Virus Neutralization Assay Research Articles

Serum AZD7442 (tixagevimab-cilgavimab) concentrations and in vitroIC50 values predict SARS-CoV-2 neutralising antibody titres.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates rapid methods for assessing monoclonal antibody (mAb) potency against emerging variants. Authentic virus neutralisation assays are considered the gold standard for measuring virus-neutralising antibody (nAb) titres in serum. However, authentic virus-based assays pose inherent practical challenges for measuring nAb titres against emerging SARS-CoV-2 variants (e.g. storing infectious viruses and testing at biosafety level-3 facilities). Here, we demonstrate the utility of pseudovirus neutralisation assay data in conjunction with serum mAb concentrations to robustly predict nAb titres in serum. SARS-CoV-2 nAb titres were determined via authentic- and lentiviral pseudovirus-based neutralisation assays using serological data from three AZD7442 (tixagevimab-cilgavimab) studies: PROVENT (NCT04625725), TACKLE (NCT04723394) and a phase 1 dose-ranging study (NCT04507256). AZD7442 serum concentrations were assessed using immunocapture. Serum-based half-maximal inhibitory concentration (IC50) values were derived from pseudovirus nAb titres and serum mAb concentrations, and compared with in vitro IC50 measurements. nAb titres measured via authentic- and lentiviral pseudovirus-based neutralisation assays were strongly correlated for the ancestral SARS-CoV-2 virus and SARS-CoV-2 Alpha. Serum AZD7442 concentrations and pseudovirus nAb titres were strongly correlated for multiple SARS-CoV-2 variants with all Spearman correlation coefficients ≥ 0.78. Serum-based IC50 values were similar to in vitro IC50 values for AZD7442, for ancestral SARS-CoV-2 and Alpha, Delta, Omicron BA.2 and Omicron BA.4/5 variants. These data highlight that serum mAb concentrations and pseudovirus in vitro IC50 values can be used to rapidly predict nAb titres in serum for emerging and historical SARS-CoV-2 variants.

Development and characterization of a recombinant Senecavirus A expressing enhanced green fluorescent protein.

Senecavirus A (SVA), belonging to the genus Senecavirus in the family Picornaviridae, is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development. In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes. We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. In mice, rSVA-eGFP also showed similar replication kinetics and pathogenicity to the parental virus, both causing mild lung lesions. In addition, a high-throughput viral neutralization assay was developed using eGFP as a surrogate readout in a fluorescence-based direct titration (FBT) assay based on rSVA-eGFP, facilitating rapid and accurate determination of the neutralizing antibody (nAb) titers. The successful establishment of an SVA reverse genetics system and the rescue of rSVA-eGFP would create a powerful tool for future studies of SVA replication mechanisms and pathogenicity as well as for antiviral development.

High diagnostic accuracy of quantitative SARS-CoV-2 spike-binding-IgG assay and correlation with in vitro viral neutralizing activity

BackgroundAntibody testing can easily evaluate the clinical status of patients, aid in the diagnosis of multisystem inflammatory syndrome, and monitor the immunity level in the population. However, the applicability of serological tests in detecting antibodies against the severe acute respiratory syndrome 2 (SARS-CoV-2) spike-binding protein remains limited. This study aimed to quantify both serum-derived neutralizing immunoglobulin-G (IgG) antibody activity and the amount of anti-SARS-CoV-2 Spike-IgG (S-IgG) in convalescent sera/plasmas and evaluate the direct correlation between the in vitro IgG-EC50 values and S-IgG values. MethodsWe evaluated the neutralizing activity of purified IgG (IgG-EC50), quantified S-IgG in the serum/plasma of consecutive COVID-19 convalescent individuals using a cell-based virus-neutralizing assay, and determined the correlation between IgG-EC50 and S-IgG. In addition, we evaluated rational cut-off values using the receiver operating characteristic (ROC) curve and calculated the sensitivity and specificity of the quantitative S-IgG assay for moderate and high IgG-EC50. ResultsA high correlation was observed between S-IgG and IgG-EC50 with a Spearman's ρ value of −0.748 (95 % confidence interval [CI]: −0.804–0.678). Using an IgG-EC50 of 50 μg/mL and 20 μg/mL as the cut-off values for moderate and high in vitro neutralizing activity, respectively, the Youden's index values of 287.5 binding antibody units (BAU)/mL and 454.1 BAU/mL determined from the ROC curve showed the highest diagnostic accuracy, with Kappa values of 0.884 (95 % CI: 0.823–0.946) and 0.920 (95 % CI: 0.681–0.979), respectively. ConclusionsQuantitative S-IgG tests are a useful and convenient tool for estimating in vitro virus-neutralizing activity, with a high correlation with IgG-EC50 when the rational cut-off value is carefully determined.

Evidence of West Nile Virus Circulation in Horses and Dogs in Libya.

West Nile virus (WNV) is a globally significant mosquito-borne Flavivirus that causes West Nile disease (WND). In Libya, evidence of WNV circulation has been reported in humans but never in animals. The aim of this study was to determine the seroprevalence of WNV infection in horses and dogs in Libya. In total, 574 and 63 serum samples were collected from apparently healthy, unvaccinated horses and dogs, respectively, between 2016 and 2019. A commercially available competitive enzyme-linked immunosorbent assay (c-ELISA) kit was initially used to test the collected samples for the presence of WNV Ig-G antibodies. Positive and doubtful sera were also tested using a more specific virus neutralisation assay to confirm whether the ELISA-positive results were due to WNV or other Flavivirus antibodies. The seroprevalence of WNV IgG antibodies according to ELISA was 13.2% out of 574 of total horses' samples and 30.2% out of 63 of total dogs' samples. The virus neutralisation test (VNT) confirmed that 10.8% (62/574) and 27% (17/63) were positive for WNV-neutralising titres ranging from 1:10 to 1:640. Univariable analysis using chi-square tests was conducted to measure the statistical significance of the association between the hypothesized risk factors including city, sex, breed, and age group and were then analyzed using the subsequent multivariable logistic regression model for horse samples. Age group was found to be the only significant risk factor in this study. The results of the present study provide new evidence about WNV circulation in Libya.

B and T Cell Responses to SARS-CoV-2 Vaccination in Kidney and Liver Transplant Recipients with and without Previous COVID-19.

(1) Background: Vulnerable populations including transplant recipients are jeopardised by COVID-19. Herein, we report on B and T cell responses among liver and kidney organ recipients at our centre. (2) Methods: 23 liver and 45 kidney (14 thereof combined kidney/pancreas) transplanted patients were vaccinated with two doses of BNT162b2 followed by a booster dose of mRNA-1273 in 28 non-responders 4 months thereafter. Anti-SARS-CoV-2-Ig was measured by specific ELISA and virus neutralisation assay; T cell responses were measured by a spike protein-specific IFN-γ release assay. (3) Results: Compared to controls, B and T cell responses were weak in transplant recipients, particularly in those without prior exposure to SARS-CoV-2. Within this group, only 15% after the first and 58.3% after the second vaccination achieved seroconversion. A total of 14 out of 28 vaccination non-responders achieved a seroconversion after a third dose. Vaccination side effects were more frequent in healthy controls. The use of mycophenolate was associated with reduced anti-SARS-CoV-2-Ig production. (4) Conclusions: Our data confirm that vaccination responses are insufficient after standard vaccination in liver and kidney transplant recipients and are affected to a variable degree by specific immunosuppressants, particularly mycophenolate. Monitoring vaccination success and re-vaccinating those who are unresponsive seems prudent to achieve sufficient titres. Overall, prospective large-scale, multinational, multicentre studies or high-quality meta-analyses will be needed to generate personalised vaccination strategies in order to achieve protective immunity in high-risk, hard-to-immunize populations.

Evaluation of humoral immune responses of experimental animals to the recombinant SARS-CoV-2 spike ectodomain with the ISCOM adjuvant

Scientific relevance. The use of recombinant antigens in vaccine production is limited because vaccines based on such antigens tend to have low immunogenicity. However, a COVID-19 vaccine that combines recombinant SARS-CoV-2 spike glycoprotein as its antigen and virus-like immune-stimulating complexes (ISCOMs) as its adjuvant (Nuvaxovid) induces a protective virus-neutralising response. The State Research Center of Virology and Biotechnology “Vector” (hereinafter, Vector) has developed the ISCOM adjuvant Matrix-V, which plays a key role in inducing virus-neutralising antibodies. Studying Matrix-V will provide for the wide use of recombinant antigens combined with this adjuvant in the development and production of novel Russian vaccines.Aim. This study aimed to evaluate the humoral immune responses of experimental animals to intramuscular injections of a complex combining the recombinant Wuhan-type SARS-CoV-2 spike RBD antigen and the virus-like ISCOM adjuvant containing Quillaja saponaria saponins.Materials and methods. The Matrix-V ISCOM adjuvant was produced using Vector’s proprietary technology, which involves cross-flow filtration through Sartorius VivaFlow cassettes. To determine the saponin and residual detergent concentrations in Matrix-V, the authors conducted high-performance liquid chromatography. Having produced the recombinant SARS-CoV-2 RBD antigen, the authors used electron microscopy to analyse the ultrastructure of the ISCOM–antigen complex. In the study of the ISCOM–antigen complex, 25 female Balb/c mice (5 groups) and 15 male and female outbred guinea pigs (3 groups) received two intramuscular injections with a 14-day interval. Serum tests relied on virus neutralisation (VN) and enzyme-linked immunosorbent assay (ELISA) methods and used antigens of 8 SARS-CoV-2 variants (State Collection of Viruses and Rickettsia, Vector). The authors used Statistica 10 to analyse the results.Results. Two injections of the SARS-CoV-2 RBD antigen (mice: 7 μg, guinea pigs: 1 μg) alone did not induce statistically significant virus-neutralising antibody responses, as shown by the VN results. Two injections of the SARS-CoV-2 RBD antigen (mice: 7 μg, guinea pigs: 1 μg) adjuvanted with Matrix-V (25 μg) resulted in geometric mean antibody titres of 1:83–1:178 (mice) and 1:174–1:587 (guinea pigs) in the VN tests with the Wuhan variant. One injection of the antigen (1 μg or 7 μg) with Matrix-V (25 μg) induced antibodies only in individual cases, as demonstrated by the VN and/or ELISA results. The most intensive immune response was observed in ELISA tests with the Delta variant after two injections of the Ecto-S-Wuhan (1 μg) and Matrix-V (25 μg) complex. Immune responses did not differ between the group that received two injections of the Ecto-S-Wuhan antigen (1 μg) without the ISCOM adjuvant and the negative control group (titres below 1:100; p=0.95). Two injections of the SARS-CoV-2 RBD antigen (7 μg) without the ISCOM adjuvant induced antibodies in mice (titres between 1:248 and 1:1477).Conclusions. Two intramuscular injections of the complex containing the recombinant SARS-CoV-2 RBD antigen and the Matrix-V ISCOM adjuvant induce virus-neutralising antibodies. The approach proposed by the authors has the potential for use in the development of immunobiological medicinal products to prevent and treat a wide range of infectious diseases.

A bead-based multiplex assay covering all coronaviruses pathogenic for humans for sensitive and specific surveillance of SARS-CoV-2 humoral immunity

Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Identification of 3 neutralizing linear epitopes on the VP8 outer capsid protein of group A equine rotavirus.

To identify protective equine rotavirus group A (ERVA) VP8 epitopes and demonstrate that immunizing hens with synthetic peptides based on these epitopes would yield high-titered, neutralizing egg yolk antibodies for potential application in foals. 26 rotavirus-positive, client-owned foals were included in the study. Five white leghorn hens were used for antibody production. Chicken antibodies were raised against 3 synthetic epitope peptides from the VP8 protein of the common ERVA P-type, P4[12] using CD40-targeted streptavidin-peptide complexes. Antipeptide serum- and egg yolk antibodies were subject to ELISA and in vitro virus neutralization assays to evaluate binding and neutralization activities. Lyophilized anti-VP8 egg yolk antibodies were orally administered (30 g; q 24 h for 5 days) to foals with rotaviral diarrhea. Physical examinations were performed daily. The duration of diarrhea and any adverse effects were recorded. CD40-targeted vaccination of hens generated high titers of anti-VP8 serum and egg yolk antibodies after just 3 immunizations. These antibodies prevented in vitro infection of ERVA with titers of 128 in the serum and 94.5 in the yolk. Oral administration (30 g; q 24 h for 5 days) of lyophilized hyperimmune egg yolk to foals with rotaviral diarrhea did not reveal any adverse effects of the treatment. This study demonstrated that antibodies raised against neutralizing epitopes of the ERVA VP8 protein could prevent ERVA infection in vitro. Based on these results and previous work in other animals, in vivo evaluation of the therapeutic efficacy of anti-VP8 egg yolk antibodies is warranted.

Application of a Receptor-Binding-Domain-Based Simple Immunoassay for Assessing Humoral Immunity against Emerging SARS-CoV-2 Virus Variants.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.

Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants

BackgroundThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display.MethodsSKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs.ResultsThe selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84.ConclusionsWe identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.

Comparisons of Pediatric and Adult SARS-CoV-2-Specific Antibodies up to 6 Months after Infection, Vaccination, or Hybrid Immunity.

Characterization of longitudinal SARS-CoV-2-specific antibody responses in children following infection and vaccination is needed to inform SARS-CoV-2 vaccine policy decisions for children, which may differ from adults. We enrolled individuals at the time of SARS-CoV-2 infection or vaccination for longitudinal serological testing and compared SARS-CoV-2-spike-specific IgG and neutralization activity in children and adults stratified by infection and vaccination status using enzyme-linked immunosorbent and virus neutralization assays. Between June 2020 and December 2022, we collected sera from 669 participants aged 40 days to 55 years, including 330 unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection, 180 vaccinated SARS-CoV-2-naïve individuals, and 159 vaccinated previously infected individuals. Half (n = 330, 49.3%) were children. SARS-CoV-2-specific IgG and neutralization activity in children < 12 years old in response to infection persisted at higher levels than those of adults through at least 6 months (spike-specific IgG levels, 2.05 [95% CI: 1.4-3.1] times higher than adults; neutralizing activity, median 88.8 vs 75.2%, respectively, p = .04). In addition, all pediatric participants had significantly higher IgG levels compared with adults at 6 months following infection or vaccination, regardless of prior infection status. Vaccine-induced SARS-CoV-2-specific IgG responses in previously infected individuals persisted at higher levels than those from infection alone at 6 months (median AUC, children 5-11 years old, 9115 vs 368; adolescents 3613 vs 475; adults 1956 vs 263, all p < .001). These data demonstrate the robust and persistent immunologic response of SARS-CoV-2 vaccination in children and emphasize the benefit of vaccination after SARS-CoV-2 infection.

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.

Humoral and cellular immunity against different SARS-CoV-2 variants in patients with chronic kidney disease

Chronic kidney disease (CKD) patients are at higher risk of severe COVID-19. Humoral and cellular immunity from prior infection or vaccination are important for protection, but the neutralizing antibody (nAb) response against SARS-CoV-2 variants is impaired. We investigated the variant-specific nAb and T cell immunity among CKD patients. Adult CKD patients were recruited between August and October 2022. nAb against the SARS-CoV-2 (ancestral strains and four Omicron sublineages) and T cell response were measured using the live virus neutralization assay and interferon-gamma release assay (IGRA). The correlation between nAb/T-cell response and subsequent infection after recruitment were also determined. Among the 88 recruited patients, 95.5% had prior infection or had completed the primary vaccine series. However, only 77.3% had detectable nAb against at least one SARS-CoV-2 strains, 59.1% tested positive in IGRA, and 52.3% had detectable nAb and tested positive in the IGRA. The nAb geometic mean titers (GMTs) against XBB.1, BA.5 and BA.2.3.20 were significantly lower than those against BA.2 and ancestral strain. Prior SARS-CoV-2 infection was associated with elevated nAb and T cell response. More kidney transplant recipients (KTRs) showed absent nAb and T cell response (36.8% vs. 10.1%), despite a higher prevalence of vaccine booster in this population (94.7% vs. 50.7%). Lower levels of nAb titer and T cell response were significantly associated with subsequent infection. A considerable proportion of CKD patients, especially KTRs, showed absence of humoral and cellular protective immunity against SARS-CoV-2. Strategies to improve immunogenicity in this population are urgently needed.

154 Antibody and Cell-Mediated Responses Associated with Bvdv and Interferon-Gamma Expression in Reciprocal Angus-Brahman First Generation Crosses

Abstract The influence of sex and reciprocal cross type were evaluated in Brahman-sired (B × A) and Angus-sired (A × B) F1 cattle following modified-live viral vaccination regarding virus neutralization titers (VNT) and cell-mediated immune (CMI) responses following in vitro exposure to Bovine Viral Diarrhea Virus (BVDV) Type 1 (BVDV1) and Type 2 (BVDV2) genotypes. VNT against BVDV were based on virus neutralization assay, and CMI responses were measured using the PrimeFlow mRNA assay for determination of the frequency of BVDV and interferon-gamma (IFN-γ) positive cells. Mixed model procedures utilized individual calf ID as a random effect with fixed effects of F1 cross type, calf sex, evaluation date, and potential 2-way interactions; models for frequency of BVDV and IFN-γ positive cells, stimulation conditions (BVDV1, BVDV2, mitogen, none) and potential two-way and three-way interactions of fixed effects. Relationships of immune responses with calf birth weight and weaning weight and steer carcass traits were also investigated. This study found several CMI traits differed due to the type of cross and calf sex; however, no differences in VNT were attributed to these effects. Cells from B × A animals consistently ranked approximately 20% greater for BVDV positive cells (BVDV+) than those from A × B animals pre-MLV vaccination; however, post-MLV vaccination, cells from A × B animals ranked 20% greater (P &amp;lt; 0.05) for CD2+, CD4+, and CD8+ cells. Post- MLV steers consistently had 30 to 35% greater (P &amp;lt; 0.05) frequencies of BVDV+ cells following viral exposure; steers had greater frequencies (P &amp;lt; 0.05) of CD4+ and CD335+ cells expressing IFN-γ and ranked greater for CD2+ and CD8+ cells when compared with heifers in both crosses. Only vaccination timing affected BVDV VNT. No significant correlations involving VNT and CMI responses were observed among A × B animals when stimulated with BVDV1, but among B × A animals, positive correlations were observed with IFN-γ in CD4+ (r = 0.406, P = 0.049) and CD335+ (r = 0.472, P = 0.020). When stimulated with BVDV2, positive correlations of 0.33 to 0.39 (P &amp;lt; 0.05) were observed between BVDV and IFN-γ frequency within cell type; these were not significant when stimulated with BVDV1, except for CD335+ cells (r = 0.41, P = 0.003). No correlations for VNT and CMI responses were observed when stimulated with BVDV2. No major correlations between immune responses and weight or carcass traits were observed, and immune response category for BVDV or IFN-γ frequency did not influence birth weight or weaning weight. Differences in CMI responses involving reciprocal cross types and sex observed in this project may relate to widely reported reciprocal cross differences in birth weight in Bos indicus - Bos taurus crossbred cattle and warrant additional study.

Neutralizing antibodies from naturally infected individuals against SARS-CoV-2 Gamma and Delta variants in the Paraguayan population

Introduction: Severe Acute Respiratory Syndrome-Coronavirus-2 Virus (SARS-CoV-2) is responsible for Coronavirus Disease 2019 (COVID-19). A substantial number of SARS-CoV-2 infection cases have been reported during the pandemic, and vaccination coverage in some regions, particularly in developing countries, remains very low. SARS-CoV-2 variants of concern (VOCs) have also emerged as some of the most pressing public health issues. In this scenario, it is crucial to know whether COVID-19 convalescent antibodies have cross-neutralizing action against VOCs to contribute to the analysis of the future progress of the pandemic. Methodology: The plasma of individuals infected with SARS-CoV-2 from June to November 2020 in Paraguay (before the first recorded infections associated with VOCs in the country) was selected. Anti-spike antibodies were determined in plasma samples (n = 626) obtained from this convalescent and unvaccinated group. Using a pseudotyped virus neutralization assay, we then investigated the neutralizing response against D614G variant and Gamma, and Delta VOCs. Results: IgG antibodies against spike were detected in 85.6% of convalescent individuals. Samples from individuals previously infected by a non-VOC showed a 6.6- and 8.1-fold reduction in neutralizing capacity to the Gamma and Delta variants, respectively, when compared to the D614G variant. Conclusions: Our findings show that antibodies generated by non-VOC infection have reduced neutralizing capabilities against Gamma and Delta variants that appeared subsequently and might have implications for immunity strategies.

Performance of SARS COV-2 IgG Anti-N as an Independent Marker of Exposure to SARS COV-2 in an Unvaccinated West African Population.

Determination of previous SARS-COV-2 infection is hampered by the absence of a standardized test. The marker used to assess previous exposure is IgG antibody to the nucleocapsid (IgG anti-N), although it is known to wane quickly from peripheral blood. The accuracies of seven antibody tests (virus neutralization test, IgG anti-N, IgG anti-spike [anti-S], IgG anti-receptor binding domain [anti-RBD], IgG anti-N + anti-RBD, IgG anti-N + anti-S, and IgG anti-S + anti-RBD), either singly or in combination, were evaluated on 502 cryopreserved serum samples collected before the COVID-19 vaccination rollout in Kumasi, Ghana. The accuracy of each index test was measured using a composite reference standard based on a combination of neutralization test and IgG anti-N antibody tests. According to the composite reference, 262 participants were previously exposed; the most sensitive test was the virus neutralization test, with 95.4% sensitivity (95% CI: 93.6-97.3), followed by 79.0% for IgG anti-N + anti-S (95% CI: 76.3-83.3). The most specific tests were virus neutralization and IgG anti-N, both with 100% specificity. Viral neutralization and IgG anti-N + anti-S were the overall most accurate tests, with specificity/sensitivity of 100/95.2% and 79.0/92.1%, respectively. Our findings indicate that IgG anti-N alone is an inadequate marker of prior exposure to SARS COV-2 in this population. Virus neutralization assay appears to be the most accurate assay in discerning prior infection. A combination of IgG anti-N and IgG anti-S is also accurate and suited for assessment of SARS COV-2 exposure in low-resource settings.

Evaluation and comparison of immune responses induced by two Mpox mRNA vaccine candidates in mice.

The epidemic of Mpox virus (MPXV) from May 2022 was once declared as a Public Health Emergency of International Concern by the World Health Organization. Vaccines play an important role in prevention of infectious diseases, and mRNA vaccine technology was proved to be a safe and effective platform with successful application in defense of coronavirus disease 2019. In this study, based on A29L, M1R, A35R, and B6R of MPXV, we developed two MPXV mRNA vaccine candidates, designated as MPXfus and MPXmix. The MPXfus was one-component, in which these four antigen proteins were linked in tandem by flexible linker and encoded by an individual mRNA as a fusion protein. The MPXmix was multicomponent containing four mRNA, and each mRNA encoded one antigen protein respectively. Mice were immunized with equal quality of MPXfus or MPXmix, delivered by lipid nanoparticles for evaluation and comparison of the immune responses induced by these two MPXV vaccine candidates. Results of immune response analyses indicated that both MPXfus and MPXmix could elicit high-level of antigen-specific antibodies and robust cellular immune response in mice. Moreover, results of virus neutralization assays suggested that sera from MPXfus- or MPXmix-immunized mice possessed high neutralizing activities against vaccinia virus. In addition, titers of antigen-specific antibody, levels of cellular immune response, and activities of neutralizing antibody against vaccinia virus induced by MPXfus and MPXmix presented no significant difference. In summary, this study provides valuable insights for further clinical development of one-component and multicomponent mRNA vaccine candidates for the prevention of MPXV and other orthomyxoviruses.

B-077 Comparison of SARS-CoV-2 Antibody Profile and Omicron-Specific Neutralizing Activity Before and After mRNA Booster Vaccines: Bivalent vs. Original Formulation

Abstract Background With entering into the era of SARS-CoV-2 vaccine boosters and shifting from pandemic to endemic, understanding how boosters change antibody levels and durability is important for vaccination strategies. This study investigated antibodies against different antigens and neutralizing antibody levels in immunocompetent healthcare workers who have received the primary 2 doses and booster mRNA vaccines. Method The study included two cohorts. Cohort 1 received 3 doses of the original vaccine and cohort 2 received the 3 original doses plus the 4th bivalent booster. Two blood samples were collected from each participant before the 3rd (pre-3) or 4th dose (pre-4) and 14–30 days after the 3rd (post-3) or 4th dose (post-4). SARS-CoV-2 IgG antibodies against the spike protein receptor-binding domain (RBD), S1, S2, and nucleocapsid protein were measured using a semi-quantitative multiplex assay on a Bioplex 2200 analyzer. Neutralizing antibodies against omicron sublineage BA.2 were determined using a surrogate virus neutralization assay. Results Among the 45 participants aged 24 to 66, 53% had natural infection evidenced by positive anti-nucleocapsid IgG or historical PCR results. In cohort 1 (n = 24), the median time between the second and third doses was 375 (range 278–439) days. In cohort 2 (n = 21), the median time between the third and fourth doses was 353 (range 102–438) days. Both the 3rd original and 4th bivalent vaccines significantly increased neutralizing, anti-RBD, anti-S1 and anti-S2 antibodies when compared to their pre-dose concentrations (P &amp;lt; 0.01). The median and interquartile range (IQR) for pre-and post-booster antibody levels were: Neutralizing antibody post-3 median: 189 U/mL (IQR 99–781) versus pre-3 median: 44 U/mL (IQR 0–180), post-4 median: 241 U/mL (IQR 148–630) versus pre-4 median: 37 U/mL (IQR 8–167); anti-RBD post-3 median: 13 100 U/mL (IQR 8001–31 850) versus pre-3 median: 3437 U/mL (IQR 201–7976), post-4 median: 16 490 U/mL (IQR 7691–24 710) versus pre-4 median: 3270 U/mL (IQR 1970–4847); anti-S1 post-3 median: 9700 U/mL (IQR 7281–27 425) versus pre-3 median: 2287 U/mL (IQR 229–7710), post-4 median: 11 000 U/mL (IQR 6935–20 665) versus pre-4 median: 2280 U/mL (IQR 1419–2833); and anti-S2 post-3 median: 148 U/mL (IQR 89–266) versus pre-3 median: 25 U/mL (IQR 9–51), post-4 median: 101 U/mL (IQR 73–280) versus pre-4 median: 28 U/mL (IQR 14–43). No difference was found between the post-4-bivalent and post-3-original antibody levels. The difference in post/pre ratios elicited by the 3rd and 4th doses was also not statistically significant (P &amp;gt; 0.05). Further analysis showed participants with natural COVID infection had much higher pre-3 and pre-4 dose antibody levels than those without natural infection (P &amp;lt; 0.01). The pre-booster neutralizing antibody levels of infected participants were not significantly different (P &amp;gt; 0.05) from the post-booster levels of the non-infected participants. This pattern was consistent across antibodies against different antigens. In addition, there was no significant difference in post-booster antibodies between previously infected and non-infected participants. Conclusion The bivalent and original mRNA vaccines are equally effective in enhancing immunity against SARS-CoV-2. Individuals with no previous COVID infection may need boosters annually to raise waning antibodies. Naturally infected individuals demonstrated long-lasting immunity and may not require annual boosters.

Development of an automated, high-throughput SARS-CoV-2 neutralization assay based on a pseudotyped virus using a vesicular stomatitis virus (VSV) vector

ABSTRACT The global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293 T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z’ factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.

Assessment of antibody dynamics and neutralizing activity using serological assay after SARS-CoV-2 infection and vaccination.

The COVID-19 antibody test was developed to investigate the humoral immune response to SARS-CoV-2 infection. In this study, we examined whether S antibody titers measured using the anti-SARS-CoV-2 IgG II Quant assay (S-IgG), a high-throughput test method, reflects the neutralizing capacity acquired after SARS-CoV-2 infection or vaccination. To assess the antibody dynamics and neutralizing potency, we utilized a total of 457 serum samples from 253 individuals: 325 samples from 128 COVID-19 patients including 136 samples from 29 severe/critical cases (Group S), 155 samples from 71 mild/moderate cases (Group M), and 132 samples from 132 health care workers (HCWs) who have received 2 doses of the BNT162b2 vaccinations. The authentic virus neutralization assay, the surrogate virus neutralizing antibody test (sVNT), and the Anti-N SARS-CoV-2 IgG assay (N-IgG) have been performed along with the S-IgG. The S-IgG correlated well with the neutralizing activity detected by the authentic virus neutralization assay (0.8904. of Spearman's rho value, p < 0.0001) and sVNT (0.9206. of Spearman's rho value, p < 0.0001). However, 4 samples (2.3%) of S-IgG and 8 samples (4.5%) of sVNT were inconsistent with negative results for neutralizing activity of the authentic virus neutralization assay. The kinetics of the SARS-CoV-2 neutralizing antibodies and anti-S IgG in severe cases were faster than the mild cases. All the HCWs elicited anti-S IgG titer after the second vaccination. However, the HCWs with history of COVID-19 or positive N-IgG elicited higher anti-S IgG titers than those who did not have it previously. Furthermore, it is difficult to predict the risk of breakthrough infection from anti-S IgG or sVNT antibody titers in HCWs after the second vaccination. Our data shows that the use of anti-S IgG titers as direct quantitative markers of neutralizing capacity is limited. Thus, antibody tests should be carefully interpreted when used as serological markers for diagnosis, treatment, and prophylaxis of COVID-19.