SARS-CoV-2 RNA and nucleocapsid antigen are blood biomarkers associated with severe disease outcomes that improve in response to remdesivir.

BACKGROUND: Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter ACTT-1 clinical trial that randomized patients to remdesivir or placebo. METHODS: Longitudinal specimens collected during hospitalization from a substudy of 642 COVID-19 patients were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. RESULTS: Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95%CI 1.40-2.71) for levels >245 pg/ml vs 1.04 (95%CI 0.76-1.42) for levels < 245 pg/ml. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. CONCLUSIONS: Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy.

Viral and host mediators of non-suppressible HIV-1 viremia.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

Long COVID After Bamlanivimab Treatment.

BACKGROUND: Prospective evaluations of long COVID in outpatients with coronavirus disease 2019 (COVID-19) are lacking. We aimed to determine the frequency and predictors of long COVID after treatment with the monoclonal antibody bamlanivimab in ACTIV-2/A5401. METHODS: Data were analyzed from participants who received bamlanivimab 700 mg in ACTIV-2 from October 2020 to February 2021. Long COVID was defined as the presence of self-assessed COVID symptoms at week 24. Self-assessed return to pre-COVID health was also examined. Associations were assessed by regression models. RESULTS: Among 506 participants, median age was 51 years. Half were female, 5% Black/African American, and 36% Hispanic/Latino. At 24 weeks, 18% reported long COVID and 15% had not returned to pre-COVID health. Smoking (adjusted risk ratio [aRR], 2.41 [95% confidence interval {CI}, 1.34- 4.32]), female sex (aRR, 1.91 [95% CI, 1.28-2.85]), non-Hispanic ethnicity (aRR, 1.92 [95% CI, 1.19-3.13]), and presence of symptoms 22-28 days posttreatment (aRR, 2.70 [95% CI, 1.63-4.46]) were associated with long COVID, but nasal severe acute respiratory syndrome coronavirus 2 RNA was not. CONCLUSIONS: Long COVID occurred despite early, effective monoclonal antibody therapy and was associated with smoking, female sex, and non-Hispanic ethnicity, but not viral burden. The strong association between symptoms 22-28 days after treatment and long COVID suggests that processes of long COVID start early and may need early intervention. CLINICAL TRIALS REGISTRATION: NCT04518410.

Evaluating Demographic Representation in Clinical Trials: Use of the Adaptive Coronavirus Disease 2019 Treatment Trial (ACTT) as a Test Case.

Background: Clinical trials initiated during emerging infectious disease outbreaks must quickly enroll participants to identify treatments to reduce morbidity and mortality. This may be at odds with enrolling a representative study population, especially when the population affected is undefined. Methods: We evaluated the utility of the Centers for Disease Control and Prevention's COVID-19-Associated Hospitalization Surveillance Network (COVID-NET), the COVID-19 Case Surveillance System (CCSS), and 2020 United States (US) Census data to determine demographic representation in the 4 stages of the Adaptive COVID-19 Treatment Trial (ACTT). We compared the cumulative proportion of participants by sex, race, ethnicity, and age enrolled at US ACTT sites, with respective 95% confidence intervals, to the reference data in forest plots. Results: US ACTT sites enrolled 3509 adults hospitalized with COVID-19. When compared with COVID-NET, ACTT enrolled a similar or higher proportion of Hispanic/Latino and White participants depending on the stage, and a similar proportion of African American participants in all stages. In contrast, ACTT enrolled a higher proportion of these groups when compared with US Census and CCSS. The proportion of participants aged ≥65 years was either similar or lower than COVID-NET and higher than CCSS and the US Census. The proportion of females enrolled in ACTT was lower than the proportion of females in the reference datasets. Conclusions: Although surveillance data of hospitalized cases may not be available early in an outbreak, they are a better comparator than US Census data and surveillance of all cases, which may not reflect the population affected and at higher risk of severe disease.

One Week of Oral Camostat Versus Placebo in Nonhospitalized Adults With Mild-to-Moderate Coronavirus Disease 2019: A Randomized Controlled Phase 2 Trial.

BACKGROUND: Camostat inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. We studied the safety and efficacy of camostat in ACTIV-2/A5401, a phase 2/3 platform trial of therapeutics for COVID-19 in nonhospitalized adults. METHODS: We conducted a phase 2 study in adults with mild-to-moderate COVID-19 randomized to oral camostat for 7 days or a pooled placebo arm. Primary outcomes were time to improvement in COVID-19 symptoms through day 28, proportion of participants with SARS-CoV-2 RNA below the lower limit of quantification (LLoQ) from nasopharyngeal swabs through day 14, and grade ≥3 treatment-emergent adverse events (TEAEs) through day 28. RESULTS: Of 216 participants (109 randomized to camostat, 107 to placebo) who initiated study intervention, 45% reported ≤5 days of symptoms at study entry and 26% met the protocol definition of higher risk of progression to severe COVID-19. Median age was 37 years. Median time to symptom improvement was 9 days in both arms (P = .99). There were no significant differences in the proportion of participants with SARS-CoV-2 RNA

Viral and Host Mediators of Non-Suppressible HIV-1 Viremia.

Non-suppressible HIV-1 viremia (NSV) can occur in persons with HIV despite adherence to combination antiretroviral therapy (ART) and in the absence of significant drug resistance. Here, we show that plasma NSV sequences are comprised primarily of large clones without evidence of viral evolution over time. We defined proviruses that contribute to plasma viremia as "producer", and those that did not as "non-producer". Compared to ART-suppressed individuals, NSV participants had a significantly larger producer reservoir. Producer proviruses were enriched in chromosome 19 and in proximity to the activating H3K36me3 epigenetic mark. CD4+ cells from NSV participants demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, NSV participants showed no elevation in HIV-specific CD8+ cell responses and producer proviruses were enriched for HLA escape mutations. We identified critical host and viral mediators of NSV that represent potential targets to disrupt HIV persistence and promote viral silencing.

Peginterferon lambda for the treatment of hospitalized patients with mild COVID-19: A pilot phase 2 randomized placebo-controlled trial.

Background: This study aimed to investigate the efficacy and safety of subcutaneous injection of peginterferon lambda in patients hospitalized with COVID-19. Methods: In this study (NCT04343976), patients admitted to hospital with COVID-19 confirmed by RT-PCR from nasopharyngeal swab were randomly assigned within 48 h to receive peginterferon lambda or placebo in a 1:1 ratio. Participants were subcutaneously injected with a peginterferon lambda or saline placebo at baseline and day 7 and were followed up until day 14. Results: We enrolled 14 participants; 6 participants (85.7%) in the peginterferon lambda group and 1 participant (14.3%) in the placebo group were treated with remdesivir prior to enrollment. Fifty percent of participants were SARS-CoV-2 RNA negative at baseline although they tested SARS-CoV-2 RNA positive within 48 h of randomization. Among participants who were SARS-CoV-2 positive at baseline, 2 out of 5 participants (40%) in the peginterferon lambda group became negative at day 14, while 0 out of 2 participants (0%) in the placebo group achieved negativity for SARS-CoV-2 by day 14 (p > 0.05). The median change in viral load (log copies per ml) was +1.72 (IQR -2.78 to 3.19) in the placebo group and -2.22 (IQR -3.24 to 0.55) in the peginterferon lambda group at day 14 (p = 0.24). Symptomatic changes did not differ between the two groups. Peginterferon lambda was well tolerated with a few treatment-related adverse effects. Conclusion: Peginterferon lambda appears to accelerate SARS-CoV-2 viral load decline and improve plasma disease progression markers in hospitalized patients with COVID-19.

Prolonged viral suppression with anti-HIV-1 antibody therapy.

HIV-1 infection remains a public health problem with no cure. Anti-retroviral therapy (ART) is effective but requires lifelong drug administration owing to a stable reservoir of latent proviruses integrated into the genome of CD4+ T cells1. Immunotherapy with anti-HIV-1 antibodies has the potential to suppress infection and increase the rate of clearance of infected cells2,3. Here we report on a clinical study in which people living with HIV received seven doses of a combination of two broadly neutralizing antibodies over 20 weeks in the presence or absence of ART. Without pre-screening for antibody sensitivity, 76% (13 out of 17) of the volunteers maintained virologic suppression for at least 20 weeks off ART. Post hoc sensitivity analyses were not predictive of the time to viral rebound. Individuals in whom virus remained suppressed for more than 20 weeks showed rebound viraemia after one of the antibodies reached serum concentrations below 10 µg ml-1. Two of the individuals who received all seven antibody doses maintained suppression after one year. Reservoir analysis performed after six months of antibody therapy revealed changes in the size and composition of the intact proviral reservoir. By contrast, there was no measurable decrease in the defective reservoir in the same individuals. These data suggest that antibody administration affects the HIV-1 reservoir, but additional larger and longer studies will be required to define the precise effect of antibody immunotherapy on the reservoir.

Preventing Infectious Complications of Immunomodulation in COVID-19 in Foreign-Born Patients.

Immunomodulating therapies for COVID-19 may carry risks of reactivating latent infections in foreign-born people. We conducted a rapid review of infection-related complications of immunomodulatory therapies for COVID-19. We convened a committee of specialists to formulate a screening and management strategy for latent infections in our setting. Dexamethasone, used in severe COVID-19, is associated with reactivation of latent tuberculosis, hepatitis B, and dissemination/hyperinfection of Strongyloides species and should prompt screening and/ or empiric treatment in appropriate epidemiologic contexts. Other immunomodulators used in COVID-19 may also increase risk, including interleukin-6 receptor antagonist (e.g., tocilizumab) and kinase inhibitors. People with specific risk factors should also be screened for HIV, Chagas disease, and endemic mycoses. Racial and ethnic minorities in North America, including foreign-born persons, who receive immunomodulating agents for COVID-19 may be at risk for reactivation of latent infections. We develop a screening and management pathway for such patients.

SARS-CoV-2 viral load is associated with increased disease severity and mortality.

The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). Here, we quantify SARS-CoV-2 viral load from participants with a diverse range of COVID-19 disease severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. We detected SARS-CoV-2 plasma RNA in 27% of hospitalized participants, and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, we report that a higher prevalence of detectable SARS-CoV-2 plasma viral load is associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, are associated with increased risk of mortality. Our data show that SARS-CoV-2 viral loads may aid in the risk stratification of patients with COVID-19, and therefore its role in disease pathogenesis should be further explored.

Maintenance of Viral Suppression in Human Immunodeficiency Virus Controllers Despite Waning T-Cell Responses During Antiretroviral Therapy.

AIDS Clinical Trials Group study A5308 found reduced T-cell activation and exhaustion in human immunodeficiency virus (HIV) controllers start antiretroviral therapy (ART). We further assessed HIV-specific T-cell responses and post-ART viral loads. Before ART, the 31% of participants with persistently undetectable viremia had more robust HIV-specific T-cell responses. During ART, significant decreases were observed in a broad range of T-cell responses. Eight controllers in A5308 and the Study of the Consequences of the Protease Inhibitor Era (SCOPE) cohort showed no viremia above the level of quantification in the first 12 weeks after ART discontinuation. ART significantly reduced HIV-specific T-cell responses in HIV controllers but did not adversely affect controller status after ART discontinuation.

On the Road to a HIV Cure: Moving Beyond Berlin and London.

The Berlin patient, a famous example for human immunodeficiency virus (HIV) cure, had received a bone marrow transplantation with an HIV resistance mutation. The authors describe his case and others that had shown HIV control, like the Mississippi baby who was started on antiretroviral therapy very early after birth, and posttreatment controllers, like the VISCONTI cohort. Moreover, the authors outline various strategies, oftentimes informed by these individuals, that have been tried in vitro, in animal models, or in human trials, to deplete the latent reservoir, which is considered the basis of HIV persistence and the obstacle to cure.

HCV induces transforming growth factor ß1 through activation of endoplasmic reticulum stress and the unfolded protein response.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-ß1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-ß1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-ß1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-ß1 expression. ROS, ER stress, NFκB, and TGF-ß1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-ß1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-ß1 activation. We conclude that HCV activates NFκB and TGF-ß1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-ß1.

The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing.

BACKGROUND &/AIMS: The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. METHODS: We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. RESULTS: We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. CONCLUSIONS: The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Entry inhibitors and future treatment of hepatitis C.

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Furthermore, HCV-induced liver disease is the leading indication for liver transplantation. The recent introduction of direct-acting antivirals (DAAs) has revolutionized HCV treatment by making possible the cure of the majority of patients. However, their efficacy and safety in difficult-to-treat patients such as patients receiving immunosuppression, those with advanced liver disease, co-morbidity and HIV/HCV-co-infection remain to be determined. Furthermore, prevention of liver graft infection remains a pressing issue. HCV entry inhibitors target the very first step of the HCV life cycle and efficiently inhibit cell-cell transmission - a key prerequisite for viral spread. Because of their unique mechanism of action on cell-cell transmission they may provide a promising and simple perspective for prevention of liver graft infection. A high genetic barrier to resistance and complementary mechanism of action compared to DAAs makes entry inhibitors attractive as a new strategy for treatment of multi-resistant or difficult-to-treat patients. Clinical studies are needed to determine the future role of entry inhibitors in the arsenal of antivirals to combat HCV infection. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication."