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Research ArticleAIDS/HIVVirology
Open Access | 
10.1172/JCI159569
1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
Find articles by Joy, J. in: JCI | PubMed | Google Scholar
1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
Find articles by Mullins, J. in: JCI | PubMed | Google Scholar
1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
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1Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA.
2Department of Microbiology, University of Washington, Seattle, Washington, USA.
3Center for Infectious Disease Research, Seattle, Washington, USA.
4Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
5Department of Medicine,
6Department of Global Health,
7Department of Pediatrics, and
8Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA.
Address correspondence to: Lisa M. Frenkel, 307 Westlake Ave N, Seattle, Washington 98109, USA. Phone: 206.987.5140; Email: lfrenkel@uw.edu.
Authorship note: JJ and AG are co–first authors.
Find articles by Frenkel, L. in: JCI | PubMed | Google Scholar
Authorship note: JJ and AG are co–first authors.
Published June 4, 2024 - More info
Published in Volume 134, Issue 14 on July 15, 2024
AbstractDespite effective antiretroviral therapy (ART), persons living with HIV harbor reservoirs of persistently infected CD4+ cells, which constitute a barrier to cure. Initiation of ART during acute infection reduces the size of the HIV reservoir, and we hypothesized that in addition, it would favor integration of proviruses in HIV-specific CD4+ T cells, while initiation of ART during chronic HIV infection would favor relatively more proviruses in herpesvirus-specific cells. We further hypothesized that proviruses in acute ART initiators would be integrated into antiviral genes, whereas integration sites (ISs) in chronic ART initiators would favor genes associated with cell proliferation and exhaustion. We found that the HIV DNA distribution across HIV-specific versus herpesvirus-specific CD4+ T cells was as hypothesized. HIV ISs in acute ART initiators were significantly enriched in gene sets controlling lipid metabolism and HIF-1α–mediated hypoxia, both metabolic pathways active in early HIV infection. Persistence of these infected cells during prolonged ART suggests a survival advantage. ISs in chronic ART initiators were enriched in a gene set controlling EZH2 histone methylation, and methylation has been associated with diminished long terminal repeat transcription. These differences that we found in antigen specificities and IS distributions within HIV-infected cells might be leveraged in designing cure strategies tailored to the timing of ART initiation.
IntroductionAntiretroviral therapy (ART) can effectively suppress HIV replication, but maintenance of virologic suppression requires that persons living with HIV adhere to the prescribed medicines over their lifetime, as current ART does not cure the infection. Initiation of ART during acute HIV infection minimizes the size of an individual’s HIV reservoir (1–5), defined variably as replication-competent (transcriptionally active or latent) proviruses or by HIV DNA that persists during ART. The HIV reservoir slowly decreases in size with time, but it persists for many years and nearly always leads to viral rebound following ART cessation (6–8). Cells that contain the HIV reservoir often undergo clonal proliferation, purportedly from homeostatic proliferation, antigen stimulation, and/or from HIV integration site–promoted (IS-promoted) proliferation/survival (reviewed in refs. 9–11).
HIV-specific CD4+ T cells, upon encountering cognate antigens during acute HIV infection, are activated, preferentially infected, and undergo several rounds of proliferation (12). During untreated chronic HIV infection, CD4+ T cells specific for human herpesviruses, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses types 1 and 2 (HSV1 and -2), as well as for HIV and other infections, become activated and serve as targets for HIV infection (13). Because antigen-driven clonal proliferation appears to be a major contributor to HIV persistence (14), targeting the antigen specificities of HIV-infected CD4+ T cells has been proposed to reverse proviral latency as part of a “shock-and-kill” strategy (15).
Evidence suggests that HIV integration into certain cancer-related genes, such as STAT5B and BACH2, promotes clonal expansion of infected cells (11, 16, 17). IS-mediated clonal expansion can be driven by the HIV 5′ long terminal repeat (LTR) promoting expression of the gene in which the provirus has integrated (18, 19). Furthermore, transcriptome analysis of HIV-infected single cells revealed that HIV can drive high aberrant host gene transcription downstream of the IS, which can in turn induce aberrant splicing, intron retention, and cryptic exon activation at the IS (20). Thus, beyond insertional activation, the IS can drive aberrant host gene expression, which may promote IS-mediated clonal expansion.
We hypothesized that individuals who initiate ART in acute infection have ISs concentrated in genes involved in antiviral pathways and individuals who initiate ART in chronic infection have ISs distributed across many different genes, especially those involved in proliferation and T cell exhaustion. Our hypotheses were based on the reasoning that the immediate immune response to HIV infection will activate HIV-specific CD4+ T cells to initiate an antiviral response and HIV will infect and integrate into these genes, and during untreated chronic HIV infection, herpesviruses will recur and HIV-specific CD4+ T cells will express high levels of exhaustion markers, such as Tim3 and PD-1, compared with healthy controls (21, 22), and HIV will infect and integrate into expressed genes.
We reasoned that both antigen specificity and functional properties of genes harboring an IS could be exploited in HIV cure strategies. To provide insights into this possibility, we compared HIV DNA load in HIV-specific and herpesvirus-specific CD4+ T cells and gene sets with ISs between ART-suppressed individuals who initiated ART during acute or chronic HIV infection.
ResultsMale participants in the Seattle Primary Infection Cohort (23–25) who initiated ART within 6 weeks (n = 7) (ART-acute-HIV) or after more than 6 months from the estimated date of HIV infection (n = 4) (ART-chronic-HIV) were studied (Table 1). The HIV DNA concentrations and ISs were compared by antigen specificities of participants’ infected CD4+ T cells from blood specimens collected after a median 10.1 years (range 2.4–17.1) of ART suppression. Initially, we measured HIV DNA in antigen-specific cells after 24 hours of peptide antigen stimulation. However, after sorting, the number of CD137+ cells was limited, and the HIV DNA measurements were near or below the limit of detection. Therefore, virus-specific CD4+ cells were expanded by stimulation of CD8+ T cell–depleted PBMCs with peptide antigen pools in media supplemented with IL-7, raltegravir, and efavirenz (day 0), IL-2 (day 3), and restimulated on day 10. Twenty-four hours after restimulation, cells were sorted based on CD137 surface expression (Figure 1, A and B, and Supplemental Tables 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/JCI159569DS1). Only one activation marker (CD137) was utilized, as the measurements were performed prior to the reporting of the activation-induced marker (AIM) assay using 2 markers (26). Serologic and cellular responses to HIV and herpesviruses were assessed for each participant (Table 2), with peptide antigen pools screened for IFN-γ, TNF-α, and IL-2 reactivity (see Methods and Supplemental Figure 1). The total number of live cells after restimulation was largely unchanged from the starting input (Figure 1C), likely due to the small number of cells activated to proliferate by each antigen. The expansion of CD8–, antigen-specific CD3+CD137+ cells varied somewhat by participant and viral antigens tested (Figure 1D and Supplemental Table 3; statistical analyses were not performed due to insufficient power from sparse data, as some participants were not uniformly reactive to herpesviruses).
Figure 1Peptide antigen stimulation and CD3+CD8–CD137+ cell expansion in vitro. (A) Experimental schema. CD8+ T cell–depleted PBMCs were incubated with anti-CD3/anti-CD28 Dynabeads (positive control), media alone (negative control), or peptide antigens derived from HIV, CMV, EBV, HSV1, or HSV2 (see Table 2) for 10 days, all with efavirenz, raltegravir, and IL-7 (ART). On day 3, IL-2 was added to the cultures. On days 5, 7, and 10, the media, ART, and growth factors were replenished. On day 10, cells were restimulated with peptide antigens to upregulate activation markers for cell sorting. Following 24 hours of restimulation, cells were harvested for intracellular cytokine staining and flow cytometry to assess reactivity to each antigen, i.e., “antigen discovery.” Cells were stained for CD3, CD8, and CD137 for cell sorting. CD8+ T cell–depleted PBMCs were plated on day 0, cultured as shown in A, and enumerated on day 11. (B) Representative flow cytometry plots for cell sorting based on CD3+CD8–CD137+ cell surface expression. (C) Fold change of total cells on day 11 versus day 0 and (D) fold change of CD3+CD8–CD137+ cells on day 11 versus day 0 are shown for individuals who initiated ART-chronic-HIV in left panels and for individuals who initiated ART-chronic-HIV in right panels. Horizontal lines indicate median of data points.
Table 1Demographics and clinical parameters of study participants
Table 2Participants’ serologic status and reactivity of PBMCs to peptide antigen pools at time of acute HIV infection
HIV DNA in antigen-specific CD4+ T cells following the 11-day antigen stimulation was measured using the viral open reading frame detection assay (VODA) (Figure 2A). HIV DNA appeared disproportionately higher in HIV-specific compared with herpesvirus-specific CD4+ T cells for participants in the ART-acute-HIV group, except in participant 97054, who had equal levels of HIV DNA in HIV-specific and CMV-specific T cells. Of note, this was the only participant in the ART-acute-HIV group who had plasma HIV RNA detected during ART (50–120 copies/mL were detected on 4 of 5 determinations in 2012–2014) (Supplemental Figure 2). In the ART-chronic-HIV group, HIV DNA appeared to be relatively evenly distributed across HIV-specific and herpesvirus-specific cells in 3 of 4 participants (Figure 2B), with participant 49021 having more HIV DNA in HIV-specific compared with his herpesvirus-specific cells (Figure 2B). This participant’s CD4+ cell count remained in the normal range during the 18 months he was untreated prior to ART initiation (Supplemental Figure 2), which may have limited recurrences of herpesviruses compared with others in the ART-chronic-HIV group, whose CD4+ counts decreased to between 200 and 400 cells/μL prior to ART initiation. To further assess the relationship between the time interval spanning from HIV acquisition to ART initiation and the proportion of proviruses in HIV-specific cells, we compared the quantity of HIV DNA in HIV-specific T cells versus herpesvirus-specific T cells and found that the shorter this interval of time, the greater the enrichment of HIV DNA in HIV-specific cells (Figure 2, C and D) (Spearman’s δ = –0.63, P = 0.04).
Figure 2HIV DNA levels in antigen-specific CD4+ T cells of participants initiating ART during acute or chronic HIV infection. (A and B) HIV DNA levels (y axis) in CD4+ T cells reactive (CD3+CD8–CD137+) to HIV or various herpesviruses (x axis) 11 days after peptide antigen stimulation are shown for participants initiating ART during acute (A) or chronic (B) HIV infection. The ART-acute-HIV cohort (n = 7) was defined as 1.5 months or less between estimated time of infection and ART initiation and the ART-chronic-HIV cohort (n = 4) was defined as more than 6 months between estimated time of infection and ART initiation. HIV DNA loads were measured by amplification of HIV genomic regions (env, gag, LTR) multiplexed with the human gene transferrin gene (hTFR) by qPCR. HIV DNA is represented as LTR copies per 106 cells. Each solid circular symbol is the mean log(LTR/hTFR) of 2 replicate measures, with bars indicating 2 standard errors. When only one replicate was above the limit of detection, an open circular symbol is shown, and when neither replicate was above the limit of detection, an open square at 1 is shown. Dotted lines represent equivalent of 1 copy of HIV per million cells (see Methods). (C) Comparison of within-participant differences of HIV DNA in HIV-specific CD4+ T cells versus highest HIV DNA among herpesviruses-specific CD4+ T cells across ART-acute-HIV and ART-chronic-HIV groups. Wilcoxon’s P = 0.79. (D) The HIV DNA enrichment factor (y axis, defined as the HIV DNA load in HIV-specific cells divided by the herpesviruses-specific cells with the greatest HIV DNA load) versus time from acute HIV infection to ART initiation (x axis); Spearman’s δ (P = 0.04) shows a negative correlation.
We considered that higher levels of HIV DNA detected in HIV-specific cells after antigen stimulation may reflect relatively greater levels of proliferation by the HIV-specific cells during the 11-day in vitro antigen stimulation instead of differences between the timing of ART initiation. To evaluate this possibility, we compared CD4+ T cell proliferation in response to peptide antigens using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay (Supplemental Figure 3). Across the ART-acute-HIV and ART-chronic-HIV groups, proliferation of HIV-specific cells exceeded that of herpesvirus-specific cells in only one (participant 97054) who had increased proliferation in response to HIV accessory protein viral protein R (Vpr). In all other individuals, proliferation of HIV-specific cells appeared similar or less than herpesviruses-specific cells, with apparent increased proliferation in response to EBV nuclear antigen 3C (EBNA3C) in participant 64428 and higher proliferation in response to pp65 in participants 86313 and 59530. Overall similar levels of proliferation between cells stimulated with HIV or herpesvirus antigens suggests that the relatively higher levels of HIV DNA detected in HIV-specific cells reflect the in vivo levels of HIV DNA in the participants and is not attributable to unequal cell expansion in culture.
To evaluate our hypothesis that the gene pathways harboring integrated HIV differ between the ART-acute-HIV versus ART-chronic-HIV groups, ISs were derived (16) from negatively selected CD4+ T cells and compared to a set of approximately 66,000 ISs derived from in vitro infection of unstimulated primary CD4+ T cells infected with HIV-1 strain BaL (HIV-1BaL) and cultured for 48 hours (11), and primary resting CD4+ T cells infected with HIV NL4-3 for 96 hours (27) (referred to as “in vitro IS”) (see Methods). To identify gene sets associated with persistence of the reservoir during ART, we separately compared 1,083 unique ISs from the ART-acute-HIV and 632 unique ISs from ART-chronic-HIV groups to unique ISs derived from the aforementioned in vitro acutely infected cells across the 1,257 gene sets curated from MSigDB (28). Significant enrichment or depletion of HIV ISs was observed for 9 gene sets by Fisher’s exact test (11, 27–29) in one or both comparisons of the ART-acute-HIV group versus in vitro HIV–infected cells and/or the ART-chronic-HIV group versus in vitro HIV–infected cells (Supplemental Table 4). Among these 9 gene sets, HIV ISs in “BILBAN_B_CLL_LPL_UP,” “GROSS_HYPOXIA_VIA_ELK3_UP,” and “IIZUKA_LIVER_CANCER_PROGRESSION_G2_G3_UP” were significantly enriched in the ART-acute-HIV compared with the ART-chronic-HIV group and “NUYTTEN_NIPP1_TARGETS_UP” was significantly enriched in the ART-chronic-HIV compared with the ART-acute-HIV group (Supplemental Table 4). The proportion of ISs in nongenic or genic regions was not associated with the time interval between HIV infection and ART initiation in our cohort (Supplemental Figure 4). Finally, a comparison of the frequency of HIV ISs in the 9 gene sets by time to ART initiation to ISs in cells acutely infected in vitro confirmed significant selection for these gene sets during ART following ART initiation during acute infection (Figure 3).
Figure 3HIV IS gene set enrichment analysis comparing ISs in gene pathways (GSEA) from in vitro acutely HIV-infected primary CD4+ T cells, ART-acute-HIV, and ART-chronic-HIV groups. Three columns of bubbles represent the proportion of HIV ISs in the 9 gene sets (see Supplemental Table 4) from acute in vitro HIV infections of cells (leftmost column) (11, 27) compared to those we detected in the ART-acute-HIV and ART-chronic-HIV IS (center and rightmost columns). The size of the bubble indicates the magnitude of overlap between the gene set and unique ISs in each group and the color of the bubble represents the statistical significance of the acute versus chronic comparison. In vitro ISs are included in this plot to highlight the statistically significant difference in representation of ISs in the gene set in either ART-acute-HIV or ART-chronic-HIV versus in vitro (see Supplemental Table 4), which implies selection for HIV ISs in these gene sets over time on ART.
DiscussionThis study of 11 men demonstrates that the interval between acquisition of HIV infection and ART initiation is roughly proportional to the size of the HIV reservoir despite years of ART suppression. Our findings include that the interval to ART initiation also appears to affect the CD4+ T cell antigen specificity and the gene pathways with HIV-infected cells that persist during ART. When ART was initiated during acute infection, HIV DNA appeared concentrated in HIV-specific CD4+ T cells, and when ART was initiated during chronic HIV infection, HIV DNA appeared more evenly distributed across HIV-specific and herpesviruses-specific CD4+ T cells. HIV ISs were significantly enriched in gene sets involved in the regulation of lipid metabolism and HIF-1α–mediated hypoxia in the ART-acute-HIV group, while ISs were enriched in enhancer of zeste homolog 2 (EZH2) histone methylation in the ART-chronic-HIV group. To our knowledge, this is the first study to compare antigen specificities and enrichment of HIV ISs across gene sets as a function of time between HIV infection and ART initiation. Our findings demonstrate that the reservoir appears relatively restricted to HIV-specific CD4+ T cells and enriched in genes of certain biological pathways when ART is initiated early in infection, suggesting that manipulation of these metabolic pathways may serve as avenues for therapeutic intervention in individuals who initiate ART during acute infection.
We believe this study is unique, as it compares reservoirs of ART-suppressed individuals who initiated ART during acute HIV infection, defined as less than 1.5 months from the estimated date of infection (25), or chronic infection, defined as more than 6 months. Previous studies that investigated antigen specificity of HIV-infected cells either evaluated only persons who initiated ART during chronic infection (14), or did not directly compare between those who initiated ART during acute or chronic infection (10, 12). The negative correlation that we observed between the duration of untreated HIV infection and enrichment of viral DNA in HIV-specific CD4+ T cells, taken together with others’ findings (10, 12, 30), support the hypothesis that acute HIV infection leads to infection of HIV-specific CD4+ T cells and active viral replication for 6 or more months leads to HIV infection of both HIV-specific CD4+ T cells and CD4+ T cells with other antigen specificities. Our finding of relatively high HIV DNA levels in HIV-specific CD4+ T cells of the ART-acute-HIV group despite years of suppressive ART demonstrates persistence of these infected cells or their clonal descendants. However, the distribution of HIV DNA across CD4+ T cells targeting HIV and multiple herpesviruses in the ART-chronic-HIV group align with previous reports of HIV-1 proviruses persisting in pp65- and gag-specific CD4+ T cells (10, 12), including clones of replication-competent proviruses in CMV-specific CD4+ T cells (14, 31).
Because our initial attempts to measure HIV DNA in antigen-specific cells after 24 hours of culture was unreliable, we expanded antigen-specific CD4+ T cells using peptide stimulation in culture over 10 days. Our CSFE dilution experiments suggest that HIV-specific and herpesviruses-specific T cells have few differences in in vitro proliferation in response to peptide antigens, and that the greater levels of HIV DNA were not due to preferential proliferation of HIV-specific CD4+ T cells. The higher proliferation in response to HIV Vpr, EBNA3C, and CMV pp65 detected on occasion could be due to central memory T cells, which are known to have greater proliferative capacities compared with effector memory T cells (32).
The gene pathways with HIV ISs that persist during ART appear to vary based on timing of ART initiation. ISs in the ART-acute-HIV group were enriched in genes associated with lipid metabolism and HIF-1α–mediated hypoxia, which are known to strongly influence functions of T lymphocytes (33, 34) and promote glycolytic metabolism in CD4+ T cells (35). CD4+ T cell activation increases both glycolysis and fatty acid metabolism to meet the energy needed for cell growth and effector functions and has been observed in acute HIV infection, with inhibition of glycolysis and fatty acid oxidation in vitro reducing HIV infection of cells (36). HIF-1α was recently shown to be hypermethylated in long-term nonprogressors and hypomethylated in individuals with detectable viral loads despite ART adherence (37). These results suggest that HIF-1α contributes to the ART suppression and possibly the persistence of infected cells. We speculate that proviruses integrated into genes controlling lipid metabolism and hypoxia-inducible factors could provide CD4+ T cells a survival advantage during years of ART suppression, perhaps through metabolic reprogramming toward a Treg phenotype (38). Gene editing studies are warranted to corroborate these findings and to assess the effects of proviral insertion on the expression of these genes.
ISs in the ART-chronic-HIV group were enriched in “NUYTTEN_NIPP1_TARGETS_UP,” which is associated with histone methylation by EZH2. EZH2 is an H3K27 methyltransferase that can lead to silencing of proviruses (39). The enrichment of this gene set in the HIV-chronic-ART group suggests that proviruses inserted in genes involved in epigenetic modifications mediated by EZH2 and other methyltransferases or deacetylases that maintain latency could lead to their increased persistence among individuals in the ART-chronic-HIV group.
Over long-term ART suppression, there is selection against transcriptionally active proviruses (40) and detection of intact proviruses in heterochromatic regions (41). Similarly, ISs in elite controllers are disproportionately increased in centromeric satellite DNA and other infrequently transcribed regions of the genome (42). While most ISs identified in our study are likely defective, defective proviruses can produce viral proteins and potentially elicit immune activation (43).
Limitations of this study include that only cisgender males were studied. It is uncertain whether antigen specificities of HIV-infected cells in females will demonstrate the same patterns, although in both males and females the amount of time on HIV treatment is associated with a smaller replication-competent HIV reservoir (44). Another limitation is that relatively few individuals were studied, especially in the HIV-chronic-ART group. The small number of individuals restricted the statistical power and limited the ability to compare expansion of antigen-specific T cells across antigens after peptide antigen stimulation, HIV DNA across antigens, and IS-enriched gene sets to the HIV-acute-ART group. Additionally, use of only CD137 to identify and sort virus-specific cells
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