Naturally Acquired SARS-CoV-2 Immunity Persists for Up to 11 Months Following Infection.

BACKGROUND: Characterizing the kinetics of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of critical importance to developing strategies that may mitigate the public health burden of coronavirus disease 2019 (COVID-19). We conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma donors at multiple time points over an 11-month period to determine how circulating antibody levels change over time following natural infection. METHODS: From April 2020 to February 2021, we enrolled 228 donors. At each study visit, subjects either donated plasma or had study samples drawn only. Anti-SARS-CoV-2 donor testing was performed using the VITROS Anti-SARS-CoV-2 Total and IgG assays and an in-house fluorescence reduction neutralization assay. RESULTS: Anti-SARS-CoV-2 antibodies were identified in 97% of COVID-19 convalescent donors at initial presentation. In follow-up analyses, of 116 donors presenting at repeat time points, 91.4% had detectable IgG levels up to 11 months after symptom recovery, while 63% had detectable neutralizing titers; however, 25% of donors had neutralizing levels that dropped to an undetectable titer over time. CONCLUSIONS: Our data suggest that immunological memory is acquired in most individuals infected with SARS-CoV-2 and is sustained in a majority of patients for up to 11 months after recovery. Clinical Trials Registration. NCT04360278.

The Calcium Channel Blocker Bepridil Demonstrates Efficacy in the Murine Model of Marburg Virus Disease.

No therapeutics are approved for the treatment of filovirus infections. Bepridil, a calcium channel blocker developed for treating angina, was identified as a potent inhibitor of filoviruses in vitro, including Ebola and Marburg viruses, and Ebola virus in vivo. We evaluated the efficacy of bepridil in a lethal mouse model of Marburg virus disease. A dose of 12 mg/kg bepridil once or twice daily resulted in 80% or 90% survival, respectively. These data confirm bepridil's broad-spectrum anti-filovirus activity warranting further investigation of bepridil, or improved compounds with a similar mechanism, as a pan-filovirus therapeutic agent.

Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures.

Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.

Targeting memory reconsolidation to prevent the return of fear in patients with fear of flying.

BACKGROUND: When a memory is recalled, it may again exist in a labile state and stored information becomes amenable to change, a psychobiological process known as reconsolidation. Exposure therapy for anxiety disorders involves accessing a fear memory and modifying it with less fearful information. A preclinical study reported that providing a reminder of a fear memory 10 min prior to extinction training in humans decreased fear up to 1 year later (Schiller et al., 2010). METHODS: For this pilot clinical study, we used virtual reality exposure therapy (VRE) for fear of flying (FoF) to determine if using a cue to reactivate the memory of the feared stimulus 10 min prior to exposure sessions leads to fewer anxiety-related behaviors and a more durable response compared to a neutral cue. FoF participants (N = 89) received four sessions of anxiety management training followed by four sessions of VRE. Participants were randomly assigned to receive an FoF cue (reactivation group) or a neutral cue (control group) prior to the VRE sessions. Heart rate (HR) and skin conductance levels (SCLs) were collected during posttreatment and 3-month follow-up assessments as objective markers of fear responding. RESULTS: Treatment was effective and all clinical measures improved equally between groups at posttreatment with maintained gains through follow-ups. Significant differences were identified with regard to HR and SCL indices. CONCLUSIONS: These results suggest that memory reactivation prior to exposure therapy did not have an impact on clinical measures but may enhance the effect of exposure therapy at the physiological level.

Teaching Medical Students to Help Patients Quit Smoking: Outcomes of a 10-School Randomized Controlled Trial.

BACKGROUND: Early in medical education, physicians must develop competencies needed for tobacco dependence treatment. OBJECTIVE: To assess the effect of a multi-modal tobacco dependence treatment curriculum on medical students' counseling skills. DESIGN: A group-randomized controlled trial (2010-2014) included ten U.S. medical schools that were randomized to receive either multi-modal tobacco treatment education (MME) or traditional tobacco treatment education (TE). SETTING/PARTICIPANTS: Students from the classes of 2012 and 2014 at ten medical schools participated. Students from the class of 2012 (N = 1345) completed objective structured clinical examinations (OSCEs), and 50 % (N = 660) were randomly selected for pre-intervention evaluation. A total of 72.9 % of eligible students (N = 1096) from the class of 2014 completed an OSCE and 69.7 % (N = 1047) completed pre and post surveys. INTERVENTIONS: The MME included a Web-based course, a role-play classroom demonstration, and a clerkship booster session. Clerkship preceptors in MME schools participated in an academic detailing module and were encouraged to be role models for third-year students. MEASUREMENTS: The primary outcome was student tobacco treatment skills using the 5As measured by an objective structured clinical examination (OSCE) scored on a 33-item behavior checklist. Secondary outcomes were student self-reported skills for performing 5As and pharmacotherapy counseling. RESULTS: Although the difference was not statistically significant, MME students completed more tobacco counseling behaviors on the OSCE checklist (mean 8.7 [SE 0.6] vs. mean 8.0 [SE 0.6], p = 0.52) than TE students. Several of the individual Assist and Arrange items were significantly more likely to have been completed by MME students, including suggesting behavioral strategies (11.8 % vs. 4.5 %, p < 0.001) and providing information regarding quitline (21.0 % vs. 3.8 %, p < 0.001). MME students reported higher self-efficacy for Assist, Arrange, and Pharmacotherapy counseling items (ps ≤0.05). LIMITATIONS: Inclusion of only ten schools limits generalizability. CONCLUSIONS: Subsequent interventions should incorporate lessons learned from this first randomized controlled trial of a multi-modal longitudinal tobacco treatment curriculum in multiple U.S. medical schools. NIH Trial Registry Number: NCT01905618.

Neural network activation during a stop-signal task discriminates cocaine-dependent from non-drug-abusing men.

Cocaine dependence is defined by a loss of inhibitory control over drug-use behaviors, mirrored by measurable impairments in laboratory tasks of inhibitory control. The current study tested the hypothesis that deficits in multiple subprocesses of behavioral control are associated with reliable neural-processing alterations that define cocaine addiction. While undergoing functional magnetic resonance imaging (fMRI), 38 cocaine-dependent men and 27 healthy control men performed a stop-signal task of motor inhibition. An independent component analysis on fMRI time courses identified task-related neural networks attributed to motor, visual, cognitive and affective processes. The statistical associations of these components with five different stop-signal task conditions were selected for use in a linear discriminant analysis to define a classifier for cocaine addiction from a subsample of 26 cocaine-dependent men and 18 controls. Leave-one-out cross-validation accurately classified 89.5% (39/44; chance accuracy = 26/44 = 59.1%) of subjects with 84.6% (22/26) sensitivity and 94.4% (17/18) specificity. The remaining 12 cocaine-dependent and 9 control men formed an independent test sample, for which accuracy of the classifier was 81.9% (17/21; chance accuracy = 12/21 = 57.1%) with 75% (9/12) sensitivity and 88.9% (8/9) specificity. The cocaine addiction classification score was significantly correlated with a measure of impulsiveness as well as the duration of cocaine use for cocaine-dependent men. The results of this study support the ability of a pattern of multiple neural network alterations associated with inhibitory motor control to define a binary classifier for cocaine addiction.

Clinical correlates of attentional bias to drug cues associated with cocaine dependence.

BACKGROUND AND OBJECTIVE: Preoccupation (attentional bias) related to drug-related stimuli has been consistently observed for drug-dependent persons with several studies reporting an association of the magnitude of measured attentional bias with treatment outcomes. The major goal of the present study was to determine if pre-treatment attentional bias to personal drug use reminders in an addiction Stroop task predicts relapse in treatment-seeking, cocaine-dependent subjects. METHODS: We sought to maximize the potential of attentional bias as a marker of risk for relapse by incorporating individualized rather than generalized drug use cues to reflect the personal conditioned associations that form the incentive motivation properties of drug cues in a sample of cocaine-dependent subjects (N = 35). RESULTS: Although a significant group Stroop interference effect was present for drug versus neutral stimuli (ie, attentional bias), the level of attentional bias for cocaine-use words was not predictive of eventual relapse in this sample (d = .56). A similar lack of prediction power was observed for a non-drug counting word Stroop task as a significant interference effect was detected but did not predict relapse outcomes (d = .40). CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: The results of the present study do not provide clear support for the predictive value of individual variation in drug-related attentional bias to forecast probability of relapse in cocaine-dependent men.

Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity 1 . This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies 1, 2 . Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe 3 . Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants.

The emergence and global spread of the SARS-CoV-2 Omicron variants, which carry an unprecedented number of mutations, raise serious concerns due to the reduced efficacy of current vaccines and resistance to therapeutic antibodies. Here, we report the generation and characterization of two potent human monoclonal antibodies, NA8 and NE12, against the receptor-binding domain of the SARS-CoV-2 spike protein. NA8 interacts with a highly conserved region and has a breadth of neutralization with picomolar potency against the Beta variant and the Omicron BA.1 and BA.2 sublineages and nanomolar potency against BA.2.12.1 and BA.4. Combination of NA8 and NE12 retains potent neutralizing activity against the major SARS-CoV-2 variants of concern. Cryo-EM analysis provides the structural basis for the broad and complementary neutralizing activity of these two antibodies. We confirm the in vivo protective and therapeutic efficacies of NA8 and NE12 in the hamster model. These results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants.

Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants.

The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

Antiviral innate immunity is diminished in the upper respiratory tract of severe COVID-19 patients.

Understanding early innate immune responses to coronavirus disease 2019 (COVID-19) is crucial to developing targeted therapies to mitigate disease severity. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection elicits interferon expression leading to transcription of IFN-stimulated genes (ISGs) to control viral replication and spread. SARS-CoV-2 infection also elicits NF-κB signaling which regulates inflammatory cytokine expression contributing to viral control and likely disease severity. Few studies have simultaneously characterized these two components of innate immunity to COVID-19. We designed a study to characterize the expression of interferon alpha-2 (IFNA2) and interferon beta-1 (IFNB1), both type-1 interferons (IFN-1), interferon-gamma (IFNG), a type-2 interferon (IFN-2), ISGs, and NF-κB response genes in the upper respiratory tract (URT) of patients with mild (outpatient) versus severe (hospitalized) COVID-19. Further, we characterized the weekly dynamics of these responses in the upper and lower respiratory tracts (LRTs) and blood of severe patients to evaluate for compartmental differences. We observed significantly increased ISG and NF-κB responses in the URT of mild compared with severe patients early during illness. This pattern was associated with increased IFNA2 and IFNG expression in the URT of mild patients, a trend toward increased IFNB1-expression and significantly increased STING/IRF3/cGAS expression in the URT of severe patients. Our by-week across-compartment analysis in severe patients revealed significantly higher ISG responses in the blood compared with the URT and LRT of these patients during the first week of illness, despite significantly lower expression of IFNA2, IFNB1, and IFNG in blood. NF-κB responses, however, were significantly elevated in the LRT compared with the URT and blood of severe patients during peak illness (week 2). Our data support that severe COVID-19 is associated with impaired interferon signaling in the URT during early illness and robust pro-inflammatory responses in the LRT during peak illness.

Anti-SARS-CoV-2 Serology persistence over time in COVID-19 Convalescent Plasma Donors.

BACKGROUND: Characterizing the kinetics of the antibody response to SARS□CoV□2 is of critical importance to developing strategies that may mitigate the public health burden of COVID-19. We sought to determine how circulating antibody levels change over time following natural infection. METHODS/MATERIALS: We conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma (CCP) donors at multiple time points over a 9-month period. At each study visit, subjects either donated plasma or only had study samples drawn. In all cases, anti-SARS-CoV-2 donor testing was performed using semi-quantitative chemiluminescent immunoassays (ChLIA) targeting subunit 1 (S1) of the SARS-CoV-2 spike (S) protein, and an in-house fluorescence reduction neutralization assay (FRNA). RESULTS: From April to November 2020 we enrolled 202 donors, mean age 47.3 ±14.7 years, 55% female, 75% Caucasian. Most donors reported a mild clinical course (91%, n=171) without hospitalization. One hundred and five (105) (52%) donors presented for repeat visits with a median 42 (12-163) days between visits. The final visit occurred at a median 160 (53-273) days post-symptom resolution. Total anti-SARS-CoV-2 antibodies (Ab), SARS-CoV-2 specific IgG and neutralizing antibodies were detected in 97.5%, 91.1%, and 74% of donors respectively at initial presentation. Neutralizing Ab titers based on FRNA 50 were positively associated with mean IgG levels (p = <0.0001). Mean IgG levels and neutralizing titers were positively associated with COVID-19 severity, increased donor age and BMI (p=0.0006 and p=0.0028, p=0.0083 and p=0.0363, (p=0.0008 and p=0.0018, respectively). Over the course of repeat visits, IgG decreased in 74.1% of donors; FRNA 50 decreased in 44.4% and remained unchanged in 33.3% of repeat donors. A weak negative correlation was observed between total Ab levels and number of days post-symptom recovery (r = 0.09). CONCLUSION: Anti-SARS-CoV-2 antibodies were identified in 97% of convalescent donors at initial presentation. In a cohort that largely did not require hospitalization. IgG and neutralizing antibodies were positively correlated with age, BMI and clinical severity, and persisted for up to 9 months post-recovery from natural infection. On repeat presentation, IgG anti-SARS-CoV-2 levels decreased in 56% of repeat donors. Overall, these data suggest that CP donors possess a wide range of IgG and neutralizing antibody levels that are proportionally distributed across demographics, with the exception of age, BMI and clinical severity.

Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Longitudinal in vivo imaging of acute neuropathology in a monkey model of Ebola virus infection.

Ebola virus (EBOV) causes neurological symptoms yet its effects on the central nervous system (CNS) are not well-described. Here, we longitudinally assess the acute effects of EBOV on the brain, using quantitative MR-relaxometry, 18F-Fluorodeoxyglucose PET and immunohistochemistry in a monkey model. We report blood-brain barrier disruption, likely related to high cytokine levels and endothelial viral infection, with extravasation of fluid, Gadolinium-based contrast material and albumin into the extracellular space. Increased glucose metabolism is also present compared to the baseline, especially in the deep gray matter and brainstem. This regional hypermetabolism corresponds with mild neuroinflammation, sporadic neuronal infection and apoptosis, as well as increased GLUT3 expression, consistent with increased neuronal metabolic demands. Neuroimaging changes are associated with markers of disease progression including viral load and cytokine/chemokine levels. Our results provide insight into the pathophysiology of CNS involvement with EBOV and may help assess vaccine/treatment efficacy in real time.

Scalable, Micro-Neutralization Assay for Qualitative Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was expanding, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5,000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Acute Late-Stage Myocarditis in the Crab-Eating Macaque Model of Hemorrhagic Smallpox.

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

Efficacy and Safety Outcomes of Cataract Surgery in Survivors of Ebola Virus Disease: 12-Month Results From the PREVAIL VII Study.

Purpose: In survivors of Ebola virus disease (EVD), intraocular viral persistence raises questions about the timing and safety of cataract surgery. To the best of our knowledge, this is the first controlled study evaluating Ebola virus persistence and cataract surgery safety and outcomes in EVD survivors. Methods: Seropositive EVD survivors and seronegative controls with vision worse than 20/40 from cataract and without active intraocular inflammation were enrolled. Aqueous humor from survivors was tested with reverse transcription-polymerase chain reaction for Ebola viral RNA. Participants underwent manual small-incision cataract surgery and 1 year of follow-up examinations. Results: Twenty-two eyes of 22 survivors and 12 eyes of eight controls underwent cataract surgery. All of the aqueous samples tested negative for Ebola viral RNA. Median visual acuity improved from 20/200 at baseline to 20/25 at 1 year in survivors and from count fingers to 20/50 in controls (overall, P < 0.001; between groups, P = 0.07). After a 1-month course of topical corticosteroids, 55% of survivors and 67% of controls demonstrated at least 1+ anterior chamber cell. Twelve months after surgery, optical coherence tomography revealed a median increase in macular central subfield thickness of 42 µm compared with baseline (overall, P = 0.029; between groups, P = 0.995). Conclusions: EVD survivors and controls demonstrated significant visual improvement from cataract surgery. The persistence of intraocular inflammation highlights the importance of follow-up. The absence of detectable intraocular Ebola viral RNA provides guidance regarding the safety of eye surgery in Ebola survivors. Translational Relevance: These findings demonstrate the safety and efficacy of cataract surgery in Ebola survivors and will inform ocular surgery guidelines in this population.

Clinical, laboratory, and temporal predictors of neutralizing antibodies against SARS-CoV-2 among COVID-19 convalescent plasma donor candidates.

BACKGROUNDSARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2-neutralizing antibodies (nAbs) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood.METHODSPotential convalescent plasma donors with virologically documented SARS-CoV-2 infection were tested for serum IgG against SARS-CoV-2 spike protein S1 domain and against nucleoprotein (NP), and for nAb.RESULTSAmong 250 consecutive persons, including 27 (11%) requiring hospitalization, who were studied a median of 67 days since symptom onset, 97% were seropositive on 1 or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titers included older age (adjusted OR [AOR] 1.03 per year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. nAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range 77-120) apart (P < 0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses.CONCLUSIONnAb titers correlated with COVID-19 severity, age, and sex. SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels declined, and a small proportion of convalescent individuals lacked adaptive immune responses.FUNDINGThe project was supported by the Frederick National Laboratory for Cancer Research with support from the NIAID under contract number 75N91019D00024, and was supported by the Fred Hutchinson Joel Meyers Endowment, Fast-Grants, a New Investigator award from the American Society for Transplantation and Cellular Therapy, and NIH contracts 75N93019C0063, 75N91019D00024, and HHSN272201800013C, and NIH grants T32-AI118690, T32-AI007044, K08-AI119142, and K23-AI140918.

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.