Salmonella enterica serovar Infantis is an emerging zoonotic pathogen increasingly linked to poultry and multidrug-resistant human infections. Existing vaccines lack serovar-specific efficacy, underscoring the need for targeted immunization strategies. In this study, we employed an immunoinformatics and reverse vaccinology pipeline to design a multiepitope subunit vaccine (MEV) against S. Infantis. A subtractive proteomics analysis of 692 poultry-derived genomes identified CsgA as a highly conserved, immunogenic, and non-host homologous antigen. Selected cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes were filtered for antigenicity, immunogenicity, allergenicity, and sequence conservation. The epitopes were assembled with appropriate linkers and a cholera toxin B (CTB) adjuvant. Structural modeling and normal mode analysis confirmed the construct’s stability, while molecular docking predicted high-affinity interactions with chicken TLR2, TLR5, MHC-I, and MHC-II. Immune simulations indicated robust humoral and cellular responses. Codon optimization yielded a codon adaptation index (CAI) of 1.0 and GC content of 54.76%, supporting efficient expression in E. coli. The optimized construct was successfully cloned in silico into the pET30a(+) vector. These findings present a rationally designed, computationally validated vaccine candidate with the potential to induce protective immunity against S. Infantis, warranting further experimental validation.

Introduction. Vibrio cholerae is a diverse species of bacteria that causes watery diarrhoea, vomiting and stomach cramps and is the aetiological agent of cholera.Gap statement. Despite the global upsurge in notifications of cholera and concerns over the impact of climate change, systematic analysis of national and international surveillance data describing the microbiology and epidemiology of V. cholerae is sparse.Aim. We reviewed the microbiology and epidemiology of V. cholerae isolated from travellers returning to the UK.Methodology. All human isolates of V. cholerae detected from 2004 to 2024 were extracted from UK Health Security Agency reference laboratory surveillance systems. Microbiological data were analysed and linked to available epidemiological data and genome sequences for all isolates from 2016 to 2024.Results. There were 984 notifications of V. cholerae from 2004 to 2024 (an average of 51 each year), of which 266 (27.0%) belonged to serogroup O1. There were over 180 different sequence types (STs), of which cholera toxin producing ST69 was the predominant type (n=99, 28.2%). The highest number of isolates was in 2010 (n=74), while the lowest was in 2020 (n=8) and 2021 (n=4) due to travel restrictions imposed during the COVID-19 pandemic. Children under the age of 10 and the middle-aged and elderly population were most susceptible to infection, and 51.6% of the cases were male. There was a seasonal peak in August and September. Travel was reported by 92.9% of cases, and the most frequently reported travel destinations were India, Pakistan and Kenya.Conclusion. From the UK perspective, to assess the risk to food safety and to more accurately determine the clinical burden of V. cholerae, we recommend (i) widespread molecular testing of shellfish to monitor the emergence of V. cholerae in UK waters due to climate change and (ii) comprehensive testing of faecal specimens from non-travellers with gastrointestinal symptoms. Public health surveillance and information sharing at the global level is essential to assess the impact of investment in water, sanitation and hygiene initiatives for the prevention of cholera.

Cholera and typhoid fever are often co-endemic, making vaccine coadministration practical. However, due to lack of immunogenicity data, current guidelines advise against coadministration of the oral inactivated whole-cell recombinant cholera toxin B-subunit vaccine (WC-rCTB) and the oral live Salmonella Typhi Ty21a vaccine. Healthy adults (18-65years) were randomized 1:1:1 to receive WC-rCTB with Ty21a (group Ch + Ty), WC-rCTB alone (group Ch) or Ty21a alone (group Ty). Peripheral blood mononuclear cells (PBMC) were isolated on Days 0, 5 and 7 from all, plus on Days 12 and 14 from WC-rCTB recipients, to assess antibody-secreting cells (ASC) specific to rCTB and to typhoidal O9,12-structures by ELISPOT. Vibriocidal antibodies were assessed, and anti-rCTB IgA/IgG and anti-S. Typhi lipopolysaccharide (LPS) IgA/IgG/IgM were measured by ELISA in Day 0 and 28 ± 3 serum samples. Adverse events (AEs) were recorded during one month. The final study population included 63 volunteers, 21 per group. A nonsignificant trend toward stronger rCTB-specific ASC (IgA + IgG + IgM) peak responses were observed in group Ch + Ty compared to group Ch (geometric mean, GM 94 versus 32 ASC/106 PBMC, p = 0.096). Serum anti-rCTB IgA and IgG fold-rises (postvaccination versus prevaccination) were higher in group Ch + Ty than in group Ch (IgA p = 0.039, IgG p = 0.028), whereas vibriocidal fold-rises were comparable between the two groups (p = 0.847). ASC (IgA + IgG + IgM) peak responses to typhoidal O9,12-structures were comparable between groups Ch + Ty and Ty (GM 183 versus 210 ASC/106 PBMC, p = 0.684). Serum anti-S. Typhi LPS IgA, IgG and IgM fold-rises were also similar across Ch + Ty and Ty groups (all p-values ≥0.145). AEs were comparable in single and coadministration groups. Coadministration of the oral cholera and typhoid vaccines demonstrated favourable safety and robust immunogenicity for both vaccines, supporting their simultaneous use without spacing precautions.

EPICERTIN, an engineered variant of cholera toxin B subunit, promotes survival and a pro-remodeling macrophage phenotype for mucosal healing in colitis.

Dual-Copy VP2 expressed with CTA1-DD in transgenic Eimeria acervulina confers partial protection against infectious bursal disease virus.

Abstract Background Impaired microbial indole production and decreased activation of the aryl hydrocarbon receptor (AhR) pathway has been reported in active coeliac disease (CeD). However, whether and how this can be targeted by specific microbial therapies is unknown. Aims We tested whether Saccharomyces boulardii CNCM I-745 (S. bou) influences indole production and AhR activation through stimulation of microbial enzymatic activity. Methods Transgenic NOD/DQ8 mice were immunized with partially digested gliadin and cholera toxin weekly for 3 weeks followed by gluten challenges (10 mg; alternate days/ 3 weeks). Non-sensitized mice were used as controls. Mice were treated daily with 3 g/kg of S. bou or water during the study. Additional immunized mice treated or not with S. bou, received daily gavage with AhR antagonist CH-223191 (10 mg/kg) or vehicle. Small intestinal pathology was analyzed by villus to crypt (V/C) ratios and CD3+ intraepithelial lymphocyte (IEL) counts. Expression levels of the AhR-activated gene Cyp1a1 were determined by RT-qPCR. Small intestinal microbiota of NOD/DQ8 mice and CeD patients were cultured in BHI media with and without 1x106 S. bou CFUs or 1x108 Lactobacillus reuteri (L. reuteri) CFUs. Free tryptophan levels, AhR activation, and total protein degradation were assayed using a commercial kit, a transfected reporter cell line, and an enzymatic assay respectively. Resulting microbiota was assessed by 16S rRNA gene sequencing and PICRUSt-predicted functions. Results S. bou increased Cyp1a1 levels, protein degradation, mucosal tryptophan and AhR activation in gluten immunized mice, paralleled by improved V/C ratios and lower IELS vs no probiotic, which was inhibited by AhR antagonist. In immunized mice treated with S. bou, microbiota analysis revealed lower PICRUSt-predicted tryptophan 2,3-dioxygenase genes, a tryptophan degrader. Mouse duodenal microbiota cultures treated with S. bou had higher total protein degradation, higher tryptophan levels and AhR activation vs cultured with no probiotic. In duodenal microbiota cultures from CeD patients the combination of S. bou and L. reuteri increased AhR activation vs cultures without S. bou. Conclusions S. bou CNCM I-745 reduced gluten immunopathology in immunized NOD/DQ8 mice through activation of the AhR pathway. In vitro experiments suggest S. bou synergizes with the microbiota to increase free tryptophan from protein sources and inhibit tryptophan degradation, leading to higher indole production and AhR activation. The results identify a specific mechanistic pathway by S. bou CNCM I-745 in celiac disease and should encourage clinical testing in this population. Funding Agencies CIHRBiocodex, France

Tunable triazole-based cholera toxin inhibitors: A QSAR-guided design and evaluation approach.

The aim of the study was to obtain a cholera toxin B-subunit producer based on the avirulent probiotic strain Escherichia coli M-17 and to study its molecular-genetic and immunogenic properties. Materials and methods . The genetically marked E. coli KS164 thy polA (pIEM3) strain, containing a recombinant plasmid with the cloned ctxB gene encoding the B-subunit of cholera toxin, and the avirulent E. coli M-17 strain, applied for the production of colicontaining probiotics, were used. The strains were analyzed using various methods: molecular genetics, biochemistry, and immunology. Results and discussion . Through introducing the recombinant cointegrate plasmid pIEM3 (KmRTcR) with the cloned ctxB gene into E. coli M-17 cells via conjugation, KmRTcR transconjugants with high levels of secreted cholera toxin B-subunit production (10 μg/ml) were obtained. One of these transconjugants (KM147) was used as the producer strain for the protective antigen. It was demonstrated that the constructed E. coli strain KM147(pIEM3) KmRTcR stably inherits the cloned ctxB gene contained in the recombinant plasmid, is capable of producing antitoxic antibodies in immunized animals, and safe for the use in model animals. These properties combined make it promising for future use in the development of prophylactic and diagnostic cholera drugs.

Background: Naegleria fowleri is a free-living amoeba that inhabits warm freshwater and causes primary amoebic meningoencephalitis (PAM), a rapidly fatal infection with >95% mortality. Due to the lack of early diagnosis and effective therapy, preventive vaccination represents a promising strategy. Methods: This study evaluated short- and long-term immune protection in BALB/c mice (20 mice per group) immunized intranasally with total N. fowleri extract co-administered with cholera toxin (CT). Mice were challenged with a lethal dose of trophozoites either 24 h (short-term) or three months (long-term) after the fourth immunization; the latter group received a booster 24 h before challenge. Serum and nasal washes were analyzed for IgA and IgG antibodies by immunoblot, and lymphocyte subsets from nasal-associated lymphoid tissue (NALT) and nasal passages (NPs) were characterized by flow cytometry. Results: Immunization conferred complete (100%) survival in the 24 h group and 60% protection in the 3-month group, whereas all control mice died. Immunoblotting showed that IgA and IgG antibodies recognized major N. fowleri antigens of 37, 45, 48 and 19, 37, and 100 kDa, respectively. Flow cytometry revealed increased activated and memory B lymphocytes, dendritic cells, and expression of CCR10, integrin α4β1, and FcγRIIB receptors, particularly in the 24 h group. Conclusions: Intranasal immunization with N. fowleri extract plus CT elicited both systemic and mucosal immune responses capable of short- and long-term protection. These findings highlight the potential of this immunization strategy as a foundation for developing effective vaccines against PAM.

Safety and immunogenicity of PanChol, a single-dose live-attenuated oral cholera vaccine: results from a phase 1a, double-blind, randomised, placebo-controlled trial.

ABSTRACT Vibrio cholerae (V. cholerae) is a type of bacterium that causes cholera, a severe diarrheal disease globally affecting hundreds of people annually. However, the effect of the V. cholerae toxin on oyster metabolite signatures has not been well studied. In this study, nuclear magnetic resonance (NMR) based metabolomics was applied to investigate the metabolic level response of eastern oysters (Crassostrea virginica) to cholera toxin (CT), under low concentrations. Our study demonstrated that the decrease of branched-chain amino acids (BCAAs) in oysters was a response to CT exposure at low concentrations (10 ng/mL) in gill and mantle extracts. Metabolites such as leucine and isoleucine were significantly decreased in gills with toxin exposure at 10 ng/mL, and similar but weaker changes were also observed at 1 ng/mL, indicating an early response to CT. However, the trend reversed at 20 ng/mL, with acetate and propionate significantly increased over control (p < 0.07), which is a sign of antioxidant defenses that could help the recovery of the BCAAs. In the hemolymph study, acetate and propionate levels correlated strongly with those in the tissue extracts at 20 ng/mL, suggesting that hemolymph metabolites begin contributing to gill metabolic perturbations. More importantly, a principal component analysis (PCA) also revealed a partial separation between the control and the 20 ng/mL CT group, indicating potential major perturbations in hemolymph metabolites. This study provides evidence that metabolites in oyster tissues resulting from exposure to Vibrio toxin can serve as a new early warning system for predicting potential human pathogen risks in both environmental and seafood exposure.

Human papillomavirus (HPV) is a major causative agent of cervical and other mucosal cancers. However, the distribution and accessibility of current prophylactic vaccines remain limited, especially in low- and middle-income countries (LMICs), due to high production costs, cold-chain dependency, and limited induction of mucosal immune responses. To addressing these challenges, we designed a rationally constructed multi-epitope HPV vaccine (HPV_MEV) incorporating conserved cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes from diverse high- and low-risk HPV genotypes. The construct includes the Toll-like receptor 4 (TLR4) agonist RS09 to enhance innate immune activation and cholera toxin B subunit (CTB) as a mucosal adjuvant to facilitate uptake and presentation at mucosal surfaces. The codon-optimized gene was stably integrated into the chloroplast genome of Nicotiana tabacum via biolistic transformation. Molecular analyses confirmed site-specific integration, homoplasmy, and high-level antigen accumulation (~3.6 mg/g fresh weight; ~20.8% of total soluble protein). Immunogenicity was evaluated in BALB/c mice following intraperitoneal administration of purified antigen or oral gavage of lyophilized transplastomic leaf tissue. Oral administration induced antigen-specific systemic IgG and mucosal IgA responses, with higher levels of vaginal IgA observed compared with parenteral delivery, reflecting enhanced mucosal antibody induction. The chloroplast-produced HPV_MEV exhibited immunogenicity comparable to its E. coli-expressed counterpart, supporting its structural and functional integrity. Overall, this study demonstrates the feasibility of plastid biotechnology as a platform for producing a thermostable, orally deliverable HPV vaccine candidate and provides preliminary immunogenicity data supporting its application as a promising vaccine candidate for accessible vaccination strategies against HPV and other mucosally transmitted pathogens in resource-limited settings.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13036-025-00618-5.

ABSTRACT Bacterial enteric pathogens are major contributors to the global burden of diarrheal diseases and the associated consequences for human health including malnutrition, growth stunting, morbidity, and mortality. While mortality from diarrhea has decreased, incidence remains high, and better interventions for preventing disease are needed. Single-domain antibodies (i.e., VHHs), functioning as target-binding proteins in the gastrointestinal tract, have been proposed as a potential approach to mitigate bacterial pathogenesis. Here, we describe a mitigation strategy where precision binding of a bivalent VHH to the receptor-binding B-pentamer of heat-labile enterotoxin aggregates the AB5 toxin and impairs enterotoxigenic Escherichia coli colonization in a flow chamber model simulating the human intestine. The VHH construct also binds to the structurally similar cholera toxin and effectively abrogates its intestinal cell cytotoxicity in vitro. Based on these results, we believe that targeting virulence could emerge as a new strategy for the management of bacterial enteric pathogens, supporting gut health in at-risk populations alongside vaccination campaigns or in populations without access to vaccines.

Introduction. Cholera, caused by Vibrio cholerae, remains a priority public health concern, particularly in developing countries. The first cholera outbreak in Zambia was documented in the 1970s, with recurring epidemics reported since then. In 2023, a cholera outbreak affected Zambia, particularly in districts bordering Malawi, Mozambique and the Democratic Republic of Congo, with significant cases reported in these neighbouring countries. This study aims to analyse cholera cases and isolates obtained during the 2023 epidemic, focusing on geographical distribution, genetic relatedness of isolates and their antibiotic resistance profiles.Methods. Stool samples were collected from patients presenting with cholera-like symptoms across three provinces of Zambia. A total of 98 samples were cultured on thiosulphate citrate bile salts sucrose agar, resulting in 32 sequenced V. cholerae isolates. Whole-genome sequencing was performed using Oxford Nanopore Technology, and phylogenetic inference was also achieved by the analysis of SNPs. Phenotypic antimicrobial resistance testing was conducted following Clinical and Laboratory Standards Institute guidelines. The genomic data were analysed for virulence factors and antimicrobial resistance profiles.Results. Of the 98 stool samples tested, 38 confirmed cholera cases were identified. A subset of 32 confirmed V. cholerae isolates, predominantly from the Eastern Province of Zambia (n=21), was selected for whole-genome sequencing. Genomic analysis revealed that all isolates belonged to the seventh pandemic El Tor lineage and the O1 serogroup, with two distinct clades identified corresponding to the 10th (T10) and 15th (T15) transmission events. Geographical analysis indicated a predominance of Ogawa serotypes in Eastern Province and Inaba in Northern Province. The virulence gene analysis confirmed the presence of key cholera toxin genes (ctxA and ctxB) and intestinal colonization factors. All isolates carried genes or mutations predicted to confer resistance to multiple antibiotics, including decreased susceptibility to ciprofloxacin, recommended for the treatment of cholera by the World Health Organization.Conclusion. The findings highlight the critical need for enhanced surveillance and targeted interventions to mitigate cholera outbreaks in Zambia. The emergence of resistant V. cholerae strains necessitates innovative strategies, including improved water sanitation, vaccination efforts and novel therapeutic approaches to combat this enduring public health threat.

BackgroundOur preliminary work revealed earlier anxiety and cognitive decline in female Alzheimer's disease (APP/PS1) mice which correlated to an unbalanced brain‐wide network. To validate these preclinical findings, we analyzed the impact of anxiety on AD in human subjects using the Alzheimer's disease neuroimaging (ADNI) dataset. Our analysis showed that that 1) female AD subjects exhibited higher anxiety; 2) female subjects with amyloid deposition transition to dementia at a faster rate compared to male subjects; 3) female subjects with anxiety have smaller brain volumes; and 4) anxiety is the best predictor of dementia transition. Here, we examine the impact of treating mice with anxiolytics, dissect the circuitry controlling anxiety, and determine whether menopause accelerates AD pathology.MethodAlprazolam (Xanax) was injected 30 minutes prior to a contextual fear memory task in control male and female mice. We then took brain tissue 60 minutes after testing to measure neuronal ensembles activated within the hippocampus. In control and AD (APP/PS1) mice, Cholera Toxin B, was injected into the amygdala and lateral hypothalamus. We measured projections to the ventral hippocampus at 2 and 6 months of age. Lastly, we induced menopause using a chemical, VCD, which depletes ovarian follicles similar to the human transitions. We then ran these mice through a battery of anxiogenic behavior paradigms and examined memory.ResultWe found that Alpazolam‐treated male and female mice exhibit a decrease in memory retention, most likely the result of less retrieval cell activation in ventral hippocampus. In mice transitioning through perimenopause we observed earlier anxiety‐like behavior and cognitive decline, but this decline rebounded in postmenopause suggesting a compensatory mechanism. We predict that there will be an increase in the number of projections in ventral hippocampus in female AD mice compared to controls and males.ConclusionThese findings suggest that Neuropsychiatric symptoms impact cognitive decline through sex‐specific pathways in the hippocampus and that benzodiazepines may not be beneficial when treating Alzheimer's disease patients. Menopause can also induce this earlier anxiety‐like phenotype, but the brain may compensate for these hormonal changes. Future studies will examine the impact of chronic BZD use on aging and Alzheimer's disease.

Background: The immunogenicity of polysaccharide conjugate vaccines is critically influenced by the choice of carrier protein, which promotes a T-cell-dependent immune response mechanism leading to strong antibody production. In this study, the cholera toxin B subunit (CTB), a non-toxic pentameric protein, was evaluated as a novel carrier protein for pneumococcal polysaccharide antigens. Methods: Recombinant CTB was produced in Escherichia coli and purified using scalable chromatographic methods. Pneumococcal polysaccharides from serotypes 7F, 22F, and 33F were chemically activated with CDAP and conjugated to CTB. Results: The resulting glycoconjugates were characterized by SEC-MALS, confirming successful conjugation, high molecular weights, consistent polysaccharide-to-protein ratios, and acceptable endotoxin levels. Immunogenicity was assessed in rabbits following immunization with alum-adjuvanted formulations. Results: Robust IgG responses were elicited by all CTB-based conjugates, with antibody levels found to be comparable to those induced by CRM197 conjugates, demonstrating the potential of CTB as a promising alternative for the next generation of conjugate vaccines.

We previously identified Alcaligenes as symbiotic bacteria residing within Peyer's patches and demonstrated that their primary components, lipopolysaccharides, and their active center, lipid A, are excellent adjuvants for mucosal vaccination. Here, we evaluated the effectiveness of Alcaligenes-derived lipid A as an adjuvant for sublingual immunization, a novel vaccination route. Mice sublingually immunized with Alcaligenes lipid A and ovalbumin (OVA) showed enhanced production of OVA-specific IgA in both the respiratory and gastrointestinal tracts. In addition, increased serum levels of OVA-specific and IgG antibodies were elicited through germinal center reactions in the draining lymph nodes without excessive inflammation at the administration sites. These results demonstrated superior efficacy not previously achieved through other routes of administration (e.g., intranasal, subcutaneous, intramuscular administration) or by existing adjuvants (e.g., CpG-ODN). In addition, sublingual immunization with cholera toxin B subunit (CTB) and lipid A led to an elevated CTB-specific IgG response in the systemic compartment and an elevated IgA response in the intestinal tract, effectively suppressing the diarrhea induced by oral challenge with cholera toxin. Furthermore, immunization with pneumococcal surface protein A (PspA) plus Alcaligenes lipid A elicited strong PspA-specific CD4+ T cell proliferation and Th17 responses, as well as IgA and IgG responses, in both the respiratory tract and the systemic compartment. These effects enhanced pneumococcal clearance in the lungs and subsequent protection against Streptococcus pneumoniae infection. Together, our findings suggest that Alcaligenes-derived lipid A is a potent sublingual vaccine adjuvant with potential efficacy against both respiratory and intestinal infectious diseases.

Background/Objectives: Sublingual vaccination offers a non-invasive route for inducing both systemic and mucosal immunity, yet the formulation properties that govern its success remain poorly defined. This study investigated the relationships among key formulation parameters for sublingual vaccines, such as viscosity, mucoadhesion, and mucosal residence, to understand their impact on in vivo immune responses in the sublingual delivery context. Methods: Ovalbumin (OVA)-based vaccine formulations containing cholera toxin B (CTB) adjuvant and mucoadhesive excipients such as hydroxypropyl methylcellulose (HPMC) or methylglycol chitosan (MGC), were evaluated for: (1) their respective rheological properties-characterized by viscosity and mucoadhesion parameters, as well as (2) in situ mucosal retention (assessed using Cy7-labeled formulations tracked by IVIS in vivo imaging system) and (3) in vivo immunogenicity via systemic (IgG) and mucosal (IgA) responses measured by ELISA, following sublingual administration to mice. Correlations between rheology, in situ/ex situ mucosal residence, and in vivo immune outcomes were determined. Results: Sublingual vaccine formulations containing HPMC exhibited the highest viscosity, mucoadhesion, and mucosal retention profiles, but paradoxically elicited the weakest systemic and mucosal antibody responses. In contrast, chitosan-based formulations enhanced immune responses even at reduced antigen and adjuvant doses, likely due to its permeation-enhancing and adjuvant effects. Correlation analyses revealed that while formulation viscosity and mucoadhesive strength were positively associated with mucosal retention, both rheological and retentive properties showed a significant inverse relationship with immunogenicity in the context of sublingual vaccine delivery. Conclusions: While viscosity and mucoadhesion are essential for in situ retention of sublingual vaccines, prolonged residence driven by excipient's excessive rheological strength was found to reduce vaccine immunogenicity-likely due to restricted antigen release and mucosal uptake. Accordingly, HPMC appears suboptimal as a sublingual vaccine excipient, while chitosan shows promise for sublingual delivery as a permeation-enhancing adjuvant. These findings may shift the design paradigm for sublingual vaccine formulations, highlighting the need to balance mucosal retention with efficient antigen absorption for maximizing immune responses.

Brucellosis, caused by the intracellular pathogen Brucella, remains a significant health challenge, alongside substantial economic impacts on livestock industries. Despite antibiotic treatments, the absence of licensed human vaccines necessitates innovative preventive strategies. In this study, we employed reverse vaccinology to design a novel multi-epitope vaccine (MEV) targeting Brucella melitensis. Three immunogenic proteins—outer membrane protein OMP31, LPS assembly protein LptE, and the type IV secretion system protein VirB2—were selected as vaccine candidates. Comprehensive bioinformatics analysis identified six cytotoxic T lymphocyte (CTL) epitopes, nine helper T lymphocyte (HTL) epitopes, seven linear B-cell epitopes, and five conformational B-cell epitopes. The incorporation of molecular adjuvants (cholera toxin B subunit and PADRE) served to further enhance the immunogenicity of the vaccine. Given that Brucella is an intracellular parasite, TAT cell-penetrating peptides were added to further enhance the intracellular delivery of MEV. The constructed MEV has been shown to have excellent antigenicity (VaxiJen score >0.8), stability (instability index <40), solubility (Protein-Sol score: 0.87) and hydrophilicity (GRAVY index: −0.319), and is non-allergenic. Structural optimization, including disulfide bond engineering (11 pairs of residues), improved molecular stability, with molecular docking and dynamics simulations confirming robust interactions with immune cell receptors (docking score: −311.85). Using SnapGene 7.1.2, we performed in silico cloning simulation of the codon-optimized multi-epitope vaccine (MEV) sequence into the pMV261 shuttle vector, generating a recombinant BCG (rBCG) construct. Immunoinformatics simulations (C-ImmSim) demonstrated potent immune activation, with significant increases in cytotoxic T cells (1050 cells/mm³), memory helper T cells (1150 cells/mm³), and IFN-γ production (2 × 10^6 ng/ml), alongside sustained IgG/IgM titers over 350 days(1 × 10^5 cells/mm3) . Furthermore, the recombinant BCG multi-epitope Brucella vaccine, developed through bioinformatics approaches, demonstrates promising characteristics and immunogenicity. Nevertheless, its immunological efficacy requires to further experimental validation.

The present study investigates the molecular mechanisms of peripheral nerve regeneration by examining the ROCK/PI3K/Akt/GSK3β pathway's role in promoting morphological and functional recovery after peripheral nerve injury (PNI). Using a mouse sciatic nerve crush (SNC) injury model and a dorsal root ganglion (DRG) explant axotomy model, mice and DRG were divided the experimental (treated with DMSO) group, Y27632 group (treated with ROCK inhibitor Y27632), Y + LY group (treated with Y27632 + PI3K inhibitor LY294002), and Y + LY + SB group (treated with Y27632 + LY294002 + GSK3β inhibitor SB216763). Immunofluorescence was used to assess axon density, diameter, myelin thickness and Schwann cell proliferation, while retrograde tracing with cholera toxin subunit B evaluated peripheral-to-central reconnection. Behavioral tests measured functional recovery, and in DRG explants, axon regeneration length and growth cone size were quantified. Protein expression analysis of RhoA, ROCK, PI3K, Akt, GSK3β, and their phosphorylated forms was conducted on day 3 post-axotomy, both in vivo and in vitro. Additionally, RSC96 Schwann cell migration and proliferation were evaluated using scratch assays and EdU staining. Results showed that ROCK inhibition with Y27632 significantly enhanced axonal regeneration, growth cone expansion, retrograde transport, and reinnervation of acetylcholine receptors and Merkel cells, and promoted Schwann cell proliferation and RSC96 migration, leading to thicker myelin sheaths after SNC. These changes mitigated gastrocnemius muscle atrophy, improved muscle strength, gait, and thermal/tactile sensitivity. Co-treatment with LY294002 blocked these effects, but adding SB216763 restored them. Protein analysis indicated that ROCK inhibition increased phosphorylated PI3K, Akt and GSK3β, whereas PI3K inhibition reduced GSK3β phosphorylation. These findings suggested that ROCK inhibition promotes axon regeneration and remyelination after PNI by enhancing PI3K/Akt phosphorylation and suppressing GSK3β activity, highlighting the therapeutic potential of targeting the ROCK/PI3K/Akt/GSK3β pathway for peripheral nerve repair.