HIV prevention and treatment research in sub-Saharan Africa: where are the adolescents?

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Monocyte, Lymphocyte and Neutrophil Ratios – Easy-to-Use Biomarkers for the Diagnosis of Pediatric Tuberculosis

Background: The neutrophil-to-lymphocyte-ratio (NLR), neutrophil-to-monocyte-plus-lymphocyte-ratio (NMLR) and monocyte-to-lymphocyte-ratio (MLR) may have diagnostic potential for tuberculosis (TB). Methods: Data of two prospective multicenter studies in Switzerland were used, which included children <18 years with TB exposure, infection or disease or with febrile non-TB lower-respiratory-tract infection (nTB-LRTI). Results: Of the 389 children included 25 (6.4%) had TB disease, 12 (3.1%) TB infection, 28 (7.2%) were healthy TB exposed and 324 (83.3%) nTB-LRTI. Median (IQR) NLR was highest with 2.0 (1.2, 2.2) in children with TB disease compared to TB exposed [0.8 (0.6, 1.3); P = 0.002] and nTB-LRTI [0.3 (0.1, 1.0); P < 0.001]. Median (IQR) NMLR was highest with 1.4 (1.2, 1.7) in children with TB disease compared to healthy exposed [0.7 (0.6, 1.1); P = 0.003] and children with nTB-LRTI [0.2 (0.1, 0.6); P < 0.001). Receiver operating characteristic curves to detect TB disease compared to nTB-LRTI for NLR and NMLR had an area under the curve of 0.82 and 0.86, the sensitivity of 88% and 88%, and specificity of 71% and 76%, respectively. Conclusion: NLR and NMLR are promising, easy-to-obtain diagnostic biomarkers to differentiate children with TB disease from other lower respiratory tract infections. These results require validation in a larger study and in settings with high and low TB endemicity

Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes.

The ratio of monocytes and lymphocytes (ML ratio) in peripheral blood is associated with tuberculosis and malaria disease risk and cancer and cardiovascular disease outcomes. We studied anti-mycobacterial function and the transcriptome of monocytes in relation to the ML ratio. Mycobacterial growth inhibition assays of whole or sorted blood were performed and mycobacteria were enumerated by liquid culture. Transcriptomes of unstimulated CD14 + monocytes isolated by magnetic bead sorting were characterised by microarray. Transcript expression was tested for association with ML ratio calculated from leucocyte differential counts by linear regression. The ML ratio was associated with mycobacterial growth in vitro (β = 2.23, SE 0.91, p = 0.02). Using sorted monocytes and lymphocytes, in vivo ML ratio (% variance explained R(2) = 11%, p = 0.02) dominated over in vitro ratios (R(2) = 5%, p = 0.10) in explaining mycobacterial growth. Expression of 906 genes was associated with the ML ratio and 53 with monocyte count alone. ML-ratio associated genes were enriched for type-I and -II interferon signalling (p = 1.2 × 10(− 8)), and for genes under transcriptional control of IRF1, IRF2, RUNX1, RELA and ESRRB. The ML-ratio-associated gene set was enriched in TB disease (3.11-fold, 95% CI: 2.28-4.19, p = 5.7 × 10(− 12)) and other inflammatory diseases including atopy, HIV, IBD and SLE. The ML ratio is associated with distinct transcriptional and anti-mycobacterial profiles of monocytes that may explain the disease associations of the ML ratio

No evidence for selection of HIV-1 with enhanced Gag-Protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial

BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

Changes in Natural Killer Cell Activation and Function during Primary HIV-1 Infection

Background: Recent reports suggest that Natural Killer (NK) cells may modulate pathogenesis of primary HIV-1 infection. However, HIV dysregulates NK-cell responses. We dissected this bi-directional relationship to understand how HIV impacts NK-cell responses during primary HIV-1 infection. Methodology/Principal Findings: Paired samples from 41 high-risk, initially HIV-uninfected CAPRISA004 participants were analysed prior to HIV acquisition, and during viraemic primary HIV-1 infection. At the time of sampling post-infection five women were seronegative, 11 women were serodiscordant, and 25 women were seropositive by HIV-1 rapid immunoassay. Flow cytometry was used to measure NK and T-cell activation, NK-cell receptor expression, cytotoxic and cytokine-secretory functions, and trafficking marker expression (CCR7, α$_4$β$_7$). Non-parametric statistical tests were used. Both NK cells and T-cells were significantly activated following HIV acquisition (p = 0.03 and p<0.0001, respectively), but correlation between NK-cell and T-cell activation was uncoupled following infection (pre-infection r = 0.68;p<0.0001; post-infection, during primary infection r = 0.074;p = 0.09). Nonetheless, during primary infection NK-cell and T-cell activation correlated with HIV viral load (r = 0.32'p = 0.04 and r = 0.35;p = 0.02, respectively). The frequency of Killer Immunoglobulin-like Receptor-expressing (KIR$_{pos}$) NK cells increased following HIV acquisition (p = 0.006), and KIR$_{pos}$ NK cells were less activated than KIR$_{neg}$ NK cells amongst individuals sampled while seronegative or serodiscordant (p = 0.001;p<0.0001 respectively). During HIV-1 infection, cytotoxic NK cell responses evaluated after IL-2 stimulation alone, or after co-culture with 721 cells, were impaired (p = 0.006 and p = 0.002, respectively). However, NK-cell IFN-y secretory function was not significantly altered. The frequency of CCR7+ NK cells was elevated during primary infection, particularly at early time-points (p<0.0001). Conclusions/Significance: Analyses of immune cells before and after HIV infection revealed an increase in both NK-cell activation and KIR expression, but reduced cytotoxicity during acute infection. The increase in frequency of NK cells able to traffic to lymph nodes following HIV infection suggests that these cells may play a role in events in secondary lymphoid tissue

Changes in natural killer cell activation and function during primary HIV-1 infection.

Background. Recent reports suggest that Natural Killer (NK) cells may modulate pathogenesis of primary HIV-1 infection. However, HIV dysregulates NK-cell responses. We dissected this bi-directional relationship to understand how HIV impacts NK-cell responses during primary HIV-1 infection. Methodology/Principal Findings. Paired samples from 41 high-risk, initially HIV-uninfected CAPRISA004 participants were analysed prior to HIV acquisition, and during viraemic primary HIV-1 infection. At the time of sampling post-infection five women were seronegative, 11 women were serodiscordant, and 25 women were seropositive by HIV-1 rapid immunoassay. Flow cytometry was used to measure NK and T-cell activation, NK-cell receptor expression, cytotoxic and cytokine-secretory functions, and trafficking marker expression (CCR7, α4β7). Non-parametric statistical tests were used. Both NK cells and T-cells were significantly activated following HIV acquisition (p = 0.03 and p<0.0001, respectively), but correlation between NK-cell and T-cell activation was uncoupled following infection (pre-infection r = 0.68;p<0.0001; post-infection, during primary infection r = 0.074;p = 0.09). Nonetheless, during primary infection NK-cell and T-cell activation correlated with HIV viral load (r = 0.32'p = 0.04 and r = 0.35;p = 0.02, respectively). The frequency of Killer Immunoglobulin-like Receptor-expressing (KIRpos) NK cells increased following HIV acquisition (p = 0.006), and KIRpos NK cells were less activated than KIRneg NK cells amongst individuals sampled while seronegative or serodiscordant (p = 0.001;p<0.0001 respectively). During HIV-1 infection, cytotoxic NK cell responses evaluated after IL-2 stimulation alone, or after co-culture with 721 cells, were impaired (p = 0.006 and p = 0.002, respectively). However, NK-cell IFN-y secretory function was not significantly altered. The frequency of CCR7+ NK cells was elevated during primary infection, particularly at early time-points (p<0.0001). Conclusions/Significance. Analyses of immune cells before and after HIV infection revealed an increase in both NK-cell activation and KIR expression, but reduced cytotoxicity during acute infection. The increase in frequency of NK cells able to traffic to lymph nodes following HIV infection suggests that these cells may play a role in events in secondary lymphoid tissue

Multi-ancestry meta-analysis of host genetic susceptibility to tuberculosis identifies shared genetic architecture

The heritability of susceptibility to tuberculosis (TB) disease has been well recognized. Over 100 genes have been studied as candidates for TB susceptibility, and several variants were identified by genome-wide association studies (GWAS), but few replicate. We established the International Tuberculosis Host Genetics Consortium to perform a multi-ancestry meta-analysis of GWAS, including 14,153 cases and 19,536 controls of African, Asian, and European ancestry. Our analyses demonstrate a substantial degree of heritability (pooled polygenic h2 = 26.3%, 95% CI 23.7–29.0%) for susceptibility to TB that is shared across ancestries, highlighting an important host genetic influence on disease. We identified one global host genetic correlate for TB at genome-wide significance (p<5 × 10-8) in the human leukocyte antigen (HLA)-II region (rs28383206, p-value=5.2 × 10-9) but failed to replicate variants previously associated with TB susceptibility. These data demonstrate the complex shared genetic architecture of susceptibility to TB and the importance of large-scale GWAS analysis across multiple ancestries experiencing different levels of infection pressure

Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function.

Understanding the role of natural killer (NK) cells in human disease pathogenesis is crucial and necessitates study of patient samples directly ex vivo. Manipulation of whole blood by density gradient centrifugation or delays in sample processing due to shipping, however, may lead to artifactual changes in immune response measures. Here, we assessed the impact of density gradient centrifugation and delayed processing of both whole blood and peripheral blood mononuclear cells (PBMC) at multiple timepoints (2–24 h) on flow cytometric measures of NK cell frequency, activation status, chemokine receptor expression, and effector functions. We found that density gradient centrifugation activated the NK cells and modified the chemokine receptor expression. Delays in processing beyond 8 h activated NK cells in PBMC but not in whole blood. Likewise, processing delays decreased chemokine receptor (CCR4 and CCR7) expression in both PBMC and whole blood. Finally, delays in processing PBMC were associated with a decreased ability of NK cells to degranulate (as measured by CD107a expression) or secrete cytokines (IFN-γ and TNF-α). In summary, our findings suggest that density gradient centrifugation and delayed processing of PBMC can alter measures of clinically relevant NK cell characteristics including effector functions; and therefore should be taken into account in designing clinical research studies

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays

Ratio of monocytes to lymphocytes in peripheral blood identifies adults at risk of incident tuberculosis among HIV-infected adults initiating antiretroviral therapy.

Background. Eight decades ago, the ratio of monocytes to lymphocytes (hereafter, the “ML ratio”) was noted to affect outcomes of mycobacterial infection in rabbits. Recent transcriptomic studies support a role for relative proportions of myeloid and lymphoid transcripts in tuberculosis outcomes. The ML ratio in peripheral blood is known to be governed by hematopoietic stem cells with distinct biases. Methods. The predictive value of the baseline ML ratio was modeled in 2 prospective cohorts of HIV-infected adults starting cART in South Africa (primary cohort, 1862 participants; replication cohort, 345 participants). Incident tuberculosis was diagnosed with clinical, radiographic, and microbiologic methods per contemporary guidelines. Kaplan-Meier survival analyses and Cox proportional hazards modeling were conducted. Results. The incidence rate of tuberculosis differed significantly by baseline ML ratio: 32.61 (95% confidence interval [CI], 15.38–61.54), 16.36 (95% CI, 12.39–21.23), and 51.80 (95% CI, 23.10–101.71) per 1000 patient-years for ML ratios of less than the 5th percentile, between the 5th and 95th percentiles, and greater than the 95th percentile, respectively (P = .007). Neither monocyte counts nor lymphocyte counts alone were associated with tuberculosis. After adjustment for sex, World Health Organization human immunodeficiency virus disease stage, CD4⁺ T-cell counts, and previous history of tuberculosis, hazards of disease were significantly higher for patients with ML ratios of less than the 5th percentile or greater than the 95th percentile (adjusted hazard ratio, 2.47; 95% CI, 1.39–4.40; P = .002). Conclusions. The ML ratio may be a useful, readily available tool to stratify the risk of tuberculosis and suggests involvement of hematopoietic stem cell bias in tuberculosis pathogenesis