remains a severe health problem in the world. The disease is transmitted via inhalation of airborne droplets carrying Mtb from TB patients. Early detection of the disease is vital to prevent transmission of the infection to people in close contact with the patients. To date, there is a need of a simple, rapid, sensitive and specific diagnostic test for TB. Previous studies showed the potential of Mtb 16 kDa antigen (Ag16) in TB diagnosis. In this study, lateral flow immunoassay, also called simple strip immunoassay or immunochromatographic test (ICT) for detection of Ag16 was developed (Mtb-strip) and assessed as a potential rapid TB diagnosis method. A monoclonal antibody against Ag16 was optimized as the capturing and detection antibody on the Mtb-strip. Parameters affecting the performance of the Mtb-strip were also optimized before a complete prototype was developed. Analytical sensitivity showed that Mtb-strip was capable to detect as low as 125 ng of purified Ag16. The analytical sensitivity of Mtb-strip suggests its potential usefulness in different clinical applications.
Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.
Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.
Humoral and cellular immune responses are associated with protection against extracellular and intracellular pathogens, respectively. In the present study, we evaluated the effect of receiving human secretory immunoglobulin A (hsIgA) on the histopathology of the lungs of mice challenged with virulent Mycobacterium tuberculosis.
Tuberculosis (TB) is the deadliest of infectious diseases. TB diagnosis, based on sputum microscopy, culture, and nucleic acid amplification tests (NAATs) to identify its main causative agent, Mycobacterium tuberculosis (MTB), remains challenging. The current available NAATs, endorsed by World Health Organization (WHO), can differentiate MTB from some MTB complex (MTBC) members. Using bioinformatics, we identified a single nucleotide polymorphism (SNP) in lprM (Rv1970) gene that differentiate MTB from other MTBC members. A forward mismatch amplification mutation assay (MAMA) primer was designed for the targeted mutation and was used in a semi-nested melt-MAMA qPCR (lprM-MAMA). Using the optimized protocol, lprM-MAMA was positive with all MTB reference and clinical strains, and negative with other MTBC members, non-tuberculous mycobacteria (NTM) and other non-mycobacterial (NM) reference strains. The limit of detection (LOD) of lprM-MAMA was 76.29 fg. Xpert® MTB/RIF (Xpert)-positive sputum samples were also positive by lprM-MAMA, except for samples classified as having "very low" bacterial load by Xpert. Xpert-negative sputum samples were also negative by lprM-MAMA. In conclusion, lprM-MAMA demonstrated to be a useful tool for specific MTB diagnosis. Further evaluation with higher number of reference strains, including NTM and NM; and sputum samples are required to determine its potential for clinical application.
B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 109 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.
Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.
The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.
TB, caused by Mycobacterium tuberculosis (MTB), is one of the major global infectious diseases. For the pandemic control, early diagnosis with sensitive and specific methods is fundamental. With the advent of bioinformatics' tools, the identification of several proteins involved in the pathogenesis of TB (TB) has been possible. In the present work, the MTB genome was explored to look for molecules with possible antigenic properties for their evaluation as part of new generation diagnostic kits based on the release of cytokines. Seven proteins from the MTB proteome and some of their combinations suited the computational test and the results suggested their potential use for the diagnosis of infection in the following population groups: Cuba, Mexico, Malaysia and sub-Saharan Africa. Our predictions were performed using public bioinformatics tools plus three computer programs, developed by our group, to facilitate information retrieval and processing.