HIV-specific CD8+ T-cell responses soon after treatment initiation during acute HIV infection are associated with viral reservoir decline

Reviewer #1 (Public review):

Summary:

In this work, van Paassen et al. have studied how CD8 T cell functionality and levels predict HIV DNA decline. The article touches on interesting facets of HIV DNA decay, but ultimately comes across as somewhat hastily done and not convincing due to the major issues.

(1) The use of only 2 time points to make many claims about longitudinal dynamics is not convincing. For instance, the fact that raw data do not show decay in intact, but do for defective/total, suggests that the present data is underpowered. The authors speculate that rising intact levels could be due to patients who have reservoirs with many proviruses with survival advantages, but this is not the parsimonious explanation vs the data simply being noisy without sufficient longitudinal follow-up. n=12 is fine, or even reasonably good for HIV reservoir studies, but to mitigate these issues would likely require more time points measured per person.

1b) Relatedly, the timing of the first time point (6 months) could be causing a number of issues because this is in the ballpark for when the HIV DNA decay decelerates, as shown by many papers. This unfortunate study design means some of these participants may already have stabilized HIV DNA levels, so earlier measurements would help to observe early kinetics, but also later measurements would be critical to be confident about stability.

(2) Statistical analysis is frequently not sufficient for the claims being made, such that overinterpretation of the data is problematic in many places.

2a) First, though plausible that cd8s influence reservoir decay, much more rigorous statistical analysis would be needed to assert this directionality; this is an association, which could just as well be inverted (reservoir disappearance drives CD8 T cell disappearance).

2b) Words like "strong" for correlations must be justified by correlation coefficients, and these heat maps indicate many comparisons were made, such that p-values must be corrected appropriately.

(3) There is not enough introduction and references to put this work in the context of a large/mature field. The impacts of CD8s in HIV acute infection and HIV reservoirs are both deep fields with a lot of complexity.

Reviewer #2 (Public review):

Summary:

This study investigated the impact of early HIV specific CD8 T cell responses on the viral reservoir size after 24 weeks and 3 years of follow-up in individuals who started ART during acute infection. Viral reservoir quantification showed that total and defective HIV DNA, but not intact, declined significantly between 24 weeks and 3 years post-ART. The authors also showed that functional HIV-specific CD8⁺ T-cell responses persisted over three years and that early CD8⁺ T-cell proliferative capacity was linked to reservoir decline, supporting early immune intervention in the design of curative strategies.

Strengths:

The paper is well written, easy to read, and the findings are clearly presented. The study is novel as it demonstrates the effect of HIV specific CD8 T cell responses on different states of the HIV reservoir, that is HIV-DNA (intact and defective), the transcriptionally active and inducible reservoir. Although small, the study cohort was relevant and well-characterized as it included individuals who initiated ART during acute infection, 12 of whom were followed longitudinally for 3 years, providing unique insights into the beneficial effects of early treatment on both immune responses and the viral reservoir. The study uses advanced methodology. I enjoyed reading the paper.

Weaknesses:

All participants were male (acknowledged by the authors), potentially reducing the generalizability of the findings to broader populations. A control group receiving ART during chronic infection would have been an interesting comparison.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this work, van Paassen et al. have studied how CD8 T cell functionality and levels predict HIV DNA decline. The article touches on interesting facets of HIV DNA decay, but ultimately comes across as somewhat hastily done and not convincing due to the major issues.

(1) The use of only 2 time points to make many claims about longitudinal dynamics is not convincing. For instance, the fact that raw data do not show decay in intact, but do for defective/total, suggests that the present data is underpowered. The authors speculate that rising intact levels could be due to patients who have reservoirs with many proviruses with survival advantages, but this is not the parsimonious explanation vs the data simply being noisy without sufficient longitudinal follow-up. n=12 is fine, or even reasonably good for HIV reservoir studies, but to mitigate these issues would likely require more time points measured per person.

(1b) Relatedly, the timing of the first time point (6 months) could be causing a number of issues because this is in the ballpark for when the HIV DNA decay decelerates, as shown by many papers. This unfortunate study design means some of these participants may already have stabilized HIV DNA levels, so earlier measurements would help to observe early kinetics, but also later measurements would be critical to be confident about stability.

We agree that in order to thoroughly investigate reservoir decay in acutely treated individuals, more participants and/or more time points measured per participant would increase the power of the study and potentially, in line with literature, show a significant decay in intact HIV DNA as well. By its design (1) the NOVA study allows for a detailed longitudinal follow-up of reservoir and immunity from start ART onwards. In the present analysis in the NOVA cohort, we decided to focus on the 24- and 156-week time points. We plan to include more individuals in our analysis in the future, so that we can better model the longitudinal dynamics of the HIV reservoir.

The main goal of the present study, however, was not to investigate the decay or longitudinal dynamics of the viral reservoir, but to understand the relationship of the HIV-specific CD8 T-cell responses early on ART with the reservoir changes across the subsequent 2.5-year period on suppressive therapy. We will revise the manuscript in order to clarify this. Moreover, we agree with the reviewer that the early time point (24 weeks) is a time at which many virological and immunological processes are ongoing and the reservoir may not have stabilized yet for every participant. We will highlight this in the revised manuscript.

(2) Statistical analysis is frequently not sufficient for the claims being made, such that overinterpretation of the data is problematic in many places.

(2a) First, though plausible that cd8s influence reservoir decay, much more rigorous statistical analysis would be needed to assert this directionality; this is an association, which could just as well be inverted (reservoir disappearance drives CD8 T cell disappearance).

The second point that was raised by reviewer 1 is the statistical analysis, which is referred to as “not sufficient for the claims being made”. Moreover, a more “rigorous statistical analysis would be needed”. At this stage, it is unclear from the reviewer's comments what specific type of additional statistical analysis is being requested. Correlation analyses, such as the one used in this study, are a well-established approach to investigate the relationship between the immune response and reservoir size. However, as we aim to perform the most rigorous analysis possible, for the revised submission we will adjust our analysis for putative confounders (e.g. age and antiretroviral regimen).

We would also like to note that the association between the CD8 T-cell response at 24 weeks and the subsequent decline (the difference between 24 and 156 weeks) in the reservoir cannot be bi-directional (that can only be the case when both variables are measured at the same time point).

(2b) Words like "strong" for correlations must be justified by correlation coefficients, and these heat maps indicate many comparisons were made, such that p-values must be corrected appropriately.

For the revised submission, we will provide correlation coefficients to justify the wording, and will adjust the p-values for multiple comparisons.

(3) There is not enough introduction and references to put this work in the context of a large/mature field. The impacts of CD8s in HIV acute infection and HIV reservoirs are both deep fields with a lot of complexity.

Lastly, reviewer 1 referred to the introduction and asked for more references and a more focused viewpoint because the field is large and complex. We aim to revise the introduction/discussion based on the suggestions from the reviewer.

Reviewer #2 (Public review):

Summary:

This study investigated the impact of early HIV specific CD8 T cell responses on the viral reservoir size after 24 weeks and 3 years of follow-up in individuals who started ART during acute infection. Viral reservoir quantification showed that total and defective HIV DNA, but not intact, declined significantly between 24 weeks and 3 years post-ART. The authors also showed that functional HIV-specific CD8⁺ T-cell responses persisted over three years and that early CD8⁺ T-cell proliferative capacity was linked to reservoir decline, supporting early immune intervention in the design of curative strategies.

Strengths:

The paper is well written, easy to read, and the findings are clearly presented. The study is novel as it demonstrates the effect of HIV specific CD8 T cell responses on different states of the HIV reservoir, that is HIV-DNA (intact and defective), the transcriptionally active and inducible reservoir. Although small, the study cohort was relevant and well-characterized as it included individuals who initiated ART during acute infection, 12 of whom were followed longitudinally for 3 years, providing unique insights into the beneficial effects of early treatment on both immune responses and the viral reservoir. The study uses advanced methodology. I enjoyed reading the paper.

Weaknesses:

All participants were male (acknowledged by the authors), potentially reducing the generalizability of the findings to broader populations. A control group receiving ART during chronic infection would have been an interesting comparison.

We thank the reviewer for their appreciation of our study. The reviewer raises the point that it would be useful to compare our data to a control group. Unfortunately, these samples are not yet available, but our study protocol allows for a control group (chronic infection) to ensure we can include a control group in the future.

(1) Dijkstra M, Prins H, Prins JM, Reiss P, Boucher C, Verbon A, et al. Cohort profile: the Netherlands Cohort Study on Acute HIV infection (NOVA), a prospective cohort study of people with acute or early HIV infection who immediately initiate HIV treatment. BMJ Open. 2021;11(11):e048582.