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.

Substantial Differences in SARS-CoV-2 Antibody Responses Elicited by Natural Infection and mRNA Vaccination (preprint)

We analyzed data from two ongoing serologic surveys, a longitudinal cohort of health care workers (HCW) from the University of California Irvine Medical Center (Orange County, CA, USA), collected from May and December 2020 through March 2021, and a cross sectional county-wide study in July 2020 (actOC; Orange County, CA) and a more focused community study in the city of Santa Ana (Santa Ana Cares; Orange County, CA, USA), collected in December 2020 - in order to compare the antibody responses to SARS-CoV-2 natural infection and vaccination. In addition, we serially tested 9 volunteers at multiple time points to analyze the time course of vaccine-induced antibody response in more detail. In May 2020, 1060 HCW were enrolled and had finger stick samples collected. Finger stick samples were again collected in December 2020, before vaccination, as well as January, February and March 2021 during vaccination campaign. A total of 8,729 finger stick blood specimens were probed and analyzed for IgG and IgM antibodies using a coronavirus antigen microarray (COVAM). The microarray contained 10 SARS-CoV-2 antigens including nucleocapid protein (NP) and several varying fragments of the spike protein, as well as 4 SARS, 3 MERS, 12 Common CoV, and 8 Influenza antigens.Based on a random forest based prediction algorithm, between May and December, prior to vaccine rollout, we observed that seropositivity in the HCW cohort increased from 4.5% to 13%. An intensive vaccination campaign with mRNA vaccines was initiated on December 16, 2020 and 6,724 healthcare workers were vaccinated within 3 weeks. The observed seropositivity of the HCW specimens taken in the last week of January 2021 jumped to 78%, and by the last week in February it reached 93%, and peaked at 98% seropositive in March. The antibody profile induced by natural exposure differed from the profile induced after mRNA vaccination. Messenger RNA vaccines induced elevated antibody (Ab) reactivity levels against the Receptor Binding Domain (RBD) domain of SARS-CoV-2 spike, and cross-reactive responses against SARS and MERS RBD domains. Nucleocapsid protein (NP), which is an immunodominant antigen induced after natural exposure, is not present in the vaccine and can be used as a biomarker of past exposure. The results show that naturally-exposed individuals mount a stronger anti-spike response upon vaccination than individuals that were not previously exposed. Longitudinal specimens taken at approximately weekly intervals from 9 individuals show variation in the response to the mRNA vaccine, with some showing a vigorous response to the first dose (prime) and others requiring a subsequent dose (boost) to reach high anti-SARS-CoV-2 levels. Antibody titers determined by serial dilution of the specimens were used to accurately compare antibody levels in these samples. mRNA vaccinees after the boost have higher Ab titers (up to 10 times higher) than convalescent plasmas from donors who recovered from natural infection. The results of this study exemplify the time course and outcomes expected from similar mRNA mass vaccination campaigns conducted in other institutions.

Epidemiology of the early COVID-19 epidemic in Orange County, California: comparison of predictors of test positivity, mortality, and seropositivity (preprint)

COVID-19 is one of the largest public health emergencies in modern history. Here we present a detailed analysis from a large population center in Southern California (Orange County, population of 3.2 million) to understand heterogeneity in risks of infection, test positivity, and death. We used a combination of datasets, including a population-representative seroprevalence survey, to assess the true burden of disease as well as COVID-19 testing intensity, test positivity, and mortality. In the first month of the local epidemic, case incidence clustered in high income areas. This pattern quickly shifted, with cases next clustering in much higher rates in the north-central area which has a lower socio-economic status. Since April, a concentration of reported cases, test positivity, testing intensity, and seropositivity in a north-central area persisted. At the individual level, several factors (e.g., age, race/ethnicity, zip codes with low educational attainment) strongly affected risk of seropositivity and death.

Antibody Response to a Nucleocapsid Epitope as a Marker for COVID-19 Disease Severity (preprint)

Characterization of antibody response to SARS-CoV-2 is urgently needed to predict COVID-19 disease trajectories. Ineffective antibodies or antibody-dependent enhancement (ADE) could derail patient immune responses, for example. ELISA and coronavirus antigen microarray (COVAM) analysis epitope-mapped plasma from 86 COVID-19 patients. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including ICU stay, requirement for ventilators, and death. Furthermore, anti-Ep9 antibodies correlate both with various comorbidities and ADE hallmarks, including increased IL-6 levels and early IgG response. Importantly, anti-Ep9 antibodies can be detected within five days post-symptom onset and sometimes within one day. The results lay the groundwork for a new type of COVID-19 diagnostic for the early prediction of disease severity to guide more effective therapeutic interventions.

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.

A Modular Microarray Imaging System for Highly Specific COVID-19 Antibody Testing (preprint)

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100x more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Plasma using a Coronavirus Antigen Microarray (preprint)

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray (preprint)

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

A serological assay to detect SARS-CoV-2 seroconversion in humans (preprint)

SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.