Search Results (140)

Background: It is still challenging to develop effective vaccines against tumorigenic human gammaherpesviruses such as Epstein–Barr virus (EBV). A major obstacle is the lack of a small animal model that reproduces the natural infection course of human gammaherpesviruses to allow for proper assessment of vaccine efficacy. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of wild rodents and laboratory mice and therefore can be used as a surrogate for human gammaherpesviruses to evaluate vaccination strategies. Methods: In this study, two mRNA vaccine candidates were generated, one encoding a fusion protein of the MHV68 gH with the gL (gHgL-mRNA) and the other expressing the MHV68 gB protein (gB-mRNA). The immunogenicity and protective efficacy of the mRNA vaccine candidates were evaluated in a mouse model of MHV68 infection. Results: The gHgL-mRNA but not the gB-mRNA candidate vaccine was able to induce neutralizing antibodies in mice, whereas both vaccines could elicit antigen-specific T-cell responses. Following MHV68 intranasal inoculation, complete blocking of the establishment of viral latency was observed in some mice immunized with individual gHgL-mRNA or gB-mRNA vaccines. Notably, co-immunization with the two mRNA vaccines appeared to be more effective than individual vaccines, achieving sterile immunity in 50% of the vaccinated mice. Conclusions: This study demonstrates that immunization with mRNA platform-based subunit vaccines is indeed capable of preventing MHV68 latent infection, thus validating a safe and efficacious vaccination strategy that may be applicable to human gammaherpesviruses. Full article

Recent outbreaks of highly pathogenic human RNA viruses from probable zoonotic origin have highlighted the relevance of epidemic preparedness as a society. However, research in vaccinology and virology, as well as epidemiologic surveillance, is often constrained by the biological risk that live virus experimentation entails. These also involve expensive costs, time-consuming procedures, and advanced personnel expertise, hampering market access for many drugs. Most of these drawbacks can be circumvented with the use of pseudotyped viruses, which are surrogate, non-pathogenic recombinant viral particles bearing the surface envelope protein of a virus of interest. Pseudotyped viruses significantly expand the research potential in virology, enabling the study of non-culturable or highly infectious pathogens in a safer environment. Most are derived from lentiviral vectors, which confer a series of advantages due to their superior efficiency. During the past decade, many studies employing pseudotyped viruses have evaluated the efficacy of vaccines or monoclonal antibodies for relevant pathogens such as HIV-1, Ebolavirus, Influenza virus, or SARS-CoV-2. In this review, we aim to provide an overview of the applications of pseudotyped viruses when evaluating the neutralization capacity of exposed individuals, or candidate vaccines and antivirals in both preclinical models and clinical trials, to further help develop effective countermeasures against emerging neutralization-escape phenotypes. Full article

Community-based serosurveillance for emerging zoonotic viruses can provide a powerful and cost-effective measurement of cryptic spillovers. Betacoronaviruses, including SARS-CoV, SARS-CoV-2 and MERS-CoV, are known to infect bats and can cause severe respiratory illness in humans, yet remain under-surveyed in high-risk populations. This study aimed to determine the seroprevalence of betacoronaviruses in an occupational cohort in contact with bats before and after the emergence of SARS-CoV-2. Serum samples from pre- and post-COVID-19 pandemic were screened using antigen-based multiplex microsphere immunoassays (MMIAs) and a multiplex surrogate virus neutralization test (sVNT). Pre-pandemic samples showed no SARS-CoV-2 antibodies, while post-pandemic samples from vaccinated participants displayed binding and neutralizing antibodies against SARS-CoV-2 and a related bat CoV. Furthermore, one participant (1/237, 0.43%) had persistent antibodies against MERS-CoV in 2017, 2018 and 2021 but was seronegative in 2023, despite reporting no history of traveling abroad or severe pneumonia. The observed sustained antibody levels indicate a possible exposure to MERS-CoV or a MERS-CoV-like virus, although the etiology and clinical relevance of this finding remains unclear. Ongoing surveillance in high-risk populations remains crucial for understanding virus epidemiology and mitigating zoonotic transmission risk. Full article

Background/Objectives: Patients suffering from inflammatory bowel diseases (IBDs) undergoing treatment with anti-TNF antibodies mount a diminished humoral immune response to vaccination against SARS-CoV-2 compared to healthy controls. The characterization of variant-specific immune responses is particularly warranted among immunosuppressed patients, where reduced responses may necessitate further medical interventions. Methods: This pilot study investigated the humoral immune response of vaccinated IBD patients on anti-TNF medication and a comparable group of healthy individuals against the viral variants Alpha, Beta, Gamma, Delta, and Omicron BA.1 and BA.5. While total IgG antibodies targeting the receptor binding site of the spike protein of SARS-CoV-2 were quantified using a chemiluminescence microparticle immunoassay (CMIA), their potential neutralizing capacity was determined using commercial and variant-specific in-house surrogate virus neutralization tests (sVNTs) against a variant-specific in-house VSV-pseudotyped virus neutralization test (pVNT) as the gold standard. Results: Employing variant-specific assays recapitulated the immune escape functions of virus variants. Conspicuously, antibody reactivity against Alpha and Omicron BA.1 and BA.5 was strikingly poor in IBD patient sera post-initial vaccination compared to healthy individuals. A comparison of the diagnostic performance of assays with the pVNT revealed that identification of patients with inadequate humoral responses by CMIA and sVNT may require adjustments to cut-off values and end-point titration of sera. Following adaptation of cut-off values, patient sera exhibited reduced reactivity against all tested variants. The assay panel used substantiated the impact of anti-TNF therapy in IBD patients as to reduced strength, function, and breadth of the immune response to several SARS-CoV-2 variants. The immune response measured following the second vaccination was comparable to the antibody response observed in healthy individuals following the first vaccination. Conclusion: Variant-specific sVNTs and pVNTs have the potential to serve as valuable tools for evaluating the efficacy of adapted vaccines and to inform clinical interventions in the care of immunosuppressed patients. Anti-TNF-treated individuals with antibody levels below the optimized CMIA threshold should be considered for early booster vaccination and/or close immunological monitoring. Full article

Hantaviruses, including the Sin Nombre virus (SNV) and Andes virus (ANDV), are associated with severe global health risks, causing high mortality rates in hantavirus pulmonary syndrome (HPS) patients. Neutralizing antibodies are essential for virus clearance and survival, making neutralization assays critical for understanding immunity and evaluating therapeutic strategies. In this study, we developed a recombinant vesicular stomatitis virus (VSV)-based surrogate system expressing SNV and ANDV glycoproteins (GPCs), enabling neutralization studies under biosafety level 2 conditions. The neutralization titers obtained with the VSV-based system closely matched the findings from authentic hantavirus assays performed under biosafety level 3 conditions, confirming its potential as a useful tool for determining immune responses and advancing hantavirus research. Full article

Background: The 2019 coronavirus disease (COVID-19) pandemic had a severe impact on frail, end-stage kidney disease (ESKD) patients, either on dialysis or transplanted, with a high mortality rate in the early waves. Vaccination against SARS-CoV-2 with mRNA vaccines has led to reduced hospitalization and mortality rates in the general population and ESKD patients. Neutralizing antibodies (NAbs) are a valuable correlate of protection after vaccination, and IgG anti-spike antibodies are considered a surrogate marker of protection. Methods: This study investigated the correlates of protection brought by NAb and anti-spike IgG antibodies against SARS-CoV-2 wild-type Wuhan strain and variants of concern in a cohort of 128 French patients on dialysis after vaccination with the BNT162b2 mRNA vaccine. The correlate was assessed using Receiver Operating Characteristic curves. Results: The level of protection for IgG anti-spike antibodies was set at 917 BAU/mL for the original Wuhan strain and 980 BAU/mL and 1450 BAU/mL, respectively, for the Delta and Omicron BA.1 variants. Conclusions: The level of protection can be regularly monitored by measuring IgG anti-spike antibody concentrations to allow tailored boosters of SARS-CoV-2 vaccination in this frail and immunocompromised ESKD population. Full article

Respiratory syncytial virus (RSV) remains a significant global health threat, especially to infants, the elderly, and immunocompromised individuals. This review comprehensively explores the progress in RSV vaccine development, the immune evaluation methods, and immunological surrogate. The RSV fusion (F) protein, a primary target for vaccine development, has been engineered in prefusion conformation to elicit potent neutralizing antibodies, while the attachment (G) glycoprotein and other immunogens are also being explored to broaden immune responses. Advances in diverse vaccine platforms, ranging from live attenuated and protein subunit vaccines to cutting-edge mRNA- and nanoparticle-based formulations, highlight the field’s progress, yet challenges in balancing safety, immunogenicity, and durability persist. Central to these efforts is the identification and validation of immunological surrogates, which may serve as critical benchmarks for vaccine efficacy. Neutralizing antibody titers, multifunctional T cell responses, and B cell memory have emerged as key correlates of protection. However, the feasibility of these surrogates depends on their ability to predict clinical outcomes across diverse populations and settings. While neutralizing antibodies block the virus directly, T cell responses are essential for clearing infected cells and preventing severe disease, and B cell memory ensures long-term immunity. Integrating these immunological markers into a cohesive framework requires standardized assays, robust clinical validation, and an in-depth understanding of RSV-induced immune response. Full article

The pathogenesis of COVID-19 highlights a complex relationship between disease severity and neutralizing antibodies (NAbs). We aimed to investigate the relationships among the total NAb (tNAb) levels, the presence of potential neutralization antibodies (pNAbs), and the clinical outcomes of COVID-19 patients. Patients aged ≥18 years diagnosed between October 2021 and September 2022 were grouped by symptom severity. Blood samples were taken at two time points and data on demographics, epidemiology, and vaccination were recorded. The tNAbs and pNAbs were measured by an enhanced chemiluminescence assay and a surrogate virus neutralization test, respectively. The tNAbs of 68 and the pNAbs of 52 patients were analyzed. Twenty-six (38.2%) patients had severe infection. The 28-day mortality rate was 16.2% (n = 11). The tNAb levels in the control blood samples (CBSs) were significantly higher than those of the admission blood samples (ABSs) (p < 0.001). The statistical analysis showed no relationship between disease severity and pNAbs. Vaccinated patients had significantly higher tNAbs in the ABSs and CBSs (p < 0.001 and p < 0.001, respectively). The presence of pNAbs in the ABSs was correlated with a lower 28-day mortality (p = 0.026) and a milder disease course (p = 0.041). Although these findings support a correlation between tNAbs and disease severity and mortality, their presence seems to be unrelated to favorable clinical outcomes. Full article

Neutralization assays have become an important tool since the beginning of the COVID-19 pandemic for testing vaccine responses and therapeutic antibodies as well as for monitoring humoral immunity to SARS-CoV-2 in epidemiological studies. The spike glycoprotein (S) present on the viral surface contains a receptor binding domain (RBD) that recognizes the angiotensin-converting enzyme 2 receptor (ACE2) in host cells, allowing virus entry. The gold standard for determining SARS-CoV-2 neutralizing antibodies is the plaque reduction neutralization test (PRNT), which relies on live-virus replication performed exclusively in biosafety level 3 (BSL-3) laboratories. Here, we report the development of a surrogate live-cell imaging-based fluorescent SARS-CoV-2 neutralization assay, applicable to BSL-1 or BSL-2 laboratories, by antibody-mediated blockage of the interaction between recombinant RBD with overexpressed ACE2 receptor in a genetically modified HEK 293T stable cell line. Our approach was able to detect neutralizing antibodies both in COVID-19-positive human serum samples and polyclonal equine formulations against SARS-CoV-2. This new cell-based surrogate neutralization assay represents a virus-free fluorescence imaging alternative to the reported approaches, which can be used to detect antibody-neutralizing capabilities toward SARS-CoV-2. This assay could also be extrapolated in the future to other established and emergent viral agents. Full article

Background: This study investigated the humoral responses to SARS-CoV-2 in hemodialysis (HD) patients. The clearance of molecules in the blood during hemodialysis is influenced by factors such as filter pore size, flow rate, operating pressure, and treatment duration. Chronic kidney disease patients often show low antibody titers for pathogens like pneumococcus, influenza virus, and hepatitis B virus. Methods: In this study, the surrogate virus neutralization test (sVNT) for the wild type (WT) and Omicron variants, as well as spike-specific IgG levels, were measured at two time points (May 2022 and December 2023). Medical records and questionnaires were used to gather participant information. Results: A total of 26 HD patients were enrolled, including 3 on immunosuppressive therapies. A total of 8 patients had COVID-19 during the first sampling, and 19 during the second. The results showed that sVNT levels for WT decreased over time, though positivity remained at 100% during both sampling periods. In contrast, sVNT levels for Omicron increased significantly, with positivity rising from 46.2% to 75.0% (p < 0.05). Spike-specific IgG levels also increased, with positivity improving from 96.2% to 100%. Patients on immunosuppressive therapies had significantly lower sVNT levels for both WT and Omicron in the second period (p < 0.05), though no significant differences were observed during the first period. Conclusion: HD patients, particularly those on immunosuppressive therapies, showed reduced and declining neutralizing responses over time. A meta-analysis of HD patients seems necessary to determine whether all dialysis patients need COVID-19 booster vaccinations, similar to the hepatitis B vaccine, highlighting the need for targeted vaccination strategies. Full article

SARS-CoV-2 (Betacoronavirus pandemicum) is responsible for the disease identified by the World Health Organization (WHO) as COVID-19. We designed “CHIVAX 2.1”, a multi-epitope vaccine, containing ten immunogenic peptides with conserved B-cell and T-cell epitopes in the receceptor binding domain (RBD) sequences of different SARS-CoV-2 variants of concern (VoCs). We evaluated the immune response of mice immunized with 20 or 60 µg of the chimeric protein with two different alum adjuvants (Alhydrogel^® and Adju-Phos^®), plus PHAD^®, in a two-immunization regimen (0 and 21 days). Serum samples were collected on days 0, 21, 31, and 72 post first immunization, with antibody titers determined by indirect ELISA, while lymphoproliferation assays and cytokine production were evaluated by flow cytometry. The presence of neutralizing antibodies was assessed by surrogate neutralization assays. Higher titers of total IgG, IgG₁, and IgG_2a antibodies, as well as increased proliferation rates of specific CD4⁺ and CD8⁺ T cells, were observed in mice immunized with 60 μg of protein plus Adju-Phos^®/PHAD^®. This formulation also generated the highest levels of TNF-α and IFN-γ, in addition to the presence of neutralizing antibodies against Delta and Omicron VoC. These findings indicate the potential of this chimeric multi-epitope vaccine with combined adjuvants as a promising platform against viral infections, eliciting a T_H1 or T_H1:T_H2 balanced cell response. Full article

Background: Cellular and humoral immunity are key to the immune response against SARS-CoV-2, but the comparability and correlation across different assays remain underexplored. This study compares three T-cell and three antibody assays in two vaccine groups. Methods: This prospective longitudinal cohort study involved 46 naïve healthcare workers: a total of 11 in the homologous mRNA-1273 group (three doses) and 35 in the heterologous ChAd group (two ChAd doses followed by a BNT booster). Blood samples were collected at five time points. Cellular immunity was assessed using ELISPOT and two commercial interferon-gamma release assays: (IGRA)-QuantiFERON SARS-CoV-2 (QF) and Covi-FERON ELISA (CoVF). Humoral immunity was evaluated using total and IgG antibody assays and a surrogate virus neutralization test. Results: The mRNA-1273 group exhibited stronger and more consistent responses than the ChAd group. The correlations between ELISPOT and IGRA varied from weak to moderate (ρ = 0.300–0.410), while QF-IGRA and CoVF-IGRA showed stronger correlations (ρ = 0.700–0.737). The ELISPOT assay showed substantial agreement with QF [Ag2]-IGRA (k = 0.697–0.774) and CoVF [O-sp]-IGRA (k = 0.641–0.718), and an 80.4% agreement rate (k = 0.608) was found between the QF [Ag2]- and CoVF [O-sp]-IGRA tests. Three antibody assays demonstrated very strong correlations with each other and substantial to near-perfect agreement with ELISPOT (k = 0.866–0.949), QF [Ag2]-IGRA (k = 0.807–0.831), and CoVF [O-sp]-IGRA (k = 0.753–0.777). Conclusions: SARS-CoV-2-specific cellular and antibody responses vary by platform and vaccine type, highlighting the importance of measuring both T-cell and B-cell responses using multiple assays to comprehensively assess immune status. Full article

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 cPass^TMTM 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. Full article

Neutralizing antibodies plays a primary role in protective immunity by preventing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from entering the cells. Therefore, characterization of antiviral immunity is important for protection against SARS-CoV-2. In this study, the neutralizing effect of the anti-SARS-CoV-2 S1 protein IgG, which was detected using the chemiluminescence microparticle immunoassay (CMIA)-based SARS-CoV-2 IgG II Quant (Abbott, Waukegan, IL, USA) test in SARS-CoV-2 infected and/or vaccinated individuals, was investigated with a surrogate virus neutralization test (sVNT). In total, 120 Seropositive individuals were included in this study. They were divided into two groups: Vaccinated (n = 60) and Vaccinated + Previously Infected (n = 60). A commercial sVNT, the ACE2–RBD Neutralization Test (Dia.Pro, Milan, Italy), was used to assess the neutralizing effect. The assay is performed in two steps: screening and titration. The screening showed positive results in all seropositive samples. Low titration in 1.7%, medium titration in 5%, and high titration in 93.3% of the Vaccinated group, and medium titration in 1.7% and high titration in 98.3% of the other group, as obtained from the ACE2-RBD titration test. A strong positive and significant correlation was found between the SARS-CoV-2 IgG II Quant test and the ACE2-RBD titration test at the 1/32 titration level for both groups (p < 0.001 for both). This study shows that the SARS-CoV-2 IgG detected using the CMIA method after SARS-CoV-2 infection and/or vaccination has a high neutralizing titration by using the sVNT. In line with these data, knowledge that seropositivity determined by CMIA also indicates a strong neutralizing effect contributes to countrywide planning for protecting the population. Full article

Amid the SARS-CoV-2 pandemic, concerns surfaced regarding the spread of the virus to wildlife. Switzerland lacked data concerning the exposure of free-ranging animals to SARS-CoV-2 during this period. This study aimed to investigate the potential exposure of Swiss free-ranging wildlife to SARS-CoV-2. From 2020 to 2023, opportunistically collected samples from 712 shot or found dead wild mustelids (64 European stone and pine martens, 13 European badgers, 10 European polecats), canids (449 red foxes, 41 gray wolves, one golden jackal) and felids (56 Eurasian lynx, 18 European wildcats), as well as from 45 captured animals (39 Eurasian lynx, 6 European wildcats) were tested. A multi-step serological approach detecting antibodies to the spike protein receptor binding domain (RBD) and N-terminal S1 subunit followed by surrogate virus neutralization (sVNT) and pseudotype-based virus neutralization assays against different SARS-CoV-2 variants was performed. Additionally, viral RNA loads were quantified in lung tissues and in oronasal, oropharyngeal, and rectal swabs by reverse transcription polymerase chain reactions (RT-qPCRs). Serologically, SARS-CoV-2 exposure was confirmed in 14 free-ranging Swiss red foxes (prevalence 3.1%, 95% CI: 1.9–5.2%), two Eurasian lynx (2.2%, 95% CI: 0.6–7.7%), and one European wildcat (4.2%, 95% CI: 0.2–20.2%). Two positive foxes exhibited neutralization activity against the BA.2 and BA.1 Omicron variants. No active infection (viral RNA) was detected in any animal tested. This is the first report of SARS-CoV-2 antibodies in free-ranging red foxes, Eurasian lynx, and European wildcats worldwide. It confirms the spread of SARS-CoV-2 to free-ranging wildlife in Switzerland but does not provide evidence of reservoir formation. Our results underscore the susceptibility of wildlife populations to SARS-CoV-2 and the importance of understanding diseases in a One Health Concept. Full article