Search Results (849)

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

Background: Buffaloes have a significant share in the agricultural economies of many developing countries; however, the industry is not well-established because of the low reproductive efficiency of buffaloes. In this study, we designed a novel strategy to enhance buffalo reproductive efficiency and examined the significance of intramuscular AMH-INH-RFRP DNA vaccine immunization on reproductive performance. Methods: Allocated into four groups (n = 30/group), female buffaloes (aged 5–8 years, n = 120) were randomly enrolled in the study. Treatment groups received 10 mL of AMH-INH-RFRP DNA vaccine intramuscularly, once daily for three consecutive days, at the following concentrations: T1—3 × 10⁸ CFU/mL, T2—3 × 10⁹ CFU/mL, and T3—3 × 10¹⁰ CFU/mL. All vaccinated groups received a booster vaccination two weeks later, while the control group received 10 mL of phosphate-buffered saline (PBS) intramuscularly on the same schedule. Serum antibody titers against anti-Müllerian hormone (AMH), inhibin (INH), and RF-amide-related peptide (RFRP) were quantified using indirect ELISA on days 14 (post-primary immunization) and 28 (post-booster immunization). Results: Following primary immunization, antibody titers against AMH, INH, and RFRP were significantly higher in the T3 group than in the T1 group (p < 0.05), and booster immunization further increased antibody positivity rates. Serum concentrations of IL-4, IFN-γ, and E₂ were significantly elevated in the T2 and T3 groups compared to the control group (p < 0.05), while P₄ levels showed no significant differences among groups. Ultrasonography revealed that the ovulatory follicle diameter and dominant follicle growth rate were significantly increased in the T2 and T3 groups compared to the control and T1 groups (p < 0.05). The estrus rate was significantly higher in the T3 group than in the control group (76.67% vs. 40.00%, p < 0.05), and ovulation rates were significantly higher in the T2 (83.33%) and T3 (86.67%) groups compared to the control group (56.67%) (p < 0.05). However, conception rates did not differ significantly between vaccinated and control groups in the primary dose comparison (p > 0.05). In exploratory post hoc analyses, antibody-positive (Ab+) buffaloes exhibited higher E₂ concentrations, larger ovulatory follicle diameters, accelerated dominant follicle growth rates, and higher estrus and ovulation rates compared to antibody-negative (Ab−) buffaloes. Furthermore, gestating buffaloes were monitored until calving, revealing no significant effect of the vaccine on newborn calf weight or body size. Conclusions: In total, intramuscular immunization with the AMH-INH-RFRP DNA vaccine, particularly at the highest dose, enhances reproductive performance in buffaloes by stimulating E₂ secretion and follicular development. Full article

Dengue fever, a rapidly spreading mosquito-borne viral disease, poses a major global health issue, particularly in tropical and subtropical regions. The absence of a universally effective vaccine against all four dengue virus serotypes necessitates continued exploration of novel vaccine strategies. This study evaluated a PrM-derived multiple antigenic peptide (MAP) vaccine candidate in BALB/c mice. Molecular docking analyses predicted favourable interactions of the selected PrM-derived B-cell epitope with both human (human BCR Fab region PDB ID: 5IFH) and mouse (mouse BCR PDB ID: 8EMA) B-cell receptors, supporting its antigenic potential. ChemSketch was used to develop the PrM–MAP vaccine construct, which was then commercially synthesized. Mice received single, double, and triple booster doses of the vaccine, while control groups received either PBS or adjuvant alone. Humoral immune responses were measured by ELISA, and safety was evaluated through in vitro cytotoxicity assays on HEK293 cells and the in vivo histopathological examination of major organs. Mice immunized with a single booster dose produced significant levels of PrM-specific antibodies compared to subsequent booster doses, suggesting variability in the humoral response elicited. Optimal coating concentrations in ELISA demonstrated the importance of antigen concentrations in the sensitivity of the assay. The safety evaluation indicated high cell viability, up to 150 µg/mL of PrM–MAP, in HEK293 cells, and an absence of significant histopathological damage in treated animals. The PrM–MAP vaccine construct elicited a detectable humoral immune response while demonstrating a high safety profile in vitro and in vivo. These findings indicate the potential of MAP-based vaccine platforms as promising candidates for further development against the dengue virus. Full article

Multidrug-resistant Klebsiella pneumoniae is an increasing global threat, and the limited availability of new antibiotics highlights vaccination as a promising strategy for infection prevention. Although different vaccine candidates have been evaluated, none is currently approved, mainly due to capsular heterogeneity among strains. Protein-based vaccines may overcome this limitation by targeting conserved epitopes. In this study, we assessed the immunogenicity of five outer membrane proteins (ChiP, LamB, RafY, OmpW, PagP) previously selected by reverse vaccinology (RV), comparing them with the well-characterized antigens OmpA and OmpK36. Mice were immunized intramuscularly with three doses of purified recombinant proteins, and antibody responses were analyzed by ELISA. All antigens elicited high, booster-induced IgG levels, with PagP slightly being less immunogenic. Regarding IgG subclasses, IgG₁ predominated, followed by IgG_2b and IgG_2c. Cross-reactivity was evaluated against six K. pneumoniae strains representing different clonal groups, and recognition by sera from previously infected mice was also examined. OmpA, ChiP and LamB showed the broadest cross-reactivity, while OmpA and LamB were most strongly recognized after infection. Overall, OmpA, LamB, ChiP and RafY emerged as the most promising vaccine candidates, although further optimization, such as a multicomponent vaccine, may be required. This work also highlights the importance of experimentally validating RV-selected antigens, as computational predictions alone do not ensure immunogenicity or in vivo relevance. Full article

Background/Objectives: We describe the rationale, design and evaluation of a novel feline combination vaccine (Nobivac^® NXT HCPChFeLV) incorporating RNA particle technology against feline leukemia virus (FeLV) alongside live attenuated components for feline herpesvirus-1 (FHV), feline calicivirus (FCV), feline panleukopenia (FPL) and Chlamydia felis. Efficacy and safety outcomes from laboratory and field studies are presented. Methods: Nobivac NXT HCPChFeLV is a 0.5 mL, non-adjuvanted, lyophilized vaccine containing live attenuated FHV, FCV, FPL, and C. felis strains, and an alphavirus replicon-derived RNA particle expressing FeLV envelope glycoprotein (gp85). The primary vaccination schedule involved subcutaneous vaccination at 8–9 weeks of age with a booster 4 weeks later. Onset-of-immunity studies were performed 1 week post primary vaccination. Duration-of-immunity studies evaluated challenge both at 1 year and 3 years after primary vaccination with a booster at 1 year (FHV, FCV, FeLV); FPL duration was assessed at 3 years after primary vaccination; C. felis duration was assessed at 1 year. Clinical signs of disease, survival, leukopenia (FPL) and pathogen excretion were assessed. Safety was assessed in laboratory overdose/repeat-dose studies and in a field study comparing concurrent (non-mixed) use with rabies vaccination vs a positive control regimen. Results: Vaccination significantly reduced clinical disease following FHV, FCV and C. felis challenge and reduced FHV-associated mortality. Vaccinated cats were protected from FPL clinical disease, mortality, leukopenia, and FPL excretion. Persistent FeLV antigenemia was significantly prevented after primary vaccination and in 1-year-duration-of-immunity studies and was reduced 3 years after revaccination. The excretion of FHV, FCV and C. felis was significantly reduced in vaccinates vs controls. No serious adverse events were observed in laboratory studies; field vaccination was well tolerated with no injection-site reactions reported. Conclusions: Nobivac NXT HCPChFeLV provides robust, long-lasting protection against multiple feline pathogens, reduced pathogen shedding and demonstrated a favorable safety profile, supporting its use in feline preventive healthcare. Full article

Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Although PCV2-Mhp bivalent inactivated vaccines are useful for simultaneous disease control, their immunogenicity may be improved by adjuvant optimization. This study evaluated a composite adjuvant consisting of porcine interleukin-2 (IL-2), IL-4, IL-6 and MnJ(beta), a colloidal manganese adjuvant, in an initial murine immunogenicity model. Thirty female Kunming mice were assigned to three groups (n = 10/group): bivalent antigen plus IL-2/IL-4/IL-6/MnJ(beta), bivalent antigen plus MnJ(beta), or phosphate-buffered saline. Body weight, complete blood count, peripheral blood T- and B-cell subsets, PCV2-specific IgG and Mhp-specific indirect hemagglutination titers were monitored after primary and booster immunization. The composite formulation did not suppress body-weight gain or induce sustained abnormalities in erythrocyte- or platelet-related indices. WBC, neutrophil, lymphocyte and monocyte counts were elevated in group A at days 7 and 28 post-primary immunization, indicating transient immune activation. Day-56 flow cytometry indicated increased CD19+IgM-IgD- B-cell and effector/memory T-cell-associated responses. PCV2-specific IgG increased from day 14 onward. At day 56, the OD450 value in group A reached 1.532 ± 0.006, compared with 1.095 ± 0.004 in group C1 and 0.102 ± 0.002 in group C2, corresponding to approximately 1.40-fold and 15.09-fold higher levels than the MnJ(beta)-adjuvanted and PBS control groups, respectively. Mhp-specific IHA titers were also maintained at high levels after booster immunization; at day 56, group A showed a log2 endpoint titer of 13.00 ± 0.00, corresponding to a GMT of 1:8192, whereas group C1 showed a log2 endpoint titer of 12.00 ± 0.00, corresponding to a GMT of 1:4096, and group C2 remained negative. These results indicate that the IL-2/IL-4/IL-6/MnJ(beta) composite adjuvant demonstrates potential for improving antibody and peripheral lymphocyte responses to PCV2-Mhp bivalent antigen, but protective efficacy must be confirmed in target-species challenge studies. Full article

Background: Despite the circulation of evolutionarily related cold-causing coronaviruses (CCCs) in the pre-COVID era, most individuals lacked pre-existing serum IgG and/or class-switched memory B cell (B_mem) reactivity for the SARS-CoV-2 Spike (S) glycoprotein expressed by the ancestral Wuhan-Hu-1 (WH1) strain. Subsequent priming of the immune system through natural infection or prophylactic COVID-19 mRNA vaccination successfully generated robust B_mem responses against the WH1-S antigen, along with eliciting cross-reactivity for the future Omicron (BA.1) variant responsible for breakthrough infections (BTIs). However, to what extent immunological imprinting of B_mem towards the WH1-S antigen detrimentally constrains the elicitation of variant-specific antibody responses following subsequent booster vaccinations or BTIs—a phenomena referred to as “original antigenic sin”—remains an unresolved and open question. Methods: Using ImmunoSpot^®, we evaluated peripheral blood mononuclear cells (PBMCs) from defined human cohorts for IgG⁺ ASC reactivity against Spike proteins representing CCCs and SARS-CoV-2. Additionally, we developed a novel dual-label inverted FluoroSpot assay to distinguish between strain-specific and cross-reactive IgG⁺ ASCs recognizing epitopes in the receptor binding domain (RBD) of SARS-CoV-2 Omicron variants. Results: Our data demonstrate a lack of appreciable back-boosting of IgG⁺ B_mem recognizing structurally conserved epitopes shared between CCCs and SARS-CoV-2. Moreover, we found evidence for immunological imprinting and the preferential expansion of B_mem recognizing cross-reactive epitopes in the RBD following BTI. Nevertheless, Omicron strain-specific B_mem were detected in PBMC donors collected in 2025. Conclusions: Our novel inverted dual-label FluoroSpot methodology evidenced preferential expansion of cross-reactive B_mem following breakthrough SARS-CoV-2 infection and supports the influence of original antigenic sin shaping the recall response. Moreover, the inverted dual-label assay provides a highly flexible and easily implementable technique for distinguishing between strain-specific and cross-reactive B cell responses and has broad applications in translational vaccine research against pathogens that undergo antigenic drift. Full article

Background: Adult susceptibility to diphtheria and tetanus may increase as vaccine-induced immunity wanes, yet repeated population-based serosurveillance data in China are limited. Methods: We analyzed annual serosurveys conducted in Pudong New Area, Shanghai, China, from 2017 to 2025 among healthy adults aged 20–49 years. Diphtheria and tetanus IgG concentrations were measured by ELISA. Seroprotection was defined as antibody concentration ≥0.1 IU/mL. Antibody concentrations were further categorized as <0.01, 0.01–<0.1, 0.1–<1.0, and ≥1.0 IU/mL, and geometric mean concentrations (GMCs) were calculated. Multivariable logistic regression models were fitted to assess factors associated with non-protection, including survey year, age group, and household registration. Sensitivity analyses excluding the 2018 survey year were conducted. Results: A total of 2376 serum samples were included. Overall seroprotection was 21.46% for diphtheria and 13.80% for tetanus. The proportion protected against both antigens was 9.05%, while 73.78% showed concurrent non-protection against both antigens. The overall GMC was 0.032 IU/mL (95% CI: 0.030–0.034) for diphtheria and 0.018 IU/mL (95% CI: 0.017–0.019) for tetanus. Concentrations ≥1.0 IU/mL were uncommon for both antigens. Adults aged 40–49 years had higher odds of non-protection than those aged 20–29 years for diphtheria (OR: 2.43, 95% CI: 1.85–3.21) and tetanus (OR: 2.94, 95% CI: 2.11–4.13). Non-local residents also had higher odds of non-protection than local residents for diphtheria (OR: 1.55, 95% CI: 1.24–1.93) and tetanus (OR: 2.81, 95% CI: 2.15–3.69). Seroprotection varied across survey years, with a marked nadir in 2018. Sensitivity analyses excluding 2018 attenuated most year-specific associations, whereas age- and residence-related differences persisted. Conclusions: Healthy adults aged 20–49 years in Pudong showed low seroprotection and low GMCs against both diphtheria and tetanus, with a high proportion concurrently non-protected against both antigens. These findings highlight a persistent adult immunity gap and support further evaluation of adult booster strategies and enhanced serosurveillance. Full article

Vaccination is one of the most important measures for infectious disease control. Recently, media-generated concern about vaccine-associated adverse effects has produced a rise in both human and animal “anti-vaccination” movements. This study aimed to understand factors involved in dog owner vaccination decisions and explore whether there has been an increase in titer testing. An online survey targeting dog owners received a total of 2585 responses, which showed 79% of respondents had their dogs vaccinated in the last 12 months. A few owners had never vaccinated their dogs, and 13% of owners used titer testing prior to booster vaccinations. The factors with the strongest positive predictors for vaccination were requirements by third party services (e.g., kennels) and, for a negative response, lack of time. For respondents that had not vaccinated, the factors with the strongest predictive powers to determine if they titer test were education/working in the veterinary industry for a positive response and not having heard of negative side effects after vaccinating for a negative response. Overall, no evidence was found that a rise in anti-vaccination attitudes was pervasive in dog owners; however, the study shows that the veterinary profession has work to do to ensure herd immunity is maintained within dog populations. Full article

Background/Objectives: Self-amplifying RNA (saRNA) vectors enable high-level transgene expression from minimal initial doses. While alphavirus-based saRNA systems are widely used, they suffer from limitations, including large genome size, complex replicase machinery, and cellular toxicity. Nodamura virus (NoV) offers a promising alternative due to its compact genome (3.2 kb) and low cytotoxicity. This study aimed to elucidate NoV RNA1 replication mechanisms and develop a novel NoV-based saRNA vector platform. Methods: We established a Schizosaccharomyces pombe system to investigate NoV RNA1 replication and protein A localization. N-terminal deletion mutants and ER-targeting chimeras were constructed to characterize membrane targeting determinants. Based on mechanistic insights, we developed NovaVec by inserting transgenes at the RNA3⁴²² site within the subgenomic RNA3 region. In vivo performance was evaluated using lipid nanoparticle-encapsulated NovaVec expressing nanoluciferase or monkeypox A33R antigen in BALB/c mice. Results: We identified redundant mitochondrial targeting domains (amino acids 2-15 and 16-33) in NoV protein A, where either domain was sufficient for proper localization and replication. The replication machinery could be functionally redirected to the endoplasmic reticulum while maintaining replication competence. Lipid nanoparticle-encapsulated NovaVec achieved sustained transgene expression for 54 days in mice, significantly outperforming conventional mRNA vectors that lost signal within 14 days. The NovaVec-based monkeypox A33R vaccine elicited robust antigen-specific humoral immunity with titers reaching approximately 1:12,800 following booster immunization. Conclusions: With its compact genome encoding only a single replicase protein, minimal cytopathic effects, and demonstrated capacity for long-term protein expression, NovaVec represents a highly promising next-generation saRNA platform for vaccines. Full article

Background: The dynamics of humoral immune responses following primary and booster COVID-19 vaccinations are crucial to understand in order to optimize vaccination strategies. This study evaluates the magnitude and durability of SARS-CoV-2-specific IgG antibody responses across different vaccines in a large cohort of Bangladeshi adults. Methods: A total of 6300 adults from nine hospitals across eight divisions of Bangladesh were enrolled. Participants received two primary doses of either ChAdOx1 nCoV-19 (Covishield, Serum Institute of India, n = 2855), mRNA-1273 (Moderna, n = 578), BNT162b2 (Pfizer-BioNTech, n = 121), or Vero-cell-inactivated (Sinopharm, n = 2746) vaccines. Booster doses were administered at one-year intervals post-primary vaccination. SARS-CoV-2 spike receptor-binding domain (RBD)-specific IgG antibody responses were measured by ELISA using serum from vaccinees at multiple time points after two primary and two booster doses. Results: A total of 3745 individuals received booster 1 (third dose), with 59% receiving heterologous boosters (a different vaccine regimen than the primary doses). Only 5.5% (n = 347) of participants received a second booster one year after the first booster (among them, 99% received BNT162b2). Our results suggest that heterologous boosters with the mRNA vaccine induced higher IgG levels than homologous boosters for individuals who received primary vaccination with adenovirus vector-based ChAdOx1 nCoV-19 or a Vero-cell-inactivated vaccine. However, in those who initially received the mRNA-based vaccine, both homologous and heterologous boosters produced comparable IgG responses. Among all vaccine types, booster immunization with the Vero-cell-inactivated vaccine induced the lowest antibody responses. Longitudinal analysis demonstrated significantly high IgG levels over the 12 months following the first booster (p < 0.0001); however, IgG levels declined significantly after the second booster dose (fourth dose). Conclusions: Heterologous boosting strategies, particularly those involving mRNA vaccines, elicit stronger and more sustained IgG responses compared to a homologous booster. However, antibody waning after the second booster highlights the need for continued monitoring and potential additional vaccine strategies. Full article

Background: Although prolonged inflammatory symptoms are an infrequent and problematic adverse effect of vaccination that can occur in some people, the transient activation of acute phase reactants (APRs) is expected with adjuvanted vaccines and helps to potentiate immune responses. Methods: This experiment examined the association between vaccine reactogenicity and immunogenicity in monkeys immunized with an adjuvanted recombinant protein including a receptor binding domain–human IgG1-Fc fusion protein (RBD-Fc) sequenced from the ancestral Wuhan strain of SARS-CoV-2. The acute inflammatory reaction to immunization was assessed by determining the decline in serum iron levels at 24 h and the increase in the neutrophil-to-lymphocyte ratio (NLR) as the adherent neutrophil pool trafficked into circulation. Results: Robust primary and secondary antibody responses were elicited. Larger decreases in serum iron and higher NLRs were associated with a stronger inhibition of RBD binding with angiotensin-converting enzyme (ACE2) when five early viral variants of SARS-CoV-2 were tested, including Wuhan, Alpha, Beta, Gamma and Delta. Inhibition of ACE2-RBD binding was less evident when the Omicron variant was tested. Individual variation in the APR was also predictive of the persistence of cell-mediated immunity based on the number of interferon-expressing mononuclear cells activated by viral antigen in ELISpot assays. Conclusions: Rapid antibody responses to primary immunization and large secondary responses to booster immunizations were elicited by this adjuvanted recombinant RBD-Fc vaccine, and our analysis affirmed the view that a transient APR can enhance antibody binding with antigen proteins. Full article

Background: SARS-CoV-2 reinfections increased substantially after the emergence of Omicron variants. Methods: We conducted a cross-sectional study of 2095 individuals with prior Omicron BA.2 infection in Shanghai, China, during the early post-zero-COVID period. Data on demographics, infection history, and lifestyle factors were collected via questionnaire, and blood samples were obtained for ancestral RBD-blocking antibody measurement. Results: Meeting WHO physical activity recommendations (≥600 MET-min/week) was associated with lower reinfection odds (OR = 0.59, 95% CI: 0.46–0.74, p < 0.001). The overall median ancestral RBD-blocking antibody level was 263.93 U/mL (IQR: 36.41–331.87). Older age was associated with lower ancestral RBD-blocking antibody levels (β = –0.0038 per year, 95% bootstrap CI: –0.0057 to –0.0019, p < 0.001). All vaccinated groups had significantly higher ancestral RBD-blocking antibody levels than unvaccinated individuals: partially vaccinated (β = 0.4440, 95% CI: 0.1569 to 0.6830, p < 0.001), fully vaccinated (β = 0.8516, 95% CI: 0.7464 to 0.9595, p < 0.001), homologous booster (β = 1.0297, 95% CI: 0.9408 to 1.1223, p < 0.001), and heterologous booster (β = 1.0838, 95% CI: 0.9387 to 1.2226, p < 0.001). Time since last immune event was inversely associated with ancestral RBD-blocking antibody levels (β = –0.0232 per month, 95% CI: –0.0385 to –0.0077, p = 0.0031). Conclusions: In this cross-sectional study, meeting WHO physical activity recommendations was associated with 41% lower odds of SARS-CoV-2 reinfection, although reverse causality cannot be ruled out. All vaccinated groups had higher ancestral RBD-blocking antibody levels than unvaccinated individuals. Older age and longer time since last immune event were associated with lower ancestral RBD-blocking antibody levels. These associations need confirmation in prospective, well-powered studies. Full article

Background: This study aimed to construct a recombinant Pseudomonas aeruginosa outer membrane vesicle (OMV) vector vaccine delivering pcrV and compare the immunological impacts of OMVs as carriers versus as adjuvants. Methods: The recombinant plasmid pBBRMCS5-pcrV was constructed and transformed into P. aeruginosa. Recombinant OMVs (OMV_PcrV) were prepared via ultracentrifugation and characterized in terms of their morphology and particle size by means of transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). After a biosafety evaluation, mice were intramuscularly immunized with PcrV or OMV_PcrV, followed by a booster immunization on day 21. On day 42, the mice were challenged subcutaneously and intranasally with PAO1. Bacterial loads in tissues and blood, pulmonary T-cell subsets, and serum antibody levels were assessed. Results: The recombinant plasmid was successfully constructed, and Western blotting confirmed the delivery of PcrV into OMVs. TEM revealed typical spherical nanostructures, and NTA showed a median particle size of 127.4 ± 5.3 nm. Upon subcutaneous challenge, the OMV, OMV_PcrV, and OMV + PcrV groups all achieved 100% protection. Both the OMV_PcrV and OMV + PcrV groups exhibited increased CD4⁺ and CD8⁺ T-cell counts and higher induction levels of specific IgM, IgG1, and IgG2a antibodies. The OMV_PcrV group showed superior clearance of respiratory bacterial colonization and reduced inflammatory injury compared with the PBS control group. Conclusions: The constructed vector successfully delivered the PcrV antigen, and the OMVPcrV vaccine induced effective immune responses. Compared with wild-type outer membrane vesicles (OMVs) and the strategy of directly mixing free PcrV antigen with OMVs (OMV + PcrV), the recombinant OMV_PcrV vaccine exhibited superior immunoprotective efficacy in terms of bacterial clearance and tissue protection, providing experimental evidence for the development of a Pseudomonas aeruginosa vaccine. Full article

Bovine viral diarrhea (BVD) is a globally significant disease that adversely affects cattle health and productivity, including in India. It is caused by three bovine pestiviruses: bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and HoBi-like pestivirus (HoBiPeV), which belong to the Pestivirus genus within the Flaviviridae family. Despite the prevalence of all three pestivirus species in India, no commercial vaccine based on the local circulating strain is currently available. This study evaluates the safety, immunogenicity, and protective efficacy of an inactivated whole-virus BVD vaccine, based on an Indian BVDV-1 strain. The virus was propagated in MDBK cells, inactivated using 3 mM binary ethylenimine (BEI) for 24 h at 37 °C, and formulated with Montanide ISA 61 VG (SEPPIC) in a 50:50 water-in-oil emulsion. Vaccine safety was confirmed in both guinea pigs and bovine calves, with no adverse effects observed. Immunogenicity testing in guinea pigs (n = 6) showed neutralizing antibody titres up to 9 log₂ (1/512). In calves aged 9–12 months (n = 3), the vaccine elicited strong humoral and cell-mediated immune responses, with mean neutralizing antibody titres against the homologous BVDV-1 strain reaching 14 log₂ (1/16,384). Neutralizing antibody levels remained detectable for up to 12 months post vaccination with sustained mean titres of 7 log₂ (1/128). Notably, titres reported to be adequate for fetal protection (≥9 log₂ or ≥1/512 were maintained for five months following vaccination. Challenge studies demonstrated complete protection of vaccinated calves against homologous BVDV-1 acute infection. In addition, the vaccine conferred partial cross-protection against heterologous strains including BVDV-2 and HoBiPeV. In a field trial involving 125 cattle, 74% of animals developed protective neutralizing titres (≥7 log₂ or ≥1/128), while 48% achieved titres reported to be adequate for fetal protection (9 log₂ or 1/512). Furthermore, 92% of vaccinated cattle maintained neutralizing antibody titres of at least 6 log₂ (≥1/64) for up to six months post-booster vaccination. A strong positive correlation was observed between guinea pig and bovine antibody responses (R² = 0.6809; p < 0.0001), indicating the potential of guinea pigs as a predictive model. Vaccine stability was confirmed for up to 8 months when stored at 4 °C, as demonstrated by the immunogenicity in guinea pigs. Collectively, these findings demonstrate that the locally developed inactivated BVDV-1 vaccine is safe, highly immunogenic, and capable of providing protective immunity against BVDV-1 infection, supporting its potential use in BVD control programs in India. Full article