Persistence of intact HIV-1 proviruses in the brain during suppressive antiretroviral therapy

Reviewer #1 (Public Review):

Despite durable viral suppression by antiretroviral therapy (ART), HIV-1 persists in cellular reservoirs in vivo. The viral reservoir in circulating memory T cells has been well characterized, in part due to the ability to safely obtain blood via peripheral phlebotomy from people living with HIV-1 infection (PWH). Tissue reservoirs in PWH are more difficult to sample and are less well understood. Sun and colleagues describe isolation and genetic characterization of HIV-1 reservoirs from a variety of tissues including the central nervous system (CNS) obtained from three recently deceased individuals at autopsy. They identified clonally expanded proviruses in the CNS in all three individuals.

Strengths of the work include the study of human tissues that are under-studied and difficult to access, and the sophisticated near-full length sequencing technique that allows for inferences about genetic intactness and clonality of proviruses. The small sample size (n=3) is a drawback. Furthermore, two individuals were on ART for just one year at the time of autopsy and had T cells compatible with AIDS, and one of these individuals had a low-level detectable viral load (Figure S1). This makes generalizability of these results to PWH who have been on ART for years or decades and have achieved durable viral suppression and immune reconstitution difficult.

While anatomic tissue compartment and CNS region accompany these PCR results, it is unclear which cell types these viruses persist in. As the authors point out, it is possible that these reservoir cells might have been infiltrating T cells from blood present at the time of autopsy tissue sampling. Cell type identification would greatly enhance the impact of this work. Several other groups have undergone similar studies (with similar results) using autopsy samples (links below). These studies included more individuals, but did not make use of the near-full length sequencing described here. In particular, the Last Gift cohort, based at UCSD and led by Sara Gianella and Davey Smith, has established protocols for tissue sampling during autopsy performed soon after death.
https://pubmed.ncbi.nlm.nih.gov/35867351/
https://pubmed.ncbi.nlm.nih.gov/37184401/

Overall, this small, thoughtful study contributes to our understanding of the tissue distribution of persistent HIV-1, and informs the ongoing search for viral eradication.

Reviewer #2 (Public Review):

The manuscript by Sun et al. applies the powerful technology of profiling viral DNA sequences in numerous anatomical sites in autopsy samples from participants who maintained their antiviral therapy up to the time of death. The sequencing is of high quality in using end-point dilution PCR to generate individual viral genomes. There is a thoughtful discussion, although there are points that we disagree with. This is an important data set that increases the scope of how the field thinks about the latent reservoir with a new look at the potential of a reservoir within the CNS.

1. The participants are very different in their exposure to HIV replication and disease progression. Participant 1 appears to have been on ART for most of the time after diagnosis of infection (16 years) and died with a high CD4 T cell count. The other two participants had only one year on ART and died with relatively low CD4 T cell counts (under 200). This could lead to differences in the nature of the reservoir. In this regard, the amount of DNA per million cells appears to be about 10-fold lower across the compartments sampled for participant 1. Also, one might expect fewer intact proviruses surviving after 16 years on ART compared to only 1 year on ART. The depth of sampling may be too limited and the number of participants too few to assess if these differences are features of these participants because of their different exposures to HIV replication. On the positive side, finding similarities across these big differences in participant profiles does reinforce the generalizability of the observations.

2. The following analysis will be limited by sampling depth but where possible it would be interesting to compare the ratio of intact to defective DNA. A sanctuary might allow greater persistence of cells with intact viral DNA even without viral replication (i.e. reduced immune surveillance). Detecting one or two intact proviruses in a tissue sample does not lend itself to a level of precision to address this question, but statistical tests could be applied to infer when there is sampling of 5 or more intact proviruses to determine if their frequency as a ratio of total DNA in different anatomical sites is similar or different. This would allow adjustment for the different amount of viral DNA in different compartments while addressing the question of the frequency of intact versus defective proviruses. One complication in this analysis is if there was clonal expansion of a cell with an intact genome which would represent a fortuitous over-representation intact genomes in that compartment.

3. The key point of this work is that the participants were on therapy up to the time of death ("enforcing" viral latency). The predominance of defective genomes is consistent with this assumption. Is there data from untreated infections to compare to as a signature of whether the viral DNA population was under selective pressure from therapy or not? Presumably untreated infections contain more intact DNA relative to total DNA. This would represent independent evidence that therapy was in place.

4. There are several points in Figure 5 to raise about V3 loop sequences. The analysis includes a large number of "undetermined" sequences that did not have a V3 loop sequence to evaluate. We would argue it is a fair assumption that the deleted proviruses have the same distribution of X4 and R5 sequences as the ones that have a V3 sequence to evaluate. In this view it would be possible to exclude the sequences for which there is no data and just look at the ratio of X4 and R5 in the different compartments, specifically does this ratio change in a statistically significant way in different compartments? The authors use "CCR5 and non-CCR5" as the two entry phenotypes. The evidence is pretty strong that the "other" coreceptor the virus routinely uses is CXCR4, and G2P is providing the FPR for X4 viruses. Perhaps the authors are trying to create some space for other coreceptors on microglia, but we are pretty sure what they are measuring is X4 viruses, especially in this late disease state of participant 2. Finally, we have previously observed that the G2P FPR score of <2 is a strong indicator of being X4, FPR scores between 2 and 10 have a 50% chance of being X4, and FPR scores above 10 are reliably R5 (PMID27226378). In addition, we observed that X4 viruses form distinct phylogenetic lineages. The authors might consider these features of X4 viruses in the evaluation of their sequences. Specifically, it would be helpful to incorporate the FPR scores of the reported X4 viruses.

5. We have puzzled over the many reports of different cell types in the CNS being infected. When we examined these cells types (both as primary cells and as iPSC-derived cells), all cells could be infected with a version of HIV that had the promiscuous VSV-G protein on the virus surface as a pseudotype. However, only macrophages and microglia could be infected using the HIV Env protein, and then only if it was the M-tropic version and not the T-tropic version (PMID35975998). RNAseq analysis was consistent with this biological readout in that only macrophages and microglia expressed CD4, neurons and astrocytes do not. From the virology point of view, astrocytes are no more infectable than neurons.

6. The brain gets exposed to virus from the earliest stages of infection but this is not synonymous with viral replication. Most of the time there is virus in the CSF but it is present at 1-10% of the level of viral load in the blood and phylogenetically it looks like the virus in the blood, most consistent with trafficking T cells, some of which are infected (PMID25811757). The fact that the virus in the blood is almost always T cell-tropic in needing a high density of CD4 for entry makes it unlikely that monocytes are infected (with their low density of CD4) and thus are not the source of virus found in the CNS. It seems much more likely that infected T cells are the "Trojan Horse" carrying virus into the CNS.

7. While all participants were taking antiretroviral therapy at the time of their death, they were not all suppressed when the tissues were collected. The authors are careful not to mention "suppressive ART" in the text, which is appreciated. However, the title should be changed to also reflect this fact.