Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots.

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure™). Results: DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants.

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays.

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

Temporal Analysis of Serial Donations Reveals Decrease in Neutralizing Capacity and Justifies Revised Qualifying Criteria for Coronavirus Disease 2019 Convalescent Plasma.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) received an Emergency Use Authorization by the US Food and Drug Administration (FDA). CCP with a signal-to-cutoff ratio of ≥12 using the Ortho VITROS severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) test (OVSARS2IgG) is permitted to be labeled "high titer." Little is known about the relationship between OVSARS2IgG ratio and neutralizing capacity of plasma/sera against genuine SARS-CoV-2. METHODS: Nine hundred eighty-one samples from 196 repeat CCP donors 0-119 days post-initial donation (DPID) were analyzed. Neutralizing capacity was assessed for 50% (PRNT50) and 90% (PRNT90) reduction of infectious virus using the gold standard plaque reduction neutralization test (PRNT). A subset of 91 donations was evaluated by OVSARS2IgG and compared to PRNT titers for diagnostic accuracy. RESULTS: Of donations, 32.7%/79.5% (PRNT90/PRNT50) met a 1:80 titer initially but only 14.0%/48.8% (PRNT90/PRNT50) met this cutoff ≥85 DPID. Correlation of OVSARS2IgG results to neutralizing capacity allowed extrapolation to CCP therapy results. CCP with OVSARS2IgG ratios equivalent to a therapeutically beneficial group had neutralizing titers of ≥1:640 (PRNT50) and/or ≥1:80 (PRNT90). Specificity and positive predictive value of the OVSARS2IgG for qualifying highly neutralizing CCP was optimal using ratios significantly greater than the FDA cutoff. CONCLUSIONS: This information provides a basis for refining the recommended properties of CCP used to treat COVID-19.

A simple protein-based surrogate neutralization assay for SARS-CoV-2.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, must adapt to host-associated environments during infection by modulating gene expression. Small regulatory RNAs (sRNAs) are key regulators of bacterial gene expression, but their roles in Mtb are not well understood. Here, we address the expression and function of the Mtb sRNA Mcr11, which is associated with slow bacterial growth and chronic infections in mice. We found that stable expression of Mcr11 requires multiple factors specific to TB-complex bacteria, including the AbmR transcription factor. Bioinformatic analyses used to predict regulatory targets of Mcr11 identified 7-11 nucleotide regions with potential for direct base-pairing with Mcr11 immediately upstream of Rv3282, fadA3, and lipB. mcr11-dependent regulation of these genes was demonstrated using qRT-PCR and found to be responsive to the presence of fatty acids. Mutation of the putative Mcr11 base-pairing site upstream of lipB in a promoter reporter strain resulted in significant de-repression of lipB expression, similar to that observed in mcr11-deleted Mtb. These studies establish Mcr11's roles in regulating growth and central metabolism in Mtb. Our finding that multiple TB-complex-specific factors are required for production of stable Mcr11 also emphasizes the need to better understand mechanisms of sRNA expression and stability in TB.

AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non-coding small RNA Mcr11.

Gene regulatory networks used by Mycobacterium tuberculosis (Mtb) during infection include many genes of unknown function, confounding efforts to determine their roles in Mtb biology. Rv1265 encodes a conserved hypothetical protein that is expressed during infection and in response to elevated levels of cyclic AMP. Here, we report that Rv1265 is a novel auto-inhibitory ATP-binding transcription factor that upregulates expression of the small non-coding RNA Mcr11, and propose that Rv1265 be named ATP-binding mcr11 regulator (AbmR). AbmR directly and specifically bound DNA, as determined by electrophoretic mobility shift assays, and this DNA-binding activity was enhanced by AbmR's interaction with ATP. Genetic knockout of abmR in Mtb increased abmR promoter activity and eliminated growth phase-dependent increases in mcr11 expression during hypoxia. Mutagenesis identified arginine residues in the carboxy terminus that are critical for AbmR's DNA-binding activity and gene regulatory function. Limited similarity to other DNA- or ATP-binding domains suggests that AbmR belongs to a novel class of DNA- and ATP-binding proteins. AbmR was also found to form large organized structures in solution and facilitate the serum-dependent association of Mtb with human lung epithelial cells. These results indicate a potentially complex role for AbmR in Mtb biology.