Introduction

Although antiretroviral therapy (ART) effectively controls HIV, it does not eradicate persisting virus from resting CD4 T cells and other cellular subsets. ART is therefore lifelong, with many side effects and may be financially unsustainable particularly in low-to middle-income countries that are most affected by the HIV epidemic. A cure is therefore necessary but difficult to achieve as the persistent HIV reservoir, which is established soon after infection [1–3] hinders these efforts through its complex nature [4].

The HIV-1 reservoir is heterogenous and dynamic in genotypic, phenotypic and cellular composition and also differs between individuals and populations, making the source of persistent infection difficult to target. The lability of the viral reservoir has been demonstrated by evidence of clonal expansion that occurs by homeostatic proliferation [5, 6], antigenic stimulation [7] or integration into genic regions controlling cell growth [8–10]. Additionally, its size and genetic composition changes over time with intact and defective proviral genomes displaying variability in their rate of decay during suppressive ART [11–13]. The heterogeneity of the HIV-1 reservoir landscape is further obscured by the integration patterns of virus-intact genomes that determine their ability to generate replication competent viral particles when stimulated [8]. Moreover, recent studies show that the viral reservoir is not restricted to CD4 T cells but persists in other cell types such as monocytes, macrophages [14–19] and follicular dendritic cells [20–23] with each having distinct biological characteristics.

Among the many studies directed towards HIV-1 cure, some have demonstrated that early ART initiation may be a crucial step in limiting the viral reservoir and achieving post-treatment viral control [24, 25]. Even though early treatment does not prevent reservoir establishment, it does result in a rapid decline of viremia and substantially reduces HIV reservoir size [11, 26–28] which delays viral load rebound and results in sustained virological remission following discontinuation of ART [25, 29, 30]. Other described benefits of starting therapy soon after primary infection include preservation of immune function and restriction of viral diversification [26, 28, 31–41]). However, there is a paucity of data that describes the impact of the timing of treatment initiation on the characteristics of the reservoir.

HIV-1 reservoir studies in African populations are limited, particularly in subtype C HIV-1 infection, which is the most prevalent form of HIV-1 globally and predominates in southern Africa. The design of a globally applicable HIV cure strategies and interventions to target the viral reservoir depend on a deeper understanding of the variability in the size, composition and characteristics of the genetic landscapes of persisting reservoir genomes in non-subtype B HIV infections. Moreover, data are lacking on reservoir characteristics in women, despite known sex differences in immune responses and in viral load during primary infection that could potentially impact the reservoir [42–44].

In this study we performed an extensive longitudinal analysis of HIV-1 subtype C reservoir characteristics in a unique hyperacute infection cohort in Durban, South Africa [45, 46]. The cohort was designed to identify acute infection before peak viremia (Fiebig stages I to III) providing HIV-1 testing twice a week to young women at high risk for HIV-1 infection in a region with high population prevalence [45]. Following changes in treatment guidelines in South Africa that allowed for ART initiation regardless of CD4 counts, all study participants were offered ART, including those who were newly detected with acute infection who received ART on average a day after first detection of plasma viremia. Study participants underwent frequent clinical follow-up and sampling following infection and initiation of ART, allowing us to study HIV-reservoir establishment and evolution from the earliest possible stages of infection. We hypothesized that the timing of ART will impact HIV-1 reservoir characteristics in terms of the size, genetic composition, and decay dynamics. The findings of this study provide insights into HIV-1 reservoirs characteristics that could inform viral targeting strategies for reservoir control in African populations.

Results

Total proviral DNA load kinetics following early and late treatment

In this analysis we included 35 participants (Supplementary Table 1), of whom 11 first initiated treatment during chronic infection at a median of 456 days (297-1203) post detection of viremia and 24 who were treated during acute infection at a median of 1 day (1-3) post detection of viremia. All participants were female and 31 (89%) were identified with acute infection at Fiebig stage I. Additional participant characteristics are shown in Table 1.

Table 1:

We first quantified total HIV-1 DNA which incorporates all forms of intracellular HIV-1 DNA, both intact and defective, including integrated and unintegrated forms, as well as linear and circularized 2-LTR and 1-LTR forms. Total HIV-1 DNA measurements were performed longitudinally, from baseline (1-3 days following detection of HIV), at the time of peak viral load and was also assessed at 6- and 12-months post-infection for untreated participants and 6- and 12-months post-treatment initiation for late and early treated participants. As expected, treatment during acute infection resulted in a significantly reduced peak plasma viral load (median = 4.18 log copies/ml, IQR, 3.40-4.87) compared to untreated acute infection (median = 7.06 log copies/ml, IQR, 6.83-7.54) (p<0.0001, Figure 1A top panel). However, at time of peak viremia, the untreated and the early treated groups did not differ in total proviral DNA load (Figure 1A, bottom panel). Longitudinal measurements showed that treatment initiated during chronic infection resulted in a significant decline in plasma viral load to undetectable levels after 1 year (p<0.0001, Figure 1B, top panel) however it did not reduce total proviral load (Figure 1B, bottom panel). In contrast, treatment initiated during acute infection resulted in both a rapid decrease of plasma viremia so that all participants had undetectable viremia at one-year post-ART (p<0.0001, Figure 1C top panel) and steady decrease of total proviral load over the same time period (p=0.0004) (Figure 1C, bottom panel). Even though treatment initiation during both chronic and acute infection resulted in complete suppression of plasma viral load after 1 year (Figure 1D, top panel), total proviral load was still detectable with the early treated group having 1.3 times lower levels of total proviral HIV DNA compared to the chronic treated group (p=0.02, Figure 1D, bottom panel). These results indicate that early treatment leads to a measurable decline in proviral DNA during the first year of treatment that is not seen when therapy is initiated during chronic infection.

Figure 1:

Factors associated with total proviral DNA load after 1 year of suppressive ART

To further understand the impact of host, virological and immunological factors, as well as timing of treatment on the establishment and maintenance of the HIV reservoir, we analysed the associations between viral and immune factors and HIV-1 proviral DNA after 1 year of treatment (Table 2). Analyses for each treatment group were performed independently using multivariate regression models with HIV-1 DNA levels as the dependent variable and other factors, specifically nadir CD4, pre-infection CD4, baseline CD4 counts and peak viral load, as the independent predictor variables. The analysis showed that when treatment was initiated during acute infection, only peak plasma viral load was significantly associated with levels of HIV-1 proviral DNA after 1 year of treatment (p=0.02). However, when treatment was initiated during chronic infection both baseline CD4 count (measured 1-3 days after detection of HIV) (p=0.002) and peak plasma viral load (p=0.03) positively associated with HIV-1 proviral DNA levels, while there was a significant inverse association with nadir CD4 (p<0.0001). Other factors such as protective HLA alleles and type of treatment regimen were not associated with HIV-1 proviral DNA (data not shown). These data indicate that both host and viral characteristics impact the establishment and maintenance of the viral reservoir.

Table 2:

Longitudinal genotypic characterisation of HIV-1 DNA

Quantification of total HIV-1 DNA by ddPCR as described above is based on the amplification of a short 127 base pair fragment of the HIV-1 genome, and thus detects defective viruses that are incapable of replication, thereby overestimating the size and functionality of the reservoir. To address this, we next performed single template near full-genome sequencing to determine potential replication competency by establishing the distribution of genome intact and genome defective latent viruses within cells. Viral genome intactness was determined by the HIVSeqinR v2.7.1 computational bioinformatics pipeline [33].

For this analysis we studied 24 participants: The chronic infection (late treatment) group (n=11) consisted of individuals who remained untreated for over one year following infection and before treatment initiation. Longitudinal sampling at untreated time points was available for 9 of these individuals whereas in two individuals, samples were only available post-treatment initiation. The acutely treated group (n=13) received treatment immediately post-detection (Figure 2A). We generated a total of 697 sequences (GenBank accession numbers OR991333-OR991737 and MK643536-MK643827) after sampling a median of 1.4 million PBMC (0.02-4.3 million) per sampling time point. Genome-intact viruses (Figure 2B) accounted for 35% (247/697) of the total pool and were detected in 23 participants (12 from the early treatment group and 11 from the late treatment group), with a median of 8 genome-intact viruses (range=1-60) per study participant. Phylogenetic analysis revealed a significant difference in the mean pairwise distances of intact viral sequences derived from early treated (median=0.12% (IQR, 0.07-0.21) compared to late treated participants (median=0.48% (IQR, 0.16-1.08)) (p=0.04) (Figure 2C). Overall, 56% of the intact genomes collected in this study were obtained from the untreated study arm, while 11% were obtained from late treated chronic infection and 33% from acutely treated infections (Figure 2D).

Figure 2:

Longitudinal studies revealed that intact proviruses accumulated rapidly during the course of HIV-1 infection with a relative contribution of 65% (450/697) to the total pool of proviral genomes detected. The majority of defective viral genomes collected in this study were detected during untreated infection (44% (199/450), left-most pie), while 39% (175/450) were detected during late treated chronic infection and 17% (76/450) during acute treated infection. Defective genomes contributed 59% to the proviral population in untreated infection, 87% in late (chronic) treated and 48% in acutely treated infection (Figure 2D). Overall, defective genomes also accumulated quickly after onset of infection and were detectable at a proportion of 47% within the first month irrespective of treatment status (data not shown). Large internal deletions within viral genomes were the most common defect with relative frequencies of 77%, 60% and 79% in untreated infection, chronic treated infection and acute treated infection respectively among the pool of defective genomes. Overall, these gene deletions occurred significantly more frequently between integrase and envelope in the integrase to envelope gene segment compared to gag (p<0.0001–0.001), with nef being similar to gag (Supplementary Figure 1). APOBEC induced hypermutations were the second most common defect observed in untreated (16%) and late (chronic) treated (31%) infection. However, in acute treated infection, hypermutations were relatively infrequent, comprising only 7% of the genome-defective pool. Premature stop codons in one of gag, pol or env occurred at a frequency of 5%, 4% and 13% as a percentage of defective genomes in sequences from untreated, chronic, and acute treated infections respectively. Internal inversions (1%, 1%, 1%), and 5’ psi defects (2%, 4%, 0%) were other types of genome defects that were detected at minor frequencies in untreated, chronically treated and acutely treated infections respectively.

To further understand the impact of ART timing on the composition, evolution, and dynamics of the HIV-1 proviral landscape over time, we next performed a stratified analysis of the relative proportions of viral genome sequences in each study arm over 1 year of follow up (Figure 3A-C). Genome-intact viruses were detectable throughout the course of untreated infection while genome-defective viruses also accumulated over this period (Figure 3A). Initiation of ART during chronic infection, at a median of 456 days after detection of plasma viremia, resulted in a decrease in the relative proportion of genome-intact viruses over 1 year of treatment (34% to 14%). However, genome-intact viruses were not completely eradicated and were easily detectable after 12 months of treatment (Figure 3B). Additionally, genomes with large deletions and hypermutations became more prominent in the chronic treated group over 1 year of treatment. In contrast, there was a more rapid decrease in the proportion of genome-intact viruses following ART initiation in acute infection such that these viruses were no longer detectable at our sampling depth after 1 year of treatment (57% to 0%) (Figure 3C). Hypermutated viruses were also less prominent before 12 months during early treated infection (Figure 3C). These data suggest that early treatment initiation facilitates faster clearance of genome-intact viruses in the blood compared to late treatment.

Figure 3:

Decay kinetics of intact and defective proviruses

Studies show that the biology and decay dynamics of genome-intact viruses within the viral reservoir likely differ from that of the genome-defective provirus pool [12, 13]. However, the effect of ART timing on the rate of decay of these different pools of viruses is not well known and has not been investigated in African populations where immune responses and viral genetic heterogeneity may well result in population-specific differences. Here we observed that the absolute proportions of both genome-intact and -defective viruses per million PBMCs sampled decreased in both early treated and late treated participants over the 1-year follow-up period of this study (Figure 4A and 4B).

Figure 4:

To estimate potential differences in the rate of change between genome-intact and -defective viruses within each treatment group we used a linear mixed effects regression model with random intercepts to account for the correlation between repeated observations from the same individual. We fit a model with log DNA copies as the response variable, and time, treatment group, and a time-group interaction as fixed effects, with participant as a random effect. The analysis was restricted to the first 6 months after starting ART as regular measurements for both groups were available over this period.

Among the acute treated, genome-intact proviruses decreased by 0.308 log copies per month in the first six months after starting ART, corresponding to a decline of 51% per month (p<0.001, Figure 4C). In contrast, among the chronic treated, intact proviruses decreased by only 0.059 log copies per month, corresponding to a decline of 13% per month; however, this decrease was not statistically significant (p=0.68) (Figure 4E).

Genome-defective proviruses also decreased significantly in the acute treated group by 0.190 log copies per month in the first six months after starting ART, corresponding to a decline of 35% (p=0.01). However, in the chronic treated group the change in the number of log copies of defective provirus in the first 6 months was only 0.015 (p=0.88) corresponding to a decline of just 2% per month (Figure 4D and 4E). These results indicate that early treatment is associated with a faster decline of both genome-intact and -defective proviruses compared to late treatment.

Contribution of clonal expansion to maintenance of proviral populations

Studies show that more than 50% of the latent HIV reservoir is maintained by clonal expansion (Lui et. al., 2020). We assessed viral genome sequences to determine the extent of persistence of infected cell clones after primary infection. Viral genome sequences sharing 100% identity by FLIP-seq was used as a marker of clonal expansion of infected cells as previous studies have shown that proviral genomes that were 100% identical share the same viral integration site whereas proviruses with different integration sites do not share 100% sequence identity (Einkauf et al., 2019). At our sampling depth, we detected clonal expansion in 3/11 (27%) participants who were treated during chronic infection and 4/13 (30%) participants treated during acute infection, showing that in subtype C infection clonal expansion of infected cells occurred as early as one day post detectable viremia (Supplementary Figure 2). Defective clones were detected in two late treated participants at proportions of 6% and 13% of total proviral population, while intact clones were identified in one late treated participant at a proportion of 6% of the total proviral pool. In contrast, a higher proportion of intact clones were detected in early treated participants at 33%, 30%, 37% and 24% of the total proviral pool. Although the data is limited and needs to be interpreted with caution, this suggests that clonal expansion of intact proviral genomes is more likely to occur when treatment is initiated early, likely due to the early inhibition of viral replication that prevents the accumulation and seeding of defective viral genomes into the viral reservoir.

CTL epitope diversity in the latent reservoir

The emergence of escape mutations in viral epitopes as a mechanism to evade human leukocyte antigen (HLA) class I-restricted immune responses, specifically of CD8+ cytotoxic T lymphocytes (CTL), drives viral diversification and is a significant challenge in developing effective therapies against HIV [47–50]. We investigated the impact of late compared to early ART initiation on CTL epitope diversity and escape in the HIV reservoir by longitudinally analysing Gag, Nef and Pol CTL epitopes, [51] from single genome viral sequences (excluding only hypermutated sequences), that are restricted by HLA genotypes B*57:02, B*57:03, B*58:01, B*81:01 and A*74:01. These HLA genotypes have been associated with protection against disease progression in HIV-1 subtype C infection [52]. CTL epitope mutations were classified according to the Los Alamos HIV Molecular Immunology Database [51]. Protective HLA genotypes were present in 7/11 (64%) late treated participants and in 7/13 (54%) early treated participants. In the presence of relevant restricting HLA genotypes, mutations compared to the Clade C consensus were detected in 12% of participants with Gag, 23% with Pol and 27% with Nef targeted epitopes after 1 year of follow-up when treatment was initiated late (Figure 5A-C) in contrast to 0%, 0% and 8% respectively when treatment was initiated early (Figure 5G-I) suggesting that chronic treatment is associated with the retention of a wide spectrum of CTL escape mutations within reservoirs compared to early treatment. Escape mutations detected at baseline (up to 1 month after infection) in the presence of restricting HLA genotypes were present in 3% of participants within Gag, 19% within Pol and 23% (Figure 5A-C) within Nef targeted epitopes when treatment was initiated later compared to and 0%, 13% and 11% respectively (Figure 5G-I) with early treatment. Escape mutations observed in early treated participants were present in the earliest sequences that were derived close to the time of infection and therefore likely represent transmitted escape variants. Similar proportions of transmitted escape mutations were present in participants who did not have a protective HLA genotype and remained unchanged after 1 year. (Figure 5D-F and 5J-L).

Figure 5:

Discussion

In this study we used a well characterised acute HIV infection longitudinal cohort of untreated, early treated and late treated study participants, to perform an extensive quantitative and qualitative analysis of the HIV-1 subtype C viral reservoir. Our aim was to determine whether the timing of treatment has an impact on the viral reservoir size, genetic landscape, and decay kinetics. This was a longitudinal study where we measured total proviral load levels by ddPCR and characterised the genetic landscape of the viral reservoir by next generation sequencing using FLIP-Seq. Total HIV-1 DNA impacts the pathophysiology of infection and is therefore clinically important [30, 53–55]. We found that HIV DNA was detectable at high levels during primary infection and even in participants who were treated during acute infection, total HIV DNA levels measured at peak viremia was very similar to untreated participants. In contrast, we found that early but not late treatment was associated with steady decline of total proviral load over the first year of ART. These observations confirm previous data that the viral reservoir is seeded at the earliest stages of infection, possibly before peak viremia [2, 56–59]. However, in contrast, studies have suggested that early ART intervention restricts the seeding of the HIV reservoir in long-lived central memory CD4 T cells [27]. Moreover, it has recently been demonstrated that a small fraction of deeply latent genetically intact proviruses are archived in CD4 T cells during the very first weeks of infection [60]. However, even though early ART initiation has been associated with continued HIV DNA reduction during long-term ART after 10 years of follow up [28], it remains detectable in most individuals indicating that early ART alone is insufficient to achieve viral eradication.

We also examined the association of clinical and virological factors with the levels of proviral DNA after 1 year of treatment. In both early and late treatment groups, peak plasma viral load correlated positively with the HIV DNA levels. Moreover, in the late treatment group there was a positive association with CD4 at enrolment (baseline) and an inverse correlation with nadir CD4 counts. It is possible that higher levels of CD4 T cells at the onset of infection (baseline) presents the availability of more target cells for HIV-1 reservoir establishment. Similar associations with nadir CD4 have been reported previously [61–63] suggesting that during untreated progressive HIV infection, ongoing viral replication may drive the accumulation of long-lived latently infected cells that repopulate the immune system by expansion during successful ART.

Even though total HIV DNA has been shown to be a clinically significant marker of the HIV reservoir it does not distinguish between replication-competent and -defective viruses that contribute to the viral reservoir. The unique design of the FRESH cohort based on frequent HIV screening and sampling intervals of high-risk uninfected participants allowed us to examine the dynamics of the proviral landscape from the earliest stages of infection (Fiebig I) up to 1 year of ART by near-full-length viral genome sequencing. We performed a comparison of the proviral populations between study participants who were treated during the acute phase of infection and those initiating ART during chronic infection. Defective genomes accumulate rapidly after the onset of infection and contributed to almost half of the proviral population within the first four weeks of infection irrespective of treatment status. Consistent with other studies [33, 56, 64] the overall viral reservoir landscape in the untreated and late treated participants was dominated by defective viruses suggesting that prolonged ongoing viral replication before treatment initiation leads to the accumulation of defective viral genomes. Genome-intact viruses were easily detectable throughout untreated infection but decreased after treatment. In participants who were treated during chronic infection, genome-intact viruses were still detectable after one year of ART compared to early treated individuals where they were no longer detectable.

With our limited sampling size and depth, of a median of 1.4 million PBMC (0.02-4.3 million) per sampling time point, we cannot rule out that intact genomes may be retrieved with further sampling and investigation into tissue reservoirs of the participants who initiate ART during acute infection. However, these findings do provide further evidence that introducing ART during acute HIV infection limits the size of the HIV reservoir considerably compared to treatment during chronic infection [11, 24–28].

The intact proviral DNA assay (IPDA) which is a more scalable method that uses multiplexed ddPCR to measure individual proviruses and differentiates intact from defective proviruses without the need for long-distance PCR, has been suggested to provide more accurate quantitative information about the size and composition of the latent reservoir compared to near full genome sequence methods [65]. However, this assay has not yet been developed and optimised for quantification of subtype C HIV.

Studies have shown that during suppressive ART, intact and defective proviruses have different rates of decay that occurs in a biphasic manner [11–13]. Our analysis of decay kinetics was limited to a linear mixed effects regression model as we were unable to fit a model for biphasic decay which requires frequent viral reservoir measurements. Our analysis was further restricted to the first six months of ART as viral genomes were difficult to detect by FLIP-seq in early treated participants after this time. We found that indeed intact genomes decay faster than defective genomes in both early and late treatment groups. Cells containing intact viral genomes likely represent productively infected cells that may be preferentially targeted for clearance by the host immune response or eliminated by viral cytopathic effects [12, 13]. Moreover, early treatment results in a faster decline of both intact and defective genomes compared to treatment initiated during chronic infection and is suggestive of a more effective immune clearance mechanism from preserved immune function. Despite this, it is estimated that 226 years of effective ART is necessary to decrease intact proviral DNA levels by 4 log₁₀ [12]. This further indicates that early ART in combination with novel interventional strategies will be needed to achieve a faster viral eradication.

Several studies show that clonal expansion of HIV-infected cells plays an important role in maintaining the HIV reservoir further contributing to the challenge of eradicating HIV [66–69]. Our findings suggest that clonal expansion of intact viral genomes detected predominantly in early treated infection may contribute to the maintenance of the HIV reservoir in these study participants. Further studies that extend beyond 1 year of treatment will help elucidate whether these clones expand further after several years of treatment. Our analysis of CTL epitope escape mutations, known to drive viral diversification, revealed that early treatment minimises the emergence of CTL escape in Gag, Pol and Nef epitopes despite these participants having protective HLA alleles. In contrast CTL escape was detected when treatment was initiated during chronic infection specifically in the well characterised TW10 gag epitope restricted by HLA B57/58. Transmitted CTL escape mutations detected within the first few weeks of infection were common in both treatment groups confirming previous data from this study population [70]. Moreover, the rapid rate of clearance of viral genomes observed with early treatment could be attributed to the lower proportion of cytotoxic T lymphocyte escape mutations in the reservoir of these participants compared with those treated later.

To our knowledge this is the first study in an African population, dominated by subtype C HIV infection, that examined the impact of the timing of ART initiation on HIV reservoirs in a longitudinal setting. Moreover, our data focused on women who are underrepresented in reservoir and cure studies globally. Our data showed that early ART initiation does not blunt proviral DNA seeding in immune reservoirs, but it nevertheless results in a more rapid decay of intact viral genomes, decreases genetic complexity and immune escape and preserves innate immune factors. Although early ART alone may not be sufficient to eradicate the persisting viral reservoir, our results suggest that when combined with interventional strategies, it is more likely to achieve an effective HIV cure.

Materials and Methods

Ethics Statement

The Biomedical Research Ethics Committee of the University of KwaZulu-Natal and the Institutional Review Board of Massachusetts General Hospital approved the study. All participants provided written informed consent.

Study Design and Participants

This was a longitudinal study of the Females Rising through Education, Support, and Health (FRESH) cohort, a prospective, observational study of 18–23-year-old HIV uninfected women at high risk for HIV acquisition, established in Umlazi, Durban, South Africa [45, 46]. Finger prick blood draws were collected from FRESH study participants twice a week and subjected to HIV-1 RNA testing, with the aim of detecting acute HIV infection during Fiebig stage I. The study included a socioeconomic intervention program and HIV prevention interventions including PrEP that coincided with study visits to address challenges faced by the young women that likely contribute to the increased risk of HIV acquisition in this setting. If a participant acquired HIV-1 infection while on the study, blood samples were collected weekly for a month, then monthly until 3 months post infection, then monthly for one year and every 3 months thereafter. Days post onset of viremia (DPOPV) was calculated as the interval between the first positive HIV test and the date of sample collection. Unique participant identifier numbers were assigned to the participants and are only known to the research group.

Study participants recruited during the first 19 months of the study did not receive antiretroviral (ARV) treatment immediately after detection of acute HIV infection but were monitored and referred for treatment when they became eligible according to national treatment guidelines at the time. The South African national treatment eligibility criteria subsequently changed allowing the immediate initiation of ART for all people living with HIV (PLWH), including those with acute HIV infection as recommended under the World Health Organisation’s universal test and treat policy [71]. The treatment schedule was a three-drug daily oral regimen of 300 mg tenofovir disoproxil fumarate, 200 mg emtricitabine, and 600 mg efavirenz. Additionally, raltegravir (400 mg twice a day) was introduced as a fourth drug and was continued for 90 days after viral suppression (<20 copies per mL).

For this study participants were categorized into 3 groups (untreated, late (chronic) treated, and early (acute) treated) where, 11 remained untreated during acute infection and later started ART during chronic infection at a median of 456 (297-1203) days post onset of viremia (DPOV), while 24 started ART at a median of 1 (1-3) DPOV. Participants were studied at 0-, 1-, 3-, 6-, 9-, 12- and 24-months post onset of viremia and up to 12 months post treatment.

Quantification of total HIV-1 DNA

Measurement of total HIV-1 DNA was performed as previously described [72]. Total DNA was extracted from total PBMC samples using DNeasy Blood & Tissue Kits (QIAGEN). Droplet digital PCR (ddPCR) (Bio-Rad) was used to measure total HIV-1 DNA and host cell concentration with primers and probes covering HIV-1 5′ LTR-gag HXB2 coordinates 684–810 (forward primer 5′- TCTCGACGCAGGACTCG-3′, reverse primer 5′-TACTGACGCTCTCGCACC-3′ probe/56-FAM/CTCTCTCCT/ZEN/TCTAGCCTC/ 31ABkFQ/, and human RPP30 gene38 forward primer 5′- GATTTGGACCTGCGAGCG-3′, reverse primer 5′-GCGGCTGTCTCCACAAGT-3′, probe/56-FAM/CTGACCTGA/ZEN/AGGCTCT/31AbkFQ/). Thermocycling conditions for ddPCR were: 95 °C for 10 min, 45 cycles of 94 °C for 30 s and 60 °C for 1 min, 72 °C for 1 min. Thereafter droplets from each sample were analyzed on the Bio-Rad QX200 Droplet Reader and data were analysed using QuantaSoft software (Bio-Rad).

Single genome amplification and deep sequencing of near-full length HIV-1 DNA

Near full-length proviral sequences were generated as previously described [72]. Total HIV-DNA copy number as determined by ddPCR was used to calculate the DNA sample dilution to achieve one PCR-positive reaction in every three reactions. This method of limiting dilution gives a Poisson probability of 85.7% that each PCR amplicon originated from a single HIV-DNA template [73]. A nested PCR approach was used to amplify the near-full genome using one unit of Platinum™ Taq DNA Polymerase High Fidelity (Invitrogen) with the following primers: first round PCR: forward primer 5′- AAATCTCTAGCAGTGGCGCCCGAACAG-3′, reverse primer 5′- TGAGGGATCTCTAGTTACCAGAGTC-3′; second round PCR: forward primer 5′- GCGCCCGAACAGGGACYTGAAARCGAAAG-3′, reverse primer 5′-GCACTCAAGGCAAGCTT TATTGAGGCTTA-3′ (HXB2 coordinates 638-9632, 8994 bp). The 20 μL reaction mix contained 1× reaction buffer, 2 mM MgSO4, 0.2mM dNTP, 0.4 μM each of forward and reverse primers. The thermal cycling conditions were 2 min at 92 °C, 10 cycles [10 s at 92 °C, 30 s at 60 °C, 10 min at 68 °C], 20 cycles [10 s at 92 °C, 30 s at 55 °C, 10 min at 68 °C], 10 min at 68 °C, 4 °C infinite hold.

Illumina Mi-Seq and Bioinformatics Analysis

All PCR amplicons detectable by gel electrophoresis were subjected to Illumina MiSeq sequencing and thereafter the resulting small reads were de novo assembled using in-house UltraCycler v1.0. (Brian Seed and Huajun Wang, unpublished) [72]. Viral genome-intactness was inferred by the computational bioinformatics pipeline HIVSeqinR v2.7.1 [33].

Statistical methods

GraphPad Prism 10 was used to perform summary statistical analyses and comparisons among study groups using Fishers’ Exact, Mann-Whitney and Kruskal-Wallis and multiple linear regression analysis.

Data Availability

All HIV sequence data used in this study have been deposited in GenBank with accession numbers OR991333-OR991737 and MK643536-MK643827.

Acknowledgements

The study cohort and sample collection were supported in part by grants from the Bill and Melinda Gates Foundation (OPP1066973 and OPP1146433), Gilead Sciences, Inc. (Grant ID #00406), the International AIDS Vaccine Initiative (IAVI) (UKZNRSA1001), the NIAID (R37AI067073), the Witten Family Foundation, the Dan and Marjorie Sullivan Foundation, the Mark and Lisa Schwartz Foundation, Ursula Brunner, the AIDS Healthcare Foundation, and the Harvard University Center for AIDS Research (CFAR, P30 AI060354, which is supported by the following institutes and centers co- funded by and participating with the US National Institutes of Health: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR.). Raltegravir used for immediate treatment was donated by Merck & Co., Inc. This work was also partially supported through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE) which is funded by the Science for Africa Foundation to the Developing Excellence in Leadership, Training and Science in Africa (DELTAS Africa) programme [Del-22-007] with support from Wellcome Trust and the UK Foreign, Commonwealth & Development Office and is part of the EDCPT2 programme supported by the European Union; the Bill & Melinda Gates Foundation [INV-033558]; and Gilead Sciences Inc., [19275]. All content contained within is that of the authors and does not necessarily reflect positions or policies of any SANTHE funder. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. The authors thank all participants in the FRESH cohort who have made this study possible. The authors thank the Massachusetts General Hospital Center for Computational & Integrative Biology DNA Core, specifically Dr. Nicole Stange-Thomann, Dr. Amy Avery, Ms. Kristina Belanger, and Mr. Huajun Wang, for providing them with the Illumina MiSeq deep sequencing service used in this manuscript.

Author Contributions

The study was conceptualized and designed by K.R., T.N., G.Q.L., M.L. and X.G.Y. and PBMC samples and clinical/demographical data were collected by K.L.D., B.D.W., K.R., and T.N. HIV-1 genotyping laboratory work was done by K.R., G.Q.L., N.R., and T.J.B.C. Results were analyzed by K.R., G.Q.L., and T.N. K.R and T.N and G.Q.L. wrote the manuscript; all authors contributed to and approved the manuscript. T.N. supervised the study.