Proviral location affects cognate peptide–induced virus production and immune recognition of HIV-1–infected T cell clones

BACKGROUND HIV-1–infected CD4+ T cells contribute to latent reservoir persistence by proliferating while avoiding immune recognition. Integration features of intact proviruses in elite controllers (ECs) and people on long-term therapy suggest that proviruses in specific chromosomal locations can evade immune surveillance. However, direct evidence of this mechanism is missing. METHODS In this case report, we characterized integration sites and full genome sequences of expanded T cell clones in an EC before and after chemoradiation. We identified the cognate peptide of infected clones to investigate cell proliferation and virus production induced by T cell activation, and susceptibility to autologous CD8+ T cells. RESULTS The proviral landscape was dominated by 2 large clones with replication-competent proviruses integrated into zinc finger (ZNF) genes (ZNF470 and ZNF721) in locations previously associated with deeper latency. A third nearly intact provirus, with a stop codon in Pol, was integrated into an intergenic site. Upon stimulation with cognate Gag peptides, infected clones proliferated extensively and produced virus, but the provirus in ZNF721 was 200-fold less inducible. While autologous CD8+ T cells decreased the proliferation of cells carrying the intergenic provirus, they had no effect on cells with the provirus in the ZNF721 gene. CONCLUSIONS We provide direct evidence that upon activation of infected clones by cognate antigen, the lower inducibility of intact proviruses in ZNF genes can result in immune evasion and persistence. FUNDING Office of the NIH Director and National Institute of Dental & Craniofacial Research; NIAID, NIH; Johns Hopkins University Center for AIDS Research.

In people on ART and ECs, reservoir stability is mostly caused by the proliferation of infected cells, which undergo clonal expansion beginning in the earliest stages of HIV-1 infection, driven by encounter with cognate antigens and survival stimuli (IL-2, IL-7, IL-15) (12)(13)(14)(15)(16)(17)(18)(19)(20)(21).In addition, HIV-1 integration into a handful of specific genes causes insertional mutagenesis, providing a survival advantage (22,23).Thus, therapeutic strategies that disrupt the proliferation of infected cells would significantly accelerate reservoir decay, contributing to the success of future curative interventions (24).However, cytoreductive and anti-proliferative compounds tested so far failed to significantly reduce reservoir size; moreover, current knowledge of the impact of anti-proliferative drugs on the heterogenous population of HIV-1-infected clones persisting on ART is limited (25,26).
The proviral landscape is dynamic, shaped by selective forces imposed by HIV-1-host interactions.Growing evidence suggests that HIV-1 integration into chromosomal locations associated with deeper latency are positively selected over time in both EC and PLWH on ART (18,27,28).Such genomic regions include centromeric and peri-centromeric repeats, laminaassociate domains (LADs), and the Zinc Finger (ZNF) gene family.Recent studies suggest that intact proviruses integrated into certain ZNF genes, often recurring across individuals, have a survival advantage due to reduced viral expression caused by the repressive epigenetic environment typical of ZNF gene clusters found in heterochromatic regions (29,30).However, direct evidence for this mechanism, and whether it affects latency reversal upon physiological stimulation, is lacking.ZNF genes and other repressive genomic locations have been linked to a survival advantage and deeper latency of intact proviruses.In a study from Halvas and colleagues, two proviruses located in ZNF721/ABCA11P could be recovered from viral outgrowth and one contributed to persistent nonsuppressible viremia (31).This signature for reservoir selection was first identified in EC, likely due to the strong CTL pressure that selects against proviruses with higher transcriptional activity (18,27).Similarly, more recent studies in people on ART found a progressive enrichment over time of intact proviruses located in these epigenetically repressed regions (28,29).Whether these proviruses are permanently "blocked and locked", and the clinical consequences, remain unclear.
In this study, we characterized HIV-1 persistence in an HLA-B*57 + EC before and after treatment with ART, chemoradiation, and anti-PDL-1 immunotherapy for metastatic lung cancer.
We performed longitudinal integration site analysis and quantified intact and total HIV-1 DNA by digital PCR.We also assessed the clonal dynamics of two intact proviruses integrated into ZNF genes that, by the end of treatment, dominated the reservoir.Finally, by PBMC stimulation experiments, we resolved the T cell specificity at the epitope level of two infected clones, which allowed us to dissect cell proliferation and provirus inducibility upon peptide-MHC class II recognition, and the impact of immune recognition by autologous CD8 + T cells.

Chemoradiation induces a transient contraction of HIV-1 infected cells.
The study participant, here referred to as ES24, is a previously described HLA-B*57 + male elite controller who maintained undetectable HIV-1 RNA without treatment for more than 10 years (15).In 2019, he was diagnosed with metastatic lung cancer for which he initiated ART and was treated with surgery and a cycle of 5 weekly doses of carboplatin and paclitaxel chemotherapy with radiotherapy (chemoradiation therapy or CRT).This was followed by a 12-month course of immunotherapy with 12 cycles of anti-PD-L1 monoclonal antibody treatment (durvalumab, additional details on his clinical history are provided in Figure 1A).Of note, CD4 + T cells reached a nadir on day 67 post CRT (304 cells/μL, 28.8%) and partially recovered by the end of study (643 cells/μL, 30.4%, at day 974).To investigate the impact of treatment on reservoir dynamics, we measured total and intact HIV-1 DNA longitudinally before, during and after CRT (Figure 1A).We observed a rapid but transient decline in both total and intact DNA (2.2-and3.0-fold reduction, respectively) at 1 month post CRT that coincided with CD4 + T cell count nadir (2.3-fold reduction).
However, HIV-1 DNA increased in the months after treatment, with a rebound from nadir that was markedly higher for intact DNA (3-versus 9.4-fold for total and intact DNA, respectively), which reached higher levels than pre-CRT (663 versus 82 copies/10 6 CD4 + T cells).Interestingly, single genome sequencing of proviral DNA and outgrowth virus showed a single dominant variant that did not evolve during the study period (Figure 1B), suggesting that the persistence of reservoir cells was driven by cell survival and proliferation, rather than low-level replication, also reflected by the persistently undetectable viral load in plasma.Thus, to further characterize changes in infected cell composition we performed integration site analysis (Figure 1C), which revealed a transient contraction in clonality during CD4 nadir (Gini index 0.41, 0.03, 0.58 at day -624, 151, and 594, respectively).Of note, some integration sites were detected at multiple timepoints, including a provirus found in the ZNF721 gene, which underwent significant expansion from day 78 to day 594 (p<0.0001).Finally, we observed a similar clonal contraction in all CD4 + T cells, measured by TCR sequencing, which preferentially affected the most abundant clones (Figure 1D), as previously described (32).Overall, these result show that although CRT perturbed the frequency and composition of infected cells, reservoir size rebounded upon CD4 + T cell recovery, as recently reported in the context of immunosuppressive therapy post-kidney transplant (33).
Clonal replacement of two intact proviruses integrated into ZNF genes.The sequencing and integration site data suggested that the increase in reservoir size was due to relatively few clones undergoing proliferation, so we sought to determine the genomic integration sites and full genome sequences of infectious proviruses (Figure 2A).We designed primers in the human genome, paired with primers in HIV-1 gag, to screen candidate integration sites from day 529, and identified two proviruses integrated into the ZNF721/ABCA11P and ZNF470 genes, termed ZNF721i and ZNF470i going forward, that were genetically intact and matched the nef region sequenced from previous time points.ZNF470i was identical to the near full-length sequences of viral outgrowth variants recovered from day -624, while ZNF721i differed only by 2 nucleotides (Figure 2B).These two ZNF genes are located in chromosomes 4 and 19, respectively, both within ZNF gene clusters where intact proviruses have been previously reported from both EC and people on long-term ART (Figure 2C and Supplemental Table S1) (18,29,(34)(35)(36).To investigate the temporal dynamics of these two intact proviruses we quantified their frequencies by integration site-specific digital PCR, and their relative abundance among all infected cells, as recently described (37,38) (see methods and Figure 2D and E).Total LTR copies and ZNF470i decreased upon CRT initiation and returned to baseline levels; in contrast, ZNF721i underwent significant proliferation and reached a frequency of 741±48 copies/10 6 CD4 + T cells, corresponding to a 69-fold increase from its nadir on day -6 (11±5 copies/10 6 cells) (p= 0.0001, Figures 2E and F).At day 1000 post CRT initiation, ZNF721i and ZNF470i together represented more than 80% of all LTR copies, with estimated total-body clone sizes of 120 and 52 million cells, respectively.Together, our results show that these two intact proviruses found in ZNF genes persisted despite profound perturbation of the HIV-1 reservoir and, in the case of ZNF721i, underwent extensive clonal selection.
Infected clones are common among cells reactive to HIV-1 antigens.To investigate the role of antigen-driven selection of infected cells in ES24, we stimulated CD8-depleted PBMCs, in the presence of antiretrovirals, with CMV and HIV-1 antigens (Figure 3), which have been involved in the persistence of HIV-1-infected clones (39)(40)(41)(42)(43). Stimulation of PBMCs with a Gag peptide pool led to a significant increase in LTR DNA copies at 9-days (23.6-fold increase, p=0.01, Figure 3A-C).To determine which proviruses expanded in culture and produced virus, we sequenced the 5'-leader-gag region from virion-associated RNA (Figure 3D).We found that 15% of sequences from cells stimulated with Gag peptides matched ZNF721i, while the remaining identified a new variant, markedly different from ZNF721i and ZNF470i, that we did not identify before from proviral DNA or QVOA.This new provirus is nearly intact but shows a premature stop codon in Pol (S311→stop).In addition, it is characterized by a 11-nucleotide deletion within the primer binding site (PBS) hairpin, located in the spacer region between the primer activation sequence and the tRNA annealing region (44) (Figure 3D and E).The provirus (named Chr7.d11sc because of its features) is located in chromosome 7p12.1, in an intergenic region distinct from those previously associated with deeper latency (30).To investigate the presence of specific histone modifications, we interrogated publicly available CHIP-seq data from CD4 + T cells (45); while ZNF721i and ZNF470i were surrounded by epigenetic signatures typical of heterochromatin and ZNF genes (H3K9me3 and H3K36me3), Chr7.d11sc was distant from activating epigenetic marks and showed little enrichment for heterochromatin (Supplemental Figure S1).
To confirm whether the cells carrying this Chr7.d11screspond to Gag peptide stimulation, we exploited its unique 11-nucleotide deletion to design an assay based on competition probes that would distinguish it from other variants in both genomic DNA and virion-associated RNA (Figure 3F).While ZNF470i did not proliferate in response to antigenic stimulation, we found that exposure to Gag peptides led to virus production and clonal expansion of both ZNF721i and Chr7.d11sc (32-and >6-fold increase, respectively) (Figure 3G and H).When we compared proviral copies and virus released at the end of culture, we noted that ZNF721i produced less virus despite the extensive proliferation (~10 3 copies/mL, ~10 4 copies/10 6 cells, respectively).
To further characterize these three proviruses of interest, we measured their frequency by integration site specific PCR and performed the qVOA from CD4 + T cells obtained at day 1400 post CRT (Supplemental Figure S2).Out of 5 wells with viral outgrowth (corresponding to 1.44 IUPM), 3 had virus matching ZNF470i and 2 wells had virus matching ZNF721i, demonstrating that these two proviruses are infectious and inducible despite being located within ZNF genes.
However, based on the high number of ZNF470i and ZNF721i proviruses screened in the qVOA (961 and 1490), we estimated that less than 0.3% (0.9-0.08%, 95% CI) of proviruses were induced and resulted in outgrowth in vitro (Supplemental Figure S2).In addition, we quantified cellassociated HIV-1 RNA isolated from total CD4 + T cells divided in small aliquots so that they would contain only 1 HIV-1 RNA producing cells, as previously described (46).We detected low-level U5-PBS RNA (average of 11 copies/10 6 cells) and recovered a single U5-gag unspliced RNA sequence, which matched ZNF470i.Taken together, these results confirm the low spontaneous transcriptional activity of ZNF721i and ZNF470i in circulating cells, in vivo (Supplemental Figure S2).
Finally, we repeated the CD8-depleted PBMC stimulation with individual Gag peptides that induced responses by ELISpot and observed increased LTR copies in response to six out of nine peptides (Supplemental Figure S3).Although these proviruses were defective by IPDA (data not shown), our results suggested that HIV-1-reactive CD4 + T cells carrying proviruses are common in this study participant.

Differential inducibility of two proviruses in cells reactive to specific Gag epitopes.
To extend our results, we sought to identify the Gag specificity of the clones carrying Chr7.d11sc and ZNF721i at the epitope level, so that we could investigate proviral inducibility upon encounter with their cognate antigen (Figure 4).As a read out for T cell response to pooled or individual Gag peptides, we used both virus production in the supernatant and infected-cell proliferation (Figure 4A-E, see methods).With this approach we found that the clone harboring Chr7.d11sc is reactive to two overlapping peptides, EKAFSPEVIPMFSAL (41, Gag 162-176) and SPEVIPMFSALSEGA (42, Gag 166-180), while cells harboring ZNF721i respond to STLQEQIGWMTNNPP (61, Gag 241-255).Conversely, ZNF470i did not proliferate or produce virus upon Gag pool stimulation (Supplemental Figure S4).Since the Chr7d11sc provirus is rare and can be detected only in cells after stimulation with peptides 41 and 42, the precise fold increase cannot be quantified; however, we can estimate a minimum of 15-to 20-fold increase using the limit of detection in untreated cells (4 copies/10 6 cells).In subsequent experiments we isolated CD4 + T cells at the end of culture, which confirmed that in the no treatment condition, the frequency of Chr7.d11sc is 2.4±3.3 copies/10 6 CD4 + T cells.Upon stimulation with peptide 61, the frequency of ZNF721i showed a 73-fold increase relative to no treatment.The stimulation with individual cognate peptides is reproducible, as shown by comparable virus production and cell expansion in triplicate experiments (Figure 4D and E, Supplemental Figure S5).In addition, we confirmed that peptides 41, 42, and 61 are recognized by CD4 + T cells, based on the increase in cells positive for intracellular TNFɑ and IFNγ after 9 days of culture (Figure 4F).Interestingly, 41 and 61 are also recognized by CD8 + T cells (Supplemental Figure S6); indeed, they overlap with well characterized epitopes binding to HLA-B*57 (KAFSPEVIPMF and TSTLQEQIGW, respectively) (47).
We hypothesized that the striking expansion of ZNF721i in vivo and its extensive proliferation ex vivo upon antigen-recognition, are favored by its chromosomal location, allowing proliferation to occur without the cytopathic effects of viral replication.To test this hypothesis, we estimated the proviral inducibility in vitro of ZNF721i and Chr7.d11sc by dividing the copies of viral RNA in the supernatant by the provirus frequency in cells at the end of culture (Figure 4G).
We found that the inducibility in vitro of ZNF721i was almost 200-fold lower (average 57 versus 0.3, p=0.021).Lastly, we used bulk TCRβ sequencing, VDJ-specific dPCR, and combinatorial statistics to identify CASSLTGGGEQFF as the putative CDR3β sequence of the clonotype carrying ZNF721i (Figure 4H and I).CASSLTGGGEQFF had a relative abundance of 0.01% in untreated cells but was among the most abundant clonotypes upon stimulation with peptide 61 (1.8%, test for differential abundance p<10 -5 ).Based on provirus and VDJ specific quantification in PBMCs stimulated with peptide 61 and the unstimulated control, we estimated that almost 100% of the clonotype carries ZNF721i, suggesting that most of its clonal selection occurred post HIV-1 integration (Figure 4I and Supplemental Figure S7) (20).

Proliferation of cells harboring ZNF721i is not affected by autologous CD8 + T cells. Clonal
expansion of infected clones is the result of opposing forces, such as the frequency of cell stimulation, proliferation rate, and cell death due to viral cytopathic effects.However, immune recognition by cytotoxic cells imposes a major selective force in shaping reservoir dynamics and composition, especially in ECs, resulting in the observed differential genomic distribution of intact proviruses (18,27).Thus, we sought to determine whether lower proviral inducibility allows infected clones to escape immune recognition.Analysis of Gag and Nef amino acid sequences revealed that Chr7.d11sc had more escape mutations in known B*57-restricted epitopes than ZNF470i and ZNF721i (Figure 5A and Supplemental Figure S8 and S9), suggesting a lower susceptibility to CD8-mediated killing for cells carrying Chr7.d11sc.PBMCs were stimulated for seven days with overlapping peptides covering the entire Nef protein and with selected Gag peptides that showed response by elispot (see methods).Of note, we excluded peptides 41, 42, and 61 to avoid stimulation of infected cells of interest (Figure 5B).In parallel, CD8-depleted PBMCs were stimulated with either peptide 42 or 61 for 24 hours; peptides were then removed, and cells were plated with stimulated CD8 + T cells at a 1:3 effector-to-target ratio and cultured for 10 days.Virus production, monitored from day 3 to day 10, was markedly reduced by CD8 + T cells; however, Chr7.d11sc was significantly more impacted than ZNF721i (1467-vs 3.7-fold reduction in HIV-1 RNA copies/mL at day 10, p=0.02, Figure 5C-E).Although CD8 + T cells had a lesser effect on cell proliferation, likely reflecting that most infected cells divide without the expression of viral antigens (13,48), cells carrying Chr7.d11sc proliferated significantly less than the ZNF721i clone (Figure 5F, G and I).Of note, cells harboring the ZNF470i provirus were not affected by CD8 + T cells (Figure 5H), indicating that in this context CTLs specifically recognized and killed only peptide-stimulated infected cells, rather than via non-specific effector molecule production and non-cytolytic effects (49)(50)(51).In the presence of activated CD8 + T cells, cells harboring Chr7.d11sc decreased to about 40% of the cells in the control arm, while the ZNF721i provirus was barely affected (95% of cells without CD8s, p=0.03; Figure 5I).Coculture with CD8 + T cells had a strikingly distinct effect on virus production and cell proliferation, suggesting that only virus producing cells were eliminated.Moreover, the amount of virus produced on a per cell level may affect the efficiency of immune recognition and the HIV RNA levels at the end of culture.Indeed, in our experiments we observed that the estimated inducibility of Chr7.d11sc was significantly reduced by CD8 + T cells (average RNA/DNA 169 versus 0.24, p=0.002, Figure 5J), while ZNF721i was only modestly affected (0.27 versus 0.08, p=ns).Together, these results demonstrate that the lower inducibility of the ZNF721i provirus allowed escape from CTL recognition by proliferating upon T cell activation with negligible expression of viral antigens, which can explain its marked clonal expansion observed in vivo after CRT and immunotherapy.

Discussion
The activation of latently infected CD4 + T cells with potent mitogens results in viral transcription and the reversal of latency in many clones (52).However, this process is stochastic and there is mounting evidence that following activation, some infected T cell clones can proliferate without producing virus (13,14,48).This process has major implications for the persistence of infected CD4 + T cell clones in the presence of HIV-1-specific CD8 + T cells (53)(54)(55).
In this case report we characterized clonal dynamics before, during, and after chemoradiation and immunotherapy in an EC with metastatic lung cancer.The contraction of individual clones is remarkable as chemotherapy and immunotherapy generally did not affect the size of the latent reservoir in prior studies (26,56,57).The same pattern of a modest but transient reduction in intact proviral DNA was recently described in kidney transplant patients receiving induction immunosuppression (33).Our results extend these findings by tracking individual clones with integration site analysis.Three clones in this study are especially interesting.The large clone which contains replication-competent ZNF470i provirus was stable during treatment.In contrast the cells carrying the ZNF721i provirus, which recognizes the Gag peptide STLQEQIGWMTNNPP (241-255), underwent a 70-fold expansion after chemotherapy was completed.This may explain why there was only a modest decline in intact proviral DNA despite the marked reduction in the number of total clones after chemoradiation.The third clone, Chr7.d11sc, contains a nearly intact provirus and recognizes the overlapping Gag peptides EKAFSPEVIPMFSAL (162-176) and SPEVIPMFSALSEGA (166-180).To the best of our knowledge, this is the first study to identify the exact epitope recognized by infected T cell clones.This is important as epitope identification could enable the specific activation of latently infected T cells as part of HIV-1 cure strategies.
While the peptides triggered both viral transcription and proliferation of both the ZNF721i and Chr7.d11sc clones, the degree of viral transcription was significantly lower for the ZNF721i provirus.This difference in viral transcription between the two clones was associated with differences in the ability of autologous CD8+ T cells to inhibit clonal expansion.While the presence of CD8+ T cells resulted in a 60% inhibition of proliferation of cells with the Chr7.d11scprovirus, CD8+ T cells had no effect on the proliferation of the clone carrying ZNF721i.This difference is particularly interesting given the fact that the Chr7.d11scprovirus has accumulated several escape mutations while the ZNF721i provirus has no escape mutations in Gag and Nef epitopes.The presence of escape mutations in Chr7.d11sc suggests that some of the reservoir cells in ES24 have been subjected to CD8 + T cell selective pressure in vivo.While these mutations are rare in proviruses from EC, they have been described and virologic control may be maintained by de novo CD8+ T cell responses that can inhibit replication of autologous escape mutants (58)(59)(60)(61).
The 60% reduction of the Chr7.11scprovirus may underestimate of the ability of CD8 + T cells to inhibit the expansion of virus producing cells given the stochastic nature of latency reversal.In a prior study that subjected CD4 + T cells to multiple rounds of potent activation, a mean of just 60% of the infected cells that eventually produced virus did so after the first round of activation (36).Thus, it is likely that only a subset of cells of the Chr7.d11scprovirus produced virus in response to 24 hours of stimulation with cognate peptide.The 3-log reduction in Chr7.d11scRNA in the presence of CD8 + T cells suggests that most of the cells that produced virus were eliminated, and the remaining cells were not targeted because they did not express viral proteins.The ZNF721i provirus is significantly less inducible than the Chr7.d11scprovirus which may contribute to immune evasion and explain the rapid expansion of cells with the ZNF721i provirus in contrast to the Chr7.d11scprovirus which is present at a much lower frequency in vivo.
Our study is limited by the fact that it involves a single patient who is an elite controller.Even though this case may not be representative of most people without spontaneous HIV-1 control, a progressive enrichment of intact proviruses in ZNF genes has been observed in people living with HIV-1 on long-term ART.It is also possible that the process of clonal expansion was affected by his malignancy.However, we describe the effects of chemoradiation and immunotherapy on individual clones and study the effects of T cell activation on infected clones using a physiologic system with individual cognate peptides.In addition, our findings can be extended to larger cohorts of ECs, whose HIV-1-reactive CD4 + clones can be easily detected (62).The highly dominant ZNF721i provirus in Gag-reactive CD4 + clones, found in this participant, has not been described before.However, the presence of HIV-1-reactive clones, albeit rarer among all infected cells, is generalizable to both EC and normal progressors on long-term ART (20,40,63).Our findings suggest that the selective recognition and elimination of transcriptionally active proviruses like Chr7.d11sc by the immune system could explain the predominance of clonally expanded proviruses integrated into transcriptionally repressed sites in HIV-1 controllers and people on long-term ART (18).The experimental evidence provided here further supports recent reports of genotypic and phenotypic signatures of intact proviruses, results of a dynamic proviral landscape shaped over time by the immune system (64).
However, additional work is needed to understand whether specific host and proviral factors contribute to the establishment and maintenance the so called "deeper latency" of some intact proviruses in ZNF genes.Whether rare proviruses are merely caught in between the evolutionary arms race against transposable elements, or specifically targeted by repressive epigenetic mediators is yet to be determined (65,66).Moreover, our data also suggest that strategies that use agents like IL-15 which induce proliferation to augment effector responses may paradoxically increase the size of the reservoir by causing expansion in the absence of virus expression of proviruses like ZNF721i that can partially evade the immune system (67).Finally, it should be noted that viral particle production does occur even from proviruses like ZNF721i, and thus immune recognition and elimination of productively infected cells would still be essential for elite control and for preventing viral rebound in most people on ART.

Methods
Human Subjects.The study participant, a 66-year-old Afro-American male, has been anonymized as ES24.He has been diagnosed HIV-1-positive in 2009.Seven total longitudinal time points have been collected for this study.The participant's PBMCs were obtained from whole blood by Ficoll-based density separation, and CD4 + T cells were isolated by magnetic bead-based negative selection (Miltenyi) Activated lymphoblasts and irradiated-PBMCs from HIV-1-negative individuals were used in the QVOA assay.
Intact Proviral DNA Assay (IPDA).The IPDA was performed as previously described (68) on CD4 + T cells, using the QX200 Droplet Digital PCR System (Bio-Rad).The assay interrogates two specific regions of the HIV-1 genome to genetically discriminate between defective and intact proviral DNA.

Quantitative Viral Outgrowth Assay (QVOA).
The QVOA was performed as previously described (58, 69) from isolated CD4 + T cells.Following identification of p24+ wells, supernatants were collected and extracted to sequence the virion-associated HIV-1 RNA in either the nef or U5-gag region.(15).Integration site analysis.Integration site analysis was performed with two different approaches.Linker-mediated PCR was performed on genomic DNA from bulk CD4+ T cells following controlled sonication, as previously described (70).PCR products obtained were purified, pooled, and sequenced on an Illumina MiSeq.Integration sites were determined from the sequence data using the INSPIIRED pipeline (https://github.com/BushmanLab/).Conversely, the Lenti-X Integration Site Analysis Kit (Clontech) was used to characterize the integration sites from limiting diluted gDNA subjected to whole genome amplification (WGA), using the Advance Single Cell Repli-G Kit (Qiagen), as previously described (35).PCR products obtained with this approach were sequenced by Sanger Sequencing (Azenta).
Provirus sequencing.Genomic DNA (gDNA) was isolated accordingly to the number of cells.
For samples lower than one million cells, DNA was extracted following Wiegand et al. (46), otherwise the QIAamp DNA Mini Kit (QIAGEN) was used.Following limiting dilution, proviral sequences of ZNF470i and ZNF721i were recovered by FLIPS (71) and MIPseq (35), respectively.Due to the limited presence of Chr7.d11sc, its viral sequence was recovered from virion-associated HIV-1 RNA in the supernatant, obtained from CD8-depleted PBMC stimulation experiment (see below).If primers commonly used for full-length proviral sequencing failed due to mismatches, specific primers were designed.Finally, to reconstruct the full proviral sequence, primers annealing in the human genome flanking the integration site were used to recover the LTR regions.All the primers used in this study are detailed in Supplemental Table S2.
Total LTR copies.To quantify all LTR copies we used primers and probe targeting the R-U5 junction, as previously described (38,72).In brief, gDNA from total CD4 + T cells undergoes controlled physical shearing so that the LTRs from the same provirus are not in linkage and are captured in separate partitions.The primers and probe anneal to the R-U5 junction, so that both LTRs are quantified regardless of 5' or 3' end.Digital PCR reactions were run using the Qiacuity One Digital PCR System (Qiagen) with an initial denaturation step of 95 °C for 2', followed by 45 cycles each including 95 °C for 15'' and 58°C for 30''.This assay can be run in a multiplex reaction with the integration site PCR described below.Oligo sequences are described in Supplemental Table S2.

Quantification of proviruses of interest by digital PCR. Specific assays to quantify ZNF470i
and ZNF721i were designed on the host-U3 junction, with the fluorescently labelled probe annealing across the site of HIV-integration, as previously described (20).In addition, exploiting the 11-nt deletion present in the primer binding site of Chr7.d11sc (HXB2 position 660-670), we designed an assay based on competition probes to discriminate the Chr7.d11scvirus from other variants.To confirm that the virus produced upon stimulation with peptide 61 was indeed from ZNF721i provirus, we performed SGS from the supernatant (Supplemental Figure S5).Copies of targets of interests were normalized based on cell genome equivalents screened, calculated by RPP30, as previously described (68).Primers and probes are detailed in Supplemental Table S2.
Digital PCR reactions were run using the Qiacuity One Digital PCR System (Qiagen) with an initial denaturation step of 95 °C for 2', followed by 45 cycles each including 95 °C for 15'' and 58°C for 30''.
Clonal expansion assay.PBMCs were obtained from blood via a standard Ficoll procedure.The cells were subjected to two rounds of CD8 + T cell depletion using negative selection beads (Miltenyi).CD8-depleted PBMCs were then cultured in RPMI (Gibco) with 10% fetal calf serum (Gibco) and 10 units/ml of IL2 at a concentration of 1 million cells per ml.The antiretroviral drugs Viral pellets underwent RNA extraction, as previously descried (73).RNA was immediately used for reverse transcription using Super Script III with a participant-specific primer located in gag.
cDNA synthesis reaction was at 55°C for 50', followed by an inactivation step at 85 °C for 5'.cDNA was quantified by a dPCR assay targeting the U5-PBS region, or used for single genome sequencing, as described above.To quantify and sequence cell-associated RNA from peripheral blood CD4 + T cells, we isolated total RNA and gDNA from small aliquots of cells (700,000) as previously described (46).HIV-1 RNA U5-PBS RNA were quantified as described above, and normalized to million cell equivalents by RPP30 copies from gDNA.
TCR sequencing and quantification of VDJ rearrangements of interest.Genomic DNA from cultured cells was subjected to TCRβ sequencing, as previously described (74).To measure CASSLTGGGEQFF, we designed a digital PCR assay with primers flanking the VDJ rearrangement of the TCR beta chain, as previously described (20).To increase specificity and avoid a polymeric region of guanosines, we designed two different probes annealing across the VD and DJ junctions, fluorescently labelled with FAM and HEX, respectively.Only double positive partitions were used to quantify the clonotype of interest (Supplemental Figure S4 and Supplemental Table S2).To confirm that the CASSLTGGGEQFF CDR3 belonged to the cells carrying ZNF721i, we distributed 800 CD8-depleted PBMCs over 96-well plates.After whole genome amplification, we identified wells positive for any provirus, ZNF721i, and CASSLTGGGEQFF by digital PCR.We used combinatorial statistics to calculate the probability that ZNF721i and CASSLTGGGEQFF would co-localize in the same wells by chance, as previously described (20) (see Supplemental Figure S4).

CD8 + T cell Assay.
To induce potent cytotoxic function, CD8 + T cells were first stimulated with peptides for 7 days (75,76).On day -7, PBMCs were stimulated with overlapping Nef peptides covering the entire protein and 10 Gag peptides he had previously responded to including Gag (PEPTAPPEESFRFGE), Gag 483-497 (KELYPLASLRSLFGN) in R10 media with 10 units/mL of IL2, efavirenz, and raltegravir.Gag peptides 41, 42, and 61 were all excluded so as not to activate and expand T cells specific for these peptides which could kill target cells that had residual peptides present on MHC molecules after activation.On day -1, fresh PBMCs were subjected to two rounds of CD8+ T cell depletion with Miltenyi micro beads.The cells were then stimulated for 24 hours with peptide 41 and 61 at a concentration of 10 ug/ml in R10 media with 10 units/mL of IL2 with efavirenz and raltegravir.On day 0, the CD8 depleted PBMCs were washed to remove peptide and plated at 3 million cells per well in a 24 well plate with 3 replicates per condition.CD8 + T cells were isolated from the PBMCs that had been stimulated with peptides at day -7 using Miltenyi micro beads.A million CD8 + T cells were added to 3 million stimulated CD8-depleted PBMCs.Half of the media was removed and fresh R10 with 10 units/ml of IL2 was added on days 3 and 7, and efavirenz and raltegravir was added on day 7. On day 10, CD4 + T cells were isolated by negative selection (Stem Cell) in 96 well plates.Genomic DNA was extracted from about 0.5 million CD4 + T cells and used for downstream dPCR assays.
Quantification and statistical analysis.Descriptive statistics, tests for normality, 2-tailed Student's t-test and one-way ANOVA tests were used to determine statistical significance using GraphPad Prism v8.0.Chi square and Fisher exact test were calculated in Excel.Combinatorial statistics for provirus and TCR co-occurrence were calculated as previously described (20,78).
A P value less than 0.05 was considered significant, unless otherwise stated.
Data and code availability.Single genome sequences and full-length proviral genome sequences retrieved for this work have been deposited to GenBank (accession numbers OR496614-OR496825 and pending), and OR496826-OR496828) and the NCI provirus database.
raltegravir and efavirenz were obtained from NIH HIV Reagent Program.Four million cells were cultured in each well of a 12-well plate and stimulated with overlapping Gag peptides from NIH AIDS Reagent Program at a concentration of 10 ug/ml.To identify the individual Gag peptides that induced clonal expansion, CD8-depleted PBMCs were stimulated with 11 different pools containing 10-13 peptides each.Each individual peptide from the 2 pools that resulted in virus production was then tested individually at a concentration of 10 µg/ml.RNA extraction and cDNA synthesis.Culture supernatants were spun at 3500g for 15' at 4 °C and transferred to clean tubes for an additional step of centrifugation at 21000g for 2h at 4 °C.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Chemoradiation and immunotherapy transiently affect the HIV-1 reservoir cell frequency and composition.(A) Clinical history of study participant ES24 before, during, and after treatment of metastatic lung cancer; values below the limit of detection are indicated with open symbols; intact and total proviruses in total CD4 + T cells were quantified by IPDA (orange and black, respectively); plasma HIV-1 RNA and CD4+ T cell counts are represented in red and aqua, respectively; TAF tenofovir alafenamide, FTC emtricitabine, BIC bictegravir, XRT radiotherapy.(B) Maximum likelihood tree of nef sequences recovered over time from viral outgrowth assays or limiting dilution PCR from CD4 + T cell -derived genomic DNA; identical sequences are indicated by the black box; HXB2 was used as outgroup.(C) Longitudinal integration site analysis shows contraction and reconstitution of proviruses in expanded clones; total integration sites recovered in each sample are indicated below stacked bars; integrations in ZNF genes relevant in subsequent analyses are highlighted in red.(D) Chemoradiation perturbs CD4 + T cell repertoire, especially causing loss of most expanded clones; rank-abundance curves indicate distribution of clonality among the top 250 TCRβ sequences; pie charts indicate distribution of clonotypes based of relative abundance; significance of differences between the two time points was assessed by chi-square test.

Figure 4 .Figure 5 .
Figure 4. PBMC stimulation with HIV-1 Gag peptides induces proliferation of infected cells and virus production.(A) CD8-depleted PBMCs from day 1111 post CRT were stimulated with 11 mini-pools, each containing 12 Gag peptides; No Tx indicates no treatment negative control; black dots indicate technical replicates of day-9 HIV-1 RNA measurement in supernatant.(B) Virus production at the end of culture of CD8-depleted PBMCs from day 1142 post CRT with individual epitopes from mini-pools showing virus production in (A).(C) Frequency of proviruses of interest by day-9 of culture; fold increase compared to no treatment is indicated above each bar; peptides with increased virus production and cell prolideration are indicated below the graph.(D and E) Validation experiments with individual peptides in triplicate.(F) Intracellular cytokine staining of CD4 + T cells re-stimulated with Gag peptides of interest after PBMC expansion; numbers within gates indicate the percentage of cells positive for both TNFa and IFNγ (G) Differential inducibility between proviruses of interest upon stimulation with cognate Gag peptides; inducibility is expressed as the ratio between HIV-1 RNA copies in the supernatant and proviral copies in cells at the end of culture.(H) Analysis of differential abundance (%) of the top 1000 productive TCRs between no treatment and stimulation with peptide 61; the differential abundance of the clonotype carrying the ZNF721i provirus (CASSLTGGGEQFF) was tested by chi-square test.(I) Frequency of both provirus and VDJ rearrangement belonging to the CASSLTGGGEQFF clonotype in CD4 + T cells at the end of culture.