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Serological assays are important diagnostic tools for surveying exposure to the pathogen, monitoring immune response post vaccination, and managing spread of the infectious agent among the population. Current serological laboratory assays are often limited because they require the use of specialized laboratory technology and/or work with a limited number of sample types. Here, we evaluate an alternative by developing time-resolved Förster resonance energy transfer (TR-FRET) homogeneous assays that exhibited exceptional versatility, scalability, and sensitivity and outperformed or matched currently used strategies in terms of sensitivity, specificity, and precision. We validated the performance of the assays measuring total immunoglobulin G (IgG) levels; antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle Eastern respiratory syndrome (MERS)-CoV spike (S) protein; and SARS-CoV-2 S and nucleocapsid (N) proteins and applied it to several large sample sets and real-world applications. We further established a TR-FRET-based ACE2-S competition assay to assess the neutralization propensity of the antibodies. Overall, these TR-FRET-based serological assays can be rapidly extended to other antigens and are compatible with commonly used plate readers., Competing Interests: E.S.F. is an equity holder and scientific advisor for Neomorph, Inc. (board member), Civetta Therapeutics, Proximity Therapeutics, Lighthorse Therapeutics, Avilar Therapeutics, and Photys Therapeutics and is a consultant to Novartis, Sanofi, AbbVie, Pfizer, Astellas, EcoR1 Capital, and Deerfield. The Fischer lab receives or has received research funding from Novartis, Ajax, Deerfield, and Astellas not related to this work. R.M. is a scientific advisory board (SAB) member and equity holder of Regenacy Pharmaceuticals, ERX Pharmaceuticals, and Frequency Therapeutics. H.Y., R.P.N., D.O., N.C.P., R.M., and E.S.F. are inventors on patent applications related to this work., (© 2023 The Author(s).)

Background: Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities., Methods: This cross-sectional study, implemented in 2020, screened PCRconfirmed coronavirus disease 2019 patients (n 86), banked prepandemic and negative samples (n 96), healthcare workers and family members (n 552), and university employees (n 327) for antiSARS-CoV-2 receptor-binding domain, trimeric spike protein, and nucleocapsid protein immunoglobulin (Ig)G and IgA antibodies with a laboratory-developed enzyme-linked immunosorbent assay and tested how antigen, isotype and threshold choices affected the seroprevalence outcomes. The following threshold methods were evaluated: (i) mean 3 standard deviations of the negative controls; (ii) 100 specificity for each antigen-isotype combination; and (iii) the maximal Youden index., Results: We found vastly different seroprevalence estimates depending on selected antigens and isotypes and the applied threshold method, ranging from 0.0 to 85.4. Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3 to 25.9., Conclusions: This study revealed the importance of evaluating serosurvey tools for antigen-, isotype-, and threshold-specific sensitivity and specificity, to interpret qualitative serosurvey outcomes reliably and consistently across studies., (The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Persistent SARS-CoV-2 replication and systemic dissemination are linked to increased COVID-19 disease severity and mortality. However, the precise immune profiles that track with enhanced viral clearance, particularly from systemic RNAemia, remain incompletely defined. To define whether antibody characteristics, specificities, or functions that emerge during natural infection are linked to accelerated containment of viral replication, we examined the relationship of SARS-CoV-2-specific humoral immune evolution in the setting of SARS-CoV-2 plasma RNAemia, which is tightly associated with disease severity and death. On presentation to the emergency department, S-specific IgG3, IgA1, and Fc-γ-receptor (Fcγ R) binding antibodies were all inversely associated with higher baseline plasma RNAemia. Importantly, the rapid development of spike (S) and its subunit (S1/S2/receptor binding domain)-specific IgG, especially FcγR binding activity, were associated with clearance of RNAemia. These results point to a potentially critical and direct role for SARS-CoV-2-specific humoral immune clearance on viral dissemination, persistence, and disease outcome, providing novel insights for the development of more effective therapeutics to resolve COVID-19. IMPORTANCE We showed that persistent SARS-CoV-2 RNAemia is an independent predictor of severe COVID-19. We observed that SARS-CoV-2-targeted antibody maturation, specifically Fc-effector functions rather than neutralization, was strongly linked with the ability to rapidly clear viremia. This highlights the critical role of key humoral features in preventing viral dissemination or accelerating viremia clearance and provides insights for the design of next-generation monoclonal therapeutics. The main key points will be that (i) persistent SARS-CoV-2 plasma RNAemia independently predicts severe COVID-19 and (ii) specific humoral immune functions play a critical role in halting viral dissemination and controlling COVID-19 disease progression.

Background: Estimating the cumulative incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for setting public health policies. We leveraged deidentified Massachusetts newborn screening specimens as an accessible, retrospective source of maternal antibodies for estimating statewide seroprevalence in a nontest-seeking population., Methods: We analyzed 72 117 newborn specimens collected from November 2019 through December 2020, representing 337 towns and cities across Massachusetts. Seroprevalence was estimated for the Massachusetts population after correcting for imperfect test specificity and nonrepresentative sampling using Bayesian multilevel regression and poststratification., Results: Statewide seroprevalence was estimated to be 0.03% (90% credible interval [CI], 0.00-0.11) in November 2019 and rose to 1.47% (90% CI: 1.00-2.13) by May 2020, following sustained SARS-CoV-2 transmission in the spring. Seroprevalence plateaued from May onward, reaching 2.15% (90% CI: 1.56-2.98) in December 2020. Seroprevalence varied substantially by community and was particularly associated with community percent non-Hispanic Black (β = .024; 90% CI: 0.004-0.044); i.e., a 10% increase in community percent non-Hispanic Black was associated with 27% higher odds of seropositivity. Seroprevalence estimates had good concordance with reported case counts and wastewater surveillance for most of 2020, prior to the resurgence of transmission in winter., Conclusions: Cumulative incidence of SARS-CoV-2 protective antibody in Massachusetts was low as of December 2020, indicating that a substantial fraction of the population was still susceptible. Maternal seroprevalence data from newborn screening can inform longitudinal trends and identify cities and towns at highest risk, particularly in settings where widespread diagnostic testing is unavailable., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of the currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from the disease, but little is known about SARS-CoV-2 antibody effector functions. Here, we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). Although the neutralizing responses to VOCs decreased in both groups, the Fc-mediated responses were distinct. In convalescent individuals, although antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued to interact with Fc-receptors and mediate antibody effector functions. These data point to a resilience in the mRNA-vaccine-induced humoral immune response that may continue to offer protection from SARS-CoV-2 VOCs independent of neutralization., Competing Interests: Declaration of interests G.A. is the founder of Seromyx Systems Inc. A.C. is an employee of Moderna Inc. D.D., P.M., A.S.M., and E.R.M. are employees of Space Exploration Technologies Corp. All other authors have declared no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Serological testing provides a record of prior infection with SARS-CoV-2, but assay performance requires independent assessment., Methods: We evaluated 3 commercial (Roche Diagnostics pan-IG, and Epitope Diagnostics IgM and IgG) and 2 non-commercial (Simoa and Ragon/MGH IgG) immunoassays against 1083 unique samples that included 251 PCR-positive and 832 prepandemic samples., Results: The Roche assay registered the highest specificity 99.6% (3/832 false positives), the Ragon/MGH assay 99.5% (4/832), the primary Simoa assay model 99.0% (8/832), and the Epitope IgG and IgM 99.0% (8/830) and 99.5% (4/830), respectively. Overall sensitivities for the Simoa, Roche pan-IG, Epitope IgG, Ragon/MGH IgG, and Epitope IgM were 92.0%, 82.9%, 82.5%, 64.5% and 47.0%, respectively. The Simoa immunoassay demonstrated the highest sensitivity among samples stratified by days postsymptom onset (PSO), <8 days PSO (57.69%) 8-14 days PSO (93.51%), 15-21 days PSO (100%), and > 21 days PSO (95.18%)., Conclusions: All assays demonstrated high to very high specificities while sensitivities were variable across assays., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The introduction of vaccines has inspired hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against SARS-CoV-2, thus we profiled the earliest humoral signatures in a large cohort of acutely ill (survivors and nonsurvivors) and mild or asymptomatic individuals with COVID-19. Although a SARS-CoV-2–specific immune response evolved rapidly in survivors of COVID-19, nonsurvivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibodies. Given the conservation of S2 across β-coronaviruses, we found that the early development of SARS-CoV-2–specific immunity occurred in tandem with preexisting common β-coronavirus OC43 humoral immunity in survivors, which was also selectively expanded in individuals that develop a paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative 1 . Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Comorbid medical illnesses, such as obesity and diabetes, are associated with more severe COVID-19, hospitalization, and death. However, the role of the immune system in mediating these clinical outcomes has not been determined. We used multiparameter flow cytometry and systems serology to comprehensively profile the functions of T cells and antibodies targeting spike, nucleocapsid, and envelope proteins in a convalescent cohort of COVID-19 subjects who were either hospitalized (n = 20) or not hospitalized (n = 40). To avoid confounding, subjects were matched by age, sex, ethnicity, and date of symptom onset. Surprisingly, we found that the magnitude and functional breadth of virus-specific CD4+ T cell and antibody responses were consistently higher among hospitalized subjects, particularly those with medical comorbidities. However, an integrated analysis identified more coordination between polyfunctional CD4+ T cells and antibodies targeting the S1 domain of spike among subjects who were not hospitalized. These data reveal a functionally diverse and coordinated response between T cells and antibodies targeting SARS-CoV-2, which is reduced in the presence of comorbid illnesses that are known risk factors for severe COVID-19.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8 + T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8 + T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2., Competing Interests: Declaration of interests R.R.A., Sailaja Gangadhara, and N.K.R. are co-inventors of the MVA/S vaccine technology. Emory University filed a patent on this technology. R.R.A. serves as a SAB member for Heat Biologics Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread relentlessly, associated with a high frequency of respiratory failure and mortality. Children experience largely asymptomatic disease, with rare reports of multisystem inflammatory syndrome in children (MIS-C). Identifying immune mechanisms that result in these disparate clinical phenotypes in children could provide critical insights into coronavirus disease 2019 (COVID-19) pathogenesis. Using systems serology, in this study we observed in 25 children with acute mild COVID-19 a functional phagocyte and complement-activating IgG response to SARS-CoV-2, similar to the acute responses generated in adults with mild disease. Conversely, IgA and neutrophil responses were significantly expanded in adults with severe disease. Moreover, weeks after the resolution of SARS-CoV-2 infection, children who develop MIS-C maintained highly inflammatory monocyte-activating SARS-CoV-2 IgG antibodies, distinguishable from acute disease in children but with antibody levels similar to those in convalescent adults. Collectively, these data provide unique insights into the potential mechanisms of IgG and IgA that might underlie differential disease severity as well as unexpected complications in children infected with SARS-CoV-2.

Antibodies serve as biomarkers of infection, but if sustained can confer long-term immunity. Yet, for most clinically approved vaccines, binding antibody titers only serve as a surrogate of protection. Instead, the ability of vaccine induced antibodies to neutralize or mediate Fc-effector functions is mechanistically linked to protection. While evidence has begun to point to persisting antibody responses among SARS-CoV-2 infected individuals, cases of re-infection have begun to emerge, calling the protective nature of humoral immunity against this highly infectious pathogen into question. Using a community-based surveillance study, we aimed to define the relationship between titers and functional antibody activity to SARS-CoV-2 over time. Here we report significant heterogeneity, but limited decay, across antibody titers amongst 120 identified seroconverters, most of whom had asymptomatic infection. Notably, neutralization, Fc-function, and SARS-CoV-2 specific T cell responses were only observed in subjects that elicited RBD-specific antibody titers above a threshold. The findings point to a switch-like relationship between observed antibody titer and function, where a distinct threshold of activity-defined by the level of antibodies-is required to elicit vigorous humoral and cellular response. This response activity level may be essential for durable protection, potentially explaining why re-infections occur with SARS-CoV-2 and other common coronaviruses.

SARS-CoV-2 infection causes more severe disease in pregnant women compared to age-matched non-pregnant women. Whether maternal infection causes changes in the transfer of immunity to infants remains unclear. Maternal infections have previously been associated with compromised placental antibody transfer, but the mechanism underlying this compromised transfer is not established. Here, we used systems serology to characterize the Fc profile of influenza-, pertussis-, and SARS-CoV-2-specific antibodies transferred across the placenta. Influenza- and pertussis-specific antibodies were actively transferred. However, SARS-CoV-2-specific antibody transfer was significantly reduced compared to influenza- and pertussis-specific antibodies, and cord titers and functional activity were lower than in maternal plasma. This effect was only observed in third-trimester infection. SARS-CoV-2-specific transfer was linked to altered SARS-CoV-2-antibody glycosylation profiles and was partially rescued by infection-induced increases in IgG and increased FCGR3A placental expression. These results point to unexpected compensatory mechanisms to boost immunity in neonates, providing insights for maternal vaccine design., Competing Interests: Declaration of interests G.A. is the founder of Seromyx. K.J.G. has consulted for BillionToOne, Quest Diagnostics, Illumina, and Aetion. A.A.B. has consulted for Microchips Biotech and is also a Scientific Advisory Board Member for Reproductive Health Investors Alliance. D.P. owns stock in Gilead Sciences, BioNano Genomics, Biogen, Bluebird Bio, ImmunoGen, Pfizer, and Bristol-Myers Squibb. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross-SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection.

The urgent need for an effective SARS-CoV-2 vaccine has forced development to progress in the absence of well-defined correlates of immunity. While neutralization has been linked to protection against other pathogens, whether neutralization alone will be sufficient to drive protection against SARS-CoV-2 in the broader population remains unclear. Therefore, to fully define protective humoral immunity, we dissected the early evolution of the humoral response in 193 hospitalized individuals ranging from moderate to severe. Although robust IgM and IgA responses evolved in both survivors and non-survivors with severe disease, non-survivors showed attenuated IgG responses, accompanied by compromised Fcɣ receptor binding and Fc effector activity, pointing to deficient humoral development rather than disease-enhancing humoral immunity. In contrast, individuals with moderate disease exhibited delayed responses that ultimately matured. These data highlight distinct humoral trajectories associated with resolution of SARS-CoV-2 infection and the need for early functional humoral immunity., Competing Interests: Declaration of Interests G.A. is a founder of SeromYx Systems, Inc. The Systems Serology platform is pending as a patent to G.A. No other authors have interests to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) in humans is often a clinically mild illness, but some individuals develop severe pneumonia, respiratory failure and death 1-4 . Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in hamsters 5-7 and nonhuman primates 8-10 have generally reported mild clinical disease, and preclinical SARS-CoV-2 vaccine studies have demonstrated reduction of viral replication in the upper and lower respiratory tracts in nonhuman primates 11-13 . Here we show that high-dose intranasal SARS-CoV-2 infection in hamsters results in severe clinical disease, including high levels of virus replication in tissues, extensive pneumonia, weight loss and mortality in a subset of animals. A single immunization with an adenovirus serotype 26 vector-based vaccine expressing a stabilized SARS-CoV-2 spike protein elicited binding and neutralizing antibody responses and protected against SARS-CoV-2-induced weight loss, pneumonia and mortality. These data demonstrate vaccine protection against SARS-CoV-2 clinical disease. This model should prove useful for preclinical studies of SARS-CoV-2 vaccines, therapeutics and pathogenesis.