Mobilization of systemic CCL4 following HIV pre-exposure prophylaxis in young men in Africa

HIV-1 pre-exposure prophylaxis (PrEP) relies on inhibition of HIV-1 replication steps. To understand how PrEP modulates the immunological environment, we derived the plasma proteomic profile of men receiving emtricitabine-tenofovir (FTC-TDF) or emtricitabine-tenofovir alafenamide (FTC-TAF) during the CHAPS trial in South Africa and Uganda (NCT03986970). The CHAPS trial randomized 144 participants to one control and 8 PrEP arms, differing by drug type, number of PrEP doses and timing from final PrEP dose to sampling. Blood was collected pre- and post-PrEP. The inflammatory profile of plasma samples was analyzed using Olink (N=92 proteins) and Luminex (N=33) and associated with plasma drug concentrations using mass spectrometry. The proteins whose levels changed most significantly from pre- to post-PrEP were CCL4, CCL3 and TNF-α; CCL4 was the key discriminator between pre- and post-PrEP samples. CCL4 and CCL3 levels were significantly increased in post-PrEP samples compared to control specimens. CCL4 was significantly correlated with FTC drug levels in plasma. Production of inflammatory chemokines CCL4 and CCL3 in response to short-term PrEP indicates the mobilization of ligands which potentially block virus attachment to CCR5 HIV-1 co-receptor. The significant correlation between CCL4 and FTC levels suggests that CCL4 increase is modulated as an inflammatory response to PrEP

Rapid, early, and potent Spike-directed IgG, IgM, and IgA distinguish asymptomatic from mildly symptomatic COVID-19 in Uganda, with IgG persisting for 28 months.

INTRODUCTION: Understanding how spike (S)-, nucleoprotein (N)-, and RBD-directed antibody responses evolved in mild and asymptomatic COVID-19 in Africa and their interactions with SARS-CoV-2 might inform development of targeted treatments and vaccines. METHODS: Here, we used a validated indirect in-house ELISA to characterise development and persistence of S- and N-directed IgG, IgM, and IgA antibody responses for 2430 SARS-CoV-2 rt-PCR-diagnosed Ugandan specimens from 320 mild and asymptomatic COVID-19 cases, 50 uninfected contacts, and 54 uninfected non-contacts collected weekly for one month, then monthly for 28 months. RESULTS: During acute infection, asymptomatic patients mounted a faster and more robust spike-directed IgG, IgM, and IgA response than those with mild symptoms (Wilcoxon rank test, p-values 0.046, 0.053, and 0.057); this was more pronounced in males than females. Spike IgG antibodies peaked between 25 and 37 days (86.46; IQR 29.47-242.56 BAU/ml), were significantly higher and more durable than N- and RBD IgG antibodies and lasted for 28 months. Anti-spike seroconversion rates consistently exceeded RBD and nucleoprotein rates. Spike- and RBD-directed IgG antibodies were positively correlated until 14 months (Spearman's rank correlation test, p-values 0.0001 to 0.05), although RBD diminished faster. Significant anti-spike immunity persisted without RBD. 64% and 59% of PCR-negative, non-infected non-contacts and suspects, exhibited baseline SARS-CoV-2 N-IgM serological cross-reactivity, suggesting undetected exposure or abortive infection. N-IgG levels waned after 787 days, while N-IgM levels remained undetectable throughout. DISCUSSION: Lower N-IgG seroconversion rates and the absence of N-IgM indicate that these markers substantially underestimate the prior exposure rates. Our findings provide insights into the development of S-directed antibody responses in mild and asymptomatic infections, with varying degrees of symptoms eliciting distinct immune responses, suggesting distinct pathogenic pathways. These longer-lasting data inform vaccine design, boosting strategies, and surveillance efforts in this and comparable settings

Short-term oral pre-exposure prophylaxis against HIV-1 modulates the transcriptome of foreskin tissue in young men in Africa

Whilst short-term oral pre-exposure prophylaxis (PrEP) with antiretroviral drugs in men who have sex with men has shown protection against HIV-1 infection, the impact of this regimen on the in vivo foreskin transcriptome is unknown. We collected foreskin tissue after voluntary medical male circumcision from 144 young men (72 from Uganda and 72 from South Africa) randomized to one to two doses of either oral tenofovir (TFV) disoproxil fumarate (FTC-TDF) or tenofovir alafenamide (FTC-TAF) or no drug (untreated controls). This novel approach allowed us to examine the impact of short-term oral PrEP on transcriptome of the male genital tract. A single dose of FTC-TDF did not affect the foreskin transcriptome in relation to control arm, however one dose of FTC-TAF induced upregulation of four genes AKAP8, KIAA0141, HSCB and METTL17. Following two doses of either FTC-TDF or FTC-TAF, there was an increase in 34 differentially expressed genes for FTC-TDF and 15 for FTC-TAF, with nine DEGs in common: KIAA0141, SAFB2, CACTIN, FXR2, AKAP8, HSCB, CLNS1A, DDX27 and DCAF15. Functional analysis of differentially expressed genes revealed modulation of biological processes related to mitochondrial stress (KIAA0141, HSCB and METTL17), anti-viral and anti-inflammatory pathways (CACTIN and AKAP8). Our results show that short-course on-demand oral PrEP in men modulates genes in foreskin tissue which are likely unfavorable to HIV acquisition and replication. We also describe an upregulated expression of genes involved in diverse mitochondria biology which may potentially result in worsened mitochondria-related. These results warrant further studies to assess the role of short-course and prolonged oral PrEP on biological processes of the foreskin mucosa

Short-term oral pre-exposure prophylaxis against HIV-1 modulates the transcriptome of foreskin tissue in young men in Africa

Whilst short-term oral pre-exposure prophylaxis (PrEP) with antiretroviral drugs in men who have sex with men has shown protection against HIV-1 infection, the impact of this regimen on the in vivo foreskin transcriptome is unknown. We collected foreskin tissue after voluntary medical male circumcision from 144 young men (72 from Uganda and 72 from South Africa) randomized to one to two doses of either oral tenofovir (TFV) disoproxil fumarate (FTC-TDF) or tenofovir alafenamide (FTC-TAF) or no drug (untreated controls). This novel approach allowed us to examine the impact of short-term oral PrEP on transcriptome of the male genital tract. A single dose of FTC-TDF did not affect the foreskin transcriptome in relation to control arm, however one dose of FTC-TAF induced upregulation of four genes AKAP8, KIAA0141, HSCB and METTL17. Following two doses of either FTC-TDF or FTC-TAF, there was an increase in 34 differentially expressed genes for FTC-TDF and 15 for FTC-TAF, with nine DEGs in common: KIAA0141, SAFB2, CACTIN, FXR2, AKAP8, HSCB, CLNS1A, DDX27 and DCAF15. Functional analysis of differentially expressed genes revealed modulation of biological processes related to mitochondrial stress (KIAA0141, HSCB and METTL17), anti-viral and anti-inflammatory pathways (CACTIN and AKAP8). Our results show that short-course on-demand oral PrEP in men modulates genes in foreskin tissue which are likely unfavorable to HIV acquisition and replication. We also describe an upregulated expression of genes involved in diverse mitochondria biology which may potentially result in worsened mitochondria-related. These results warrant further studies to assess the role of short-course and prolonged oral PrEP on biological processes of the foreskin mucosa

Dose finding study for on-demand HIV pre-exposure prophylaxis for insertive sex in sub-Saharan Africa: results from the CHAPS open label randomised controlled trial

BACKGROUND: The efficacy of on-demand HIV pre-exposure prophylaxis (PrEP) for men in sub-Saharan Africa has not been evaluated, and the on-demand PrEP dosing requirement for insertive sex remains unknown. METHODS: HIV-negative males 13-24 years, requesting voluntary medical male circumcision (VMMC), were enrolled into an open-label randomised controlled trial (NCT03986970), and randomised 1:1:1:1:1:1:1:1:1 to control arm or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) over one or two days, and circumcised 5 or 21 h thereafter. The primary outcome was foreskin p24 concentrations following ex vivo HIV-1BaL challenge. Secondary outcomes included peripheral blood mononuclear cell (PBMC) p24 concentration, and drug concentrations in foreskin tissue, PBMCs, plasma and foreskin CD4+/CD4-cells. In the control arm, post-exposure prophylaxis (PEP) activity of non-formulated tenofovir-emtricitabine (TFV-FTC) or TAF-FTC was assessed with ex vivo dosing 1, 24, 48 or 72 h post-HIV-1 challenge. FINDINGS: 144 participants were analysed. PrEP with F/TDF or F/TAF prevented ex vivo infection of foreskins and PBMCs both 5 and 21 h after PrEP dosing. There was no difference between F/TDF and F/TAF (p24day15 geometric mean ratio 1.06, 95% confidence interval: 0.65-1.74). Additional ex vivo dosing did not further increase inhibition. In the control arm, PEP ex vivo dosing was effective up to 48 post-exposure diminishing thereafter, with TAF-FTC showing prolonged protection compared to TFV-FTC. Participants receiving F/TAF had higher TFV-DP concentrations in foreskin tissue and PBMCs compared with F/TDF, irrespective of dose and sampling interval; but F/TAF did not confer preferential TFV-DP distribution into foreskin HIV target cells. FTC-TP concentrations with both drug regimens were equivalent and ∼1 log higher than TFV-DP in foreskin. INTERPRETATION: A double dose of either F/TDF or F/TAF given once either 5 or 21 h before ex vivo HIV-challenge provided protection across foreskin tissue. Further clinical evaluation of pre-coital PrEP for insertive sex is warranted. FUNDING: EDCTP2, Gilead Sciences, Vetenskapsrådet

Pre-pandemic SARS-CoV-2-specific IFN-γ and antibody responses were low in Ugandan samples and significantly reduced in HIV-positive specimens

IntroductionWe investigated whether prior SARS-CoV-2-specific IFN-γ and antibody responses in Ugandan COVID-19 pre-pandemic specimens aligned to this population's low disease severity.MethodsWe used nucleoprotein (N), spike (S), NTD, RBD, envelope, membrane, SD1/2-directed IFN-γ ELISpots, and an S- and N-IgG antibody ELISA to screen for SARS-CoV-2-specific cross-reactivity.ResultsHCoV-OC43-, HCoV-229E-, and SARS-CoV-2-specific IFN-γ occurred in 23, 15, and 17 of 104 specimens, respectively. Cross-reactive IgG was more common against the nucleoprotein (7/110, 15.5%; p = 0.0016, Fishers' Exact) than the spike (3/110, 2.72%). Specimens lacking anti-HuCoV antibodies had higher rates of pre-epidemic SARS-CoV-2-specific IFN-γ cross-reactivity (p-value = 0.00001, Fishers’ exact test), suggesting that exposure to additional factors not examined here might play a role. SARS-CoV-2-specific cross-reactive antibodies were significantly less common in HIV-positive specimens (p=0.017; Fishers' Exact test). Correlations between SARS-CoV-2- and HuCoV-specific IFN-γ responses were consistently weak in both HIV negative and positive specimens.DiscussionThese findings support the existence of pre-epidemic SARS-CoV-2-specific cellular and humoral cross-reactivity in this population. The data do not establish that these virus-specific IFN-γ and antibody responses are entirely specific to SARS-CoV-2. Inability of the antibodies to neutralise SARS-CoV-2 implies that prior exposure did not result in immunity. Correlations between SARS-CoV-2 and HuCoV-specific responses were consistently weak, suggesting that additional variables likely contributed to the pre-epidemic cross-reactivity patterns. The data suggests that surveillance efforts based on the nucleoprotein might overestimate the exposure to SARS-CoV-2 compared to inclusion of additional targets, like the spike protein. This study, while limited in scope, suggests that HIV-positive people are less likely than HIV-negative people to produce protective antibodies against SARS-CoV-2

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB

Neuropsychiatric manifestations and sleep disturbances with dolutegravir-based antiretroviral therapy versus standard of care in children and adolescents: a secondary analysis of the ODYSSEY trial

BACKGROUND: Cohort studies in adults with HIV showed that dolutegravir was associated with neuropsychiatric adverse events and sleep problems, yet data are scarce in children and adolescents. We aimed to evaluate neuropsychiatric manifestations in children and adolescents treated with dolutegravir-based treatment versus alternative antiretroviral therapy. METHODS: This is a secondary analysis of ODYSSEY, an open-label, multicentre, randomised, non-inferiority trial, in which adolescents and children initiating first-line or second-line antiretroviral therapy were randomly assigned 1:1 to dolutegravir-based treatment or standard-of-care treatment. We assessed neuropsychiatric adverse events (reported by clinicians) and responses to the mood and sleep questionnaires (reported by the participant or their carer) in both groups. We compared the proportions of patients with neuropsychiatric adverse events (neurological, psychiatric, and total), time to first neuropsychiatric adverse event, and participant-reported responses to questionnaires capturing issues with mood, suicidal thoughts, and sleep problems. FINDINGS: Between Sept 20, 2016, and June 22, 2018, 707 participants were enrolled, of whom 345 (49%) were female and 362 (51%) were male, and 623 (88%) were Black-African. Of 707 participants, 350 (50%) were randomly assigned to dolutegravir-based antiretroviral therapy and 357 (50%) to non-dolutegravir-based standard-of-care. 311 (44%) of 707 participants started first-line antiretroviral therapy (ODYSSEY-A; 145 [92%] of 157 participants had efavirenz-based therapy in the standard-of-care group), and 396 (56%) of 707 started second-line therapy (ODYSSEY-B; 195 [98%] of 200 had protease inhibitor-based therapy in the standard-of-care group). During follow-up (median 142 weeks, IQR 124–159), 23 participants had 31 neuropsychiatric adverse events (15 in the dolutegravir group and eight in the standard-of-care group; difference in proportion of participants with ≥1 event p=0·13). 11 participants had one or more neurological events (six and five; p=0·74) and 14 participants had one or more psychiatric events (ten and four; p=0·097). Among 14 participants with psychiatric events, eight participants in the dolutegravir group and four in standard-of-care group had suicidal ideation or behaviour. More participants in the dolutegravir group than the standard-of-care group reported symptoms of self-harm (eight vs one; p=0·025), life not worth living (17 vs five; p=0·0091), or suicidal thoughts (13 vs none; p=0·0006) at one or more follow-up visits. Most reports were transient. There were no differences by treatment group in low mood or feeling sad, problems concentrating, feeling worried or feeling angry or aggressive, sleep problems, or sleep quality. INTERPRETATION: The numbers of neuropsychiatric adverse events and reported neuropsychiatric symptoms were low. However, numerically more participants had psychiatric events and reported suicidality ideation in the dolutegravir group than the standard-of-care group. These differences should be interpreted with caution in an open-label trial. Clinicians and policy makers should consider including suicidality screening of children or adolescents receiving dolutegravir

Mobilization of systemic CCL4 following HIV pre-exposure prophylaxis in young men in Africa.

HIV-1 pre-exposure prophylaxis (PrEP) relies on inhibition of HIV-1 replication steps. To understand how PrEP modulates the immunological environment, we derived the plasma proteomic profile of men receiving emtricitabine-tenofovir (FTC-TDF) or emtricitabine-tenofovir alafenamide (FTC-TAF) during the CHAPS trial in South Africa and Uganda (NCT03986970). The CHAPS trial randomized 144 participants to one control and 8 PrEP arms, differing by drug type, number of PrEP doses and timing from final PrEP dose to sampling. Blood was collected pre- and post-PrEP. The inflammatory profile of plasma samples was analyzed using Olink (N=92 proteins) and Luminex (N=33) and associated with plasma drug concentrations using mass spectrometry. The proteins whose levels changed most significantly from pre- to post-PrEP were CCL4, CCL3 and TNF-α; CCL4 was the key discriminator between pre- and post-PrEP samples. CCL4 and CCL3 levels were significantly increased in post-PrEP samples compared to control specimens. CCL4 was significantly correlated with FTC drug levels in plasma. Production of inflammatory chemokines CCL4 and CCL3 in response to short-term PrEP indicates the mobilization of ligands which potentially block virus attachment to CCR5 HIV-1 co-receptor. The significant correlation between CCL4 and FTC levels suggests that CCL4 increase is modulated as an inflammatory response to PrEP

Optimisation and Validation of a conventional ELISA and cut-offs for detecting and quantifying anti-SARS-CoV-2 Spike, RBD, and Nucleoprotein IgG, IgM, and IgA antibodies in Uganda

There is an urgent need for better immunoassays to measure antibody responses as part of immune-surveillance activities and to profile immunological responses to emerging SARS-CoV-2 variants. We optimised and validated an in-house conventional ELISA to identify and quantify SARS-CoV-2 spike- (S-), receptor binding domain- (RBD-), and nucleoprotein- (N-) directed IgG, IgM, and IgA binding antibodies in the Ugandan population and similar settings. Pre- and post-pandemic specimens were used to compare the utility of mean ± 2SD, mean ± 3SD, 4-fold above blanks, bootstrapping, and receiver operating characteristic (ROC) analyses in determining optimal cut-off optical densities at 450 nm (OD) for discriminating between antibody positives and negatives. “Limits of detection” (LOD) and “limits of quantitation” (LOQ) were validated alongside the assay’s uniformity, accuracy, inter-assay and inter-operator precision, and parallelism. With spike-directed sensitivity and specificity of 95.33 and 94.15%, respectively, and nucleoprotein sensitivity and specificity of 82.69 and 79.71%, ROC was chosen as the best method for determining cutoffs. Accuracy measurements were within the expected CV range of 25%. Serum and plasma OD values were highly correlated (r = 0.93, p=0.0001). ROC-derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA were 0.432, 0.356, 0.201 (S), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N). The sensitivity and specificity of the S-IgG cut-off were equivalent to the WHO 20/B770-02 S-IgG reference standard at 100% level. Spike negative IgG, IgM, and IgA ODs corresponded to median antibody concentrations of 1.49, 3.16, and 0 BAU/mL, respectively, consistent with WHO low titre estimates. Anti-spike IgG, IgM, and IgA cut-offs were equivalent to 18.94, 20.06, and 55.08 BAU/mL. For the first time, we provide validated parameters and cut-off criteria for the in-house detection of subclinical SARS-CoV-2 infection and vaccine-elicited binding antibodies in the context of Sub-Saharan Africa and populations with comparable risk factors