Associations of HIV persistence, cigarette smoking, inflammation, and pulmonary dysfunction in people with HIV on antiretroviral therapy.

We aimed to investigate the relationship between measures of HIV persistence with antiretroviral therapy (ART) and cigarette smoking, systemic markers of inflammation, and pulmonary function. Retrospective study of 82 people with HIV (PWH) on ART for a median of 6.9 years (5.6-7.8) and plasma HIV RNA levels <50 copies/mL. HIV DNA and cell-associated HIV RNA (CA-RNA) were measured in peripheral blood mononuclear cells (PBMC) and plasma HIV RNA was measured by single-copy assay (SCA). Plasma levels of 17 inflammatory mediators were measured by Bio-Plex, and standard pulmonary function tests (PFT) were performed in all participants. Median age was 52 years and 41% were women. Most had preserved CD4+ T cell counts (median (IQR) 580 (361-895) cells/mm3). Median plasma HIV RNA was 1.3 (0.7-4.6) copies/mL, and median levels of HIV DNA and CA-RNA in PBMC were 346 (140-541) copies and 19 (3.7-49) copies per 1 million PBMC, respectively. HIV DNA was higher in smokers than in nonsmokers (R = 0.3, P < 0.05), and smoking pack-years positively correlated with HIV DNA and CA-RNA (R = 0.3, P < 0.05 and R = 0.4, P < 0.01, respectively). HIV DNA, CA-RNA, and plasma HIV RNA were not significantly associated with any measure of pulmonary function or inflammation. Cigarette smoking was associated with HIV DNA and CA-RNA levels in blood, but measures of HIV persistence were not associated with pulmonary function or inflammation.

Contribution of Coronavirus-Specific Immunoglobulin G Responses to Complement Overactivation in Patients with Severe Coronavirus Disease 2019.

BACKGROUND: Excessive complement activation has been implicated in the pathogenesis of coronavirus disease 2019 (COVID-19), but the mechanisms leading to this response remain unclear. METHODS: We measured plasma levels of key complement markers, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antibodies against SARS-CoV-2 and seasonal human common cold coronaviruses (CCCs) in hospitalized patients with COVID-19 of moderate (n = 18) and critical severity (n = 37) and in healthy controls (n = 10). RESULTS: We confirmed that complement activation is systemically increased in patients with COVID-19 and is associated with a worse disease outcome. We showed that plasma levels of C1q and circulating immune complexes were markedly increased in patients with severe COVID-19 and correlated with higher immunoglobulin (Ig) G titers, greater complement activation, and higher disease severity score. Additional analyses showed that the classical pathway was the main arm responsible for augmented complement activation in severe patients. In addition, we demonstrated that a rapid IgG response to SARS-CoV-2 and an anamnestic IgG response to the nucleoprotein of the CCCs were strongly correlated with circulating immune complex levels, complement activation, and disease severity. CONCLUSIONS: These findings indicate that early, nonneutralizing IgG responses may play a key role in complement overactivation in severe COVID-19. Our work underscores the urgent need to develop therapeutic strategies to modify complement overactivation in patients with COVID-19.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

Comparison of methods to quantify inducible HIV-1 outgrowth.

The quantitative viral outgrowth assay (qVOA) is the gold standard for measuring inducible, replication-competent HIV-1. Using MOLT4-R5 and SupT1-R5 cell lines instead of allogeneic blasts and HIV-1 RNA detection rather than p24 enzyme-immunoassay (EIA) has been proposed to improve the sensitivity of the qVOA. It is unclear, however, how these alternative approaches affect qVOA performance. We compared three qVOAs methods across 15 persons with HIV-1 on suppressive antiretroviral therapy and found that the MOLT4-R5 method yielded a significantly higher proportion of p24-positive wells (42%) than both the allogeneic blast (29%) and SupT1-R5 (32%) assays. Additionally, 5 of 7 qVOAs that were negative by p24 EIA showed viral outgrowth by HIV-1 RNA quantification (>10-fold increase within 7 days). These findings reveal the potential for underestimation of the latent, inducible reservoir by qVOA depending on the target cells used and the measure of viral outgrowth. Use of MOLT4-R5 cells with both p24 EIA and HIV-1 RNA to detect viral outgrowth was the most sensitive method.

Intractable Coronavirus Disease 2019 (COVID-19) and Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Replication in a Chimeric Antigen Receptor-Modified T-Cell Therapy Recipient: A Case Study.

A chimeric antigen receptor-modified T-cell therapy recipient developed severe coronavirus disease 2019, intractable RNAemia, and viral replication lasting >2 months. Premortem endotracheal aspirate contained >2 × 1010 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intrahost virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.

Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy.

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual integrase-targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.