Differential Performance of CoronaCHEK SARS-CoV-2 Lateral Flow Antibody Assay by Geographic Origin of Samples.

We assessed the performance of the CoronaCHEK lateral flow assay on samples from Uganda and Baltimore to determine the impact of geographic origin on assay performance. Plasma samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR-positive individuals (Uganda, 78 samples from 78 individuals, and Baltimore, 266 samples from 38 individuals) and from prepandemic individuals (Uganda, 1,077, and Baltimore, 532) were evaluated. Prevalence ratios (PR) were calculated to identify factors associated with a false-positive test. After the first positive PCR in Ugandan samples, the sensitivity was 45% (95% confidence interval [CI], 24,68) at 0 to 7 days, 79% (95% CI, 64 to 91) at 8 to 14 days, and 76% (95% CI, 50 to 93) at >15 days. In samples from Baltimore, sensitivity was 39% (95% CI, 30 to 49) at 0 to 7 days, 86% (95% CI, 79 to 92) at 8 to 14 days, and 100% (95% CI, 89 to 100) at 15 days after positive PCR. The specificity of 96.5% (95% CI, 97.5 to 95.2) in Ugandan samples was significantly lower than that in samples from Baltimore, 99.3% (95% CI, 98.1 to 99.8; P < 0.01). In Ugandan samples, individuals with a false-positive result were more likely to be male (PR, 2.04; 95% CI, 1.03,3.69) or individuals who had had a fever more than a month prior to sample acquisition (PR, 2.87; 95% CI, 1.12 to 7.35). Sensitivity of the CoronaCHEK was similar in samples from Uganda and Baltimore. The specificity was significantly lower in Ugandan samples than in Baltimore samples. False-positive results in Ugandan samples appear to correlate with a recent history of a febrile illness, potentially indicative of a cross-reactive immune response in individuals from East Africa.

Computational MHC-I epitope predictor identifies 95% of experimentally mapped HIV-1 clade A and D epitopes in a Ugandan cohort.

BACKGROUND: Identifying immunogens that induce HIV-1-specific immune responses is a lengthy process that can benefit from computational methods, which predict T-cell epitopes for various HLA types. METHODS: We tested the performance of the NetMHCpan4.0 computational neural network in re-identifying 93 T-cell epitopes that had been previously independently mapped using the whole proteome IFN-γ ELISPOT assays in 6 HLA class I typed Ugandan individuals infected with HIV-1 subtypes A1 and D. To provide a benchmark we compared the predictions for NetMHCpan4.0 to MHCflurry1.2.0 and NetCTL1.2. RESULTS: NetMHCpan4.0 performed best correctly predicting 88 of the 93 experimentally mapped epitopes for a set length of 9-mer and matched HLA class I alleles. Receiver Operator Characteristic (ROC) analysis gave an area under the curve (AUC) of 0.928. Setting NetMHCpan4.0 to predict 11-14mer length did not improve the prediction (37-79 of 93 peptides) with an inverse correlation between the number of predictions and length set. Late time point peptides were significantly stronger binders than early peptides (Wilcoxon signed rank test: p = 0.0000005). MHCflurry1.2.0 similarly predicted all but 2 of the peptides that NetMHCpan4.0 predicted and NetCTL1.2 predicted only 14 of the 93 experimental peptides. CONCLUSION: NetMHCpan4.0 class I epitope predictions covered 95% of the epitope responses identified in six HIV-1 infected individuals, and would have reduced the number of experimental confirmatory tests by > 80%. Algorithmic epitope prediction in conjunction with HLA allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort.

Association between serotonin transporter gene polymorphisms and increased suicidal risk among HIV positive patients in Uganda.

BACKGROUND: Persons living with HIV/AIDS (PLWHA) are at an increased risk of suicide. Increased suicidal risk is a predictor of future attempted and completed suicides and has been associated with poor quality of life and poor adherence with antiretroviral therapy. Clinical risk factors have low predictive value for suicide, hence the interest in potential neurobiological correlates and specific heritable markers of suicide vulnerability. The serotonin transporter gene has previously been implicated in the aetiology of increased suicidal risk in non-HIV infected study populations and its variations may provide a platform for identifying genetic risk for suicidality among PLWHA. The present cross-sectional study aimed at identifying two common genetic variants of the serotonin transporter gene and their association with increased suicidal risk among human immunodeficiency virus (HIV)-positive adults in Uganda. RESULTS: The prevalence of increased suicidal risk (defined as moderate to high risk suicidality on the suicidality module of the Mini Neuropsychiatric Interview (M.I.N.I) was 3.3% (95% CI, 2.0-5.3). The 5-HTTLPR was found to be associated with increased suicidal risk before Bonferroni correction (p-value = 0.0174). A protective effect on increased suicidal risk was found for the 5-HTTLPR/rs25531 S A allele (p-value = 0.0046)- which directs reduced expression of the serotonin transporter gene (5-HTT). CONCLUSION: The S A allele at the 5-HTTLPR/rs25531 locus is associated with increased suicidal risk among Ugandan PLWHA. Further studies are needed to validate this finding in Ugandan and other sub-Saharan samples.

Quantitative and qualitative differences in the T cell response to HIV in uninfected Ugandans exposed or unexposed to HIV-infected partners.

HIV-exposed and yet persistently uninfected individuals have been an intriguing, repeated observation in multiple studies, but uncertainty persists on the significance and implications of this in devising protective strategies against HIV. We carried out a cross-sectional analysis of exposed uninfected partners in a Ugandan cohort of heterosexual serodiscordant couples (37.5% antiretroviral therapy naive) comparing their T cell responses to HIV peptides with those of unexposed uninfected individuals. We used an objective definition of exposure and inclusion criteria, blinded ex vivo and cultured gamma interferon (IFN-γ) enzyme-linked immunospot assays, and multiparameter flow cytometry and intracellular cytokine staining to investigate the features of the HIV-specific response in exposed versus unexposed uninfected individuals. A response rate to HIV was detectable in unexposed uninfected (5.7%, 95% confidence interval [CI] = 3.3 to 8.1%) and, at a significantly higher level (12.5%, 95% CI = 9.7 to 15.4%, P = 0.0004), in exposed uninfected individuals. The response rate to Gag was significantly higher in exposed uninfected (10/50 [20.%]) compared to unexposed uninfected (1/35 [2.9%]) individuals (P = 0.0004). The magnitude of responses was also greater in exposed uninfected individuals but not statistically significant. The average number of peptide pools recognized was significantly higher in exposed uninfected subjects than in unexposed uninfected subjects (1.21 versus 0.47; P = 0.0106). The proportion of multifunctional responses was different in the two groups, with a higher proportion of single cytokine responses, mostly IFN-γ, in unexposed uninfected individuals compared to exposed uninfected individuals. Our findings demonstrate both quantitative and qualitative differences in T cell reactivity to HIV between HESN (HIV exposed seronegative) and HUSN (HIV unexposed seronegative) subject groups but do not discriminate as to whether they represent markers of exposure or of protection against HIV infection.

The subdued post-boost spike-directed secondary IgG antibody response in Ugandan recipients of the Pfizer-BioNTech BNT162b2 vaccine has implications for local vaccination policies.

Introduction: This study aimed to delineate longitudinal antibody responses to the Pfizer-BioNTech BNT162b2 COVID-19 vaccine within the Ugandan subset of the Sub-Saharan African (SSA) demographic, filling a significant gap in global datasets. Methods: We enrolled 48 participants and collected 320 specimens over 12 months after the primary vaccination dose. A validated enzyme-linked immunosorbent assay (ELISA) was used to quantify SARS-CoV-2-specific IgG, IgM, and IgA antibody concentrations (ng/ml) and optical densities (ODs). Statistical analyses included box plots, diverging bar graphs, and the Wilcoxon test with Bonferroni correction. Results: We noted a robust S-IgG response within 14 days of the primary vaccine dose, which was consistent with global data. There was no significant surge in S-IgG levels after the booster dose, contrasting trends in other global populations. The S-IgM response was transient and predominantly below established thresholds for this population, which reflects its typical early emergence and rapid decline. S-IgA levels rose after the initial dose then decreased after six months, aligning with the temporal patterns of mucosal immunity. Eleven breakthrough infections were noted, and all were asymptomatic, regardless of the participants' initial S-IgG serostatus, which suggests a protective effect from vaccination. Discussion: The Pfizer-BioNTech BNT162b2 COVID-19 vaccine elicited strong S-IgG responses in the SSA demographic. The antibody dynamics distinctly differed from global data highlighting the significance of region-specific research and the necessity for customised vaccination strategies.

The single-dose Janssen Ad26.COV2.S COVID-19 vaccine elicited robust and persistent anti-spike IgG antibody responses in a 12-month Ugandan cohort.

Introduction: The study investigation examined the immune response to the Janssen Ad26.COV2.S COVID-19 vaccine within a Ugandan cohort, specifically targeting antibodies directed against spike (S) and nucleocapsid (N) proteins. We aimed to examine the durability and robustness of the induced antibody response while also assessing occurrences of breakthrough infections and previous anti-Spike seropositivity to SARS-CoV-2. Methods: The study included 319 specimens collected over 12 months from 60 vaccinees aged 18 to 64. Binding antibodies were quantified using a validated ELISA method to measure SARS-CoV-2-specific IgG, IgM, and IgA levels against the S and N proteins. Results: The results showed that baseline seropositivity for S-IgG was high at 67%, increasing to 98% by day 14 and consistently stayed above 95% for up to 12 months. However, S-IgM responses remained suboptimal. A raised S-IgA seropositivity rate was seen that doubled from 40% at baseline to 86% just two weeks following the initial vaccine dose, indicating sustained and robust peripheral immunity. An increase in N-IgG levels at nine months post-vaccination suggested breakthrough infections in eight cases. Baseline cross-reactivity influenced spike-directed antibody responses, with individuals harbouring S-IgG antibodies showing notably higher responses. Discussion: Robust and long lasting vaccine and infection-induced immune responses were observed, with significant implications for regions where administering subsequent doses poses logistical challenges.

Sustained S-IgG and S-IgA antibodies to Moderna's mRNA-1273 vaccine in a Sub-Saharan African cohort suggests need for booster timing reconsiderations.

Introduction: This study sought to elucidate the long-term antibody responses to the Moderna mRNA-1273 COVID-19 vaccine within a Ugandan cohort, aiming to contribute to the sparse data on m-RNA vaccine immunogenicity in Sub-Saharan Africa. Methods: We tracked the development and persistence of the elicited antibodies in 19 participants aged 18 to 67, who received two doses of the mRNA-1273 vaccine. A validated enzyme-linked immunosorbent assay (ELISA) was used to quantify SARS-CoV-2-specific IgG, IgM, and IgA antibodies against the spike (S) and nucleoproteins (N). The study's temporal scope extended from the baseline to one year, capturing immediate and long-term immune responses. Statistical analyses were performed using the Wilcoxon test to evaluate changes in antibody levels across predetermined intervals with the Hochberg correction for multiple comparisons. Results: Our results showed a significant initial rise in spike-directed IgG (S-IgG) and spike-directed IgA (S-IgA) levels, which remained elevated for the duration of the study. The S-IgG concentrations peaked 14 days afterboosting, while spike-directed IgM (S-IgM) levels were transient, aligning with their early response role. Notably, post-booster antibody concentrations did not significantly change. Prior S-IgG status influenced the post-priming S-IgA dynamics, with baseline S-IgG positive individuals maintaining higher S-IgA responses, a difference that did not reach statistical difference post-boost. Three instances of breakthrough infections: two among participants who exhibited baseline seropositivity for S-IgG, and one in a participant initially seronegative for S-IgG. Discussion: In conclusion, the mRNA-1273 vaccine elicited robust and persistent S-IgG and S-IgA antibody responses, particularly after the first dose, indicating potential for long-term immunity. Prior viral exposure enhances post-vaccination S-IgA responses compared to naive individuals, which aligned with the prior-naïve, post-boost. The stable antibody levels observed post-booster dose, remaining high over an extended period, with no significant secondary rise, and no difference by baseline exposure, suggest that initial vaccination may sufficiently prime the immune system for prolonged protection in this population, allowing for potential to delay booster schedules as antibody responses remained high at the time of boosting. This finding calls for a reassessment of the booster dose scheduling in this demographic.

An Optimised Indirect ELISA Protocol for Detection and Quantification of Anti-viral Antibodies in Human Plasma or Serum: A Case Study Using SARS-CoV-2.

Advanced immunoassays are crucial in assessing antibody responses, serving immune surveillance goals, characterising immunological responses to evolving viral variants, and guiding subsequent vaccination initiatives. This protocol outlines an indirect ELISA protocol to detect and quantify virus-specific antibodies in plasma or serum after exposure to viral antigens. The assay enables the measurement of IgG, IgA, and IgM antibodies specific to the virus of interest, providing qualitative and quantitative optical densities and concentration data. Although this protocol refers to SARS-CoV-2, its methodology is versatile and can be modified to assess antibody responses for various viral infections and to evaluate vaccine trial outcomes. Key features • This protocol builds upon previously described methodology [1] explicitly tailored for SARS-CoV-2 and broadens its applicability to other viral infections. • The protocol outlines establishing antibody responses to SARS-CoV-2 infections by determining optical densities and concentrations from blood plasma or serum.

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.

A phase I COVID-19 vaccine trial among SARS-CoV-2 seronegative and seropositive individuals in Uganda utilizing a self-amplifying RNA vaccine platform: Screening and enrollment experiences.

We report the screening and enrollment process for a phase I vaccine trial in Masaka, Uganda that investigated the safety and immunogenicity of a self-amplifying SARS-CoV-2 RNA vaccine amongst individuals with and without antibodies to SARS-CoV-2. Participant screening and enrollment were conducted between December 2021 and April 2022. Individuals were eligible if they were aged between 18 and 45 years, healthy, and never vaccinated against COVID-19. SARS-CoV-2 antibody status was determined using two point-of-care rapid tests, i.e. Multi G (MGFT3) and Standard Q (Standard Q COVID-19 IgM/IgG Plus). Data were entered and managed in OpenClinica. Analyses were performed and presented descriptively. A total of 212 individuals were screened and 43(20.3%) enrolled. The most common reasons for exclusion were ≥ grade 1 laboratory abnormalities (39, 18.4%), followed by discordant SARS-CoV-2 antibody results (23, 10.9%). While the first 38 participants were quickly enrolled over a period of 9 weeks, it took another 9 weeks to enroll the remaining five, as antibody negative participants became scarce during the surge of the Omicron variant. The SARS-CoV-2 antibody positivity rate was determined to be 60.8% and 84.4% in each half of the 18 months of screening respectively. The mean age (±Standard Deviation, SD) of screened and enrolled participants was 27.7 (±8.1) and 30.2 (±8.3) years respectively. We demonstrated that it is feasible to successfully screen and enroll participants for COVID-19 vaccine trials in Uganda in the time of a pandemic. Our experiences may be useful for investigators planning to undertake similar work in Africa.

Seroprevalence and durability of antibody responses to AstraZeneca vaccination in Ugandans with prior mild or asymptomatic COVID-19: implications for vaccine policy.

Introduction: The duration and timing of immunity conferred by COVID-19 vaccination in sub-Saharan Africa are crucial for guiding pandemic policy interventions, but systematic data for this region is scarce. This study investigated the antibody response after AstraZeneca vaccination in COVID-19 convalescent Ugandans. Methods: We recruited 86 participants with a previous rt-PCR-confirmed mild or asymptomatic COVID-19 infection and measured the prevalence and levels of spike-directed IgG, IgM, and IgA antibodies at baseline, 14 and 28 days after the first dose (priming), 14 days after the second dose (boosting), and at six- and nine-months post-priming. We also measured the prevalence and levels of nucleoprotein-directed antibodies to assess breakthrough infections. Results: Within two weeks of priming, vaccination substantially increased the prevalence and concentrations of spike-directed antibodies (p < 0.0001, Wilcoxon signed rank test), with 97.0% and 66% of vaccinated individuals possessing S-IgG and S-IgA antibodies before administering the booster dose. S-IgM prevalence changed marginally after the initial vaccination and barely after the booster, consistent with an already primed immune system. However, we also observed a rise in nucleoprotein seroprevalence, indicative of breakthroughs six months after the initial vaccination. Discussion: Our results suggest that vaccination of COVID-19 convalescent individuals with the AstraZeneca vaccine induces a robust and differential spike-directed antibody response. The data highlights the value of vaccination as an effective method for inducing immunity in previously infected individuals and the importance of administering two doses to maintain protective immunity. Monitoring anti-spike IgG and IgA when assessing vaccine-induced antibody responses is suggested for this population; assessing S-IgM will underestimate the response. The AstraZeneca vaccine is a valuable tool in the fight against COVID-19. Further research is needed to determine the durability of vaccine-induced immunity and the potential need for booster doses.

Sustained spike-specific IgG antibodies following CoronaVac (Sinovac) vaccination in sub-Saharan Africa, but increased breakthrough infections in baseline spike-naive individuals.

Introduction: This study investigated the antibody responses to the inactivated COVID-19 vaccine, CoronaVac (Sinovac Biotech) in the African population to provide valuable insights into long-term immunity and breakthrough infections against SARS-CoV-2 in individuals with varying prior IgG seropositivity. Methods: Real-life cohorts were used to longitudinally track antibody levels against the SARS-CoV-2 spike and nucleoprotein in 60 participants over 12 months to examine the levels of multiple antibody isotypes (S-IgG, S-IgM, S-IgA, N-IgG, and N-IgM). Results: Throughout the 12 months, we observed consistently high and stable seropositivity rates for spike-IgG antibodies, spike-IgM antibodies showed a decline in frequencies over time, and spike-IgA levels remained moderate and stable. Vaccinated individuals previously positive for spike-IgG antibodies demonstrated strong and persistent seropositivity, while those initially negative experienced a gradual and delayed increase in seropositivity rates. The fold change analysis of S- and N- antibody responses demonstrated a consistently stable and comparable profile over time, indicating that vaccine-induced antibody responses remain constant and lack significant fluctuations beyond the initial boost. The study emphasized that individuals lacking previous IgG positivity showed reduced vaccine-induced spike-IgG antibodies and were more susceptible to breakthrough infections, highlighting their higher vulnerability. All cases of breakthrough infections were asymptomatic, indicating the conferred protection to the vaccinated individuals. Discussion: The findings corroborated earlier studies on the effectiveness of the CoronaVac vaccine and emphasized the significance of accounting for pre-existing seropositivity in vaccine assessments. This study effectively demonstrated durable antibody responses against SARS-CoV-2 in the African population following the CoronaVac vaccination, providing crucial insights for informing vaccination strategies and safeguarding vulnerable populations. Continuous surveillance is imperative for tracking breakthrough infections and monitoring waning immunity. The insights gained offer crucial direction for public health strategies and enhance comprehension of vaccine effectiveness in sub-Saharan Africa. Further research should explore functional outcomes, cellular immune responses, and the vaccine's effectiveness against different variants to enhance our understanding and optimize vaccine strategies.

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

Introduction: We 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. Methods: We 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. Results: HCoV-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. Discussion: These 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.

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.

Spike protein is a key target for stronger and more persistent T-cell responses-a study of mild and asymptomatic SARS-CoV-2 infection.

OBJECTIVES: Understanding the immune response in very mild and asymptomatic COVID-19 is crucial for developing effective vaccines and immunotherapies, yet remains poorly characterized. This longitudinal study examined the evolution of interferon (IFN)-γ responses to SARS-CoV-2 peptides in 109 asymptomatic or mildly symptomatic Ugandan COVID-19 patients across 365 days and explored their association with antibody generation. METHODS: T-cell responses to spike-containing clusters of differentiation (CD4)-S and CD8 nCoV-A (CD8-A) megapools, and the non-spike CD4-R and CD8 nCoV-B (CD8-B) megapools, were assessed and correlated with demographic and temporal variables. RESULTS: SARS-CoV-2-specific IFN-γ responses were consistently detected in all peptide pools and time points, with the spike-targeted response exhibiting higher potency and durability than the non-spike responses. Throughout the entire 365-day infection timeline, a robust positive correlation was observed between CD4 T-cell responses to the spike-derived peptides and anti-spike immunoglobulin G antibody levels, underscoring their interdependent dynamics in the immune response against SARS-CoV-2; in contrast, CD8 T-cell responses exhibited no such correlation, highlighting their distinctive, autonomous role in defense. No meaningful variations in complete blood count parameters were observed between individuals with COVID-19 infection and those without, indicating clinical insignificance. CONCLUSIONS: This study highlights the dominant role of spike-directed T-cell responses in mild and asymptomatic disease and provides crucial longitudinal data from Sub-Saharan African settings. The findings provide valuable insights into the dynamics of T-cell responses and their potential significance in developing effective strategies for combating COVID-19.

Bacterial microbiome and host inflammatory gene expression in foreskin tissue.

The penile epithelial microbiome remains underexplored. We sequenced human RNA and a segment of the bacterial 16S rRNA gene from the foreskin tissue of 144 adolescents from South Africa and Uganda collected during penile circumcision after receipt of 1-2 doses of placebo, emtricitabine + tenofovir disoproxil fumarate, or emtricitabine + tenofovir alafenamide to investigate the microbiome of foreskin tissue and its potential changes with antiretroviral use. We identified a large number of anaerobic species, including Corynebacterium acnes, which was detected more frequently in participants from South Africa than Uganda. Bacterial populations did not differ by treatment received, and no differentially abundant taxa were identified between placebo versus active drug recipients. The relative abundance of specific bacterial taxa was negatively correlated with expression of genes downstream of the innate immune response to bacteria and regulation of inflammation. Our results show no difference in the tissue microbiome of the foreskin with short-course antiretroviral use but that bacterial taxa were largely inversely correlated with inflammatory gene expression, consistent with commensal colonization.

A randomized clinical trial of on-demand oral pre-exposure prophylaxis does not modulate lymphoid/myeloid HIV target cell density in the foreskin.

OBJECTIVES: As topical pre-exposure prophylaxis (PrEP) has been shown to cause immune modulation in rectal or cervical tissue, our aim was to examine the impact of oral PrEP on lymphoid and myeloid changes in the foreskin in response to dosing and timing of drug administration. DESIGN: HIV-negative male individuals ( n  = 144) were recruited in South Africa and Uganda into an open-label randomized controlled trial in a 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 ratio to control arm (with no PrEP) or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) at one of two different doses, 5 or 21 h before undergoing voluntary medical male circumcision (VMMC). METHODS: After dorsal-slit circumcision, foreskin tissue sections were embedded into Optimal Cutting Temperature media and analysed, blinded to trial allocation, to determine numbers of CD4 + CCR5 + , CD1a + cells and claudin-1 expression. Cell densities were correlated with tissue-bound drug metabolites and p24 production after ex-vivo foreskin challenge with HIV-1 bal . RESULTS: There was no significant difference in CD4 + CCR5 + or CD1a + cell numbers in foreskins between treatment arms compared with the control arm. Claudin-1 expression was 34% higher ( P  = 0.003) in foreskin tissue from participants receiving PrEP relative to controls, but was no longer statistically significant after controlling for multiple comparisons. There was neither correlation of CD4 + CCR5 + , CD1a + cell numbers, or claudin-1 expression with tissue-bound drug metabolites, nor with p24 production after ex-vivo viral challenge. CONCLUSION: Oral doses and timing of on-demand PrEP and in-situ drug metabolite levels in tissue have no effect on numbers or anatomical location of lymphoid or myeloid HIV target cells in foreskin tissue.

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.

Optimization and validation of an ELISA assay for the determination of antibody responses to CN54gp140 and AIDSVAX BE for use in the Phase IIb PrEPVacc vaccine trial.

PrEPVacc is an international, multi-centre, double-blind vaccine study comparing experimental combination vaccine regimens including DNA/AIDSVAX BE and DNA/CN54gp140 with placebo control. Simultaneously, daily oral PrEP is compared for efficacy against daily Truvada in the context of the current PrEP availability situation at the study sites. An important clinical trial outcome is the accurate measurement of in vivo antibody titer induced through vaccination. Here we report the validation of two ELISAs for CN54gp140 and AIDSVAX BE at Uganda Virus Research Institute that demonstrates precision, specificity, and robustness for assessing the reciprocal antibody end point titer in human serum. This is a critical endpoint for determining whether vaccination can provide any protection against HIV in populations at risk of acquiring HIV.

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.