Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation

<div><p>Mucosal Th17 cells play an important role in maintaining gut epithelium integrity and thus prevent microbial translocation. Chronic HIV infection is characterized by mucosal Th17 cell depletion, microbial translocation and subsequent immune-activation, which remain elevated despite antiretroviral therapy (ART) correlating with increased mortality. However, when Th17 depletion occurs following HIV infection is unknown. We analyzed mucosal Th17 cells in 42 acute HIV infection (AHI) subjects (Fiebig (F) stage I-V) with a median duration of infection of 16 days and the short-term impact of early initiation of ART. Th17 cells were defined as IL-17+ CD4+ T cells and their function was assessed by the co-expression of IL-22, IL-2 and IFNγ. While intact during FI/II, depletion of mucosal Th17 cell numbers and function was observed during FIII correlating with local and systemic markers of immune-activation. ART initiated at FI/II prevented loss of Th17 cell numbers and function, while initiation at FIII restored Th17 cell numbers but not their polyfunctionality. Furthermore, early initiation of ART in FI/II fully reversed the initially observed mucosal and systemic immune-activation. In contrast, patients treated later during AHI maintained elevated mucosal and systemic CD8+ T-cell activation post initiation of ART. These data support a loss of Th17 cells at early stages of acute HIV infection, and highlight that studies of ART initiation during early AHI should be further explored to assess the underlying mechanism of mucosal Th17 function preservation.</p></div

Clinical, immunological and virological baseline characteristics and demographics of study participants.

A<p>range;</p>B<p>MHAbce: Multi-region hybridization assay distinguishing between subtypes B, C and CRF01_AE. One subject was CRF01_AE/B, 8 were non typable and results for 3 subjects were not typed by the time the manuscript was written;</p>C<p>Western blot positive with p31 band; MSM: Men who have sex with men; Fiebig I - positive HIV RNA, negative p24 antigen, negative 3rd generation EIA; Fiebig II – positive HIV RNA, positive p24 antigen, negative 3rd generation EIA; Fiebig III - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, negative western blot; Fiebig IV - positive HIV RNA, positive or negative p24 antigen, positive 3rd generation EIA, indeterminate western blot; Fiebig V - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, positive western blot except p31; NA: Not Applicable; ND: Not Determined</p><p>Clinical, immunological and virological baseline characteristics and demographics of study participants.</p

Frequency of IL-17 and/or IL-22 expressing mucosal CD4+T cells decreases by progression of Fiebig stage.

<p>To quantify the expression of Il-17 and IL-22 in CD4+T cells, mucosal and peripheral mononuclear cells were stimulated for 5 hours with 40 ng/mL PMA and 1 µM Ionomycin. Gating strategy of sigmoid colon Th17 and Th22 CD4+T cells is shown for a FI subject (<b>a</b>) and representative flow cytometry plots including unstimulated controls (unstim) gated on CD4+T cells showing expression of IL-17 and/or IL-22 in FI (<b>b</b>), FIII (<b>c</b>) and FV (<b>d</b>) in the sigmoid colon. A decrease in the frequency of IL-17 (<b>e</b>), IL-22 (<b>f</b>), IL-17/IL-22 (<b>g</b>)-producing cells was seen as well as in the subpopulation of triple-cytokine producing (IL-2, IL-22, IFNγ) Th17 cells (<b>h</b>) from FI (black circle)/FII (red circle) to FIII and FIV (red square)/FV (black square). Frequency of single and double cytokine producing cells is calculated from percentage CD4+T cells, while triple-cytokine producing cells are calculated from percentage of CD4+IL17+T cells. All comparisons were made to FI/II: *p≤0.05, **p≤0.01 and ***p≤0.001.</p

Impact of early ART initiation on CD4+ and CD8+T cells as well as Th17 cells.

<p>Subjects that initiated ART during FI (black circle)/FII (red circle) for 6 months were able to maintain mucosal CD4+T cells (<b>a</b>), CD4+CCR5+ (<b>b</b>), Th17 cells (<b>c</b>), triple cytokine-producing Th17 cells (<b>d</b>), Th22 cells (<b>e</b>), and IL-17 and/or IL-22 (<b>f</b>) producing CD4+T cells with no differences when compared to HIV-. In addition their CD8 activation in the mucosa (<b>g</b>) and periphery (<b>h</b>) normalized after 6 months of ART. Frequency of single and double cytokine producing cells was calculated from percentage CD4+T cells, while triple-cytokine producing cells were calculated from percentage of CD4+IL17+T cells. *p≤0.05, **p≤0.01 and ***p≤0.001; DR: HLA-DR; blue dotted line: median of HIV- individuals; purple dotted line: median of CHI individuals.</p

Proportion and absolute number of CD4+, CD4+CCR5+ and CD8+ T cells in sigmoid colon and peripheral blood at baseline in HIV-, FI/II, FIII, FIV/V and CHI subjects.

<p>All data are median (interquartile rang); All comparisons were made to FI/II; *p≤0.05, **p≤0.01 and ***p≤0.001; CHI: Chronically HIV-infected patients; ND: Not Determined; abs numbers in the sigmoid colon are shown as 10⁶ cells per gram tissue and in the peripheral blood as cells/mm^{3; &}Percentage of CD4+ and CD8+T cells based on population of CD3+T cells.</p><p>Proportion and absolute number of CD4+, CD4+CCR5+ and CD8+ T cells in sigmoid colon and peripheral blood at baseline in HIV-, FI/II, FIII, FIV/V and CHI subjects.</p

Percentage area of lamina propria CD4+ staining (% Area LP) decreases with progression of Fiebig stage.

<p>(<b>a</b>) The percent area of the lamina propria that stained for CD4+T cells (representative images shown in e) decreases by Fiebig stage in the sigmoid colon when compared to FI/II (*p≤0.05, **p≤0.01 and ***p≤0.001). The percent area of the lamina propria that stained for CD4+T cells correlated inversely with the colonic (<b>b</b>) and plasma (<b>c</b>) HIV RNA in FI/II, FIII and FIV/V. HIV viral replication during different Fiebig stages is shown by in <i>situ hybridization</i> displaying HIV-1 vRNA+ cells within the sigmoid mucosa, indicated by blue/black stained cells in nuclear fast red counterstained tissue sections (black arrows highlighting examples of HIV vRNA+ cells) of patients in FI, FII and FIII (<b>d</b>). CD4+T cell depletion is shown by immunohistochemical staining of CD4+T cells (brown) and macrophages (red) in sigmoid mucosa of patients in FI, FII and FIII (<b>e</b>). FI (black circle)/FII (red circle) and FIV (red square)/FV (black square).</p

Clinical, immunological and virological baseline characteristics and demographics of study participants.

A<p>range;</p>B<p>MHAbce: Multi-region hybridization assay distinguishing between subtypes B, C and CRF01_AE. One subject was CRF01_AE/B, 8 were non typable and results for 3 subjects were not typed by the time the manuscript was written;</p>C<p>Western blot positive with p31 band; MSM: Men who have sex with men; Fiebig I - positive HIV RNA, negative p24 antigen, negative 3rd generation EIA; Fiebig II – positive HIV RNA, positive p24 antigen, negative 3rd generation EIA; Fiebig III - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, negative western blot; Fiebig IV - positive HIV RNA, positive or negative p24 antigen, positive 3rd generation EIA, indeterminate western blot; Fiebig V - positive HIV RNA, positive p24 antigen, positive 3rd generation EIA, positive western blot except p31; NA: Not Applicable; ND: Not Determined</p><p>Clinical, immunological and virological baseline characteristics and demographics of study participants.</p

Safety and efficacy of VRC01 broadly neutralising antibodies in adults with acutely treated HIV (RV397): a phase 2, randomised, double-blind, placebo-controlled trial

Background: HIV-1-specific broadly neutralising antibodies such as VRC01 could promote HIV remission by halting viral replication and clearing infected cells. We investigated whether VRC01 could promote sustained viral control off antiretroviral therapy (ART) in adults who initiated ART during acute HIV infection. Methods: We did a randomised, double-blind, placebo-controlled trial at the Thai Red Cross AIDS Research Centre in Bangkok, Thailand. Eligible participants were aged 20–50 years, had initiated ART during acute infection (ie, Fiebig stages I–III), had been taking ART for more than 24 months, had fewer than 50 HIV-1 RNA copies per mL on three consecutive measurements, had more than 400 CD4 cells per μL, had fewer than ten copies of integrated HIV-1 DNA per 10 6 peripheral blood mononuclear cells, and were in generally good health. Eligible participants were randomly assigned (3:1) based on computer-generated lists with a blocking factor of 4 to receive VRC01 (40 mg/kg) or placebo (saline) intravenously every 3 weeks for up to 24 weeks during analytic interruption of ART, followed by continued observation off all therapies. Randomisation was stratified by Fiebig stage (I vs II vs III) at HIV diagnosis. Participants were monitored closely and resumed ART if 1000 or more HIV-1 RNA copies were detected per mL of plasma. The primary outcomes were the frequency of serious adverse events and the proportion of participants with fewer than 50 HIV-1 RNA copies per mL 24 weeks after treatment interruption. Efficacy analyses included all participants who received at least one full dose of study product, and safety analyses included all participants exposed to any study product. The trial was registered with ClinicalTrials.gov, number NCT02664415. This trial is completed. Findings: Between Aug 8, 2016, and Jan 9, 2017, 19 men were randomly assigned, 14 to the VRC01 group and five to the placebo group. One participant in the VRC01 group received a partial infusion without undergoing treatment interruption. The other 18 participants all received at least one full study infusion and underwent ART interruption. No serious adverse events were reported in either group. Only one participant in the VRC01 group achieved the primary efficacy endpoint of viral suppression 24 weeks after ART interruption. The other 17 restarted ART because of a confirmed recording of 1000 or more HIV-1 RNA copies per mL before 24 weeks. Interpretation: VRC01 monotherapy in individuals who initiated ART during acute HIV infection was well tolerated but did not significantly increase the number of participants with viral suppression 24 weeks after ART interruption. Further development of VRC01 and other immunotherapies for HIV will probably occur as part of combination regimens that include several treatments directed against unique therapeutic targets. Funding: US Department of the Army, US National Institutes of Health, and the Thai Red Cross AIDS Research Centre