COVID-19: Challenges of Viral Variants.

The COVID-19 pandemic has been accompanied by SARS-CoV-2 evolution and emergence of viral variants that have far exceeded initial expectations. Five major variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) have emerged, each having both unique and overlapping amino acid substitutions that have affected transmissibility, disease severity, and susceptibility to natural or vaccine-induced immune responses and monoclonal antibodies. Several of the more recent variants appear to have evolved properties of immune evasion, particularly in cases of prolonged infection. Tracking of existing variants and surveillance for new variants are critical for an effective pandemic response.

Prospective assessment of humoral and cellular immune responses to a 3rd COVID-19 mRNA vaccine dose among immunocompromised individuals.

BACKGROUND: Improved COVID-19 prevention is needed for immunocompromised individuals. METHODS: Prospective study of healthcare workers (HCW) and immunocompromised participants with baseline serology following 2 mRNA vaccines and who were retested after dose 3 (D3); multivariable regression was used to identify predictors of serological responses. IFNγ/TNFα T-cell responses were assessed in a subset. RESULTS: 536 participants were included: 492 immunocompromised [(206 solid organ transplant (SOT), 128 autoimmune, 80 hematologic malignancy (HM), 48 solid tumor, 25 HIV], 44 HCW. D3 significantly increased Spike IgG levels among all, but SOT and HM participants had the lowest median antibody levels post-D3 (increase from 0.09 to 0.83 and 0.27 to 1.92, respectively), versus HCW and persons with HIV, autoimmune conditions, and solid tumors (increases from 4.44 to 19.79, 2.9 to 15.75, 3.82 to 16.32, and 4.1 to 25.54, respectively). Seropositivity post-D3 was lowest for SOT (49.0%) and HM (57.8%), versus others (>90% seropositive). Neutralization post-D3 was lowest among SOT and HM. Predictors of lower antibody levels included low baseline levels and shorter intervals between vaccines. T-cell responses against Spike increased significantly among HCW and non-significantly among immunocompromised individuals. CONCLUSIONS: D3 significantly improves serological but not T-cell responses among immunocompromised individuals. SOT and HM patients have suboptimal responses to D3.

Therapy With Allogeneic Severe Acute Respiratory Syndrome Coronavirus-2-Specific T Cells for Persistent Coronavirus Disease 2019 in Immunocompromised Patients.

We administered severe acute respiratory syndrome coronavirus-2 viral-specific T cells (VSTs) under emergency investigational new drug applications to 6 immunocompromised patients with persistent coronavirus disease 2019 (COVID-19) and characterized clinical and virologic responses. Three patients had partial responses after failing other therapies but then died. Two patients completely recovered, but the role of VSTs in recovery was unclear due to concomitant use of other antivirals. One patient had not responded to 2 courses of remdesivir and experienced sustained recovery after VST administration. The use of VSTs in immunocompromised patients with persistent COVID-19 requires further study.

Plasma SARS-CoV-2 RNA Levels as a Biomarker of Lower Respiratory Tract SARS-CoV-2 Infection in Critically Ill Patients With COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in serially collected samples from mechanically ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (n = 33, r = 0.83, P < 10-9) and then declined in parallel in available serial samples except in nonsurvivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early after ICU admission.

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.

Prospective Evaluation of Coronavirus Disease 2019 (COVID-19) Vaccine Responses Across a Broad Spectrum of Immunocompromising Conditions: the COVID-19 Vaccination in the Immunocompromised Study (COVICS).

BACKGROUND: We studied humoral responses after coronavirus disease 2019 (COVID-19) vaccination across varying causes of immunodeficiency. METHODS: Prospective study of fully vaccinated immunocompromised adults (solid organ transplant [SOT], hematologic malignancy, solid cancers, autoimmune conditions, human immunodeficiency virus [HIV]) versus nonimmunocompromised healthcare workers (HCWs). The primary outcome was the proportion with a reactive test (seropositive) for immunoglobulin G to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain. Secondary outcomes were comparisons of antibody levels and their correlation with pseudovirus neutralization titers. Stepwise logistic regression was used to identify factors associated with seropositivity. RESULTS: A total of 1271 participants enrolled: 1099 immunocompromised and 172 HCW. Compared with HCW (92.4% seropositive), seropositivity was lower among participants with SOT (30.7%), hematological malignancies (50.0%), autoimmune conditions (79.1%), solid tumors (78.7%), and HIV (79.8%) (P < .01). Factors associated with poor seropositivity included age, greater immunosuppression, time since vaccination, anti-CD20 monoclonal antibodies, and vaccination with BNT162b2 (Pfizer) or adenovirus vector vaccines versus messenger RNA (mRNA)-1273 (Moderna). mRNA-1273 was associated with higher antibody levels than BNT162b2 or adenovirus vector vaccines after adjusting for time since vaccination, age, and underlying condition. Antibody levels were strongly correlated with pseudovirus neutralization titers (Spearman r = 0.89, P < .0001), but in seropositive participants with intermediate antibody levels, neutralization titers were significantly lower in immunocompromised individuals versus HCW. CONCLUSIONS: Antibody responses to COVID-19 vaccines were lowest among SOT and anti-CD20 monoclonal recipients, and recipients of vaccines other than mRNA-1273. Among those with intermediate antibody levels, pseudovirus neutralization titers were lower in immunocompromised patients than HCWs. Additional SARS-CoV-2 preventive approaches are needed for immunocompromised persons, which may need to be tailored to the cause of immunodeficiency.

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.

A Simpler and More Sensitive Single-Copy HIV-1 RNA Assay for Quantification of Persistent HIV-1 Viremia in Individuals on Suppressive Antiretroviral Therapy.

A real-time quantitative reverse transcriptase PCR assay with single-copy sensitivity targeting the integrase region of HIV-1 (integrase single-copy assay [iSCA] v1.0) has been widely used to quantify plasma viremia in individuals on antiretroviral therapy (ART). iSCA v1.0 requires the use of an ultracentrifuge, and only about half of the nucleic acid extracted from plasma is assayed for HIV-1 RNA. We sought to simplify sample processing using microcentrifugation and improve assay sensitivity by testing more than 75% of the total extracted nucleic acid for HIV-1 RNA (iSCA v2.0). We evaluated the limit of detection (LoD) of iSCA v2.0 by testing replicates of low-copy plasma HIV-1 RNA standards. By probit analysis, the 95% LoD was 1 copy of HIV-1 RNA per milliliter for a 5-ml plasma sample. To compare the sensitivity of iSCA v1.0 and v2.0, we tested plasma samples with both assays from 60 participants on ART with HIV-1 RNA below 20 cps/ml. Of the 31 samples that had no detectable HIV-1 RNA by iSCA v1.0, 17 (55%) were detectable by v2.0 with an HIV-1 RNA mean value of 3.5 cps/ml. Twenty-nine samples were detectable with both assay versions, but average values of HIV-1 RNA cps/ml were 2.7-fold higher for v2.0 than v1.0. These results support the adoption of a new, more sensitive and simpler single-copy HIV-1 RNA assay (iSCA v2.0) to quantify residual viremia on ART and to assess the impact of experimental interventions designed to decrease HIV-1 reservoirs.

Regulation of mitochondrial antiviral signaling (MAVS) expression and signaling by the mitochondria-associated endoplasmic reticulum membrane (MAM) protein Gp78.

In a previous study, we identified the E3 ubiquitin ligase Gp78 by RNAi high-throughput screening as a gene whose depletion restricted enterovirus infection. In the current study, we show that Gp78, which localizes to the ER-mitochondria interface, is a regulator of RIG-I-like receptor (RLR) antiviral signaling. We show that depletion of Gp78 results in a robust decrease of vesicular stomatitis virus (VSV) infection and a corresponding enhancement of type I interferon (IFN) signaling. Mechanistically, we show that Gp78 modulates type I IFN induction by altering both the expression and signaling of the mitochondria-localized RLR adaptor mitochondrial antiviral signaling (MAVS). Expression of mutants of Gp78 that abolish its E3 ubiquitin ligase and its participation in ER-associated degradation (ERAD) lost their ability to degrade MAVS, but surprisingly maintained their ability to repress RLR signaling. In contrast, Gp78 lacking its entire C terminus lost both its ability to degrade MAVS and repress RLR signaling. We show that Gp78 interacts with both the N- and C-terminal domains of MAVS via its C-terminal RING domain, and that this interaction is required to abrogate Gp78-mediated attenuation of MAVS signaling. Our data thus implicate two parallel pathways by which Gp78 regulates MAVS signaling; one pathway requires its E3 ubiquitin ligase and ERAD activity to directly degrade MAVS, whereas the other pathway occurs independently of these activities, but requires the Gp78 RING domain and occurs via a direct association between this region and MAVS.

A human claudin-1-derived peptide inhibits hepatitis C virus entry.

UNLABELLED: Hepatitis C virus (HCV) entry is a complicated process that requires multiple host factors, such as CD81, scavenger receptor BI, claudin-1 (CLDN1), and occludin. The interaction of virus and cellular entry factors represents a promising target for novel anti-HCV drug development. In this study, we sought to identify peptide inhibitors for HCV entry by screening a library of overlapping peptides covering the four above-mentioned entry factors. An 18-amino acid peptide (designated as CL58) that was derived from the CLDN1 intracellular and first transmembrane region inhibited both de novo and established HCV infection in vitro. Unlike previously reported peptides corresponding to CLDN1 extracellular loops, CL58 did not alter the normal distribution of CLDN1 and was not cytotoxic in vitro at concentrations nearly 100-fold higher than the effective antiviral dose. The inhibitory effect of CL58 appeared to occur at a late step during viral entry, presumably after initial binding. Finally, overexpressed CL58 was able to interact with HCV envelope proteins. CONCLUSION: We identified a novel CLDN1-derived peptide that inhibits HCV entry at a postbinding step. The findings expand our knowledge of the roles that CLDN1 play in HCV entry and highlight the potential for developing a new class of inhibitors targeting the viral entry process.

TIM-3 signaling contributes to the suppressive capacity of Tregs from people with HIV on antiretroviral therapy.

TIM-3 expression is increased on peripheral regulatory T cells (Tregs) of virally suppressed persons with HIV-1 on antiretroviral therapy (PWH-ART). However, the relevance of TIM-3 expression in this setting is unclear. We used flow cytometry to evaluate the suppressive phenotype and signaling pathways in peripheral TIM-3- vs TIM-3+ Tregs in PWH-ART. TIM-3+ Tregs showed increased expression of IL-10 compared with persons without HIV-1. In addition, TIM-3+ Tregs displayed elevated signaling and activation, relative to TIM-3- Tregs from the same PWH-ART. Dramatically, TIM-3 blockade restrained the in vitro suppressive capacity of peripheral Tregs. Therefore, our data demonstrate not only that TIM-3 expression by Tregs is associated with an immunosuppressive response among PWH-ART, but also that TIM-3 contributes directly to the enhanced suppressive activity of Tregs in this setting.

Rapid determination of SARS-CoV-2 antibody neutralization titer using Bio-Rad Bio-Plex correlates strongly with pseudovirus-determined neutralization titer.

Accurate and rapid evaluation of SARS-CoV-2 half-maximal neutralizing antibody (nAb) titer (NT50) is an important research tool for measuring nAb responses after prophylaxis or therapeutics for COVID-19 prevention and management. Compared with ACE2-competitive enzyme immunoassays for nAb detection, pseudovirus assays remain low-throughput and labor intensive. A novel application of the Bio-Rad Bio-Plex Pro Human SARS-CoV-2 D614G S1 Variant nAb Assay was used to determine NT50 from COVID-19-vaccinated individuals and showed strong correlation to a laboratory-developed SARS-CoV-2 pseudovirus nAb assay. The Bio-Plex nAb assay could provide a rapid, high-throughput, culture-free method for NT50 determination in sera.

Rapid Emergence of Potentially Transmissible Severe Acute Respiratory Syndrome Coronavirus 2 With Resistance to Combination Monoclonal Antibody Therapy.

Prolonged coronavirus disease 2019 may generate new viral variants. We report an immunocompromised patient treated with monoclonal antibodies who experienced rebound of viral RNA and emergence of an antibody-resistant (>1000-fold) variant containing 5 mutations in the spike gene. The mutant virus was isolated from respiratory secretions, suggesting the potential for secondary transmission.

Noninvasive diagnosis of secondary infections in COVID-19 by sequencing of plasma microbial cell-free DNA.

Secondary infection (SI) diagnosis in severe COVID-19 remains challenging. We correlated metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) with clinical SI assessment, immune response, and outcomes. We classified 42 COVID-19 inpatients as microbiologically confirmed-SI (Micro-SI, n = 8), clinically diagnosed-SI (Clinical-SI, n = 13, i.e., empiric antimicrobials), or no-clinical-suspicion-for-SI (No-Suspected-SI, n = 21). McfDNA-Seq was successful in 73% of samples. McfDNA detection was higher in Micro-SI (94%) compared to Clinical-SI (57%, p = 0.03), and unexpectedly high in No-Suspected-SI (83%), similar to Micro-SI. We detected culture-concordant mcfDNA species in 81% of Micro-SI samples. McfDNA correlated with LRT 16S rRNA bacterial burden (r = 0.74, p = 0.02), and biomarkers (white blood cell count, IL-6, IL-8, SPD, all p < 0.05). McfDNA levels were predictive of worse 90-day survival (hazard ratio 1.30 [1.02-1.64] for each log10 mcfDNA, p = 0.03). High mcfDNA levels in COVID-19 patients without clinical SI suspicion may suggest SI under-diagnosis. McfDNA-Seq offers a non-invasive diagnostic tool for pathogen identification, with prognostic value on clinical outcomes.

Development of a robust cytopathic effect-based high-throughput screening assay to identify novel inhibitors of dengue virus.

We have developed a robust cytopathic effect-based high-throughput screening assay to identify inhibitors of dengue virus (DENV) infection. Screening of a small natural product library yielded 11 hits. Four of these were found to be potent inhibitors of DENV, although serotype differences were noted. Taken together, these data suggest that screening of larger and more complex molecule libraries may result in the identification of more potent and specific DENV inhibitors.

Plasma SARS-CoV-2 RNA levels as a biomarker of lower respiratory tract SARS-CoV-2 infection in critically ill patients with COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in simultaneously collected longitudinal samples from mechanically-ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (r=0.83, p<10 -8 ) and then declined in parallel except in non-survivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early in severe disease.

SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization.

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (VH ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains.

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human VH domain, F6, which we generated by sequentially panning large phage-displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants.