Administration of vaccine-boosted COVID-19 convalescent plasma to SARS-CoV-2 infected hamsters decreases virus replication in lungs and hastens resolution of the infection despite transiently enhancing disease and lung pathology. (preprint)

The utility of COVID-19 convalescent plasma (CCP) for treatment of immunocompromised patients who are not able to mount a protective antibody response against SARS-CoV-2 and who have contraindications or adverse effects from currently available antivirals remains unclear. To better understand the mechanism of protection in CCP, we studied viral replication and disease progression in SARS-CoV-2 infected hamsters treated with CCP plasma obtained from recovered COVID patients that had also been vaccinated with an mRNA vaccine, hereafter referred to as Vaxplas. We found that Vaxplas dramatically reduced virus replication in the lungs and improved infection outcome in SARS-CoV-2 infected hamsters. However, we also found that Vaxplas transiently enhanced disease severity and lung pathology in treated animals likely due to the deposition of immune complexes, activation of complement and recruitment of increased numbers of macrophages with an M1 proinflammatory phenotype into the lung parenchyma.

COVID-19 vaccine antibody response is associated with side-effects, chronic health conditions, and vaccine type in a large Northern California cohort (preprint)

As vaccines have become available for COVID-19, it is important to understand factors that may impact response. The objective of this study is to describe vaccine response in a well-characterized Northern California cohort, including differences in side-effects and antibody response by vaccine type, sex, and age, as well as describe responses in subjects with pre-existing health conditions that are known risk factors for more severe COVID-19 infection. From July 2020 to March 2021, ~5,500 adults from the East Bay Area in Northern California were followed as part of a longitudinal cohort study. Comprehensive questionnaire data and biospecimens for COVID-19 antibody testing were collected at multiple time-points. All subjects were at least 18 years of age and members of the East-Bay COVID-19 cohort who answered questionnaires related to vaccination status and side-effects at two time-points. Three vaccines, Moderna (2 doses), Pfizer-BioNTech (2 doses), and Johnson & Johnson (single dose), were examined as exposures. Additionally, pre-existing health conditions were assessed. The main outcomes of interest were anti-SARS-CoV-2 Spike antibody response (measured by S/C ratio in the Ortho VITROS assay) and self-reporting of 11 potential vaccine side effects. When comparing both doses of the Moderna vaccine to respective doses of Pfizer-BioNTech, participants receiving the Moderna vaccine had higher odds of many reported side-effects. The same was true comparing the single-dose Johnson & Johnson vaccine to dose 2 of the Pfizer-BioNTech vaccine. The antibody S/C ratio also increased with each additional side-effect after the second dose. S/C ratios after vaccination were lower in participants aged 65 and older, and higher in females. At all vaccination timepoints, Moderna vaccine recipients had a higher S/C ratio. Individuals who were fully vaccinated with Pfizer-BioNTech had a 72.4% lower S/C ratio compared to those who were fully vaccinated with Moderna. Subjects with asthma, diabetes, and cardiovascular disease all demonstrated more than a 20% decrease in S/C ratio. In support of previous findings, we show that antibody response to the Moderna vaccine is higher than the Pfizer-BioNTech vaccine. We also observed that antibody response was associated with side-effects, and participants with a history of asthma, diabetes, and cardiovascular disease had lower antibody responses. This information is important to consider as further vaccines are recommended.

Evaluation of Ortho VITROS and Roche Elecsys S and NC Immunoassays for SARS‑CoV‑2 Serosurveillance Applications (preprint)

SARS-CoV-2 seroprevalence studies are instrumental in monitoring epidemic activity and require well-characterized, high-throughput assays, and appropriate testing algorithms. The U.S. Nationwide Blood Donor Seroprevalence Study performed monthly cross-sectional serological testing from July 2020 to December 2021, implementing evolving testing algorithms in response to changes in pandemic activity. With high vaccine uptake, anti-Spike (S) reactivity rates reached >80% by May 2021, and the study pivoted from reflex Roche anti-nucleocapsid (NC) testing of Ortho S-reactive specimens to parallel Ortho S/NC testing. We evaluated the performance of the Ortho NC assay as a replacement for the Roche NC assay and compared performance of parallel S/NC testing on both platforms. Qualitative and quantitative agreement of Ortho NC with Roche NC assays was evaluated on pre-selected S/NC concordant and discordant specimens. All 200 Ortho S+/Roche NC+ specimens were reactive on the Ortho NC assay; 36% of 383 Ortho S+/Roche NC- specimens collected prior to vaccine availability and 52% of 101 Ortho S-/Roche NC+ specimens were reactive on the Ortho NC assay. Performance of parallel S/NC testing using Ortho and Roche platforms was evaluated on 200 specimens collected in 2019 and 3,903 study specimens collected in 2021. All 200 pre-COVID 2019 specimens tested negative on the four assays. Agreement of S and NC reactivity on specimens was 96.4% (3,769/3,903); most discordant results had reactivity close to the cutoffs on the alternate assays. These findings, and higher efficiency and throughput, support use of parallel S/NC testing on either Roche or Ortho platforms for large serosurveillance studies.

COVID-19 vaccine effectiveness against SARS-CoV-2 infection in the United States prior to the Delta and Omicron-associated surges: a retrospective cohort study of repeat blood donors (preprint)

To inform public health policy, it is critical to monitor COVID-19 vaccine effectiveness (VE), including against acquiring infection. We estimated VE using a retrospective cohort study among repeat blood donors who donated during the first half of 2021, demonstrating a viable approach for monitoring of VE via serological surveillance. Using Poisson regression, we estimated overall VE was 88.8% (95% CI: 86.2-91.1), adjusted for demographic covariates and variable baseline risk. Time since first reporting vaccination, age, race-ethnicity, region, and calendar time were statistically significant predictors of incident infection. Studies of VE during periods of Delta and Omicron spread are underway.

COVID-19 vaccine effectiveness against SARS-CoV-2 infection in the United States prior to the Delta and Omicron-associated surges: a retrospective cohort study of repeat blood donors (preprint)

To inform public health policy, it is critical to monitor COVID-19 vaccine effectiveness (VE), including against acquiring infection. We estimated VE using a retrospective cohort study among repeat blood donors who donated during the first half of 2021, demonstrating a viable approach for monitoring of VE via serological surveillance. Using Poisson regression, we estimated overall VE was 88.8% (95% CI: 86.2–91.1), adjusted for demographic covariates and variable baseline risk. Time since first reporting vaccination, age, race-ethnicity, region, and calendar time were statistically significant predictors of incident infection. Studies of VE during periods of Delta and Omicron spread are underway.

Impact of individual-level characteristics and transmission mitigation behaviors on SARS-CoV-2 infection and seroprevalence in a large Northern California Bay Area cohort (preprint)

Comprehensive data on transmission mitigation behaviors and SARS-CoV-2 infection and serostatus are needed from large, community-based cohorts to identify SARS-CoV-2 risk factors and impact of public health measures. From July 2020 to March 2021, {approx}5,500 adults from the East Bay Area, California were followed over three data collection rounds. We estimated the prevalence of antibodies from SARS-CoV-2 infection and COVID-19 vaccination, and self-reported COVID-19 test positivity. Population-adjusted SARS-CoV-2 seroprevalence was low, increasing from 1.03% (95% CI: 0.50-1.96) in Round 1 (July-September 2020), to 1.37% (95% CI: 0.75-2.39) in Round 2 (October-December 2020), to 2.18% (95% CI: 1.48-3.17) in Round 3 (February-March 2021). Population-adjusted seroprevalence of COVID-19 vaccination was 21.64% (95% CI: 19.20-24.34) in Round 3. Despite >99% of participants reporting wearing masks, non-Whites, lower-income, and lower-educated individuals had the highest SARS-CoV-2 seroprevalence and lowest vaccination seroprevalence. Our results demonstrate that more effective policies are needed to address these disparities and inequities.

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation (preprint)

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses. Author summaryThe results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

Use of U.S. Blood Donors for National Serosurveillance of SARS-CoV-2 Antibodies: Basis for an Expanded National Donor Serosurveillance Program. (preprint)

Introduction: The REDS-IV-P Epidemiology, Surveillance and Preparedness of the Novel SARS-CoV-2 Epidemic (RESPONSE) seroprevalence study conducted monthly cross-sectional testing for SARS-CoV-2 antibodies on blood donors in six U.S. metropolitan regions to estimate the extent of SARS-COV-2 infections over time. Study Design/Methods During March-August 2020, approximately [≥]1,000 serum specimens were collected monthly from each region and tested for SARS-CoV-2 antibodies using a well-validated algorithm. Regional seroprevalence estimates were weighted based on demographic differences with the general population. Seroprevalence was compared with reported COVID-19 case rates over time. Results/Findings: For all regions, seroprevalence was <1.0% in March 2020. New York experienced the biggest increase (peak seroprevalence, 15.8 % in May). All other regions experienced modest increases in seroprevalence(1-2% in May-June to 2-4% in July-August). Seroprevalence was higher in younger, non-Hispanic Black, and Hispanic donors. Temporal increases in donor seroprevalence correlated with reported case rates in each region. In August, 1.3-5.6 estimated cumulative infections (based on seroprevalence data) per COVID-19 case reported to CDC. Conclusion: Increases in seroprevalence were found in all regions, with the largest increase in New York. Seroprevalence was higher in non-Hispanic Black and Hispanic blood donors than in non-Hispanic White blood donors. SARS-CoV-2 antibody testing of blood donor samples can be used to estimate the seroprevalence in the general population by region and demographic group. The methods derived from the RESPONSE seroprevalence study served as the basis for expanding SARS-CoV-2 seroprevalence surveillance to all 50 states and Puerto Rico.

SARS-CoV-2 Antibody persistence in COVID-19 convalescent plasma donors (preprint)

Background Antibody response duration following SARS-CoV-2 infection tends to be variable and depends on severity of disease and method of detection. Study design and methods COVID-19 convalescent plasma (CCP) from 18 donors was collected longitudinally for a maximum of 63 - 129 days following resolution of symptoms. All the samples were initially screened by the Ortho Total Ig test to confirm positivity and subsequently tested with 7 additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, RBD, and NC), along with 2 neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute Pseudovirus RVPN). Results The direct detection assays (Ortho Total Ig total and Roche Total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay. Discussion These data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag-sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1 and NC based assays can help reduce the rate of false-positivity and discriminate between natural infection and vaccine-derived seroreactivity.

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay (preprint)

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

COVID-19 herd immunity in the Brazilian Amazon (preprint)

The herd immunity threshold is the proportion of a population that must be immune to an infectious disease, either by natural infection or vaccination such that, in the absence of additional preventative measures, new cases decline and the effective reproduction number falls below unity. This fundamental epidemiological parameter is still unknown for the recently-emerged COVID-19, and mathematical models have predicted very divergent results. Population studies using antibody testing to infer total cumulative infections can provide empirical evidence of the level of population immunity in severely affected areas. Here we show that the transmission of SARS-CoV-2 in Manaus, located in the Brazilian Amazon, increased quickly during March and April and declined more slowly from May to September. In June, one month following the epidemic peak, 44% of the population was seropositive for SARS-CoV-2, equating to a cumulative incidence of 52%, after correcting for the false-negative rate of the antibody test. The seroprevalence fell in July and August due to antibody waning. After correcting for this, we estimate a final epidemic size of 66%. Although non-pharmaceutical interventions, plus a change in population behavior, may have helped to limit SARS-CoV-2 transmission in Manaus, the unusually high infection rate suggests that herd immunity played a significant role in determining the size of the epidemic.

Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection (preprint)

The COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans, from asymptomatic or mild disease to severe disease that can require mechanical ventilation. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from indiviuals that go on to become infected with SARS-CoV-2. Here, we utilized data from a screen of genetically diverse mice from the Collaborative Cross (CC) infected with SARS-CoV to determine whether circulating baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Further, early control of virus in the lung correlates with an increased abundance of activated CD4 and CD8 T cells and regulatory T cells prior to infections across strains. A basal propensity of T cells to express IFNg and IL17 over TNFa also correlated with early viral control. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. While future studies of human samples prior to infection with SARS-CoV-2 are required, our studies in mice with SARS-CoV serve as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry (preprint)

Coronavirus interaction with viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter the host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkeys, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants of susceptibility of ACE2 orthologs to viral entry, we compared koala and mouse ACE2 sequences with human ortholog, and identified the key residues in koala or mouse ACE2 that restrict its viral receptor activity. Humanization of these critical residues could render the capabilities of koala and mouse ACE2 to bind viral spike protein and facilitate the viral entry. Our work identifies the genetic determinant of ACE2 for SARS-CoV-2 susceptibility, and a single mutation could restore the mouse ACE2 receptor activity, providing a potential avenue for the development of mouse model of SARS-CoV-2.

Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19 (preprint)

Less than a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 22 million people worldwide with a death toll approaching 1 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Angstrom and 2.6 Angstrom respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.

SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism (preprint)

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5 untranslated region (5 UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5 UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5 UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5 UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5 UTR may be targeted for anti-COVID-19 therapeutics.

An immunodominance hierarchy exists in CD8+ T cell responses to HLA-A*02:01-restricted epitopes identified from the non-structural polyprotein 1a of SARS-CoV-2. (preprint)

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-{gamma}-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. ImportanceFor the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

A soluble ACE2 microbody protein fused to a single immunoglobulin Fc domain is a potent inhibitor of SARS-CoV-2 infection in cell culture (preprint)

Soluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. We report on an improved soluble form of ACE2, termed a "microbody" in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody inhibited entry of SARS-CoV-2 spike protein pseudotyped virus and live SARS-CoV-2 with a potency 10-fold higher than unmodified soluble ACE2 and retained activity even after the virus had bound to the cell. The ACE2 microbody inhibited entry of ACE2-utilizing {beta} coronaviruses and entry of viruses with the high infectivity variant D614G spike. The ACE2 microbody may be a valuable therapeutic for COVID-19 that is active against SARS-CoV-2 variants and against coronaviruses that may arise in the future.

Distinct SARS-CoV-2 Antibody Reactivity Patterns in Coronavirus Convalescent Plasma Revealed by a Coronavirus Antigen Microarray (preprint)

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and Random Forest (RF) prediction model, to classify individual specimens as either Reactive or Non-Reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS(R) Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the 3 assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 95%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed 3 main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified according to these reactivity patterns may be better associated with CCP treatment efficacy than antibody levels alone. The use of a SARS-CoV-2 antigen array may be useful to qualify CCP for administration as a treatment for acute COVID-19 and to interrogate vaccine immunogenicity and performance in preclinical and clinical studies to understand and recapitulate antibody responses associated with protection from infection and disease.

SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques (preprint)

CD4 T follicular helper (Tfh) cells are important for the generation of long-lasting and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that, following infection with SARS-CoV-2, adult rhesus macaques exhibited transient accumulation of activated, proliferating Tfh cells in their peripheral blood on a transitory basis. The CD4 helper cell responses were skewed predominantly toward a Th1 response in blood, lung, and lymph nodes, reflective of the interferon-rich cytokine environment following infection. We also observed the generation of germinal center Tfh cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies but delayed or absent IgA antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area (preprint)

We report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seropositivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors. We additionally describe the longitudinal dynamics of immunoglobulin-G, immunoglobulin-M, and in vitro neutralizing antibody titers in COVID-19 patients. Neutralizing antibodies rise in tandem with immunoglobulin levels following symptom onset, exhibiting median time to seroconversion within one day of each other, and there is >93% positive percent agreement between detection of immunoglobulin-G and neutralizing titers.