Virus Neutralization Test Research Articles

Orthoflavivirus circulation in South-East Queensland, Australia, before and during the 2021–2022 incursion of Japanese encephalitis virus assessed through sero-epidemiological survey of a sentinel equine population

An incursion and outbreak of Japanese encephalitis virus (JEV) was reported in Australia in 2021 and 2022, respectively. There was speculation that JEV may have been circulating in Australia unknowingly prior to the detection. In this study, we determined sero-prevalence and transmission of West Nile virus (WNV), Murray Valley encephalitis virus (MVEV) and JEV, prior to and post JEV incursion in a sentinel equine population in south-east Queensland (SEQ), Australia, using blocking ELISAs (screening test) and virus neutralisation test (confirmatory). Serum samples collected between 2018 and 2020 (prior to JEV incursion; n = 607) from horses residing in SEQ revealed that sero-prevalence to pathogenic orthoflaviviruses was low, specifically WNV (1.3 %; 8/607), MVEV (1.2 %; 7/607), and JEV (4.9 %; 30/607). The significantly higher prevalence of JEV (P < 0.05) was skewed by the high proportion of horses previously enrolled in one or more JEV vaccine studies (17/30; 56.7 %) and the unknown JEV vaccination history due to international travel (6/30; 20 %). Thirty-two foals were enrolled as sentinels to monitor for arbovirus transmissions in SEQ between 2020 and 2023. Results showed that JEV seroconversion was first detected in April 2022 (n = 4), with seven more seroconversions detected in the following months until November 2022. This study (i) confirms that it is highly unlikely that JEV incursion in SEQ occurred prior to February 2022; (ii) circulation of WNV in SEQ remains very low; and (iii) highlights the complexity in the interpretation of orthoflavivirus serological results. The authors propose that horses should be included as sentinels for arbovirus transmission monitoring in Australia.

Impact of Different Foot and Mouth Disease Vaccine Schemes in Cross-Neutralization Against Heterologous Serotype O Strains in Cattle

The high antigenic variability of the foot-and-mouth disease virus (FMDV) represents a challenge for developing prophylactic strategies, stressing the need for research into vaccines offering broad protection against a range of virus strains. Here, the heterotypic cross-reaction using different vaccine schemes against serotype O strains was studied, evaluating the impact of revaccination, antigen dose, and incorporation of additional FMDV serotypes. Naïve cattle were immunized with seven distinct FMDV vaccines, receiving three doses of the same formulation at 0, 28, and 56 days post-primary vaccination (dpv). Serum samples were collected up to 70 dpv and tested by a virus-neutralizing test against serotype O strains from a South American lineage and two strains representative of two Asian lineages. Our results showed that vaccines containing the ME-SA topotype O1/Campos strain developed cross-neutralizing responses against the two Asian viruses after the first vaccination. In contrast, significant heterotypic neutralizing antibody titers against the homologous topotype strain were only found after the second vaccination, indicating that the phylogenic relationship may differ from the antigenic profiles for these two viruses. The amount of the O1/Campos strain and the revaccination were essential factors for neutralization against the homologous- and heterologous-type O FMDV viruses. The strain composition of the vaccine was only relevant for cross-neutralization against one of the Asian strains, suggesting potential intra-serotypic divergences for this pattern.

Cattle, sheep, and goat humoral immune responses against SARS-CoV-2

Following the emergence of SARS-CoV-2 in late 2019, several species of domestic and wild animals have been found to be susceptible to SARS-CoV-2 infection through experimental inoculation and animal surveillance activities. Detection of SARS-CoV-2 specific antibodies in animals is an important surveillance tool since viral shedding in animals can only be detected for a short period of time. In this study, convenience serum samples were collected from 691 cattle, 698 sheep, and 707 goats from several regions in the United States, between 2019 to 2022. The samples were evaluated for the presence of SARS-CoV-2 specific antibodies using two commercial enzyme-linked immunosorbent assays (ELISA); one based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) and the other based on the nucleocapsid protein (N-ELISA) of SARS-CoV-2. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT) employing the Wuhan-like SARS-CoV-2 USA/WA1/2020 isolate. Our results indicate that ∼1% (6/691) of cattle, ∼2% (13/698) of sheep, and ∼2.5% (18/707) of goat serum samples were positive when using the sVNT, whereas ∼4% of cattle (25/691) and sheep (27/698), and 2.5% (18/707) of goat serum samples tested positive with the N-ELISA. None of the sVNT positive cattle, sheep, or goat serum samples had detectable neutralizing antibody activity (<1:8) against the SARS-CoV-2 USA/WA1/2020 isolate by the VNT. Our results indicate low seropositivity in cattle, sheep, and goats in the U.S., indicating the importance to continue monitoring for SARS-CoV-2 prevalence in animal species that are in close contact with humans.

Cilgavimab and tixagevimab as pre-exposure prophylaxis in vaccine non-responder kidney transplant recipients during a period of prevalent SARS-CoV-2 BA.2 and BA.4/5 variants—a prospective cohort study (RESCUE-TX)

The response to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination is severely impaired in patients on maintenance immunosuppression after kidney transplantation. We conducted a prospective cohort study of 194 kidney transplant recipients (KTR) who exhibited no response to SARS-CoV-2 vaccinations (i.e., SARS-CoV-2 spike protein antibodies ≤264 U/mL) and had no prior documented infection. Patients received 300mg of cilgavimab/tixagevimab as SARS-CoV-2 pre-exposure prophylaxis (PrEP) between March 4, 2022, and May 3, 2022 and were contrasted to a matched cohort of 186 KTRs also without immunization again defined as SARS-CoV-2 spike protein antibodies ≤264 U/mL and no documented prior infection. The primary outcome was the serum kinetics of cilgavimab/tixagevimab, the secondary endpoints were time to SARS-CoV-2 breakthrough infection, severity of disease and variant specific live viral invitro neutralization tests of patient sera. Longitudinal serum level monitoring showed a half-life of 91 days for both antibodies (95% CI 86-95 days for cilgavimab and 85-96 days for tixagevimab) in KTRs. Invitro neutralization tests showed effectiveness against the BA.2 omicron subvariant but not BA.5. The cumulative incidence of SARS-CoV-2 infections until May 15, 2022, (BA.2 dominance) was 15/194 vs 36/186 in the PrEP and control group respectively (OR=0.35, 95% CI 0.18-0.66) but was not different thereafter (BA.4/5 dominance). The number of severe infections during the BA.2 period was lower in the prophylaxis than in the control group (OR=0.37, 95% CI 0.17-0.79). This study showed that SARS-CoV-2 PrEP with cilgavimab/tixagevimab demonstrated clinical effectiveness against variants that are neutralised (BA.2) but not against BA.4/5. This study was funded by the Medical University of Vienna and an unrestricted grant from AstraZeneca (ESR-21-21585).

Improvement of RBD-FC Immunogenicity by Using Alum-Sodium Alginate Adjuvant Against SARS-COV-2.

Adjuvants use several mechanisms to boost immunogenicity and to modulate immune response. The strength of adsorption of antigen by adjuvants can be a determinant factor for significant improvement of immunopotentiation. We expressed recombinant RBD-FC in PichiaPink Strain 4 and examined the vaccination of mice by vaccine formulation with different adjuvants (sodium alginate and aluminum hydroxide, alone and together). Sodium alginate significantly increased the immunogenicity and stability of RBD-FC antigen, so RBD-FC formulated with combined alginate and alum (AlSa) and sodium alginate alone showed higher antibody titer and stability. Immunogenicity of RBD-FC:AlSa was determined by serological assays including direct enzyme-linked immunosorbent assay (ELISA) and surrogate virus neutralization test (sVNT). High levels of IgGs and neutralizing antibodies were measured in serum of mice immunized with the RBD-FC:AlSa formulation. On the other hand, cytokines IL-10 and INF-γ were severely accumulated in response to RBD-FC:AlSa, and after 10 days, their accumulation was significantly declined, whereas IL-4 showed the highest and the lowest accumulation in response to alum and alginate, respectively. Our data may suggest that combination of alum and sodium alginate has a better compatibility with RBD-FC in vaccine formulation.

Humoral Immunity Profiling to Pandemic and Bat‐Derived Coronavirus Variants: A Geographical Comparison

AbstractDynamic pathogen exposure may impact the immunological response to SARS‐CoV‐2 (SCV2). One potential explanation for the lack of severe SCV2‐related morbidity and mortality in Southeast Asia is prior exposure to related betacoronaviruses. Recent discoveries of SCV2‐related betacoronaviruses from horseshoe bats (Rhinolophus sinicus) in Thailand, Laos, and Cambodia suggest the potential for bat‐to‐human spillover exposures in the region. In this work, serum antibodies to protein constructs from SCV2 and a representative bat coronavirus isolated in Cambodia (RshSTT182) are measured in pre‐pandemic Cambodian human sera using ELISA assays. Of 293 Cambodian samples tested (N = 131 with acute malaria, n = 162 with acute undifferentiated febrile illness), 32 (10.9%) are seropositive for SCV2 based on established Spike and receptor‐binding domain (RBD) cutoffs. Within SCV2 seropositive samples, 16 (50%) have higher antibody levels to antigens from the representative virus RshSTT182 versus SCV2 antigens; competitive binding ELISA assays demonstrate inhibition of reactivity to SCV2 Spike after pre‐incubation with RshSTT182 Spike. Surrogate virus neutralization tests demonstrate that 8/30 (26.7%) SCV2 ELISA positive pre‐pandemic Cambodian samples have neutralizing activity against SCV2, while 14/30 (46.7%) have activity against other SCV2‐related betacoronaviruses. These data suggest that exposure to related betacoronaviruses may elicit cross‐reactive immunity to SCV2 prior to the global pandemic.

Development and optimization of a double antigen sandwich ELISA detecting for Senecavirus A antibodies based on VP2 protein.

Senecavirus A (SVA) is an emerging viral pathogen that threatens the global swine industry significantly. The major clinical symptoms of SVA-infected animals are vesicular lesions, but various diseases can cause the same symptoms, which makes it difficult to distinguish SVA from other vesicular diseases clinically. The absence of an effective and safe vaccine necessitates the development of a simple, specific, and sensitive serological detection method for SVA antibodies. The VP2 protein of SVA, characterized by high immunogenicity and sequence conservatism, is an essential target for serological diagnosis. In this study, a double-antigen sandwich enzyme-linked immunosorbent assay [ELISA (DAgS-ELISA)] based on VP2 protein expressed by Escherichia coli was established for SVA antibody detection. With a cutoff value of 0.237, this assay demonstrated outstanding performance, showing high sensitivity and sharp specificity, which is manifested in the absence of cross-reaction with classical swine fever virus (CSFV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV), and foot-and-mouth disease virus (FMDV) serotype A and O. Additionally, the repeatability of the method is remarkable, as shown by the coefficients variation (CV) of both the intra- and inter-assay below 10%. By detecting 166 clinical sera, it was found that the kappa value of the DAgS-ELISA was 0.78 compared with that of the virus neutralization test (VNT), indicating a high level of consistency. In general, this method has high sensitivity, sharp specificity, remarkable repeatability, sound consistency, and low cost, making it a reliable and effective tool for detecting SVA antibodies.IMPORTANCESVA has rapidly become prevalent in many countries, and its outbreaks have threatened the global swine industry significantly. The major clinical symptoms of SVA-infected animals are vesicular lesions that are similar to other vesicular diseases, making it difficult to distinguish SVA. Currently, no commercial vaccines are available for SVA; therefore, effective diagnosis of SVA infection is vital for its prevention and control. In this study, VP2 protein of SVA was expressed by E. coli, and a double-antigen sandwich enzyme-linked immunosorbent assay [ELISA (DAgS-ELISA)] for SVA antibodies detection was successfully established based on the VP2 protein. The DAgS-ELISA has a high sensitivity, sharp specificity, remarkable repeatability, sound consistency, and low cost for detecting SVA antibodies. Therefore, the DAgS-ELISA established in this study may be a reliable and effective tool for detecting SVA antibodies and may be used to strengthen the monitoring and prevention of SVA epidemic in the long run.

Camelpox Virus in Western Kazakhstan: Assessment of the Role of Local Fauna as Reservoirs of Infection.

This article investigates the role of local fauna in Western Kazakhstan as potential reservoirs of the camelpox virus (CMLV). The study emphasizes analyzing possible sources and transmission pathways of the virus using polymerase chain reaction (PCR) and serological methods, including virus neutralization tests and enzyme-linked immunosorbent assays (ELISA). Samples were collected from both young and adult camels, as well as rodents, ticks and blood-sucking insects in the Mangystau and Atyrau regions. The PCR results revealed the absence of viral DNA in rodents, ticks and blood-sucking insects; also, the ELISA test did not detect specific antibodies in rodents. These findings suggest that these groups of fauna likely do not play a significant role in the maintenance and spread of CMLV. Consequently, the primary sources of transmission are likely other factors, potentially including the camels themselves. The study's results indicate the need to reassess current hypotheses regarding infection reservoirs and to explore alternative sources to enhance strategies for the control and prevention of the camelpox virus.

Characterisation of the antibody-mediated selective pressure driving intra-host evolution of SARS-CoV-2 in prolonged infection.

Neutralising antibodies against the SARS-CoV-2 spike (S) protein are major determinants of protective immunity, though insufficient antibody responses may cause the emergence of escape mutants. We studied the humoral immune response causing intra-host evolution in a B-cell depleted, haemato-oncologic patient experiencing clinically severe, prolonged SARS-CoV-2 infection with a virus of lineage B.1.177.81. Following bamlanivimab treatment at an early stage of infection, the patient developed a bamlanivimab-resistant mutation, S:S494P. After five weeks of apparent genetic stability, the emergence of additional substitutions and deletions within the N-terminal domain (NTD) and the receptor binding domain (RBD) of S was observed. Notably, the composition and frequency of escape mutations changed in a short period with an unprecedented dynamic. The triple mutant S:Delta141-4 E484K S494P became dominant until virus elimination. Routine serology revealed no evidence of an antibody response in the patient. A detailed analysis of the variant-specific immune response by pseudotyped virus neutralisation test, surrogate virus neutralisation test, and immunoglobulin-capture enzyme immunoassay showed that the onset of an IgM-dominated antibody response coincided with the appearance of escape mutations. The formation of neutralising antibodies against S:Delta141-4 E484K S494P correlated with virus elimination. One year later, the patient experienced clinically mild re-infection with Omicron BA.1.18, which was treated with sotrovimab and resulted in an increase in Omicron-reactive antibodies. In conclusion, the onset of an IgM-dominated endogenous immune response in an immunocompromised patient coincided with the appearance of additional mutations in the NTD and RBD of S in a bamlanivimab-resistant virus. Although virus elimination was ultimately achieved, this humoral immune response escaped detection by routine diagnosis and created a situation temporarily favouring the rapid emergence of various antibody escape mutants with known epidemiological relevance.

Analysis of the immunological response elicited by a polyvalent foot and mouth disease vaccine and its compatibility with a diva test in Jimma Town, Ethiopia

The research was conducted in Jimma town, Oromiya Regional State, from October 2022 to June 2023, with the aim of assessing the immune response of polyvalent FMD (Foot and Mouth Disease) vaccine. The study involved 34 cattle in a longitudinal study, divided into two groups: 29 vaccinated and 5 unvaccinated. The vaccinated cattle received an inactivated polyvalent FMD virus vaccine produced by the National Veterinary Institute. Blood samples were collected on days 0, 14, 21, 35, 80, and 125 after vaccination and tested using Virus Neutralization Test and 3ABC ELISA. The results showed a significant increase in neutralizing antibodies against structural proteins in all vaccinated cattle on day 14 after vaccination for all three serotypes. (A/ETH/21/2000, p = 0.015; O/ETH/38/2005, p = 0.017; SAT2/ETH/64/2009, p = 0.007). On day, fourteen of post-vaccination vaccinated group showed immune response equal or above 1.5 log10 in a proportion of 69%, 73% and 94% for serotype A/ETH/21/2000, O/ETH/38/2005 and SAT2/ETH/64/2009 respectively. The status of raised antibody titer on day 125 post-vaccination showed decreasing by 14%, 18% and 4% for serotype A/ETH/21/2000, O/ETH/38/2005 and SAT2/ETH/64/2009 respectively. The DIVA test, or 3ABC ELISA, used to differentiate infected from vaccinated animals, revealed the absence of immune response to the Non-structural protein in the vaccinated cattle group. Conversely, the unvaccinated group showed no recorded antibody titer to both structural and non-structural proteins. In summary, the commercially available FMD vaccine, comprising serotype A, O, and SAT2, triggers an immune response to the structural protein rather than the non-structural protein after the initial administration. This outcome implies that FMD vaccines from the National Veterinary Institute align with the DIVA test. Nevertheless, additional efforts may be necessary to bolster the strength and duration of the vaccine-induced immune response.

B-086 Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure

Abstract Background The immune response to SARS-CoV-2 becomes increasingly complex as individuals receive different combinations of vaccines and encounter breakthrough infections. Our study aims to describe the immunogenicity observed over a two-year period in healthy individuals who completed two-dose series and then experienced booster and/or Omicron infection. Methods In June 2023, we recruited 78 healthcare workers who had previously participated in clinical research initiated in March 2021 at a single medical center. At 1, 5, 11, and 25 months after second dose, we assessed SARS-CoV-2-specific humoral and cellular immune responses using commercially available immunoassays and surrogate virus neutralization tests, along with interferon gamma (IFN-γ)-based enzyme-linked immunosorbent spot (ELIspot) assay using SARS-CoV-2 spike peptide pools. Results As the humoral immune response was monitored longitudinally, robust antibody production persisted after the third antigen exposure, in contrast to the rapid decline observed between the second and third dose injections. All immune metrics tended to increase as the number of vaccine doses increased. We also found a trend toward increased humoral immune response in the homologous mRNA-based vaccine group and enhanced cellular immune response in the vector-based primary series with mRNA booster heterologous combination group. Interestingly, no significant difference was found between Omicron exposure through breakthrough infection and bivalent vaccination. Omicron exposure significantly increased the breadth of neutralizing activity, while it did not significantly alter the cross-reactivity in the cellular immune response. Conclusions We identified individuals with diminished neutralization, aiding decisions for future vaccination prioritization. In addition, our findings suggest the role of cellular immunity as an important correlate of long-term protection through cross-reactivity to emerging variants.

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.

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.

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.

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.

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.

Obtaining from the cell walls of Saccharomyces cerevisiae yeast and assessing their adjuvant properties in a subunit vaccine model

Background. Polysaccharides are known to possess adjuvant properties, they are biodegradable, safe, and are of low-labor production. In this regard, the development of polysaccharide-based adjuvants is an urgent task.The aim. To develop a method for obtaining mannans from the cell walls of Saccharomyces cerevisiae yeast and to study their adjuvant properties using subunit vaccine model.Materials and methods. The preparation of mannans was obtained from the Saccharomyces cerevisiae yeast by enzymatic and alkaline hydrolysis. Its adjuvant properties were assessed in BALB/c mice immunized with the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 (S) protein (Delta (B.1.617.2)). The titers of specific antibodies in the blood sera were determined by ELISA assays using the recombinant RBD (Wuhan-Hu-1 and Delta), and the recombinant (S) protein (Wuhan-Hu-1, Delta and Omicron) as antigens. The titers of virus-neutralizing antibodies were determined using virus-neutralization tests with the SARS-CoV-2 virus strains Wuhan – hCoV19/Australia/VIC01/2020 (Wuhan-Hu-1), Delta – hCoV-19/Russia/PSK-2804/2021 (Delta (B.1.617.2)), and Omicron 1 – hCoV-19/Russia/Moscow171619-031221/2021 (Omicron (B.1.1.529)).Results. The developed scheme allowed for obtaining up to 200 mg of mannans from 10 g of yeast cell debris. Double, with a two-week interval, immunization with RBD (50 μg) in combination with mannans (40 μg and 10 μg) induced the production of specific antibodies in titers from 1:2477330 to 1:188360. The titer of virus-neutralizing antibodies to the Delta – hCoV-19/Russia/PSK-2804/2021 was 1:485 (40 μg of mannans per mouse).Conclusions. We developed a scheme for obtaining a low-toxic preparation of mannans from the Saccharomyces cerevisiae yeast. The highest adjuvant activity was achieved when using mannans at the dose of 40 µg per mouse. Blood sera obtained from the immunized animals neutralized both homologous and heterologous SARS-CoV-2 strains.

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.

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.