Search Results (965)

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

Nipah virus (NiV) is a highly pathogenic bat-borne zoonotic pathogen that poses a significant threat to human and animal health, with fatality rates exceeding 70% in some outbreaks. Despite its significant public health impact, there are currently no licensed vaccines or specific therapeutics available. Various virological tools—such as reverse genetics systems, replicon particles, VSV-based pseudoviruses, and recombinant Cedar virus chimeras—have been widely used to study the molecular mechanisms of NiV and to support vaccine development. Building upon these platforms, we developed a replication-competent recombinant vesicular stomatitis virus (rVSVΔG-eGFP-NiV_BD F/G) expressing NiV attachment (G) and fusion (F) glycoproteins. This recombinant virus serves as a valuable tool for investigating NiV entry mechanisms, cellular tropism, and immunogenicity. The virus was generated by replacing the VSV G protein with NiV F/G through reverse genetics, and protein incorporation was confirmed via immunofluorescence and electron microscopy. In vitro, the virus exhibited robust replication, characteristic cell tropism, and high viral titers in multiple cell lines. Neutralization assays showed that monoclonal antibodies HENV-26 and HENV-32 effectively neutralized the recombinant virus. Furthermore, immunization of golden hamsters with inactivated rVSVΔG-eGFP-NiV_BD F/G induced potent neutralizing antibody responses, demonstrating its robust immunogenicity. These findings highlight rVSVΔG-eGFP-NiV_BD F/G as an effective platform for NiV research and vaccine development. 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

Viral oncolysis is considered a promising cancer treatment method because of its good tolerability and durable anti-tumor effects. Compared with other oncolytic viruses, Newcastle disease virus (NDV) has some distinct advantages. As an RNA virus, NDV does not recombine with the host genome, making it safer compared with DNA viruses and retroviruses; NDV can induce syncytium formation, allowing the virus to spread among cells without exposure to host neutralizing antibodies; and its genome adheres to the hexamer genetic code rule (genome length as a multiple of six nucleotides), ensuring accurate replication, low recombination rates, and high genetic stability. Although wild-type NDV has a killing effect on various tumor cells, its oncolytic effect and working mechanism are diverse, increasing the complexity of generating engineered oncolytic viruses with NDV. This study aims to employ whole-genome CRISPR-Cas9 knockout screening and RNA sequencing to identify putative key regulatory factors involved in the interaction between NDV and human colon cancer HCT116 cells and map their global interaction networks. The results suggests that NDV infection disrupts cellular homeostasis, thereby exerting oncolytic effects by inhibiting cell metabolism and proliferation. Meanwhile, the antiviral immune response triggered by NDV infection, along with the activation of anti-apoptotic signaling pathways, may be responsible for the limited oncolytic efficacy of NDV against HCT116 cells. These findings not only enhance our understanding of the oncolytic mechanism of NDV against colonic carcinoma but also provide potential strategies and targets for the development of NDV-based engineered oncolytic viruses. Full article

Co-infections of Orthopoxviruses (OPVs), such as vaccinia virus (VACV) and monkeypox virus (MPXV), and the human immunodeficiency virus (HIV) can be associated with severe outcomes. Serro’s dairy region, located in Minas Gerais, southeastern Brazil, is an endemic area for VACV, where zoonotic outbreaks affect rural communities. This epidemiological context is especially relevant for at-risk populations, such as people living with HIV (PLHIV). This study aimed to assess the presence of neutralizing antibodies (NAbs) against OPV in PLHIV in this endemic setting. Serum samples were collected from 177 PLHIV in treatment at the specialized service between December 2021 and August 2022. VACV and MPXV NAbs were measured using the plaque reduction neutralization test (PRNT) and VACV-infected cells. The overall occurrence of OPV NAbs was 27.7%. NAbs were higher in individuals born before 1980 (53.3%) than those born after 1980 (1.1%). Among anti-VACV-seropositive individuals, 40.8% also had MPXV NAbs, suggesting cross-immunity. These findings indicate the circulation of VACV in PLHIV and highlight the increased susceptibility to OPV infections among individuals born after the cessation of smallpox vaccination. The results reinforce the importance of continued surveillance of OPV, especially in endemic regions and vulnerable populations. Full article

The 2016 Zika virus (ZIKV) epidemic has largely subsided, but a key question remains. How did ZIKV evolve to become a virulent human pathogen compared to the virus of its original discovery? What specific virologic and pathologic changes contributed to increased pathogenicity in humans? Phylogenetic studies have identified two genetically distinct ZIKV, the African and Asian lineages, which differ in their pathogenicity. Previous studies including ours suggest that the envelope (E) protein plays a key role in viral entry, immune activation, and neuropathogenesis. This study aimed to further elucidate virologic and pathogenic differences between these lineages by assessing their ability to bind and replicate in host cells, induce apoptotic cell death, trigger inflammatory responses, and influence human neural progenitor cell (hNPC)-derived neurosphere formation. We compared a historic African ZIKV strain (MR766) with an epidemic Brazilian strain (BR15) and evaluated the effects of the E protein inhibitor quercetin-3-β-O-D-glucoside (Q3G) and an E protein-neutralizing antibody (AbII). Our results revealed distinct virologic properties and that MR766 exhibited stronger inhibition of neurosphere formation due to enhanced viral binding to neuronal SH-SY5Y cells, while BR15 infection triggered a heightened pro-inflammatory cytokine response with reduced viral binding. Chimeric virus studies suggested that the E protein likely influences viral binding, replication efficiency, immune activation, and neuropathogenesis. Notably, Q3G exhibited antiviral activities against both MR766 and BR15, whereas AbII preferentially inhibited MR766. These findings highlight the virological differences between ancestral and epidemic viral strains, as well as the critical role of E protein in viral permissiveness, immune response, and neuropathogenesis, providing insights for developing targeted antiviral strategies. Full article

Adeno-associated virus (AAV) vectors face a critical translational challenge in ocular gene therapy due to pre-existing neutralizing antibodies (NAbs) whose seroprevalence limits patient eligibility. Standard NAb detection using non-ocular cell models (Human Embryonic Kidney 293T) may inadequately predict retinal transduction inhibition due to cell type-related variations in receptor usage and immunogenicity. This study established parallel NAb detection platforms utilizing human retinal pigment epithelial (ARPE-19) cells and standard 293T cells to systematically evaluate clinical serum samples against ophthalmologically relevant AAV serotypes (2, 5, 8, 9) via luciferase reporter-based transduction inhibition assays. Comparative analysis demonstrated ARPE-19 exhibited 42–48% higher NAb titers against AAV5/9 compared to 293T cells, with distinct serotype-biased neutralization hierarchies observed between cellular models. Furthermore, female-derived sera exhibited significantly elevated NAbs against particular serotypes in the ARPE-19 system. Critically, inter-serotype cross-neutralization correlation patterns differed substantially between cellular platforms. These findings demonstrate that physiologically relevant retinal cellular models provide essential immunological profiling data, revealing NAb characteristics obscured in standard assays. Consequently, employing retinal cell-based platforms is crucial for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes in ocular gene therapy. Full article

Although vaccine development has primarily focused on inducing neutralizing antibodies, increasing evidence supports an important role of CD8⁺ T cell responses in vaccine effectiveness. Routine assays, which are mainly based on antibody titers, may therefore not accurately reflect the full immune response elicited by vaccination. Assessing antigen-specific T cell responses upon vaccination poses several challenges. A common issue in studying T cells specific to a vaccine antigen is their low frequency in circulation, which can limit their ex vivo analysis. Moreover, the use of human cell-based models is crucial for studying and optimizing the induction of T cell responses to design effective vaccines. We developed an innovative in vitro approach of human CD8⁺ T cell priming, based on the rapid mobilization of dendritic cells (DCs) directly from unfractionated peripheral blood mononuclear cells (PBMCs). This simple and original method allows for side-by-side comparisons of multiple test parameters in a standardized system, providing both quantitative and qualitative readouts of primed antigen-specific CD8⁺ T cells. Here, we discuss the genesis of this approach and its versatile applications, including monitoring antigen-specific T cell responses, evaluating an individual’s T cell priming capacity, and conducting preclinical studies on potential adjuvants and vaccine candidates. Full article

Wildlife serves as a significant reservoir for various pathogens transmissible to domestic animals and humans. Vector-borne diseases represent an increasing concern in Europe, affecting both animal and human health. This pilot study investigated the circulation of endemic and emerging vector-borne viruses in wild ungulates in Hungary, utilizing a One Health approach. Serum samples were obtained from European fallow deer (Dama dama), red deer (Cervus elaphus), and roe deer (Capreolus capreolus) during routine national game management activities between 2020 and 2023. Samples were analyzed for antibodies against the Bluetongue virus (BTV), West Nile virus (WNV), and Epizootic hemorrhagic disease virus (EHDV) using ELISA and neutralization tests. The results revealed a WNV seroprevalence of 22.3% in fallow deer and 31.8% in red deer, while BTV seroprevalence was 2.5% in fallow deer. All samples were negative for EHDV antibodies. These findings confirm the circulation of WNV and BTV in Hungarian wild ungulates. While the study’s design precludes statistical analysis due to non-random sampling, it demonstrates the potential of integrating wild ungulate serosurveillance into disease monitoring programs, leveraging established wildlife management activities for a cost-effective and complementary approach to One Health surveillance, particularly considering the ongoing spread of EHDV in Europe and the importance of BTV serotype monitoring for effective vaccination strategies. Full article

Lymphotoxin-alpha (LTα3) is a soluble cytokine of the TNF superfamily. Its role in inflammation and arthritis is not well known. Macrophages are important in K/BxN Serum-Transfer Arthritis (STA) and rheumatoid arthritis (RA). Anti-TNF monoclonal antibodies as well as etanercept (ETA), a soluble TNF receptor II that also neutralizes LTα3, are efficient in the treatment of RA. Objectives: To evaluate the role of LTα3 in macrophage phenotypes and in arthritis. Methods: Macrophages were cultured in the presence of recombinant LTα3, and their phenotypes were studied. The clinical effect of blocking LTα3 in STA was evaluated, as well as the effect of switching from anti-TNF monoclonal antibodies to etanercept in the “ROC” register of RA patients. Results: We showed that recombinant LTα3 was capable of directing mouse and human macrophages towards a pro-inflammatory “M1” phenotype. In K/BxN STA, ETA decreased clinical score and joint swelling. Anti-LTα3 reduced arthritis only in TNF-KO mice, indicating that the effect of LTα3 was visible in the absence of TNF. The “ROC” register indicated that switching anti-TNF mAb to ETA did not induce clinical and biological improvement in RA. Conclusion: We show a pro-inflammatory role for LTα3 in murine and human macrophages. The neutralization of both TNF and LTα3 is not beneficial in the treatment of RA. Full article

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses, therapeutic treatments, and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring spike protein mutations linked to immune evasion. In this study, we characterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants, particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC), we established both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-β, and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying important implications for therapeutic development and public health preparedness. Full article

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development. Full article

Severe acute respiratory syndrome coronavirus 2 has undergone several mutations since 2020, and novel variants continue to emerge to this day. The immune escape ability of the emerging mutants is enhanced and results in robust transmissibility. The neutralizing ability of the antibodies produced in the human body during previous infections is decreased against some of these mutants, which poses a severe challenge to the preventive and therapeutic effectiveness of vaccines and antibody drugs. The nucleocapsid protein is one of the main structural proteins of the coronavirus and plays an important role in the life cycle of the novel coronavirus. This protein is one of the key targets for drug development, and the first major step in drug development is to obtain pure nucleocapsid proteins. However, since nucleocapsid proteins have a nucleic acid-binding function and automatically undergo liquid–liquid phase separation and agglomeration, the purification of full-length nucleocapsids is challenging. In this context, a set of easy-to-operate processes was developed in this study for the purification of nucleocapsid proteins. Finally, a pure full-length nucleocapsid protein without nucleic acid contamination was obtained, which exhibited significantly enhanced accessibility for structural and functional virological studies, vaccine development, and related research applications. Further, the nucleic acid-binding domain of the nucleocapsid protein was targeted, and potential severe acute respiratory syndrome coronavirus 2 inhibitors were identified using virtual screening and biolayer interferometry technology. Notably, the eukaryotically expressed nucleocapsid protein demonstrated a significantly greater binding affinity for Light Green SF Yellowish (K_D = 119.7 nM) compared to that demonstrated by its prokaryotic counterpart (K_D = 19.9 × 10³ nM). The findings of this study suggest the importance of considering both protein source and post-translational modifications of the target proteins to be used in drug screening workflows. Therefore, this compound not only represents a novel therapeutic candidate for COVID-19 but also a critical tool for elucidating antiviral mechanisms. Full article

Background: Tetanus toxin, produced by Clostridium tetani, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD₅₀) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD₅₀ TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD₅₀ TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs. Full article

Neuroinvasive arboviruses such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Usutu virus (USUV), and Toscana virus (TOSV) have (re-)emerged with increasing incidence and geographic range. We analyzed the epidemiology of arboviral infections in Croatia during the 2024 transmission season. A total of 154 patients with neuroinvasive diseases (NID), 1596 horses, 69 dead birds, and 7726 mosquitoes were tested. Viral RNA was detected using RT-qPCR. IgM/IgG-specific antibodies were detected using commercial ELISA or IFA, with confirmation of cross-reactive samples by virus neutralization test. RT-qPCR-positive samples were Sanger sequenced. Arboviral etiology was confirmed in 33/21.42% of patients with NID. WNV was most frequently detected (17/11.03%), followed by TBEV (10/6.49%), USUV (5/3.24%), and TOSV (1/0.64%). WNV infections were reported in regions previously known as endemic, while in one continental county, WNV was recorded for the first time. USUV infections re-emerged after a six-year absence. In addition to human cases, acute WNV infections were recorded in 11/395 (2.78%) of horses and two dead crows. WNV IgG seropositivity was detected in 276/1168 (23.63%) and TBEV IgG seropositivity in 68/428 (15.88%) horses. None of the tested mosquito pools were positive for WNV and USUV RNA. Phylogenetic analysis showed the circulation of WNV lineage 2 and Usutu Europe 2 lineage. Climate conditions in 2024 in Croatia were classified as extremely warm, which could, at least in part, impact the quite intense arboviral season. The spreading of flaviviruses in Croatia highlights the need for continuous surveillance in humans, animals, and vectors (“One Health”). Full article