Baseline and end-of-treatment host serum biomarkers predict relapse in adults with pulmonary tuberculosis.

BACKGROUND: There is a need for new tools for monitoring of the response to TB treatment. Such tools may allow for tailored treatment regimens, and stratify patients initiating TB treatment into different risk groups. We evaluated combinations between previously published host biomarkers and new candidates, as tools for monitoring TB treatment response, and prediction of relapse. METHODS: Serum samples were collected at multiple time points, from patients initiating TB treatment at research sites situated in South Africa (ActionTB study), Brazil and Uganda (TBRU study). Using a multiplex immunoassay platform, we evaluated the concentrations of selected host inflammatory biomarkers in sera obtained from clinically cured patients with and without subsequent relapse within 2 years of TB treatment completion. RESULTS: A total of 130 TB patients, 30 (23%) of whom had confirmed relapse were included in the study. The median time to relapse was 9.7 months in the ActionTB study (n = 12 patients who relapsed), and 5 months (n = 18 patients who relapsed) in the TBRU study. Serum concentrations of several host biomarkers changed during TB treatment with IL-6, IP-10, IL-22 and complement C3 showing potential individually, in predicting relapse. A six-marker signature comprising of TTP, BMI, sICAM-1, IL-22, IL-1ß and complement C3, predicted relapse, prior to the onset of TB treatment with 89% sensitivity and 94% specificity. Furthermore, a 3-marker signature (Apo-CIII, IP-10 and sIL-6R) predicted relapse in samples collected at the end of TB treatment with sensitivity of 71% and specificity of 74%. A previously identified baseline relapse prediction signature (TTP, BMI, TNF-ß, sIL-6R, IL-12p40 and IP-10) also showed potential in the current study. CONCLUSION: Serum host inflammatory biomarkers may be useful in predicting relapse in TB patients prior to the initiation of treatment. Our findings have implications for tailored patient management and require prospective evaluation in larger studies.

Safety and immunogenicity of booster vaccination and fractional dosing with Ad26.COV2.S or BNT162b2 in Ad26.COV2.S-vaccinated participants.

We report the safety and immunogenicity of fractional and full dose Ad26.COV2.S and BNT162b2 in an open label phase 2 trial of participants previously vaccinated with a single dose of Ad26.COV2.S, with 91.4% showing evidence of previous SARS-CoV-2 infection. A total of 286 adults (with or without HIV) were enrolled >4 months after an Ad26.COV2.S prime and randomized 1:1:1:1 to receive either a full or half-dose booster of Ad26.COV2.S or BNT162b2 vaccine. B cell responses (binding, neutralization and antibody dependent cellular cytotoxicity-ADCC), and spike-specific T-cell responses were evaluated at baseline, 2, 12 and 24 weeks post-boost. Antibody and T-cell immunity targeting the Ad26 vector was also evaluated. No vaccine-associated serious adverse events were recorded. The full- and half-dose BNT162b2 boosted anti-SARS-CoV-2 binding antibody levels (3.9- and 4.5-fold, respectively) and neutralizing antibody levels (4.4- and 10-fold). Binding and neutralizing antibodies following half-dose Ad26.COV2.S were not significantly boosted. Full-dose Ad26.COV2.S did not boost binding antibodies but slightly enhanced neutralizing antibodies (2.1-fold). ADCC was marginally increased only after a full-dose BNT162b2. T-cell responses followed a similar pattern to neutralizing antibodies. Six months post-boost, antibody and T-cell responses had waned to baseline levels. While we detected strong anti-vector immunity, there was no correlation between anti-vector immunity in Ad26.COV2.S recipients and spike-specific neutralizing antibody or T-cell responses post-Ad26.COV2.S boosting. Overall, in the context of hybrid immunity, boosting with heterologous full- or half-dose BNT162b2 mRNA vaccine demonstrated superior immunogenicity 2 weeks post-vaccination compared to homologous Ad26.COV2.S, though rapid waning occurred by 12 weeks post-boost. Trial Registration: The study has been registered to the South African National Clinical Trial Registry (SANCTR): DOH-27-012022-7841. The approval letter from SANCTR has been provided in the up-loaded documents.

Safety and immunogenicity of booster vaccination and fractional dosing with Ad26.COV2.S or BNT162b2 in Ad26.COV2.S-vaccinated participants.

Background: We report the safety and immunogenicity of fractional and full dose Ad26.COV2.S and BNT162b2 in an open label phase 2 trial of participants previously vaccinated with a single dose of Ad26.COV2.S, with 91.4% showing evidence of previous SARS-CoV-2 infection. Methods: A total of 286 adults (with or without HIV) were enrolled >4 months after an Ad26.COV2.S prime and randomized 1:1:1:1 to receive either a full or half-dose booster of Ad26.COV2.S or BNT162b2 vaccine. B cell responses (binding, neutralization and antibody dependent cellular cytotoxicity-ADCC), and spike-specific T-cell responses were evaluated at baseline, 2, 12 and 24 weeks post-boost. Antibody and T-cell immunity targeting the Ad26 vector was also evaluated. Results: No vaccine-associated serious adverse events were recorded. The full- and half-dose BNT162b2 boosted anti-SARS-CoV-2 binding antibody levels (3.9- and 4.5-fold, respectively) and neutralizing antibody levels (4.4- and 10-fold). Binding and neutralizing antibodies following half-dose Ad26.COV2.S were not significantly boosted. Full-dose Ad26.COV2.S did not boost binding antibodies but slightly enhanced neutralizing antibodies (2.1-fold). ADCC was marginally increased only after a full-dose BNT162b2. T-cell responses followed a similar pattern to neutralizing antibodies. Six months post-boost, antibody and T-cell responses had waned to baseline levels. While we detected strong anti-vector immunity, there was no correlation between anti-vector immunity in Ad26.COV2.S recipients and spike-specific neutralizing antibody or T-cell responses post-Ad26.COV2.S boosting. Conclusion: In the context of hybrid immunity, boosting with heterologous full- or half-dose BNT162b2 mRNA vaccine demonstrated superior immunogenicity 2 weeks post-vaccination compared to homologous Ad26.COV2.S, though rapid waning occurred by 12 weeks post-boost. Trial Registration: South African National Clinical Trial Registry (SANCR): DOH-27-012022-7841. Funding: South African Medical Research Council (SAMRC) and South African Department of Health (SA DoH).

Blood and Site of Disease Inflammatory Profiles Differ in Patients With Pericardial Tuberculosis and Human Immunodeficiency Virus Type 1.

Background: To better understand the pathogenesis of pericardial tuberculosis (PCTB), we sought to characterize the systemic inflammatory profile in people with human immunodeficiency virus type 1 (HIV-1) with latent TB infection (LTBI), pulmonary TB (PTB), or PCTB. Methods: Using Luminex, we measured the concentration of 39 analytes in pericardial fluid (PCF) and paired plasma from 18 PCTB participants, and plasma from 16 LTBI and 20 PTB participants. Follow-up plasma samples were also obtained from PTB and PCTB participants. HLA-DR expression on Mycobacterium tuberculosis-specific CD4 T cells was measured in baseline samples using flow cytometry. Results: Assessment of the overall systemic inflammatory profile by principal component analysis showed that the inflammatory profile of active TB participants was distinct from the LTBI group, while PTB patients could not be distinguished from those with PCTB. When comparing the inflammatory profile between PCF and paired blood, we found that the concentrations of most analytes (25/39) were elevated at site of disease. However, the inflammatory profile in PCF partially mirrored inflammatory events in the blood. After TB treatment completion, the overall plasma inflammatory profile reverted to that observed in the LTBI group. Lastly, HLA-DR expression showed the best performance for TB diagnosis compared to previously described biosignatures built from soluble markers. Conclusions: Our results show that the inflammatory profile in blood was comparable between PTB and PCTB. However, at the site of infection (PCF), inflammation was significantly elevated compared to blood. Additionally, our data emphasize the potential role of HLA-DR expression as a biomarker for TB diagnosis.

Protocol to quantify and phenotype SARS-CoV-2-specific T cell response using a rapid flow-cytometry-based whole blood assay.

Monitoring antigen-specific T cell frequency, function, and phenotype is essential to assess the host immune response to pathogens or novel vaccines. Here, we describe a rapid and simple ex vivo whole blood assay to detect and phenotype the SARS-CoV-2-specific T cell response. We detail steps for whole blood stimulation with SARS-CoV-2 spike peptide and subsequent cell fixation and cryopreservation. We further describe thawing and cell staining steps for flow cytometry analysis. This approach minimizes sample manipulation and has a quick turnaround time. For complete details on the use and execution of this protocol, please refer to Riou et al. (2021).

Escape from recognition of SARS-CoV-2 variant spike epitopes but overall preservation of T cell immunity.

SARS-CoV-2 variants that escape neutralization and potentially affect vaccine efficacy have emerged. T cell responses play a role in protection from reinfection and severe disease, but the potential for spike mutations to affect T cell immunity is incompletely understood. We assessed neutralizing antibody and T cell responses in 44 South African COVID-19 patients either infected with the Beta variant (dominant from November 2020 to May 2021) or infected before its emergence (first wave, Wuhan strain) to provide an overall measure of immune evasion. We show that robust spike-specific CD4 and CD8 T cell responses were detectable in Beta-infected patients, similar to first-wave patients. Using peptides spanning the Beta-mutated regions, we identified CD4 T cell responses targeting the wild-type peptides in 12 of 22 first-wave patients, all of whom failed to recognize corresponding Beta-mutated peptides. However, responses to mutated regions formed only a small proportion (15.7%) of the overall CD4 response, and few patients (3 of 44) mounted CD8 responses that targeted the mutated regions. Among the spike epitopes tested, we identified three epitopes containing the D215, L18, or D80 residues that were specifically recognized by CD4 T cells, and their mutated versions were associated with a loss of response. This study shows that despite loss of recognition of immunogenic CD4 epitopes, CD4 and CD8 T cell responses to Beta are preserved overall. These observations may explain why several vaccines have retained the ability to protect against severe COVID-19 even with substantial loss of neutralizing antibody activity against Beta.

Identification of novel salivary candidate protein biomarkers for tuberculosis diagnosis: A preliminary biomarker discovery study.

BACKGROUND: There is an urgent need for new, accurate, rapid, and affordable tuberculosis (TB) diagnostic tests. The aim of the present study was to use mass spectrometry to identify new preliminary candidate TB diagnostic protein biomarkers in saliva obtained from individuals with TB, and patients with other respiratory diseases (ORD). METHODS: Saliva samples were collected from 22 individuals who self-presented with symptoms suggestive of TB as part of a larger TB biomarker project. Purified salivary proteins were subjected to tryptic digestion peptides were analyzed using a QExactive Orbitrap Mass Spectrometer. Data are available via ProteomeXchange with identifier PXD027294. Identified proteins were subjected to gene ontology and ingenuity pathway analysis for functional enrichment analysis. RESULTS: 26 of the 652 identified proteins significantly discriminated individuals with TB from those with ORD after Benjamini Hochberg correction (5% FDR), with five of these proteins diagnosing TB with an AUC ≥ 0.80. A 5-protein biosignature comprising of P01011, Q8NCW5, P28072, A0A2Q2TTZ9, and Q99574 diagnosed TB with an AUC of 1.00 (95% CI, 1.00-1.00), sensitivity of 100% (95% CI, 76.2-100%) and specificity of 90.9% (95% CI, 58.7-99.8%) after leave-one-out cross validation. CONCLUSIONS: We identified novel candidate salivary protein biomarkers and biosignatures with strong potential as TB diagnostic candidates. Our results are preliminary and require validation in larger studies.

Serum and cerebrospinal fluid host proteins indicate stroke in children with tuberculous meningitis.

INTRODUCTION: Stroke is a common complication in children with tuberculous meningitis (TBM). Host proteins may give us insight into the mechanisms of stroke in TBM and serve as biomarkers for detection of stroke, however, they have not been widely explored. In this study, we compared the concentrations of cerebrospinal fluid (CSF) and serum proteins between children who had TBM-related stroke and children with TBM without stroke. METHODS: We collected CSF and serum from 47 children consecutively admitted to the Tygerberg Academic Hospital in Cape Town, South Africa between November 2016, and November 2017, on suspicion of having TBM. A multiplex platform was used to measure the concentrations of 69 host proteins in CSF and serum from all study participants. RESULTS: After classification of study participants, 23 (48.9%) out of the 47 study participants were diagnosed with TBM, of which 14 (60.9%) demonstrated radiological arterial ischemic infarction. The levels of lipocalin-2, sRAGE, IP-10/ CXCL10, sVCAM-1, MMP-1, and PDGF-AA in CSF samples and the levels of D-dimer, ADAMTS13, SAA, ferritin, MCP-1/ CCL2, GDF-15 and IL-13 in serum samples were statistically different between children who had TBM-related stroke and children with TBM without stroke. After correcting for multiple testing, only the levels of sVCAM-1, MMP-1, sRAGE, and IP-10/ CXCL10 in CSF were statistically different between the two groups. CSF and serum protein biosignatures indicated stroke in children diagnosed with TBM with up to 100% sensitivity and 88.9% specificity. CONCLUSION: Serum and CSF proteins may serve as biomarkers for identifying individuals with stroke amongst children diagnosed with TBM at admission and may guide us to understand the biology of stroke in TBM. This was a pilot study, and thus further investigations in larger studies are needed.

Validation and Optimization of Host Immunological Bio-Signatures for a Point-of-Care Test for TB Disease.

The development of a non-sputum-based, point-of-care diagnostic test for tuberculosis (TB) is a priority in the global effort to combat this disease, particularly in resource-constrained settings. Previous studies have identified host biomarker signatures which showed potential, but there is a need to validate and refine these for development as a test. We recruited 1,403 adults presenting with symptoms suggestive of pulmonary TB at primary healthcare clinics in six countries from West, East and Southern Africa. Of the study cohort, 326 were diagnosed with TB and 787 with other respiratory diseases, from whom we randomly selected 1005 participants. Using Luminex® technology, we measured the levels of 20 host biomarkers in serum samples which we used to evaluate the diagnostic accuracy of previously identified and novel bio-signatures. Our previously identified seven-marker bio-signature did not perform well (sensitivity: 89%, specificity: 60%). We also identified an optimal, two-marker bio-signature with a sensitivity of 94% and specificity of 69% in patients with no history of previous TB. This signature performed slightly better than C-reactive protein (CRP) alone. The cut-off value for a positive diagnosis differed for human immuno-deficiency virus (HIV)-positive and -negative individuals. Notably, we also found that no signature was able to diagnose TB adequately in patients with a prior history of the disease. We have identified a two-marker, pan-African bio-signature which is more robust than CRP alone and meets the World Health Organization (WHO) target product profile requirements for a triage test in both HIV-negative and HIV-positive individuals. This signature could be incorporated into a point-of-care device, greatly reducing the necessity for expensive confirmatory diagnostics and potentially reducing the number of cases currently lost to follow-up. It might also potentially be useful with individuals unable to provide sputum or with paucibacillary disease. We suggest that the performance of TB diagnostic signatures can be improved by incorporating the HIV-status of the patient. We further suggest that only patients who have never had TB be subjected to a triage test and that those with a history of previous TB be evaluated using more direct diagnostic techniques.

Potential of Host Serum Protein Biomarkers in the Diagnosis of Tuberculous Meningitis in Children.

Background: Tuberculous meningitis (TBM) is the most severe form of tuberculosis and results in high morbidity and mortality in children. Diagnostic delay contributes to the poor outcome. There is an urgent need for new tools for the rapid diagnosis of TBM, especially in children. Methods: We collected serum samples from children in whom TBM was suspected at a tertiary hospital in Cape Town, South Africa. Children were subsequently classified as having TBM or no TBM using a published uniform research case-definition. Using a multiplex cytokine array platform, we investigated the concentrations of serum biomarkers comprising biomarkers that were previously found to be of value in the diagnosis of adult pulmonary TB (CRP, SAA, CFH, IFN-γ, IP-10, Apo-AI, and transthyretin) plus other potentially useful host biomarkers as diagnostic candidates for TBM. Findings: Out of 47 children included in the study, 23 (48.9%) had a final diagnosis of TBM and six were HIV infected. A modified version of the adult 7-marker biosignature in which transthyretin was replaced by NCAM1, diagnosed TBM in children with AUC of 0.80 (95% CI, 0.67-0.92), sensitivity of 73.9% (95% CI, 51.6-89.8%) and specificity of 66.7% (95% CI, 44.7-84.4%), with the other six proteins in the signature (CRP, IFN-γ, IP-10, CFH, Apo-A1, and SAA) only achieving and AUC of 0.75 (95% CI, 0.61-0.90) when used in combination. A new childhood TBM specific 3-marker biosignature (adipsin, Aß42, and IL-10) showed potential in the diagnosis of TBM, with AUC of 0.84 (95% CI, 0.73-0.96), sensitivity of 82.6% (95 CI, 61.2-95.0%) and specificity of 75.0% (95% CI, 53.3-90.2%) after leave-one-out cross validation. Conclusion: A previously described adult 7-marker serum protein biosignature showed potential in the diagnosis of TBM in children. However, a smaller childhood TBM-specific 3-marker signature demonstrated improved performance characteristics. Our data indicates that blood-based biomarkers may be useful in the diagnosis of childhood TBM and requires further validation in larger cohort studies.

Prospective evaluation of host biomarkers other than interferon gamma in QuantiFERON Plus supernatants as candidates for the diagnosis of tuberculosis in symptomatic individuals.

BACKGROUND: There is an urgent need for new tools for the diagnosis of TB. We evaluated the usefulness recently described host biomarkers in supernatants from the newest generation of the QuantiFERON test (QuantiFERON Plus) as tools for the diagnosis of active TB. METHODS: We recruited individuals presenting at primary health care clinics in Cape Town, South Africa with symptoms requiring investigation for TB disease, prior to the establishment of a clinical diagnosis. Participants were later classified as TB or other respiratory diseases (ORD) based on the results of clinical and laboratory tests. Using a multiplex platform, we evaluated the concentrations of 37 host biomarkers in QuantiFERON Plus supernatants from study participants as tools for the diagnosis of TB. RESULTS: Out of 120 study participants, 35(29.2%) were diagnosed with active TB, 69(57.5%) with ORD whereas 16(13.3%) were excluded. 14(11.6%) of the study participants were HIV infected. Although individual host markers showed potential as diagnostic candidates, the main finding of the study was the identification of a six-marker biosignature in unstimulated supernatants (Apo-ACIII, CXCL1, CXCL9, CCL8, CCL-1, CD56) which diagnosed TB with sensitivity and specificity of 73.9%(95% CI; 51.6-87.8) and 87.6%(95% CI; 77.2-94.5), respectively, after leave-one-out cross validation. Combinations between TB-antigen specific biomarkers also showed potential (sensitivity of 77.3% and specificity of 69.2%, respectively), with multiple biomarkers being significantly different between TB patients, Quantiferon Plus Positive and Quantiferon Plus negative individuals with ORD, regardless of HIV status. CONCLUSIONS: Biomarkers detected in QuantiFERON Plus supernatants may contribute to adjunctive diagnosis of TB.

Application of Cerebrospinal Fluid Host Protein Biosignatures in the Diagnosis of Tuberculous Meningitis in Children from a High Burden Setting.

BACKGROUND: The diagnosis of tuberculous meningitis (TBM) especially in children is challenging. New tests are urgently needed for the diagnosis of the disease, especially in resource-limited settings. METHODS: We collected cerebrospinal fluid (CSF) samples from children presenting with symptoms requiring investigation for meningitis at a tertiary hospital in Cape Town, South Africa. Children were later classified as TBM or no TBM using published case definitions. Using a multiplex platform, we investigated the concentrations of biomarkers comprising a previously established 3-marker biosignature (VEGF, IL-13, and LL-37) and other potentially useful host biomarkers as diagnostic candidates for TBM. FINDINGS: Out of 47 children, age, 3 months to 13 years, 23 were diagnosed with TBM and six (16%) were HIV-infected. We validated the previously identified CSF biosignature (sensitivity of 95.7% (95% CI, 79.0-99.2%) and specificity of 37.5% (95% CI, 21.2-57.3%)). However, substitution of IL-13 and LL-37 with IFN-γ and MPO, respectively, resulted in improved accuracy (area under the ROC curve (AUC) = 0.97, 95% CI, 0.92-1.00, up to 91.3% (21/23) sensitivity and up to 100% (24/24) specificity). An alternative four-marker biosignature (sICAM-1, MPO, CXCL8, and IFN-γ) also showed potential, with an AUC of 0.97. CONCLUSION: We validated a previously identified CSF biosignature and showed that refinement of this biosignature by incorporation of other biomarkers diagnosed TBM with high accuracy. Incorporation of these biomarkers into a point-of-care or bedside diagnostic test platform may result in the improved management of TBM in children.