COVID-19 Antigen Results Correlate with the Quantity of Replication-Competent SARS-CoV-2 in a Cross-Sectional Study of Ambulatory Adults during the Delta Wave.

Appropriate interpretation of various diagnostic tests for COVID-19 is critical, yet the association among rapid antigen tests, reverse transcription (RT)-PCR, and viral culture has not been fully defined. To determine whether rapid antigen testing correlates with the presence and quantity of replication-competent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in ambulatory adults, 626 adult participants were enrolled in a cross-sectional diagnostic study. Each participant had two anterior nasal swabs obtained for rapid antigen and RT-PCR testing and SARS-CoV-2 viral culture. The primary outcomes were the presence and quantification of SARS-CoV-2 growth in VeroE6-ACE2-TMPRSS2 cells in asymptomatic and symptomatic ambulatory adults. In this cross-sectional study of 626 adult outpatients, the sensitivity of a single positive antigen test to identify replication-competent SARS-CoV-2 was 63.6% in asymptomatic and 91.0% in symptomatic participants. Viral culture titers were the highest at the onset of symptoms and rapidly declined by 7 days after symptom onset. The positive agreement of the rapid antigen test with RT-PCR at a cycle threshold CT less than 30 was 66.7% in asymptomatic and 90.7% in symptomatic participants. Among symptomatic participants a with a CT less than 30, a single antigen test had a positive agreement of 90.7% (95% confidence interval [CI], 84.8% to 94.8%). There was 100% negative agreement as all 425 RT-PCR-negative participants had a negative antigen test. A positive antigen test in symptomatic adults with COVID-19 has a strong correlation with replication-competent SARS-CoV-2. Rapid antigen test results may be a suitable proxy for infectiousness. IMPORTANCE Do rapid antigen test results correlate with replication-competent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (i.e., infectious) virus? In this cross-sectional diagnostic study of 626 adults, the sensitivity of the antigen test to identify replication-competent SARS-CoV-2 was 63.6% in asymptomatic and 91.0% in symptomatic participants. Viral culture titers were the highest at the onset of symptoms and rapidly declined by 7 days after symptom onset. The positive agreement of the rapid antigen test with reverse transcription (RT)-PCR at a CT of less than 30 was 66.7% in asymptomatic participants and 90.7% in symptomatic participants. A positive antigen test may be an appropriate surrogate for identifying replication-competent virus in symptomatic individuals with COVID-19.

Extended Remdesivir Infusion for Persistent Coronavirus Disease 2019 Infection.

Persistent severe acute respiratory syndrome coronavirus 2 infection is difficult to treat. Here, we report a case of 5-month persistent coronavirus disease 2019 in an immunocompromised patient who was successfully treated with 30 consecutive days of remdesivir. Prolonged remdesivir infusion with concurrent cycle threshold monitoring might provide a potential solution to cure these patients with difficult-to-treat infections.

Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study

Background Guidelines for SARS-CoV-2 have relied on limited data on duration of viral infectiousness and correlation with COVID-19 symptoms and diagnostic testing. Methods We enrolled ambulatory adults with acute SARS-CoV-2 infection and performed serial measurements of COVID-19 symptoms, nasal swab viral RNA, nucleocapsid (N) and spike (S) antigens, and replication-competent SARS-CoV-2 by viral growth in culture. We determined average time from symptom onset to a first negative test result and estimated risk of infectiousness, as defined by positive viral growth in culture. Results Among 95 adults, median [interquartile range] time from symptom onset to first negative test result was 9 [5] days, 13 [6] days, 11 [4] days, and >19 days for S antigen, N antigen, culture growth, and viral RNA by RT-PCR, respectively. Beyond two weeks, virus growth and N antigen titers were rarely positive, while viral RNA remained detectable among half (26/51) of participants tested 21-30 days after symptom onset. Between 6-10 days from symptom onset, N antigen was strongly associated with culture positivity (relative risk=7.61, 95% CI: 3.01-19.22), whereas neither viral RNA nor symptoms were associated with culture positivity. During the 14 days following symptom onset, the presence of N antigen remained strongly associated (adjusted relative risk=7.66, 95% CI: 3.96-14.82) with culture positivity, regardless of COVID-19 symptoms. Conclusions Most adults have replication-competent SARS-CoV-2 for 10-14 after symptom onset. N antigen testing is a strong predictor of viral infectiousness and may be a more suitable biomarker, rather than absence of symptoms or viral RNA, to discontinue isolation within two weeks from symptom onset. Funding Bill and Melinda Gates Foundation

Cytotoxic T Cells Targeting Spike Glycoprotein Are Associated with Hybrid Immunity to SARS-CoV-2.

mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared with vaccination in the absence of prior infection. However, the immune mechanisms by which this hybrid immunity is generated and maintained are unknown. Whereas genetic variation in spike glycoprotein effectively subverts neutralizing Abs, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled human T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive (n = 13) or -convalescent (n = 17) individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells and polyfunctional Th1 responses was similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multimodal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.

Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study.

BACKGROUND: Guidelines for SARS-CoV-2 have relied on limited data on duration of viral infectiousness and correlation with COVID-19 symptoms and diagnostic testing. METHODS: We enrolled ambulatory adults with acute SARS-CoV-2 infection and performed serial measurements of COVID-19 symptoms, nasal swab viral RNA, nucleocapsid (N) and spike (S) antigens, and replication-competent SARS-CoV-2 by viral growth in culture. We determined average time from symptom onset to a first negative test result and estimated risk of infectiousness, as defined by positive viral growth in culture. RESULTS: Among 95 adults, median [interquartile range] time from symptom onset to first negative test result was 9 [5] days, 13 [6] days, 11 [4] days, and >19 days for S antigen, N antigen, culture growth, and viral RNA by RT-PCR, respectively. Beyond two weeks, virus growth and N antigen titers were rarely positive, while viral RNA remained detectable among half (26/51) of participants tested 21-30 days after symptom onset. Between 6-10 days from symptom onset, N antigen was strongly associated with culture positivity (relative risk=7.61, 95% CI: 3.01-19.22), whereas neither viral RNA nor symptoms were associated with culture positivity. During the 14 days following symptom onset, the presence of N antigen remained strongly associated (adjusted relative risk=7.66, 95% CI: 3.96-14.82) with culture positivity, regardless of COVID-19 symptoms. CONCLUSIONS: Most adults have replication-competent SARS-CoV-2 for 10-14 after symptom onset. N antigen testing is a strong predictor of viral infectiousness and may be a more suitable biomarker, rather than absence of symptoms or viral RNA, to discontinue isolation within two weeks from symptom onset.

Angiopoietin-Like4 Is a Novel Marker of COVID-19 Severity.

Vascular dysfunction and capillary leak are common in critically ill COVID-19 patients, but identification of endothelial pathways involved in COVID-19 pathogenesis has been limited. Angiopoietin-like 4 (ANGPTL4) is a protein secreted in response to hypoxic and nutrient-poor conditions that has a variety of biological effects including vascular injury and capillary leak. OBJECTIVES: To assess the role of ANGPTL4 in COVID-19-related outcomes. DESIGN SETTING AND PARTICIPANTS: Two hundred twenty-five COVID-19 ICU patients were enrolled from April 2020 to May 2021 in a prospective, multicenter cohort study from three different medical centers, University of Washington, University of Southern California and New York University. MAIN OUTCOMES AND MEASURES: Plasma ANGPTL4 was measured on days 1, 7, and 14 after ICU admission. We used previously published tissue proteomic data and lung single nucleus RNA (snRNA) sequencing data from specimens collected from COVID-19 patients to determine the tissues and cells that produce ANGPTL4. RESULTS: Higher plasma ANGPTL4 concentrations were significantly associated with worse hospital mortality (adjusted odds ratio per log2 increase, 1.53; 95% CI, 1.17-2.00; p = 0.002). Higher ANGPTL4 concentrations were also associated with higher proportions of venous thromboembolism and acute respiratory distress syndrome. Longitudinal ANGPTL4 concentrations were significantly different during the first 2 weeks of hospitalization in patients who subsequently died compared with survivors (p for interaction = 8.1 × 10-5). Proteomics analysis demonstrated abundance of ANGPTL4 in lung tissue compared with other organs in COVID-19. ANGPTL4 single-nuclear RNA gene expression was significantly increased in pulmonary alveolar type 2 epithelial cells and fibroblasts in COVID-19 lung tissue compared with controls. CONCLUSIONS AND RELEVANCE: ANGPTL4 is expressed in pulmonary epithelial cells and fibroblasts and is associated with clinical prognosis in critically ill COVID-19 patients.

Dynamics of SARS-CoV-2 VOC Neutralization and Novel mAb Reveal Protection against Omicron.

New variants of SARS-CoV-2 continue to emerge and evade immunity. We isolated SARS-CoV-2 temporally across the pandemic starting with the first emergence of the virus in the western hemisphere and evaluated the immune escape among variants. A clinic-to-lab viral isolation and characterization pipeline was established to rapidly isolate, sequence, and characterize SARS-CoV-2 variants. A virus neutralization assay was applied to quantitate humoral immunity from infection and/or vaccination. A panel of novel monoclonal antibodies was evaluated for antiviral efficacy. We directly compared all variants, showing that convalescence greater than 5 months post-symptom onset from ancestral virus provides little protection against SARS-CoV-2 variants. Vaccination enhances immunity against viral variants, except for Omicron BA.1, while a three-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a two-dose. A novel Mab neutralizes Omicron BA.1 and BA.2 variants better than the clinically approved Mabs, although neither can neutralize Omicron BA.4 or BA.5. Thus, the need remains for continued vaccination-booster efforts, with innovation for vaccine and Mab improvement for broadly neutralizing activity. The usefulness of specific Mab applications links with the window of clinical opportunity when a cognate viral variant is present in the infected population.

A C57BL/6 Mouse Model of SARS-CoV-2 Infection Recapitulates Age- and Sex-Based Differences in Human COVID-19 Disease and Recovery.

We present a comprehensive analysis of SARS-CoV-2 infection and recovery using wild type C57BL/6 mice and a mouse-adapted virus, and we demonstrate that this is an ideal model of infection and recovery that phenocopies acute human disease arising from the ancestral SARS-CoV-2. Disease severity and infection kinetics are age- and sex-dependent, as has been reported for humans, with older mice and males in particular exhibiting decreased viral clearance and increased mortality. We identified key parallels with human pathology, including intense virus positivity in bronchial epithelial cells, wide-spread alveolar involvement, recruitment of immune cells to the infected lungs, and acute bronchial epithelial cell death. Moreover, older animals experienced increased virus persistence, delayed dispersal of immune cells into lung parenchyma, and morphologic evidence of tissue damage and inflammation. Parallel analysis of SCID mice revealed that the adaptive immune response was not required for recovery from COVID disease symptoms nor early phase clearance of virus but was required for efficient clearance of virus at later stages of infection. Finally, transcriptional analyses indicated that induction and duration of key innate immune gene programs may explain differences in age-dependent disease severity. Importantly, these data demonstrate that SARS-CoV-2-mediated disease in C57BL/6 mice phenocopies human disease across ages and establishes a platform for future therapeutic and genetic screens for not just SARS-CoV-2 but also novel coronaviruses that have yet to emerge.

Combinations of Host- and Virus-Targeting Antiviral Drugs Confer Synergistic Suppression of SARS-CoV-2.

Three directly acting antivirals (DAAs) demonstrated substantial reduction in COVID-19 hospitalizations and deaths in clinical trials. However, these agents did not completely prevent severe illness and are associated with cases of rebound illness and viral shedding. Combination regimens can enhance antiviral potency, reduce the emergence of drug-resistant variants, and lower the dose of each component in the combination. Concurrently targeting virus entry and virus replication offers opportunities to discover synergistic drug combinations. While combination antiviral drug treatments are standard for chronic RNA virus infections, no antiviral combination therapy has been approved for SARS-CoV-2. Here, we demonstrate that combining host-targeting antivirals (HTAs) that target TMPRSS2 and hence SARS-CoV-2 entry, with the DAA molnupiravir, which targets SARS-CoV-2 replication, synergistically suppresses SARS-CoV-2 infection in Calu-3 lung epithelial cells. Strong synergy was observed when molnupiravir, an oral drug, was combined with three TMPRSS2 (HTA) oral or inhaled inhibitors: camostat, avoralstat, or nafamostat. The combination of camostat plus molnupiravir was also effective against the beta and delta variants of concern. The pyrimidine biosynthesis inhibitor brequinar combined with molnupiravir also conferred robust synergistic inhibition. These HTA+DAA combinations had similar potency to the synergistic all-DAA combination of molnupiravir plus nirmatrelvir, the protease inhibitor found in paxlovid. Pharmacodynamic modeling allowed estimates of antiviral potency at all possible concentrations of each agent within plausible therapeutic ranges, suggesting possible in vivo efficacy. The triple combination of camostat, brequinar, and molnupiravir further increased antiviral potency. These findings support the development of HTA+DAA combinations for pandemic response and preparedness. IMPORTANCE Imagine a future viral pandemic where if you test positive for the new virus, you can quickly take some medicines at home for a few days so that you do not get too sick. To date, only single drugs have been approved for outpatient use against SARS-CoV-2, and we are learning that these have some limitations and may succumb to drug resistance. Here, we show that combinations of two oral drugs are better than the single ones in blocking SARS-CoV-2, and we use mathematical modeling to show that these drug combinations are likely to work in people. We also show that a combination of three oral drugs works even better at eradicating the virus. Our findings therefore bode well for the development of oral drug cocktails for at home use at the first sign of an infection by a coronavirus or other emerging viral pathogens.

Extended Remdesivir Infusion for Persistent COVID-19 Infection

Persistent SARS-CoV-2 infection patients are difficult to treat. Here, we report a case of 5-month persistent COVID-19 in an immunocompromised patient who was successfully treated with 30 consecutive days of Remdesivir. Prolonged remdesivir infusion with concurrent cycle threshold monitoring might provide a potential solution to cure these difficult patients.

IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

T cell receptor sequencing identifies prior SARS-CoV-2 infection and correlates with neutralizing antibodies and disease severity.

BACKGROUNDMeasuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T cell responses, but these responses vary with disease severity and individual characteristics.METHODSA T cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T cell testing was assessed and compared with serologic testing.RESULTSSARS-CoV-2-specific T cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T cell testing was most apparent in recovered, nonhospitalized individuals sampled > 150 days after initial illness, suggesting greater sensitivity than serology at later time points and in individuals with less severe disease. T cell testing identified SARS-CoV-2 infection in 68% (55 of 81) of samples with undetectable nAb titers (<1:40) and in 37% (13 of 35) of samples classified as negative by 3 antibody assays.CONCLUSIONThese results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology.TRIAL REGISTRATIONSpecimens were accrued under trial NCT04338360 accessible at clinicaltrials.gov.FUNDINGThis work was funded by Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, NIAID, Fred Hutchinson Joel Meyers Endowment, Fast Grants, and American Society for Transplantation and Cell Therapy.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease following challenge with heterologous variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Since 2019, the SARS-CoV-2 virus has spread worldwide and caused hundreds of millions of cases of COVID-19. Vaccines were rapidly developed to protect people from becoming severely ill from the virus and decrease the risk of death. However, new variants ­ such as Alpha, Beta and Omicron ­ have emerged that the vaccines do not work as well against, contributing to the ongoing spread of the virus. One way to overcome this is to create a vaccine that can be quickly and easily updated to target new variants, like the vaccine against influenza. Many of the vaccines made against COVID-19 use a new technology to introduce the RNA sequence of the spike protein on the surface of SARS-CoV-2 into our cells. Once injected, our cells use their own machinery to build the protein, or 'antigen', so the immune system can learn how to recognize and destroy the virus. Here, Hawman et al. have renovated an RNA vaccine they made in 2020 which provides immunity against the original strain of SARS-CoV-2 in monkeys and mice. In the newer versions of the vaccine, the RNA was updated with a sequence that matches the spike protein on the Beta or Alpha variant of the virus. Both the original and updated vaccines were then administered to mice and hamsters to see how well they worked against SARS-CoV-2 infections. The experiment showed that all three vaccines caused the animals to produce antibodies that can neutralize the original, Alpha and Beta strains of the virus. Vaccinated hamsters were then infected with one of the three variants ­ either matched or mismatched to their vaccination ­ to see how much protection each vaccine provided. All the vaccines reduced the amount of virus in the animals after infection and mitigated damage in their lungs. But animals that received a vaccine which corresponded to the SARS-CoV-2 strain they were infected with had slightly better protection. These findings suggest that these vaccines work best when their RNA sequence matches the strain responsible for the infection; however, even non-matched vaccines still provide a decent degree of protection. Furthermore, the data demonstrate that the vaccine platform created by Hawman et al. can be easily updated to target new strains of SARS-CoV-2 that may emerge in the future. Recently, the Beta variant of the vaccine entered clinical trials in the United States (led by HDT Bio) to evaluate whether it can be used as a booster in previously vaccinated individuals as well as unvaccinated participants.

Validation of Home Visual Acuity Tests for Telehealth in the COVID-19 Era.

Importance: Visual acuity (VA) is one of the most important clinical data points in ophthalmology. However, few options for validated at-home VA assessments are currently available. Objective: To validate 3 at-home visual acuity tests in comparison with in-office visual acuity. Design, Setting, and Participants: Between July 2020 and April 2021, eligible participants with VA of 20/200 or better were recruited from 4 university-based ophthalmology clinics (comprehensive, cornea, glaucoma, and retina clinics). Participants were prospectively randomized to self-administer 2 of 3 at-home VA tests (printed chart, mobile phone app, and website) within 3 days before their standard-of-care clinic visit. Participants completed a survey assessing usability of the at-home tests. At the clinic visit, best-corrected Snellen distance acuity was measured as the reference standard. Main Outcomes and Measures: The at-home VA test results were compared with the in-office VA test results using paired and unpaired t tests, Pearson correlation coefficients, analysis of variance, χ2 tests, and Cohen κ agreement. The sensitivity, specificity, positive predictive value, and negative predictive value of each at-home test were calculated to detect significant VA changes (≥0.2 logMAR) from the in-office baseline. Results: A total of 121 participants with a mean (SD) age of 63.8 (13.0) years completed the study. The mean in-office VA was 0.11 logMAR (Snellen equivalent 20/25) with similar numbers of participants from the 4 clinics. Mean difference (logMAR) between the at-home test and in-office acuity was -0.07 (95% CI, -0.10 to -0.04) for the printed chart, -0.12 (95% CI, -0.15 to -0.09) for the mobile phone app, and -0.13 (95% CI, -0.16 to -0.10) for the website test. The Pearson correlation coefficient for the printed chart was 0.72 (95% CI, 0.62-0.79), mobile phone app was 0.58 (95% CI, 0.46-0.69), and website test was 0.64 (95% CI, 0.53-0.73). Conclusions and Relevance: The 3 at-home VA test results (printed chart, mobile phone app, and website) appeared comparable within 1 line to in-office VA measurements. Older participants were more likely to have limited access to digital tools. Further development and validation of at-home VA testing modalities is needed with the expansion of teleophthalmology care.

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4+ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

T cell response to intact SARS-CoV-2 includes coronavirus cross-reactive and variant-specific components.

SARS-CoV-2 provokes a robust T cell response. Peptide-based studies exclude antigen processing and presentation biology, which may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DCs to activate CD8 and CD4 T cells from convalescent people. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory tract cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the Alpha, Beta, Gamma, and Delta variant lineages.

Performance of anterior nares and tongue swabs for nucleic acid, Nucleocapsid, and Spike antigen testing for detecting SARS-CoV-2 against nasopharyngeal PCR and viral culture.

OBJECTIVES: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture. METHODS: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs. RESULTS: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing. CONCLUSIONS: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.