Assessing SARS-CoV-2 Neutralizing Antibodies after BNT162b2 Vaccination and Their Correlation with SARS-CoV-2 IgG Anti-S1, Anti-RBD and Anti-S2 Serological Titers

The humoral response through neutralizing antibodies (NAbs) is a key component of the immune response to COVID-19. However, the plaque reduction neutralization test (PRNT), the gold standard for determining NAbs, is technically demanding, time-consuming and requires BSL-3 conditions. Correlating the NAbs and total antibodies levels, assessed by generalized and automated serological tests, is crucial. Through a commercial surrogate virus neutralization test (sVNT), we aimed to evaluate the production of SARS-CoV-2 NAbs in a set of vaccinated healthcare workers and to correlate these NAbs with the SARS-CoV-2 IgG anti-S1, anti-RBD and anti-S2 serological titers. We found that 6 months after vaccination, only 74% maintain NAbs for the Wuhan strain/UK variant (V1) and 47% maintain NAbs for the South African and Brazil variants (V2). Through Spearman’s correlation, we found the following correlations between the percentage of inhibition of NAbs and the SARS-CoV-2 IgG II Quant (Abbott Laboratories, Chicago, IL, USA) and BioPlex 2200 SARS-CoV-2 IgG Panel (Bio-Rad, Hercules, CA, USA) immunoassays: rho = 0.87 (V1) and rho = 0.73 (V2) for anti-S1 assessed by Abbott assay; rho = 0.77 (V1) and rho = 0.72 (V2) for anti-S1, rho = 0.88 (V1) and rho = 0.82 (V2) for anti-RBD, and rho = 0.68 (V1) and rho = 0.60 (V2) for anti-S2 assessed by BioPlex assay (p < 0.001 for all). In conclusion, we found a strong correlation between this fast, user-friendly, mobile and bio-safe sVNT and the serological immunoassays.

Humoral Immune Response in IBD Patients Three and Six Months after Vaccination with the SARS-CoV-2 mRNA Vaccines mRNA-1273 and BNT162b2

Severe acute respiratory syndrome coronovirus-2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID-19) pandemic. Vaccination is considered the core approach to containing the pandemic. There is currently insufficient evidence on the efficacy of these vaccines in immunosuppressed inflammatory bowel disease (IBD) patients. The aim of this study was to investigate the humoral response in immunosuppressed IBD patients after COVID-19 mRNA vaccination. In this prospective study, IgG antibody levels (AB) against the SARS-CoV-2 receptor-binding domain (spike-protein) were quantitatively determined. For assessing the potential neutralizing capacity, a SARS-CoV-2 surrogate neutralization test (sVNT) was employed in IBD patients (n = 95) and healthy controls (n = 38). Sera were examined prior to the first/second vaccination and 3/6 months after second vaccination. Patients showed lower sVNT (%) and IgG-S (AU/mL) AB both before the second vaccination (sVNT p < 0.001; AB p < 0.001) and 3 (sVNT p = 0.002; AB p = 0.001) and 6 months (sVNT p = 0.062; AB p = 0.061) after the second vaccination. Although seroconversion rates (sVNT, IgG-S) did not differ between the two groups 3 months after second vaccination, a significant difference was seen 6 months after second vaccination (sVNT p = 0.045). Before and three months after the second vaccination, patients treated with anti-tumor necrosis factor (TNF) agents showed significantly lower AB than healthy subjects. In conclusion, an early booster shot vaccination should be discussed for IBD patients on anti-TNF therapy.

RADIOIMMUNOASSAY FOR DETECTION OF ANTI-RINDERPEST ANTIBODIES IN CATTLES

Liquid Phase Radioimmunoassay (RIA) was developed to detect and measure anti-rinderpest immunoglobulins in field animals sera, within two hours. Rinderpest virus adapted on Vero cell line culture and antigen purified by treatment with Triton-Gentron 13 Butanol, and, labeled with I isotope, using chloramin T iodination method. Comparative studies for detecting anti-rinderpest immunoglobulin in 80 calves sera samples, using the developed assay in parallel with virus neutralization test (VNT). The study showed 58.75 % agreement between the two methods. However, 71 % of seven months old, non-vaccinated calves showed anti rinderpest antibodies in their sera, also 81 % of 10 months old vaccinated calves were developed antibodies in their blood. These results demonstrate the development of sensitive, specific and rapid quantitative / qualitative radioimmunoassay, necessary for screening the development of immunity against rinderpest in cattle.

Development of specific immunity in laboratory animals after co-immunization against seasonal influenza and COVID-19

Introduction. In clinical practice, the differential diagnosis of COVID-19 can be challenging during the flu season, entailing serious consequences such as delays in appropriate control measures against the SARS-CoV-2 pandemic. Another problem is posed by co-infection of SARS-CoV-2 and influenza virus (IV), which significantly contributes to the severity of the COVID-19 disease. This study was aimed to explore the cross-impact of co-administration of Russian influenza and COVID-19 vaccines on development of specific immunity in laboratory animals.Materials and methods. The study was conducted on BALB/c mice. The animals were inoculated intramuscularly with the vaccine for COVID-19 prevention (CoviVac) and the vaccine for influenza prevention (Flu-M). The sera from the immunized animals were examined separately. Three IV strains were used in the hemagglutination inhibition assay. Antibodies (Abs) against SARS-CoV-2 were detected by an enzyme-linked immunosorbent assay (ELISA). The neutralization test was performed to detect virus neutralizing antibodies against SARS-CoV-2 and IV.Results. Relatively high titers of specific Abs were found in the groups of animals inoculated with one vaccine and with two vaccines concurrently. In the groups of animals inoculated with CoviVac and with two vaccines concurrently, both in the ELISA test and in the neutralization test, the average titers of specific Abs against SARSCoV- 2 did not demonstrate any statistical difference. The group of animals inoculated concurrently with two vaccines demonstrated statistically higher titers of Abs against IV after the second immunization compared to the group of animals inoculated with Flu-M.Discussion. The study has shown that post-vaccination immunity both to IV and to SARS-CoV-2 develops after co-vaccination with two vaccines. The observed enhanced post-vaccination immune response to IV in the coimmunized laboratory animals needs further research.Conclusion. The performed studies suggest the possibility of co-administration of two vaccines to prevent influenza and COVID-19.

Evaluation of Commercial Anti-SARS-CoV-2 Neutralizing Antibody Assays in seropositive subjects

The virus neutralization test (VNT) is the reference for the assessment of the functional ability of neutralizing antibodies (NAb) to block SARS-CoV-2 entry into cells. New competitive immunoassays measuring antibodies preventing interaction between the spike protein and its cellular receptor are proposed as surrogate VNT (sVNT). We tested three commercial sVNT (a qualitative immunochromatographic test and two quantitative immunoassays named YHLO and TECO) together with a conventional anti-spike IgG assay (bioMerieux) in comparison with an in-house plaque reduction neutralization test (PRNT50) using the original 19A strain and different variants of concern (VOC), on a panel of 306 sera from naturally-infected or vaccinated patients. The qualitative test was rapidly discarded because of poor sensitivity and specificity. Areas under the curve of YHLO and TECO assays were, respectively, 85.83 and 84.07 (p-value >0.05) using a positivity threshold of 20 for PRNT50, and 95.63 and 90.35 (p-value =0.02) using a threshold of 80. However, the performances of YHLO and bioMerieux were very close for both thresholds, demonstrating the absence of added value of sVNT compared to a conventional assay for the evaluation of the presence of NAb in seropositive subjects. In addition, the PRNT50 assay showed a reduction of NAb titers towards different VOC in comparison to the 19A strain that could not be appreciated by the commercial tests. Despite the good correlation between the anti-spike antibody titer and the titer of NAb by PRNT50, our results highlight the difficulty to distinguish true NAb among the anti-RBD antibodies with commercial user-friendly immunoassays.

Neutralizing antibody titres to SARS-CoV-2 Omicron variant and wild-type virus in those with past infection or vaccinated or boosted with mRNA BNT162b2 or inactivated CoronaVac vaccines

Omicron, a novel SARS-CoV-2 variant has emerged and is rapidly becoming the dominant SARS-CoV-2 virus circulating globally. It is important to define reductions in virus neutralizing activity in serum of convalescent or vaccinated individuals to understand potential loss of protection from infection or re-infection. Two doses of BNT162b2 or CoronaVac vaccines provided little 50% plaque reduction neutralization test (PRNT50) antibody immunity against the Omicron variant, even at one-month post vaccination. Booster doses with BNT162b2 in those with two doses of either BNT162b2 or CoronaVac provided acceptable neutralizing immunity against Omicron variant at 1-month post-booster dose. However, three doses of BNT162b2 elicited higher levels of PRNT50 antibody to Omicron variant suggesting longer duration of protection. Convalescent from SARS-CoV-2 infection did not have protective PRNT50 antibody levels to Omicron, but a single dose of BNT162b2 vaccine provided protective immunity. Field vaccine-efficacy studies against Omicron variant against different vaccines are urgently needed.

Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a solid correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive ELISA formats or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) show excellent correlation to a cVNT with fully infectious SARS-CoV-2 and allow to estimate the reduced neutralization capacity of COVID-19 vaccinee sera with variants of concern of SARS-CoV-2.Author summaryThe current pandemic caused by SARS-CoV-2 is a major challenge not only for COVID-19 patients, medical staff, healthcare systems and the general public, but also virologists and clinical laboratories. A particular challenge are safety issues which require biological safety level 3 to work with and study the pathogen. An alternative are virus-like particles (VLPs) of SARS-CoV-2, which are authentic in terms of viral structure and function but are harmless bioproducts in nature. We engineered VLPs which are close-to-perfect mimics of SARS-CoV-2 by all structural, biochemical, physical and functional criteria tested. SARS-CoV-2 VLPs were used in virus neutralization tests (VNTs). Because high concentrations of neutralizing antibodies correlate with protection from COVID-19 practical VNTs are urgently needed. We developed an authentic, virus-free, thus safe yet very fast in vitro diagnostic test with SARS-CoV-2 VLPs. Virus neutralizing antibodies induced by natural infection or vaccination but also certain monoclonal antibodies inhibit VLP fusion with recipient cells carrying ACE2. Quantitative results from a conventional neutralization test with fully infectious SARS-CoV-2 and results from the VLP-based neutralization test correlate perfectly. The setup of the test is very flexible and allows to analyze sera for their neutralizing capacity against different variants of concern and in a standardized assay format.

Timeline of SARS-CoV-2 Spread in Italy: Results from an Independent Serological Retesting

The massive emergence of COVID-19 cases in the first phase of pandemic within an extremely short period of time suggest that an undetected earlier circulation of SARS-CoV-2 might have occurred. Given the importance of this evidence, an independent evaluation was recommended by the World Health Organization (WHO) to test a subset of samples selected on the level of positivity in ELISA assays (positive, low positive, negative) detected in our previous study of prepandemic samples collected in Italy. SARS-CoV-2 antibodies were blindly retested by two independent centers in 29 blood samples collected in the prepandemic period in Italy, 29 samples collected one year before and 11 COVID-19 control samples. The methodologies used included IgG-RBD/IgM-RBD ELISA assays, a qualitative micro-neutralization CPE-based assay, a multiplex IgG protein array, an ELISA IgM kit (Wantai), and a plaque-reduction neutralization test. The results suggest the presence of SARS-CoV-2 antibodies in some samples collected in the prepandemic period, with the oldest samples found to be positive for IgM by both laboratories collected on 10 October 2019 (Lombardy), 11 November 2019 (Lombardy) and 5 February 2020 (Lazio), the latter with neutralizing antibodies. The detection of IgM and/or IgG binding and neutralizing antibodies was strongly dependent on the different serological assays and thresholds employed, and they were not detected in control samples collected one year before. These findings, although gathered in a small and selected set of samples, highlight the importance of harmonizing serological assays for testing the spread of the SARS-CoV-2 virus and may contribute to a better understanding of future virus dynamics.

Global Seroprevalence of West Nile Virus in Horses of Morocco

West Nile Fever (WNF) is a viral emerging mosquito-borne disease causing mortality and morbidity with varying severity (from mild fever to severe neuroinvasive disease) among human and animal populations in many parts of the world. The current study aimed to confirm the virus circulation and assess the disease seroprevalence in horses of Morocco. A sample of 1171 healthy non-vaccinated (against-WNF) horses, taken from 11 (out of 12) regions of the country during July-December 2016, was primarily tested using competitive ELISA assay (cELISA). All cELISA-reactive positive and doubtful sera (n= 269) were further tested by virus neutralization test (VNT). The results of cELISA test revealed an overall WNF seroprevalence in 21.8% (255/1171) of sampled horses. This rate decreased to 18.8% (220/1171) after the confirmation of VNT. The WNF seroprevalence in the current study varied significantly by age, gender, and breed of the tested horses. Indeed, the higher seropositivity rates were found in the oldest (27.7%), female (22.0%), and saddle (32.1%) horses. However, the origin of animals did not show any significant effect on the West Nile virus infection. The obtained results of the present study, therefore, provided serological and epidemiological evidence of the endemicity of the WNV in horse populations of Morocco.

A high-throughput neutralization assay for yellow fever serodiagnostics

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks, for surveillance and to evaluate vaccine efficacy in population-wide studies. All this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming and limited in throughput, classical plaque reduction neutralization test (PRNT) is still considered gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika and dengue viruses amenable for differential diagnostics.