Virus Neutralization Assay Research Articles

Correlation between pseudotyped virus and authentic virus neutralisation assays, a systematic review and meta-analysis of the literature.

The virus neutralization assay is a principal method to assess the efficacy of antibodies in blocking viral entry. Due to biosafety handling requirements of viruses classified as hazard group 3 or 4, pseudotyped viruses can be used as a safer alternative. However, it is often queried how well the results derived from pseudotyped viruses correlate with authentic virus. This systematic review and meta-analysis was designed to comprehensively evaluate the correlation between the two assays. Using PubMed and Google Scholar, reports that incorporated neutralisation assays with both pseudotyped virus, authentic virus, and the application of a mathematical formula to assess the relationship between the results, were selected for review. Our searches identified 67 reports, of which 22 underwent a three-level meta-analysis. The three-level meta-analysis revealed a high level of correlation between pseudotyped viruses and authentic viruses when used in an neutralisation assay. Reports that were not included in the meta-analysis also showed a high degree of correlation, with the exception of lentiviral-based pseudotyped Ebola viruses. Pseudotyped viruses identified in this report can be used as a surrogate for authentic virus, though care must be taken in considering which pseudotype core to use when generating new uncharacterised pseudotyped viruses.

Targeting spike glycans to inhibit SARS-CoV2 viral entry

SARS-CoV-2 spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical approaches, we demonstrate that the interaction is avidity driven and that BOA cross-links the spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that multivalent glycan-targeting molecules have the potential to act as pan-coronavirus inhibitors.

The evaluation of five serological assays in determining seroconversion to peste des petits ruminants virus in typical and atypical hosts

Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015–2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0–88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4–62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.

A Subunit Vaccine Candidate Composed of Mpox Virus A29L, M1R, A35R, and B6R Elicits Robust Immune Response in Mice.

With no specific antiviral drugs and preventive vaccines against Mpox virus (MPXV), the epidemic has led to the declaration of a Public Health Emergency of International Concern. As a developmental direction for new vaccines, studies of subunit vaccines based upon MPXV antigen proteins are lacking. In this study, A29L, M1R, A35R, and B6R of MPXV were expressed and purified from a prokaryotic system. The four MPXV antigen proteins in combination were mixed with aluminum hydroxide or CpG7909 as adjuvant, and subsequently used to inoculate mice. The results of enzyme-linked immunosorbent assay (ELISA), flow cytometry analyses, and enzyme-linked immunospot (ELISPOT) assays indicated that A29L, M1R, A35R, and B6R elicited high-level antigen-specific antibodies and CD4+ T cells-based cellular immune response in mice. Moreover, the results of virus neutralization assays suggested that sera from the mice immunized with four proteins elicited high neutralizing activities against the vaccinia virus. Notably, the results of ELISA, ELISPOT, and virus neutralization assays also showed that the CpG7909 adjuvant was more effective in inducing an immune response compared with the aluminum adjuvant. In summary, this study offers valuable insights for further studies of subunit vaccine candidates for the prevention of MPXV and other orthomyxoviruses.

Cache a Killer: Cache Valley virus seropositivity and associated farm management risk factors in sheep in Ontario, Canada.

Cache Valley virus (CVV) disease is a mosquito-borne zoonosis endemic to North America. CVV disease is reported most often in sheep, causing lethal congenital deformities. There are limited data on CVV in Ontario, which is the largest sheep producing province in Canada. This study aimed to determine CVV seroprevalence in Ontario sheep flocks and investigate farm management factors associated with CVV exposure. A cross-sectional study was performed including 364 mature ewes across 18 farms selected from the five largest sheep districts in the province. A questionnaire was administered at each farm to determine farm management practices pertinent to the flock and ewes specifically sampled. Mixed multivariable logistic regression with a random effect for farm was conducted to assess associations between CVV seropositivity (outcome variable) and farm management risk factors (predictor variables). CVV seroprevalence was 33.2% in individual ewes (95% CI: 28.4%-38.1%) as determined by a virus neutralization assay with a titre > 4. Sixteen of the eighteen flocks (88.9%) had at least one CVV seropositive ewe. Increased age, smaller flock size, and sheep housing near wetlands, lakes, or ponds were found to be significantly associated with higher odds of CVV seropositivity. These findings are valuable in guiding breeding practices and housing during mosquito season to minimize infection and, ultimately, CVV disease in the flock.

Infant antibody and B-cell responses following confirmed pediatric GII.17 norovirus infections functionally distinguish GII.17 genetic clusters.

Genogroup II (GII) noroviruses are a major cause of diarrheal disease burden in children in both high- and low-income countries. GII.17 noroviruses are composed of distinct genetic clusters (I, II, IIIa, and IIIb) and have shown potential for replacing historically more prevalent GII.4 strains, but the serological basis for GII.17 antigenic diversity has not been studied in children. Utilizing samples from a birth cohort, we investigated antibody and B-cell responses to GII.17 cluster variants in confirmed GII.17 infections in young children as well as demonstrated that the distinct genetic clusters co-circulate. Polyclonal serum antibodies bound multiple clusters but showed cluster-specific blockade activity in a surrogate virus neutralization assay. Antibodies secreted by immortalized memory B cells (MBCs) from an infant GII.17 case were highly specific to GII.17 and exhibited blockade activity against this genotype. We isolated an MBC-derived GII.17-specific Immunoglobulin A (IgA) monoclonal antibody called NVA.1 that potently and selectively blocked GII.17 cluster IIIb and recognized an epitope targeted in serum from cluster IIIb-infected children. These data indicate that multiple antigenically distinct GII.17 variants co-circulate in young children, suggesting retention of cluster diversity alongside potential for immune escape given the existence of antibody-defined cluster-specific epitopes elicited during infection.

Effectiveness of Bivalent Omicron-Containing Booster Vaccines against SARS-CoV-2 Omicron Variant among Individuals with and without Prior SARS-CoV-2 Infection.

In this study, we evaluated the effectiveness of the bivalent mRNA COVID-19 vaccines against the Omicron variant in individuals with or without prior SARS-CoV-2 infection history. We assessed the SARS-CoV-2-specific neutralizing antibody in serum samples by surrogate virus neutralizing assay (sVNT) and determined the serum's neutralizing capacity against the Omicron BA.5 by a plaque reduction neutralizing test (PRNT50). The results of the sVNT assay demonstrate a higher percentage of inhibition of the serum samples from the infected group than from the uninfected group (p = 0.01) before the bivalent vaccination but a similarly high percentage of inhibition after the vaccination. Furthermore, the results of the PRNT50 assay demonstrate a higher neutralizing capacity of the serum samples against Omicron BA.5 in the infected group compared to the uninfected group, both before and after the bivalent vaccine administration (p < 0.01 and p = 0.02 for samples collected before and after the bivalent vaccination, respectively). A higher neutralizing capacity of the serum samples against BA.5 following bivalent vaccination compared to those before vaccination suggests the efficacy of bivalent mRNA COVID-19 vaccines in triggering an immune response against the Omicron variant, particularly BA.5, regardless of infection history.

Subcutaneous and intramuscular administration of a SARS-CoV-2 vaccine are similarly effective in generating a humoral response in domestic goats (Capra hircus).

To determine severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) serum antibody titers in domestic goats after SC and IM administration of an experimental, veterinary SARS-CoV-2 vaccine. 31 healthy adult domestic goats from 4 zoological institutions. On day 0, blood was collected for baseline serum titer before vaccination with 1 mL SARS-CoV-2 recombinant S protein vaccine SC (n = 22) or IM (n = 9). A booster vaccination was administered 21 (SC group) or 28 days (IM group) after the initial vaccine and blood samples were collected at days 21 (SC group) or 28 (IM group), 42, 90, and 180 postvaccinations. The study took place between September 27, 2021, and June 01, 2022. Seroconversion for SARS-CoV-2 was assessed by a SARS-CoV-2 virus neutralization (VN) assay. Before vaccination, no goats had detectable antibodies. On day 42, 100% of goats had detectable serum titers. Serum titers peaked at day 42 for 94% of goats vaccinated by either route of administration. There was a significant difference between SC and IM groups regarding the proportion of goats with detectable titers on day 21/28 (68% vs 0%, respectively) and day 180 (50% vs 89%, respectively), relative to day 0. The 2 vaccination protocols (SC 21 days apart and IM 28 days apart) were similarly effective in mounting serum antibody response in goats. The SC route of administration appeared to have a more rapid onset of immunity, while the IM route may have produced a longer duration of immunity. These data may be useful in determining appropriate SARS-CoV-2 vaccination schedules in ruminants.

Humoral Immune Response in Calves Vaccinated with Monovalent Vaccines or a Trivalent Combination Thereof and Matching of These Vaccines to the Selected Circulating Foot-and-Mouth Disease Viruses in Ethiopia.

Foot-and-mouth disease (FMD) is an endemic, highly contagious, and devastating disease of livestock production in Ethiopia. Control of this disease relies mainly on prophylactic vaccination by willing farmers without a countrywide vaccination program. The objectives of this study were to quantify the humoral immune response and evaluation of the serological relationship of the vaccine strain used with representative field strain isolates. This was performed by primo vaccination of 6-9-month-old Holstein Friesian calves (35 treatment and 4 control calves) on day one and booster vaccination on day 28. Calves were vaccinated using the locally available National Veterinary Institute (NVI), Bishoftu, Ethiopia, inactivated aluminum hydroxide adjuvant monovalent (either O, A, SAT-2 alone) or trivalent (combination of A, O, SAT-2) vaccine (A/ETH/6/2000 (G-VII, O/ETH/38/2005(EA-3) and SAT-2/ETH/64/2009(XIII)). A 2 mL or 4 mL dose was used to vaccinate all calves except the animals that served as a control. In the case of the trivalent vaccine, a 4 mL dose was used to vaccinate calves. The serum was collected at 7, 14, 21, 28, and 56 days post-vaccination (d.p.v.). The humoral immune response was quantified by the solid-phase competitive enzyme-linked immunosorbent assay (SPC ELISA) and the virus-neutralization test (VNT). The serological relationship of heterologous and homologous viruses was also evaluated by adjuvant vaccine matching tests. The r1-value was determined using serum collected 21 d.p.v. An increase in immune response was observed from 7 d.p.v. to 28 d.p.v. in calves who received a 4 mL dose containing a 107.24 antigen load of 100 tissue culture infective dose (100TCID50) virus titer in the formulation. Upon receiving a booster dose on day 28, the humoral immune response was checked on the 56th day post-initial vaccination. Amounts of 54%, 72%, 79%, and 72% of inhibition for A, O, SAT-2, and trivalent vaccine in the three serotypes SPCE, respectively, was measured. Here, it was found that the immune response of calves increased from day 7 to 56, as evidenced by SPCE analysis. Likewise, an increase in antibody titer measured by a one-dimensional virus neutralization assay was also in line with SPCE analysis. This indicates that the vaccine is capable of inducing a neutralizing antibody that confers a protective immune response in 70%, 62%, and 100% heterologous field strains of A, O, and SAT-2 isolates, respectively, and has an average antigenic relationship of >0.3 with a standard deviation of +0.05 (N = 3) to the vaccine strains A/ETH/6/2000, O/ETH/38/2005 and SAT-2/ETH/64/2009, respectively, when using the one-dimensional virus neutralization test. The contribution and importance of this study is a confirmation of the vaccine and the field strain serological relationship for serotype SAT-2 strain and further research/change of vaccination strategy/ improvement in the currently used vaccine to cover a wide range of prevailing genotypes/lineages and induction of sound immune response after vaccination for serotype A and O strain. This study suggests that the trivalent vaccine produced by the National Veterinary Institute containing viral isolates from serotype O, A, and SAT-2 has a good serological relationship with the majority of circulating field strains in Ethiopia.

Seroprevalence of anti-SARS-CoV-2 antibodies in household domestic ferrets (Mustela putorius furo) in Spain, 2019–2023

SARS-CoV-2 is the causal agent of Coronavirus Disease 2019 (COVID-19) in humans that emerged in late 2019. This virus is able to infect humans and different animal species. Among pets, cats and ferrets are more susceptible to be infected by the SARS-CoV-2. Epidemiological studies are an important tool to provide information under natural conditions of exposure to SARS-CoV-2 virus. In comparison to cats, limited epidemiological studies have been performed in domestic ferrets (Mustela putorius furo) reporting the presence of antibodies in this species. This study analysed the presence of anti-SARS-CoV-2 antibodies in 432 cliend-owned ferrets from different geographical areas of Spain during the different waves of COVID-19 outbreaks from December 2019 to May 2023 (42 months). For this purpose, anti-SARS-CoV-2 antibodies were detected by an enzyme-linked immunosorbent method (ELISA) using the receptor binding domain (RBD) of Spike antigen and confirmed by serum virus neutralization assay. Eighteen of the 432 ferrets included were seroreactive by the in-house ELISA (4.17%, 95% Confidence Interval (CI): 2.65–6.49). In this sense, the wave of COVID-19 with the higher number of seropositive ferrets occurred during the seventh wave when the different Omicron subvariants were the dominant virus variants. Our results suggest that the risk of SARS-CoV-2 transmission in domestic ferrets in natural conditions is low. Further research is need to evaluate the potential risk of transmission of SARS-CoV-2 from human to pets.

Clinical assessment of a point-of-care assay to determine protective vaccinal antibody titers to canine viral diseases

Guidelines recommend that dogs are vaccinated for canine distemper virus (CDV), canine parvovirus (CPV), and canine adenovirus (CAV) every 3 years. Alternatively, their antibody titers are measured and vaccines given when titers fall below a protective threshold. In this study, a point-of-care (POC) assay was compared to hemagglutination inhibition (for CPV) and virus neutralization (for CAV and CDV) assays to predict the need for revaccination Ninety-two dogs presented for vaccination were enrolled. The POC assay indicated protective titers against CDV in 79/80, CPV in 89/90, and CAV in 91/91 dogs with reference standard antibody measurements that were over a protective threshold. The sensitivity of the POC assay for to detect protective concentrations of CDV antibodies was 99% (95% confidence interval [CI 95%], 93.3–99.9%). Ten dogs were falsely considered protected against CDV by the POC assay with a specificity of 17% (CI 95%, 3.0–44.8%). The sensitivity of the POC assay for protective concentrations of CPV titers was 99% (CI 95%, 93.9–99.9%). The sensitivity of the POC assay to detect protective concentrations of CAV antibodies was 100% (CI 95%, 95.9–100%). Only classifying high-positive CDV and CPV titers on the POC assay as protective improved assay specificity to 100%, but sensitivity decreased to 51% and 76% respectively. This POC assay had a high sensitivity for the detection of protective antibody titers; however, some dogs were falsely categorized as protected, especially for CDV.

First evidence of schmallenberg virus infection in southern Italy

BackgroundSchmallenberg virus (SBV) is a vector-borne pathogen that mainly affects ruminants. Schmallenberg disease has never been described in southern Italy, although this geographic area displays climatic features suitable for Culicoides biting midges, which transmit the pathogen. An observational study was carried out in the Campania region in 2020 to evaluate the seroprevalence in cattle and water buffalo as well as to identify the risk factors involved in the distribution of SBV.ResultsRelatively high seroprevalences of 38.2% (cattle) and 43% (water buffalo) were found by using a commercial SBV ELISA, which is comparable to the prevalence obtained in other countries under post-epidemic conditions. A virus neutralization assay performed on positive samples showed high titers in a large percentage of animals which is assumed to indicate recent exposure. Bivariate analysis of several variables revealed some environmental factors associated with higher seroprevalence, such as mean annual temperature, distance from the coast, and altitude. Multivariate logistic regression confirmed the statistical association only for mean annual temperature, that was found to be the main factor responsible for the distribution of the virus in southern Italy. In addition, molecular diagnosis attempts were performed on serum samples and resulted in the detection of SBV RNA in two herds and six animals.ConclusionsIn this work we have demonstrated the circulation of SBV in southern Italy using both molecular and serological assays. This study emphasized the essential role of monitoring in preventing the re-emergence of vector-borne diseases in ruminants.

A Pseudovirus-Based Entry Assay to Evaluate Neutralizing Activity against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) infection can cause life-threatening pneumonia and bronchiolitis, posing a significant threat to human health worldwide, especially to children and the elderly. Currently, there is no specific treatment for RSV infection. The most effective measures for preventing RSV infection are vaccines and prophylactic medications. However, not all population groups are eligible for the approved vaccines or antibody-based preventive medications. Therefore, there is an urgent need to develop novel vaccines and prophylactic drugs available for people of all ages. High-throughput assays that evaluate the efficacy of viral entry inhibitors or vaccine-induced neutralizing antibodies in blocking RSV entry are crucial for evaluating vaccine and prophylactic drug candidates. We developed an efficient entry assay using a lentiviral pseudovirus carrying the fusion (F) protein of type A or B RSV. In addition, the essential parameters were systematically optimized, including the number of transfected plasmids, storage conditions of the pseudovirus, cell types, cell numbers, virus inoculum, and time point of detection. Furthermore, the convalescent sera exhibited comparable inhibitory activity in this assay as in the authentic RSV virus neutralization assay. We established a robust pseudovirus-based entry assay for RSV, which holds excellent promise for studying entry mechanisms, evaluating viral entry inhibitors, and assessing vaccine-elicited neutralizing antibodies against RSV.

Breakthrough SARS-CoV-2 Omicron Variant in Individuals Primed with Heterologous Vaccines Enhances Inhibition Performance of Neutralizing Antibody to BA.2 Parental Lineage.

This study aims to analyze the neutralization ability against Omicron parental variants in five clusters of individuals with different Coronavirus disease (COVID-19) immunity backgrounds, including individuals receiving a homologous or heterologous vaccine without prior infection, recovered patients with homologous or heterologous vaccination, and recovery patients without vaccination. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogate virus neutralization assay was performed on serum samples. Spearman correlation analysis showed that the percent inhibition against Omicron B.1.1.529 and BA.2 was significantly related to the period of serum collection (r = 0.730 and 0.787, p < 0.001, respectively). Very strong correlation between percent inhibition of neutralizing antibody against Omicron B.1.1.529 and BA.2 variants (rs = 0.973, p < 0.001) was also observed. The neutralizing activity of the sera from recovery patients receiving homologous and heterologous vaccine against the wild-type, B.1.1.529, and BA.2 Omicron variants was significantly higher (p < 0.001) than that of recovery patients without vaccination. This study robustly showed that the breakthrough SARS-CoV-2 Omicron variant in individuals who received homologous and heterologous vaccines had a high level of neutralizing activity against B.1.1.529 and BA.2 parental lineage of XBB subvariants. Therefore, the next-generation COVID-19 vaccine against emerging variants is needed to improve resilience against ongoing variants, particularly for persons who have never been infected.

A Multivariant Surrogate Neutralization Assay Identifies Variant-Specific Neutralizing Antibody Profiles in Primary SARS-CoV-2 Omicron Infection.

Primary infection with the Omicron variant of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) can be serologically identified with distinct profiles of neutralizing antibodies (nAbs), as indicated by high titers against the Omicron variant and low titers against the ancestral wild-type (WT). Here, we evaluated whether a novel surrogate virus neutralization assay (sVNT) that simultaneously quantifies the binding inhibition of angiotensin-converting enzyme 2 (ACE2) to the proteins of the WT- and Omicron-specific receptor-binding domains (RBDs) can identify nAb profiles after primary Omicron infection with accuracy similar to that of variant-specific live-virus neutralization tests (NTs). Therefore, we comparatively tested 205 samples from individuals after primary infection with the Omicron variant and the WT, and vaccinated subjects with or without Omicron breakthrough infections. Indeed, variant-specific RBD-ACE2 binding inhibition levels significantly correlated with respective NT titers (p < 0.0001, Spearman's r = 0.92 and r = 0.80 for WT and Omicron, respectively). In addition, samples from individuals after primary Omicron infection were securely identified with the sVNT according to their distinctive nAb profiles (area under the curve = 0.99; sensitivity: 97.2%; specificity: 97.84%). Thus, when laborious live-virus NTs are not feasible, the novel sVNT we evaluated in this study may serve as an acceptable substitute for the serological identification of individuals with primary Omicron infection.

A fully human monoclonal antibody possesses antibody-dependent cellular cytotoxicity (ADCC) activity against the H1 subtype of influenza A virus by targeting a conserved epitope at the HA1 protomer interface.

The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCCactivity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.

Rabies neutralizing antibody titers in Korean dogs and cats intended for overseas travel

Animals imported from abroad are a cause of rabies outbreaks in many countries. Therefore, rabies serology testing for dogs and cats traveling abroad is an important measure to reduce the incidence of rabies. Rabies virus antibodies were measured in sera collected from 2,367 dogs and 894 cats between 2017 and 2021. A serum sample with a value of 0.5 IU/mL or higher was considered a pass. The overall pass rates for rabies virus were 96.4% in dogs and 98.4% in cats. The mean rabies virus neutralization assay titers were higher in cats than in dogs and in female than in male animals. According to age, 6-year-old dogs and 9-year-old cats had the highest virus neutralization assay titers. Of the failure cases, 53.0% (53/100) were dogs or cats less than 1 year old. Although the average failure rates in dogs and cats were low at 3.5% and 1.6%, respectively, the factors influencing failure were age and vaccine manufacturer. Therefore, it is necessary to observe the vaccination interval and timing of blood collection after boosting.

SARS-CoV-2 Prevalence and Variant Surveillance among Cats in Pittsburgh, Pennsylvania, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects many mammals, and SARS-CoV-2 circulation in nonhuman animals may increase the risk of novel variant emergence. Cats are highly susceptible to SARS-CoV-2 infection, and there were cases of virus transmission between cats and humans. The objective of this study was to assess the prevalence of SARS-CoV-2 variant infection of cats in an urban setting. We investigated the prevalence of SARS-CoV-2 variant infections in domestic and community cats in the city of Pittsburgh (n = 272). While no cats tested positive for SARS-CoV-2 viral RNA, 35 cats (12.86%) tested SARS-CoV-2-antibody-positive. Further, we compared a cat-specific experimental lateral flow assay (eLFA) and species-agnostic surrogate virus neutralization assay (sVNT) for SARS-CoV-2 antibody detection in cats (n = 71). The eLFA demonstrated 100% specificity compared to sVNT. The eLFA also showed 100% sensitivity for sera with >90% inhibition and 63.63% sensitivity for sera with 40-89% inhibition in sVNT. Using a variant-specific pseudovirus neutralization assay (pVNT) and antigen cartography, we found the presence of antibodies to pre-Omicron and Omicron SARS-CoV-2 variants. Hence, this approach proves valuable in identifying cat exposure to different SARS-CoV-2 variants. Our results highlight the continued exposure of cats to SARS-CoV-2 and warrant coordinated surveillance efforts.

Accelerating drug target inhibitor discovery with a deep generative foundation model.

Inhibitor discovery for emerging drug-target proteins is challenging, especially when target structure or active molecules are unknown. Here, we experimentally validate the broad utility of a deep generative framework trained at-scale on protein sequences, small molecules, and their mutual interactions-unbiased toward any specific target. We performed a protein sequence-conditioned sampling on the generative foundation model to design small-molecule inhibitors for two dissimilar targets: the spike protein receptor-binding domain (RBD) and the main protease from SARS-CoV-2. Despite using only the target sequence information during the model inference, micromolar-level inhibition was observed in vitro for two candidates out of four synthesized for each target. The most potent spike RBD inhibitor exhibited activity against several variants in live virus neutralization assays. These results establish that a single, broadly deployable generative foundation model for accelerated inhibitor discovery is effective and efficient, even in the absence of target structure or binder information.

Immune response after two doses of the BNT162b2 COVID-19 vaccine and risk of SARS-CoV-2 breakthrough infection in Tyrol, Austria: an open-label, observational phase 4 trial

Correlates of protection could help to assess the extent to which a person is protected from SARS-CoV-2 infection after vaccination (so-called breakthrough infection). We aimed to clarify associations of antibody and T-cell responses after vaccination against COVID-19 with risk of a SARS-CoV-2 breakthrough infection and whether measurement of these responses enhances risk prediction. We did an open-label, phase 4 trial in two community centres in the Schwaz district of the Federal State of Tyrol, Austria, before the emergence of the omicron (B.1.1.529) variant of SARS-CoV-2. We included individuals (aged ≥16 years) a mean of 35 days (range 27-43) after they had received a second dose of the BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine. We quantified associations between immunological parameters and breakthrough infection and assessed whether information on these parameters improves risk discrimination. The study is registered with the European Union Drug Regulating Authorities Clinical Trials Database, 2021-002030-16. 2760 individuals (1682 [60·9%] female, 1078 [39·1%] male, mean age 47·4 years [SD 14·5]) were enrolled into this study between May 15 and May 21, 2021, 712 (25·8%) of whom had a previous SARS-CoV-2 infection. Over a median follow-up of 5·9 months, 68 (2·5%) participants had a breakthrough infection. In models adjusted for age, sex, and previous infection, hazard ratios for breakthrough infection for having twice the immunological parameter level at baseline were 0·72 (95% CI 0·60-0·86) for anti-spike IgG, 0·80 (0·70-0·92) for neutralising antibodies in a surrogate virus neutralisation assay, 0·84 (0·58-1·21) for T-cell response after stimulation with a CD4 peptide pool, and 0·77 (0·54-1·08) for T-cell response after stimulation with a combined CD4 and CD8 peptide pool. For neutralising antibodies measured in a nested case-control sample using a pseudotyped virus neutralisation assay, the corresponding odds ratio was 0·78 (0·62-1·00). Among participants with previous infection, the corresponding hazard ratio was 0·73 (0·61-0·88) for anti-nucleocapsid Ig. Addition of anti-spike IgG information to a model containing information on age and sex improved the C-index by 0·085 (0·027-0·143). In contrast to T-cell response, higher levels of binding and neutralising antibodies were associated with a reduced risk of breakthrough SARS-CoV-2 infection. The assessment of anti-spike IgG enhances the prediction of incident breakthrough SARS-CoV-2 infection and could therefore be a suitable correlate of protection in practice. Our phase 4 trial measured both humoral and cellular immunity and had a 6-month follow-up period; however, the longer-term protection against emerging variants of SARS-CoV-2 remains unclear. None.