Africa-specific human genetic variation near CHD1L associates with HIV-1 load.

HIV-1 remains a global health crisis1, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.

Admission COVID-19 clinical risk assessment for guiding patient placement and diagnostic testing strategy.

INTRODUCTION: Without universal access to point-of-care SARS-CoV-2 testing, many hospitals rely on clinical judgement alone for identifying cases of COVID-19 early. METHODS: Cambridge University Hospitals NHS Foundation Trust introduced a 'traffic light' clinical judgement aid to the COVID-19 admissions unit in mid-March 2020. Ability to accurately predict COVID-19 was audited retrospectively across different stages of the epidemic. RESULTS: One SARS-CoV-2 PCR positive patient (1/41, 2%) was misallocated to a 'green' (non-COVID-19) area during the first period of observation, and no patients (0/32, 0%) were mislabelled 'green' during the second period. 33 of 62 (53%) labelled 'red' (high risk) tested SARS-CoV-2 PCR positive during the first period, while 5 of 22 (23%) 'red' patients were PCR positive in the second. CONCLUSION: COVID-19 clinical risk stratification on initial assessment effectively identifies non-COVID-19 patients. However, diagnosing COVID-19 is challenging and risk of overcalling COVID-19 should be recognised, especially when background prevalence is low.

Point of Care Nucleic Acid Testing for SARS-CoV-2 in Hospitalized Patients: A Clinical Validation Trial and Implementation Study.

There is an urgent need for rapid SARS-CoV-2 testing in hospitals to limit nosocomial spread. We report an evaluation of point of care (POC) nucleic acid amplification testing (NAAT) in 149 participants with parallel combined nasal and throat swabbing for POC versus standard lab RT-PCR testing. Median time to result is 2.6 (IQR 2.3-4.8) versus 26.4 h (IQR 21.4-31.4, p < 0.001), with 32 (21.5%) positive and 117 (78.5%) negative. Cohen's κ correlation between tests is 0.96 (95% CI 0.91-1.00). When comparing nearly 1,000 tests pre- and post-implementation, the median time to definitive bed placement from admission is 23.4 (8.6-41.9) versus 17.1 h (9.0-28.8), p = 0.02. Mean length of stay on COVID-19 "holding" wards is 58.5 versus 29.9 h (p < 0.001). POC testing increases isolation room availability, avoids bed closures, allows discharge to care homes, and expedites access to hospital procedures. POC testing could mitigate the impact of COVID-19 on hospital systems.

A novel, sensitive dual-indicator cell line for detection and quantification of inducible, replication-competent latent HIV-1 from reservoir cells.

Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials.

HIV Silencing and Inducibility Are Heterogeneous and Are Affected by Factors Intrinsic to the Virus.

Transcriptionally silent HIV proviruses form the major obstacle to eradicating HIV. Many studies of HIV latency have focused on the cellular mechanisms that maintain silencing of proviral DNA. Here we show that viral sequence variation affecting replicative ability leads to variable rates of silencing and ability to reactivate. We studied naturally occurring and engineered polymorphisms in a recently identified exonic splice enhancer (ESEtat) that regulates tat mRNA splicing and constructed viruses with increased (strain M1), reduced (strain M2), or completely absent (strain ERK) binding of splicing factors essential for optimal production of tat mRNA resulting in a corresponding change in Tat activity. The mutations affected viral replication, with M1 having wild-type (WT) kinetics, M2 exhibiting reduced kinetics, and ERK showing completely abrogated replication. Using single-round infection with green fluorescent protein (GFP)-expressing viruses to study proviral gene expression, we observed progressively greater rates of silencing relating to the degree of ESEtat disruption, with the WT strain at 53%, strain M2 at 69%, and strain ERK at 94%. By stimulating infected cells with a latency reversal agent (phorbol myristate acetate [PMA], panobinostat, or JQ1), we observed that the dose required to achieve 50% of the maximum signal was lowest in the WT, intermediate in M2, and highest in ERK, indicating progressively higher thresholds for reactivation. These results suggest that the ability of silent proviruses to reactivate from latency is variable and that minor differences in the viral sequence can alter the proportion of silenced viruses as well as the threshold required to induce silenced viruses to reactivate and express.IMPORTANCE A reservoir of infected cells in which the HIV genome is transcriptionally silent is acknowledged to be the principal barrier to eradicating the virus from an infected person. A number of cellular processes are implicated in this silencing; however, the viral factors that may contribute remain underexplored. Here we examined mutations altering the correct splicing of HIV gene products as a model to study whether differences in viral sequence can affect either the proportion of viruses that are active or silent or their ability to reactivate. We found that some naturally occurring variations result in viruses that are silenced at a higher rate and require a proportionally increased stimulus for reactivation from latency. These data suggest that the silencing and reactivation behavior of HIV exists in a spectrum, influenced by factors intrinsic to the virus.

HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation.

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual-who is known as the 'Berlin patient'-underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin's lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression.

No evidence of ongoing evolution in replication competent latent HIV-1 in a patient followed up for two years.

The persistence of infected T cells harbouring intact HIV proviruses is the barrier to the eradication of HIV. This reservoir is stable over long periods of time despite antiretroviral therapy. There has been controversy on whether low level viral replication is occurring at sanctuary sites periodically reseeding infected cells into the latent reservoir to account its durability. To study viral evolution in a physiologically relevant population of latent viruses, we repeatedly performed virus outgrowth assays on a stably treated HIV positive patient over two years and sequenced the reactivated latent viruses. We sought evidence of increasing sequence pairwise distances with time as evidence of ongoing viral replication. 64 reactivatable latent viral sequences were obtained over 103 weeks. We did not observe an increase in genetic distance of the sequences with the time elapsed between sampling. No evolution could be discerned in these reactivatable latent viruses. Thus, in this patient, the contribution of low-level replication to the maintenance of the latent reservoir detectable in the blood compartment is limited.

Innovations in the quantitative virus outgrowth assay and its use in clinical trials.

A robust measure of the size of the latent HIV reservoir is essential to quantifying the effect of interventions designed to deplete the pool of reactivatable, replication competent proviruses. In addition to the ability to measure a biologically relevant parameter, any assay designed to be used in a clinical trial needs to be reproducible and scalable. The need to quantify the number of resting CD4+ T cells capable of releasing infectious virus has led to the development of the quantitative viral outgrowth assay (VOA). The assay as originally described has a number of features that limit its scalability for use in clinical trials; however recent developments reducing the time and manpower requirements of the assay, while importantly improving reproducibility mean that it is becoming much more practical for it to enter into more widespread use. This review describes the background to VOA development and the practical issues that they present in utilising them in clinical trials. It describes the innovations that have made their usage more practical and the limitations that still exist.

A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4+ T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4+ T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.