Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir.

The HIV-1 entry inhibitor temsavir prevents the viral receptor CD4 (cluster of differentiation 4) from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this, temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveals that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad antiviral activity.

Structure-function Analyses Reveal Key Molecular Determinants of HIV-1 CRF01_AE Resistance to the Entry Inhibitor Temsavir.

The HIV-1 entry inhibitor temsavir prevents CD4 from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir-resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveal that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad-antiviral activity.

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity.

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4+ T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD.

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

Evaluation of the performance of multiple immunoassay diagnostic platforms on the National Microbiology Laboratory SARS-CoV-2 National Serology Panel.

BACKGROUND: Serological assays designed to detect SARS-CoV-2 antibodies are being used in serological surveys and other specialized applications. As a result, and to ensure that the outcomes of serological testing meet high quality standards, evaluations are required to assess the performance of these assays and the proficiency of laboratories performing them. METHODS: A panel of 60 plasma/serum samples from blood donors who had reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections and 21 SARS-CoV-2 negative samples were secured and distributed to interested laboratories within Canada (n = 30) and the United States (n = 1). Participating laboratories were asked to provide details on the diagnostic assays used, the platforms the assays were performed on, and the results obtained for each panel sample. Laboratories were blinded with respect to the expected outcomes. RESULTS: The performance of the different assays evaluated was excellent, with the high-throughput platforms of Roche, Ortho, and Siemens demonstrating 100% sensitivity. Most other high-throughput platforms had sensitivities of >93%, with the exception of the IgG assay using the Abbott ARCHITECT which had an average sensitivity of only 87%. The majority of the high-throughput platforms also demonstrated very good specificities (>97%). CONCLUSION: This proficiency study demonstrates that most of the SARS-CoV-2 serological assays utilized by provincial public health or hospital laboratories in Canada have acceptable sensitivity and excellent specificity.

HISTORIQUE: Les dosages sérologiques conçus pour dépister les anticorps anti-SRAS-CoV-2 sont utilisés dans les études sérologiques et d'autres applications spécialisées. Par conséquent, et pour s'assurer que leurs résultats respectent des normes de qualité, il faut procéder à des évaluations de leur performance et de la compétence des laboratoires à les effectuer. MÉTHODOLOGIE: Les chercheurs ont obtenu une batterie de 60 prélèvements de plasma et de sérum chez des donneurs dont l'amplification en chaîne par polymérase après transcription inverse (RT-PCR) avait confirmé des infections par le SRAS-CoV-2 et de 21 prélèvements dont les résultats étaient négatifs au SRAS-CoV-2 et les ont distribués aux laboratoires intéressés du Canada (n = 30) et des États-Unis (n = 1). Ils ont invité les laboratoires participants à fournir de l'information détaillée sur les dosages diagnostiques utilisés, les plateformes sur lesquelles les dosages étaient exécutés et les résultats obtenus pour chaque échantillon. Les chercheurs ont demandé aux laboratoires participants de fournir de l'information détaillée sur les dosages diagnostiques utilisés, les plateformes sur lesquelles les dosages ont été effectués, et les résultats obtenus à l'égard de chaque échantillon. Les laboratoires ont mené les études à l'insu des résultats escomptés. RÉSULTATS: Les divers dosages avaient une excellente exécution, les plateformes à haut débit de Roche, d'Ortho et de Siemens démontrant une sensibilité de 100 %. La plupart des autres plateformes à haut débit avaient des sensibilités de plus de 93 %, à l'exception des dosages des IgG faisant appel à l'analyseur ARCHITECT d'Abbott, dont la sensibilité moyenne était de seulement 87 %. La majorité des plateformes à haut débit avaient également une très bonne spécificité (plus de 97 %). CONCLUSION: La présente étude de compétence démontre que la plupart des dosages sérologiques du SRAS-CoV-2 évalués dans des laboratoires sanitaires provinciaux ou les laboratoires hospitaliers du Canada possèdent une sensibilité acceptable et une excellente spécificité.

Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen.

Spacing of BNT162b2 mRNA doses beyond 3 weeks raises concerns about vaccine efficacy. We longitudinally analyze B cell, T cell, and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously infected donors. This regimen elicits robust RBD-specific B cell responses whose kinetics differs between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting does not increase magnitude of CD4+ T cell responses further compared with the first dose, unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. Integrated analysis shows longitudinal immune component-specific associations, with early T helper responses post first dose correlating with B cell responses after the second dose, and memory T helper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.

A Fc-enhanced NTD-binding non-neutralizing antibody delays virus spread and synergizes with a nAb to protect mice from lethal SARS-CoV-2 infection.

Emerging evidence indicates that both neutralizing and Fc-mediated effector functions of antibodies contribute to protection against SARS-CoV-2. It is unclear whether Fc-effector functions alone can protect against SARS-CoV-2. Here, we isolated CV3-13, a non-neutralizing antibody, from a convalescent individual with potent Fc-mediated effector functions. The cryoelectron microscopy structure of CV3-13 in complex with the SARS-CoV-2 spike reveals that the antibody binds from a distinct angle of approach to an N-terminal domain (NTD) epitope that only partially overlaps with the NTD supersite recognized by neutralizing antibodies. CV3-13 does not alter the replication dynamics of SARS-CoV-2 in K18-hACE2 mice, but its Fc-enhanced version significantly delays virus spread, neuroinvasion, and death in prophylactic settings. Interestingly, the combination of Fc-enhanced non-neutralizing CV3-13 with Fc-compromised neutralizing CV3-25 completely protects mice from lethal SARS-CoV-2 infection. Altogether, our data demonstrate that efficient Fc-mediated effector functions can potently contribute to the in vivo efficacy of anti-SARS-CoV-2 antibodies.

Detection of the HIV-1 Accessory Proteins Nef and Vpu by Flow Cytometry Represents a New Tool to Study Their Functional Interplay within a Single Infected CD4+ T Cell.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

Structural basis and mode of action for two broadly neutralizing antibodies against SARS-CoV-2 emerging variants of concern.

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here, we elucidate the structural basis and mode of action for two potent SARS-CoV-2 spike (S)-neutralizing monoclonal antibodies, CV3-1 and CV3-25, which remain effective against emerging variants of concern in vitro and in vivo. CV3-1 binds to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggers potent shedding of the S1 subunit. In contrast, CV3-25 inhibits membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among ß-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in the RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Strong humoral immune responses against SARS-CoV-2 Spike after BNT162b2 mRNA vaccination with a 16-week interval between doses.

The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration.

Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early predictor of COVID-19 mortality.

Despite advances in COVID-19 management, identifying patients evolving toward death remains challenging. To identify early predictors of mortality within 60 days of symptom onset (DSO), we performed immunovirological assessments on plasma from 279 individuals. On samples collected at DSO11 in a discovery cohort, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA), low receptor binding domain­specific immunoglobulin G and antibody-dependent cellular cytotoxicity, and elevated cytokines and tissue injury markers were strongly associated with mortality, including in patients on mechanical ventilation. A three-variable model of vRNA, with predefined adjustment by age and sex, robustly identified patients with fatal outcome (adjusted hazard ratio for log-transformed vRNA = 3.5). This model remained robust in independent validation and confirmation cohorts. Since plasma vRNA's predictive accuracy was maintained at earlier time points, its quantitation can help us understand disease heterogeneity and identify patients who may benefit from new therapies.

Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays.

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Contribution of single mutations to selected SARS-CoV-2 emerging variants spike antigenicity.

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37°C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

Across Functional Boundaries: Making Nonneutralizing Antibodies To Neutralize HIV-1 and Mediate Fc-Mediated Effector Killing of Infected Cells.

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, "closed" Env trimer present on HIV-1 virions or expressed on HIV-1-infected cells. Antibodies targeting this region are therefore nonneutralizing and their potential as mediators of antibody-dependent cellular cytotoxicity (ADCC) of HIV-1-infected cells diminished by a lack of available binding targets. Here, we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or cluster A specificity to the extracellular domains 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties, confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics that are effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening "closed" Env trimers on HIV-1 particles/infected cells to expose the cluster A region and activate ADCC and neutralization against these nonneutralizing targets. IMPORTANCE Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1-infected cells through antibody-dependent cellular cytotoxicity and inactivate HIV-1 virus through neutralization.

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern.

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among ß-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2.

The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Evaluation of a Commercial Culture-Free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison With an Antireceptor-Binding Domain Enzyme-Linked Immunosorbent Assay.

BACKGROUND: Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript) is the first such commercially available assay that detects antibodies that block receptor-binding domain (RBD)/angiotensin-converting enzyme (ACE)-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared with anti-RBD enzyme-linked immunosorbent assays (ELISAs). METHODS: Serum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD immunoglobulin (Ig)G, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection. RESULTS: Compared with PRNT-50, cPass sensitivity ranged from 77% to 100% and specificity was 95% to 100%. Sensitivity was also high compared with the pseudotyped lentiviral neutralization assay (93%; 95% confidence interval [CI], 85-97), but specificity was lower (58%; 95% CI, 48-67). Highest agreement between cPass and ELISA was for anti-RBD IgG (r = 0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99%; 95% CI, 94-100) was very similar to that of cPass, but overall specificity was lower (37%; 95% CI, 28-47). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical. CONCLUSIONS: The added value of cPass compared with an IgG anti-RBD ELISA was modest.