Alveolar T-helper 17 responses to streptococcus pneumoniae are preserved in ART-untreated and treated HIV-infected Malawian adults

Objective: We explored if HIV infection is associated with impaired T-Helper 17 responses against Streptococcus pneumoniae in the lung. Methods: We recruited 30 HIV-uninfected healthy controls, 23 asymptomatic HIV-infected adults not on ART, and 40 asymptomatic HIV-infected adults on ART (Median time 3.5yrs), in whom we collected bronchoalveolar lavage fluid. We measured alveolar CD4+ T cell immune responses following stimulation with pneumococcal cell culture supernatant using flow cytometry-based intracellular cytokine staining. Results: We found that the proportion of alveolar CD4+ T cells producing IL-17A following stimulation with pneumococcal cell culture supernatant (CCS) was similar between HIV-uninfected controls and ART-naïve HIV-infected adults (0.10% vs. 0.14%; p = 0.9273). In contrast, the proportion and relative absolute counts of CD4+ T cells producing IL-17A in response to pneumococcal CCS were higher in ART-treated HIV-infected adults compared HIV-uninfected controls (0.22% vs. 0.10%, p = 0.0166; 5420 vs. 1902 cells/100 ml BAL fluid; p = 0.0519). The increase in relative absolute numbers of IL-17A-producing alveolar CD4+ T cells in ART-treated individuals was not correlated with the peripheral blood CD4+ T cell count (r=–0.1876, p = 0.1785). Conclusion: Alveolar Th17 responses against S. pneumoniae are preserved in HIV-infected adults. This suggests that there are other alternative mechanisms that are altered in HIV-infected individuals that render them more susceptible to pneumococcal pneumonia

Performance of molecular methods for the detection of Salmonella in human stool specimens [version 2; peer review: 2 approved]

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of Salmonella in the stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of Salmonella ttr and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same ttr and InvA genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: Ttr and InvA primers were both able to detect all the different Salmonella serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. Ttr sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex-ttr had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance, although stool culture and monoplexed ttr primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events

Circulating microparticles are increased amongst people presenting with HIV and advanced immune suppression in Malawi and correlate closely with arterial stiffness: a nested case control study

Background: We aimed to investigate whether circulating microparticle (CMPs) subsets were raised amongst people presenting with human immunodeficiency virus (HIV) and advanced immune suppression in Malawi, and whether they associated with arterial stiffness. Methods: Antiretroviral therapy (ART)-naïve adults with a new HIV diagnosis and CD4 <100 cells/µL had microparticle characterisation and carotid femoral Pulse Wave Velocity (cfPWV) at 2 weeks post ART initiation. HIV uninfected controls were matched on age, systolic blood pressure (BP) and diastolic BP in a 1:1 ratio.  Circulating microparticles were identified from platelet poor plasma and stained for endothelial, leucocyte, monocyte and platelet markers. Results: The median (IQ) total CMP count for 71 participants was 1 log higher in HIV compared to those without (p<0.0001) and was associated with arterial stiffness (spearman rho 0.47, p<0.001). In adjusted analysis, every log increase in circulating particles showed a 20% increase in cfPWV (95% confidence interval [CI] 4 – 40%, p=0.02). In terms of subsets, endothelial and platelet derived microparticles were most strongly associated with HIV. Endothelial derived E-selectin+ CMPs were 1.3log-fold higher and platelet derived CD42a+ CMPs were 1.4log-fold higher (both p<0.0001). Endothelial and platelet derived CMPs also correlated most closely with arterial stiffness (spearman rho: E-selectin+ 0.57 and CD42a 0.56, both p<0.0001). Conclusions: Circulating microparticles associate strongly with arterial stiffness among people living with HIV in Malawi. Endothelial damage and platelet microparticles are the predominant cell origin types and future translational studies could consider prioritising these pathways

AstraZeneca COVID-19 vaccine induces robust broadly cross-reactive antibody responses in Malawian adults previously infected with SARS-CoV-2

Background: Binding and neutralising anti-Spike antibodies play a key role in immune defence against SARS-CoV-2 infection. Since it is known that antibodies wane with time and new immune-evasive variants are emerging, we aimed to assess the dynamics of anti-Spike antibodies in an African adult population with prior SARS-CoV-2 infection and to determine the effect of subsequent COVID-19 vaccination. Methods: Using a prospective cohort design, we recruited adults with prior laboratory-confirmed mild/moderate COVID-19 in Blantyre, Malawi, and followed them up for 270 days (n = 52). A subset of whom subsequently received a single dose of the AstraZeneca COVID-19 vaccine (ChAdOx nCov-19) (n = 12). We measured the serum concentrations of anti-Spike and receptor-binding domain (RBD) IgG antibodies using a Luminex-based assay. Anti-RBD antibody cross-reactivity across SARS-CoV-2 variants of concern (VOC) was measured using a haemagglutination test. A pseudovirus neutralisation assay was used to measure neutralisation titres across VOCs. Ordinary or repeated measures one-way ANOVA was used to compare log10 transformed data, with p value adjusted for multiple comparison using Šídák's or Holm-Šídák's test. Results: We show that neutralising antibodies wane within 6 months post mild/moderate SARS-CoV-2 infection (30–60 days vs. 210–270 days; Log ID50 6.8 vs. 5.3, p = 0.0093). High levels of binding anti-Spike or anti-RBD antibodies in convalescent serum were associated with potent neutralisation activity against the homologous infecting strain (p < 0.0001). A single dose of the AstraZeneca COVID-19 vaccine following mild/moderate SARS-CoV-2 infection induced a 2 to 3-fold increase in anti-Spike and -RBD IgG levels 30 days post-vaccination (both, p < 0.0001). The anti-RBD IgG antibodies from these vaccinated individuals were broadly cross-reactive against multiple VOCs and had neutralisation potency against original D614G, beta, and delta variants. Conclusions: These findings show that the AstraZeneca COVID-19 vaccine is an effective booster for waning cross-variant antibody immunity after initial priming with SARS-CoV-2 infection. The potency of hybrid immunity and its potential to maximise the benefits of COVID-19 vaccines needs to be taken into consideration when formulating vaccination policies in sub-Saharan Africa, where there is still limited access to vaccine doses

Surfactant protein D inhibits HIV-1 infection of target cells via interference with gp120-CD4 interaction and modulates pro-inflammatory cytokine production

© 2014 Pandit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SPD against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection. © 2014 Pandit et al.The work (Project no. 2011-16850) was supported by Medical Innovation Fund of Indian Council of Medical Research, New Delhi, India (www.icmr.nic.in/)

Naturally-Acquired Influenza-Specific CD4+ T-Cell Proliferative Responses Are Impaired in HIV-Infected African Adults

BACKGROUND Seasonal influenza has been associated with greater morbidity and mortality in AIDS patients. Highly-active antiretroviral therapy (HAART) has led to some reduction in influenza-related complications but the nature of naturally-acquired T-cell immunity to influenza virus in an African setting, and how this changes with immune reconstitution following HAART is unknown. We measured influenza-specific CD4(+) T-cell immunity in unimmunized HIV-infected Malawian adults and then investigated immune reconstitution following HAART. METHODS Peripheral blood mononuclear cells were isolated from HIV-infected and HIV-uninfected Malawian adults. CFSE proliferation and CD154 expression flow cytometry-based assays were used to measure influenza-specific CD4(+) T-cell immunity. RESULTS We found lower naturally-acquired proliferative influenza-specific CD4(+) T-cell responses in AIDS patients that was also present in asymptomatic HIV-infected adults with relatively high CD4 counts (>350 cells/µl). Influenza-specific CD4(+) T-cell immune reconstitution in HIV-infected patients on HAART for 12 months was poor despite a marked reduction in viral load and an increase in CD4 count. This poor immune reconstitution was characterised by a low influenza-specific proliferative CD4(+) T-cell response and reduced proportions of CD154-expressing influenza-specific CD4(+) T-cells in peripheral blood. CONCLUSION Our data suggest that asymptomatic HIV-infected adults may also be at risk of influenza-related complications and that HAART alone may not circumvent this risk in AIDS patients. This study highlights the need to identify possible interventions early in HIV infection to reduce the risk of influenza and to intensify influenza surveillance in these susceptible African populations

Evaluating the reactivation of herpesviruses and inflammation as cardiovascular and cerebrovascular risk factors in antiretroviral therapy initiators in an African HIV-infected population (RHICCA): a protocol for a longitudinal cohort study.

INTRODUCTION In Sub-Saharan Africa, the rising rates of cerebrovascular and cardiovascular diseases (CBD/CVD) are intersecting with an ageing HIV-infected population. The widespread use of antiretroviral therapy (ART) may confer an additive risk and may not completely suppress the risk associated with HIV infection. High-quality prospective studies are needed to determine if HIV-infected patients in Africa are at increased risk of CBD/CVD and to identify factors associated with this risk. This study will test the hypothesis that immune activation and dysfunction, driven by HIV and reactivation of latent herpesvirus infections, lead to increased CBD/CVD risk in Malawian adults aged ≥35 years. METHODS AND ANALYSIS We will conduct a single-centre, 36-month, prospective cohort study in 800 HIV-infected patients initiating ART and 190 HIV-uninfected controls in Blantyre, Malawi. Patients and controls will be recruited from government ART clinics and the community, respectively, and will be frequency-matched by 5-year age band and sex. At baseline and follow-up visits, we will measure carotid intima-media thickness and pulse wave velocity as surrogate markers of vasculopathy, and will be used to estimate CBD/CVD risk. Our primary exposures of interest are cytomegalovirus and varicella zoster reactivation, changes in HIV plasma viral load, and markers of systemic inflammation and endothelial function. Multivariable regression models will be developed to assess the study's primary hypothesis. The occurrence of clinical CBD/CVD will be assessed as secondary study endpoints. ETHICS AND DISSEMINATION The University of Malawi College of Medicine and Liverpool School of Tropical Medicine research ethics committees approved this work. Our goal is to understand the pathogenesis of CBD/CVD among HIV cohorts on ART, in Sub-Saharan Africa, and provide data to inform future interventional clinical trials. This study runs between May 2017 and August 2020. Results of the main trial will be submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER ISRCTN42862937