CD8+ T-Cell Responses before and after Structured Treatment Interruption in Ugandan Adults Who Initiated ART with CD4+ T Cells <200 Cell/μL: The DART Trial STI Substudy

Objective. To better understand attributes of ART-associated HIV-induced T-cell responses that might be therapeutically harnessed. Methods. CD8+ T-cell responses were evaluated in some HIV-1 chronically infected participants of the fixed duration STI substudy of the DART trial. Magnitudes, breadths, and functionality of IFN-γ and Perforin responses were compared in STI (n = 42) and continuous treatment (CT) (n = 46) before and after a single STI cycle when the DART STI trial was stopped early due to inferior clinical outcome in STI participants. Results. STI and CT had comparable magnitudes and breadths of monofunctional CD8+IFNγ+ and CD8+Perforin+ responses. However, STI was associated with significant decline in breadth of bi-functional (CD8+IFNγ+Perforin+) responses; P = .02, Mann-Whitney test. Conclusions. STI in individuals initiated onto ART at <200 CD4+ T-cell counts/μl significantly reduced occurrence of bifunctional CD8+IFNγ+/Perforin+ responses. These data add to others that found no evidence to support STI as a strategy to improve HIV-specific immunity during ART

Interferon gamma (IFN-γ) negative CD4+ and CD8+ T-cells can produce immune mediators in response to viral antigens.

Evaluation of antigen-specific T-cell responses to viral antigens is frequently performed on IFN-γ secreting cells. However, T-cells are capable of producing many more functions than just IFN-γ, some of which, like Perforin, are associated with immune protection in HIV-1 disease elite controllers. We evaluated the extent of missed T-cell functions when IFN-γ secretion is used as a surrogate marker for further evaluation of T-cell functions. Intracellular cytokine staining assay and flow cytometry were used to assess peripheral blood mononuclear cells (PBMCs) from 31 HIV-infected ART-naive individuals for the extent to which gated CD4+ and CD8+ IFN-γ producing and non-producing T-cells also secreted IL-2, Perforin, and TNF-α functions. Similarly, the extent of missed virus-specific responses in IFN-γ ELISpot assay negative T-cells from 5 HIV-1 uninfected individuals was evaluated. Cells from HIV-infected individuals were stimulated with pooled consensus group M (Con M) peptides; and those from healthy individuals were stimulated with pooled adenovirus (Ad) peptides. Overall, frequencies of virus-specific IFN-γ secreting CD4+ and CD8+ cells were low. Proportions of IFN-γ negative CD4+ expressing IL-2, Perforin, or TNF-α to Con M were significantly higher (5 of 7 functional profiles) than the corresponding IFN-γ positive CD4+ (0 of 7) T-cell phenotype, p = 0.02; Fisher's Exact test. Likewise, proportions of CD8+ T-cells expressing other functions were significantly higher in 4 of the 7 IFN-γ negative CD8+ T-cells. Notably, newly stimulated Perforin, identified as Perforin co-expression with IL-2 or TNF-α, was significantly higher in IFN-γ negative CD8+ T-cell than in the positive CD8+ T-cells. Using SEB, lower responses in IFN-γ positive cells were most associated with CD4+ than CD8+ T-cells. These findings suggest that studies evaluating immunogenicity in response to HIV and Adenovirus viral antigens should not only evaluate T-cell responsiveness among IFN-γ producing cells but also among those T-cells that do not express IFN-γ

Absence of in vivo selection for K13 mutations after artemether–lumefantrine treatment in Uganda

Additional file 1. Eligibility criteria for recruitment into the therapeutic efficacy study and the molecular study

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

Distribution of Gag T-cell recognition among slow progressors.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g004" target="_blank">Figure 4</a> illustrates the frequency of Gag T-cell recognition across the p17, p24 and p15 Gag region among the slow progressors. The frequency is presented as the proportion of SP individuals with Gag T cell recognition. The horizontal axis represents the peptides recognised.</p

Relationship between multiclade Gag T-cell recognition and HIV disease progression.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g005" target="_blank">Figure 5</a> illustrates the total magnitudes of HIV Gag-induced IFNγ responses evaluated using ELISpot assay. A test was considered positive when response was ≥50 SFU/million PBMCs and at least twice the mean background response (6 wells of cells and media response only). The data is presented as net response; all background values have been subtracted. For purposes of statistical analysis and graphical representation, all negative responses (less than 50 net SFU/million PBMCs) were equated to zero SFU/ml PBMCs. Because the Y axis is presented in log, negative responses are not represented on these graphs. The X-axis represents individual participants. The horizontal dotted lines parallel to the X-axis represent the cutoff for a positive response. Slow progressors (SPs) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g005" target="_blank">figure 5A</a>; rapid progressors (RPs) are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g005" target="_blank">figure 5B</a> while normal progressors are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g005" target="_blank">figure 5C</a>.</p

Breadth of HIV-induced IFN-γ T-cell recognition.

<p>The peptides sets evaluated were dictated by availability from the NIH AIDS reagent programme; clades A, B, C and D Gag peptides were obtainable while only clade B was available for Nef, Tat, Rev, Vif, Vpr, Pol and Vpu proteins. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g003" target="_blank">Figure 3</a> compares the proportion of participants (%) inducing HIV-specific IFN-γ responses using clade B peptides only, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g003" target="_blank">figure 3A</a>; as opposed to multiple clade Gag peptide sets, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-g003" target="_blank">figure 3B</a>.</p

Study population demographics, host genetics and multi-clade gag recognition.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004188#pone-0004188-t002" target="_blank">Table 2</a> illustrates study participant demographics and IFNγ response to clades A, B, C and D gag peptides. CD4+ T –cell slopes were derived from multilevel regression analysis of retrospective 6-monthly CD4+ T-cell counts. Under annual CD4 slope, symbol (−) indicates model-derived decreasing CD4 slopes while (+).indicates decreasing CD4 slopes over time. Under Gag-induced IFN-γ, areas marked + indicate induction of Gag-induced IFN-γ responses to the respective clade of Gag peptides, blank areas indicate lack of IFN-γ response. Clade of the infecting HIV virus was determined from partial sequences of the Gag region, “nd” indicates not done. Bold highlights indicate HLA alleles that have been reported to confer protection from HIV disease.</p