Virus Neutralization Test Research Articles

West Nile virus seropositivity in Alanya, a coastal city in the Mediterranean region of Turkey.

West Nile virus (WNV)-related illness is a global health problem. Understanding the seropositivity rates and identifying the risk factors related to WNV in various animal species including humans is crucial for the implementation of effective prevention strategies. Assess the rate of seropositivity and the risk factors associated with WNV seropositivity. Descriptive, cross-sectional. Microbiology and virology departments in a veterinary college. In a sample of healthy human participants in Alanya, located close to regions where WNV activity has been detected, anti-WNV IgG antibody detection was performed using enzyme-linked immunosorbent assays. The positive results were confirmed by virus neutralization tests (VNTs). The sample was compared with a second group of age- and gender-matched healthy subjects selected from a previous cross-sectional study. Determination of the seropositivity and risk factors that were associated with WNV in healthy humans. 87 in current study; 356 in previous study. The first group of 87, which had a high risk of encountering vector mosquitoes, had a positivity rate of 8% (7/87), whereas positivity in the second group was 4.5% (16/356; P=.181). In the entire sample, the anti-WNV IgG antibody was positive in 23 out of 443 (5.2%) samples by the ELISA test. Among these 23 samples, ten were confirmed as positive using VNTs. Therefore, the WNV IgG seropositivity was 2.3% (10/442). Confirmed IgG seropositivity rates were higher among male (3.8%) than female participants (0.9%; P=.054) and among adults aged ≥45 years (4%) than those aged 18-44 years (0.8%; P=.048). This study highlights the presence of WNV infection in the research region. More comprehensive and multidisciplinary studies are required to increase our knowledge about this zoonotic infection including risk factors in line with the One Health approach. Small sample size.

Infectious bovine rhinotracheitis: Unveiling the hidden threat to livestock productivity and global trade.

An infectious disease called infectious bovine rhinotracheitis (IBR) can lead to a number of disorders affecting cattle's respiratory system. The disease is caused by bovine alphaherpesvirus type 1 (BoAHV-1). Based on antigenic and genetic characteristics, BoAHV-1 strains are divided into subtypes 1.1, 1.2a, 1.2b, and 1.3. IBR is currently widespread throughout the world, with the exception of a few nations that have achieved eradication. The most significant characteristic of this illness is that, after a clinical or subclinical infection, the virus typically establishes a latent condition that can later be reactivated in the presence of stress, immunosuppressive conditions/substances, or other diseases. Primarily, the virus spreads by direct or indirect contact between animals. It may also be transmitted via the reproductive system, causing infectious balanoposthitis or vulvovaginitis. Most virus subtypes are associated with reproductive failure, such as fetal or embryonic resorption and abortions. The virus may also be transmitted through semen, which could lead to genital transfer. Bovine herpesvirus type 1 (BoHV-1) infection produces a variety of lesions. Lesion in the mucosal surface usually consists of white necrotic material. Regular methods for diagnosing BoHV-1 infections include isolation in cell culture, enzyme linked immunosorbent assay, virus neutralisation test, and methods based on identification of nucleic acids, like PCR. The interplay of several host, pathogen, environmental, and management factors affects the spread of IBR. Through its impacts on health and fitness, IBR can lead to production losses. In order to minimize the severity of clinical signs and stop the infection from spreading, the veterinarian may advise that sick or at-risk animals be placed under immediate isolation and vaccinated (such as intranasal vaccination, including the use of both killed and live attenuated virus vaccines) as soon as an IBR diagnosis is obtained.

Characterization of infectious laryngotracheitis virus isolated from commercial layer chickens in Bangladesh during the year 2021-2022.

Infectious laryngotracheitis virus (ILTV) is responsible for causing infectious laryngotracheitis (ILT), which is a rapidly spreading and extremely transmissible disease in chickens. The current research aims to isolate and characterize ILTV from layer chickens in Bangladesh. A total of 345 samples (trachea, larynx, and lungs) were collected from ILT-suspected dead and sick layer chickens of 32 ILT-suspected farms in three different outbreak districts (Gazipur, Tangail, and Mymensingh) of Bangladesh during the outbreak year 2021-2022. Rapid detection kits examined the samples for avian influenza virus (AIV) and Newcastle disease virus (NDV). ILTV-specific primers were used to screen 72 NDV- and AIV-negative samples by polymerase chain reaction (PCR). Using chorioallantoic membrane (CAM), the study isolated the ILT virus from 9 to 10-day-old seronegative embryonated chicken eggs (ECEs) using selected PCR-positive samples. The virus was confirmed using nucleotide sequencing, agar gel immunodiffusion test (AGIDT), viral neutralization test (VNT), and pathogenicity evaluations using mortality index for chicken embryos (MICEs) and intra-tracheal pathogenicity index (ITPI). The results indicated that among the PCR-positive 10 samples, only two (Alim_ILT_1001 and Alim_ILT_1,000) were found positive using ECEs. There were two field isolates of ILTVs, as shown by the amplicon size of the ICP4 gene-based PCR. A phylogenetic study of the ICP4 gene revealed that the recent isolates have a close similarity with the ILTV isolates of Turkey, Bangladesh, and Australia. AGIDT revealed strong precipitation lines due to ILTV-specific antibodies reacting with field viruses, while VNT neutralized both isolates with conventional ILTV antibodies. The pathogenicity testing indicated that Alim_ILT_1001 had MICE and ITPI values of 0.77 and 0.63, whereas Alim_ILT_1,000 had 0.71 and 0.57. Both the ILTV isolates have similarities with the isolates of Turkey, Bangladesh, and Australia, and they are highly virulent for chickens.

Resposta anamnéstica contra o vírus da diarreia viral bovina e herpesvírus bovino tipo 1 em novilhas Holandesas jovens vacinadas com quatro diferentes formulações comerciais

ABSTRACT: This study evaluated the vaccine-induced serological response after administering four commercial formulations containing Bovine Viral Diarrhea Virus (BVDV) type-1, BVDV-2, and Bovine Herpesvirus type 1 (BoHV-1) to young heifers with circulating maternal antibody titers. The study also determined the anamnestic response to vaccinations after the complete metabolization of maternal antibodies when the calves reached six months. Eighty-seven Holstein heifers were selected and randomly distributed into four experimental groups based on the vaccine administered. The four vaccine-based experimental groups were as follows: vaccine A (n = 22), vaccine B (n = 22), vaccine C (n = 24), and vaccine D (n = 19), given on the 60th, 90th, and 180th day of life, respectively. Virus-neutralization (VN) tests were performed at baseline (day 60) and 30 days after administering the second (day 120) and third doses (day 210). We analyzed the effect of vaccine group (P ≤ 0.001), day of vaccination (P ≤ 0.001), and group versus vaccine day interaction (P ≤ 0.001) for antibody titers produced against BVDV-1, BVDV-2, and BoHV-1 using the PROC MIXED method (Statistical Analysis System -SAS 9.4). Antibody titers against BVDV-1, BVDV-2, and BoHV-1 were similar at baseline and on day 60 of life. The mean antibody titers were constant and persisted against BVDV-1 in heifers immunized with vaccines A and C. Heifers immunized with vaccine A alone had a similar effect against BVDV-2. Regarding BoHV-1, the antibody titers decreased between days 60 and 210 in groups B, C, and D. The antibody titer for heifers in group A also decreased between days 60 and day 120, and an intense increase in titers was observed on day 210. After being immunized with formulations B, C, and D, the frequency of animals with titers above protective levels for BVDV-1, BVDV-2, and BoHV-1 was very low or null. Indicators of anamnestic response were observed in heifers vaccinated with formulation A only. Therefore, it can be concluded that passive immunity negatively interferes with antibody production after vaccination.

Anti-severe acute respiratory syndrome coronavirus 2 spike receptor-binding domain antibody levels in patients with systemic lupus erythematosus based on vaccination status and related factors in Indonesia.

We aim to analyze the proportion and level of coronavirus disease 2019 (COVID-19) seropositivity in patients with systemic lupus erythematosus (SLE) and explore factors associated with lower anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (S-RBD) antibody levels. A cross-sectional study involving patients with SLE was conducted. We included those aged 18-60 years, either unvaccinated or had received inactivated vaccine (CoronaVac; Sinovac Biotech Ltd., China). Furthermore, participants were tested for anti-SARS-CoV-2 S-RBD antibody levels and SARS-CoV-2 surrogate virus neutralization test, and comparative test analysis was employed. This study included 159 subjects of whom 92 and 67 were SLE subjects and controls, respectively. Significantly higher seropositive results were noted in patients with SLE receiving vaccine (96.9% versus 3.1%). Unvaccinated SLE patients receiving cyclophosphamide (CYC) had higher anti-RBD levels compared to unvaccinated SLE patients not receiving CYC (23.81 [interquartile range (IQR), 2.26-78.85] versus 2.13 [IQR, 0.1-12.5]), whereas vaccinated SLE patients receiving CYC had lower anti-RBD levels compared to vaccinated SLE patients not receiving CYC (15.5 [IQR, 6.62-35.09] and 69.77 [IQR, 17.48-201]). In the vaccinated SLE group, a lower value of anti-SARS-CoV-2 RBD antibody levels was observed in patients receiving mycophenolate mofetil and those with chronic kidney disease. No correlation was noted between disease activity and organ involvement with lower antibody response. The increase in COVID-19 antibody levels in patients with SLE may be affected by exposure to hospital settings and vaccine. Furthermore, CYC treatment is associated with lower antibody response after receiving vaccine.

A STUDY ON HOMOLOGY AND HETEROLOGY BETWEEN PANASIA-II & PAK-98 LINEAGES OF FOOT AND MOUTH DISEASE VIRUS SEROTYPE-O

To understand the dynamics of epidemics, it is crucial to evaluate prior exposure to infectious diseases as a possible defense against newly developing strains. The cross-protection prediction is unresolved matter that holds great importance, particularly in the field of Foot-and-Mouth Disease Virus (FMDV). The new methods for controlling FMD involve the implementation of vaccine, limited movement, containment and elimination of cattle that are either affected or vulnerable to the disease. Therefore, it is necessary to use a serological analysis strategy that applies association coefficients (r1-values) to identify effective vaccines. The primary objective of this study was aimed to explain the genetic homologous and heterologous association among two ancestries of FMDV serotype O found in Pakistan (Pk) and to find a suitable strain from field for selection of vaccinated seed that provide cross-protection against both ancestries. Virus neutralization tests (VNTs) were used to evaluate an antigenic association among the two groups of viruses, indicates a significant degree of antigenic similarity between stains within a particular cluster. Conversely, r1 values ranging from 0.07-0.2 were obtained from cross-combinations among clusters, suggesting antigenic variation amongst virus strains. These results were further supported by in vitro tests, which showed that heterologous combinations of PanAsia-II and PAK-98 isolates showed antigenic variation, except for 25-06-PK-19, SDG-43-FSD-PK, and TANWL-34PK, which showed a 0.3 (r1 value). In contrast, when considering homologous combination of strains from both lineages showed antigenic similarity. Furthermore, it was projected that these combinations would provide protective titers during in-vivo. As a conclusion, it is recommended that all strains, except 25-06-PK-19, SDG-43-FSD-PK, and TANWL-34PK considered as potential vaccine candidates for effectively managing outbreaks of FMDV serotype-O, due to their ability to confer broad-spectrum immunity.

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

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

Investigation of the immunomodulatory effect of inactive parapoxvirus (iPPVO) on infectious bovine rhinotracheitis (IBR) vaccine in cattle

In the study, alterations in antibody titers, proinflammatory and antiinflammatory cytokine levels were determined in serum samples collected at various periods before and after administration of inactive IBR and iPPVO to cattle. It was aimed to investigate the immunomodulatory effects of inactivated parapoxvirus ovis (iPPVO) in cattle vaccinated with inactivated-IBR vaccine. In the study, 40 unvaccinated, clinically healthy cattle of different breeds aged older then 3 months were used. Three groups of cattle were formed as control group 1 (n=10), control group 2 (n=10) and experimental group (n=20). iPPVO was applied to the cattle in the control group 1 and experimental groups on the 0, 2nd, and 4th days. Blood samples were colleted from all the animals after 6th hours of the injections applied on 0th and 4th days of the study. Commercially available ELISA kits were used to determine serum levels of of IL-2, IL-6, IL-12 and IFN-γ. Furthermore, virus neutralization test was also performed to detect virus neutralizing antibody titres. In the present study, serum levels of IL-2, IL-6 IL-12, and IFN-γ levels were found to be significantly higher in the experimental group compared to that of the control group 1 and control group 2 (p<0,05). The differences between control group 1 and control group 2 groups were not statistically significant. In conclusion, iPPVO increased the levels of cytokines in IBR vaccinated cattle due to its immunomodulatory effects. In addition, virüs neutralizing antibody titers were found to be significantly higher in cattle that received vaccine and iPPVO.

Evaluation of hemagglutination inhibition test for canine respiratory coronavirus antibodies and seroprevalence in Korean dogs

Canine respiratory coronavirus (CRCoV) is a significant pathogen that causes respiratory diseases in dogs, collectively known as a canine infectious respiratory disease. The virus is highly contagious and exhibits high seroprevalence worldwide. Currently, bovine coronavirus (BCoV) enzyme-linked immunosorbent assay (ELISA) kits are used to detect CRCoV antibodies. However, BCoV-ELISA kits cannot differentiate between infections caused by BCoV and those caused by CRCoV. In this study, we evaluated the hemagglutination inhibition (HI) test for CRCoV by comparing it with the virus neutralization (VN) test. Subsequently, we evaluated the seroprevalence of CRCoV in 383 dog serum samples collected from South Korea utilizing the HI test. The HI test for CRCoV showed a strong correlation with the VN test (R = 0.83, p < 0.001). The analysis of seroprevalence revealed that 52.2% (95% confidence interval [CI], 47.2%–57.1%) of the Korean dog serum samples were positive. The seroprevalence exhibited varied with age, with a positivity rate of 43.9% in dogs under 1 year of age and 66.7% in dogs aged 3 to 5 years (odds ratio [OR], 2.54; 95% CI, 1.43–4.59). In conclusion, the HI test to monitor CRCoV antibody proved to be closely related to the VN test. Furthermore, over half of the dogs in Korea tested positive for CRCoV antibodies. These findings contribute to a better understanding of the sero-epidemiology of CRCoV.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Performance of a Point-of-Care Fluorescence Immunoassay Test to Measure the Anti-Severe Acute Respiratory Syndrome Corona Virus 2 Spike, Receptor Binding Domain Antibody Level.

Quantitative determination of anti-SARS-CoV2-S-RBD is necessary for the evaluation of vaccination effectiveness. The surrogate viral neutralization test (SVNT) is approved for measuring anti-SARS-CoV2-S-RBD, but a point-of-care platform is needed to simplify anti-SARS-CoV-2-S-RBD measurement. We aimed to evaluate the performance of a rapid fluorescent immunoassay-based kit, FastBio-RBDTM, compared to the SVNT. During April-September 2021, we enrolled two groups of subjects, convalescent subjects and subjects without a COVID-19 history. The subjects were tested for the anti-SARS-CoV2-S-RBD antibody using FastBio-RBDTM and the GenScript-cPASSTM SVNT. We measured the correlation coefficient and conducted an ROC analysis to determine the best cut-off value of anti-SARS-CoV2-S-RBD against the SVNT percent inhibition levels of 30% and 60%. We included 109 subjects. Anti-SARS-CoV-2-S-RBD strongly correlated to SVNT % inhibition with an R value of 0.866 (p < 0.0001). The ROC analysis showed that the anti-SARS-CoV-2-S-RBD of 6.71 AU/mL had 95.7% sensitivity and 87.5% specificity to detect a percentage inhibition of 30%. The anti-SARS-CoV-2-S-RBD of 59.76 AU/mL had a sensitivity of 88.1% and specificity of 97.0% to detect a percentage inhibition of 60%. FastBio-RBDTM could determine the presence and level of anti-SARS-CoV-2-S-RBD with good sensitivity and specificity. It has the potential to be deployed in health facilities with limited resources.

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

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

Dimeric ACE2-FC is equivalent to monomeric ACE2 in the surrogate virus neutralization test

Angiotensin converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of the protective humoral immune response to a viral infection or vaccine using a surrogate virus neutralization test (sVNT). Soluble ACE2 is also being considered as an antiviral therapy option potentially insensitive to changes in the SARS-CoV-2 spike protein. For widespread sVHT testing, it is necessary to use ACE2 preparations or ACE2 conjugates with constant properties. Previously, we obtained a cell line that produces soluble monomeric ACE2 and showed that this variant of ACE2 can be used in sBHT, preferably in the form of a conjugate with horseradish peroxidase. To obtain a stable and universally applicable form of soluble ACE2, a cell line was obtained that produced the ACE2-Fc fusion protein with high productivity, more than 150 mg/l of the target protein during cultivation in a stirred flask. Affinity-purified ACE2-Fc is a mixture of dimeric and tetrameric forms, but allows one to obtain linearizable antibody inhibition curves for complexation with the receptor-binding domain of the SARS-CoV-2 spike protein. The ACE2-Fc-HRP based sVHT testing system can be used to practically measure the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

Superior antibody and membrane protein-specific T-cell responses to CoronaVac by intradermal versus intramuscular routes in adolescents

BackgroundOptimising the immunogenicity of COVID-19 vaccines to improve their protection against disease is necessary. Fractional dosing by intradermal (ID) administration has been shown to be equally immunogenic as intramuscular (IM) administration for several vaccines, but the immunogenicity of ID inactivated whole severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the full dose is unknown. This study (NCT04800133) investigated the superiority of antibody and T-cell responses of full-dose CoronaVac by ID over IM administration in adolescents.MethodsParticipants aged 11–17 years received two doses of IM or ID vaccine, followed by the 3rd dose 13–42 days later. Humoral and cellular immunogenicity outcomes were measured post-dose 2 (IM-CC versus ID-CC) and post-dose 3 (IM-CCC versus ID-CCC). Doses 2 and 3 were administered to 173 and 104 adolescents, respectively.ResultsSpike protein (S) immunoglobulin G (IgG), S-receptor-binding domain (RBD) IgG, S IgG Fcγ receptor IIIa (FcγRIIIa)-binding, SNM [sum of individual (S), nucleocapsid protein (N), and membrane protein (M) peptide pool]-specific interleukin-2 (IL-2)+CD4+, SNM-specific IL-2+CD8+, S-specific IL-2+CD8+, N-specific IL-2+CD4+, N-specific IL-2+CD8+ and M-specific IL-2+CD4+ responses fulfilled the superior and non-inferior criteria for ID-CC compared to IM-CC, whereas IgG avidity was inferior. For ID-CCC, S-RBD IgG, surrogate virus neutralisation test, 90% plaque reduction neutralisation titre (PRNT90), PRNT50, S IgG avidity, S IgG FcγRIIIa-binding, M-specific IL-2+CD4+, interferon-γ+CD8+ and IL-2+CD8+ responses were superior and non-inferior to IM-CCC. The estimated vaccine efficacies were 49%, 52%, 66% and 79% for IM-CC, ID-CC, IM-CCC and ID-CCC, respectively. The ID groups reported more local, mild adverse reactions.ConclusionThis is the first study to demonstrate superior antibody and M-specific T-cell responses by ID inactivated SARS-CoV-2 vaccination and serves as the basis for future research to improve the immunogenicity of inactivated vaccines.Graphical abstract

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

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

Surrogate Virus Neutralisation Test Based on Nanoluciferase-Tagged Antigens to Quantify Inhibitory Antibodies against SARS-CoV-2 and Characterise Omicron-Specific Reactivity in a Vaccination Cohort.

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.

Dual-Function Janus Nanozymes for Performance Evaluation and Application in a Surrogate Virus Neutralization Test with Vaccinated Samples.

The need exists for biosensing technologies capable of sensitively and accurately detecting various biomarkers. In response, the development of nanozymes is actively underway; they have advantages in stability, cost, performance, and functionalization over natural enzymes commonly used for signal amplification in sensing technologies. However, the performance of nanozymes is interdependent with factors such as shape, size, and surface functional moiety, making it challenging to perform quantitative performance comparisons based on the nanozyme material. In this study, we propose a physical synthetic approach to fabricate double-layered bimetallic nanozymes with identical shapes, sizes, and surfaces but different material compositions. These Janus nanozymes consist of a nanozymatic layer responsible for catalytic activity and a gold layer responsible for quantification and efficient surface modification. Based on their identical physicochemical properties, the synthesized double-layered bimetallic nanozymes allow, for the first time, a quantitative comparison of nanozymatic activities in terms of various kinetic parameters. We compared several candidates and found that the Ir-Au nanozyme exhibited the best performance. Subsequently, we applied this nanozyme to detect neutralizing antibodies against SARS-CoV-2 based on a surrogate virus neutralization test. The results demonstrated a limit of detection as low as 2 pg/mL and selectivity specifically toward MERS-CoV. The performance of this assay was further validated using vaccinated samples, demonstrating the potential of our approach as a cost-effective, rapid, and sensitive diagnostic tool for neutralizing antibody detection against viruses such as SARS-CoV-2.

The Immunogenicity of a Foot-and-Mouth Disease Virus Serotype O Vaccine in Commercial and Subsistence Cattle Herds in Zambia.

The recent introduction of foot-and-mouth disease (FMD) virus serotype O (O/EA-2 topotype) in Southern Africa has changed the epidemiology of the disease and vaccine requirements of the region. Commercial and subsistence cattle herds in Zambia were vaccinated with an FMD virus serotype O Manisa vaccine according to a double- or single-dose vaccination schedule. Heterologous antibody responses induced by this vaccine against a representative O/EA-2 virus from Zambia were determined. Virus neutralisation tests (VNTs) showed double-dosed cattle had a mean reciprocal log virus neutralisation titre of 2.02 (standard error [SE] = 0.16, n = 9) for commercial herds and 1.65 (SE = 0.17, n = 5) for subsistence herds 56 days after the first vaccination (dpv). Significantly lower mean titres were observed for single-dosed commercial herds (0.90, SE = 0.08, n = 9) and subsistence herds (1.15, SE = 0.18, n = 3) 56 dpv. A comparison of these results and those generated by solid-phase competitive ELISA (SPCE) tests showed a statistically significant positive correlation by Cohen's kappa coefficient. Therefore, SPCE might be used in assessing the immunogenicity of vaccines in place of VNT. Furthermore, for this vaccine and field strain, a vaccination regime employing a two-dose primary course and revaccination after 4-6 months is likely to be appropriate.

Clinicopathological characteristics of 3 probable pediatric cases with acute severe hepatitis of unknown aetiology

BackgroundAcute severe hepatitis with unknown aetiology in children (ASHep-UA) has become a global health alert. This article reported clinicopathological characteristics of 3 probable ASHep-UA cases. MethodsWe respectively collected serological data and liver biopsies of 3 suspected cases of ASHep-UA. Neutralizing antibodies titer for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants were determined by virus neutralization test (VNT). Histological assessment, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for cytomegalovirus (CMV), Epstein-Barr virus (EBV), human adenoviruses (HAdV), adeno-associated virus (AAV2), human herpes virus type 6 (HHV-6) were performed to identify possible aetiologies. ResultsRemarkable elevation of transaminase (median ALT level, 1100 IU/liter; median AST level, 500 IU/liter) were revealed with undetectable hepatitis A-E and non-hepatotropic virus in both sera and tissues. Weakness, jaundice, pale stools and splenomegaly were observed. Interestingly, two individuals had SARS-CoV-2 Omicron variants infection. Histologically, moderate or severe lobular necroinflammation, active interface hepatitis and portal inflammatory infiltrate with lymphocytic, plasma cells, neutrophils and eosinophilic cells were noted. ConclusionsThe exact aetiology of ASHep-UA was still unknown. By reporting the 3 probable cases, we expect to enrich the clinical experience in diagnosis and treatment of ASHep-UA as well as the pathological characteristics.