Abstract
Objective:
To assess how antiretroviral therapy (ART) initiation during acute or early HIV infection (AEHI) affects the viral reservoir and host immune responses.
Design:
Single-arm trial of ART initiation during AEHI at 30 sites in the Americas, Africa, and Asia.
Methods:
HIV DNA was measured at week 48 of ART in 5 million CD4+ T cells by sensitive qPCR assays targeting HIV gag and pol. Peripheral blood mononuclear cells were stimulated with potential HIV T cell epitope peptide pools consisting of env, gag, nef and pol peptides and stained for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines.
Results:
From 2017–2019, 188 participants initiated ART during Fiebig stages I (n=6), II (n=43), III (n=56), IV (n=23), and V (n=60). Median age was 27 years (interquartile range 23–38), 27 (14%) participants were female, and 180 (97%) cisgender. Among 154 virally-suppressed participants at week 48, 100% had detectable HIV gag or pol DNA. Participants treated during Fiebig I had the lowest HIV DNA levels (p<0.001). Week 48 HIV DNA mostly did not correlate with concurrent CD4+ or CD8+ T cell HIV-specific immune responses (rho range −0.11 to +0.19, all p>0.025). At week 48, the magnitude, but not polyfunctionality, of HIV-specific T cell responses was moderately reduced among participants who initiated ART earliest.
Conclusions:
Earlier ART initiation during AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood. These findings explain the rapid viral rebound observed after ART cessation in early-treated individuals with undetectable HIV DNA by less sensitive methods.
Keywords: Acute Retroviral Syndrome, Acquired Immunodeficiency Syndrome, Anti-Retroviral Agents, HIV, T-Lymphocytes
INTRODUCTION
Antiretroviral therapy (ART) initiated during the chronic phase of human immunodeficiency virus (HIV) infection suppresses viral replication and reduces morbidity.[1] However, it does not eradicate infection in cellular and anatomic reservoirs.[2] These viral reservoirs contribute to rebound viremia within weeks after stopping ART[3, 4] and represent the major barrier to achieving durable viral control without lifelong ART.
Prior studies suggest that early ART initiation limits HIV reservoir establishment,[5–7] enhances reservoir decay,[8, 9] and limits viral genetic diversification.[10–12] In some cases, early ART has been associated with post-treatment viral control.[13, 14] However, clinical trials that included analytic treatment interruption after ART initiation during acute or early HIV infection (AEHI) have mostly found that viral rebound occurred, regardless of the intervention tested.[14–18]
Treatment during the earliest stages of AEHI may minimize HIV reservoir size, which might facilitate post-treatment control. Paradoxically, participants with post-treatment control in the VISCONTI cohort were mainly those treated in later Fiebig stages of AEHI.[13] Very early ART preserves HIV-specific CD4+ and CD8+ T cell responses measured by enzyme-linked immunospot assays[19–21], but virus-controlling polyfunctional T cell responses may be diminished.[22] Characterizing the virologic and immunologic impact of therapy during AEHI is a high priority for defining HIV pathogenesis, characterizing reservoirs, and developing novel interventions for HIV control or eradication. Prior studies have been limited by small numbers of participants, limited sensitivity of HIV persistence assays, and incomplete characterization of AEHI stage at treatment initiation.
We conducted a single-arm, open-label, multinational clinical trial of ART initiation during AEHI to evaluate whether the timing of ART predicts the magnitude of HIV persistence and HIV-specific immune responses.
METHODS
Participants
The AIDS Clinical Trials Group (ACTG) A5354 study (“Early ART to Limit Infection and Establishment of Reservoir” [EARLIER]; ClinicalTrials.gov NCT02859558) enrolled participants from January 2017 through December 2019 at 30 sites in the United States, Brazil, Thailand, Malawi, Zimbabwe, and Peru.
Participants were aged 18 years or older, able and willing to initiate ART, and had presumptive AEHI based on one of six study-specific criteria designed to support a likely HIV diagnosis and establish relative recency of infection as previously described.[23] These criteria yielded an estimated AEHI stage according to protocol-defined groupings (Table S1). The study was approved by ethics committees and institutional review boards at all participating institutions. Participants provided written informed consent prior to enrollment.
Procedures
Participants were encouraged to initiate ART on the day of enrollment, either with study-provided single-tablet elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide fumarate (EVG/COBI/FTC/TAF) or any other combination ART regimen. Single-tablet bictegravir/emtricitabine/tenofovir alafenamide fumarate (BIC/FTC/TAF) became available as an alternative study-provided regimen after enrollment was completed. ART changes were allowed at participant or provider discretion.
Samples from the enrollment visit prior to ART initiation underwent retrospective, centralized HIV confirmatory testing and staging, including plasma HIV RNA quantification and IgG-antibody discrimination (Supplementary Appendix p 2). Participants were assigned to study groups based on this centralized testing: Group 1 (G1; Fiebig stages I/II), Group 2 (G2; Fiebig stages III/IV), and Group 3 (G3; Fiebig stage V).[24] Participants without confirmed HIV were withdrawn from the study immediately and participants in Fiebig stage VI were withdrawn by week 24, after confirming ART access outside the study.
Primary endpoint assessment occurred at week 48 and key secondary endpoints were evaluated through week 72. Plasma HIV RNA was measured in real-time using standard PCR-based clinical assays at weeks 1, 4, 12, 24, 36, 48, 60, and 72. Viral failure was defined by two consecutive HIV RNA measurements greater than 200 copies/mL 24 weeks or more after study entry, or at any time after achieving plasma HIV RNA <50 copies/mL. Any measurement greater than 200 copies/mL prompted repeat testing as soon as possible. Participants with confirmed viral failure or who interrupted ART for 7 or more consecutive days prior to achieving viral suppression were withdrawn from the study.
Outcomes
We hypothesized that ART initiation prior to HIV antibody detection (Fiebig I/II) would be associated with limited or no detectable cell-associated HIV DNA or HIV-specific immune responses after 48 weeks of ART, while later ART initiation would be associated with larger reservoirs and quantitatively and qualitatively different HIV-specific immune responses.
The primary objective of the study was to compare, among participants who achieved and maintained plasma HIV RNA <50 copies/mL, the amount of cell-associated HIV DNA at 48 weeks after ART initiation during Fiebig I/II (G1), Fiebig III/IV (G2), or Fiebig V (G3). We isolated and tested up to 5 million purified CD4+ T cells using sensitive qPCR assays that targeted conserved regions in HIV gag or pol as previously described.[25, 26] Each assay run included a negative control of peripheral blood mononuclear cell (PBMC) DNA from donors without HIV.
Protocol-specified secondary outcomes included pre-ART levels of cell-associated HIV DNA; the magnitude and distribution of CD4+ and CD8+ T cell responses during suppressive ART; signs and symptoms of acute retroviral syndrome; and absolute CD4+ and CD8+ T cell counts, CD4/CD8 ratio, and plasma HIV RNA levels prior to and after ART initiation.
HIV-specific immune responses were assessed by stimulation of PBMC with T cell epitope peptide pools (env, gag, nef, and pol peptides) and staining for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines (CD40L, MIP-1β, IFN-γ, and TNF) as described in the Supplementary Appendix (pp 2–3).
Dates of onset and resolution of signs and symptoms potentially associated with acute retroviral syndrome[27] were recorded at enrollment. The case definition of acute retroviral syndrome required fever >38.0°C plus three or more other signs and/or symptoms, spanning five days, without an alternative explanation, within 30 days of enrollment (Supplementary Appendix p 3).
Absolute CD4+ and CD8+ T cell counts were measured in real-time using standard clinical assays at enrollment and study weeks 4, 12, 24, and 48. All real-time testing was conducted at local laboratories certified by the Division of AIDS (DAIDS) Virology Quality Assurance Program.
Statistical Analyses
With estimated enrollment and retention of 42 virally-suppressed participants in each study group through week 48, this study had greater than 95% power to detect the hypothesized difference in the proportion of participants with undetectable HIV DNA (hypothesized 75% in Fiebig I/II, 25% Fiebig III/IV, <5% Fiebig V).
The primary analysis was restricted to participants with no ART interruption of ≥7 consecutive days, HIV RNA <50 copies/mL at week 48, and available HIV DNA results at week 48. The primary endpoint of undetectable HIV DNA was defined as an undetectable result for both the gag- and pol-targeting HIV DNA assays. Pairwise comparisons of the proportion with undetectable HIV DNA between groups used Fisher’s exact tests. A 95% confidence interval (95%CI) was calculated using the exact binomial distribution. Wilcoxon rank sum tests were used to compare HIV DNA on the continuous scale, with undetectable results imputed as 0.01 copies/million CD4+ T cells. Jonckheere–Terpstra tests were used to assess potential trends across study groups. Pre-ART HIV DNA was analyzed similarly for all participants with available assay data.
Analyses of HIV-specific CD4+ and CD8+ T cell responses were restricted to the primary analysis population. Single cytokine and total responses were calculated by subtracting the corresponding background control value (media control) from the value measured for a specific participant. A positive result was defined as any functional response above the background (unstimulated condition). Results less than zero, after background subtraction, were set to zero. If the result of the active (positive) control was less than the result of the background control, the observation was excluded from analyses. Comparisons of HIV-specific immune responses followed the same approaches used for the primary outcome measure. Spearman correlation coefficients were calculated between selected HIV-specific CD4+ and CD8+ T cell responses and HIV DNA at week 48, both unadjusted and adjusted for study group.
For all analyses, a two-sided type I error of 5% was considered statistically significant. No adjustment was made for multiple comparisons. Analyses were performed using SAS 9.4 (Cary, NC).
RESULTS
Study Population
A total of 195 participants enrolled and initiated ART either on the day of enrollment (n=171, 87.7%) or the following day (n=24, 12.3%). Centralized testing identified 3 (1.5%) participants without HIV and 4 (2.0%) with Fiebig VI (chronic) HIV who were withdrawn from the study and excluded from these analyses. AEHI was confirmed in 188 participants, including 6 who initiated ART during Fiebig stage I, 43 during Fiebig II, 56 Fiebig III, 23 Fiebig IV, and 60 Fiebig V (Figure 1; Table 1). The median age of these participants was 27 years (interquartile range 23–38), 27 (14%) were female, 180 (96%) were cisgender, 93 (54%) were Black or African American, and 65 (35%) were Hispanic or Latino.
Figure 1.
CONSORT Diagram. This diagram displays the progression of participants through screening, enrollment, allocation, and follow-up in the A5354 single-arm, open-label study to evaluate the impact of ART initiation during AEHI on viral persistence and development of HIV-specific immune responses, using the standard recommended by the CONSORT Group. The study had a target enrollment of 50 participants in each of Study Group 1 (Fiebig I/II), Study Group 2 (Fiebig III/IV), and Study Group 3 (Fiebig V). Fiebig staging and group assignment were conducted retrospectively using centralized testing of blood specimens from the enrollment visit. Participants without confirmed HIV or in Fiebig stage VI were withdrawn from the study. The primary analysis population was restricted to participants with no ART interruption of ≥7 consecutive days, HIV-1 RNA <50 copies/mL at week 48, and available HIV DNA results at week 48. Abbreviations: LTFU, lost to follow-up
Table 1.
Characteristics of 188 Participants with Acute or Early HIV Infection in the A5354/EARLIER Study
| Group 1 [Fiebig I/II] (n=49) | Group 2 [Fiebig III/IV] (n=79) | Group 3 [Fiebig V] (n=60) | Total (n=188) | |
|---|---|---|---|---|
| Age (years) | ||||
| Median (Q1, Q3) | 26 (22, 35) | 30 (24, 40) | 26 (23, 38) | 27 (23, 38) |
| Sex, n (%) | ||||
| Female | 4 (8%) | 12 (15%) | 11 (18%) | 27 (14%) |
| Male | 45 (92%) | 67 (85%) | 49 (82%) | 161 (86%) |
| Gender Identity, n (%) | ||||
| Cisgender | 47 (98%) | 73 (94%) | 60 (100%) | 180 (97%) |
| Transgender Spectrum | 1 (2%) | 5 (6%) | 0 (0%) | 6 (3%) |
| Missing | 1 | 1 | 0 | 2 |
| Race, n (%) | ||||
| Black/African American | 19 (42%) | 35 (49%) | 39 (70%) | 93 (54%) |
| White | 14 (31%) | 36 (50%) | 16 (29%) | 66 (38%) |
| Asian | 12 (27%) | 1 (1%) | 1 (2%) | 14 (8%) |
| Missing | 4 | 7 | 4 | 15 |
| Ethnicity, n (%) | ||||
| Hispanic or Latino | 17 (35%) | 30 (38%) | 18 (30%) | 65 (35%) |
| Not Hispanic or Latino | 32 (65%) | 48 (62%) | 42 (70%) | 122 (65%) |
| Missing | 0 | 1 | 0 | 1 |
| Country, n (%) | ||||
| United States | 25 (51%) | 65 (82%) | 42 (70%) | 132 (70%) |
| Brazil | 6 (12%) | 10 (13%) | 8 (13%) | 24 (13%) |
| Thailand | 12 (24%) | 1 (1%) | 0 (0%) | 13 (7%) |
| Malawi | 4 (8%) | 3 (4%) | 3 (5%) | 10 (5%) |
| Zimbabwe | 0 (0%) | 0 (0%) | 6 (10%) | 6 (3%) |
| Peru | 2 (4%) | 0 (0%) | 1 (2%) | 3 (2%) |
| Body Mass Index (kg/m2), n (%) | ||||
| Underweight (<18) | 5 (10%) | 6 (8%) | 3 (5%) | 14 (7%) |
| Normal (18–24.9) | 30 (61%) | 31 (39%) | 31 (52%) | 92 (49%) |
| Overweight (25–29.9) | 7 (14%) | 24 (30%) | 14 (23%) | 45 (24%) |
| Obese (30+) | 7 (14%) | 18 (23%) | 12 (20%) | 37 (20%) |
| HIV RNA (log10 copies/mL) | ||||
| Median (Q1, Q3) | 6.4 (5.3, 7.0) | 6.5 (6.0, 7.0) | 5.4 (5.0, 6.4) | 6.3 (5.3, 6.8) |
| CD4+ T-Cell Count (cells/mm3) | ||||
| Median (Q1, Q3) | 348 (211, 493) | 383 (264, 538) | 490 (366, 652) | 402 (281, 570) |
| Initial Antiretroviral Regimen, n (%) | ||||
| EVG/COBI/FTC/TAF | 31 (63%) | 65 (82%) | 50 (83%) | 146 (78%) |
| DTG/3TC/TDF | 18 (37%) | 10 (13%) | 8 (13%) | 36 (19%) |
| EFV/3TC/TDF | 0 (0%) | 0 (0%) | 2 (3%) | 2 (1%) |
| BIC/FTC/TAF | 0 (0%) | 1 (1%) | 0 (0%) | 1 (<1%) |
| DTG/FTC/TAF | 0 (0%) | 1 (1%) | 0 (0%) | 1 (<1%) |
| DRV/COBI/FTC/TAF | 0 (0%) | 1 (1%) | 0 (0%) | 1 (<1%) |
| MVC/DTG/3TC/TDF | 0 (0%) | 1 (1%) | 0 (0%) | 1 (<1%) |
| Symptoms at AEHI Diagnosis, n (%) | ||||
| Asymptomatic | 12 (25%) | 16 (20%) | 25 (42%) | 53 (28%) |
| Symptomatic | 36 (75%) | 63 (80%) | 35 (58%) | 134 (72%) |
| Missing | 1 | 0 | 0 | 1 |
Abbreviations: 3TC, lamivudine; AEHI, acute or early HIV infection; BIC, bictegravir; COBI, cobicistat; DRV, darunavir; DTG, dolutegravir; EFV, efavirenz; EVG, elvitegravir; FTC, emtricitabine; MVC, maraviroc; TAF, tenofovir alafenamide fumarate; TDF, tenofovir disoproxil fumarate.
A total of 195 participants enrolled and initiated antiretroviral therapy at 30 sites in six countries. Retrospective centralized testing identified 3 (1.5%) participants without HIV and 4 (2.0%) with Fiebig VI (chronic) HIV who were withdrawn from the study and excluded from analyses. Demographic and other characteristics for the remaining 188 participants with Fiebig I-V (acute or early) HIV are summarized here.
Clinical and Safety Outcomes
Among the 188 participants who started ART during AEHI, 134 (72%) were symptomatic at diagnosis, 68 (36%) were hospitalized (including for reasons other than HIV), and 49 (26%) met the case definition for acute retroviral syndrome.
Modifications to the initial ART regimen were made for 29 (15%) participants. Most modifications occurred due to co-enrollment in a local protocol for ART (NCT00796263) in Bangkok, Thailand, which prescribed a change from initial site-provided dolutegravir (DTG)/lamivudine (3TC)/tenofovir disoproxil fumarate (TDF) to abacavir/DTG/3TC if HLA-B5701 was not detected (n=13). Additional modification reasons included adverse events (n=6), clinician decision/preference (n=5), non-adherence to initial regimen (n=4), and a discretionary switch to study-provided BIC/FTC/TAF upon availability (n=1).
Fifty-six (30%) participants experienced at least one grade 3 or 4 adverse event. The most common event was decreased estimated glomerular filtration rate (n=20). Other common grade 3 or 4 events included increased alanine aminotransferase (n=8), increased aspartate aminotransferase (n=8), decreased weight (n=4), and suicide attempt (n=4). There were 16 (9%) participants who experienced serious adverse events. One ectopic pregnancy occurred at week 40, which required left salpingectomy.
Study Adherence
The protocol-defined 72 weeks of Step 1 follow-up were completed by 153 (81%) participants. Reasons for premature study discontinuation included loss to follow-up (n=16, 9%), non-adherence to study drug or requirements (n=8, 4%), viral failure (n=4, 2%), withdrawal by participant (n=4, 2%), participant relocation (n=2, 1%), and incarceration (n=1, <1%).
Plasma HIV RNA
Four (2%) participants experienced protocol-defined viral failure, none of whom had new evidence of drug resistance as compared to pre-ART sequencing. An additional seven (4%) had a single HIV RNA >200 copies/mL and were lost to follow-up or withdrew from the study before a confirmatory measurement could be obtained. Most participants who remained in the study achieved HIV RNA <50 copies/mL (Figure 2A) and target not detected (Figure 2B). Participants in G1 were more likely to have target not detected at week 24 (71% [95%CI 56–84%]) than participants in G2 and G3 combined (46% [95%CI 37–56%], p=0.005). Results were similar by Fiebig stage (Figures 2C and 2D).
Figure 2.
Proportion of participants who achieved plasma HIV RNA suppression targets, by study groups and Fiebig stages. Panel A displays the proportion of participants with plasma HIV-1 RNA less than 50 copies/mL by study group. Panel B displays the proportion of participants with plasma HIV-1 RNA target not detected by study group. Panel C displays the proportion of participants with plasma HIV-1 RNA less than 50 copies/mL by Fiebig stage. Panel D displays the proportion of participants with plasma HIV-1 RNA target not detected by Fiebig stage. Vertical bars represent 95% confidence intervals calculated using the exact binomial distribution.
HIV DNA in CD4+ T cells Purified from PBMC
Among 154 participants with HIV RNA <50 copies/mL and without protocol-defined ART interruption through week 48, 100% had detectable HIV gag or pol DNA. Compared to Groups 2 and 3, participants in G1 had lower HIV gag DNA (Figure 3A) and HIV pol DNA (Figure 3B) levels after 48 weeks of effective ART. Participants who started ART during Fiebig I, as compared to later Fiebig stages, had significantly lower HIV gag and pol DNA levels at 48 weeks (all trend tests p<0.001, Figures 3C and 3D).
Figure 3.
HIV DNA (copies per million CD4+ T-cells) after 48 weeks of antiretroviral therapy, by Study Group and Fiebig Stage. Panel A displays the Week 48 HIV gag DNA by study group. Panel B displays the Week 48 HIV pol DNA by study group. Panel C displays the Week 48 HIV gag DNA by Fiebig stage. Panel D displays the Week 48 HIV pol DNA by Fiebig stage. Undetectable measurements are indicated with open symbols. Vertical bars represent the interquartile range and are bisected with a horizontal line at the median. *p<0.05, **p<0.01.
Pre-ART HIV gag and pol DNA levels tended to be lowest in participants with the earliest stages of AEHI (Figure S1). From pre-ART to week 48, the greatest fold-change decrease in HIV gag and pol DNA levels was observed in G1 and the smallest decrease in G3, with a similar trend towards larger decreases with ART in earlier Fiebig stages (Figure 4). HIV gag and pol DNA measurements were correlated pre-ART (rho=0.42, p<0.001) and after 48 weeks of ART (rho=0.34, p<0.001; Figure S2).
Figure 4.
Change in HIV DNA (copies per million CD4+ T-cells) from prior to ART initiation and after 48 weeks of antiretroviral therapy, by Study Group and Fiebig Stage. Panel A displays the HIV gag DNA by study group. Panel B displays the HIV pol DNA by study group. Panel C displays the HIV gag DNA by Fiebig stage. Panel D displays the HIV pol DNA by Fiebig stage. Vertical bars represent interquartile ranges.
CD4+ and CD8+ T cell Counts
Median pre-ART CD4+ T cells counts were significantly lower in G1 (348 cells/mm3) and G2 (383 cells/mm3) as compared to G3 (552 cells/mm3; p<0.001 and p=0.004, respectively) and demonstrated significantly greater increases in CD4+ T cell counts through study week 72 (Median +401, +392 and +239 cells/mm3; p=0.014 and p=0.002, respectively; Figure S3A). Trends were similar for CD8+ T cell counts (Figure S3B). Pre-ART CD4/CD8 ratio was highest in G1 (median 1.24, 0.73, 0.41; all pairwise comparison p<0.001), but by week 72 was similar across groups (Figure S3C). Results were similar by Fiebig stage (Figure S3D-F).
HIV-specific Immune Responses
At week 48, HIV-specific T cell responses tended to be diminished among participants receiving the earliest treatment intervention (G1) compared to the other groups. Significant differences (p<0.05) were observed for nef-specific (G1 vs G3 and G2 vs G3) and pol-specific (G1 vs G3) CD8+ T cell responses and for gag-specific (G1 vs G2) and pol-specific (G1 vs G3) CD4+ T cell responses (Figure 5). The frequency of HIV-specific T cells that were induced by the pol peptide pool was weakly correlated with week 48 HIV pol DNA (rho=0.19, p=0.025 unadjusted for study groups) but frequencies of HIV-specific T cells stimulated by other peptide pools were not correlated with week 48 HIV pol or gag DNA (|rho| ≤ 0.15, p>0.07; unadjusted and adjusted for study groups), suggesting that the size of the HIV reservoir was not a determining factor in T cell frequencies observed.
Figure 5.
CD4+ and CD8+ T-cell-specific immune responses during suppressive antiretroviral therapy, by Study Group. HIV-specific immune responses were assessed by stimulation of peripheral blood mononuclear cells with T-cell epitope peptide pools (env, gag, nef, and pol peptides) and staining for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines (CD40L, Mip1b, IFN-g, and TNF-a). Responses were calculated by subtracting the corresponding background control value (media control) from the value measured for a specific participant. A positive result was defined as any functional response above the background (unstimulated condition) to one or more intracellular cytokines/chemokines. Panel A displays week 48 total percent CD4+ T-cell-specific immune responses to env, gag, nef, and pol peptides by Study Group. Panel B displays week 48 total percent CD8+ T-cell-specific immune responses by Study Group. Vertical bars represent interquartile ranges. *p<0.05, **p<0.01.
T cell polyfunctionality, measured as the proportions of CD4+ or CD8+ T cells that could express 2 to 5 functions, was generally similar between groups. Modest and marginally significant differences were noted comparing G1 vs G2 for percentages of HIV-reactive CD4+ T cells expressing two functions after stimulation with env (median: 10.96% vs. 9.13%, p=0.047), nef (9.84% vs. 8.87%, p=0.051), or pol (10.48% vs. 8.62%, p=0.043) peptide pools. A similar difference comparing G1 vs G2 was seen for percentages of HIV-reactive CD8+ T cells expressing 3 functions after stimulation with pol (7.40% vs. 4.81%, p=0.006). No other differences in T cell polyfunctionality were observed.
DISCUSSION
This single arm, open-label study demonstrated that ART initiation in earlier stages of AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood, explaining why rapid viral rebound has been observed after ART cessation in early-treated individuals across multiple studies despite undetectable HIV DNA by less sensitive methods. Early initiation of ART was associated with a modest reduction in the magnitude of HIV-specific T cell responses. This study also demonstrated the feasibility of identifying, treating, and characterizing a multinational cohort of individuals with AEHI.
Prior studies have shown that some early-treated people with HIV had undetectable cell-associated HIV in peripheral blood; however, nearly all experienced viral rebound after ART cessation [14–17]. In our study, after 48 weeks of ART, a highly sensitive and specific assay performed on a large number of purified CD4+ T cells detected HIV DNA in the peripheral blood of all 188 participants regardless of Fiebig stage at ART initiation. These results suggest that, without further intervention, even early-treated individuals are likely to experience viral rebound after treatment interruption. However, our study did not evaluate persons treated within a few hours to days of HIV acquisition as has been described in some pediatric cases of early ART initiation and subsequent viral control.[26, 28, 29] We observed that earlier treatment during AEHI was associated with lower levels of HIV DNA in peripheral blood, reinforcing the hypothesis that although early treatment alone does not facilitate clearance of HIV reservoirs, it could restrict viral diversity[10–12] sufficiently to enhance the efficacy of interventions to potentially establish ART-free immune control. Previously published results from 89 participants from our study with full-length env sequencing showed low viral diversity during AEHI and no viral evolution during approximately 60 weeks of ART.[30]
In our study, ART initiation in the earliest Fiebig stages (I/II) tended to result in reduced frequencies, but not polyfunctionality, of HIV-specific T cells at week 48. It is possible that interventions during the earliest Fiebig stages may limit the magnitude of memory HIV-specific T cell responses that can be detected at later time points. Most participants who started ART in Fiebig I/II had detectable HIV-specific T cell responses at week 48, consistent with other studies of ART initiation during AEHI.[19–21] It is important to note that some potential approaches to achieve HIV remission would rely on the existence and enhancement of HIV-specific immune responses, so ART initiation at the earliest Fiebig stages that abrogates or impairs the development of HIV-specific immune responses may be counterproductive.[22, 31, 32] Whether the reduced HIV-specific T cell frequencies observed with early ART initiation will adversely affect outcomes of future interventions, such as therapeutic vaccines or immune-based cure strategies, is unclear.
The magnitude or make-up of persistent HIV during ART may influence HIV-specific T cell responses. A previous study described a direct relationship between HIV DNA levels and nef-specific T cell frequencies among ART-treated individuals with chronic HIV.[33] Theoretically, this relationship might stem from nef being an early product of HIV re-activation, leading to immune recognition during ART. In contrast, we did not detect a relationship between HIV DNA levels and HIV-specific T cell frequencies, including nef-specific T cells 48 weeks after ART initiation. Our observation suggests that HIV DNA does not appear to be a major driver of HIV-specific T cell responses during the first year of ART started during AEHI. A longer duration of ART may be required to detect a potential relationship between HIV DNA and HIV-specific T cell responses.[33]
Research involving individuals with AEHI is critical to defining HIV pathogenesis and testing novel treatment strategies, particularly those intended to induce HIV remission. This study showed that the identification and rapid initiation of ART during AEHI was feasible across a large, multinational network of clinical research sites. Historically, most studies of AEHI were conducted at one or a few affiliated research centers, leveraging either serial testing of people at risk for HIV acquisition or technologically advanced screening algorithms.[34–37] These approaches are potentially costly, labor-intensive, and slow to accrue new participants. More efficient methods are needed to identify people with AEHI and initiate early ART to support the testing of new interventions to achieve durable suppression of HIV.
A critical aspect of studying people treated during AEHI is maintaining high levels of adherence to therapy, recognizing that treatment interruptions may reestablish or enlarge HIV reservoirs. In our study, viral failure was observed in only 2% of participants and premature study discontinuation for other reasons occurred in 16%. While any loss to follow-up is suboptimal, this amount was similar to that observed in randomized clinical trials of the “test and treat” strategy for HIV management; retention in care at one year after ART initiation at the time of HIV diagnosis was around 65–80% in studies from sub-Saharan Africa.[38, 39] Importantly, participants who remained engaged in this study tended to achieve and maintain viral suppression, as has been seen in prior studies of ART initiation during AEHI.[40]
Our study has some limitations. We used sensitive methods for assessing the presence of HIV DNA in 5M isolated CD4+ T cells but did not define how much is intact or replication competent, nor did we examine tissues or the expressed reservoir by cell-associated HIV RNA. The measures used to assess HIV-specific T cell responses may not be all encompassing. The clinical relevance of some of the virologic and immunologic measures and the implications of quantitative differences across groups are uncertain. Our study did not evaluate the effects of early ART initiation on non-specific markers of chronic inflammation and immune activation, some of which have been shown to be reduced with earlier ART initiation during AEHI.[41] While stringent criteria were used for exclusion of participants from the analysis population in the event of treatment interruption, adherence assessment relied on self-report. While participants were enrolled in all stages of AEHI, only six participants were in the earliest stage (Fiebig I). Finally, assessments were all made after approximately 48 weeks of therapy, and results might differ with further follow-up. Future research may include the use of other assays, such as the intact proviral DNA assay, to further characterize HIV reservoirs before and after ART initiation. In addition, it will be important to characterize compartments outside of the blood, such as lymphoid tissues.[42]
In conclusion, we found that ART initiation in earlier stages of AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood from a diverse global cohort. Sensitive assays detected HIV DNA in all participants after 48 weeks of ART, regardless of Fiebig stage at ART initiation. HIV DNA did not appear to be a major driver of HIV-specific immune responses. This study demonstrated the feasibility of strategies to identify and recruit people with AEHI in a multinational setting which can be leveraged to conduct future clinical trials in this unique population. Findings from this study enhance our understanding of the viral and immune barriers to achieving durable viral control of HIV without ART, even after early treatment.
Supplementary Material
ACKNOWLEDGMENTS
We would like to thank the study participants who made this research possible. This work was supported by the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health (grant numbers UM1AI068634, UM1AI068618, UM1AI106701, and P30AI027757). Antiretroviral therapy for the study was donated by Gilead Sciences (Rich Clark). We gratefully acknowledge the following members of the ACTG A5354 Study Team: Oladapo Alli, Deborah Anisman-Posner, LuAnn Borowski, Benjamin Chi, Morgan Gapara, Tydie Higgins, Brenda Hoagland, Andrew Kaytes, Dimas Kliemann, Eugène Kroon, Mwenda Kudumu, Linda Naini, Gonasagrie Nair, Deborah Persaud, Magdalena Sobieszczyk, Jennifer Tiu, Kyle Whitson. The ACTG A5354 study was conducted at the following clinical research sites supported by grants from the U.S. National Institutes of Health: Alabama CRS (Site 31788, UM1AI69452; Sonya Heath), Barranco CRS (Site 11301, AI069438; Luis Limo and Mey Leon), Brigham and Women’s Hospital Therapeutics CRS (Site 107, UM1AI69412; Paul Sax), Chapel Hill CRS (Site 3201, UL1TR002489; Cynthia Gay, Brittney Soderman, and Mandy Tipton), Columbia Physicians and Surgeons CRS (Site 30329, UM1AI068636; Jolene Noel Connor and Brett Gray), Cincinnati CRS (Site 2401, UM1AI69501; Carl J. Fichtenbaum and Linda Hinds), Greensboro CRS (Site 3203, UM1AI69423; Cornelius Van Dam and Kimberly Epperson), Harbor University of California Los Angeles Center CRS (Site 603, UM1A1069424 and UL1TR000124; Sadia Shaik and Mario Guerrero), Hospital Nossa Senhora da Conceição CRS (Site 12201, UM1AI69424; Breno Riegel Santos and Rita de Cassia Alves Lira), Houston AIDS Research Team CRS (Site 31473, UM1AI069432; Jesus A. Lara and Romina Chinchay Collahua), Instituto de Pesquisa Clinica Evandro Chagas CRS (Site 12101, UM1AI69476; Sandra Wagner Cardoso and Brenda Hoagland), Malawi CRS (Site 12001, UM1AI069423; Cecilia Kanyama and Cornelius Munyanga), Massachusetts General Hospital CRS (Site 101, UM1AI69412; Rajesh Gandhi, Amy Sbrolla, and Teri Flynn), Milton Park CRS (Site 30313, UM1AI069436; Wadzanai P. Samaneka and Pamela G. Mukwekwerere), New Jersey Medical School Clinical Research Center CRS (Site 31786, UM1AI69419; Shobha Swaminathan and Susana Rivera), Northwestern University CRS (Site 2701, UM1AI69471; Baiba Berzins and Shannon Galvin), Ohio State University CRS (Site 2301, UM1AI69494; Susan Koletar and Heather Harber), Penn Therapeutics CRS (Site 6201, UM1AI69534; Pablo Tebas), Ponce de Leon Center CRS (Site 5802, P30AI050409; Ericka R. Patrick and Clifford Gunthel), Rush University CRS (Site 2702, UM1AI69471; Beverly E. Sha and Shivanjali Shankaran), San Miguel CRS (Site 11302, UM1AI69438; John Thays), Thai Red Cross AIDS Research Centre CRS (Site 31802, UM1AI69399; Pornphen Tantivitayakul, Eugène Kroon, and Kiat Ruxrungtham), The Miriam Hospital CRS (Site 2951, UM1AI69412; Karen Tashima and Pamela Poethke), Trinity Health and Wellness Center CRS (Site 31443, UM1AI69471; Roger Bedimo), UCSD Antiviral Research Center CRS (Site 701, UM1AI069432; Alina Burgi and Yvette Longduriyang), University of Washington Positive Research CRS (Site 1401, UM1AI69481; Janine Maenza), Washington University Therapeutics CRS (Site 2101, UM1AI69439; Rachel Presti), Weill Cornell Chelsea CRS (Site 7804, UL1TR000457; Shaun Barcavage and Tiina Ilmet), Whitman-Walker Institute, Inc., CRS (Site 31791, UM1AI069465; W. David Hardy and Lynsay MacLaren).
The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army, the Department of Defense, the National Institutes of Health, or the Department of Health and Human Services.
Source of Funding:
This work was supported by the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health (grant numbers UM1AI068634, UM1AI068618, UM1AI106701, and P30AI027757). Antiretroviral therapy for the study was donated by Gilead Sciences. TAC reports research grants from the U.S. Army. SAR reports research grants from Gilead Sciences and Merck. JFR is an employee and reports stock in Gilead Sciences. JJE reports research grants from ViiV Healthcare, Gilead Sciences, and Janssen as well as personal fees from ViiV Healthcare, Gilead Sciences, and Merck. JWM reports research grants from Gilead Sciences; consultant fees from Gilead Sciences, Allovir, and Infectious Diseases Connect; and financial interests in Galapangos NV, MingMed, and Infectious Diseases Connect. ESD reports research grants from ViiV Healthcare and Gilead Sciences as well as personal fees from ViiV Healthcare, Gilead Sciences, and Theratechnologies.
Footnotes
Conflicts of Interest The remaining authors declare no relevant conflicts of interest.
Prior Presentation: This work was presented, in part, at the 29th Conference on Retroviruses and Opportunistic Infections, held virtually, 12–16 February 2022.
ACTG A5354 STUDY TEAM
Oladapo Alli
Deborah Anisman-Posner
LuAnn Borowski
Benjamin Chi
Morgan Gapara
Tydie Higgins
Brenda Hoagland
Andrew Kaytes
Dimas Kliemann
Eugène Kroon
Mwenda Kudumu
Linda Naini
Gonasagrie Nair
Deborah Persaud
Magdalena Sobieszczyk
Jennifer Tiu
Kyle Whitson
DATA SHARING STATEMENT
The authors confirm that all data underlying the findings are fully available. Due to ethical restrictions, study data are available upon request from sdac.data@sdac.harvard.edu with the written agreement of the AIDS Clinical Trials Group.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The authors confirm that all data underlying the findings are fully available. Due to ethical restrictions, study data are available upon request from sdac.data@sdac.harvard.edu with the written agreement of the AIDS Clinical Trials Group.