In response to the COVID-19 pandemic, we developed and implemented a theory-informed process to adapt a comprehensive pre-clerkship medical school curriculum to run in the virtual learning environment utilizing sociocultural learning theory and cognitive load theory. Of 124 student respondents, 45% rated virtual learning as very or extremely effective, and 49% as moderately effective. Positive aspects of virtual learning included effectiveness of chat moderators, displaying pronouns on Zoom, active learning technology, and captioning and transcription. Negative aspects included access to technology and feeling isolated from community. Overall course ratings, examination performance, and work hours did not differ pre- and post-implementation.

Objectives

The aim of this study was to assess soluble CD30 (sCD30), a protein that colocalises with HIV-1 RNA and DNA in lymphoid cells and tissues, in cerebrospinal fluid (CSF) as a marker of HIV-1 infection in the central nervous system (CNS).

Methods

This was a cross-sectional study using archived samples from two clinical cohorts. Soluble CD30 concentrations were measured in paired CSF and plasma from untreated viraemic individuals (n=52), individuals on suppressive antiretroviral therapy (ART) (n=33), HIV-1 controllers (n=10), participants with CSF HIV-1 'escape' (n=11) and controls without HIV-1 infection (n=16). Nonparametric tests were used to compare levels across groups and evaluate correlations with HIV-1 RNA, CSF neurofilament light chain protein (NFL) and neopterin.

Results

Compared with controls (median 30 ng/mL, interquartile range [IRQ] 23-50), plasma sCD30 levels were elevated in viraemic participants (75 ng/mL, 52-116; P<0.001), but not in those on suppressive ART (38 ng/mL, 32-62). In contrast, CSF sCD30 levels were elevated in ART-suppressed individuals (34 ng/mL, 19-46; P=0.001) and in those with CSF 'escape' (33 ng/mL, 27-40; P=0.004) compared with controls (18 ng/mL, 11-23), but not in untreated viraemic individuals. No association was observed between CSF sCD30 and plasma HIV-1 RNA, concurrent or nadir CD4+ T cell count, duration of infection or plasma sCD30. CSF sCD30 correlated with CSF NFL (r=0.34, P=0.001).

Conclusions

In contrast to plasma, sCD30 levels are elevated in the CSF of individuals with HIV-1 infection who are on suppressive ART. Elevated levels of sCD30 in the CSF may be an indicator of persistent CNS HIV-1 infection, although the mechanism underlying this elevation warrants further investigation.

Anti-CD30 therapy for Hodgkin lymphoma led to transient loss of detectable CD4+ T-cell HIV RNA and a decrease in residual plasma viremia. Targeting nonviral markers expressed on HIV-1 transcriptionally active cells may lead to reduced measures of HIV-1 persistence.

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

Background

Identification of nonviral markers of human immunodeficiency virus (HIV) infection that increase before viral rebound during analytical treatment interruption (ATI) may affect HIV persistence research. We previously showed that HIV ribonucleic acid (RNA) is enriched in CD30+CD4+ T cells in many individuals. Here, we studied CD30+CD4+ T-cell dynamics before ATI, during ATI (before detectable plasma RNA), and after HIV rebound.

Methods

Peripheral blood mononuclear cells from 23 participants collected longitudinally from 5 Adult AIDS Clinical Trials Group studies incorporating ATI were included in this study. Flow cytometric characterization of expression of CD30 and markers of T-cell activation and exhaustion were performed along with HIV-1 RNA and deoxyribonucleic acid quantification and measurement of soluble plasma CD30 and CD30 ligand.

Results

The percentage of CD4+ T cells expressing CD30 significantly increased from pre-ATI to postinterruption time points before detectible viremia (1.65 mean relative increase, P = .005). Seventy-seven percent of participants experienced an increase in CD30+ cells before viral rebound. In contrast, there were no significant differences between pre-ATI and postinterruption pre-rebound time points in percentages of lymphocytes expressing CD69, CD38/HLA-DR, or PD-1 until after HIV recrudescence.

Conclusions

CD30 may be a surrogate marker of early replication or viral transcriptional activity before detection by routine peripheral blood sampling.

Background

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.

Case presentation

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.

Conclusion

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.

After publication of the original article [1], we were notified in Table 1 a column should be removed.

Objective

Prior genomewide association studies have identified variation in major histocompatibility complex (MHC) class I alleles and C-C chemokine receptor type 5 gene (CCR5Δ32) as genetic predictors of viral control, especially in 'elite' controllers, individuals who remain virally suppressed in the absence of therapy.

Design

Cross-sectional genomewide association study.

Methods

We analyzed custom whole exome sequencing and direct human leukocyte antigen (HLA) typing from 202 antiretroviral therapy (ART)-suppressed HIV+ noncontrollers 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.

Results

Previously 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, the CCR5Δ32 deletion was associated with smaller HIV tDNA (P = 4.3 × 10-3) and usRNA (P = 8.7 × 10-3). In addition, genomewide analysis identified several single nucleotide polymorphisms in MX1 (an interferon stimulated gene) that were significantly associated with HIV tDNA (q = 0.02), and the direction of these associations paralleled MX1 gene eQTL expression.

Conclusions

We observed a significant association between previously reported 'protective' MHC class I alleles and CCR5Δ32 with the HIV reservoir size in noncontrollers. We also found a novel association between MX1 and HIV total DNA (in addition to other interferon signaling relevant genes, PPP1CB, DDX3X). These findings warrant further investigation in future validation studies.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.

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.