Neutralising antibodies (NAbs) against SARS-CoV-2 in BNT162b2 vaccine recipients with a history of advanced HIV infection: cPass as a simpler alternative to gold-standard tests

Background: Established SARS-CoV-2 NAb tests are labor-intensive. We prospectively measured NAbs vs Wuhan-1 and Omicron BA.2 using the novel GenScript cPass assay and examined correlations with responses measured by gold-standard plaque reduction neutralisation test (PRNT) (Cotugno, Ruggiero et al. Cell Rep 2021) and with anti-Spike IgG quantified by Roche Elecsys. Given the paucity of data, we selected BNT162b2 vaccine recipients with a history of advanced HIV infection (prior AIDS-defining conditions and/or nadir CD4 <200 cells). Method(s): In Mar 2021-Apr 2022, 55 PWH received 2 vaccine doses median 3 weeks apart [IQR 3-3] and a 3rd dose 27 weeks later [23-31]. Plasma samples (n=147) were stored immediately before dose-1 (T0), median 4 weeks [3-5] after dose-2 (T1) and median 13 weeks [9-19] after dose-3 (T2) for batch testing. Result(s): Participants' characteristics: 74% male, 85% white, all on ART, 82% HIV-RNA <50 cps/ml;median age 55 years, ART duration 7 years, nadir CD4 83 cells [36-211], current CD4 440 cells [270-710], CD4:CD8 ratio 0.6 [0.4-1.0];73% had a history of advanced HIV infection;15% received a COVID-19 diagnosis during the study. At T0, T1 and T2, proportions with quantifiable anti-S IgG (>0.8 U/ml) were 11/49 (22%), 50/54 (93%) and 43/43 (100%), respectively;their median anti-S IgG titres were 30 [15-124], 15949 [596-3389] and 8527 [3146-17190] U/ml. Proportions showing Wuhan-1 neutralisation by cPass were 6/50 (12%), 45/53 (85%) and 40/43 (93%), with median neutralisations of 67% [47-70], 97% [91-98] and 98% [98-98] and corresponding NAb titres of 1332 [792-1436], 5354 [3529-6187] and 6242 [5765-6766] U/ml. At T2, 25/28 (89%) showed BA.2 neutralisation by cPass (median 83% [68-93];NAb titre 7836 [3172-12173] U/ml) (Fig 1A). Two participants lacking NAbs at T2 had a history of advanced HIV infection. cPass data were highly correlated with anti-S IgG titres (rho 0.82;p<0.0001) and with PRNT data for both Wuhan-1 (n=27, Fig 1B) and Omicron BA.2 (n=28, Fig 1C). Conclusion(s): cPAss offers a simple methodology for measuring SARS-CoV-2 NAbs. Despite prior advanced HIV infection, neutralising activity improved with successive vaccinations and most participants showed NAbs against both Wuhan-1 and Omicron BA.2 after 3 vaccine doses. (Figure Presented).

Effectiveness of two dose Covaxin, an Inactivated COVID-19 vaccine in protection against SARS-CoV-2 infection: Six-month prospective cohort study

Introduction: SARS-CoV-2 affected millions of lives globally and led to devastating impact on public health. India had also witnessed the dreadful effect of SARS-CoV-2 pandemic. Within a short span of time, various SARS-CoV-2 vaccines were developed using different platforms across the world. India has also developed one such indigenous whole-virion inactivated SARSCoV-2 vaccine named as BBV152 (Covaxin). The Covaxin has been found to be immunogenic and second most widely used vaccine in India. Recent studies have also shown significant increase in the humoral and neutralizing antibody response post the administration of booster dose against Omicron variant. Apparently, there is limited data on the long-term persistence of the immune response against the Covaxin in Indian context. Method(s): We evaluated an effectiveness of the Covaxin and comparing its specific immune responses in two categories through prospective cohorts recruited at the vaccination centre, Pune during June 2021 to March 2022. We defined the study population in two groups who were COVID-19 naive individuals (group-1) and COVID-19 recovered individuals (group-2) prior to the immunization with Covaxin. The two cohorts and the study participants were decided considering the baseline antibody titres against SARS-CoV-2, the COVID-19 positivity rate, sample power and loss to follow up. The study population was assessed during three follow-ups at second dose, one and six months post second dose to determine the immune response and effectiveness using S1-RBD IgG ELISA and neutralizing antibody response (NAbs) by plaque reduction neutralization test (PRNT). Result(s): We enrolled participants between age group of 18-80 year (median 32 years). In group-1 and group-2, we recruited 118 and 128 participants respectively. The cohort retention was found to be> 85%,>70% and>40% in 1st, 2nd and 3rd follow up respectively. Loss to the 3rd follow up was coincided with third wave with omicron variant. A rise in geometrical mean titre (GMT) of S1-RBD IgG were observed amongst the participants of both the groups at one-month post immunization (Group 1: S1-RBD: 154.4 to 446.3, Group 2 S1- RBD: 918 to 1127). However, the GMTs at six months post vaccination found to be slightly raised in Group 1 compared to one-month follow-up. Considering the hybrid immunity in group 2 participants, the GMTs of NAbs were higher than group 1 participants at each follow-up against B.1, Delta, Omicron BA.1 and BA.2. Both the groups had shown significant reduction in the levels of NAbs against Delta, Omicron BA.1 and BA.2 compared to B.1. The lowest GMTs of NAbs was observed against BA.1 variant. The IgG and NAbs persisted till six months in 90% participants in both categories except BA.1 variant. Breakthrough cases were reported at one-month (n = 1) and six-months (n = 2) post vaccination respectively from group 1. While reinfection cases (n = 3) were detected at six months post vaccination from group 2 due to Omicron BA.1 variant. Conclusion(s): A two-dose regimen of the Covaxin vaccine enhanced humoral immune response in adults with/without past COVID-19 infection and protected more than 90% adults against SARSCoV-2 infection. Additionally, IgG and NAb responses persisted for six months postvaccination.

Performance of multiplex bead based assay for detection of neutralizing antibodies in SARS CoV-2 infected and vaccinated individuals

Background: After the pandemic of SARS COV2 it is evolving and causing a threat and concern worldwide. Studies on variants are crucial in understanding the change in virulence and transmissibility of the virus and further vaccine efficacy. Plaque reduction neutralization test (PRNT) is the gold standard to detect neutralizing antibodies. Consequently, it is essential to explore other neutralization platforms which give promising results with quick turnaround time and safety compared to live neutralization assay. Objective(s): To compare and evaluate the neutralizing antibody responses of a multiplex bead-based assay (MBA), using the SARSCoV- 2 Variants Neutralizing Antibody Human 5-Plex ProcartaPlexTM Panel, with PRNT and further evaluate it to estimate the neutralizing responses in infected and vaccinated individuals. Material(s) and Method(s): Confirmed RT PCR covid positive and serum samples from vaccinated individuals (Covaxin and Covishield) were assesssed for the presence of neutralizing antibodies using Multiplex bead assay Human 5-Plex ProcartaPlexTM kit. Result(s): The sensitivity and specificity of MBA was less as compared to PRNT. In our study, we observed that qualitative assay was more comparable than quantitative assay. Conclusion(s): This study demonstrated the utility of MBA for simultaneous measurement of neutralizing antibodies against multiple SARS-CoV-2 strains in serum. The qualitative assay performance was imparted excellent with high sensitivity and specificity but variation in quantitative titres was observed compared to live neutralization assay titres.

mRNA VACCINES INDUCES A HIGHER ANTIBODIES RESPONSE IN CHILDREN WITH PREVIOUS COVID-19

Background: mRNA vaccines elicit a durable humoral response to SARS-CoV-2 in adults, whereas evidence in children is lacking. This study aimed to evaluate the early and long-term immunological response after the BNT162b2 vaccine in children with or without a previous SARS-CoV-2 infection. Method(s): In a multicenter, prospective, observational study we profiled the immune response to the BNT162b2 vaccine in children aged 5-11 years attending the Pediatric Departments at the University of Padua and Bambino Gesu Children's Hospital in Rome (Italy). Forty-four healthy children (HC), 20 immune compromised (IC), and 18 children who previously developed MIS-C (MIS-C) were included in the study. Blood samples were collected pre-, 1, and 6 months after a 2-doses vaccination schedule. Neutralizing antibodies (NAbs) and anti-S-RBD IgG titers were analyzed through Plaque Reduction Neutralization Test (PRNT) and chemiluminescent immune-enzymatic assay (CLIA), respectively. B and T cell phenotypes were analyzed by flow cytometry. Geometric mean titers (GMTs) and 95% confidence intervals and median and interquartile range (IQR) of variables were evaluated according to pre-existing confirmed COVID-19. Result(s): Eighty-two children were studied;60 with a molecular-documented previous COVID-19 (Group A) and 22 without previous infection defined as the absence of antigen-specific antibodies before the vaccination (Group B). Overall, in Group A we observed higher NAbs GMTs, anti-S-RBD titers, and T- and B-reg cells than in Group A, at both 1 and 6 mo after vaccination (table);Nabs against the parental virus resulted to be greater in Group A than in Group B by a factor of 18 and 11, at 1 and 6 mo after vaccination, respectively. Both Groups recorded a decrease in antibody titers of approximately 50-70% between 1 and 6 months. A significant difference for Omicron NAbs (p=0.02) and anti-S-RBD (p=0.07) titers decay was observed between Group A and B;in contrast, Parental NAbs titers appeared to have similar trends in the 2 groups (p=0.47). Comparable antibody titers at 1 and 6 mo. (p=0.37) were detected across the three categories of HC, IC, and MIS-C (table). Conclusion(s): mRNA vaccination triggers a higher humoral response in children with a previous history of COVID-19, regardless of the immune deficiency or previous MIS-C, at least up to 6 mo, providing insight into boosting preexisting immunity with mRNA vaccines.

Validation and Establishment of the SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a Prescreening Tool for the Plaque Reduction Neutralization Test.

Neutralization assays are important for understanding and quantifying neutralizing antibody responses toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and prescreening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity assays produced acceptable results, with 100% (95% confidence interval [CI], 94% to 100%) specificity and 100% (95% CI, 94% to 100%) sensitivity against ancestral Wuhan spike-pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike-pseudotyped lentivirus were 88.3% (95% CI, 77.8% to 94.2%) and 100% (95% CI, 94% to 100%), respectively. Assay precision measuring intra-assay variability produced acceptable results for high (50% PRNT [PRNT50], 1:≥640), mid (PRNT50, 1:160), and low (PRNT50, 1:40) antibody titer concentration ranges based on the PRNT50, with coefficients of variation (CVs) of 14.21%, 12.47%, and 13.28%, respectively. Intermediate precision indicated acceptable ranges for the high and mid concentrations, with CVs of 15.52% and 16.09%, respectively. However, the low concentration did not meet the acceptance criteria, with a CV of 26.42%. Acceptable ranges were found in the robustness evaluation for both intra-assay and interassay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT. IMPORTANCE Neutralization studies play an important role in providing guidance and justification for vaccine administration and helping prevent the spread of diseases. The neutralization data generated in our laboratory have been included in the decision-making process of the National Advisory Committee on Immunization (NACI) in Canada. During the coronavirus 2019 (COVID-19) pandemic, the plaque reduction neutralization test (PRNT) has been the gold standard for determining neutralization of SARS-CoV-2. We validated a SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) as an alternative method to help support the PRNT. The advantages of using the SCLSNA is that it can process more samples, is less tedious to perform, and can be used in laboratories with a lower biosafety level. The use of the SCLSNA can further expand our capabilities to help fulfill the requirements for NACI and other important collaborations.

Plaque reduction neutralization test as a method for detecting functional neutralizing antibodies against live SARS-CoV-2 virus

Introduction and objective: Since 2020, little is known about neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus emerging and causing COVID-19 in humans. Here, we established a plaque reduction neutralization test (PRNT) to use as a method for detecting functional NAbs to SARS-CoV-2. Material(s) and Method(s): Vero cells were used as target cells for infection with the new coronavirus and cytopathic effects were obviously exhibited. Result(s): The development of plaque reduction after neutralization of virus with diluted specific antiserum was assessed according to a dose-response effect and consistency of test results with pooled antibody serum, which illustrated the robustness and dynamic reduction of plaque. In addition, the PRNT was used for evaluating functional NAb responses in Thai patients after symptom onset. Conclusion(s): The PRNT can be a method for measuring antibodies against live SARS-CoV-2 and future studies will be planned to investigate functional NAb responses elicited by any COVID-19 vaccine and to evaluate long-term protection of Thai COVID-19 vaccination. Copyright © 2022, Faculty of Pharmaceutical Sciences, Chulalongkorn University. All rights reserved.

Serological and Molecular Surveillance for SARS-CoV-2 Infection in Captive Tigers (Panthera tigris), Thailand.

Coronavirus disease (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Given the emergence of SARS-CoV-2 variants, continuous surveillance of SARS-CoV-2 in animals is important. To monitor SARS-CoV-2 infection in wildlife in Thailand, we collected 62 blood samples and nine nasal- and rectal-swab samples from captive tigers (Panthera tigris) in Ratchaburi province in Thailand during 2020-2021. A plaque reduction neutralization test (PRNT) was employed to detect SARS-CoV-2 neutralizing antibodies. A real-time RT-PCR assay was performed to detect SARS-CoV-2 RNA. Our findings demonstrated that four captive tigers (6.5%, 4/62) had SARS-CoV-2 neutralizing antibodies against Wuhan Hu-1 and the Delta variant, while no SARS-CoV-2 RNA genome could be detected in all swab samples. Moreover, a low-level titer of neutralizing antibodies against the Omicron BA.2 subvariant could be found in only one seropositive tiger. The source of SARS-CoV-2 infection in these tigers most likely came from close contact with the infected animals' caretakers who engaged in activities such as tiger petting and feeding. In summary, we described the first case of natural SARS-CoV-2 infection in captive tigers during the COVID-19 outbreak in Thailand and provided seroepidemiological-based evidence of human-to-animal transmission. Our findings highlight the need for continuous surveillance of COVID-19 among the captive tiger population and emphasize the need to adopt a One Health approach for preventing and controlling outbreaks of COVID-19 zoonotic disease.

Evolution of neutralizing antibodies and cross-activity against different variants of SARS-CoV-2 in patients recovering from COVID-19.

BACKGROUND: Patients recovering from COVID-19 may need vaccination against SARS-CoV-2 because acquired immunity from primary infection may wane, given the emergence of new SARS-CoV-2 variants. Understanding the trends of anti-spike IgG and neutralizing antibody titers in patients recovering from COVID-19 may inform the decision made on the appropriate interval between recovery and vaccination. METHODS: Participants aged 20 years or older and diagnosed with COVID-19 between January and December, 2020 were enrolled. Serum specimens were collected every three months from 10 days to 12 months after the onset of symptom for determinations of anti-spike IgG and neutralizing antibody titers against SARS-CoV-2 Wuhan strain with D614G mutation, alpha, gamma and delta variants. RESULTS: Of 19 participants, we found a decreasing trend of geometric mean titers of anti-spike IgG from 560.9 to 217 and 92 BAU/mL after a 4-month and a 7-month follow-up, respectively. The anti-spike IgG titers declined more quickly in the ten participants with severe or critical disease than the nine participants with only mild to moderate disease between one month and seven months after SARS-CoV-2 infection (-8.49 vs - 2.34-fold, p < 0.001). The neutralizing activity of the convalescent serum specimens collected from participants recovering from wild-type SARS-CoV-2 infection against different variants was lower, especially against the delta variants (p < 0.01 for each variant with Wuhan strain as reference). CONCLUSION: Acquired immunity from primary infection with SARS-CoV-2 waned within 4-7 months in COVID-19 patients, and neutralizing cross-activities against different SARS-CoV-2 variants were lower compared with those against wild-type strain.

Correlation between In Vitro Neutralization Assay and Serological Tests for Protective Antibodies Detection.

Serological assays are useful in investigating the development of humoral immunity against SARS-CoV-2 in the context of epidemiological studies focusing on the spread of protective immunity. The plaque reduction neutralization test (PRNT) is the gold standard method to assess the titer of protective antibodies in serum samples. However, to provide a result, the PRNT requires several days, skilled operators, and biosafety level 3 laboratories. Therefore, alternative methods are being assessed to establish a relationship between their outcomes and PRNT results. In this work, four different immunoassays (Roche Elecsys® Anti SARS-CoV-2 S, Snibe MAGLUMI® SARS-CoV-2 S-RBD IgG, Snibe MAGLUMI® 2019-nCoV IgG, and EUROIMMUN® SARS-CoV-2 NeutraLISA assays, respectively) have been performed on individuals healed after SARS-CoV-2 infection. The correlation between each assay and the reference method has been explored through linear regression modeling, as well as through the calculation of Pearson's and Spearman's coefficients. Furthermore, the ability of serological tests to discriminate samples with high titers of neutralizing antibodies (>160) has been assessed by ROC curve analyses, Cohen's Kappa coefficient, and positive predictive agreement. The EUROIMMUN® NeutraLISA assay displayed the best correlation with PRNT results (Pearson and Spearman coefficients equal to 0.660 and 0.784, respectively), as well as the ROC curve with the highest accuracy, sensitivity, and specificity (0.857, 0.889, and 0.829, respectively).

Non-RBD binding antibody neutralize SARSCoV- 2

Since November 2019, the COVID-19 pandemic has been going on around the world, according to the WHO, more 5.5 million people have died. The main strategy for developing therapeutic antibodies is to obtain human viral neutralizing antibodies directed to the receptor-binding domains (RBD) of the SARS-CoV-2 S-protein. However, it is known that the immune response of humans and mice to different antigens is different, therefore, studies of B-cell epitopes of SARS-CoV-2 S-protein with mouse monoclonal antibodies may allow us to find new virus neutralizing epitopes. Eighteen monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained using hybridoma technology from mice immunized with inactivated SARS-CoV-2. ELISA demonstrated that selected 16 mAbs bound recombinant spike (S) protein and 2 mAbs bound recombinant nucleocapsid (N) protein. The equilibrium dissociation constants of the obtained mAbs against S protein ranged from 0.08 to 10 nM. Three mAbs bound recombinant RBD of S protein, the equilibrium dissociation constants of the mAbs against RBD ranged from 0.2 to 3 nM. Anti-RBD mAbs did not neutralize SARS-CoV-2 in the plaque reduction neutralization test. mAbs RS2 demonstrated a dose-dependent inhibition of plaque formation after infection with SARS-CoV-2. The kD and IC50 values for this antibody were 0.2 nM and 400 mcg/ml, respectively. To determine the S protein region responsible for binding to mAb RS2 S1, S2 and RBD subunit of S protein SARS-CoV-2 were expressed in CHO cells. Unfortunately, the localization of the epitope recognized by neutralizing mAb RS2 was not identified using ELISA or western blot analysis. Moreover, mAb RS2 do not recognized full sized recombinant S-protein in western blot analysis. The obtained results demonstrated that the epitope recognized by neutralizing mAb RS2 were discontinuous and have quaternary structure.

Evaluation of Antiviral Effects and Toxicity of Herbal Medicine Vipdervir Capsules

Background: Antiviral vaccine is not effective, synthetic antiviral drugs are highly toxic, leading to increased interest in herbal medicines as promising antiviral drugs. Recently, Vipdervir has been developed from medicinal herbs with the aim to support and treat diseases caused by viruses such as H5N1 and SARSCoV- 2. In the present study, we assessed Vipdervir's antiviral activity against H5N1 and SARS-CoV-2. In addition, we also evaluated the acute toxicity and repeated dose toxicity of Vipdervir in mice and rabbits, respectively. Methods: H5N1 inhibitory effect of Vipdervir was assessed using hemagglutination inhibition assay. Vipdervir's SARS-CoV-2 inhibitory effect was evaluated by Plaque Reduction Neutralization assay. Acute and repeated dose oral toxicities of Vipdervir were determined according to OECD 423 and OECD 407 guidelines, respectively. Results: Data show that Vipdervir is effective against both H5N1 and SARSCoV- 2. At concentrations of 3 mg/mL and 5 mg/mL Vipdervir completely inhibits H5N1. At a concentration of 50 μg/mL Vipdervir showed an inhibitory effect on SARS-CoV-2. Acute toxicity data revealed that the LD50 of Vipdervir is greater than 35200 mg/kg, b.wt. in mice. Repeated toxicity data indicated that Vipdervir did not induce significant differences in body weight gain, hematology and clinical biochemistry in compared to the control group. The No Observed Adverse Effect Level of Vipdervir is greater than 613.8 mg/kg b.wt./day in rabbits. No delayed toxicity effects of Vipdervir were observed. Conclusion: Vipdervir capsules were found to be antiviral effective and relatively safe in the tested doses and experimental conditions.

Correlation of the VITROS® anti-SARS-CoV-2 Igg quantitative assay with SARS-CoV-2 plaque reduction neutralization test

Background: The VITROS IgG Quant assay∗ is for the quantitative detection of SARS-CoV-2 IgG antibodies with calibration traceable to the first World Health Organization International Standard for Anti- SARS-CoV-2 antibody. Results are reported in both qualitative (reactive/ non-reactive) and quantitative values (Binding Antibody Units [BAU]/ml). PRNT is considered the gold standard method for determining neutralizing antibody (nAb) titers. Aims: This study was designed to assess the correlation of the VITROS Immunodiagnostic Products Anti-SARS-CoV-2 IgG Quantitative Assay (VITROS IgG Quant) to a plaque reduction neutralization test developed at Colorado State University (CSU PRNT). Methods: VITROS IgG Quant: The VITROS IgG Quant assay is a fully automated, high-throughput method run on the VITROS family of immunoassay analyzers. First, antibodies to SARS-CoV-2 present in the sample bind with the S1 subunit of the Spike protein coated on wells. After washing, horseradish peroxidase (HRP)- labelled murine monoclonal anti-human IgG antibodies are added. Following a final wash, bound HRP conjugates are detected using the VITROS signal reagent. The amount of conjugate directly correlates to the amount of SARS-CoV-2 IgG antibody present and is reported in BAU/ml. CSU PRNT: Samples were heat-inactivated for 30 min at 56°C, and serial two-fold dilutions were prepared in a 96-well plate. Viral stock (strain hCoV-19/USA/WA1/2020) containing ∼200 pfu per 0.1 ml was added to each well containing serum dilutions. Following incubation at 37°C in 5% CO2, 6-well plates containing recently confluent Vero cells were inoculated with the virus-serum mixtures. After a second incubation at 37°C, 2 ml of overlay was added to each well. After 24 h incubation at 37°C, a second overlay containing neutral red was dispensed into each well and the number of plaques was counted 48-72 h after initial inoculation. The highest dilution of serum that inhibited plaque formation by 50% (PRNT50) was determined based upon the titre of the samples and the number of plaques present at each dilution. Samples with PRNT50 titers less than or equal to 1:20 are considered negative for nAbs. One hundred forty-nine samples were blind-tested with both the VITROS IgG Quant assay and the CSU PRNT, 74 known positive for SARS-CoV-2 antibody and 75 negatives. The correlation of the VITROS IgG Quant values to CSU PRNT50 was determined. Results: The VITROS IgG Quant results ranged from <2 to 2009 BAU/ml. The CSU PRNT50 results ranged from <1:20 to >1:2560. Pearson correlation coefficient was calculated to be 0.867, demonstrating a good correlation between the VITROS IgG Quant results and the CSU PRNT50 titers. Summary/Conclusions: The VITROS Anti-SARS-CoV-2 IgG Quant assay demonstrates a strong correlation to PRNT50 for the measurement of SARS-CoV-2 nAb titers.

Comparison Of Different Methods For Detection Of Ig G Antibodies Against The Spike (S1) Receptor-Binding Domain (RBD) Of SARS-COV-2

The aim of this study was to know, the antibody response following infection with SARS-CoV-2 virus and post vaccination using kits from two different manufacturers and on two different platforms were carried out. This study was conducted by the department of Microbiology, over a period of 4 months from 18 January to May 2021 from the samples received in Neuberg Ehrlich Laboratory. Samples from 50 vaccinated (Covishied) subjects were taken for this study. We found that the quantitative assay was more sensitive than the qualitative assay in detecting the Ig G antibodies against the spike receptor-binding domain (RBD) of SARS-CoV-2.

Neutralization Assay for SARS-CoV-2 Infection: Plaque Reduction Neutralization Test.

Neutralization assays are often used as part of research and diagnostics to detect neutralizing antibodies and to determine a possible protective antibody titer after infection or vaccination. Here we describe a conventional plaque reduction neutralization test (PRNT) to check the presence of antibodies against SARS-CoV-2 in patient samples (serum or plasma).

For better or for worse: COVID-19 vaccination during or early after (immuno-) chemotherapy or hematopoietic progenitor cell transplantation

Background: Patients with hematologic conditions have a high mortality rate when infected with SARS-CoV-2 (Williamson, Nature 2020). Protection of this group from severe COVID-19 is therefore important. However, according to available vaccination guidelines, one should consider to postpone vaccination of patients on or early after chemotherapy, hematopoietic progenitor cell transplantation (HCT) or with graft versus host disease, because of anticipated poor efficacy. Based on previous (non-COVID-19) vaccination studies among hematology patients, we hypothesized that a significant group of patients may acquire sufficient protection following COVID-19 vaccination, despite disease and therapy related immunodeficiencies. Methods: We conducted a prospective cohort study with 17 cohorts of hematology patients of particular risk for severe COVID-19 who are considered to have no or limited benefit from vaccination. We evaluated humoral immune responses following 2 doses (28 days apart) of the mRNA-1273 vaccine (Moderna/Spikevax) in 722 patients, at baseline and 28 days after each vaccination as SARS-COV-2 S1- (spike)-specific serum IgG antibody concentrations by bead-based multiplex immune assay. The threshold for adequate antibody response is set at ≥300 binding antibody units (BAU)/ml according to the international WHO standard, and is associated with virus plaque reducing neutralization test titers of ≥40 PRNT 50. This study is registered as EudraCT 2021-001072-41, NL76768.029.21. Results: Patient cohorts and corresponding vaccine responses are depicted in Table 1. Vaccine efficacy, as measured by antibody concentration, 4 weeks after the 2nd mRNA-1273 vaccination was available for 691 out of 722 participants. The majority of patients (389/691;56%) obtained an S1 antibody titer that is considered adequate (≥300 BAU/ml). Twenty-nine percent of patients (198/691) did not seroconvert (S1 antibody titer <10 BAU/ml), while the remaining 15% (104/691) did seroconvert but not to sufficient levels (10-300 BAU/ml). Adequate responses were observed in the majority of patients with sickle cell disease using hydroxyurea, chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitor therapy, acute myeloid leukemia (AML) on or early after high dose chemotherapy, patients with myeloproliferative disorders on ruxolitinib, patients with multiple myeloma (MM), including those on daratumumab and those early after high-dose melphalan and autologous HCT, patients with untreated chronic lymphocytic leukemia (CLL), and patients with chronic GvHD. Insufficient or absent antibody responses were observed in the majority of AML patients receiving hypomethylating agents, CLL patients on ibrutinib, patients with B-cell non-Hodgkin's Lymphoma (NHL) during or shortly after rituximab-chemotherapy or following BEAM chemotherapy and autologous HCT, allogeneic HCT recipients <6 months after transplantation, and CAR-T cell therapy recipients. However, even in these low-responder groups considerable numbers of patients did mount sufficient antibody titers. In others, titers increased after each of both vaccinations, suggesting that booster vaccination may enhance antibody titers to sufficient levels (Figure 1). Conclusion: Vaccination with mRNA-1273 had significant efficacy in severely immunocompromised hematology patients. Adequate humoral immune responses after two dose vaccination were reached in the majority of patients receiving therapy for sickle cell disease, MPD, MM, CML and AML, in patients early after HCT and in patients with GvHD. We are currently evaluating clinical and immunologic parameters that correlate with sufficient antibody responses, pseudovirus neutralization and SARS-COV-2-specific B and T cell numbers, phenotype and function. Per study design, all participants with absent or insufficient antibody responses (<300 BAU/ml) will receive a booster vaccination 5 months after initial vaccination, and antibody responses to booster vaccinations will be presented as well. Unlike currently available guidelines, COVID-19 vaccination should not be postponed. Moreover, as antibody titers increased after each of both vaccinations, booster vaccination of patients with absent or insufficient antibody responses seems warranted. (Figure Presented) .

Differences in the Humoral Response to SARS-CoV-2 Infection in Children vs. Adult

Background. One of the most striking observations of the COVID-19 pandemic has been the difference in infection among children vs. adults. Overall, children with SARS-CoV-2 infection generally had milder disease compared to adults, though the cause is not clear. The objective of this study was to compare the humoral response to infection in children vs. adults of a same family. Methods. We performed a prospective cohort study at Sainte-Justine University Health Center in Montreal, Canada from July 2020 to March 2021. Children with a positive SARS-CoV-2 PCR were recruited from the COVID-19 clinic (index case), enrollment was offered to all household members. Serum IgG against SARS-CoV-2 native S1/S2 spike proteins was measured using the Diasorin (Liaison XL) assay, 4-6 months following a positive PCR. A mean antibody threshold of 15 Arbitrary unit per ml (AU/ml) was considered seropositive, with 94.4% positive agreement to plaque reduction neutralization tests (PRNT90) at a 1:40 ratio. Antibody titer was compared between children and adults. Results. 111 participants (52 adults and 59 children) were recruited from 50 separate families. Characteristic of participants and their clinical symptoms are described in Table 1. Among all participants, 76.3% children were SARS-CoV-2 seropositive vs. 51.9% of adults (p=0.007). Median antibody titer was significantly higher in children vs. adults (82.8 AU, [IQR: 18.4-130], vs 17.0 AU, [IQR: 6.8-77.8], p=0.006);findings were similar among SARS-CoV-2 PCR positive participants only. Overall, 13 participants were PCR positive but seronegative, 7 were PCR negative and seropositive, while 61 were both PCR positive and seropositive. Older participants and those with any comorbidity. Among the PCR positive group, the seropositive participants were younger (median age 31±17 vs 19±17 years, p=0.003) and more likely to have comorbidity (69% vs 29%, p=0.007). Conclusion. These results suggest that children have a stronger antibody response to SARS-CoV-2 infection than adults, and that older age and presence of comorbidity are associated with a less robust humoral response. Further work on the differences in response between children and adults may help elucidate mechanisms underlying the severity of disease.

SARS-CoV-2 Spike Protein S1/S2 Antibodies after Vaccination with Sinopharm in Peruvian Physicians

Background. Peru started its national vaccination campaign in February 2021 using Sinopharm vaccine, targeting healthcare personnel on its initial phase. Although the immunogenicity of this vaccine was tested in clinical trials, there are no studies that evaluated the humoral response post vaccination in Peru. Methods. We conducted a cross sectional study, which objective was to evaluate the humoral immunogenicity triggered by the Sinopharm vaccine in Peruvian physicians. We collected demographic and epidemiologic data via an electronic. The SARS-CoV-2 spike protein S1/S2 antibodies were measured by chemiluminescense (Liaison®). A positive test was defined as >15 U/ml, which has correlation of 95% with neutralizing antibodies measured by plaque reduction neutralizing test. Results. 92 participants were enrolled in the study. The epidemiologic characteristics are described in table 1. The mean level of antibodies measured at least 2 weeks from the second vaccine dose was 67.5 ± 70.5 U/ml. 85.7% of the study cohort had positive S1/S2 antibodies. In the univariate analysis, an imperfect negative correlation was found between the level of antibodies and participants' age (r= -0.24;regression F test 5.25;p = 0.0242). A weak negative correlation was observed between the antibody titer and the time elapsed from the second vaccine dose and the day of antibody measurement (r= -0.17). A higher antibody level post vaccine was found in individuals who worked in COVID units (105.5 U / mL vs 58.2 U / mL;p = 0.0125), and in participants with history of COVID (216.5 U / mL vs 81.2 U / mL;p = < 0.0000). Hypertension was associated with lower antibody titers (36.9 U / mL vs. 74.6;p = 0.0464). In the multivariate analysis, working in COVID units, having previous COVID infection and shorter time from second vaccine dose and day of antibody measurement were associated with higher antibody levels post vaccine (table 2). Conclusion. Our study showed that the time elapsed from the second vaccine dose and the day of antibody measurement, having previous COVID-19 infection and working in COVID -19 units may help to predict higher antibody titers post vaccine. Larger studies to evaluate the humoral response post Sinopharm vaccine and its clinical implications are still needed in Peru.

Preliminary Results from a Phase 1 Single Ascending-Dose Study Assessing Safety, Serum Viral Neutralizing Antibody Titers (sVNA), and Pharmacokinetic (PK) Profile of ADG20: An Extended Half-Life Monoclonal Antibody Being Developed for the Treatment and Prevention of Coronavirus Disease (COVID-19)

Background. ADG20 is a fully human IgG1 monoclonal antibody engineered to have high potency and broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like CoVs with pandemic potential by binding to a highly conserved epitope in the receptor-binding domain (RBD) of the spike protein. The Fc region of ADG20 has been modified to provide an extended half-life. ADG20 is in clinical development for the treatment and prevention of COVID-19. Methods. This is an ongoing Phase 1, randomized, placebo (PBO)-controlled, single ascending-dose study of ADG20 administered intramuscularly (IM) or intravenously (IV) to healthy adults aged 18-50 years with no evidence of prior or current SARS-CoV-2 infection. Participants were randomized 8:2 in 3 cohorts (N=10/cohort: n=8 ADG20, n=2 PBO): ADG20 300 mg IM, 500 mg IV, and 600 mg IM. Safety, tolerability, PK, and sVNA titers were assessed up to 3 months post dose. Serum ADG20 concentrations were measured with a validated hybrid ligand binding liquid chromatography-mass spectrometry (MS)/MS assay. sVNA titers against authentic SARS-CoV-2 were determined by a plaque reduction neutralization assay. Results. Overall, 30 participants received ADG20 (n=24) or PBO (n=6). Blinded safety data for all cohorts and PK/sVNA titer data for the 300 mg IM cohort are reported. Through a minimum of 10 weeks post dose, no study drug-related adverse events (AEs), serious AEs, injection site reactions, or hypersensitivity reactions were reported. The observed preliminary PK profile was dose proportional, consistent with an extended half-life monoclonal antibody, and well predicted by translational physiologically-based PK modeling. The measured 50% sVNA titer (MN50;geometric mean [coefficient of variation, %]) was 1382 (32.7%) 13 days after a single 300 mg IM dose. These values are within the range of peak serum neutralizing antibody titers reported for COVID-19 mRNA vaccines. Conclusion. A single dose of ADG20, up to 600 mg IM, was well tolerated. Preliminary PK and sVNA titer profiles support the ongoing Phase 2/3 trials of ADG20 at a 300 mg IM dose for the prevention of COVID-19 (EVADE: NCT04859517) and treatment of ambulatory patients with mild to moderate COVID-19 (STAMP: NCT04805671).

Use of a Whole-Body Quantitative System Pharmacology Physiologically-Based Pharmacokinetic (QSP/PBPK) Model to Support Dose Selection of ADG20: An Extended Half-Life Monoclonal Antibody Being Developed for the Prevention of Coronavirus Disease (COVID-19)

Background. ADG20 is a fully human IgG1 monoclonal antibody engineered to have potent and broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like CoVs with pandemic potential as well as an extended-half-life. ADG20 is administered intramuscularly (IM). A QSP/PBPK model was constructed to support dose selection for a COVID-19 Phase 2/3 prevention trial (EVADE: NCT04859517). Methods. A QSP/PBPK model and a CDC reference adult body weight distribution (45-150 kg) were used to simulate 1000 concentration-time profiles for candidate single-dose regimens of ADG20 (150-450 mg IM). As serum virus neutralizing antibody (sVNA) titers are reportedly a key correlate of protection from COVID-19, a regression equation between time-matched serum ADG20 concentrations (following a 300 mg IM dose) and sVNA titers was developed using measured titers against authentic SARS-CoV-2 determined by a plaque reduction neutralization assay. Projected ADG20 serum concentrations relative to neutralization potency in vitro (90% inhibitory concentration [IC90]) for authentic SARSCoV-2 were also evaluated. Results. The measured 50% neutralization titer (MN50;geometric mean [coefficient of variation, %]) was 1382 (32.7%) 13 days after a single 300 mg IM dose of ADG20. This was within the range of peak sVNA titers reported for COVID-19 vaccine recipients. Using the linear equation relating serum ADG20 concentration to time matched individual MN50 titers and the QSP/PBPK median PK prediction, the anticipated median MN50 exceeded the threshold for protection from SARS-CoV-2 infection established in a non-human primate adoptive transfer model for up to 52 weeks. Based on the QSP/PBPK median PK prediction, median ADG20 serum concentrations are projected to remain >100-fold above the ADG20 IC90 value of 0.011 mg/L against authentic SARS-CoV-2 for up to 52 weeks (Figure). Conclusion. Following administration of a single 300 mg IM dose, sVNA titers and concentrations of ADG20 are projected to remain above thresholds anticipated to be required for protection against COVID-19 for up to 52 weeks. These data support the evaluation of a single ADG20 300 mg IM dose for the prevention of COVID-19.