Differences in HIV-1 reservoir size, landscape characteristics and decay dynamics in acute and chronic treated HIV-1 Clade C infection

Reviewer #1 (Public Review):

The authors sought to determine the impact of early antiretroviral treatment on the size, composition, and decay of the HIV latent reservoir. This reservoir represents the source of viral rebound upon treatment interruption and therefore constitutes the greatest challenge to achieving an HIV cure. A particular strength of this study is that it reports on reservoir characteristics in African women, a significantly understudied population, of whom some have initiated treatment within days of acute HIV diagnosis. With the use of highly sensitive and current technologies, including digital droplet PCR and near full-length genome next-generation sequencing, the authors generated a valuable dataset for investigation of proviral dynamics in women initiating early treatment compared to those initiating treatment in chronic infection. The authors confirm previous reports that early antiretroviral treatment restricts reservoir size, but further show that this restriction extends to defective viral genomes, where late treatment initiation was associated with a greater frequency of defective genomes. Furthermore, an additional strength of this study is the longitudinal comparison of viral dynamics post-treatment, wherein early treatment was shown to be associated with a more rapid rate of decay in proviral genomes, regardless of intactness, over a period of one year post-treatment. While it is indicated that intact genomes were not detected after one year following early treatment initiation, caution should be taken with interpretation where sequence numbers are low. Defective genomes are more abundant than intact genomes and are therefore more likely to be sampled. Early treatment was also associated with reduced proviral diversity and fewer instances of polymorphisms associated with cytotoxic T-lymphocyte immune selection. This is expected given that rapid evolution and extensive immune selection are synonymous with HIV infection in the absence of treatment, yet points to an additional benefit of early treatment in the context of immune therapies to restrict the reservoir.

Given that this is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C, the manuscript would benefit from placing these findings in the context of what has been reported in other populations, for example, how decay rates of intact and defective genomes compare with that of other subtypes where known. While not a primary outcome of the study, the comparisons of peak viremia in the hyperacute and chronic-treated groups may be confounded by the fact that peak viremia may have been pre-empted by early treatment i.e., the true peak was not reached in early-treated individuals. Indeed, in the abstract, the authors indicate that treatment was initiated before the peak. The use of the term 'peak' viremia in the hyperacute-treated group could perhaps be replaced with 'highest recorded viral load'. The statistical comparison of this measure in the two groups is perhaps more relevant with regards to viral burden over time or area under the curve viral load as these are previously reported as correlates of reservoir size. The analysis of clonal expansion of proviral genomes may be limited by higher sequence homogeneity in hyperacute infection i.e., cells with different proviral integration sites may have a higher likelihood of containing identical genomes than chronic infection.

Overall, these data demonstrate the distinct benefits of early treatment initiation at reducing the barrier to a functional cure for HIV, not only by restricting viral abundance and diversity but also potentially through the preservation of immune function and limiting immune escape. It therefore provides clues to curative strategies even in settings where early diagnosis and treatment may be unlikely.

Reviewer #2 (Public Review):

HIV infection is characterized by viral integration into permissive host cells - an event that occurs very early in viral-host encounter. This constitutes the HIV proviral reservoir and is a feature of HIV infection that provides the greatest challenge for eradicating HIV-1 infection once an individual is infected.

This study looks at how starting HIV treatment very early after infection, which substantially reduces the peak viral load detectable (compared to untreated infection), affects the amount and characteristics of the viral reservoir. The authors studied 35 women in South Africa who were at high risk of getting HIV. Some of these women started HIV treatment very soon after getting infected, while others started later. This study is well-designed and has as its focus a very well-characterized cohort. Comparison groups are appropriately selected to address reservoir characterization and dynamics in the context of acute and chronic treated HIV-1. The amount of HIV and various characteristics of the genetic makeup of the virus (intact/defective proviral reservoir) were evaluated over one year of treatment. Methods employed for reservoir characterization are state-of-the-art and provide in-depth insights into the reservoir in peripheral blood.

While starting treatment early didn't reduce the amount of HIV DNA at the outset, it did lead to a gradual decrease in total HIV DNA quantity over time. In contrast, those who started treatment later didn't see much change in this parameter. Starting treatment early led to a faster decrease in intact provirus (a measure of replication-competence), compared to starting treatment later. Additionally, early treatment reduced the genetic diversity of the viral DNA and resulted in fewer immune escape variants within intact genomes. This suggests that collectively having a smaller intact replication-competent reservoir, less viral variability, and less opportunity for the virus to evade the immune system - are all features that are likely to facilitate more effective clearance of viral reservoir, especially when combined with other intervention strategies.

Major strengths of the study include the cohort of very early treated persons with HIV and the depth of study. These are important findings, particularly as the study was conducted in HIV-1 subtype C infected women (more cure studies have focussed on men and with subtype B infection)- and in populations most affected by HIV and in need of HIV cure interventions. This is highly relevant because it cannot be assumed that any interventions employed for reducing/clearing the HIV reservoir would perform similarly in men and women or across different populations. Other factors also deserve consideration and include age, and environment (e.g. other comorbidities and coinfections).

Reviewer #3 (Public Review):

Summary:

This paper assesses the size and clearance kinetics of proviral HIV DNA (intact and total) in women in South Africa with clade C virus. who started ART at different time points of infection (very early vs late).

Strengths:

The cohort is excellent. The paper is easy to read. The methodology is appropriate. Some conclusions, particularly the differing kinetics of total HIV DNA despite a similar amount of virus in early vs late treated women are novel and thought-provoking. I really enjoyed reading this paper!

Weaknesses:

There are several areas in the paper that could be explicated a bit more accurately with more detailed references to past work.

(1) The word reservoir should not be used to describe proviral DNA soon after ART initiation. It is generally agreed upon that there is still HIV DNA from actively infected cells (phase 1 & 2 decay of RNA) during the first 6-12 months of ART. Only after a full year of uninterrupted ART is it really safe to label intact proviral HIV DNA as an approximation of the reservoir. This should be amended throughout.

(2) All raw, individualized data should be made available for modelers and statisticians. It would be very nice to see the RNA and DNA data presented in a supplementary figure by an individual to get a better grasp of intra-host kinetics.

(3) The legend of Supplementary Figure 2 should list when samples were taken.

Author response:

Reviewer #1 (Public Review):

Given that this is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C, the manuscript would benefit from placing these findings in the context of what has been reported in other populations, for example, how decay rates of intact and defective genomes compare with that of other subtypes where known.

Most published studies are from men living with HIV-1 subtype B and the studies are not from the hyperacute infection phase and therefore a direct head-to-head comparison with the FRESH study is difficult. However, we can cite/highlight and contrast our study with a few examples from other acute infection studies as follows.

(1) Peluso et. al., JCI, 2020, showed that in Caucasian men (SCOPE study), with subtype B infection, initiating ART during chronic infection virus intact genomes decayed at a rate of 15.7% per year, while defective genomes decayed at a rate of 4% per year. In our study we showed that in chronic treated participants genomes decreased at a rate of 25% (intact) and 3% (defective) per month for the first 6 months of treatment.

(2) White et. al., PNAS, 2021, demonstrated that in a cohort of African, white and mixed-race American men treated during acute infection, the rate of decay of intact viral genomes in the first phase of decay was <0.3 logs copies in the first 2-3 weeks following ART initiation. In the FRESH cohort our data from acute treated participants shows a comparable decay rate of 0.31 log copies per month for virus intact genomes.

(3) A study in Thailand (Leyre et. al., 2020, Science Translational Medicine), of predominantly HIV-1 CRF01-AE subtype compared HIV-reservoir levels in participants starting ART at the earliest stages of acute HIV infection (in the RV254/SEARCH 010 cohort) and participants initiating ART during chronic infection (in SEARCH 011 and RV304/SEARCH 013 cohorts). In keeping with our study, they showed that the frequency of infected cells with integrated HIV DNA remained stable in participants who initiated ART during chronic infection, while there was a sharp decay in these infected cells in all acutely treated individuals during the first 12 weeks of therapy. Rates of decay were not provided and therefore a direct comparison with our data from the FRESH cohort is not possible.

(4) A study by Bruner et. al., Nat. Med. 2016, described the composition of proviral populations in acute treated (within 100 days) and chronic treated (>180 days), predominantly male subtype B cohort. In comparison to the FRESH chronic treated group, they showed that in chronic treated infection 98% (87% in FRESH) of viral genomes were defective, 80% (60% in FRESH) had large internal deletions and 14% (31% in FRESH) were hypermutated. In acute treated 93% (48% in FRESH) were defective and 35% (7%) in FRESH were hypermutated. The differences frequency of hypermutations could be explained by the differences in timing of infection specifically in the acute treated groups were FRESH participants initiate ART at a median of 1 day after infection. It is also possible that sex- or race-based differences in immunological factors that impact the reservoir may play a role.

This study also showed that large deletions are non-random and occur at hotspots in the HIV-1 genome. The design of the subtype B IPDA assay (Bruner et. al., Nature, 2019) is based on optimal discrimination between intact and deleted sequences - obtained with a 5′ amplicon in the Ψ region and a 3′ amplicon in Envelope. This suggest that Envelope is a hotspot for large while deletions in Ψ is the site of frequent small deletions and is included in larger 5′ deletions. In the FRESH cohort of HIV-1 subtype C, genome deletions were most frequently observed between Integrase and Envelope relative to Gag (p<0.0001–0.001).

(5) In 2017, Heiner et. al., in Cell Rep, also described genetic characteristics of the latent HIV-1 reservoir in 3 acute treated and 3 chronic treated male study participants with subtype B HIV. Their data was similar to Bruner et. al. above showing proportions of intact proviruses in participants who initiated therapy during acute/early infection at 6% (94% defective) and chronic infection at 3% (97% defective). In contrast the frequencies in FRESH in acute treated were 52% intact and 48% defective and in chronic infection were 13% intact and 87% defective. These differences could be attributed to the timing of treatment initiation where in the aforementioned study early treatment ranged from 0.6-3.4 months after infection.

Indeed, in the abstract, the authors indicate that treatment was initiated before the peak. The use of the term 'peak' viremia in the hyperacute-treated group could perhaps be replaced with 'highest recorded viral load'. The statistical comparison of this measure in the two groups is perhaps more relevant with regards to viral burden over time or area under the curve viral load as these are previously reported as correlates of reservoir size.

We will edit the manuscript text to describe the term peak viraemia in hyperacute treated participants more clearly. We will perform an analysis of area under the curve to compare viral burden in the two study groups.

Reviewer #2 (Public Review):

Other factors also deserve consideration and include age, and environment (e.g. other comorbidities and coinfections.)

We agree that these factors could play a role however participants in this study were of similar age (18-23), and information on co-morbidities and coinfections are not known.

Reviewer #3 (Public Review):

The word reservoir should not be used to describe proviral DNA soon after ART initiation. It is generally agreed upon that there is still HIV DNA from actively infected cells (phase 1 & 2 decay of RNA) during the first 6-12 months of ART. Only after a full year of uninterrupted ART is it really safe to label intact proviral HIV DNA as an approximation of the reservoir. This should be amended throughout.

We agree and will amend the use of the word reservoir to only refer to the proviral DNA load after full viral suppression, i.e., during undetectable viral load.

All raw, individualized data should be made available for modelers and statisticians. It would be very nice to see the RNA and DNA data presented in a supplementary figure by an individual to get a better grasp of intra-host kinetics.

We will make all relevant data available and accessible to interested parties.

The legend of Supplementary Figure 2 should list when samples were taken.

The data in this figure represents an overall analysis of all sequences available for each participant at all time points. This will be explained more clearly in the manuscript and added to the figure legend.