Programs, Origins, and Niches of Immunomodulatory Myeloid Cells in Gliomas.

Gliomas are incurable malignancies notable for an immunosuppressive microenvironment with abundant myeloid cells whose immunomodulatory properties remain poorly defined. Here, utilizing scRNA-seq data for 183,062 myeloid cells from 85 human tumors, we discover that nearly all glioma-associated myeloid cells express at least one of four immunomodulatory activity programs: Scavenger Immunosuppressive, C1Q Immunosuppressive, CXCR4 Inflammatory, and IL1B Inflammatory. All four programs are present in IDH1 mutant and wild-type gliomas and are expressed in macrophages, monocytes, and microglia whether of blood or resident myeloid cell origins. Integrating our scRNA-seq data with mitochondrial DNA-based lineage tracing, spatial transcriptomics, and organoid explant systems that model peripheral monocyte infiltration, we show that these programs are driven by microenvironmental cues and therapies rather than myeloid cell type, origin, or mutation status. The C1Q Immunosuppressive program is driven by routinely administered dexamethasone. The Scavenger Immunosuppressive program includes ligands with established roles in T-cell suppression, is induced in hypoxic regions, and is associated with immunotherapy resistance. Both immunosuppressive programs are less prevalent in lower-grade gliomas, which are instead enriched for the CXCR4 Inflammatory program. Our study provides a framework to understand immunomodulatory myeloid cells in glioma, and a foundation to develop more effective immunotherapies.

Geographically skewed recruitment and COVID-19 seroprevalence estimates: a cross-sectional serosurveillance study and mathematical modelling analysis.

OBJECTIVES: Convenience sampling is an imperfect but important tool for seroprevalence studies. For COVID-19, local geographic variation in cases or vaccination can confound studies that rely on the geographically skewed recruitment inherent to convenience sampling. The objectives of this study were: (1) quantifying how geographically skewed recruitment influences SARS-CoV-2 seroprevalence estimates obtained via convenience sampling and (2) developing new methods that employ Global Positioning System (GPS)-derived foot traffic data to measure and minimise bias and uncertainty due to geographically skewed recruitment. DESIGN: We used data from a local convenience-sampled seroprevalence study to map the geographic distribution of study participants' reported home locations and compared this to the geographic distribution of reported COVID-19 cases across the study catchment area. Using a numerical simulation, we quantified bias and uncertainty in SARS-CoV-2 seroprevalence estimates obtained using different geographically skewed recruitment scenarios. We employed GPS-derived foot traffic data to estimate the geographic distribution of participants for different recruitment locations and used this data to identify recruitment locations that minimise bias and uncertainty in resulting seroprevalence estimates. RESULTS: The geographic distribution of participants in convenience-sampled seroprevalence surveys can be strongly skewed towards individuals living near the study recruitment location. Uncertainty in seroprevalence estimates increased when neighbourhoods with higher disease burden or larger populations were undersampled. Failure to account for undersampling or oversampling across neighbourhoods also resulted in biased seroprevalence estimates. GPS-derived foot traffic data correlated with the geographic distribution of serosurveillance study participants. CONCLUSIONS: Local geographic variation in seropositivity is an important concern in SARS-CoV-2 serosurveillance studies that rely on geographically skewed recruitment strategies. Using GPS-derived foot traffic data to select recruitment sites and recording participants' home locations can improve study design and interpretation.

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

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.

Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations.

The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.

Glioblastoma stem cells reprogram chromatin in vivo to generate selective therapeutic dependencies on DPY30 and phosphodiesterases.

Glioblastomas are universally fatal cancers and contain self-renewing glioblastoma stem cells (GSCs) that initiate tumors. Traditional anticancer drug discovery based on in vitro cultures tends to identify targets with poor therapeutic indices and fails to accurately model the effects of the tumor microenvironment. Here, leveraging in vivo genetic screening, we identified the histone H3 lysine 4 trimethylation (H3K4me3) regulator DPY30 (Dpy-30 histone methyltransferase complex regulatory subunit) as an in vivo­specific glioblastoma dependency. On the basis of the hypothesis that in vivo epigenetic regulation may define critical GSC dependencies, we interrogated active chromatin landscapes of GSCs derived from intracranial patient-derived xenografts (PDXs) and cell culture through H3K4me3 chromatin immunoprecipitation and transcriptome analyses. Intracranial-specific genes marked by H3K4me3 included FOS, NFκB, and phosphodiesterase (PDE) family members. In intracranial PDX tumors, DPY30 regulated angiogenesis and hypoxia pathways in an H3K4me3-dependent manner but was dispensable in vitro in cultured GSCs. PDE4B was a key downstream effector of DPY30, and the PDE4 inhibitor rolipram preferentially targeted DPY30-expressing cells and impaired PDX tumor growth in mice without affecting tumor cells cultured in vitro. Collectively, the MLL/SET1 (mixed lineage leukemia/SET domain-containing 1, histone lysine methyltransferase) complex member DPY30 selectively regulates H3K4me3 modification on genes critical to support angiogenesis and tumor growth in vivo, suggesting the DPY30-PDE4B axis as a specific therapeutic target in glioblastoma.

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.

Comparative immunogenicity and effectiveness of mRNA-1273, BNT162b2 and Ad26.COV2.S COVID-19 vaccines.

BACKGROUND: Understanding immunogenicity and effectiveness of SARS-CoV-2 vaccines is critical to guide rational use. METHODS: We compared the immunogenicity of mRNA-1273, BNT-162b2 or Ad26.COV2.S in ambulatory adults in Massachusetts, USA. To correlate immunogenicity with effectiveness of the three vaccines, 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 negative neutralization titers. Bulk and cytotoxic T-cell responses were higher in mRNA1273 and BNT162b2 than Ad26.COV2.S recipients, and <50% of vaccinees demonstrate CD8+ T-cell responses to spike peptides. 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. 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 three FDA EUA vaccines deployed in the USA.

Mosaicism for Receptor Tyrosine Kinase Activation in a Glioblastoma Involving Both PDGFRA Amplification and NTRK2 Fusion.

Rearrangements involving the neurotrophic receptor tyrosine kinase (NTRK) gene family have been reported in diverse tumor types, and NTRK-targeted therapies have recently been approved. In this article, we report a case of a 26-year-old man with an NTRK2-rearranged isocitrate dehydrogenase-wild-type glioblastoma who showed a robust but temporary response to the NTRK inhibitor larotrectinib. Rebiopsy after disease progression showed elimination of the NTRK2-rearranged tumor cell clones, with secondary emergence of a PDGFRA-amplified subclone. Retrospective examination of the initial biopsy material confirmed rare cells harboring PDGFRA amplification. Although mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma has been previously described, mosaicism involving a fusion gene driver event has not. This case highlights the potential efficacy of NTRK-targeted treatment in glioblastoma and the implications of molecular heterogeneity in the setting of targeted therapy. KEY POINTS: This case highlights the efficacy of the NTRK inhibitor larotrectinib in treating NTRK-rearranged glioblastoma. This is the first case to demonstrate mosaicism in glioblastoma involving both a fusion gene and amplification for receptor tyrosine kinases. Intratumoral heterogeneity in glioblastoma has significant implications for tumor resistance to targeted therapies.

Recruitment location influences bias and uncertainty in SARS-CoV-2 seroprevalence estimates.

The initial phase of the COVID-19 pandemic in the US was marked by limited diagnostic testing, resulting in the need for seroprevalence studies to estimate cumulative incidence and define epidemic dynamics. In lieu of systematic representational surveillance, venue-based sampling was often used to rapidly estimate a community's seroprevalence. However, biases and uncertainty due to site selection and use of convenience samples are poorly understood. Using data from a SARS-CoV-2 serosurveillance study we performed in Somerville, Massachusetts, we found that the uncertainty in seroprevalence estimates depends on how well sampling intensity matches the known or expected geographic distribution of seropositive individuals in the study area. We use GPS-estimated foot traffic to measure and account for these sources of bias. Our results demonstrated that study-site selection informed by mobility patterns can markedly improve seroprevalence estimates. Such data should be used in the design and interpretation of venue-based serosurveillance 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.

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.

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, and high anti-RBD antibody levels. While anti-RBD 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 protection from reinfection by this strain. However, SARS-CoV-2 sera was unable to cross-neutralize a highly-homologous pre-emergent bat coronavirus, WIV1-CoV, that 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.

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.

High Seroprevalence of Anti-SARS-CoV-2 Antibodies in Chelsea, Massachusetts.

SARS-CoV-2 antibody testing allows quantitative determination of disease prevalence, which is especially important in high-risk communities. We performed anonymized convenience sampling of 200 currently asymptomatic residents of Chelsea, the epicenter of COVID-19 illness in Massachusetts, by BioMedomics SARS-CoV-2 combined IgM-IgG point-of-care lateral flow immunoassay. The seroprevalence was 31.5% (17.5% IgM+IgG+, 9.0% IgM+IgG-, and 5.0% IgM-IgG+). Of the 200 participants, 50.5% reported no symptoms in the preceding 4 weeks, of which 24.8% (25/101) were seropositive, and 60% of these were IgM+IgG-. These data are the highest seroprevalence rates observed to date and highlight the significant burden of asymptomatic infection.

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.

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.

SARS-CoV-2-specific ELISA development.

Critical to managing the spread of COVID-19 is the ability to diagnose infection and define the acquired immune response across the population. While genomic tests for the novel Several Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) detect the presence of viral RNA for a limited time frame, when the virus is shed in the upper respiratory tract, tests able to define exposure and infection beyond this short window of detectable viral replication are urgently needed. Following infection, antibodies are generated within days, providing a durable read-out and archive of exposure and infection. Several antibody tests have emerged to diagnose SARS-CoV-2. Here we report on a qualified quantitative ELISA assay that displays all the necessary characteristics for high-throughput sample analysis. Collectively, this test offers a quantitative opportunity to define both exposure and levels of immunity to SARS-CoV-2.

Test performance evaluation of SARS-CoV-2 serological assays.

BACKGROUND: Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. METHOD: We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. RESULTS: Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. CONCLUSION: Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.