Lectin Fingerprinting Distinguishes Antibody Neutralization in SARS-CoV-2.

Enveloped viruses co-opt host glycosylation pathways to decorate their surface proteins. As viruses evolve, emerging strains can modify their glycosylation patterns to influence host interactions and subvert immune recognition. Still, changes in viral glycosylation or their impact on antibody protection cannot be predicted from genomic sequences alone. Using the highly glycosylated SARS-CoV-2 Spike protein as a model system, we present a lectin fingerprinting method that rapidly reports on changes in variant glycosylation state, which are linked to antibody neutralization. In the presence of antibodies or convalescent and vaccinated patient sera, unique lectin fingerprints emerge that distinguish neutralizing versus non-neutralizing antibodies. This information could not be inferred from direct binding interactions between antibodies and the Spike receptor-binding domain (RBD) binding data alone. Comparative glycoproteomics of the Spike RBD of wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) variants reveal O-glycosylation differences as a key determinant of immune recognition differences. These data underscore the interplay between viral glycosylation and immune recognition and reveal lectin fingerprinting to be a rapid, sensitive, and high-throughput assay to distinguish the neutralization potential of antibodies that target critical viral glycoproteins.

Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

Cross-reactive SARS-CoV-2 epitope targeted across donors informs immunogen design.

The emergence of the antigenically distinct and highly transmissible Omicron variant highlights the possibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape due to viral evolution. This continued evolution, along with the possible introduction of new sarbecoviruses from zoonotic reservoirs, may evade host immunity elicited by current SARS-CoV-2 vaccines. Identifying cross-reactive antibodies and defining their epitope(s) can provide templates for rational immunogen design strategies for next-generation vaccines. Here, we characterize the receptor-binding-domain-directed, cross-reactive humoral repertoire across 10 human vaccinated donors. We identify cross-reactive antibodies from diverse gene rearrangements targeting two conserved receptor-binding domain epitopes. An engineered immunogen enriches antibody responses to one of these conserved epitopes in mice with pre-existing SARS-CoV-2 immunity; elicited responses neutralize SARS-CoV-2, variants, and related sarbecoviruses. These data show how immune focusing to a conserved epitope targeted by human cross-reactive antibodies may guide pan-sarbecovirus vaccine development, providing a template for identifying such epitopes and translating to immunogen design.

Repertoires of SARS-CoV-2 epitopes targeted by antibodies vary according to severity of COVID-19.

Antibodies to SARS-CoV-2 are central to recovery and immunity from COVID-19. However, the relationship between disease severity and the repertoire of antibodies against specific SARS-CoV-2 epitopes an individual develops following exposure remains incompletely understood. Here, we studied seroprevalence of antibodies to specific SARS-CoV-2 and other betacoronavirus antigens in a well-annotated, community sample of convalescent and never-infected individuals obtained in August 2020. One hundred and twenty-four participants were classified into five groups: previously exposed but without evidence of infection, having no known exposure or evidence of infection, seroconverted without symptoms, previously diagnosed with symptomatic COVID-19, and recovered after hospitalization with COVID-19. Prevalence of IgGs specific to the following antigens was compared between the five groups: recombinant SARS-CoV-2 and betacoronavirus spike and nucleocapsid protein domains, peptides from a tiled array of 22-mers corresponding to the entire spike and nucleocapsid proteins, and peptides corresponding to predicted immunogenic regions from other proteins of SARS-CoV-2. Antibody abundance generally correlated positively with severity of prior illness. A number of specific immunogenic peptides and some that may be associated with milder illness or protection from symptomatic infection were identified. No convincing association was observed between antibodies to Receptor Binding Domain(s) (RBDs) of less pathogenic betacoronaviruses HKU1 or OC43 and COVID-19 severity. However, apparent cross-reaction with SARS-CoV RBD was evident and some predominantly asymptomatic individuals had antibodies to both MERS-CoV and SARS-CoV RBDs. Findings from this pilot study may inform development of diagnostics, vaccines, and therapeutic antibodies, and provide insight into viral pathogenic mechanisms.

T cell reactivity to the SARS-CoV-2 Omicron variant is preserved in most but not all individuals.

The SARS-CoV-2 Omicron variant (B.1.1.529) contains mutations that mediate escape from antibody responses, although the extent to which these substitutions in spike and non-spike proteins affect T cell recognition is unknown. In this study, we show that T cell responses in individuals with prior infection, vaccination, both prior infection and vaccination, and boosted vaccination are largely preserved to Omicron spike and non-spike proteins. However, we also identify a subset of individuals (∼21%) with a >50% reduction in T cell reactivity to the Omicron spike. Evaluation of functional CD4+ and CD8+ memory T cell responses confirmed these findings and revealed that reduced recognition to Omicron spike is primarily observed within the CD8+ T cell compartment potentially due to escape from HLA binding. Booster vaccination enhanced T cell responses to Omicron spike. In contrast to neutralizing immunity, these findings suggest preservation of T cell responses to the Omicron variant, although with reduced reactivity in some individuals.

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant.

Recent surveillance has revealed the emergence of the SARS-CoV-2 Omicron variant (BA.1/B.1.1.529) harboring up to 36 mutations in spike protein, the target of neutralizing antibodies. Given its potential to escape vaccine-induced humoral immunity, we measured the neutralization potency of sera from 88 mRNA-1273, 111 BNT162b, and 40 Ad26.COV2.S vaccine recipients against wild-type, Delta, and Omicron SARS-CoV-2 pseudoviruses. We included individuals that received their primary series recently (<3 months), distantly (6-12 months), or an additional "booster" dose, while accounting for prior SARS-CoV-2 infection. Remarkably, neutralization of Omicron was undetectable in most vaccinees. However, individuals boosted with mRNA vaccines exhibited potent neutralization of Omicron, only 4-6-fold lower than wild type, suggesting enhanced cross-reactivity of neutralizing antibody responses. In addition, we find that Omicron pseudovirus infects more efficiently than other variants tested. Overall, this study highlights the importance of additional mRNA doses to broaden neutralizing antibody responses against highly divergent SARS-CoV-2 variants.

Neutralization breadth of SARS-CoV-2 viral variants following primary series and booster SARS-CoV-2 vaccines in patients with cancer.

Patients with cancer are more likely to have impaired immune responses to SARS-CoV-2 vaccines. We study the breadth of responses against SARS-CoV-2 variants after primary vaccination in 178 patients with a variety of tumor types and after booster doses in a subset. Neutralization of alpha, beta, gamma, and delta SARS-CoV-2 variants is impaired relative to wildtype, regardless of vaccine type. Regardless of viral variant, mRNA1273 is the most immunogenic, followed by BNT162b2, and then Ad26.COV2.S. Neutralization of more variants (breadth) is associated with a greater magnitude of wildtype neutralization, and increases with time since vaccination; advancing age associates with a lower breadth. The concentrations of anti-spike protein antibody are a good surrogate for breadth (positive predictive value of =90% at >1,000 U/mL). Booster SARS-CoV-2 vaccines confer enhanced breadth. These data suggest that achieving a high antibody titer is desirable to achieve broad neutralization; a single booster dose with the current vaccines increases the breadth of responses against variants.

Comparative Immunogenicity and Effectiveness of mRNA-1273, BNT162b2, and Ad26.COV2.S COVID-19 Vaccines.

BACKGROUND: Understanding immunogenicity and effectiveness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines is critical to guide rational use. METHODS: We compared the immunogenicity of mRNA-1273, BNT-162b2, and Ad26.COV2.S in healthy ambulatory adults. We performed an inverse-variance meta-analysis of population-level effectiveness from public health reports in > 40 million individuals. RESULTS: A single dose of either mRNA vaccine yielded comparable antibody and neutralization titers to convalescent individuals. Ad26.COV2.S yielded lower antibody concentrations and frequently undetectable neutralization titers. Bulk and cytotoxic T-cell responses were higher in mRNA1273 and BNT162b2 than Ad26.COV2.S recipients. Regardless of vaccine, <50% of vaccinees demonstrated CD8+ T-cell responses. Antibody concentrations and neutralization titers increased comparably after the first dose of either vaccine, and further in recipients of a second dose. Prior infection was associated with high antibody concentrations and neutralization even after a single dose and regardless of vaccine. Neutralization of Beta, Gamma, and Delta strains were poorer regardless of vaccine. In meta-analysis, relative to mRNA1273 the effectiveness of BNT162b2 was lower against infection and hospitalization, and Ad26COV2.S was lower against infection, hospitalization, and death. CONCLUSIONS: Variation in the immunogenicity correlates with variable effectiveness of the 3 vaccines deployed in the United States.

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity.

Vaccination elicits immune responses capable of potently neutralizing SARS-CoV-2. However, ongoing surveillance has revealed the emergence of variants harboring mutations in spike, the main target of neutralizing antibodies. To understand the impact of these variants, we evaluated the neutralization potency of 99 individuals that received one or two doses of either BNT162b2 or mRNA-1273 vaccines against pseudoviruses representing 10 globally circulating strains of SARS-CoV-2. Five of the 10 pseudoviruses, harboring receptor-binding domain mutations, including K417N/T, E484K, and N501Y, were highly resistant to neutralization. Cross-neutralization of B.1.351 variants was comparable to SARS-CoV and bat-derived WIV1-CoV, suggesting that a relatively small number of mutations can mediate potent escape from vaccine responses. While the clinical impact of neutralization resistance remains uncertain, these results highlight the potential for variants to escape from neutralizing humoral immunity and emphasize the need to develop broadly protective interventions against the evolving pandemic.

Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape convalescent and vaccine-induced antibody responses has renewed focus on the development of broadly protective T-cell-based vaccines. Here, we apply structure-based network analysis and assessments of HLA class I peptide stability to define mutationally constrained CD8+ T cell epitopes across the SARS-CoV-2 proteome. Highly networked residues are conserved temporally among circulating variants and sarbecoviruses and disproportionately impair spike pseudotyped lentivirus infectivity when mutated. Evaluation of HLA class I stabilizing activity for 18 globally prevalent alleles identifies CD8+ T cell epitopes within highly networked regions with limited mutational frequencies in circulating SARS-CoV-2 variants and deep-sequenced primary isolates. Moreover, these epitopes elicit demonstrable CD8+ T cell reactivity in convalescent individuals but reduced recognition in recipients of mRNA-based vaccines. These data thereby elucidate key mutationally constrained regions and immunogenic epitopes in the SARS-CoV-2 proteome for a global T-cell-based vaccine against emerging variants and SARS-like coronaviruses.

Remote Fingerstick Blood Collection for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Testing.

The rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has propelled the rapid development of serologic tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, and helping establish a recent diagnosis of coronavirus disease 2019 (COVID-19), and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serologic assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large-scale SARS-CoV-2 serologic studies by combining self or office collection of fingerprick blood with a volumetric absorptive microsampling device (Mitra, Neoteryx LLC) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys, Roche Diagnostics Inc) that is emergency use authorization approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Mitra devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies compared with neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Mitra dried blood collection device with the Roche Elecsys SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.

COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

Dynamics and significance of the antibody response to SARS-CoV-2 infection.

BACKGROUND: Characterizing the humoral immune response to SARS-CoV-2 and developing accurate serologic assays are needed for diagnostic purposes and estimating population-level seroprevalence. METHODS: We measured the kinetics of early antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of 259 symptomatic North American patients infected with SARS-CoV-2 (up to 75 days after symptom onset) compared to antibody levels in 1548 individuals whose blood samples were obtained prior to the pandemic. RESULTS: Between 14-28 days from onset of symptoms, IgG, IgA, or IgM antibody responses to RBD were all accurate in identifying recently infected individuals, with 100% specificity and a sensitivity of 97%, 91%, and 81% respectively. Although the estimated median time to becoming seropositive was similar across isotypes, IgA and IgM antibodies against RBD were short-lived with most individuals estimated to become seronegative again by 51 and 47 days after symptom onset, respectively. IgG antibodies against RBD lasted longer and persisted through 75 days post-symptoms. IgG antibodies to SARS-CoV-2 RBD were highly correlated with neutralizing antibodies targeting the S protein. No cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies was observed with several known circulating coronaviruses, HKU1, OC 229 E, OC43, and NL63. CONCLUSIONS: Among symptomatic SARS-CoV-2 cases, RBD-targeted antibodies can be indicative of previous and recent infection. IgG antibodies are correlated with neutralizing antibodies and are possibly a correlate of protective immunity.

Analytical performance of lateral flow immunoassay for SARS-CoV-2 exposure screening on venous and capillary blood samples.

OBJECTIVES: We validate the use of a lateral flow immunoassay (LFI) intended for rapid screening and qualitative detection of anti-SARS-CoV-2 IgM and IgG in serum, plasma, and whole blood, and compare results with ELISA. We also seek to establish the value of LFI testing on blood obtained from a capillary blood sample. METHODS: Samples collected by venous blood draw and finger stick were obtained from patients with SARS-CoV-2 detected by RT-qPCR and control patients. Samples were tested with Biolidics 2019-nCoV IgG/IgM Detection Kit lateral flow immunoassay, and antibody calls were compared with ELISA. RESULTS: Biolidics LFI showed clinical sensitivity of 92% with venous blood at 7 days after PCR diagnosis of SARS-CoV-2. Test specificity was 92% for IgM and 100% for IgG. There was no significant difference in detecting IgM and IgG with Biolidics LFI and ELISA at D0 and D7 (p = 1.00), except for detection of IgM at D7 (p = 0.04). Capillary blood of SARS-CoV-2 patients showed 93% sensitivity for antibody detection. CONCLUSIONS: Clinical performance of Biolidics 2019-nCoV IgG/IgM Detection Kit is comparable to ELISA and was consistent across sample types. This provides an opportunity for decentralized rapid testing and may allow point-of-care and longitudinal self-testing for the presence of anti-SARS-CoV-2 antibodies.

Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients.

We measured plasma and/or serum antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in 343 North American patients infected with SARS-CoV-2 (of which 93% required hospitalization) up to 122 days after symptom onset and compared them to responses in 1548 individuals whose blood samples were obtained prior to the pandemic. After setting seropositivity thresholds for perfect specificity (100%), we estimated sensitivities of 95% for IgG, 90% for IgA, and 81% for IgM for detecting infected individuals between 15 and 28 days after symptom onset. While the median time to seroconversion was nearly 12 days across all three isotypes tested, IgA and IgM antibodies against RBD were short-lived with median times to seroreversion of 71 and 49 days after symptom onset. In contrast, anti-RBD IgG responses decayed slowly through 90 days with only 3 seropositive individuals seroreverting within this time period. IgG antibodies to SARS-CoV-2 RBD were strongly correlated with anti-S neutralizing antibody titers, which demonstrated little to no decrease over 75 days since symptom onset. We observed no cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies with other widely circulating coronaviruses (HKU1, 229 E, OC43, NL63). These data suggest that RBD-targeted antibodies are excellent markers of previous and recent infection, that differential isotype measurements can help distinguish between recent and older infections, and that IgG responses persist over the first few months after infection and are highly correlated with neutralizing antibodies.

Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.