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The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.

Background. The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in SARS-CoV-2 infected patients and vaccinees. Methods. Samples were collected from 42 SARS-CoV-2 infected patients during the first pandemic wave, 50 subjects who received 2 doses of mRNA vaccine before the Omicron wave, 44 subjects who received 3 doses of mRNA vaccine, and 35 subjects who received heterologous vaccination (2 doses of adenovirus-based vaccine plus mRNA vaccine) during the Omicron wave. Samples were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results. In patients, RBD-binding IgG titers against the wild-type virus increased with time, but remained low. High neutralizing titers against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions. Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.

Background Over the past few decades, World Health Organization (WHO) has made massive efforts to promote the development of a vaccine against Lassa virus (LASV), one of the top ten priority pathogens for research and development under the WHO R&D Blueprint for Emerging Infections. To date, several vaccines are at different stages of development. In this scenario, a validated and standardised assay to measure LASV neutralising antibodies is urgently needed for vaccine development and comparison. Methods The neutralisation assay remains the gold standard for determining antibody efficacy. Here we have proposed a safe and validated pseudotyped neutralisation assay for LASV, taking advantage of the development of the first WHO International Standard and Reference Panel for Anti-Lassa Fever (NIBSC code 21/332). Results and Conclusions The proposed results demonstrate that the pseudotyped luciferase neutralisation assay is a specific serological test for the measurement of LASV neutralising antibodies without cross-reacting with standard sera specific for heterologous viral infections. In addition, the assay is accurate, precise, and linear according to criteria and statistical analyses defined and accepted by international guidelines.

Emerging viral diseases pose a threat to the global population as intervention strategies are mainly limited to basic containment due to the lack of efficacious and approved vaccines and antiviral drugs. The former was the only available intervention when the current unprecedented Ebolavirus (EBOV) outbreak in West Africa began. Prior to this, the development of EBOV vaccines and anti-viral therapies required time and resources that were not available. Therefore, focus has turned to re-purposing of existing, licenced medicines that may limit the morbidity and mortality rates of EBOV and could be used immediately. Here we test three such medicines and measure their ability to inhibit pseudotype viruses (PVs) of two EBOV species, Marburg virus (MARV) and avian influenza H5 (FLU-H5). We confirm the ability of chloroquine (CQ) to inhibit viral entry in a pH specific manner. The commonly used proton pump inhibitors, Omeprazole and Esomeprazole were also able to inhibit entry of all PVs tested but at higher drug concentrations than may be achieved in vivo. We propose CQ as a priority candidate to consider for treatment of EBOV.

The SARS-CoV-2 Omicron variant has rapidly replaced the Delta variant of concern. This new variant harbors worrisome mutations on the spike protein, which are able to escape the immunity elicited by vaccination and/or natural infection. To evaluate the impact and susceptibility of different serum samples to the Omicron variant BA.1, samples from COVID-19 patients and vaccinated individuals were tested for their ability to bind and neutralize the original SARS-CoV-2 virus and the Omicron variant BA.1. COVID-19 patients show the most drastic reduction in Omicron-specific antibody response in comparison with the response to the wild-type virus. Antibodies elicited by a triple homologous/heterologous vaccination regimen or following natural SARS-CoV-2 infection combined with a two-dose vaccine course, result in highest neutralization capacity against the Omicron variant BA.1. Overall, these findings confirm that vaccination of COVID-19 survivors and booster dose to vaccinees with mRNA vaccines is the correct strategy to enhance the antibody cross-protection against Omicron variant BA.1. Antibodies elicited by a triple homologous or heterologous vaccination regimen or following natural SARS-CoV-2 infection combined with a two-dose vaccine course, result in highest neutralization capacity against the Omicron variant BA.1.

The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide.

The human population is constantly exposed to multiple influenza A subtypes due to zoonotic spillover and rapid viral evolution driven by intrinsic error-prone replication and immunological pressure. In this context, antibody responses directed against the HA protein are of importance since they have been shown to correlate with protective immunity. Serological techniques, detecting these responses, play a critical role for influenza surveillance, vaccine development, and assessment. As the recent human pandemics and avian influenza outbreaks have demonstrated, there is an urgent need to be better prepared to assess the contribution of the antibody response to protection against newly emerged viruses and to evaluate the extent of preexisting heterosubtypic immunity in populations. In this study, 68 serum samples collected from the Italian population between 1992 and 2007 were found to be positive for antibodies against H5N1 as determined by single radial hemolysis (SRH), but most were negative when evaluated using haemagglutination inhibition (HI) and microneutralisation (MN) assays. As a result of these discordant serological findings, the increased sensitivity of lentiviral pseudotypes was exploited in pseudotype-based neutralisation (pp-NT) assays and the results obtained provide further insight into the complex nature of humoral immunity against influenza A viruses.

The pseudotype particle neutralization test (pp-NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses against hemagglutinin (HA), including stalk-directed antibodies. However, a validation of this assay has yet to be performed, and this limits its use to primarily research laboratories. To identify possible serological standards to be used in optimization and validation of the pp-NT, we have evaluated the cross-reactivity of hyperimmune chicken reference antisera in this assay. Our findings show that the cross-reactivity detected by the pp-NT is only partly explained by phylogenetic relationships and protein homology between the HA subtypes analysed; further studies are necessary to understand the origin of the cross-reactivity detected, and reference standards with higher specificity should be evaluated or generated de novo for future use in pp-NT.

Shigella is a major cause of diarrheal disease in young children worldwide, particularly in low- and middle-income countries (LMICs). The emergence of antibiotic resistance and limited access to healthcare in LMICs underscores the urgent need for an effective Shigella vaccine. Several next-generation Shigella vaccines are under development, many of which target the O-antigen component of lipopolysaccharide (LPS) from multiple serotypes to achieve broad protective coverage. Reliable, multiplexed assays are needed to evaluate vaccine immunogenicity across Shigella serotypes and vaccine platforms. A Luminex-based multiplex immunoassay was developed incorporating purified LPS from five Shigella serotypes (S. sonnei, S. flexneri 1b, 2a, 3a, and 6) along with Shigella IpaB protein and common vaccine carrier proteins. Assay performance was evaluated using pooled post-vaccination serum samples with qualification parameters including linearity, accuracy, precision, and specificity. Dilutional linearity was confirmed in all antigens across a broad dynamic range (R2 ≥ 0.98), with slopes ranging from 0.91 to 1.16. Within-run and between-run accuracy and intra-assay and inter-assay precision met predefined criteria for all antigens, with variability primarily observed at high dilutions for S. flexneri 1b and 3a. The assigned lower and upper limits of quantitation (LLOQ–ULOQ) spanned up to two orders of magnitude per antigen. The multiplex assay demonstrated specificity for homologous antigens, with some cross-reactivity patterns reflecting known structural similarities among LPS serotypes. This multiplex immunoassay enabled accurate and efficient measurement of vaccine-induced antibody responses across multiple Shigella antigens and carrier proteins. It therefore offers a qualified scalable solution for evaluating multivalent Shigella vaccines in clinical trials, particularly in resource-limited settings.