Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration.

OBJECTIVES: To assess the long-term humoral immunity induced by booster administration, as well as the ability of binding antibody and surrogate virus neutralization tests (sVNT) to predict neutralizing antibodies (NAbs) against the SARS-CoV-2 Omicron variant. METHODS: A total of 269 sera samples were analyzed from 64 healthcare workers who had received a homologous booster dose of BNT162b2. Neutralizing antibodies assessed by sVNT and anti-RBD IgG measured with the sCOVG assay (Siemens Healthineers®) were analyzed at five timepoints; before and up to 6 months following the booster. Antibody titers were correlated with neutralizing antibodies against the Omicron BA.1 variant obtained by pseudovirus neutralization test (pVNT) as a reference method. RESULTS: While Wild-type sVNT percentage of inhibition (POI) remained above 98.6% throughout the follow-up period after booster administration, anti-RBD IgG and NAbs assessed by Omicron BA.1 pVNT showed respectively a 3.4-fold and 13.3-fold decrease after 6 months compared to the peak reached at day 14. NAbs assessed by Omicron sVNT followed a steady decline until reaching a POI of 53.4%. Anti-RBD IgG and Omicron sVNT assays were strongly correlated (r=0.90) and performed similarly to predict the presence of neutralizing antibodies with Omicron pVNT (area under the ROC: 0.82 for both assays). In addition, new adapted cut-off values of anti-RBD IgG (>1,276 BAU/mL) and Omicron sVNT (POI>46.6%) were found to be better predictors of neutralizing activity. CONCLUSIONS: This study showed a significant drop in humoral immunity 6 months after booster administration. Anti-RBD IgG and Omicron sVNT assays were highly correlated and could predict neutralizing activity with moderate performance.

Clinical performance evaluation of the Fluorecare® SARS-CoV-2 & Influenza A/B & RSV rapid antigen combo test in symptomatic individuals

Background : A SARS-CoV-2+Flu A/B+RSV Combo Rapid test may be more relevant than Rapid Antigen Diagnostic (RAD) tests targeting only SARS-CoV-2 since we are facing a concurrent circulation of these viruses during the winter season. Objectives : To assess the clinical performance of a SARS-CoV-2+Flu A/B+RSV Combo test in comparison to a multiplex RT-qPCR. Study Design : Residual nasopharyngeal swabs issued from 178 patients were included. All patients, adults and children, were symptomatic and presented at the emergency department with flu-like symptoms. Characterization of the infectious viral agent was done by RT-qPCR. The viral load was expressed as cycle threshold (Ct). Samples were then tested using the multiplex RAD test Fluorecare®ฏ SARS-CoV-2 & Influenza A/B & RSV Antigen Combo Test. Data analysis was carried out using descriptive statistics. Results : The sensitivity of the test varies according to the virus, with the highest sensitivity observed for Influenza A (80.8.% [95%CI: 67.2 - 94.4]) and the lowest sensitivity observed for RSV (41.5% [95%CI: 26.2 - 56.8]). Higher sensitivities were observed for samples with high viral loads (Ct < 20) and decrease with low viral loads. The specificity for SARS-CoV-2, RSV and Influenza A and B was >95%. Conclusions : The Fluorecare® combo antigenic presents satisfying performance in real-life clinical setting for Influenza A and B in samples with high viral load. This could be useful to allow a rapid (self-)isolation as the transmissibility of these viruses increase with the viral load. According to our results, its use to rule-out SARS-CoV-2 and RSV infection is not sufficient.

Peri-infection titers of neutralizing and binding antibodies as a predictor of COVID-19 breakthrough infections in vaccinated healthcare professionals: importance of the timing.

OBJECTIVES: The BNT162b2 messenger RNA vaccine is highly effective in reducing COVID-19 infection, hospitalization and death. However, many subjects developed a breakthrough infection despite a full vaccination scheme. Since the waned efficacy of mRNA vaccines is correlated with the decrease of antibodies occurring over time, we aimed at evaluating whether lower levels of antibodies were associated with an increased risk of breakthrough infection in a cohort of breakthrough subjects that received three vaccine doses. METHODS: Total binding antibodies against the RBD of the S1 subunit (Roche Diagnostics, Machelen, Belgium) and neutralizing antibodies using the Omicron B.1.1.529 variant pseudovirus were measured. Based on individual kinetic curves, the antibody titer of each subject was interpolated just before the breakthrough infection and compared to a matched-control group that did not develop a breakthrough infection. RESULTS: Lower levels of total binding and neutralizing antibodies were observed compared to the control group (6.900 [95% CI; 5.101-9.470] vs. 11.395 BAU/mL [8.627-15.050] [p=0.0301] and 26.6 [18.0-39.3] vs. 59.5 dilution titer-1 [32.3-110] [p=0.0042], respectively). The difference between breakthrough and control subjects was mostly observed for neutralizing antibodies before three months after the homologous booster administration (46.5 [18.2-119] vs. 381 [285-509] [p=0.0156]). Considering the measurement of total binding antibodies before 3 months, there was no significant difference (p=0.4375). CONCLUSIONS: In conclusion, our results showed that subjects that developed a breakthrough infection had lower levels of neutralizing and total binding antibodies compared to controls. The difference was mostly noticeable considering neutralizing antibodies, especially for infections occurring before 3 months after the booster administration.

Clinical performance evaluation of the Fluorecare® SARS-CoV-2 & Influenza A/B & RSV rapid antigen combo test in symptomatic individuals.

BACKGROUND: A SARS-CoV-2+Flu A/B+RSV Combo Rapid test may be more relevant than Rapid Antigen Diagnostic (RAD) tests targeting only SARS-CoV-2 since we are facing a concurrent circulation of these viruses during the winter season. OBJECTIVES: To assess the clinical performance of a SARS-CoV-2+Flu A/B+RSV Combo test in comparison to a multiplex RT-qPCR. STUDY DESIGN: Residual nasopharyngeal swabs issued from 178 patients were included. All patients, adults and children, were symptomatic and presented at the emergency department with flu-like symptoms. Characterization of the infectious viral agent was done by RT-qPCR. The viral load was expressed as cycle threshold (Ct). Samples were then tested using the multiplex RAD test Fluorecare®à¸ SARS-CoV-2 & Influenza A/B & RSV Antigen Combo Test. Data analysis was carried out using descriptive statistics. RESULTS: The sensitivity of the test varies according to the virus, with the highest sensitivity observed for Influenza A (80.8.% [95%CI: 67.2 - 94.4]) and the lowest sensitivity observed for RSV (41.5% [95%CI: 26.2 - 56.8]). Higher sensitivities were observed for samples with high viral loads (Ct < 20) and decrease with low viral loads. The specificity for SARS-CoV-2, RSV and Influenza A and B was >95%. CONCLUSIONS: The Fluorecare® combo antigenic presents satisfying performance in real-life clinical setting for Influenza A and B in samples with high viral load. This could be useful to allow a rapid (self-)isolation as the transmissibility of these viruses increase with the viral load. According to our results, its use to rule-out SARS-CoV-2 and RSV infection is not sufficient.

Clinical Chemistry and Laboratory Medicine celebrates 60 years - narrative review devoted to the contribution of the journal to the diagnosis of SARS-CoV-2.

This review is an integral part of the special issue for the 60 years of the journal Clinical Chemistry and Laboratory Medicine (CCLM). The aim of the review is to highlight the role of the clinical laboratory since the emergence of the "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), which causes Coronavirus disease 2019 (COVID-19), with special focus on the contribution of the journal in generating knowledge in SARS-CoV-2 diagnosis. As of October 30, 2022, a total of 186 CCLM publications were dedicated to COVID-19. Of importance, major International Federation of Clinical Chemistry (IFCC) guidelines related to the diagnosis of COVID-19 were published in CCLM. Between early-2020 and late October 2022, COVID-19 publications represented around 27% of all articles in CCLM, highlighting the willingness of the editorial board to help the field in order to better describe and diagnose this new emerging disease. First launched in 1963 under the name "Zeitschrift für Klinische Chemie", the Journal was entirely devoted to clinical chemistry in the strict sense. The various topics published in relation to COVID-19 including its diagnosis, its impact on biochemical or hematological measures, as well as biosafety measures, is the perfect example that shows that the journal has greatly diversified over time.

Vaccine-induced binding and neutralizing antibodies against Omicron 6 months after a homologous BNT162b2 booster.

Evidence about the long-term persistence of the booster-mediated immunity against Omicron is mandatory for pandemic management and deployment of vaccination strategies. A total of 155 healthcare professionals (104 COVID-19 naive and 51 with a history of SARS-CoV-2 infection) received a homologous BNT162b2 booster. Binding antibodies against the spike protein and neutralizing antibodies against Omicron were measured at several time points before and up to 6 months after the booster. Geometric mean titers of measured antibodies were correlated to vaccine efficacy (VE) against symptomatic disease. Compared to the highest response, a significant 10.2- and 11.5-fold decrease in neutralizing titers was observed after 6 months in participants with and without history of SARS-CoV-2 infection. A corresponding 2.5- and 2.9-fold decrease in binding antibodies was observed. The estimated T1/2 of neutralizing antibodies in participants with and without history of SARS-CoV-2 infection was 42 (95% confidence interval [CI]: 25-137) and 36 days (95% CI: 25-65). Estimated T1/2 were longer for binding antibodies: 168 (95% CI: 116-303) and 139 days (95% CI: 113-180), respectively. Both binding and neutralizing antibodies were strongly correlated to VE (r = 0.83 and 0.89). However, binding and neutralizing antibodies were modestly correlated, and a high proportion of subjects (36.7%) with high binding antibody titers (i.e., >8434 BAU/ml) did not have neutralizing activity. A considerable decay of the humoral response was observed 6 months after the booster, and was strongly correlated with VE. Our study also shows that commercial assays available in clinical laboratories might require adaptation to better predict neutralization in the Omicron era.

Resistance towards ChadOx1 nCoV-19 in an 83 Years Old Woman Experiencing Vaccine Induced Thrombosis with Thrombocytopenia Syndrome.

BACKGROUND: in this report, we describe the case of an 83-year-old woman vaccinated with ChadOx1 nCoV-19 who developed a so-called vaccine-induced thrombosis with thrombocytopenia syndrome and who did not develop any antibodies against the spike protein of SARS-CoV-2 at 30 days following the administration of her first dose of ChadOx1 nCoV-19. Experimental section: two serum samples from the patient and 5 serum samples from 5 control individuals having received the two-dose regimen vaccination with ChadOx1 nCoV-19 were evaluated. In order to investigate the lack of response to the vaccination, a cell model was developed. This model permits to evaluate the interaction between responsive cells (A549) possessing the Coxsackievirus and Adenovirus Receptor (CAR), a defined concentration of ChadOx1 nCoV-19 and serial dilution of the patient or the control serum. The aim was to assess the impact of these sera on the production of the spike (S) protein induced by the transfection of the genetic material of ChadOx1 nCoV-19 into the A549 cells. The S protein is measured in the supernatant using an ELISA technique. RESULTS: interestingly, the serum from the patient who developed the vaccine-induced thrombosis with thrombocytopenia syndrome impaired the production of S protein by the A549 cells transfected with ChadOx1 nCoV-19. This was not observed with the controls who did not interfere with the transfection of ChadOx1 nCoV-19 into A549 cells since the S protein is retrieved in the supernatant fraction. CONCLUSION: based on the data coming from the clinical and the cell model information, we found a possible explanation on the absence of antibody response in our patient. She has, or has developed, characteristics that prevent the production of the S protein in contrast to control subjects. We were not able to investigate the entire mechanism behind this resistance which deserve further investigations. A link between this resistance and the development of the thrombosis with thrombocytopenia syndrome following vaccination with ChadOx1 nCoV-19 cannot be excluded.

Serum SARS-CoV-2 Antigens for the Determination of COVID-19 Severity.

The diagnostic of SARS-CoV-2 infection relies on reverse transcriptase polymerase chain reactions (RT-PCRs) performed on nasopharyngeal (NP) swabs. Nevertheless, false-negative results can be obtained with inadequate sampling procedures, making the use of other biological matrices worthy of investigation. This study aims to evaluate the kinetics of serum N antigens in severe and non-severe patients and compare the clinical performance of serum antigenic assays with NP RT-PCR. Ninety patients were included in the study and monitored for several days. Disease severity was determined according to the WHO clinical progression scale. Serum N antigen levels were measured with a chemiluminescent assay (CLIA) and the Single Molecular Array (Simoa) assay. Viremia thresholds for severity were determined and proposed. In severe patients, the peak antigen response was observed 7 days after the onset of symptoms, followed by a decline. No real peak response was observed in non-severe patients. Severity thresholds for the Simoa and the CLIA provided positive likelihood ratios of 30.0 and 10.9 for the timeframe between day 2 and day 14, respectively. Sensitive detection of N antigens in serum may thus provide a valuable new marker for COVID-19 diagnosis and evaluation of disease severity. When assessed during the first 2 weeks since the onset of symptoms, it may help in identifying patients at risk of developing severe COVID-19 to optimize better intensive care utilization.

NETosis and Nucleosome Biomarkers in Septic Shock and Critical COVID-19 Patients: An Observational Study.

Background: Neutrophil extracellular traps' (NETs') formation is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of microorganisms. Aim: The aim was to evaluate biomarkers of NETs' formation in a patient cohort admitted to intensive care unit (ICU) due to infection. Methods: Forty-six septic shock patients, 22 critical COVID-19 patients and 48 matched control subjects were recruited. Intact nucleosomes containing histone 3.1 (Nu.H3.1), or citrullinated histone H3R8 (Nu.Cit-H3R8), free citrullinated histone (Cit-H3), neutrophil elastase (NE) and myeloperoxidase (MPO) were measured. Results: Significant differences in Nu.H3.1 and NE levels were observed between septic shock and critical COVID-19 subjects as well as with controls (p-values < 0.05). The normalization of nucleosome levels according to the neutrophil count improved the discrimination between septic shock and critical COVID-19 patients. The ratio of Nu.Cit-H3R8 to Nu.H3.1 allowed the determination of nucleosome citrullination degree, presumably by PAD4. Conclusions: H3.1 and Cit-H3R8 nucleosomes appear to be interesting markers of global cell death and neutrophil activation when combined. Nu.H3.1 permits the evaluation of disease severity and differs between septic shock and critical COVID-19 patients, reflecting two distinct potential pathological processes in these conditions.

Lung Transplant Recipients Immunogenicity after Heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA Vaccination.

(1) Background: High immunosuppressive regimen in lung transplant recipients (LTRs) hampers the immune response to vaccination. We prospectively investigated the immunogenicity of heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA vaccination in an LTR cohort. (2) Methods: Forty-nine COVID-19 naïve LTRs received a two-dose regimen ChAdOx1 nCoV-19 vaccine. A subset of 32 patients received a booster dose of BNT162b2 mRNA vaccine 18 weeks after the second dose. (3) Results: Two-doses of ChAdOx1 nCoV-19 induced poor immunogenicity with 7.2% seropositivity at day 180 and low neutralizing capacities. The BNT162b2 mRNA vaccine induced significant increases in IgG titers with means of 197.8 binding antibody units per milliliter (BAU/mL) (95% CI 0-491.4) and neutralizing antibodies, with means of 76.6 AU/mL (95% CI 0-159.6). At day 238, 32.2% of LTRs seroconverted after the booster dose. Seroneutralization capacities against Delta and Omicron variants were found in only 13 and 9 LTRs, respectively. Mycophenolate mofetil and high-dose corticosteroids were associated with a weak serological response. (4) Conclusions: The immunogenicity of a two-dose ChAdOx1 nCoV-19 vaccine regimen was very poor in LTRs, but was significantly enhanced after the booster dose in one-third of LTRs. In immunocompromised individuals, the administration of a fourth dose may be considered to increase the immune response against SARS-CoV-2.

Identification of SARS-CoV-2 Neutralizing Antibody with Pseudotyped Virus-based Test on HEK-293T hACE2 Cells.

Neutralizing antibodies (NAbs) are of particular importance because they can prevent binding of the receptor binding domain (RBD) of the spike protein (S protein) to the angiotensin-converting enzyme 2 (ACE2) receptor present at the surface of human cells, preventing virus entry into the host cells. The gold standard method for detection of NAbs is the plaque reduction neutralization test (PRNT). Based on the measurement of cell lysis due to viral infection, this test is able to detect antibodies that prevent cell infection (Muruato et al., 2020; Lau et al., 2021). This technique requires the use of live pathogens, i.e., SARS-CoV-2 in this case, and must be done in a biosafety level 3 (BL3) laboratory. In addition, it requires expensive installations, skillful and meticulous staff, and a high workload, which prevents its wide implementation even in research laboratories. A SARS-CoV-2 pseudovirus will express the S protein responsible for cell entrance, but will not express the pathogenic genetic material of the virus, making them less dangerous for laboratory staff and the environment. Graphic abstract.

Ad interim recommendations for diagnosing SARS-CoV-2 infection by the IFCC SARS-CoV-2 variants working group.

This document, endorsed by the IFCC Working Group on SARS-CoV-2 Variants, aims to update previous indications for diagnosing acute SARS-CoV-2 infection, taking into consideration the evidence that has emerged after the origin and spread of new lineages and sub-lineages of the virus characterized by mutated genetics and altered biochemical, biological and clinical characteristics. These indications encompass the use of different diagnostic strategies in specific clinical settings, such as high risk of SARS-CoV-2 infection (symptomatic patients), low risk of SARS-CoV-2 infection (asymptomatic subjects) at hospital admission/contact tracing, testing in asymptomatic subjects, in epidemiologic surveys and/or population screening, along with tentative indications for identification of new lineages and/or sub-lineages of SARS-CoV-2.

Analytical Sensitivity of Six SARS-CoV-2 Rapid Antigen Tests for Omicron versus Delta Variant.

Rapid antigen detection (RAD) tests are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC), presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One hundred and twenty samples were selected and tested with both RT-qPCR and six commercial RAD tests that are commonly sold in Belgian pharmacies. Of these, direct whole-genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron (BA.1 and BA.1.1 sub-lineages, respectively). The sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) ranged from 70.0% to 92.9% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25, corresponding to <4.9 log10 copies/mL), only the Roche RAD test showed acceptable performances for the Delta strains (80.0%), while poor performances were observed for the other RAD tests (20.0% to 40.0%). All the tested devices had poor performances for the Omicron samples with a low viral load (0.0% to 23.1%). The poor performances observed with low viral loads, particularly for the Omicron strain, is an important limitation of RAD tests, which is not sufficiently highlighted in the instructions for use of these devices.

Dynamics of Neutralizing Antibody Responses Following Natural SARS-CoV-2 Infection and Correlation with Commercial Serologic Tests. A Reappraisal and Indirect Comparison with Vaccinated Subjects.

Neutralising antibodies (NAbs) represent the real source of protection against SARS-CoV-2 infections by preventing the virus from entering target cells. The gold standard in the detection of these antibodies is the plaque reduction neutralization test (PRNT). As these experiments must be done in a very secure environment, other techniques based on pseudoviruses: pseudovirus neutralization test (pVNT) or surrogate virus neutralization test (sVNT) have been developed. Binding assays, on the other hand, measure total antibodies or IgG, IgM, and IgA directed against one epitope of the SARS-CoV-2, independently of their neutralizing capacity. The aim of this study is to compare the performance of six commercial binding assays to the pVNT and sVNT. In this study, we used blood samples from a cohort of 62 RT-PCR confirmed COVID-19 patients. Based on the results of the neutralizing assays, adapted cut-offs for the binding assays were calculated. The use of these adapted cut-offs does not permit to improve the accuracy of the serological assays and we did not find an adapted cut-off able to improve the capacity of these tests to detect NAbs. For a part of the population, a longitudinal follow-up with at least two samples for the same patient was performed. From day 14 to day 291, more than 75% of the samples were positive for NAbs (n = 87/110, 79.1%). Interestingly, 6 months post symptoms onset, the majority of the samples (N = 44/52, 84.6%) were still positive for NAbs. This is in sharp contrast with the results we obtained 6 months post-vaccination in our cohort of healthcare workers who have received the two-dose regimens of BNT162b2. In this cohort of vaccinated subjects, 43% (n = 25/58) of the participants no longer exhibit NAbs activity 180 days after the administration of the first dose of BNT162b2.

Waning of IgG, Total and Neutralizing Antibodies 6 Months Post-Vaccination with BNT162b2 in Healthcare Workers.

Data about the long-term duration of antibodies after SARS-CoV-2 vaccination are still scarce and are important to design vaccination strategies. In this study, 231 healthcare professionals received the two-dose regimen of BNT162b2. Of these, 158 were seronegative and 73 were seropositive at baseline. Samples were collected at several time points. The neutralizing antibodies (NAbs) and antibodies against the nucleocapsid and the spike protein of SARS-CoV-2 were measured. At day 180, a significant antibody decline was observed in seronegative (-55.4% with total antibody assay; -89.6% with IgG assay) and seropositive individuals (-74.8% with total antibody assay; -79.4% with IgG assay). The estimated half-life of IgG from the peak humoral response was 21 days (95% CI: 13-65) in seronegative and 53 days (95% CI: 40-79) in seropositive individuals. The estimated half-life of total antibodies was longer and ranged from 68 days (95% CI: 54-90) to 114 days (95% CI: 87-167) in seropositive and seronegative individuals, respectively. The decline of NAbs was more pronounced (-98.6%) and around 45% of the subjects tested were negative at day 180. Whether this decrease correlates with an equivalent drop in the clinical effectiveness against the virus would require appropriate clinical studies.