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SARS-CoV-2 variants have continuously emerged even as highly effective vaccines have been widely deployed. Reduced neutralization observed against variants of concern (VOC) raises the question as to whether other antiviral antibody activities are similarly compromised, or if they might compensate for lost neutralization activity. In this study, the breadth and potency of antibody recognition and effector function was surveyed in both healthy individuals as well as immunologically vulnerable subjects following either natural infection or receipt of an mRNA vaccine. Considering pregnant women as a model cohort with higher risk of severe illness and death, we observed similar binding and functional breadth for healthy and immunologically vulnerable populations. In contrast, considerably greater functional antibody breadth and potency across VOC was associated with vaccination than prior infection. However, greater antibody functional activity targeting the endemic coronavirus OC43 was noted among convalescent individuals, illustrating a dichotomy in recognition between close and distant human coronavirus strains that was associated with exposure history. Probing the full-length spike and receptor binding domain (RBD) revealed that antibody-mediated Fc effector functions were better maintained against full-length spike as compared to RBD. This analysis of antibody functions in healthy and vulnerable populations across a panel of SARS-CoV-2 VOC and extending through endemic alphacoronavirus strains suggests the differential potential for antibody effector functions to contribute to protecting vaccinated and convalescent subjects as the pandemic progresses and novel variants continue to evolve. One Sentence SummaryAs compared to natural infection with SARS-CoV-2, vaccination drives superior functional antibody breadth raising hopes for candidate universal CoV vaccines.
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Neutralizing antibody responses are attenuated in many solid organ transplant recipients (SOTRs) despite SARS-CoV-2 vaccination. Pre-exposure prophylaxis (PrEP) with the monoclonal antibody combination Tixagevimab and Cilgavimab (T+C) might augment immunoprotection, yet activity against Omicron sublineages in vaccinated SOTRs is unknown. Vaccinated SOTRs who received 300+300mg T+C (either single dose or two 150+150mg doses) within a prospective observational cohort submitted pre- and post-injection samples between 1/10/2022-4/4/2022. Binding antibody (anti-receptor binding domain [RBD], Roche) and surrogate neutralization (%ACE2 inhibition; [≥]20% connoting neutralizing inhibition, Meso Scale Discovery) were measured against variants including Omicron sublineages BA.1 and BA.2. Data were analyzed using the Wilcoxon matched-pairs signed-rank test and McNemars test. Among 61 participants, median (IQR) anti-RBD increased from 424 (IQR <0.8-2322.5) to 3394.5 (IQR 1403.9-7002.5) U/ml post T+C (p<0.001). The proportion demonstrating vaccine strain neutralizing inhibition increased from 46% to 100% post-T+C (p<0.001). BA.1 neutralization was low and did not increase (8% to 16% of participants post-T+C, p=0.06). In contrast, BA.2 neutralization increased from 7% to 72% of participants post-T+C (p<0.001). T+C increased anti-RBD levels, yet BA.1 neutralizing activity was minimal. Encouragingly, BA.2 neutralization was augmented and in the current variant climate T+C PrEP may serve as a useful complement to vaccination in high-risk SOTRs.
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The ongoing evolution of SARS-Co-V2 variants to omicron severely limits available effective monoclonal antibody therapies. Effective drugs are also supply limited. Covid-19 convalescent plasma (CCP) qualified for high antibody levels effectively reduces immunocompetent outpatient hospitalization. The FDA currently allows outpatient CCP for the immunosuppressed. Viral specific antibody levels in CCP can range ten-to hundred-fold between donors unlike the uniform viral specific monoclonal antibody dosing. Limited data are available on the efficacy of polyclonal CCP to neutralize variants. We examined 108 pre-delta/pre-omicron donor units obtained before March 2021, 20 post-delta COVID-19/post-vaccination units and one pre-delta/pre-omicron hyperimmunoglobulin preparation for variant specific virus (vaccine-related isolate (WA-1), delta and omicron) neutralization correlated to Euroimmun S1 IgG antibody levels. We observed a 2-to 4-fold and 20-to 40-fold drop in virus neutralization from SARS-CoV-2 WA-1 to delta or omicron, respectively. CCP antibody levels in the upper 10% of the 108 donations as well as 100% of the post-delta COVID-19/post-vaccination units and the hyperimmunoglobulin effectively neutralized all three variants. High-titer CCP neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants. Key pointsAll of the post-delta COVID-19/post vaccination convalescent plasma effectively neutralizes the omicron and delta variants. High-titer CCP and hyperimmunoglobulin neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants.
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The association between COVID-19 symptoms and antibody responses against SARS-CoV-2 is poorly characterized. We analyzed antibody levels in individuals with known SARS-CoV-2 infection to identify potential antibody-symptom associations. Convalescent plasma from 216 SARS-CoV-2 RNA+ individuals with symptomatology information were tested for the presence of IgG to the spike S1 subunit (Euroimmun ELISA), IgG to receptor binding domain (RBD, CoronaCHEK rapid test), and for IgG, IgA, and IgM to nucleocapsid (N, Bio-Rad ELISA). Logistic regression was used to estimate the odds of having a COVID-19 symptom from the antibody response, adjusting for sex and age. Cough strongly associated with antibodies against S1 (adjusted odds ratio [aOR]= 5.33; 95% CI from 1.51 to 18.86) and RBD (aOR=4.36; CI 1.49, 12.78). In contrast, sore throat significantly associated with the absence of antibodies to S1 and N (aOR=0.25; CI 0.08, 0.80 and aOR=0.31; 0.11, 0.91). Similarly, lack of symptoms associated with the absence of antibodies to N and RBD (aOR=0.16; CI 0.03, 0.97 and aOR=0.16; CI 0.03, 1.01). Cough appeared to be correlated with a seropositive result, suggesting that SARS-CoV-2 infected individuals exhibiting lower respiratory symptoms generate a robust antibody response. Conversely, those without symptoms or limited to a sore throat while infected with SARS-CoV-2 were likely to lack a detectable antibody response. These findings strongly support the notion that severity of infection correlates with robust antibody response.
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BACKGROUNDThe efficacy of polyclonal high titer convalescent plasma to prevent serious complications of COVID-19 in outpatients with recent onset of illness is uncertain. METHODSThis multicenter, double-blind randomized controlled trial compared the efficacy and safety of SARS-CoV-2 high titer convalescent plasma to placebo control plasma in symptomatic adults [≥]18 years positive for SARS-CoV-2 regardless of risk factors for disease progression or vaccine status. Participants with symptom onset within 8 days were enrolled, then transfused within the subsequent day. The measured primary outcome was COVID-19-related hospitalization within 28 days of plasma transfusion. The enrollment period was June 3, 2020 to October 1, 2021. RESULTSA total of 1225 participants were randomized and 1181 transfused. In the pre-specified modified intention-to-treat analysis that excluded those not transfused, the primary endpoint occurred in 37 of 589 (6.3%) who received placebo control plasma and in 17 of 592 (2.9%) participants who received convalescent plasma (relative risk, 0.46; one-sided 95% upper bound confidence interval 0.733; P=0.004) corresponding to a 54% risk reduction. Examination with a model adjusting for covariates related to the outcome did not change the conclusions. CONCLUSIONEarly administration of high titer SARS-CoV-2 convalescent plasma reduced outpatient hospitalizations by more than 50%. High titer convalescent plasma is an effective early outpatient COVID-19 treatment with the advantages of low cost, wide availability, and rapid resilience to variant emergence from viral genetic drift in the face of a changing pandemic. Trial RegistrationClinicalTrials.gov number, NCT04373460.
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There is a growing concern that ongoing evolution of SARS-CoV-2 could lead to variants of concern (VOC) that are capable of avoiding some or all of the multi-faceted immune response generated by both prior infection or vaccination, with the recently described B.1.1.529 (Omicron) VOC being of particular interest. Peripheral blood mononuclear cell samples from PCR-confirmed, recovered COVID-19 convalescent patients (n=30) infected with SARS-CoV-2 in the United States collected in April and May 2020 who possessed at least one or more of six different HLA haplotypes were selected for examination of their anti-SARS-CoV-2 CD8+ T-cell responses using a multiplexed peptide-MHC tetramer staining approach. This analysis examined if the previously identified viral epitopes targeted by CD8+ T-cells in these individuals (n=52 distinct epitopes) are mutated in the newly described Omicron VOC (n=50 mutations). Within this population, only one low-prevalence epitope from the Spike protein restricted to two HLA alleles and found in 2/30 (7%) individuals contained a single amino acid change associated with the Omicron VOC. These data suggest that virtually all individuals with existing anti-SARS-CoV-2 CD8+ T-cell responses should recognize the Omicron VOC, and that SARS-CoV-2 has not evolved extensive T-cell escape mutations at this time. ImportanceThe newly identified Omicron variant of concern contains more mutations than any of the previous variants described to date. In addition, many of the mutations associated with the Omicron variant are found in areas that are likely bound by neutralizing antibodies, suggesting that the first line of immunological defense against COVID-19 may be compromised. However, both natural infection and vaccination develop T-cell based responses, in addition to antibodies. This study examined if the parts of the virus, or epitopes, targeted by the CD8+ T-cell response in thirty individuals who recovered from COVID-19 in 2020 were mutated in the Omicron variant. Only one of 52 epitopes identified in this population contained an amino acid that was mutated in Omicron. These data suggest that the T-cell immune response in previously infected, and most likely vaccinated individuals, should still be effective against Omicron.
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Pre-existing antibodies to endemic coronaviruses (CoV) that cross-react with SARS-CoV-2 have the potential to influence the antibody response to COVID-19 vaccination and infection for better or worse. In this observational study of mucosal and systemic humoral immunity in acutely infected, convalescent, and vaccinated subjects, we tested for cross reactivity against endemic CoV spike (S) protein at subdomain resolution. Elevated responses, particularly to the {beta}-CoV OC43, were observed in all natural infection cohorts tested and were correlated with the response to SARS-CoV-2. The kinetics of this response and isotypes involved suggest that infection boosts preexisting antibody lineages raised against prior endemic CoV exposure that cross react. While further research is needed to discern whether this recalled response is desirable or detrimental, the boosted antibodies principally targeted the better conserved S2 subdomain of the viral spike and were not associated with neutralization activity. In contrast, vaccination with a stabilized spike mRNA vaccine did not robustly boost cross-reactive antibodies, suggesting differing antigenicity and immunogenicity. In sum, this study provides evidence that antibodies targeting endemic CoV are robustly boosted in response to SARS-CoV-2 infection but not to vaccination with stabilized S, and that depending on conformation or other factors, the S2 subdomain of the spike protein triggers a rapidly recalled, IgG-dominated response that lacks neutralization activity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/21265574v1_figA1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@168d38aorg.highwire.dtl.DTLVardef@1183afcorg.highwire.dtl.DTLVardef@1c88b77org.highwire.dtl.DTLVardef@13c6e0a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Antibody responses to SARS-CoV-2 and endemic CoV spike proteins were measured in diverse cohorts. While antibodies to SARS-CoV-2 were induced across all isotypes, only IgA and IgG responses to endemic CoV were robustly boosted, and only among naturally-infected but not vaccinated individuals. These recalled, cross-reactive responses to endemic CoV primarily recognized the better conserved S2 domain and were non-neutralizing. While other antiviral activities of broadly cross-reactive S2-specifc antibodies are not known, the differing antigenicity of natural infection and vaccination with stabilized pre-fusion spike has potential implications for the breadth and level of protection afforded by each. C_FIG
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BackgroundEmergency Departments (EDs) can serve as surveillance sites for infectious diseases. Our purpose was to determine the burden of SARS-CoV-2 infection and prevalence of vaccination against COVID-19 among patients attending an urban ED in Baltimore City. MethodsUsing 1914 samples of known exposure status, we developed an algorithm to differentiate previously infected, vaccinated, and unexposed individuals using a combination of antibody assays. We applied this testing algorithm to 4360 samples ED patients obtained in the springs of 2020 and 2021. Using multinomial logistic regression, we determined factors associated with infection and vaccination. ResultsFor the algorithm, sensitivity and specificity for identifying vaccinated individuals was 100% and 99%, respectively, and 84% and 100% for naturally infected individuals. Among the ED subjects, seroprevalence to SARS-CoV-2 increased from 2% to 24% between April 2020 and March 2021. Vaccination prevalence rose to 11% by mid-March 2021. Marked differences in burden of disease and vaccination coverage were seen by sex, race, and ethnicity. Hispanic patients, though 7% of the study population, had the highest relative burden of disease (17% of total infections) but similar vaccination rates. Women and White individuals were more likely to be vaccinated than men or Black individuals (adjusted odds ratios [aOR] 1.35 [95% CI: 1.02, 1.80] and aOR 2.26 [95% CI: 1.67, 3.07], respectively). ConclusionsIndividuals previously infected with SARS-CoV-2 can be differentiated from vaccinated individuals using a serologic testing algorithm. SARS-CoV-2 exposure and vaccination uptake frequencies reflect gender, race and ethnic health disparities in this urban context. SummaryUsing an antibody testing algorithm, we distinguished between immune responses from SARS-CoV-2-infected and vaccinated individuals. When applied to blood samples from an emergency department in Baltimore, disparities in disease burden and vaccine uptake by sex, race, and ethnicity were identified.
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Vaccine-induced SARS-CoV-2 antibody responses are attenuated in solid organ transplant recipients (SOTRs) and breakthrough infections are more common. Additional SARS-CoV-2 vaccine doses increase anti-spike IgG in some SOTRs, but it is uncertain whether neutralization of variants of concern (VOCs) is enhanced. We tested 47 SOTRs for clinical and research anti-spike IgG, pseudoneutralization (ACE2 blocking), and live-virus neutralization (nAb) against VOCs before and after a third SARS-CoV-2 vaccine dose (70% mRNA, 30% Ad26.COV2.S) with comparison to 15 healthy controls after two mRNA vaccine doses. We used correlation analysis to compare anti-spike IgG assays and focused on thresholds associated with neutralizing activity. A third SARS-CoV-2 vaccine dose increased median anti-spike (1.6-fold) and receptor-binding domain (1.5-fold) IgG, as well as pseudoneutralization against VOCs (2.5-fold versus Delta). However, IgG and neutralization activity were significantly lower than healthy controls (p<0.001); 32% of SOTRs had zero detectable nAb against Delta after third vaccination. Correlation with nAb was seen at anti-spike IgG >4 AU on the clinical assay and >10^4 AU on the research assay. These findings highlight benefits of a third vaccine dose for some SOTRs and the need for alternative strategies to improve protection in a significant subset of this population.
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While antibodies provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined. In this study, we employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action. To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These predictive models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion. Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.
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This study examined whether CD8+ T-cell responses from COVID-19 convalescent individuals(n=30) potentially maintain recognition of the major SARS-CoV-2 variants. Out of 45 mutations assessed, only one from the B.1.351 Spike overlapped with a low-prevalence CD8+ epitope, suggesting that virtually all anti-SARS-CoV-2 CD8+ T-cell responses should recognize these newly described variants.
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Oral fluid (hereafter saliva) offers a non-invasive sampling method for the detection of SARS-CoV-2 antibodies. However, data comparing performance of salivary tests against commercially-available serologic and neutralizing antibody (nAb) assays are lacking. This study compared the performance of a multiplex salivary SARS-CoV-2 IgG assay targeting antibodies to nucleocapsid (N), receptor binding domain (RBD) and spike (S) antigens to three commercially-available SARS-CoV-2 serology enzyme immunoassays (EIAs) (Ortho Vitros, Euroimmun, and BioRad) and nAb. Paired saliva and plasma samples were collected from 101 eligible COVID-19 convalescent plasma (CCP) donors >14 days since PCR+ confirmed diagnosis. Concordance was evaluated using positive (PPA) and negative (NPA) percent agreement, overall percent agreement (PA), and Cohens kappa coefficient. The range between salivary and plasma EIAs for SARS-CoV-2-specific N was PPA: 54.4-92.1% and NPA: 69.2-91.7%, for RBD was PPA: 89.9-100% and NPA: 50.0-84.6%, and for S was PPA: 50.6-96.6% and NPA: 50.0-100%. Compared to a plasma nAb assay, the multiplex salivary assay PPA ranged from 62.3% (N) and 98.6% (RBD) and NPA ranged from 18.8% (RBD) to 96.9% (S). Combinations of N, RBD, and S and a summary algorithmic index of all three (N/RBD/S) in saliva produced ranges of PPA: 87.6-98.9% and NPA: 50-91.7% with the three EIAs and ranges of PPA: 88.4-98.6% and NPA: 21.9-34.4% with the nAb assay. A multiplex salivary SARS-CoV-2 IgG assay demonstrated comparable performance to three commercially-available plasma EIAs and a nAb assay, and may be a viable alternative to assist in screening CCP donors and monitoring population-based seroprevalence and vaccine antibody response.
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Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.
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Convalescent plasma has emerged as a promising COVID-19 treatment. However, the humoral factors that contribute to efficacy are poorly understood. This study functionally and phenotypically profiled plasma from eligible convalescent donors. In addition to viral neutralization, convalescent plasma contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis and antibody-dependent cellular cytotoxicity against SARS-CoV-2. These activities expand the antiviral functions associated with convalescent plasma and together with neutralization efficacy, could be accurately and robustly from antibody phenotypes. These results suggest that high-throughput profiling could be used to screen donors and plasma may provide benefits beyond neutralization.
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It remains unclear why some patients infected with SARS-CoV-2 readily resolve infection while others develop severe disease. To address this question, we employed a novel assay to interrogate immune-metabolic programs of T cells and myeloid cells in severe and recovered COVID-19 patients. Using this approach, we identified a unique population of T cells expressing high H3K27me3 and the mitochondrial membrane protein voltage-dependent anion channel (VDAC), which were expanded in acutely ill COVID-19 patients and distinct from T cells found in patients infected with hepatitis c or influenza and in recovered COVID-19. Increased VDAC was associated with gene programs linked to mitochondrial dysfunction and apoptosis. High-resolution fluorescence and electron microscopy imaging of the cells revealed dysmorphic mitochondria and release of cytochrome c into the cytoplasm, indicative of apoptosis activation. The percentage of these cells was markedly increased in elderly patients and correlated with lymphopenia. Importantly, T cell apoptosis could be inhibited in vitro by targeting the oligomerization of VDAC or blocking caspase activity. In addition to these T cell findings, we also observed a robust population of Hexokinase II+ polymorphonuclear-myeloid derived suppressor cells (PMN-MDSC), exclusively found in the acutely ill COVID-19 patients and not the other viral diseases. Finally, we revealed a unique population of monocytic MDSC (M-MDSC) expressing high levels of carnitine palmitoyltransferase 1a (CPT1a) and VDAC. The metabolic phenotype of these cells was not only highly specific to COVID-19 patients but the presence of these cells was able to distinguish severe from mild disease. Overall, the identification of these novel metabolic phenotypes not only provides insight into the dysfunctional immune response in acutely ill COVID-19 patients but also provide a means to predict and track disease severity as well as an opportunity to design and evaluate novel metabolic therapeutic regimens. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/20186064v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@89f509org.highwire.dtl.DTLVardef@1362640org.highwire.dtl.DTLVardef@940aeorg.highwire.dtl.DTLVardef@175792b_HPS_FORMAT_FIGEXP M_FIG C_FIG
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Accurate serological assays to detect antibodies to SARS-CoV-2 are needed to characterize the epidemiology of SARS-CoV-2 infection and identify potential candidates for COVID-19 convalescent plasma (CCP) donation. This study compared the performance of commercial enzyme immunoassays (EIAs) to detect IgG or total antibodies to SARS-CoV-2 and neutralizing antibodies (nAb). The diagnostic accuracy of five commercially available EIAs (Abbott, Euroimmun, EDI, ImmunoDiagnostics, and Roche) to detect IgG or total antibodies to SARS-CoV-2 was evaluated from cross-sectional samples of potential CCP donors that had prior molecular confirmation of SARS-CoV-2 infection for sensitivity (n=214) and pre-pandemic emergency department patients for specificity (n=1,102). Of the 214 potential CCP donors, all were sampled >14 days since symptom onset and only a minority had been hospitalized due to COVID-19 (n=16 [7.5%]); 140 potential CCP donors were tested by all five EIAs and a microneutralization assay. When performed according to the manufacturers protocol to detect IgG or total antibodies to SARS-CoV-2, the sensitivity of each EIA ranged from 76.4% to 93.9%, and the specificity of each EIA ranged from 87.0% to 99.6%. Using a nAb titer cutoff of [≥]160 as the reference positive test (n=140 CCP donors), the empirical area under receiver operating curve of each EIA ranged from 0.66 (Roche) to 0.90 (Euroimmun). Commercial EIAs with high diagnostic accuracy to detect SARS-CoV-2 antibodies did not necessarily have high diagnostic accuracy to detect high nAbs. Some but not all commercial EIAs may be useful in the identification of individuals with high nAbs in convalescent individuals.
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BackgroundRapid point-of-care tests (POCTs) for SARS-CoV-2-specific antibodies vary in performance. A critical need exists to perform head-to-head comparison of these assays. MethodsPerformance of fifteen different lateral flow POCTs for the detection of SARS-CoV-2-specific antibodies was performed on a well characterized set of 100 samples. Of these, 40 samples from known SARS-CoV-2-infected, convalescent individuals (average of 45 days post symptom onset) were used to assess sensitivity. Sixty samples from the pre-pandemic era (negative control), that were known to have been infected with other respiratory viruses (rhinoviruses A, B, C and/or coronavirus 229E, HKU1, NL63 OC43) were used to assess specificity. The timing of seroconversion was assessed on five POCTs on a panel of 272 longitudinal samples from 47 patients of known time since symptom onset. ResultsFor the assays that were evaluated, the sensitivity and specificity for any reactive band ranged from 55%-97% and 78%-100%, respectively. When assessing the performance of the IgM and the IgG bands alone, sensitivity and specificity ranged from 0%-88% and 80%-100% for IgM and 25%-95% and 90%-100% for IgG. Longitudinal testing revealed that median time post symptom onset to a positive result was 7 days (IQR 5.4, 9.8) for IgM and 8.2 days (IQR 6.3 to 11.3). ConclusionThe testing performance varied widely among POCTs with most variation related to the sensitivity of the assays. The IgM band was most likely to misclassify pre-pandemic samples. The appearance of IgM and IgG bands occurred almost simultaneously.
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The COVID-19 pandemic has brought the world to a halt, with cases observed around the globe causing significant mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess the prevalence of infection, as well as ascertain individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which may lack scalability to meet the demand of hundreds of millions of antibody tests in the coming year. Herein, we present an alternative method of antibody testing that just depends on one protein reagent being added to patient serum/plasma or whole blood and a short five-minute assay time. A novel fusion protein was designed that binds red blood cells (RBC) via a single-chain variable fragment (scFv) against the H antigen and displays the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Upon mixing of the fusion protein, RBD-scFv with recovered COVID-19 patient serum and RBCs, we observed agglutination of RBCs, indicating the patient developed antibodies against SARS-CoV-2 RBD. Given that the test uses methods routinely used in hospital clinical labs across the world, we anticipate the test can be rapidly deployed with only the protein reagent required at projected manufacturing cost at U.S. cents per test. We anticipate our agglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.Competing Interest StatementR.L.K. is an inventor on a provisional patent application related to the work described in the manuscript. All other authors have no competing interests.View Full Text