The interplay of PTEN and AKT nexus in breast cancer: a molecular perspective.

BACKGROUND: Breast cancer is a highly prevalent and life-threatening ailment that is commonly detected among the females. The downregulation of PTEN in breast cancer is associated with a poor prognosis, aggressive tumor type, and metastasis to lymph nodes, as it activates the pro-survival pathway PI3K/AKT, which is considered the ultimate proliferative pathway. MATERIAL AND METHODS: The mRNA expression of PTEN and AKT genes was investigated using RT-qPCR and TaqMan primer probe chemistry. Moreover DNA was also isolated from the same tissue samples and exonic regions of both genes were amplified for mutational analysis. The proteins expression of PTEN and AKT from seven human breast cancer cell lines was checked through western blot experiments. RESULT: The study revealed a decrease in PTEN expression in 73.3% of the samples, whereas an increase in AKT expression in 40% of samples was observed when compared to the distant normal breast tissue. Conversely, the remaining 60% of samples exhibited a decrease in AKT mRNA expression. There was no observed alteration in the genetic sequence of AKT and PTEN within the targeted amplified regions of breast cancer samples. The high levels of PTEN protein in T-47D and MDA-MB-453 resulted in a lower p-AKT. Two cell lines ZR-75-1 and MDA-MB-468 appeared to be PTEN negative on western blot but mRNA was detected on RT-qPCR. CONCLUSION: In breast cancer the status/expression of PTEN & AKT at mRNA and protein level might be obliging in forecasting the path of disease progression, treatment and prognosis.

Dynamics of temporal immune responses in nonhuman primates and humans immunized with COVID-19 vaccines.

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1-4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.

The Rv3874-Rv3875 chimeric protein shows a promiscuous serodiagnostic potential for tuberculosis.

Tuberculosis (TB) stays a major cause of death globally after COVID-19 and HIV. An early diagnosis to control TB effectively, needs a fast reliable diagnostic method with high sensitivity. Serodiagnosis involving polyclonal antibodies detection against an antigen of Mycobacterium tuberculosis (Mtb) in serum samples can be instrumental. In our study, Rv3874 and Rv3875 antigens were cloned, expressed, and purified individually and as a chimeric construct in Escherichia coli BL21. Enzyme-Linked Immunosorbent Assay (ELISA) based findings revealed that the Rv3874-Rv3875 chimeric construct was two-fold more sensitive (59.7%) than the individual sensitivities of Rv3874 (28.4%) and Rv3875 (24.9%) for 201 serum TB positive samples. Furthermore, the fusion construct was a little more sensitive (60.4%) for male subjects than that for females (58.8%). Lastly, our preliminary findings, molecular insights of secondary structure, and statistical and in silico analysis of each construct also advocate that CEP can be considered a better immunodiagnostic tool in addition to previously reported EC skin test.

Peripheral γδ T Cells Regulate Neutrophil Expansion and Recruitment in Experimental Psoriatic Arthritis.

OBJECTIVE: This study was undertaken to identify the mechanistic role of γδ T cells in the pathogenesis of experimental psoriatic arthritis (PsA). METHODS: In this study, we performed interleukin-23 (IL-23) gene transfer in wild-type (WT) and T cell receptor δ-deficient (TCRδ-/- ) mice and conducted tissue phenotyping in the joint, skin, and nails to characterize the inflammatory infiltrate. We further performed detailed flow cytometry, immunofluorescence staining, RNA sequencing, T cell repertoire analysis, and in vitro T cell polarization assays to identify regulatory mechanisms of γδ T cells. RESULTS: We demonstrated that γδ T cells support systemic granulopoiesis, which is critical for murine PsA-like pathology. Briefly, γδ T cell ablation inhibited the expression of neutrophil chemokines CXCL1 and CXCL2 and neutrophil CD11b+Ly6G+ accumulation in the aforementioned PsA-related tissues. Although significantly reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A was detected systemically in TCRδ-/- mice, no GM-CSF+/IL-17A+ γδ T cells were detected locally in the inflamed skin or bone marrow in WT mice. Our data showed that nonresident γδ T cells regulate the expansion of an CD11b+Ly6G+ neutrophil population and their recruitment to joint and skin tissues, where they develop hallmark pathologic features of human PsA. CONCLUSION: Our findings do not support the notion that tissue-resident γδ T cells initiate the disease but demonstrate a novel role of γδ T cells in neutrophil regulation that can be exploited therapeutically in PsA patients.

Prediction of Tuberculosis Using an Automated Machine Learning Platform for Models Trained on Synthetic Data.

High-quality medical data is critical to the development and implementation of machine learning (ML) algorithms in healthcare; however, security, and privacy concerns continue to limit access. We sought to determine the utility of "synthetic data" in training ML algorithms for the detection of tuberculosis (TB) from inflammatory biomarker profiles. A retrospective dataset (A) comprised of 278 patients was used to generate synthetic datasets (B, C, and D) for training models prior to secondary validation on a generalization dataset. ML models trained and validated on the Dataset A (real) demonstrated an accuracy of 90%, a sensitivity of 89% (95% CI, 83-94%), and a specificity of 100% (95% CI, 81-100%). Models trained using the optimal synthetic dataset B showed an accuracy of 91%, a sensitivity of 93% (95% CI, 87-96%), and a specificity of 77% (95% CI, 50-93%). Synthetic datasets C and D displayed diminished performance measures (respective accuracies of 71% and 54%). This pilot study highlights the promise of synthetic data as an expedited means for ML algorithm development.

Enhanced serodiagnostic potential of a fusion molecule consisting of Rv1793, Rv2628 and a truncated Rv2608 of Mycobacterium tuberculosis.

Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population.

Automated machine learning for endemic active tuberculosis prediction from multiplex serological data.

Serological diagnosis of active tuberculosis (TB) is enhanced by detection of multiple antibodies due to variable immune responses among patients. Clinical interpretation of these complex datasets requires development of suitable algorithms, a time consuming and tedious undertaking addressed by the automated machine learning platform MILO (Machine Intelligence Learning Optimizer). MILO seamlessly integrates data processing, feature selection, model training, and model validation to simultaneously generate and evaluate thousands of models. These models were then further tested for generalizability on out-of-sample secondary and tertiary datasets. Out of 31 antigens evaluated, a 23-antigen model was the most robust on both the secondary dataset (TB vs healthy) and the tertiary dataset (TB vs COPD) with sensitivity of 90.5% and respective specificities of 100.0% and 74.6%. MILO represents a user-friendly, end-to-end solution for automated generation and deployment of optimized models, ideal for applications where rapid clinical implementation is critical such as emerging infectious diseases.

Immune response dynamics in COVID-19 patients to SARS-CoV-2 and other human coronaviruses.

COVID-19 serological test must have high sensitivity as well as specificity to rule out cross-reactivity with common coronaviruses (HCoVs). We have developed a quantitative multiplex test, measuring antibodies against spike (S) proteins of SARS-CoV-2, SARS-CoV, MERS-CoV, and common human coronavirus strains (229E, NL63, OC43, HKU1), and nucleocapsid (N) protein of SARS-CoV viruses. Receptor binding domain of S protein of SARS-CoV-2 (S-RBD), and N protein, demonstrated sensitivity (94% and 92.5%, respectively) in COVID-19 patients (n = 53), with 98% specificity in non-COVID-19 respiratory-disease (n = 98), and healthy-controls (n = 129). Anti S-RBD and N antibodies appeared five to ten days post-onset of symptoms, peaking at approximately four weeks. The appearance of IgG and IgM coincided while IgG subtypes, IgG1 and IgG3 appeared soon after the total IgG; IgG2 and IgG4 remained undetectable. Several inflammatory cytokines/chemokines were found to be elevated in many COVID-19 patients (e.g., Eotaxin, Gro-α, CXCL-10 (IP-10), RANTES (CCL5), IL-2Rα, MCP-1, and SCGF-b); CXCL-10 was elevated in all. In contrast to antibody titers, levels of CXCL-10 decreased with the improvement in patient health suggesting it as a candidate for disease resolution. Importantly, anti-N antibodies appear before S-RBD and differentiate between vaccinated and infected people-current vaccines (and several in the pipeline) are S protein-based.

Plasma Linoleate Diols Are Potential Biomarkers for Severe COVID-19 Infections.

Polyunsaturated fatty acids are metabolized into regulatory lipids important for initiating inflammatory responses in the event of disease or injury and for signaling the resolution of inflammation and return to homeostasis. The epoxides of linoleic acid (leukotoxins) regulate skin barrier function, perivascular and alveolar permeability and have been associated with poor outcomes in burn patients and in sepsis. It was later reported that blocking metabolism of leukotoxins into the vicinal diols ameliorated the deleterious effects of leukotoxins, suggesting that the leukotoxin diols are contributing to the toxicity. During quantitative profiling of fatty acid chemical mediators (eicosanoids) in COVID-19 patients, we found increases in the regioisomeric leukotoxin diols in plasma samples of hospitalized patients suffering from severe pulmonary involvement. In rodents these leukotoxin diols cause dramatic vascular permeability and are associated with acute adult respiratory like symptoms. Thus, pathways involved in the biosynthesis and degradation of these regulatory lipids should be investigated in larger biomarker studies to determine their significance in COVID-19 disease. In addition, incorporating diols in plasma multi-omics of patients could illuminate the COVID-19 pathological signature along with other lipid mediators and blood chemistry.

Serodiagnostic evaluation of fusion proteins from multiple antigens of Mycobacterium tuberculosis for active TB.

Tuberculosis (TB) is a global health problem, being prevalent in the developing countries. A rapid, reliable and cost effective diagnostic method would help in controlling TB in the endemic populations. Development of suitable fusion molecules detecting multiple antibodies produced against Mycobacterium tuberculosis antigens would enhance sensitivity of serodiagnostic assays. In this study, EspC, CFP7 and PPE57 antigens of M. tuberculosis were selected for constructing fusion molecules after prediction of B-cell epitopes using in silico tools. Fusion proteins EspC-CFP7, HspX-EspC-CFP7 and HspX-EspC-CFP7-PPE57 were expressed in E.coli (BL21). The serodiagnostic potential of the individual antigens and their fusions was analyzed by screening 230 plasma samples of pulmonary TB patients. The single antigens HspX, EspC, CFP7, PPE57 showed sensitivities of 30%, 31%, 22% and 35%, respectively. The fusion protein EspC-CFP7 showed sensitivity of 43%. Linking of HspX antigen to the N-terminus of EspC-CFP7 fusion molecule increased sensitivity to 58%, while joining PPE57 antigen to the C-terminus of HspX-EspC-CFP7 increased sensitivity to 69%. The fusion protein HspX-EspC-CFP7-PPE57 seems to be a promising molecule for use in the development of fusions with higher sensitivity.

Gut microbiome dysbiosis and correlation with blood biomarkers in active-tuberculosis in endemic setting.

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment.

Designing fusion molecules from antigens of Mycobacterium tuberculosis for detection of multiple antibodies in plasma of TB patients.

Tuberculosis (TB) is amongst the deadliest diseases worldwide. For effective control of TB a rapid, reliable and sensitive method for its diagnosis is essential. Serodiagnosis detecting multiple antibodies against antigens of Mycobacterium tuberculosis (Mtb) in blood samples could prove beneficial. Based on the epitope position in the molecule, two truncated variants of Rv1984c, i.e., Tn1Rv1984c and Tn2Rv1984c were expressed in Escherichia coli. Screening of the Rv1984c, Tn1Rv1984c and Tn2Rv1984c against 231 sera samples from the culture positive TB patients showed sensitivities of 34.2%, 49.4% and 26.8%, respectively. Another antigen Rv1352 was analyzed for the location of epitopes, which had not been reported before. A fusion molecule consisting of Tn1Rv1984c and Rv1352, expressed in E. coli, showed enhanced sensitivity of 62.8%. Joining another antigen Rv2031c to the N-terminus of Tn1Rv1984c-Rv1352, improved sensitivity to 71.4%. The fusion construct Rv2031c-Tn1Rv1984c-Rv1352 showed comparatively higher sensitivity of 73.4% in the male group as compared to 67% in the female group. Data derived for the secondary structure analysis through Circular Dichroism (CD) spectroscopy and prediction on the basis of molecular modelling was also in agreement. This construct can be a potential base for producing constructs with greater sensitivity through fusion of epitopes from additional Mtb antigens.

Evaluation of multi-antigen serological screening for active tuberculosis among people living with HIV.

Better triage tests for screening tuberculosis (TB) disease are needed for people living with HIV (PLHIV). We performed the first evaluation of a previously-validated 8-antigen serological panel to screen PLHIV for pulmonary TB in Kampala, Uganda. We selected a random 1:1 sample with and without TB (defined by sputum culture) from a cohort of PLHIV initiating antiretroviral therapy. We used a multiplex microbead immunoassay and an ensemble machine learning classifier to determine the area under the receiver operating characteristic curve (AUC) for Ag85A, Ag85B, Ag85C, Rv0934-P38, Rv3881, Rv3841-BfrB, Rv3873, and Rv2878c. We then assessed the performance with the addition of four TB-specific antigens ESAT-6, CFP-10, Rv1980-MPT64, and Rv2031-HSPX, and every antigen combination. Of 262 participants (median CD4 cell-count 152 cells/µL [IQR 65-279]), 138 (53%) had culture-confirmed TB. The 8-antigen panel had an AUC of 0.53 (95% CI 0.40-0.66), and the additional 4 antigens did not improve performance (AUC 0.51, 95% CI 0.39-0.64). When sensitivity was restricted to ≥90% for the 8- and 12-antigen panel, specificity was 2.2% (95% CI 0-17.7%) and 8.1% (95% CI 0-23.9%), respectively. A three-antigen combination (Rv0934-P38, Ag85A, and Rv2031-HSPX) outperformed both panels, with an AUC of 0.60 (95% CI 0.48-0.73), 90% sensitivity (95% CI 78.2-96.7%) and 29.7% specificity (95% CI 15.9-47%). The multi-antigen panels did not achieve the target accuracy for a TB triage test among PLHIV. We identified a new combination that improved performance for TB screening in an HIV-positive sample compared to an existing serological panel in Uganda, and suggests an approach to identify novel antigen combinations specifically for screening TB in PLHIV.

A Chemical Probe for Dehydrobutyrine.

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Diagnostic performance of blood inflammatory markers for tuberculosis screening in people living with HIV.

BACKGROUND: Approaches to screening for active tuberculosis (TB) among people living with HIV are inadequate, leading to missed diagnoses and poor implementation of preventive therapy. METHODS: Consecutive HIV-infected adults hospitalized at Mulago Hospital (Kampala, Uganda) between June 2011 and July 2013 with a cough ≥ 2 weeks were enrolled. Patients underwent extensive evaluation for pulmonary TB. Concentrations of 43 cytokines/chemokines were measured at the same time point as C-reactive protein (CRP) in banked plasma samples using commercially-available multiplex kits. Advanced classification algorithms were used to rank cytokines/chemokines for their ability to identify TB, and to model the specificity of the top-ranked cytokines/chemokines individually and in combination with sensitivity constrained to ≥ 90% as recommended for TB screening. RESULTS: The median plasma level of 5 biomarkers (IL-6, INF-γ, MIG, CRP, IL-18) was significantly different between patients with and without TB. With sensitivity constrained to 90%, all had low specificity with IL-6 showing the highest specificity (44%; 95% CI 37.4-49.5). Biomarker panels were found to be more valuable than any biomarker alone. A panel combining IFN-γ and IL-6 had the highest specificity (50%; 95% CI 46.7-53.3). Sensitivity remained high (>85%) for all panels among sputum smear-negative TB patients. CONCLUSIONS: Direct measurement of unstimulated plasma cytokines/chemokines in peripheral blood is a promising approach to TB screening. Cytokine/chemokine panels retained high sensitivity for smear-negative TB and achieved improved specificity compared to individual cytokines/chemokines. These markers should be further evaluated in outpatient settings where most TB screening occurs and where other illnesses associated with systematic inflammation are less common.

Synthesis, In silico Molecular Docking and Pharmacokinetic Studies, In vitro Antimycobacterial and Antimicrobial Studies of New Imidozolones Clubbed with Thiazolidinedione.

BACKGROUND: A series of (E)-5-(4-((Z)-4-substitutedbenzylidene-2-thienylmethylene-5-oxo- 2-phenyl-4,5-dihydro-1H-imidazol-1-yl) benzylidene)thiazolidine-2,4-diones were synthesized and evaluated for antimycobacterial and antimicrobial activity. All these ligands were docked against protein (InhA) Enoyl-ACP reductase of the type II fatty acid syntase (FAS-II) system, (PDB ID: 4COD). OBJECTIVE: In this report, we have designed and synthesized azole scaffolds with good antitubercular activities as there is a real need to develop new candidates with less toxicity and more efficiency toward pathogen. The obtained antimycobacterial activity data have been validated in the terms of ligand-protein interaction and were also analyzed for ADME properties to determine their potential to build up as good oral drug candidates. METHODS: All the synthesized compounds have been established by elemental analysis, IR, 1H NMR, 13C NMR and Mass spectral data. In vitro antimycobacterial activity was carried out against (M. tuberculosis) H37Rv strain using Lowenstein-Jensen medium and antimicrobial activity against two gram-positive bacteria (S. aureus, S. pyogenes), two gram-negative bacteria (E. coli, P. aeruginosa) and three fungal species (C. albicans, A. niger, A. clavatus) using the broth microdilution method. In silico molecular docking studies were carried out using Glide (grid-based ligand docking) program incorporated in the Schrödinger molecular modeling package by Maestro 11.0 and ADME properties of synthesized compounds was performed using DruLito software. RESULTS: Compounds 3a, 3b, 3d, 3g, 3i and 3n exhibited promising antimicrobial activity whereas compound 3n showed very good antimycobacterial activity along with Gilde docking score (-8.864) and with the one violation in Lipinski's rule of five. CONCLUSION: The wet lab result for compound 3n along with Glide XP docking score and the calculated ADME parameters give the best choice for the preparation of new derivatives in order to improve antitubercular activity in future with more improved potency.

Evaluation of antibody responses to panels of M. tuberculosis antigens as a screening tool for active tuberculosis in Uganda.

BACKGROUND: Improved systematic screening of high-risk groups is a key component of the tuberculosis (TB) elimination strategy endorsed by the World Health Organization (WHO). We used a multiplex microbead immunoassay to measure antibody responses to 28 M. tuberculosis (M.tb) antigens, and assessed whether combinations of antibody responses achieve accuracy thresholds required for a TB screening test. METHODS: A random selection of plasma samples obtained from consecutive HIV-negative adults who were admitted to Mulago Hospital in Kampala, Uganda with cough ≥2 weeks' but <6 months' duration were analyzed for serological response to 28 M.tb antigens using an in-house multiplex microbead immunoassay. We compared the median difference of the antibody response to each antigen between patients with and without culture-confirmed TB, ranked each antigen according to variable importance (VIM), and assessed the sensitivity and specificity of combinations of antibody responses using an advanced classification algorithm, SuperLearner. RESULTS: Among the 237 patients included in the analysis, 119 (50%) were female, median age was 32 years (IQR 25, 46), and 113 (48%) had TB. Median antibody levels to eight antigens were significantly different between patients with and without TB. A panel including eight of the top ranked antigens had a sensitivity of 90.6% (95% CI 89.4, 93.8) and a specificity of 88.6% (95% CI 78.2, 97.6) (Ag85B, Ag85A, Ag85C, Rv0934-P38, Rv3881, BfrB, Rv3873, and Rv2878c). With sensitivity constrained to be >90%, specificity remained close to 70% with as few as 3 antigens included in the panels. CONCLUSIONS: Measuring antibody responses to combinations of antigens could facilitate TB screening and should be further evaluated in populations being targeted for systematic screening.

Field evaluation of a blood based test for active tuberculosis in endemic settings.

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year are unaccounted for, and 1.5 million die. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB, including state-of-the-art molecular tests, have failed in delivering the capacity needed in endemic countries to curtail this ongoing pandemic. Efficient, cost effective and scalable diagnostic approaches are critically needed. We report a multiplex TB serology panel using microbead suspension array containing a combination of 11 M.tb. antigens that demonstrated overall sensitivity of 91% in serum/plasma samples from TB patients confirmed by culture. Group wise sensitivities for sputum smear positive and negative patients were 95%, and 88%, respectively. Specificity of the test was 96% in untreated COPD patients and 91% in general healthy population. The sensitivity of this test is superior to that of the frontline sputum smear test with a comparable specificity (30-70%, and 93-99%, respectively). The multiplex serology test can be performed with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput, thus enabling a scalable clinical work flow model for TB endemic countries. Taken together, the above results suggest that well defined antibody profiles in blood, analyzed by an appropriate technology platform, offer a valuable approach to TB diagnostics in endemic countries.

Development of immune-biomarkers of pulmonary tuberculosis in a rabbit model.

Tuberculosis (TB) causes extensive morbidity and mortality worldwide with approximately 10 million new cases of active disease emerging mostly from a pool of two billion individuals latently infected with Mycobacterium tuberculosis (M. tb) every year. The underlying host immune responses that drive M. tb infection to active disease or latency are not well understood. We propose that identification and characterization of host immune biomarkers will be helpful to better understand the mechanisms that drive this process, and may, in addition, lead to the development of better diagnostic tools for TB. We have previously reported the profiles of plasma immune biomarkers in pulmonary TB patients in endemic countries, and in M. tb-infected nonhuman primates. However, biomarker profiling for a cost-effective and user-friendly animal model relevant to human disease, such as rabbit, has not been developed. One challenge in the analysis of circulating cytokines/chemokines for rabbit model of TB is the limited availability of validated immune-reagents. Here we report the use of a commercially available multiplex microbead human cytokine/chemokine panels as development platform for rabbit immune reagents. The results demonstrate their utility to determine circulating analytes and define their profiles related to TB in the rabbit model. In addition, we report the profiles of circulating anti-M. tb antibodies in the plasma of rabbits with active pulmonary TB. These studies show that the pattern of expression of circulating immune biomarkers correlate with TB pathology in rabbits, and are similar to those defined in pulmonary TB patients.

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and ß-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.