Search Results (2,185)

Background/Objectives: Nipah virus (NiV) is a highly pathogenic zoonotic virus with fatality rates exceeding 70% and causes recurring outbreaks in South and Southeast Asia. Reliable serological assays are critical for outbreak surveillance, diagnosis, and evaluation of vaccine-induced immune responses. This study aimed to develop and qualify an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant NiV glycoprotein G for the detection of virus-specific IgG antibodies in human serum. Methods: An indirect ELISA was developed and optimized for antigen concentration, blocking conditions, and serum dilution. The assay performance was evaluated using convalescent human sera from Bangladesh, along with the World Health Organization (WHO) International Standard for anti-Nipah virus antibodies, maintained and distributed by the National Institute for Biological Standards and Control (NIBSC). Analytical validation was conducted in accordance with ICH Q2 (R2) guidelines, including assessments of sensitivity, specificity, Precision, Linearity, and detection limits. Results: The assay demonstrated 100% sensitivity and specificity relative to reference sera. Intra-assay coefficients of variation ranged from 0.36% to 5.73%, and inter-assay variation was 4.16%, indicating high precision. The ELISA showed excellent Linearity (R² > 0.995). The lower limit of detection was 0.51 IU/mL, and the lower limit of quantification was 0.98 IU/mL. Conclusions: The developed ELISA is a BSL-2-compatible, robust, and scalable platform suitable for serosurveillance and the assessment of vaccine-induced immunity in endemic regions. Calibration against an international standard supports its applicability for standardized antibody measurement. This assay provides a practical tool for NiV outbreak response and vaccine evaluation. Full article

Background/Objectives: Strangles, caused by Streptococcus equi subspecies equi (S. equi), remains a common and severe equine infectious disease. Strangvac^®, a recombinant fusion protein vaccine licenced in Europe, contains the antigens (Ag) CCE, Eq85, IdeE and a saponin adjuvant. Although its efficacy is high (94% in clinical trials and 100% in some natural outbreaks), immune correlates of protection have not been defined. This study determined the antibody (Ab) thresholds predictive of protection against clinical disease following high-dose experimental S. equi infection and the expected levels of protection at 6 and 12 months after V2. Methods: This study was a retrospective analysis of six independent double-blinded placebo-controlled experimental infection studies involving 129 ponies (80 vaccinated controls and 49 placebo controls) and a serology study (12 vaccinated ponies). Ponies received two to five vaccine doses before being experimentally challenged with S. equi strain Se4047. Ponies in the serology study were not experimentally infected. The onset of pyrexia (≥39 °C for at least 2 of 3 consecutive days, OOT) was used as a disease marker. Serology to IdeE, Eq85 and CCE was analysed with standardised clinical outcomes to define protective thresholds through correlation and Receiver Operating Characteristic (ROC) analyses. The predicted level of protection up to one year after V2 was then calculated (duration of immunity: DOI). Results: A protection threshold of ≥10 days to OOT, derived from the control distribution, was used for ROC modelling. Predictive performance (e.g., accuracy, precision, specificity) was calculated for individual and combined Ab thresholds. All controls developed pyrexia (median 6 days, IQR 5–7), with 46 out of 49 (93.9%) within 9 days of the challenge. Vaccinated ponies showed significantly delayed or absent OOT compared with controls (p < 0.0001), with 37 vaccinated ponies (46.25%) reaching the end of the studies without developing pyrexia. The Ab titre to all antigens was significantly associated with the level of protection (p < 0.0001). ROC analyses demonstrated high discriminative power (AUC 0.86–0.88). Optimal Ab titre boundaries yielded high precision (≥80%) for all Ags (IdeE: 3.5–4.3; Eq85: 2.65–3.7 and CCE: 2.66–3.2). Both precision and accuracy remained above 80% for levels of IdeE and Eq85 Ab titres superior or equal to those measured up to one year after V2, with an estimated level of protection of 78.9% to 81.2% in vaccinated animals. Conclusions: Ab titres to all three Ags represent robust correlates of protection against pyrexia following high-dose experimental S. equi challenge in Strangvac^®-vaccinated ponies. Ab titres measured up to one year after V2 were estimated to continue to provide significant protection in vaccinated animals. These findings support the observed levels of protection conferred by Strangvac^® against natural infection with S. equi. Full article

Background: Brucellosis is a global zoonosis caused by Brucella. Histidine biosynthesis is essential for bacterial growth, but its role in Brucella melitensis virulence remains unclear. HisD catalyzes the final two steps of histidine synthesis and is absent in mammals, making it a potential drug target. Results: We constructed a hisD deletion mutant (ΔhisD) and complemented strain (ChisD) via homologous recombination. ΔhisD failed to grow in medium without histidine supplementation. It showed reduced survival under polymyxin B and SDS stress, and impaired outer membrane integrity under polymyxin B challenge, though no defect was observed under non-stressed conditions. Intracellularly, ΔhisD replicated poorly in HeLa and RAW264.7 cells, and this defect was rescued by exogenous histidine. In a mouse model, ΔhisD exhibited lower bacterial loads in liver and spleen, reduced splenomegaly, and attenuated hepatic granuloma formation. Conclusions: Histidine biosynthesis deficiency attenuates Brucella virulence by restricting nutritional acquisition and conditionally compromising outer membrane stability. HisD is a promising target for anti-brucellosis drug development, and ΔhisD holds potential as a live attenuated vaccine candidate. 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: 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

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating swine disease. To date, no commercial ASF vaccine has been authorized for global marketing except in Vietnam, and emerging genotype I/II recombinant ASFV strains pose severe new challenges to ASF control. Live-attenuated vaccines (LAVs) are widely recognized as the most promising strategy for ASF control. This review systematically summarizes three conventional development strategies for ASF LAVs, dissects the molecular mechanisms of two core bottlenecks—intergenotypic ASFV recombination and vaccine strain reversion to virulence—and elaborates rational design strategies for next-generation LAVs based on cutting-edge technologies. These strategies can fundamentally mitigate the aforementioned risks, offering promising solutions for addressing the major limitations of conventional ASF LAVs. Full article

Background/Objectives: Canine papillomavirus (CPV) is an important viral pathogen associated with papillomatosis in dogs, with canine papillomavirus type 1 (CPV1) and type 2 (CPV2) among the most prevalent and clinically relevant genotypes. The L1 capsid protein is a major immunogenic antigen of papillomaviruses; however, conserved linear B-cell epitopes shared between CPV genotypes remain poorly defined. This study aimed to identify conserved cross-reactive B-cell epitopes within CPV1 and CPV2 L1 proteins and to evaluate their preliminary immunoreactivity. Methods: Conserved linear B-cell epitopes were predicted through integrated bioinformatic and structural analyses based on sequence conservation and surface accessibility. Three candidate epitopes were selected. Recombinant CPV1 and CPV2 L1 proteins were expressed in Escherichia coli (E. coli), purified, used as recombinant L1 antigens, together with BSA-conjugated synthetic epitope peptides for mouse immunization. Antigen-specific IgG responses were assessed by ELISA, antigen-associated IFN-γ responses were evaluated by ELISpot, and cross-reactive antibody recognition was assessed by Western blot. Results: Recombinant L1 proteins induced strong antigen-specific IgG responses in mice. The selected peptides induced detectable but weaker humoral responses compared with the recombinant L1 proteins. Among the three epitopes, TPSGSLV and TVVDNTR elicited antibodies that recognized both CPV1 and CPV2 L1 proteins, while the epitope VIVPKVS showed minimal or no detectable immunoreactivity. ELISpot analysis showed only modest antigen-associated IFN-γ responses, particularly in peptide-immunized groups. Conclusions: This study identified conserved cross-reactive linear B-cell epitope candidates within CPV1 and CPV2 L1 proteins and provided preliminary immunological evidence supporting their potential relevance for CPV antigen design. However, peptide-induced responses were weaker than those induced by recombinant L1 proteins, and VLP formation, antibody neutralizing activity, and protective efficacy were not evaluated. Further studies in dogs, including optimized antigen-display platforms, neutralization assays, and protection studies, are required to determine the practical value of these epitopes for CPV vaccine development. Full article

The extraordinary genetic diversity of human immunodeficiency virus type 1 (HIV-1), driven by high mutation and recombination rates, poses significant challenges for diagnostics, therapy, and vaccine development. While variable regions enable immune escape, hyperconserved regions are critical for viral function and represent promising targets for novel therapeutic interventions. This study aimed to develop and validate a bioinformatic algorithm for quantitative assessment of sequence conservation and automated identification of functionally significant conserved regions across all major HIV-1 proteins. A total of 1119 full-length HIV-1 genome sequences representing major subtypes (A1, A2, A6, B, C, D, F1, F2, G, H, J, K) were analyzed. Normalized Shannon entropy (S-index) was calculated for each alignment column. Statistical thresholds for conserved regions were established using 95% confidence intervals derived from bootstrap resampling. Two complementary algorithms, clustering and local maxima detection, were applied to identify conserved regions, which were subsequently mapped to known functional domains based on literature data. Protein conservation varied markedly, with S_m values ranging from 0.784 (Vpu) to 0.920 (Pol). Gag, Pol, and Vpr demonstrated the highest overall conservation, while Env, Rev, Tat, and Vpu exhibited pronounced variability interspersed with conserved domains. In total, 25 conserved regions in Gag, 49 in Pol, 28 in Env, and 6–4 regions in accessory proteins (Vif, Vpr, Rev, Tat, Nef, Vpu) were identified. These regions corresponded to critical functional elements including enzyme catalytic centers, zinc fingers, receptor-binding sites, protein interaction interfaces, and membrane-anchoring domains. The developed computational framework enables statistically grounded identification of evolutionarily constrained regions across analyzed HIV-1 subtypes. The identified conserved regions represent candidate sites for further investigation and may inform downstream studies focused on antiviral target prioritization, immunogen design, and diagnostic assay development. However, their translational applicability requires additional analytical, structural, and experimental validation. Full article

Therapeutic vaccines are a key strategy to achieve the goal of “functional cure” of chronic viral infections, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papillomavirus (HPV), and Epstein–Barr virus (EBV). Various platforms (such as viral vectors, nucleic acid vaccines, recombinant proteins, etc.) have successfully induced strong virus-specific T-cell responses in early trials, but their clinical efficacy is still limited by the immunosuppressive environment formed by the host. The core bottlenecks are severe T-cell exhaustion, viral immune escape, and various forms of local immune tolerance. Therefore, the field is moving toward combination therapies, including reduction of viral load, targeting of immune activation, and inhibition of inhibitory signaling pathways. This article summarizes the preclinical and clinical progress of therapeutic vaccines in the past decade, analyzes the major challenges in vaccine development, and discusses the future development directions in this field. Full article

Porcine epidemic diarrhea (PED), caused by the PED virus (PEDV), remains one of the most devastating diseases in the swine industry, with a mortality rate approaching 90–100% in suckling piglets due to severe dehydration and electrolyte imbalances. Passive immunization with egg yolk antibodies (IgY) represents a promising therapeutic strategy. In this study, we developed a novel recombinant adenovirus, rADM-IFN-G-ped, co-expressing selected antigenic regions of the PEDV S protein and chicken interferon-gamma (ChIFN-γ) as a molecular adjuvant. Laying hens were immunized with this construct to produce PEDV-specific IgY, which was subsequently purified from eggs using a polyethylene glycol (PEG-6000) precipitation method. The induced IgY demonstrated potent neutralizing activity against PEDV in vitro, with a neutralization titer (NT₅₀) of 1:96, which was significantly higher than that of IgY derived from hens immunized with a commercial inactivated PEDV G2b vaccine (NT₅₀ = 1:52). In a passive immunization and challenge trial, piglets treated with the rADM-IFN-G-ped-derived IgY exhibited significantly reduced fecal viral RNA shedding following challenge with the virulent PEDV-NX-2022 strain, compared to control groups. Crucially, while all piglets in the challenge control group succumbed to infection within 72 h, a 50% survival rate was achieved in the IgY-treated group. Histopathological examination of intestinal tissues further confirmed the protective efficacy, showing that IgY treatment markedly alleviated villous atrophy, epithelial necrosis, and inflammatory cell infiltration in the small intestine. These findings demonstrate that vaccination of laying hens with the rADM-IFN-G-ped recombinant adenovirus elicits a robust immune response, enabling the production of protective IgY. This proof-of-concept study establishes the viability of the multi-epitope adenoviral IgY platform as a passive immunization strategy against PEDV. Full article

Porcine enteric coronaviruses (PECs), including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), remain major causes of neonatal diarrhea, dehydration, mortality, and economic loss in swine production. Despite substantial progress in vaccine development, durable field protection is still inconsistent. In this narrative review, this narrative review synthesizes current knowledge on PEC vaccine design from three connected perspectives: antigenic breadth, mucosal immunity, and translational evaluation. The economic and virological context of PEC vaccine development is first summarized, including the recurrent production burden of PECs, coronavirus genome organization, structural proteins, and the central role of the spike protein in receptor engagement, membrane fusion, and neutralizing antibody induction. Key issues are then discussed, including how spike diversity, conformational stability, epitope accessibility, glycan shielding, and antigen matching influence protective breadth; why intestinal secretory IgA, mucosal immune-cell trafficking, local memory responses, and lactogenic immunity should be prioritized as biologically relevant endpoints; and how delivery route, adjuvant selection, and platform design shape response quality. Current evidence on recombinant protein, viral-vectored, nanoparticle, virus-like particle, probiotic, plant-derived, and mRNA-based approaches is compared with attention to both promise and current evidentiary and translational limitations. The available literature suggests that future progress in PEC vaccinology is likely to depend less on platform novelty alone than on integrated vaccine designs that align antigen selection, mucosal delivery, maternal–neonatal protection, heterologous challenge, manufacturability, and field applicability. Full article

Background/Objectives: Respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza virus type 3 (PIV3) are leading causes of acute respiratory infections in children and the elderly, yet no licensed T-cell vaccines are available. This study aimed to develop multivalent T-cell vaccine candidates against these pathogens using a live attenuated influenza virus (LAIV) vector platform. Methods: Conserved F, N, and M proteins of RSV, hMPV, and PIV3 were identified through multiple sequence alignments. Fragments enriched with experimentally confirmed and predicted T-cell epitopes were selected using the IEDB and NetMHCpan servers. These fragments were assembled into polyepitope immunogenic cassettes, and their selected order was determined by thermodynamic analysis of mRNA secondary structures using the RNAfold Web Server. The selected cassettes were cloned into the neuraminidase (NA) gene of a cold-adapted LAIV vector. Recombinant viruses were rescued by reverse genetics and assessed for replicative fitness in embryonated chicken eggs and MDCK cells, NA enzymatic activity and genetic stability upon serial passaging. Results: Four cassettes were designed for RSV, three for hMPV, and one for PIV3, all containing fragments with multiple T-cell epitopes. Three recombinant viruses of LAIV/RSV type and three of LAIV/hMPV type were successfully rescued, while attempts to recover the remaining recombinant viruses, i.e., LAIV/RSV and LAIV/PIV3, were not successful. All rescued recombinant viruses replicated to titers comparable to the parental LAIV strain and retained the full-length insert for at least eight passages in eggs. Importantly, NA enzymatic activity of the LAIV vector was not compromised by the insertion of the polyepitope T-cell cassettes. Conclusions: We developed a panel of recombinant T cell-based vaccine candidates against RSV and hMPV using the LAIV vector platform. These recombinant viruses encode conserved T-cell epitopes of the target viruses while retaining the biological properties of LAIV strains. Taken together, these characteristics warrant further evaluation of these recombinant viruses in appropriate relevant in vitro models to directly assess their immunogenicity in terms of stimulating a T-cell response against target pathogens. Full article

Porcine circovirus type 3 (PCV3) capsid protein (Cap) is a key antigen for immunological studies and vaccine development. Different optimized PCV3 ORF2 sequences were used to construct six baculovirus transfer plasmids, with the pOET1.1-based design yielding the highest Cap level. Cap expression was confirmed by Western blot, IPMA and IFA. Recombinant baculovirus amplification was optimized, achieving the highest titer at an MOI of 0.1 with a 72 h harvest to 10^7.5TCID₅₀/0.1 mL, while maximal Cap production was obtained at an MOI of 0.1 with a 96 h harvest. PCV3 Cap virus-like particles (VLPs) were purified by sucrose density-gradient ultracentrifugation and cation-exchange chromatography, and TEM revealed spherical particles of approximately 17–20 nm. In mice, VLP immunization induced increasing antigen-specific IgG from day 14. Immunization increased both IgG1 and IgG2a without a significant difference, and post-immunization serum specifically recognized PCV3-positive passaged PK-15 cells in an indirect immunofluorescence assay. In splenic lymphocytes, IFN-γ, TNF-α, IL-4, and IL-10 mRNA levels were significantly upregulated (p < 0.01). Moreover, pig challenge data supported the protective potential of PCV3 Cap VLPs in the natural host. In our study, Cap assembled into VLPs and induced immune responses, providing a basis for PCV3 subunit vaccine development. Full article

The Getah virus (GETV) is a mosquito-borne pathogen that infects diverse hosts, including pigs, horses, and humans, which can cause swine reproductive disorders such as abortion and stillbirth, posing a potential threat to animal and public health. Therefore, there is an urgent need for efficient and accurate serological diagnostic methods for surveillance and control of GETV. However, commercial diagnostic kits for swine GETV infection remain unavailable. In this study, we developed a novel enzyme-linked immunosorbent assay (ELISA) based on a GETV-specific epitope peptide (E2EP3) for serological detection. The N-terminally biotinylated E2EP3 peptide was synthesized, and the reaction conditions were systematically optimized, resulting in a cut-off value of 0.363. The assay exhibited no cross-reactivity with Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), pseudorabies virus (PRV), or classical swine fever virus (CSFV). It demonstrated good reproducibility and high sensitivity, detecting GETV-positive serum diluted up to 1:640. The overall agreement rate reached 95%, consistent with a conventional recombinant GETV E2 protein-based ELISA. Benefiting from the biotin–streptavidin system, this assay achieved strong signal amplification and low background. Moreover, the procedure is simple, cost-effective, and stable, making it suitable for GETV large-scale serological surveillance and vaccine evaluation. Full article

As a naturally self-assembling protein nanocarrier, ferritin enables multivalent antigen display and functions as an intrinsic adjuvant to enhance vaccine-induced immune responses. Streptococcus equi subsp. equi (S. equi) is the causative agent of equine strangles, an acute and highly contagious respiratory disease responsible for substantial economic losses worldwide. However, currently available vaccines often show suboptimal immunogenicity and limited protective efficacy. In this study, we developed a recombinant equine ferritin (rHF)-based nanoparticle vaccine, rSE5Mix, presenting five core protective antigens (EQ8, EQ5, CNE, IdeE, EAG). The fusion proteins efficiently assembled into uniform nanoparticles. Immunization of BALB/c mice elicited rapid, high-titer antigen-specific IgG antibodies and a balanced Th1/Th2 immune response without additional adjuvants. Following lethal challenge with S. equi, rSE5Mix-immunized mice showed 100% survival and markedly milder clinical signs. Histopathological analysis demonstrated significantly alleviated organ damage, and bacterial loads in major tissues were reduced to nearly undetectable levels. Importantly, compared with the octavalent tandem vaccine rSE8, rSE5Mix induced faster elevation of partial antigen-specific antibodies, especially for EQ8 and CNE. Their antibody titers were comparable at later stages. This study developed a safe and effective ferritin nanoparticle vaccine candidate against equine strangles and verified that equine ferritin is a promising candidate delivery platform for veterinary bacterial vaccines. Full article