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The major barrier to an HIV cure is the persistence of infected cells that evade host immune surveillance despite effective antiretroviral therapy (ART). Most prior host genetic HIV studies have focused on identifying DNA polymorphisms (e.g.,CCR5Δ32, MHC class I alleles) associated with viral load among untreated “elite controllers” (~1% of HIV+ individuals who are able to control virus without ART). However, there have been few studies evaluating host genetic predictors of viral control for the majority of people living with HIV (PLWH) on ART. We performed host RNA sequencing and HIV reservoir quantification (total DNA, unspliced RNA, intact DNA) from peripheral CD4+ T cells from 191 HIV+ ART-suppressed non-controllers. Multivariate models included covariates for timing of ART initiation, nadir CD4+ count, age, sex, and ancestry. Lower HIV total DNA (an estimate of the total reservoir) was associated with upregulation of tumor suppressor genesNBL1(q=0.012) andP3H3(q=0.012). Higher HIV unspliced RNA (an estimate of residual HIV transcription) was associated with downregulation of several host genes involving inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9,CXCL3, CXCL10) and innate immune (TLR7) signaling, as well as novel associations with potassium (KCNJ2) and gap junction (GJB2) channels, all qPLGLB1) and glucose metabolism (AGL) genes, but data were (HIV intact DNA detected in only 42% of participants). Our findings demonstrate that among treated PLWH, that inflammation, innate immune responses, bacterial translocation, and tumor suppression/cell proliferation host signaling play a key role in the maintenance of the HIV reservoir during ART. Further data are needed to validate these findings, including functional genomic studies, and expanded epidemiologic studies in female, non-European cohorts.Author SummaryAlthough lifelong HIV antiretroviral therapy (ART) suppresses virus, the major barrier to an HIV cure is the persistence of infected cells that evade host immune surveillance despite effective ART, “the HIV reservoir.” HIV eradication strategies have focused on eliminating residual virus to allow for HIV remission, but HIV cure trials to date have thus far failed to show a clinically meaningful reduction in the HIV reservoir. There is an urgent need for a better understanding of the host-viral dynamics during ART suppression to identify potential novel therapeutic targets for HIV cure. This is the first epidemiologic host gene expression study to demonstrate a significant link between HIV reservoir size and several well-known immunologic pathways (e.g., IL-1β, TLR7, TNF-α signaling pathways), as well as novel associations with potassium and gap junction channels (Kir2.1, connexin 26). Further data are needed to validate these findings, including functional genomic studies and expanded epidemiologic studies in female, non-European cohorts.

ObjectivePrior genomewide association studies have identified variation in MHC Class I alleles andCCR5Δ32as genetic predictors of viral control, especially in “elite” controllers, individuals who remain virally suppressed in the absence of therapy.DesignCross-sectional genomewide association study.MethodsWe analyzed custom whole exome sequencing and direct HLA typing from 202 ART-suppressed HIV+ non-controllers in relation to four measures of the peripheral CD4+ T cell reservoir: HIV intact DNA, total (t)DNA, unspliced (us)RNA, and RNA/DNA. Linear mixed models were adjusted for potential covariates including age, sex, nadir CD4+ T cell count, pre-ART HIV RNA, timing of ART initiation, and duration of ART suppression.ResultsPreviously reported “protective” host genetic mutations related to viral setpoint (e.g., among elite controllers) were found to predict smaller HIV reservoir size. The HLA “protective” B*57:01 was associated with significantly lower HIV usRNA (q=3.3×10−3), and among the largest subgroup, European ancestry individuals, theCCR5Δ32deletion was associated with smaller HIV tDNA (p=4.3×10−3) and usRNA (p=8.7×10−3). In addition, genomewide analysis identified several SNPs inMX1(an interferon stimulated gene) that were significantly associated with HIV tDNA (q=0.02), and the direction of these associations paralleledMX1gene eQTL expression.ConclusionsWe observed a significant association between previously reported “protective” MHC class I alleles andCCR5Δ32with the HIV reservoir size in non-controllers. We also found a novel association betweenMX1and HIV total DNA (in addition to other interferon signaling relevant genes,PPP1CB,DDX3X). These findings warrant further investigation in future validation studies.

Elite controllers (EC), a small subset of the HIV-positive population ( 1%), suppress HIV viremia below the limit of quantification of clinical viral load assays in the absence of antiretroviral therapy (ART). However, there is a paucity of longitudinal data detailing the viral and immune dynamics or HIV reservoir seeding during acute infection in individuals that go on to become Elite Controllers.In this report, we describe a case of a 42 year old woman diagnosed during acute infection who rapidly and permanently suppressed her viremia in the absence of antiretroviral therapy (ART). Rapid antibody/antigen testing was either negative or equivocal during acute infection, despite subsequent viral load testing at that time point with 71,550 plasma HIV RNA copies/mL, making initial diagnosis challenging. The patient subsequently developed detectable anti-HIV antibodies and an increase in HIV-specific CD8+ T cell responses to overlapping subtype C HIV gag peptide; very low-level plasma viremia (0.84 RNA copies/mL) was detected by an ultrasensitive assay 2 years following infection. Subsequently, she was started on ART for multifocal furunculosis despite continued suppression of virus and stable CD4+ T cell counts. Following ART initiation, HIV specific antibody levels and CD8+ T cell responses increased, but no HIV DNA or RNA was able to be isolated from large numbers of peripheral blood CD4+ T cells.This case provides important information regarding the establishment of elite HIV control during acute infection and also demonstrates an increase in HIV-specific immune responses following ART despite undetectable peripheral blood cellular measures of HIV persistence. This case also highlights the challenges in diagnosing acute HIV infection without the use of viral load testing in this rare elite controller phenotype.

Key Points Graft-versus-host effects may lead to HIV-1 reactivation and cell death of infected pre-HCT CD4+ T cells. Natural killer cell activation correlates with in vitro HIV-1 transcriptional activity in the setting of HCT.

Background It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. Methods and findings Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. Conclusions We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission., Timothy Henrich and colleagues study the effect of very early antiretroviral treatment on the reservoir of HIV-infected cells in two patients., Author summary Why was this study done? Early initiation of ART following infection may limit the total body burden of HIV. It is not known if starting ART extremely early after HIV infection will lead to ART-free remission or cure. We studied 2 individuals who started ART an estimated 10 and 12 days after HIV infection with very low peak viral load measurement; extensive testing of blood and tissue for HIV persistence was performed. One participant stopped ART in order to test if and when HIV would rebound. What did the researchers find? No HIV could be definitively detected for up to 2 years in the participant who initiated ART approximately 10 days after HIV infection. Intermittent, very low levels of HIV were detected in blood but not tissue in the participant who initiated ART an estimated 12 days following infection. The participant with no detectable HIV following ART experienced viral rebound 225 days after stopping ART. What do these findings mean? HIV relapsed despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission.

Background Human Herpes Virus 8 (HHV8) can cause Kaposi’s Sarcoma (KS) in immunosuppressed individuals. However, little is known about the association between chemotherapy or hematopoietic stem cell transplantation (HSCT), circulating HHV8 DNA levels, and clinical KS in HIV-1-infected individuals with various malignancies. Therefore, we examined the associations between various malignancies, systemic cancer chemotherapy, T cell phenotypes, and circulating HHV8 DNA in 29 HIV-1-infected participants with concomitant KS or other cancer diagnoses. Methods We quantified HHV8 plasma viral loads and cell-associated HHV8 DNA and determined the relationship between circulating HHV8 DNA and lymphocyte counts, and markers of early and late lymphocyte activation, proliferation and exhaustion. Results There were no significant differences in plasma HHV8 DNA levels between baseline and post-chemotherapy time points or with the presence or absence of clinical KS. However, in two participants circulating HHV8 DNA increased following treatment for KS or HSCT for lymphoma,. We observed an approximately 2-log10 reduction in plasma HHV8 DNA in an individual with KS and multicentric Castleman disease following rituximab monotherapy. Although individuals with clinical KS had lower mean CD4+ T cell counts and percentages as expected, there were no significant associations with these factors and plasma HHV8 levels. We identified increased proportions of CD8+ and CD4+ T cells expressing CD69 (P = 0.01 & P = 0.04 respectively), and increased CD57 expression on CD4+ T cells (P = 0.003) in participants with detectable HHV8. Conclusion These results suggest there is a complex relationship between circulating HHV8 DNA and tissue-based disease in HIV-1 and HHV8 co-infected individuals with various malignancies.

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4 + T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies., Highlights • A common approach to HIV cure involves reactivating HIV-infected cells. • Developed single cell assay to directly quantify HIV transcriptionally reactivated cells. • Single cell effects of latency reversing agents on HIV reactivation are heterogeneous. • Bulk cell-associated HIV RNA levels are divergent from the number of RNA-producing cells. • Assay allows for single-cell quantification of genomic DNA and mRNA following target cell identification. We designed and implemented a microfluidic, single-cell assay to directly measure the number of individual cells from individuals on antiretroviral therapy that produce HIV-1 RNA. The assay also allows for single-cell quantification of human genomic DNA and messenger RNA following identification and isolation of actively HIV-1-infected cells. The ability to directly measure transcriptional activity of HIV-1 in individual cells followed by downstream characterization of human and viral genetic information within these cells has the potential to provide a greater mechanistic understanding of experimental strategies to purge residual HIV-1 reservoirs.

Background Systemic chemotherapies for various malignancies have been shown to significantly, yet transiently, decrease numbers of CD4+ T lymphocytes, a major reservoir for human immunodeficiency virus type 1 (HIV-1) infection. However, little is known about the impact of cytoreductive chemotherapy on HIV-1 reservoir dynamics, persistence, and immune responses. Methods We investigated the changes in peripheral CD4+ T-cell-associated HIV-1 DNA and RNA levels, lymphocyte activation, viral population structure, and virus-specific immune responses in a longitudinal cohort of 15 HIV-1-infected individuals receiving systemic chemotherapy or subsequent autologous stem cell transplantation for treatment of hematological malignancies and solid tumors. Results Despite a transient reduction in CD4+ T cells capable of harboring HIV-1, a 1.7- and 3.3-fold increase in mean CD4+ T-cell-associated HIV-1 RNA and DNA, respectively, were observed months following completion of chemotherapy in individuals on antiretroviral therapy. We also observed changes in CD4+ T-cell population diversity and clonal viral sequence expansion during CD4+ T-cell reconstitution following chemotherapy cessation. Finally, HIV-1 DNA was preferentially, and in some cases exclusively, detected in cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-responsive CD4+ T cells following chemotherapy. Conclusions Expansion of HIV-infected CMV/EBV-specific CD4 + T cells may contribute to maintenance of the HIV DNA reservoir following chemotherapy.

Assays that can verify full viral eradication are essential in the context of achieving a cure for HIV/AIDS. In vitro quantitative viral out growth assays (qVOA) are currently the gold standard for measuring latent HIV-1 but these assays often fail to detect very low levels of replication-competent virus. Here we investigated an alternative in vivo approach for sensitive viral detection using humanized mice (hmVOA). Peripheral blood CD4+ T cell samples from HIV subjects on stable ART with undetectable viral loads by RT-PCR were first assayed by in vitro qVOA. Corresponding patient samples in which no virus was detected by traditional qVOA were injected into humanized mice to allow viral outgrowth. Of the five in vitro qVOA virus negative samples, four gave positive viral outgrowth in the in vivo hmVOA assay suggesting that it is more sensitive in detecting latent HIV-1.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection., Author summary Previous studies have shown that higher levels of soluble CD30 are associated with HIV-1 disease progression. Many of these studies, however, were performed prior to the implementation of combination ART, and the relationship between surface CD30 expression, soluble CD30 and HIV-1 infection in ART suppressed individuals, or those with viremic control off ART, is not known. We demonstrate that cell-associated HIV-1 RNA is highly enriched in CD4+ T cells expressing CD30, a member of the tumor necrosis factor receptor superfamily. These findings were observed in several HIV-1 infected donor groups, regardless of whether or not the participants were receiving suppressive ART. Furthermore, we demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate that targets CD30, reduces the total amount of HIV-1 DNA in PBMC obtained from infected individuals. Finally, we show through in situ RNA hybridization studies that CD30 and HIV transcriptional activity co-localize in cells from gut biopsies obtained from HIV-1 infected donors. These data suggest that CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART.

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.