INVESTIGATING ANTIGENIC FEATURES OF THE SARS-CoV-2 ISOLATED IN RUSSIAN FEDERATION IN 2021-2022 BY HYPERIMMUNE MOUSE SERUM NEUTRALISATION.

Introduction. The rapid spread of a new coronavirus infection among populations in many countries worldwide has contributed to the genetic evolution of the virus, resulting in the emergence of multiple genetic variants of the SARSCoV-2 coronavirus. Mutations in the viral genome can affect the ability of the virus to bypass the immune system and complicate development of diagnostic and prophylactic drugs. Data on the neutralizing activity of the sera obtained against previously circulating genetic variants of the virus in relation to current SARS-CoV-2 strains may serve as a scientific basis for the selection of the antigens in vaccine development. The aim of this work was to study cross-reactivity of SARSCoV-2 coronavirus strains belonging to different genetic variants, which were isolated in the territory of the Russian Federation during 2020-2022 in the neutralization reaction using mouse hyperimmune sera. Materials and methods. Ten strains of SARS-CoV-2 coronavirus belonging to different genetic variants were used (three non-VOC strains, alpha, beta, gamma, delta, delta+AY, omicron 1 and omicron 2). The hCoV-19/Australia/VIC01/2020 strain (Wuhan) was included in the study as a prototypical variant. BALBc mice were immunized with inactivated concentrated antigen mixed with a 1:1 adjuvant, which was a virus-like immunostimulatory complex based on Quillaja saponaria (Quillaja saponaria). The antibody titer was determined in the neutralization reaction. Results. Essential decrease of neutralizing ability of antibodies specific to non-vOC genetic variants of SARS-CoV-2 coronavirus was revealed against beta VOC and to a lesser degree against alpha and gamma VOC variants. The differences in the neutralizing activity level of antibodies for alpha and beta VOC variants are not significant among themselves, and with gamma VOC variants - there are no significant differences. Neutralizing ability of antibodies specific to delta VOC against alpha and beta VOC variants decreased 4-fold. Neutralizing activity of sera obtained to omicron 1 and 2 variants in relation to the prototype coronavirus variant was reduced 18-fold, to the gamma variant - 12-fold, to delta variants - more than 30-fold;for other variants it was even lower. Conclusions. The results obtained testify to the presence of cross-reactivity between strains of coronavirus belonging to genetic lines Wuhan, alpha, beta, gamma;it is weaker for delta variants. Mutations in the genome of VOC omicron variants led to a significant decrease in antigenic cross-links with earlier genetic variants of the coronavirus. These findings explain the low efficacy of vaccines based on the Wuhan strain, synthetic immunogens, and recombinant proteins based on it against omicron VOC variants, which have caused a rise in morbidity since early 2022, as well as cases of re-infection of humans with new genetic variants of the coronavirus.Copyright © 2023 Saint Petersburg Pasteur Institute. All rights reserved.

INVESTIGATING ANTIGENIC FEATURES OF THE SARS-CoV-2 ISOLATED IN RUSSIAN FEDERATION IN 2021–2022 BY HYPERIMMUNE MOUSE SERUM NEUTRALISATION

Introduction. The rapid spread of a new coronavirus infection among populations in many countries worldwide has contributed to the genetic evolution of the virus, resulting in the emergence of multiple genetic variants of the SARSCoV-2 coronavirus. Mutations in the viral genome can affect the ability of the virus to bypass the immune system and complicate development of diagnostic and prophylactic drugs. Data on the neutralizing activity of the sera obtained against previously circulating genetic variants of the virus in relation to current SARS-CoV-2 strains may serve as a scientific basis for the selection of the antigens in vaccine development. The aim of this work was to study cross-reactivity of SARSCoV-2 coronavirus strains belonging to different genetic variants, which were isolated in the territory of the Russian Federation during 2020–2022 in the neutralization reaction using mouse hyperimmune sera. Materials and methods. Ten strains of SARS-CoV-2 coronavirus belonging to different genetic variants were used (three non-VOC strains, alpha, beta, gamma, delta, delta+AY, omicron 1 and omicron 2). The hCoV-19/Australia/VIC01/2020 strain (Wuhan) was included in the study as a prototypical variant. BALBc mice were immunized with inactivated concentrated antigen mixed with a 1:1 adjuvant, which was a virus-like immunostimulatory complex based on Quillaja saponaria (Quillaja saponaria). The antibody titer was determined in the neutralization reaction. Results. Essential decrease of neutralizing ability of antibodies specific to non-vOC genetic variants of SARS-CoV-2 coronavirus was revealed against beta VOC and to a lesser degree against alpha and gamma VOC variants. The differences in the neutralizing activity level of antibodies for alpha and beta VOC variants are not significant among themselves, and with gamma VOC variants — there are no significant differences. Neutralizing ability of antibodies specific to delta VOC against alpha and beta VOC variants decreased 4-fold. Neutralizing activity of sera obtained to omicron 1 and 2 variants in relation to the prototype coronavirus variant was reduced 18-fold, to the gamma variant — 12-fold, to delta variants — more than 30-fold;for other variants it was even lower. Conclusions. The results obtained testify to the presence of cross-reactivity between strains of coronavirus belonging to genetic lines Wuhan, alpha, beta, gamma;it is weaker for delta variants. Mutations in the genome of VOC omicron variants led to a significant decrease in antigenic cross-links with earlier genetic variants of the coronavirus. These findings explain the low efficacy of vaccines based on the Wuhan strain, synthetic immunogens, and recombinant proteins based on it against omicron VOC variants, which have caused a rise in morbidity since early 2022, as well as cases of re-infection of humans with new genetic variants of the coronavirus. © 2023 Saint Petersburg Pasteur Institute. All rights reserved.

Sensitivity of SARS-CoV-2 antibody tests with late convalescent sera.

SARS-CoV-2-specific IgM antibodies wane during the first three months after infection and IgG antibody levels decline. This may limit the ability of antibody tests to identify previous SARS-CoV-2 infection at later time points. To examine if the diagnostic sensitivity of antibody tests falls off, we compared the sensitivity of two nucleoprotein-based antibody tests, the Roche Elecsis II Anti-SARS-CoV-2 and the Abbott SARS-CoV-2 IgG assay and three glycoprotein-based tests, the Abbott SARS-CoV-2 IgG II Quant, Siemens Atellica IM COV2T and Euroimmun SARS-CoV-2 assay with 53 sera obtained 6 months after SARS-CoV-2 infection. The sensitivity of the Roche, Abbott SARS-CoV-2 IgG II Quant and Siemens antibody assays was 94.3% (95% confidence interval (CI) 84.3-98.8%), 98.1 % (95% CI: 89.9-100%) and 100 % (95% CI: 93.3-100%). The sensitivity of the N-based Abbott SARS-CoV-2 IgG and the glycoprotein-based Euroimmun ELISA was 45.3 % (95% CI: 31.6-59.6%) and 83.3% (95% CI: 70.2-91.9%). The nucleoprotein-based Roche and the glycoprotein-based Abbott receptor binding domain (RBD) and Siemens tests were more sensitive than the N-based Abbott and the Euroimmun antibody tests (p = 0.0001 to p = 0.039). The N-based Abbott antibody test was less sensitive 6 months than 4-10 weeks after SARS-CoV-2 infection (p = 0.0001). The findings show that most SARS-CoV-2 antibody assays correctly identified previous infection 6 months after infection. The sensitivity of pan-Ig antibody tests was not reduced at 6 months when IgM antibodies have usually disappeared. However, one of the nucleoprotein-based antibody tests significantly lost diagnostic sensitivity over time.

An Overview of the Treatment Contributions Measured Globally for the COVID-19 Outbreak

Background: SARS CoV2 is a newly emerged animal beta coronavirus that causes respiratory illness. This infection has affected 212 countries to date and has been declared a pandemic by the World Health Organization. Due to the high transmission rate and lack of availability of any approved anti-viral drug, the formulation of a specific anti-viral therapy has now become a global emergency. Genomic studies have revealed a 79% identity of SARS CoV2 with SARS CoV and 50% identity with MERS CoV, which has given a clue point to test the drugs that were efficient against previously encoun-tered beta coronaviruses. For this purpose, several clinical trials based on the knowledge of existing drugs are moving ahead. These therapies include chloroquine and hydroxychloroquine, remdesivir, cor-ticosteroids therapy, favipiravir, ribavirin, lopinavir/ritonavir, anti-cytokine therapy, and convalescent sera. Aim of the study: The purpose of this review is to give a pointer of contributions conducted globally, including strategies utilized for treatments, the pattern of dosage, adverse reactions, and effective outcomes from different drugs. Methodology: Literature has been retrieved from PubMed, PubMed Central, ResearchGate, ScienceDi-rect, and Google Scholar, using a combination of keywords for extensive information. Conclusion(s): Among all the drug options, Remdesivir and the use of Convalescent Sera have been con-sidered as the safest options for treatment against COVID-19. Data from the ongoing clinical trials will be required for the formulation of a specific and approved anti-viral drug,.Copyright © 2021 Bentham Science Publishers.

Comparative Pharmacological Efficacy of COVID-19 Vaccines against the Variants of Concerns (VOCs) of SARS-CoV-2: Recent Clinical Studies on Booster Dose.

Sera obtained from convalescent individuals, and vaccinated individuals can induce low neutralizing efficacy against variants of concerns (VOCs) of SARS-CoV-2. In addition, the majority of COVID-19 vaccines are less efficacious against VOCs when compared to their efficacy against the original virus. Immune escape is one of the significant mechanisms observed during SARS-CoV-2 infection due to the substantial mutational capacity of VOCs such as B.1.1.7, P.1, B.1.351, B.1.617.2, C.37, and B.1.621. Omicron, a novel strain of SARS-CoV-2, also referred to as B.1.1.529, was identified in South Africa. This variant is a potential new VOC by the World Health Organization (WHO), and confirmed cases have been arising across several nations due to its rapid spreading ability. Omicron variant can acquire substantial immune escape following Delta, Beta/Gamma D614G VOCs and subsequently facilitating potential infectivity due to its enhanced ACE2 binding ability. The Omicron variant is a highly mutated variant accompanied by higher transmissibility and immune evasion. This mini review describes the ability of VOCs to acquire immune escape and also describes the comparative neutralization efficacy of several vaccines, including Booster doses against SARS-CoV-2.

Longitudinal neutralization activities on authentic Omicron variant provided by three doses of BBIBP-CorV vaccination during one year.

The majority of people in China have been immunized with the inactivated viral vaccine BBIBP-CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one-year longitudinal neutralization data of BBIBP-CorV on authentic Omicron, Delta, and wild-type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP-CorV. The sera were also subjected for monitoring the SARS-CoV-2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP-CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.

Estimated Infection and Vaccine Induced SARS-CoV-2 Seroprevalence in Israel among Adults, January 2020-July 2021.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in Israel in February 2020 and spread from then. In December 2020, the FDA approved an emergency use authorization of the Pfizer-BioNTech vaccine, and on 20 December, an immunization campaign began among adults in Israel. We characterized seropositivity for IgG anti-spike antibodies against SARS-CoV-2 between January 2020 and July 2021, before and after the introduction of the vaccine in Israel among adults. We tested 9520 serum samples, collected between January 2020 and July 2021. Between January and August 2020, seropositivity rates were lower than 5.0%; this rate increased from September 2020 (6.3%) to April 2021 (84.9%) and reached 79.1% in July 2021. Between January and December 2020, low socio-economic rank was an independent, significant correlate for seropositivity. Between January and July 2021, the 40.00-64.99-year-old age group, Jews and others, and residents of the Northern district were significantly more likely to be seropositive. Our findings indicate a slow, non-significant increase in the seropositivity rate to SARS-CoV-2 between January and December 2020. Following the introduction of the Pfizer-BioNTech vaccine in Israel, a significant increase in seropositivity was observed from January until April 2021, with stable rates thereafter, up to July 2021.

Pre-Omicron Vaccine Breakthrough Infection Induces Superior Cross-Neutralization against SARS-CoV-2 Omicron BA.1 Compared to Infection Alone.

SARS-CoV-2 variants raise concern because of their high transmissibility and their ability to evade neutralizing antibodies elicited by prior infection or by vaccination. Here, we compared the neutralizing abilities of sera from 70 unvaccinated COVID-19 patients infected before the emergence of variants of concern (VOCs) and of 16 vaccine breakthrough infection (BTI) cases infected with Gamma or Delta against the ancestral B.1 strain, the Gamma, Delta and Omicron BA.1 VOCs using live virus. We further determined antibody levels against the Nucleocapsid (N) and full Spike proteins, the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the Spike protein. Convalescent sera featured considerable variability in the neutralization of B.1 and in the cross-neutralization of different strains. Their neutralizing capacity moderately correlated with antibody levels against the Spike protein and the RBD. All but one convalescent serum failed to neutralize Omicron BA.1. Overall, convalescent sera from patients with moderate disease had higher antibody levels and displayed a higher neutralizing ability against all strains than patients with mild or severe forms of the disease. The sera from BTI cases fell into one of two categories: half the sera had a high neutralizing activity against the ancestral B.1 strain as well as against the infecting strain, while the other half had no or a very low neutralizing activity against all strains. Although antibody levels against the spike protein and the RBD were lower in BTI sera than in unvaccinated convalescent sera, most neutralizing sera also retained partial neutralizing activity against Omicron BA.1, suggestive of a better cross-neutralization and higher affinity of vaccine-elicited antibodies over virus-induced antibodies. Accordingly, the IC50: antibody level ratios were comparable for BTI and convalescent sera, but remained lower in the neutralizing convalescent sera from patients with moderate disease than in BTI sera. The neutralizing activity of BTI sera was strongly correlated with antibodies against the Spike protein and the RBD. Together, these findings highlight qualitative differences in antibody responses elicited by infection in vaccinated and unvaccinated individuals. They further indicate that breakthrough infection with a pre-Omicron variant boosts immunity and induces cross-neutralizing antibodies against different strains, including Omicron BA.1.

Development of an efficient reproducible cell-cell transmission assay for rapid quantification of SARS-CoV-2 spike interaction with hACE2.

Efficient quantitative assays for measurement of viral replication and infectivity are indispensable for future endeavors to develop prophylactic or therapeutic antiviral drugs or vaccines against SARS-CoV-2. We developed a SARS-CoV-2 cell-cell transmission assay that provides a rapid and quantitative readout to assess SARS-CoV-2 spike hACE2 interaction in the absence of pseudotyped particles or live virus. We established two well-behaved stable cell lines, which demonstrated a remarkable correlation with standard cell-free viral pseudotyping for inhibition by convalescent sera, small-molecule drugs, and murine anti-spike monoclonal antibodies. The assay is rapid, reliable, and highly reproducible, without a requirement for any specialized research reagents or laboratory equipment and should be easy to adapt for use in most investigative and clinical settings. It can be effectively used or modified for high-throughput screening for compounds and biologics that interfere with virus-cell binding and entry to complement other neutralization assays currently in use.

Potency of Fusion-Inhibitory Lipopeptides against SARS-CoV-2 Variants of Concern.

The ability of SARS-CoV-2 to evolve in response to selective pressures poses a challenge to vaccine and antiviral efficacy. The S1 subunit of the spike (S) protein contains the receptor-binding domain and is therefore under selective pressure to evade neutralizing antibodies elicited by vaccination or infection. In contrast, the S2 subunit of S is only transiently exposed after receptor binding, which makes it a less efficient target for antibodies. As a result, S2 has a lower mutational frequency than S1. We recently described monomeric and dimeric SARS-CoV-2 fusion-inhibitory lipopeptides that block viral infection by interfering with S2 conformational rearrangements during viral entry. Importantly, a dimeric lipopeptide was shown to block SARS-CoV-2 transmission between ferrets in vivo. Because the S2 subunit is relatively conserved in newly emerging SARS-CoV-2 variants of concern (VOCs), we hypothesize that fusion-inhibitory lipopeptides are cross-protective against infection with VOCs. Here, we directly compared the in vitro efficacies of two fusion-inhibitory lipopeptides against VOC, in comparison with a set of seven postvaccination sera (two doses) and a commercial monoclonal antibody preparation. For the beta, delta, and omicron VOCs, it has been reported that convalescent and postvaccination sera are less potent in virus neutralization assays. Both fusion-inhibitory lipopeptides were equally effective against all five VOCs compared to ancestral virus, whereas postvaccination sera and therapeutic monoclonal antibody lost potency to newer VOCs, in particular to omicron BA.1 and BA.2. The neutralizing activity of the lipopeptides is consistent, and they can be expected to neutralize future VOCs based on their mechanism of action. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, with waves resulting from new variants that evade immunity generated by vaccines and previous strains and escape available monoclonal antibody therapy. Fusion-inhibitory peptides may provide an intervention strategy that is not similarly affected by this viral evolution.

Assessment of specific human antibodies against SARS-CoV-2 receptor binding domain by rapid in-house ELISA.

BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88% specificity, 78.5% agreement, a positive predictive value (PPV) of 92.3%, and a negative predictive value of 56.5%. Moreover, the optical density (OD) values of positive samples significantly correlated with the commercial kit titers. CONCLUSIONS: Specific human antibodies against SARS-CoV-2 spike protein were detected by rapid in-house ELISA in sera of human COVID-19-infected patients. The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications, particularly in developing countries. Future studies are planned for the use of the generated SARS-CoV-2 rRBD-6×His protein in vaccine development and other diagnostic applications.

Sera Metabolomics Characterization of Patients at Different Stages in Wuhan Identifies Critical Biomarkers of COVID-19.

We have witnessed the 2-year-long global rampage of COVID-19 caused by the wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, knowledge about biomarkers of the entire COVID-19 process is limited. Identification of the systemic features of COVID-19 will lead to critical biomarkers and therapeutic targets for early intervention and clinical disease course prediction. Here, we performed a comprehensive analysis of clinical measurements and serum metabolomics in 199 patients with different stages of COVID-19. In particular, our study is the first serum metabolomic analysis of critical rehabilitation patients and critical death patients. We found many differential metabolites in the comparison of metabolomic results between ordinary, severe, and critical patients and uninfected patients. Through the metabolomic results of COVID-19 patients in various stages, and critical rehabilitation patients and critical death patients, we identified a series of differential metabolites as biomarkers, a separate queue and precise distinction, and predicted COVID-19 verification. These differentially expressed metabolites, included 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphate, propylparaben, 20-hydroxyeicosatetraenoic acid, triethanolamine, chavicol, disialosyl galactosyl globoside, 1-arachidonoylglycerophosphoinositol, and alpha-methylstyrene, all of which have been identified for the first time as biomarkers in COVID-19 progression. These biomarkers are involved in many pathological and physiological pathways of COVID-19, for example, immune responses, platelet degranulation, and metabolism which might result in pathogenesis. Our results showed valuable information about metabolites obviously altered in COVID-19 patients with different stages, which could shed light on the pathogenesis as well as serve as potential therapeutic agents of COVID-19.

Comparative Investigation of Methods for Analysis of SARS-CoV-2-Spike-Specific Antisera.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

SARS-CoV-2 E484K Mutation Narrative Review: Epidemiology, Immune Escape, Clinical Implications, and Future Considerations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly over the world and claimed million lives. The virus evolves constantly, and a swarm of mutants is a now major concern globally. Distinct variants could have independently converged on same mutation, despite being detected in different geographic regions, which suggested it could confer an evolutionary advantage. E484K has rapidly emerged and has frequently been detected in several SARS-CoV-2 variants of concern. In this study, we review the epidemiology and impact of E484K, its effects on neutralizing effect of several monoclonal antibodies, convalescent plasma, and post-vaccine sera.

Sensitivity of SARS-CoV-2 Variants to Neutralization by Convalescent Sera and a VH3-30 Monoclonal Antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel coronavirus disease (COVID-19). Though vaccines and neutralizing monoclonal antibodies (mAbs) have been developed to fight COVID-19 in the past year, one major concern is the emergence of SARS-CoV-2 variants of concern (VOCs). Indeed, SARS-CoV-2 VOCs such as B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil), and B.1.617.1 (India) now dominate the pandemic. Herein, we found that binding activity and neutralizing capacity of sera collected from convalescent patients in early 2020 for SARS-CoV-2 VOCs, but not non-VOC variants, were severely blunted. Furthermore, we observed evasion of SARS-CoV-2 VOCs from a VH3-30 mAb 32D4, which was proved to exhibit highly potential neutralization against wild-type (WT) SARS-CoV-2. Thus, these results indicated that SARS-CoV-2 VOCs might be able to spread in convalescent patients and even harbor resistance to medical countermeasures. New interventions against these SARS-CoV-2 VOCs are urgently needed.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals.

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen's kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Evaluation of a multi-species SARS-CoV-2 surrogate virus neutralization test.

Assays to measure SARS-CoV-2-specific neutralizing antibodies are important to monitor seroprevalence, to study asymptomatic infections and to reveal (intermediate) hosts. A recently developed assay, the surrogate virus-neutralization test (sVNT) is a quick and commercially available alternative to the "gold standard" virus neutralization assay using authentic virus, and does not require processing at BSL-3 level. The assay relies on the inhibition of binding of the receptor binding domain (RBD) on the spike (S) protein to human angiotensin-converting enzyme 2 (hACE2) by antibodies present in sera. As the sVNT does not require species- or isotype-specific conjugates, it can be similarly used for antibody detection in human and animal sera. In this study, we used 298 sera from PCR-confirmed COVID-19 patients and 151 sera from patients confirmed with other coronavirus or other (respiratory) infections, to evaluate the performance of the sVNT. To analyze the use of the assay in a One Health setting, we studied the presence of RBD-binding antibodies in 154 sera from nine animal species (cynomolgus and rhesus macaques, ferrets, rabbits, hamsters, cats, cattle, mink and dromedary camels). The sVNT showed a moderate to high sensitivity and a high specificity using sera from confirmed COVID-19 patients (91.3% and 100%, respectively) and animal sera (93.9% and 100%), however it lacked sensitivity to detect low titers. Significant correlations were found between the sVNT outcomes and PRNT50 and the Wantai total Ig and IgM ELISAs. While species-specific validation will be essential, our results show that the sVNT holds promise in detecting RBD-binding antibodies in multiple species.

History repeats itself: horse originated hyperimmune sera production against SARS CoV-2

Background/aim/AIM: SARS-CoV-2 disease was announced as a pandemic by The World Health Organization in early 2020. It is still threatening the world population. Here, we aimed to produce hyperimmune sera that contain immunoglobulin G and F(ab')2 fragments sourced from horse antibodies as an urgent response to the pandemic. Materials and methods: SARS-CoV-2 was produced and inactivated with three different methods [formaldehyde (FA), formaldehyde, and binary ethylene amine (FA + BEI), and heat treatment]. After in vitro inactivation control, immunogens were mixed with Freund's adjuvant, thereafter horses (n: 2 for FA, 4 for FA + BEI, 2 for heat inactivation) and New Zealand rabbits (n: 6 for FA, 6 fo r FA + BEI, 6 for heat inactivation) were immunized four times. Neutralizing antibody levels of the sera were measured at the 4th, 6th, and 8th weeks. When the antibodies were detected at the peak level, plasma was collected from horses and hyperimmune sera procured after the purification process. Results: Horses and rabbits produced highly neutralizing antibodies against the SARS-CoV-2 in FA and FA + BEI inactivation groups, foreign proteins were removed effectively after purification. Conclusion: This study presents a profitable practice to develop specific antisera in horses against SARS-CoV-2 for emergency and low-cost response. In further studies, new purification methods can be used to increase the efficiency of the final product.

Unexpected high frequency of unspecific reactivities by testing pre-epidemic blood specimens from Europe and Africa with SARS-CoV-2 IgG-IgM antibody rapid tests points to IgM as the Achilles heel.

We aimed to evaluate the rates of false-positive test results of three rapid diagnostic tests (RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G (IgG) and IgM detection. Two serum panels from patients hospitalized in Paris, France, and from patients living in Bangui, Central African Republic, acquired before the 2019 COVID-19 outbreak, were tested by 3 CE IVD-labeled RDTs for SARS-CoV-2 serology (BIOSYNEX® COVID-19 BSS [IgG/IgM]; SIENNA™ COVID-19 IgG/IgM Rapid Test Cassette; NG-Test® IgG-IgM COVID-19). Detectable IgG or IgM reactivities could be observed in 31 (3.43%) of the 902 IgG and IgM bands of the 3 RDTs used with all pre-epidemic sera. The frequencies of IgG/IgM reactivities were similar for European (3.20%) and African (3.55%) sera. IgM reactivities were observed in 9 European and 14 African sera, while IgG reactivity was observed in only 1 African serum (15.1% vs. 0.66%). The test NG-Test® IgG-IgM COVID-19 showed the highest rates of IgG or IgM reactivities (6.12% [18/294]), while the test BIOSYNEX® COVID-19 BSS (IgG/IgM) showed the lowest rate (1.36% [4/294]). Some combinations of 2 RDTs in series allowed decreasing significantly the risk of false-positive test results. Our observations point to the risk of false-positive reactivities when using currently available RDT for SARS-CoV-2 serological screening, especially for the IgM band, even if the test is CE IVD-labeled and approved by national health authorities, and provide the rational basis for confirmatory testing by another RDT in case of positive initial screening.

No Evidence for Human Monocyte-Derived Macrophage Infection and Antibody-Mediated Enhancement of SARS-CoV-2 Infection.

Vaccines are essential to control the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and to protect the vulnerable population. However, one safety concern of vaccination is the possible development of antibody-dependent enhancement (ADE) of SARS-CoV-2 infection. The potential infection of Fc receptor bearing cells such as macrophages, would support continued virus replication and inflammatory responses, and thereby potentially worsen the clinical outcome of COVID-19. Here we demonstrate that SARS-CoV-2 and SARS-CoV neither infect human monocyte-derived macrophages (hMDM) nor induce inflammatory cytokines in these cells, in sharp contrast to Middle East respiratory syndrome (MERS) coronavirus and the common cold human coronavirus 229E. Furthermore, serum from convalescent COVID-19 patients neither induced enhancement of SARS-CoV-2 infection nor innate immune response in hMDM. Although, hMDM expressed angiotensin-converting enzyme 2, no or very low levels of transmembrane protease serine 2 were found. These results support the view that ADE may not be involved in the immunopathological processes associated with COVID-19, however, more studies are necessary to understand the potential contribution of antibodies-virus complexes with other cells expressing FcR receptors.