Virus Neutralization Research Articles

RSV Neutralizing Antibodies Following Nirsevimab and Palivizumab Dosing.

Data describing respiratory syncytial virus (RSV) neutralizing antibody (nAb) levels for nirsevimab, a recently approved, extended half-life, anti-RSV fusion protein (F protein) monoclonal antibody, relative to the previous standard of care, palivizumab, have not been reported. The MEDLEY trial was a randomized, palivizumab-controlled, phase 2/3 study of nirsevimab during 2 RSV seasons (season 1 and 2) in infants born preterm (≤35 weeks' gestational age; dosed season 1 only) or with congenital heart disease or chronic lung disease of prematurity (dosed seasons 1 and 2). Participants were randomly assigned to receive a single dose of nirsevimab followed by 4 monthly placebo doses, or 5 once-monthly doses of palivizumab. Anti-RSV fusion protein serology (ie, levels of prefusion [pre-F]/postfusion [post-F] conformation antibodies), nirsevimab and palivizumab serum concentrations, and RSV nAbs were measured in participant samples collected at baseline (pre-dose) and days 31, 151, and 361. Serologic data were similar in seasons 1 and 2. Nirsevimab predominately conferred pre-F antibodies, whereas palivizumab conferred pre-F and post-F antibodies. Nirsevimab and palivizumab serum concentrations were highly correlated with nAb levels in both seasons. In season 1, the nAb levels in nirsevimab recipients were highest in day 31 samples and gradually declined but remained 17-fold above baseline at day 361. nAb levels in palivizumab recipients increased incrementally with monthly doses to day 151. nAb levels followed similar patterns in season 2. nAb levels were ∼10-fold higher with nirsevimab compared with palivizumab across both seasons. Nirsevimab prophylaxis confers ∼10-fold higher and more sustained RSV nAb levels relative to palivizumab.

A novel inactivated oral rabies vaccine with the incorporation of U-OMP19 enhances the immunogenicity by reducing viral proteins degradation and activating dendritic cells in a mouse model

Currently, dogs, especially stray dogs, and/or wild animals are the main sources of rabies transmission, and oral vaccination is the most practical way to control rabies in these animals. Safety and efficacy are two key criteria for developing oral vaccines. Concerning the efficacy of oral vaccines, degradation of immunogens by gastrointestinal fluid is a major challenge, resulting in suboptimal immune responses after vaccination. For safety reasons, inactivated vaccines are the most optimal choice. In the present study, a recombinant rabies virus (RABV) with un-lipidated outer membrane protein 19 (U-OMP19) of Brucella spp incorporated into RABV virions, designated as LBNSE-OMP19-G, was constructed and rescued. We found that U-OMP19 was incorporated into LBNSE-OMP19-G virion, which could protect RABV G protein from digestion by gastrointestinal fluids in vitro. Moreover, the immunogenicity of LBNSE-OMP19-G as an inactivated oral vaccine was evaluated, and the inactivated LBNSE-OMP19-G could activate more dendritic cells (DCs) and promote the generation of follicular helper T (TFH) cells, germinal center (GC) B cells, and plasma cells in immunized mice compared with those in mice immunized with parent virus LNBSE, which consequently induced a higher level of virus neutralizing antibody and provided better protection after a lethal challenge of rabies. These data indicate that LBNSE-OMP19-G, which has good safety and immunogenicity, could be a potential inactivated oral rabies vaccine candidate.

Performance of an envelope glycoprotein-based multiplex immunoassay for Ebola virus antibody detection in a cohort of Ebola virus disease survivors

Serological surveillance in animal and human hosts can be a cost-effective strategy for orthoebolavirus detection, but is challenged by accurate estimates of seroprevalence, potential pauci-symptomatic disease presentation, and antigenic cross-reactivity. Here, we describe the use of an envelope glycoprotein (GP)-based multiplex microsphere immunoassay, consisting of nine filovirus GP antigens, for the detection of anti-Ebola virus (EBOV) antibodies in a well-characterized cohort of Guinean Ebola virus disease (EVD) survivors and contacts from the 2013 – 2016 West African EVD outbreak. We examined sensitivity and specificity for the detection of anti-EBOV antibodies by GP expressed as recombinant trimeric ectodomains, yielding an assay performance of 95.96% sensitivity and 98.61% specificity. Additionally, agreement between the multiplex test and a whole virus ELISA and virus neutralization test showed strong correlations. The results demonstrate this filovirus multiplex test is a sensitive tool for high-throughput serosurveillance

Serologic Evidence for Early SARS-CoV-2 Circulation in Lima, Peru, 2020.

During early 2021, Peru had the highest COVID-19-associated per-capita mortality rate. Socioeconomic inequality, insufficiently prepared healthcare, and surveillance systems are factors explaining the mortality rate, which can be severely worsened by early undetected SARS-CoV-2 circulation. We tested 1,441 individuals with fever sampled during August 2019-May 2021, several months before the first SARS-CoV-2 seroprevalence study available so far in Lima, Peru, for SARS-CoV-2-specific antibodies. The testing algorithm included a chemiluminescence immunoassay and surrogate virus neutralization test. Early positive samples (N = 24) from January-March 2020 were further tested using a plaque-reduction neutralization test (PRNT) and avidity test against the SARS-CoV-2 spike and nucleoprotein. None of the early samples were PRNT-confirmed, in contrast to 81.8% (18/22) of a subsample from April 2020 onward (Fisher exact test; P <0.0001). Therefore, we excluded non-PRNT-confirmed samples from subsequent analyses. The SARS-CoV-2 antibody detection rate was 0.9% in mid-April 2020 (1/104; 95% CI: 0.1-5.8%), suggesting viral circulation in early-middle March 2020, consistent with the first molecular detection of SARS-CoV-2 in Peru on March 2020. Mean avidity increase of 62-77% to 81-94% from all PRNT-confirmed SARS-CoV-2-positive samples during early 2020 were consistent with onset of SARS-CoV-2 circulation during late February/March 2020. Early circulation was also confirmed in a susceptible, exposed, infected, and recovered mathematical model that calculated an effective reproduction number >1 during February-March 2020. Early introduction of SARS-CoV-2 thus contributed to the high COVID-19 mortality rate in Peru. Emphasizing the role of diagnostic confirmation in understanding the pandemic's trajectory, this study highlights the importance of early detection and accurate testing in managing infectious disease outbreaks.

Modelling the mechanisms of antibody mixtures in viral infections: the cases of sequential homologous and heterologous dengue infections.

Antibodies play an essential role in the immune response to viral infections, vaccination or antibody therapy. Nevertheless, they can be either protective or harmful during the immune response. Moreover, competition or cooperation between mixed antibodies can enhance or reduce this protective or harmful effect. Using the laws of chemical reactions, we propose a new approach to modelling the antigen-antibody complex activity. The resulting expression covers not only purely competitive or purely independent binding but also synergistic binding which, depending on the antibodies, can promote either neutralization or enhancement of viral activity. We then integrate this expression of viral activity in a within-host model and investigate the existence of steady-states and their asymptotic stability. We complete our study with numerical simulations to illustrate different scenarios: firstly, where both antibodies are neutralizing and secondly, where one antibody is neutralizing and the other enhancing. The results indicate that efficient viral neutralization is associated with purely independent antibody binding, whereas strong viral activity enhancement is expected in the case of purely competitive antibody binding. Finally, data collected during a secondary dengue infection were used to validate the model. The dataset includes sequential measurements of virus and antibody titres during viremia in patients. Data fitting shows that the two antibodies are in strong competition, as the synergistic binding is low. This contributes to the high levels of virus titres and may explain the antibody-dependent enhancement phenomenon. Besides, the mortality of infected cells is almost twice as high as that of susceptible cells, and the heterogeneity of viral kinetics in patients is associated with variability in antibody responses between individuals. Other applications of the model may be considered, such as the efficacy of vaccines and antibody-based therapies.

Development of a blocking ELISA for detection of Japanese encephalitis virus antibodies in pig and horse sera

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA.

Facile quantitative diagnostic testing for neutralizing antibodies against Chikungunya virus

BackgroundViral neutralization (NT) assays can be used to determine the immune status of patients or assess the potency of candidate vaccines or therapeutic monoclonal antibodies (mAbs). Focus reduction neutralization test (FRNT) is a conventional neutralization test (cVNT) with superior specificity for measurement of neutralizing antibodies against a specific virus. Unfortunately, the application of FRNT to the chikungunya virus (CHIKV) involves a highly pathogenic bio-agent requiring biosafety level 3 (BSL3) facilities, which inevitably imposes high costs and limits accessibility. In this study, we evaluated a safe surrogate virus neutralization test (sVNT) that uses novel CHIKV replicon particles (VRPs) expressing eGFP and luciferase (Luc) to enable the rapid detection and quantification of neutralizing activity in clinical human serum samples.MethodsThis unmatched case-control validation study used serum samples from laboratory-confirmed cases of CHIKV (n = 19), dengue virus (DENV; n = 9), Japanese encephalitis virus (JEV; n = 5), and normal individuals (n = 20). We evaluated the effectiveness of sVNT, based on mosquito cell-derived CHIK VRPs (mos-CHIK VRPs), in detecting (eGFP) and quantifying (Luc) neutralizing activity, considering specificity, sensitivity, and reproducibility. We conducted correlation analysis between the proposed rapid method (20 h) versus FRNT assay (72 h). We also investigated the correlation between sVNT and FRNT in NT titrations in terms of Pearson’s correlation coefficient (r) and sigmoidal curve fitting.ResultsIn NT screening assays, sVNT-eGFP screening achieved sensitivity and specificity of 100%. In quantitative neutralization assays, we observed a Pearson’s correlation coefficient of 0.83 for NT50 values between sVNT-Luc and FRNT.ConclusionsFacile VRP-based sVNT within 24 h proved highly reliable in the identification and quantification of neutralizing activity against CHIKV in clinical serum samples.

Isolation and genetic characterization of canine adenovirus type 2 variant from raccoon dog (Nyctereutes procynoide koresis) in Republic of Korea

Canine adenovirus type 2 (CAV-2) is a common causative agent of respiratory disease in canines. There have been no reports of CAV-2 variants isolated from raccoon dogs. This study aims to investigate the biological and genetic characteristics of a novel Korean CAV-2 variant. Madin-Darby canine kidney cells were used to isolate the CAV-2 variant from 45 fecal swab samples. Diagnostic tools such as the cytopathic effect (CPE) assay, electron microscopy, polymerase chain reaction, and immunofluorescence and hemagglutination assays were used to confirm the presence of the CAV-2 isolate. A cross-virus neutralization assay was performed to verify the novelty of this CAV variant. Genetic analysis was performed using nucleotide sequences obtained through next-generation sequencing. The isolate was confirmed to be a CAV-2 variant based on the aforementioned methods and designated CAV2232. The number of bases in the fiber and E3 genes of CAV2232 were 1,626 and 414, respectively. Phylogenetic analysis of the fiber and E3 genes confirmed that CAV2232 was classified into a different clade from the known CAV-1 and CAV-2 strains. Mice inoculated with the CAV2232 vaccine developed high virus neutralization antibody titers of 1,024 (210) against CAV2232, while mice inoculated with CAV-1 and CAV-2 vaccines had low virus neutralization antibody titers of 12.9 (23.7) and 6.5 (22.7), respectively, against CAV2232. CAV2232 isolated from wild raccoon dog feces was classified as a novel CAV-2 variant. CAV2232 may therefore be used as an antigen for new vaccine development and serological investigations.

Analysis of colostral antibodies to herpesvirus 1 and 5 in Murrah buffalo calves

Familiar agriculture is predominant in the production of milk buffaloes in São Paulo southwest, Brazil. The production of buffaloes’ milk is sold almost exclusively to cheese production inside and out São Paulo State. This search was done in a proprerty located in Alambari, São Paulo, specialized in the production of Murrah buffaloes, with 21 animals, born between March and May in the year 2017. Blood was collected at 24 to 48 h from the birth, every 15 days and at the completetion of weaning (91 days of age). The samples were collected in vaccum tubes of 10 mL with coagulum activator, by a puncture of the jugular vein. With the application of the virus neutralization tests, we could see 90.47% reagent animals for BoHV-5 and or BoHV-1 in the first two days of life. The geometric medium titles (GMT) of antibodies reduced significantly from the time 0 (1.4563) to the time 6 (05721) (P=0.0001), showing adequate transference of passive immunity through the ingestion of colostrum by buffalo calves, with the persistence of antibodies until 3 months old. Considering that most buffalo calves were reagents after sucking the colostrum, it is possible to conclude that there is a great occurrence of BoHV-5 and or BoHV-1 once there is a crossed reaction among these and the cattle with no historic of vaccination for the agents.

Bison, Elk, and Other Captive Wildlife Species Humoral Immune Responses against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has been found to infect various domestic and wild animal species. In this study, convenience serum samples from 575 bison, 180 elk, and 147 samples from various wildlife species collected between 2020 and 2023 from several regions in the United States were analyzed for the presence of SARS-CoV-2-specific antibodies. Two commercial ELISA assays based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) or the nucleocapsid protein (N-ELISA) of SARS-CoV-2 were used. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT). Our results indicated that 1.2% of bison, 2.2% of elk, and 4.1% of the other wildlife species serum samples were seropositive in the sVNT, whereas 4.2% of bison, 3.3% of elk, and 1.4% of the other captive wildlife species serum samples tested positive by the N-ELISA. Among the sVNT serum samples, two samples from bison, one sample from elk, and five serum samples from other wildlife species (one cheetah, one gorilla, two lions, and one hippopotamus) had neutralizing antibody titers in the VNT, indicating these species are susceptible to SARS-CoV-2 infection. These findings highlight the importance of broad surveillance efforts for the effective monitoring of SARS-CoV-2 in non-human hosts.

Protective role of antibodies in enteric virus infections: Lessons from primary and secondary immune deficiencies.

Enteric viruses are the main cause of acute gastroenteritis worldwide with a significant morbidity and mortality, especially among children and aged adults. Some enteric viruses also cause disseminated infections and severe neurological manifestations such as poliomyelitis. Protective immunity against these viruses is not well understood in humans, with most knowledge coming from animal models, although the development of poliovirus and rotavirus vaccines has extended our knowledge. In a classical view, innate immunity involves the recognition of foreign DNA or RNA by pathogen recognition receptors leading to the production of interferons and other inflammatory cytokines. Antigen uptake and presentation to T cells and B cells then activate adaptive immunity and, in the case of the mucosal immunity, induce the secretion of dimeric IgA, the more potent immunoglobulins in viral neutralization. The study of Inborn errors of immunity (IEIs) offers a natural opportunity to study nonredundant immunity toward pathogens. In the case of enteric viruses, patients with a defective production of antibodies are at risk of developing neurological complications. Moreover, a recent description of patients with low or absent antibody production with protracted enteric viral infections associated with hepatitis reinforces the prominent role of B cells and immunoglobulins in the control of enteric virus.

DNA methylation profiling identifies TBKBP1 as potent amplifier of cytotoxic activity in CMV-specific human CD8+ T cells.

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. Although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions (DMRs) when compared to memory CD8+ T cells. Among the top demethylated DMRs in cytomegalovirus-specific CD8+ T cells was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with higher TBKBP1 expression at both mRNA and protein level, independent of alternative splicing of TBKBP1 transcripts. Notably, the distinct DNA methylation patterns in CD8+ T cell subsets was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies.

Detection of anti-enterovirus IgG in human sera by ELISA method using the KTL-510 peptide

Enterovirus (EV) infections occur frequently in humans. In some geographical areas they are more common. These viruses cause diseases with varying degrees of severity, from a simple respiratory tract infection to severe diseases. Since EVs include more than 70 serotypes currently circulating in the population, a methodology that detects most of them is needed. ELISA is a rapid, sensitive, and economical diagnostic method for the identification of EV serotypes and can also be used as a retrospective diagnostic tool or in the investigation of outbreaks of infection. Commercial EV-ELISAs often appear and gradually disappear from the market supply. We have used the KTL-510 peptide, a synthetic viral protein of poliovirus VP1, as an antigen in a peptide-based ELISA for the detection of a broader spectrum of anti-EV antibodies. We aimed to design, optimize, and standardize this in-house ELISA with the peptide, and implement the method for routine detection of anti-EV IgG in human sera. For determining the cut-off value, we used 100 patients’ sera which were previously tested negative for IgG antibodies against EVs using a commercial ELISA kit available. We monitored patients’ sera samples sent for serological testing of anti-coxsackievirus antibodies to the National Reference Center for the Identification of Enteric Viruses between 2018–2022. These serum samples were examined using a standard virus neutralization test as well as the newly developed ELISA method.

Navigating the Landscape of B Cell Mediated Immunity and Antibody Monitoring in SARS-CoV-2 Vaccine Efficacy: Tools, Strategies and Clinical Trial Insights

Correlates of Protection (CoP) are biomarkers above a defined threshold that can replace clinical outcomes as primary endpoints, predicting vaccine effectiveness to support the approval of new vaccines or follow up studies. In the context of COVID-19 vaccination, CoPs can help address challenges such as demonstrating vaccine effectiveness in special populations, against emerging SARS-CoV-2 variants or determining the durability of vaccine-elicited immunity. While anti-spike IgG titres and viral neutralising capacity have been characterised as CoPs for COVID-19 vaccination, the contribution of other components of the humoral immune response to immediate and long-term protective immunity is less well characterised. This review examines the evidence supporting the use of CoPs in COVID-19 clinical vaccine trials, and how they can be used to define a protective threshold of immunity. It also highlights alternative humoral immune biomarkers, including Fc effector function, mucosal immunity, and the generation of long-lived plasma and memory B cells and discuss how these can be applied to clinical studies and the tools available to study them.

SARS-CoV-2 Infection Enhances Humoral Immune Response in Vaccinated Liver Transplant Recipients.

In the spring of 2020, the SARS-CoV-2 pandemic presented a formidable challenge to national and global healthcare systems. Immunocompromised individuals or those with relevant pre-existing conditions were particularly at risk of severe coronavirus disease 2019 (COVID-19). Thus, understanding the immunological processes in these patient groups is crucial for current research. This study aimed to investigate humoral immunity following vaccination and infection in liver transplant recipients. Humoral immunity analysis involved measuring IgG against the SARS-CoV-2 spike protein (anti-S IgG) and employing a surrogate virus neutralization test (sVNT) for assessing the hACE2 receptor-binding inhibitory capacity of antibodies. The study revealed that humoral immunity post-vaccination is well established, with positive results for anti-S IgG in 92.9% of the total study cohort. Vaccinated and SARS-CoV-2-infected patients exhibited significantly higher anti-S IgG levels compared to vaccinated, non-infected patients (18,590 AU/mL vs. 2320 AU/mL, p < 0.001). Additionally, a significantly elevated receptor-binding inhibitory capacity was observed in the cPassTMTM sVNT (96.4% vs. 91.8%, p = 0.004). Furthermore, a substantial enhancement of anti-S IgG levels (p = 0.034) and receptor-binding inhibition capacity (p < 0.001) was observed with an increasing interval post-transplantation (up to 30 years), calculated by generalized linear model analysis. In summary, fully vaccinated liver transplant recipients exhibit robust humoral immunity against SARS-CoV-2, which significantly intensifies following infection and with increasing time after transplantation. These findings should be considered for booster vaccination schemes for liver transplant recipients.

Assessment of twelve echovirus virus-neutralisation assays in Europe: recommendations for harmonisation of non-polio enterovirus sero-surveillance studies

Non-polio enteroviruses (NPEV) cause significant disease worldwide. Population-based sero-surveillance, by measuring antibodies against specific NPEV types, provides additional information on past circulation and the prediction for future upsurges. Virus neutralisation assays (VNA), the current method of choice for measuring NPEV type specific antibodies, are not entirely standardised. Via the European Non-Polio Enterovirus Network, we organised a VNA quality assessment in which twelve laboratories participated. We provided five echovirus (E) types (E1, E18, E30 G2, E30 G6 and E6) and intravenous immunoglobulins (IVIG) as a sample for the NPEV VNA quality assessment. Differences in VNA protocols and neutralising Ab (nAb) titres were found between the participating laboratories with geometric coefficients of variation ranging from 10.3–62.9 %. Mixed-effects regression analysis indicated a small but significant effect of type of cell line used. Harmonisation of cell line passage number, however, did not improve variation between laboratories. Calibration by making use of a reference sample, reduced variation between laboratories but differences in nAb titres remained higher than two log2 dilution steps. In conclusion, sero-surveillance data from different laboratories should be compared with caution and standardised protocols are needed.

The comparison of pathogenicity among SARS-CoV-2 variants in domestic cats

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected or isolated from domestic cats. It is unclear whether cats play an important role in the SARS-CoV-2 transmission cycle. In this study, we examined the susceptibility of cats to SARS-CoV-2, including wild type and variants, by animal experiments. Cats inoculated with wild type, gamma, and delta variants secreted a large amount of SARS-CoV-2 for 1 week after the inoculation from nasal, oropharyngeal, and rectal routes. Only 100 TCID50 of virus could infect cats and replicate well without severe clinical symptoms. In addition, one cat inoculated with wild type showed persistent virus secretion in feces for over 28 days post-inoculation (dpi). The titer of virus-neutralizing (VN) antibodies against SARS-CoV-2 increased from 11 dpi, reaching a peak at 14 dpi. However, the omicron variant could not replicate well in cat tissues and induced a lower titer of VN antibodies. It is concluded that cats were highly susceptible to SARS-CoV-2 infection, but not to the Omicron Variant, which caused the attenuated pathogenicity.

Bovine Respiratory Syncytial Virus Nanovaccine Induces Long-Lasting Humoral Immunity in Mice.

With limited therapies and vaccines available, human respiratory syncytial virus (HRSV) has a significant negative health impact on all age groups but particularly on infants, young children, and older adults. Bovine respiratory syncytial virus (BRSV) is pathogenically and antigenically similar to HRSV. Building upon previous studies using a BRSV nanovaccine coencapsulating multiple proteins, this work demonstrates the development and comparative evaluation of a coencapsulated nanovaccine to a cocktail nanovaccine formulation composed of polyanhydride nanoparticles encapsulating BRSV postfusion (F) glycoprotein and CpG ODN 1668 coadjuvant delivered simultaneously with nanoparticles encapsulating BRSV attachment glycoprotein (G) and CpG ODN 1668. These nanovaccine formulations were administered to C57BL/6 mice by one of two prime-boost regimens (i.e., intranasal/intranasal or intranasal/subcutaneous) followed by assessment of humoral immunity. The cocktail nanovaccine induced sustained anti-F and anti-G serum IgG antibody responses for 12 weeks postprimary immunization. Using polyanhydride particles to deliver G protein in a prime-boost regime also significantly induced serum anti-G antibodies compared to protein and coadjuvant alone. Serum IgG induced by the nanovaccine demonstrated virus-neutralizing capability from 42 to 119 days postprimary immunization. Further, anti-F IgG antibodies were detected in the bronchoalveolar lavage fluid of vaccinated animals. Finally, the nanovaccine induced long-lived anti-F antibody secreting plasma cells that were detectable in the bone marrow 205 days postprimary immunization. Overall, the BRSV nanovaccine(s) successfully induced long-lived humoral immune responses capable of virus neutralization, making this a promising vaccine candidate for further evaluation in other relevant animal models.

PSLBI-30 SARS-CoV-2 Screening and Detection: Assessment of surrogate virus neutralization test (svNT) diagnostic appropriateness in multispecies SARS-CoV-2 virus screening and detection

Abstract The National Veterinary Services Laboratories (NVSL) have been testing animal samples since the 2020 SARS-CoV-2 outbreak. The current “gold standard” for serodetection of SARS-CoV-2 is the virus neutralization (VN) test. This method requires specialized training, more time to complete, and specialized facilities (BSL 3). Therefore, the SARS-CoV-2 VN test is not widely run outside of NVSL. To facilitate increased testing capacity during the outbreak, the surrogate virus neutralization test (sVNT) was selected mid-2020. This commercially available kit uses an ELISA format, giving a percent inhibition (%I) cut-off value to indicate “negative” or “positive,” allowing for faster testing turnaround. Despite its widespread use in many species, an in-depth, multispecies analysis of the data comparing sVNT to VN results has not been reported. Such an analysis is useful to assess the diagnostic appropriateness of the sVNT test for SARS-CoV-2 virus screening and detection across multiple species. Results were considered from all serological samples submitted to NVSL from March 2020 to May 2023 for routine SARS-CoV-2 diagnostic testing. Of these, samples with results for both VN and sVNT testing were considered for further analysis (n = 942). Samples were sorted by the species and family levels, and were assessed for rates of “true positive,” “false positive,” “true negative,” and “false negative” by sVNT, using paired VN results as the indicator of the true status of the sample. Additionally, the increased sVNT cut-off threshold for %I (20%I to 30%I for “positive”) was assessed for appropriateness by species using mean %I values for VN-positive samples. Increasing sVNT %I cut-off threshold for “positive” results from 20%I to 30%I resulted in 58 samples being correctly re-classified as “negative” by sVNT, rather than “positive” at the 20%I cut-off threshold. The mean %I values for most species fell between 40 to 80%I, except for Felidae, which had a mean %I of 93%I, and a minimum %I of 40.6%I for VN-positive samples. The true negative (sVNT/VN-NEG) rates for most species fell between 97to 100%, with 0 to 3% being false negatives (svNT-NEG, VN-POS), with very few exceptions. However, the least false positive (sVNT-POS;VN-NEG) rate was 8.8%, ranging up to 100% false positives for Equidae and one species of Cervidae. While the mean %I for most species indicates the cut-off value is appropriate (for those species where sVNT is an appropriate diagnostic), overall, the results highlight a lack of precision and specificity of the sVNT for many animal species. This indicates a lack of diagnostic appropriateness of the sVNT as a SARS-CoV-2 screening tool for certain species and a need for refinement in cut-off values for other species. The sVNT as a screening tool varies considerably by species for serodetection of SARS-CoV-2, results should be interpreted with caution, and use of other confirmatory tests is recommended.

Discovery of three novel neutralizing antibody epitopes on the human astrovirus capsid spike and mechanistic insights into virus neutralization.

Human astroviruses infect nearly every child in the world, causing diarrhea, vomiting, and fever. Despite the prevalence of human astroviruses, little is known about how antibodies block virus infection. Here, we determined high-resolution structures of the astrovirus capsid protein in complex with three virus-neutralizing antibodies. The antibodies bind distinct sites on the capsid spike domain. We find that the antibodies block virus attachment to human cells and prevent capsid spike interaction with the human neonatal Fc receptor. These findings support the use of the human astrovirus capsid spike as an antigen in a vaccine to prevent astrovirus disease.