Similar Frequency and Inducibility of Intact Human Immunodeficiency Virus-1 Proviruses in Blood and Lymph Nodes.

BACKGROUND: The human immunodeficiency virus (HIV)-1 latent reservoir (LR) in resting CD4+ T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. METHODS: We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from 10 HIV-1-infected patients on antiretroviral therapy (ART) using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. RESULTS: The frequencies of CD4+ T cells with intact proviruses were not significantly different in PB versus LN (61/106 vs 104/106 CD4+ cells), and they were substantially lower than frequencies of CD4+ T cells with defective proviruses. The frequencies of CD4+ T cells induced to produce high levels of viral RNA were not significantly different in PB versus LN (4.3/106 vs 7.9/106), but they were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. CONCLUSIONS: These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs.

Progress Toward HIV Eradication: Case Reports, Current Efforts, and the Challenges Associated with Cure.

An estimated 35 million people worldwide are infected with HIV, yet a widely applicable cure strategy remains elusive. Recent case reports have suggested that curing HIV infection is possible, renewing excitement about research efforts. We describe those cases and discuss their relevance to the global HIV epidemic. We also review ongoing cure strategies that are transitioning from the lab to the clinic, and the assays and clinical assessments that can be used to evaluate cure interventions.

Characterization of Elite Suppressors Cell-Associated HIV-1 mRNA at Baseline and with T Cell Activation

.

Objective: Elite Controllers or Suppressors (ES) are patients who control HIV replication without antiretroviral therapy. In this study, we compared baseline and inducible HIV-1 mRNA levels in CD4+ T cells from ES and chronic progressors (CPs) receiving suppressive antiretroviral therapy. Methods: We quantified basal levels of cell associated HIV-1 mRNA in CD4+ T cells isolated from CPs and ES. Additionally, we measured the fold upregulation of intracellular HIV-mRNA after stimulation of CD4+ T cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and quantified the amount of HIV-mRNA levels released into culture supernatant. Results: ES have significantly less cell associated HIV-mRNA per 5x106 cells (p = 0.003); 8 of 10 CPs had quantifiable HIV-1 mRNA at baseline, whereas this was present in only 2 of 10 ES. Upon stimulation with PMA and ionomycin, 4 of 5 CPs and 7 of 9 ES showed increased cell associated HIV-mRNA. Interestingly, released HIV-1 mRNA could be detected in supernatants of CD4+ T cells stimulated with PMA/ionomycin from 5 of 8 ES. Conclusion: Our results demonstrate that while the baseline levels of cell associated HIV-1 mRNA are significantly lower in ES compared to CPs, stimulation of CD4+ T cells results in a comparable relative upregulation of viral transcription.

Analysis of HER2 expression changes from breast primary to brain metastases and the impact of HER2-low expression on overall survival.

BACKGROUND: There are limited data regarding HER2-low expression dynamics between matched primary tumors and brain metastases (BrMs) in breast cancer. HER2-low expression has emerged as a new therapeutic biomarker for highly active antibody-drug conjugates with emerging intracranial activity. METHODS: Patients with metastatic breast cancer (MBC) and BrMs seen at an NCI-designated center between 2003-2023 were identified. HER2 expression was defined as HER2-positive (3+,2+/ISH amplified), HER2-low (1+,2+/ISH negative), or HER2-0 by ASCO-CAP guidelines. Estrogen receptor (ER) status was defined as ER≥1%. Multivariate survival analyses by Cox proportional hazard models were determined from time of BrM resection to death or last follow-up between the 3 subtypes, controlling for ER and age. RESULTS: Among 197 matched primary and resected BrMs, 81% exhibited HER2 expression in the brain:61% HER2-positive, 20% HER2-low, and 19% HER2-0. Concordance was high in HER2-positive primary tumors with 100% retaining HER2 expression (97% retained HER2+ expression and 2.7% switched to HER2-low). HER2-0 primaries frequently showed HER2 gain in BrMs to HER2-low (35%) or HER2-positive (5.4%) status. Among 48 HER2-low primary tumors, 52% were discordant for HER2 status in the brain with 21% testing HER2-positive and 31% testing HER2-0. In adjusted analyses, patients with HER2-positive BrMs had significantly lower death risk than patients with HER2-low BrMs (HR=0.41, P=0.0006); no difference was observed between HER2-0 and HER2-low. CONCLUSIONS: In this retrospective analysis, HER2 expression is common in breast cancer BrMs, emphasizing the need for improved, non-invasive diagnostics. Patients with HER2-low and HER2-0 BrMs face inferior survival, presenting an unmet clinical need.

Racial differences in α4ß7 expression on CD4+ T cells of HIV-negative men and women who inject drugs.

INTRODUCTION: We performed a cross-sectional study of HIV-uninfected men and women who inject drugs from the ALIVE cohort to examine if black men and women who inject drugs have higher levels of CD4+ T cells expressing the integrin heterodimer α4ß7 compared to white men and women. MATERIALS AND METHODS: Flow cytometry was used to examine expression of α4ß7 and other markers associated with different functional CD4+ T cell subsets in both men and women who inject drugs. RESULTS: Higher levels of α4ß7, CCR5, and CCR6 were observed on CD4+ T cells from black participants compared with white participants. In a multivariable model, α4ß7 expression differed by race, but not sex, age, or other factors. DISCUSSION: Black men and women express higher percentages of α4ß7 expressing CD4+ T cells, which may play a role in HIV disease.

The association of α4ß7 expression with HIV acquisition and disease progression in people who inject drugs and men who have sex with men: Case control studies.

BACKGROUND: α4ß7 is a gut-homing integrin heterodimer that can act as a non-essential binding molecule for HIV. A previous study in heterosexual African women found that individuals with higher proportions of α4ß7 expressing CD4+ T cells were more likely to become infected with HIV, as well as present with faster disease progression. It is unknown if this phenomenon is also observed in men who have sex with men (MSM) or people who inject drugs (PWID). METHODS: MSM and transgender women who seroconverted as part of the HVTN 505 HIV vaccine trial and PWID who seroconverted during the ALIVE cohort study were selected as cases and matched to HIV-uninfected controls from the same studies (1:1 and 1:3, respectively). Pre-seroconversion PBMC samples from cases and controls in both studies were examined by flow cytometry to measure levels of α4ß7 expression on CD4+ T cells. Multivariable conditional logistic regression was used to compare α4ß7 expression levels between cases and controls. A Kaplan-Meier curve was used to examine the association of α4ß7 expression pre-seroconversion with HIV disease progression. FINDINGS: In MSM and transgender women (n = 103 cases, 103 controls), there was no statistically significant difference in the levels of α4ß7 expression on CD4+ T cells between cases and controls (adjusted odds ratio [adjOR] =1.10, 95% confidence interval [CI]=0.94,1.29; p = 0.246). Interestingly, in PWID (n = 49 cases, 143 controls), cases had significantly lower levels of α4ß7 expression compared to their matched controls (adjOR = 0.80, 95% CI = 0.68, 0.93; p = 0.004). Among HIV-positive PWID (n = 47), there was no significant association in HIV disease progression in individuals above or below the median level of α4ß7 expression (log-rank p = 0.84). INTERPRETATION: In contrast to findings in heterosexual women, higher α4ß7 expression does not predict HIV acquisition or disease progression in PWID or MSM. FUNDING: This study was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. The study was also supported by extramural grants from NIAID T32AI102623 (E.U.P.), and UM1AI069470.

Rapamycin-mediated mTOR inhibition uncouples HIV-1 latency reversal from cytokine-associated toxicity.

Current strategies for HIV-1 eradication require the reactivation of latent HIV-1 in resting CD4+ T cells (rCD4s). Global T cell activation is a well-characterized means of inducing HIV-1 transcription, but is considered too toxic for clinical applications. Here, we have explored a strategy that involves a combination of immune activation and the immunosuppressive mTOR inhibitor rapamycin. In purified rCD4s from HIV-1-infected individuals on antiretroviral therapy, rapamycin treatment downregulated markers of toxicity, including proinflammatory cytokine release and cellular proliferation that were induced after potent T cell activation using αCD3/αCD28 antibodies. Using an ex vivo assay for HIV-1 mRNA, we demonstrated that despite this immunomodulatory effect, rapamycin did not affect HIV-1 gene expression induced by T cell activation in these rCD4s. In contrast, treating activated rCD4s with the immunosuppressant cyclosporin, a calcineurin inhibitor, robustly inhibited HIV-1 reactivation. Importantly, rapamycin treatment did not impair cytotoxic T lymphocyte (CTL) recognition and killing of infected cells. These findings raise the possibility of using rapamycin in conjunction with T cell-activating agents in HIV-1 cure strategies.

Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4+ T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal.

Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations.

Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs.