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Parvovirus infection causes severe diseases in both feline and canine species. It primarily affects adult cats and dogs but poses a higher risk to kittens and puppies. This virus is highly contagious and is easily transmitted through contaminated food, shared shelter environments, as well as the hands and clothing of people. The recovered species may continue to shed parvovirus in their feces for an extended period, leading to severe environmental contamination. There is no universal vaccine available that protects dogs and cats against parvovirus infections. The main goal of this study is to design a pan-parvovirus multiepitope-based vaccine that could be administered to dogs and cats. We utilized AI-machine learning-incorporated server tools such as IEDB and NetMHCpan to predict B-cell and T-cell epitopes. VaxiJen and ToxinPred were used to analyze immune characteristic features and docking with feline alleles using the HADDOCK server. Following this, the immune response and stability of the vaccine construct were confirmed with disulfide engineering, normal mode analysis, and molecular docking performed with toll-like receptors of both feline and canine (TLR4 and TLR5), and molecular dynamics simulation was performed for 10 ns. The triggered immune response was determined with immuno-simulation (ImmSim), and their activity in a biological environment was reinforced with in silico cloning. The B-cell epitopes (NS1-9, NS2-4, VP1-12 and VP2-9) predicted with the IEDB database were subjected to antigenicity prediction. MHC class I and IFN prediction and MHC class II and IL-4 prediction were performed with IEDB and NetMHCpan. The T-cell epitopes showed high binding affinities with the feline alleles. The final vaccine was designed by combining the top-ranked B-cell epitopes and T-cell epitopes, filtered for high antigenicity, non-allergic, non-toxic, and good solubility, and with the better binding affinity score of the structural and non-structural proteins (NS1, NS2, VP1, and VP2) of feline and canine parvoviruses through linkers and adjuvants. The disulfide bond prediction and normal mode analysis showed that our vaccine construct is stable and flexible. The molecular docking analysis was performed between the designed vaccine epitopes and the TLRs (TLR4–feline and TLR5–canine) with Biovia Discovery Studio using Zdock; it showed better binding interactions with a value of 22.26 (Zdock score), −47.409 (Zrank score) for feline and 16.54 (Zdock score), −134.295 (Zrank score) for canine. A pan-multi-epitope-based vaccine based on the two structural and non-structural proteins (NS1, NS2, VP1, and VP2) was designed and constructed to provide dual protection against parvovirus in both feline and canine species. The molecular docking and molecular dynamics simulation analysis showed higher binding affinities and stable conformations with canine (TLR5) and feline (TLR4) toll-like receptors. Although computational analysis supports the prediction of top-ranked epitopes and their immunogenic properties with greater precision, further experimental validation is required before they can be used against these viruses. Full article

Hantaviruses, the etiological agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), represent a high-risk zoonotic threat with substantial global health impact. Currently, there is no FDA-approved vaccine. The viral surface glycoprotein (GP) is crucial for host cell entry and is regarded as a key target for vaccine development. However, its variability among hantavirus species limits the effectiveness of conventional vaccine strategies. Epitope-based vaccines offer a promising alternative by enabling the design of broadly protective constructs. In this study, we applied immunoinformatics approaches to design a universal multi-epitope vaccine candidate targeting both HFRS- and HPS-associated hantaviruses through a multi-layered workflow integrating B-cell and T-cell epitope prediction, antigenicity scoring, IFN-γ induction potential, conservation analysis, and population coverage assessment. Viral GPs from SEOV, PUUV, SNV, and ANDV were analyzed using algorithms for B-cell and T-cell epitope prediction. Predicted epitopes were assessed for allergenicity, toxicity, conservation, and population coverage. Two vaccine constructs incorporating β-defensin or 50S ribosomal protein L7/L12 as adjuvants were assessed for physicochemical properties, structural stability and immunogenic potential. Molecular docking analyses provided exploratory ectodomain-compatibility screening, suggesting potential interactions with TLR4 that require future experimental confirmation. The in silico immune simulations suggested potential robust and long-lasting responses with memory cell persistence exceeding one year. Simulations also indicated balanced humoral and cellular responses, robust antibody production, and long-term memory formation suggestive of durable protective immunity. These findings support the rational design of broad-spectrum multi-epitope vaccines against genetically diverse hantaviruses, offering a rational framework for preclinical development of next-generation universal vaccines against hantavirus-associated diseases. Full article

African swine fever virus (ASFV) is a highly pathogenic DNA virus that continues to circulate globally and poses a serious threat to the swine industry. However, the antigenic complexity of ASFV poses formidable challenges for vaccine and diagnostic development. The pE146L protein, which serves as a structural component of the inner envelope, is vitally required for efficient ASFV replication. In this study, the pE146L protein was expressed in E. coli Rosetta (DE3) cells, and five monoclonal antibodies (mAbs) were generated by immunizing BALB/c mice with the purified pE146L. Epitope fine mapping using a panel of truncated overlapping fragments identified five B-cell linear epitopes: aa37–45, aa65–80, aa86–94, aa86–101, and aa115–124, recognized by mAbs 8F3, 1B8, 5C10, 10F4, and 4B7, respectively. Furthermore, synthesized peptides SP1–SP5 were designed based on these sequences, wherein SP5 was prepared as an extended peptide spanning aa110–124 to potentially enhance antibody binding or peptide presentation. These synthetic peptides reacted not only with their respective mAbs but also with ASFV-positive pig sera, among which SP3 exhibited the strongest immunoreactivity. Sequence alignment revealed that all five epitopes were highly conserved among ASFV genotypes I, II, and the emerging I/II recombinant strains. Collectively, these findings identify novel antigenic epitopes of pE146L, providing preliminary candidates that may contribute to the future development of ASFV serological diagnostics. Full article

Inclusion body hepatitis (IBH), a disease predominantly associated with fowl adenovirus serotypes 8a (FAdV-8a) and 8b (FAdV-8b), poses a significant challenge to poultry production on a global scale. This study reports a severe outbreak of IBH that occurred in 2021 at a chicken layer farm in Hunan Province, China. Liver tissues were collected from visibly affected layer chickens for viral isolation in LMH cell cultures. Subsequent phylogenetic analysis was conducted based on DNA sequencing and bioinformatics data to determine the genetic relationships of the isolated virus. Pathogenicity trials were performed on specific-pathogen-free (SPF) chickens. A novel FAdV-8, designated HuN21, with recombination between serotypes FAdV-8a/FAdV-8b, was isolated. Recombination and phylogenetic analyses revealed that this recombinant strain harbored a genomic backbone and fiber gene from FAdV-8a and FAdV-8b, respectively. HuN21’s pathogenicity differed significantly by age groups, showing high pathogenicity (100% mortality, classic IBH signs) in 1-day-old SPF chickens, compared with no mortality despite viral replication in 28-day-old chickens. These data provide robust evidence for recombination between different serotypes of FAdV-8. Notably, the unique genetic and pathogenic characteristics of the HuN21 strain indicate potential as a candidate strain for the development of a bivalent inactivated vaccine or a natural vaccine against FAdV-8a/FAdV-8b. Full article

Seasonal influenza vaccine strain selection remains challenging because circulating viruses may drift after vaccine recommendations are made. This study presents FluEvoFormer, a structure-guided generative foundation model for prospective influenza antigenic evolution forecasting and vaccine strain ranking. The framework jointly encodes hemagglutinin and neuraminidase sequences and incorporates residue-contact graphs. Separate prediction heads estimate future viral dominance and the vaccine–virus antigenic match. Controlled future-like variant stress testing, uncertainty adjustments, and clade balancing are then used to rank vaccine candidates. A rolling retrospective evaluation was performed for target seasons involving the influenza A(H1N1)pdm09 virus and influenza A(H3N2) virus. The evaluation used cutoff-restricted sequence records, hemagglutination inhibition data, vaccine-composition records, protein-structure resources, and vaccine-effectiveness indicators. The historical training corpus for the influenza A(H1N1) virus also contained pre-2009 seasonal records. FluEvoFormer achieved the lowest held-out antigenicity prediction error, with mean absolute error (MAE) values of 0.389 for the combined historical influenza A(H1N1) virus corpus and 0.456 for the influenza A(H3N2) virus corpus. It also improved the future dominance prediction, with Kullback–Leibler (KL) divergence values of 0.255 and 0.289, respectively. The model selected candidates with higher empirical normalized coverage scores in seven out of 10 influenza A(H1N1)pdm09 virus seasons and nine out of 10 influenza A(H3N2) virus seasons. The predicted coverage score showed a strong positive correlation with external vaccine-effectiveness estimates. These findings support FluEvoFormer as a computational decision-support framework for prioritizing influenza vaccine candidates before downstream laboratory and public health evaluations. Full article

Background: The simultaneous circulation of highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4b) and virulent Newcastle disease virus (NDV) genotype VII creates a cumulative risk for the poultry industry, rendering monovalent vaccination insufficient. This study aimed to develop and evaluate a combined inactivated vaccine against AIV H5N8 and NDV genotype VII based on local endemic strains. Methods: The vaccine was formulated as a water-in-oil emulsion (30:70) using Montanide™ ISA 78 VG. Evaluation included physicochemical characterization, safety assessment in chickens, immunogenicity evaluation via the hemagglutination inhibition (HI) test, and protective efficacy following challenge with virulent isolates. Results: The formulation demonstrated high stability and complete safety without adverse reactions. By 28 days post-immunization, the vaccine induced a robust, balanced humoral response with antibody titers reaching 9.10 ± 0.23 log₂ against NDV and 9.00 ± 0.26 log₂ against AIV, indicating no antigenic interference. Upon challenge, the vaccinated group exhibited 100% clinical protection and survival, compared to 100% mortality in controls. Furthermore, vaccination significantly reduced viral shedding, limiting horizontal spread. Conclusions: The developed combined inactivated vaccine demonstrates high safety, stability, and protective efficacy. It represents a promising candidate for comprehensive specific prophylaxis in endemic regions, effectively limiting horizontal pathogen transmission and mitigating economic losses in poultry production. Full article

African swine fever virus (ASFV) causes African swine fever (ASF), a highly lethal disease in pigs. Vietnam has approved two ASF live-attenuated vaccines (LAVs), but their efficacy and safety remain controversial, and no reliable, highly effective commercial ASF vaccine is available yet. Humoral immunity plays an important role in protection against ASFV infection. However, there is still controversy regarding whether ASFV infection can induce antibodies with neutralizing activity. Antibody-dependent cellular cytotoxicity (ADCC), as an antibody-mediated protective mechanism, offers a novel perspective for screening protective ASFV antigens. This study evaluated five structural proteins (pCP312R, pA104R, pA151R, p17, and pF317L) as subunit vaccine candidates based on their ability to induce antibodies that inhibit viral replication and mediate ADCC. The recombinant proteins were expressed in Escherichia coli, purified, and used to immunize pigs. Immune sera collected two weeks after the third immunization were tested for their ability to inhibit ASFV replication in porcine alveolar macrophages (PAMs) using rASFV-Gluc/EGFP. ADCC activity was assessed using a stable HEK293T-p17 cell line as target cells and porcine peripheral blood mononuclear cells (PBMCs) as effectors, with cytotoxicity measured by lactate dehydrogenase release. All five recombinant proteins were successfully expressed and purified. Immunization with pCP312R, pA104R, p17, and pF317L induced the production of specific antibodies in pigs, but only anti-p17 antibodies significantly inhibited ASFV replication in PAMs. The p17 is highly conserved across different ASFV genotypes and is predicted to contain a transmembrane domain. Anti-p17 antibodies effectively mediated PBMCs to specifically kill target cells, demonstrating significant ADCC activity. Moreover, the HEK293T-p17 cell line was specifically recognized by anti-ASFV sera. These findings indicate that p17 is a dual-functional antigen capable of eliciting antibodies that both inhibit viral replication and mediate ADCC in vitro. Furthermore, we have developed an in vitro platform for screening protective ASFV antibodies based on viral inhibition and ADCC, providing candidate targets for the development of next-generation ASF subunit vaccines. Full article

Neonatal calf diarrhea (NCD) is one of the most important problems of calf breeding across the world. It causes deaths in calves in the first 10 days of their life, and it is mainly caused by Escherichia coli(E. coli), Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV). The lack of vaccines with consistently high protective efficacy against the main causes of NCD makes disease control highly challenging. The current study aims to design a multi-epitope mRNA-based vaccine targeting the major pathogens responsible for NCD using immunoinformatic tools and molecular modeling approaches. BRV capsid protein VP6, BCoV Spike glycoprotein and E. coli F5 fimbrial protein were used as antigenic proteins to predict potential epitopes. Fifteen selected epitopes were linked with suitable linkers and conjugated with a built-in adjuvant, resulting in the design of a stable, antigenic and non-allergenic vaccine candidate against NCD pathogens. Furthermore, molecular docking analysis shows strong binding affinity between the vaccine candidate and the bovine toll-like receptors TLR2 and TLR4 at low energy and high stability. Based on these findings, the proposed multi-epitope vaccine represents a promising approach for the prevention and control of neonatal calf diarrhea and provides a solid scientific foundation for future experimental studies to validate its efficacy and safety in vivo. Full article

Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D²) and antigen concentration (R² = 0.956–0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625–10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations. Full article

Acinetobacter baumannii, a Gram-negative opportunistic bacterium in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter spp.), has emerged as a leading cause of nosocomial infections worldwide. It is known to possess diverse virulence traits and antimicrobial resistance, making it a critical priority pathogen on the World Health Organization’s 2024 Bacterial Priority Pathogens List. Carbapenem-resistant A. baumannii (CRAB) is currently endemic across several continents, with global carbapenem resistance exceeding 70% in healthcare settings and multidrug-resistant infections being associated with alarming mortality rates. This review comprehensively discusses the molecular underpinnings of A. baumannii pathogenesis and virulence, detailing the array of factors coordinated by complex regulatory networks. The convergence of this pathogen’s virulence and antimicrobial resistance traits, resulting in multidrug resistance, leaves clinicians with only a handful of therapeutic options. The review also discusses upcoming therapeutic strategies, including phage therapy, antimicrobial peptides, monoclonal antibodies, photodynamic therapy, and vaccine candidates in the pipeline. While emerging therapeutics show promise, several challenges remain, and integrated approaches are warranted to efficiently combat A. baumannii’s virulence and resistance armamentarium. Full article

Pharyngeal infection with high-risk human papillomavirus (HPV), particularly HPV16, is biologically distinct from cervical infection because it occurs within the specialized lymphoepithelial environment of Waldeyer’s ring. This review evaluates nanoparticle materials for the prevention, detection, and treatment of pharyngeal HPV, with an emphasis on structure–property–function relationships, mucosal performance, and translational feasibility. Lipid nanoparticle platforms, polymeric nanoparticle platforms, inorganic systems, and hybrid platforms are compared with respect to composition, particle size distribution, surface charge, colloidal stability, biodegradability, payload compatibility, release behavior, and manufacturing complexity. Evidence suggests that lipid and polymeric systems are the most credible near-future candidates for mucosal vaccination and localized nucleic acid delivery because they offer the best balance between controllable fabrication, analytical tractability, and biologically plausible performance in mucus-exposed tissue. By contrast, the development of inorganic theranostics and CRISPR-enabled platforms remains at an earlier stage because repeated mucosal dosing, retention in lymphoid tissue, and combined product regulation impose substantial burdens. A translational roadmap is proposed in which material selection is guided by clinically relevant quality attributes, standardized saliva- and mucus-relevant assays, human tonsil organoid testing, and early attention to manufacturability, safety, and regulatory strategy. The field is promising, but direct pharyngeal HPV data remain limited; accordingly, there is an urgent need for comparative studies that connect nanoparticle architecture to measurable outcomes such as tonsillar deposition, epithelial uptake, immune activation, and local tolerability. Full article

Porcine epidemic diarrhea virus (PEDV) is a major enteric coronavirus that causes severe economic losses to the global swine industry. Current vaccines suffer from weak immunogenicity, insufficient mucosal immunity, and a short duration of protection. Nanoparticle delivery systems have emerged as a promising strategy for next-generation vaccine development. In the present study, we constructed an S1-Ferritin nanoparticle vaccine using the SpyTag-SpyCatcher modular conjugation system. The morphology, particle size, and uniformity of the nanoparticle vaccine were systematically characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After two immunizations, the S-Ferritin nanoparticle vaccine elicited potent humoral and cellular immune responses in both mice and piglets. In piglets, at 2 weeks post booster vaccination, PEDV-specific serum IgG endpoint titers peaked at 1:2560, virus-neutralizing antibody titers reached 1:256 against the JS-2/2015 strain, and serum IFN-γ levels reached 274 pg/mL. All these immunological indicators were significantly higher than those observed in the PEDV-inactivated whole-virus vaccine group. Furthermore, challenge tests showed that the S1-Ferritin nanoparticle vaccine provided complete protection against PEDV infection and significantly reduced the severity of diarrhea and intestinal damage in piglets after challenge. These findings suggest that the S1-Ferritin nanoparticle vaccine constitutes a promising candidate against PEDV infection, though our study is only a preliminary step and subsequent field trials in pigs are still required. Full article

H9N2 avian influenza virus (AIV) continues to mutate, leading to immunosuppression and secondary infections in poultry. Traditional inactivated vaccines mainly induce humoral immunity and have limited cross-protection efficacy against various subtypes of virus strains. In this study, we targeted the HA2 and M1 proteins of H9N2 as antigens and used immunoinformatics methods to design a broad-spectrum multi-epitope vaccine (MEV) that can simultaneously activate humoral and cellular immunity. Firstly, through systematic evolutionary analysis and sequence comparison, highly conserved amino acid sequence regions were selected from HA2 and M1 proteins. B-cell epitopes were predicted in the HA2 conserved sequence, and cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes were predicted in the M1 conserved sequence. Three candidate vaccines containing different epitope combinations were constructed. After secondary structure and physicochemical property comparisons, HM1 was determined as the optimal scheme. HM1 contains three B cell epitopes, two CTL epitopes, and three HTL epitopes, and was connected to chicken β-defensin at the N-terminus as a molecular adjuvant; a dendritic cell-targeting peptide was added at the C-terminus. The HM1 tertiary structure optimized by GalaxyRefine met the standards of a reliable model. The molecular docking results indicated that HM1 can form stable binding with chicken TLR2, TLR4, MHC I, and MHC II molecules, with binding free energies of −7.1 kcal/mol and −6.1 kcal/mol, respectively, and can form multiple hydrogen bonds and salt bridges. Normal mode analyses revealed that the HM1–TLR complex exhibits favorable dynamic properties at the computational level. The immune simulation prediction results showed that after vaccination with HM1, specific antibodies can be induced, B cells, helper T cells, and cytotoxic T cells can be activated, and IFN-γ and IL-2 can be secreted. In summary, the HM1 designed based on the conserved regions of HA2 and M1 proteins has good physicochemical stability and immunogenicity, providing a theoretical basis for the development of broad-spectrum and highly effective H9N2 vaccines. Full article

Background: Adenoviral vectors are widely used in vaccine development; however, pre-existing immunity to common human adenovirus serotypes can limit their effectiveness. Canine adenovirus type 2 (CAV-2) is a non-human adenovirus with low seroprevalence in humans, making it a suitable alternative vector. Despite its promise, comprehensive preclinical safety data for CAV-2 vector-based vaccine platforms remain limited. In this study, we evaluated the safety profile of a replication-defective CAV-2 vector expressing the Omicron BA.4 SARS-CoV-2 spike immunogen in BALB/c mouse models. Methods: The SARS-CoV-2 CAV-2 vector-based vaccine was expressed and propagated in AD293 cells. The mice received intramuscular prime-boost immunisations with low (1 × 10⁶ PFU), moderate (0.5 × 10¹⁰ PFU), or high (1 × 10¹⁰ PFU) vaccine doses, alongside empty CAV-2 vector and physiological buffer control groups, and were monitored longitudinally up to Day 72. The mice were clinically assessed at days 0, 7, 21, 42, and 72 for any deviations from normal conditions in comparison to the control groups. Biochemical analyses were performed to evaluate liver and kidney function, as well as any tissue injury due to the vaccine candidate. Hematological parameters were assessed by conducting complete blood counts. Body temperature and weight changes were also monitored as an indicator of systemic toxicity. Results: The biochemical and haematological parameters remained within physiological reference ranges across all dose groups and timepoints, with no dose-related deviations, indicating that the vaccine candidate did not show evidence of hepatotoxicity, nephrotoxicity, tissue or haematological toxicity. Body temperatures remained within normal physiological ranges following both prime and booster immunisations, and body weights increased normally across all groups as the animals grew throughout the study period without any abnormal weight gain or loss. Conclusions: These results suggest that the replication-defective CAV-2-vectored SARS-CoV-2 vaccine candidate was well tolerated and did not demonstrate evidence of systemic toxicity under the conditions tested. These findings demonstrate that the CAV-2 vector exhibits a favourable safety profile in murine models when used as a vaccine delivery platform, supporting its translational potential as an alternative adenoviral vector-based vaccine platform. Further studies incorporating additional safety endpoints, including histopathological, vector persistence and shedding evaluation, are warranted to support continued development of the platform. Full article

Orf virus (ORFV) is a major pathogen in goats and sheep, and control currently depends mainly on commercial live vaccines. Although mRNA vaccines have revolutionized human medicine, their use in veterinary settings is largely unexplored. In this study, an mRNA vaccine candidate encoding the ORFV F1L protein (F1L-mRNA-LNP) was developed via in vitro transcription and encapsulated in lipid nanoparticles. BALB/c mice were divided into five groups (n = 14 each): three receiving different doses of F1L-mRNA-LNP (5, 10, or 15 μg), one receiving a commercial live vaccine (CV), and a PBS control group. Mice were immunized intramuscularly and boosted after 14 days; immune responses were assessed 14 days later following ARRIVE 2.0 guidelines. Both the F1L-mRNA-LNP and CV vaccines induced specific antibodies versus PBS (p < 0.01). The 10 μg mRNA group showed Th1 cytokine and CD8⁺ T cell responses comparable to CV (p > 0.05), whereas IL-4 (Th2) was significantly higher in the CV group (p < 0.05). Neutralizing antibody titers did not differ between groups, indicating that the mRNA vaccine induces comparable Th1 cellular immunity but weaker Th2 humoral immunity. Upon ORFV challenge, the 10 μg F1L-mRNA-LNP vaccine protected BALB/c mice, as evidenced by stable body weight, no clinical symptoms, and reduced viral load, with efficacy comparable to CV (p > 0.05). This study provides strong evidence supporting the optimization of ORFV mRNA vaccines and highlights the translational potential of the F1L-mRNA-LNP candidate vaccine for veterinary applications. Full article