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Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens causing severe human infections including hemorrhagic colitis and hemolytic uremic syndrome, particularly in children. Ruminants are the main reservoir of EHEC which colonize their intestinal tract through a mechanism involving the bacterial adhesin intimin. Vaccination of cattle has shown efficacy in reducing EHEC O157:H7 shedding in feces. However, most of these vaccines rely on purified proteins and/or adjuvants, making them expensive and not used by breeders. This study introduced the development of a new type of vaccine based on Outer Membrane Vesicles (OMVs) carrying the C-terminal domain of intimin (Int280). A vaccine which combines OMVs carrying luminal Int280 and OMVs displaying surface-exposed Int280 was produced using two addressing systems based on PelB peptide signal and Lpp-OmpA hybrid protein, respectively. Dot blot experiments on OMVs combined with FAS assay with bacteria confirmed the correct localization of the fusion proteins and the functionality of Lpp-OmpA-Int280, respectively. As a proof of concept, the efficiency of the mixed vaccine was tested in a mouse model using the pathogen Citrobacter rodentium which shares a similar intimin-based adhesion mechanism with EHEC. Intraperitoneal vaccination of mice, at two-week intervals with 1 μg of the mixture of OMV-Int280, elicited a strong anti-intimin IgG response. Interestingly, we observed a shortened C. rodentium fecal shedding duration in immunized mice compared to the control unvaccinated group, with significant reduction of C. rodentium colonization from day 14 (q < 0.0001) to day 18 (q = 0.0068). This OMV-Int280 vaccine therefore represents a promising candidate for the control of EHEC intestinal carriage and fecal shedding in ruminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pasteurella multocida (P. multocida), a pathogenic bacterium known to induce duck cholera, stands as a significant contributor to bacterial diseases afflicting the duck industry, causing substantial annual economic losses on a global scale. In this study, the genes encoding the lipoproteins PlpE of P. multocida strain PMWSG-4 was cloned, inserted into the pBAD-ClyA vector, and the recombinant outer membrane vesicles (OMVs) fused with PlpE antigen of P. multocida was expressed by Escherichia coli (E. coli). Ducks immunized with OMV-PlpE had significantly (P < 0.001) increased production of antigen-specific antibodies. Moreover, at 28 days post-immunization, the expression of genes associated with immune response, including interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ in the spleen tissue of immunized ducks were significantly (P < 0.001) up-regulated compared to unimmunized ducks in the control group. And the active serum had significant bactericidal effects against the PMWSG-4 strain (P < 0.001). The protective efficacy of the vaccines was evaluated by leg muscle challenge with 20 LD50 doses of P. multocida, with the recombinant OMV-PlpE conferring 100 % protection. Histopathological examination and tissue bacterial load detection revealed that OMV-PlpE mitigated tissue damage and bacterial colonization to a statistically significant extent (P < 0.001). These findings serve as a valuable reference for the development of vaccines against P. multocida., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests in relation to this study., (Copyright © 2024. Published by Elsevier Ltd.)

Bacterial extracellular vesicles (EVs) are cytosol-containing membrane spheres providing a chassis for the removal and delivery of cargoes in a highly dynamic and cue-responsive manner. EVs play important roles in cell-to-cell communication, including the dialogue between recipient microbial and plant cells. Bacterial EVs are well-studied in the medical field, but their relevance for plant infection is only now being recognized. Recent studies have demonstrated the role of EVs from phytobacteria in modulating plant immunity and the outcome of disease or in symbiosis. In this review, we highlight the composition of EVs and discuss their role in the interaction with plants. Knowledge of EV composition and functions will aid their use in biotechnology and agriculture., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Brucellosis is the most common zoonotic disease worldwide and still there is no vaccine for human use. The commercial animal vaccines also have major problems that limit their use. Therefore, there is a need for an effective Brucella vaccine which is multivalent and produces a good protective immunity with minimal disadvantages. Due to their heterogeneous composition and diverse functions, OMVs are promising acellular vaccine candidates against brucellosis. In the present study, the potential of Poly(I:C) or CpG ODN 1826+ Montanide ISA 70 VG adjuvant formulations were evaluated to enhance the immunity and protection levels conferred by OMVs against Brucella challenge in mice. The results indicated that both vaccine regimens were able to induce strong Th1-biased responses and confer protective levels significantly higher than REV.1 live vaccine. With regard to the results, it is concluded that OMVs in either adjuvant can be introduced as a new vaccine candidate against B. melitensis infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacterial infections cause acute and chronic diseases. Antimicrobial resistance and aging-related immune weakness remain challenging in therapy of infectious diseases. Vaccines are however an alternative to prevent bacterial infections. Here we report a facile method to rapidly generate bacterium-membrane-formed nanovesicles as a vaccine using nitrogen cavitation. The vaccine is comprised of double-layered membrane vesicles (DMVs) characterized by cryo-TEM, biochemistry and proteomics, showing DMVs possess the integrity of bacterial membrane and contain a wide range of membrane proteins required for vaccination. In the mouse sepsis model induced by Pseudomonas aeruginosa, we found that DMVs can improve mouse survival after mice were immunized with DMVs. The increased adaptive immunity and unique biodistribution of DMVs were responsible for enhanced protection of bacterial infection. Our studies demonstrate that this simple and innovative approach using nitrogen cavitation would be a promising technology for vaccine developments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cholera is caused by toxigenic Vibrio cholerae. It is a significant health problem and an important cause of mortality of children in developing countries. Annually, about 5-7 million people are being infected worldwide, leading to death of 100,000 to 120,000. Immunization using the currently available cholera vaccines has been recommended by World Health Organization (WHO) in areas where cholera is endemic or at risk of outbreaks. Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play important roles in virulence and host-pathogen interaction. The content of protein and lipid in OMVs are affected by purification methods and bacterial growth condition. OMVs released from V. cholerae are an appropriate candidate for vaccine development. The protection conferred by a new vaccine candidate prepared using different methods and in two different growth conditions with nanoparticles in an experimental model of cholera in mice was investigated. OMVs were encapsulated in chitosan-tripolyphosphate (TPP) nanoparticles prepared by an ionic gelation method and coated with Eudragit as an enteric polymer. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 417nm. BALB/c mice (male, 20-24g) were immunized via intraperitoneal (10µg) or oral route (50µg) with free or encapsulated OMVs. Seventy-eight days after first administration, serum of mice was infected with infection dose of V. cholerae (≥10 7 CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intraperitoneal route, the unpolymerized OMVs increased the protection against these bacteria., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bacterial outer membrane vesicles have been extensively investigated and considered as a next generation vaccine. Recently, we have demonstrated that the cholera pentavalent outer membrane vesicles (CPMVs) immunogen induced adaptive immunity and had a strong protective efficacy against the circulating V. cholerae strains in a mouse model. In this present study, we are mainly focusing on reducing outer membrane vesicle (OMV) -mediated toxicity without altering its antigenic property. Therefore, we have selected All-trans Retinoic Acid (ATRA), active metabolites of vitamin A, which have both anti-inflammatory and mucosal adjuvant properties. Pre-treatment of ATRA significantly reduced CPMVs induced TLR2 mediated pro-inflammatory responses in vitro and in vivo. Furthermore, we also found ATRA pre-treatment significantly induced mucosal immune response and protective efficacy after two doses of oral immunization with CPMVs (75µg). This study can help to reduce OMV based vaccine toxicity and induce better protective immunity where children and men suffered from malnutrition mainly in developing countries., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

For the development of a third generation of pertussis vaccine that could improve the control of the disease, it was proposed that the immune responses induced by the classic whole cell vaccine (wP) or after infection should be used as a reference point. We have recently identified a vaccine candidate based on outer membrane vesicles (OMVs) derived from the disease etiologic agent that have been shown to be safe and protective in mice model of infection. Here we characterized OMVs-mediated immunity and the safety of our new candidate. We also deepen the knowledge of the induced humoral response contribution in pertussis protection. Regarding the safety of the OMVs based vaccine (TdapOMVsBp,) the in vitro whole blood human assay here performed, showed that the low toxicity of OMVs-based vaccine previously detected in mice could be extended to human samples. Stimulation of splenocytes from immunized mice evidenced the presence of IFN-γ and IL-17-producing cells, indicated that OMVs induces both Th1 and Th17 response. Interestingly TdapOMVsBp-raised antibodies such as those induced by wP and commercial acellular vaccines (aP) which contribute to induce protection against Bordetella pertussis infection. As occurs with wP-induced antibodies, the TdapOMVsBp-induced serum antibodies efficiently opsonized B. pertussis. All the data here obtained shows that OMVs based vaccine is able to induce Th1/Th17 and Th2 mixed profile with robust humoral response involved in protection, positioning this candidate among the different possibilities to constitute the third generation of anti-pertussis vaccines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pertussis has resurged during the last two decades in different countries. In particular in the 2010-2013 period large outbreaks were detected in US, Australia, UK and The Netherlands with significant mortality in infants. The epidemiological situation of pertussis points out the need to develop new vaccines and in this regard we previously developed a new vaccine based on outer membrane vesicles (OMVs) which have been shown to be safe and to induce protection in mice. Here we have further investigated the properties of OMVs vaccines; in particular we studied the contribution of pertussis toxin (PTx) and pertactin (Prn) in OMVs-mediated protection against pertussis. PTx-deficient OMVs and Prn-deficient OMVs were obtained from defective Bordetella pertussis mutants. The absence of PTx or Prn did compromise the protective capacity of the OMVs formulated as Tdap vaccine. Whereas the protective efficacy of the PTx-deficient OMVs in mice was comparable to Prn-deficient OMVs, the protective capacity of both of them was significantly impaired when it was compared with the wild type OMVs. Interestingly, using OMVs obtained from a B. pertussis strain which does not express any of the virulence factors but expresses the avirulent phenotype; we observed that the protective ability of such OMVs was lower than that of OMVs obtained from virulent B. pertussis phase. However, it was surprising that although the protective capacity of avirulent OMVs was lower, they were still protective in the used mice model. These results allow us to hypothesize that OMVs from avirulent phase shares protective components with all OMVs assayed. Using an immune proteomic strategy we identified some common components that could play an important role in protection against pertussis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Despite high vaccination coverage rates, pertussis continues to be a global concern, with increased incidence widely noted. The current pertussis epidemiologic situation has been mainly attributed to waning immunity and pathogen adaptation. To improve the disease control, a new generation of vaccines capable to overcome those weaknesses associated to the current vaccines need to be developed. Previously we have demonstrated that the outer membrane vesicles obtained from the recombinant Bordetella pertussis strain expressing PagL enzyme (OMVs(BpPagL)) are good vaccine candidates to protect against pertussis. In this work the OMVs(BpPagL) formulated with diphtheria and tetanus toxoids (Tdap(OMVsBpPagL)) was used to evaluate its capacity to offer protection against Argentinean clinical isolates and to induce long-term immunity. To these aims BALB/c mice were immunized with Tdap(OMVsBpPagL) and challenged with sublethal doses of the clinical isolate Bp106 selected as a representative circulating isolate. Comparisons with a current commercial Tdap vaccine used at a dose in which pertussis toxin level was equivalent to that of Tdap(OMVsBpPagL) were performed. With the normalized doses of both vaccines we observed that Tdap(OMVsBpPagL) protected against the clinical isolate infection, whereas current commercial Tdap vaccine showed little protection against such pathogen. Regarding long-term immunity we observed that the Tdap(OMVsBpPagL) protective capacity against the recommended WHO reference strain persisted at least 9 months. In agreement with these results Tdap(OMVsBpPagL) induced Th1 and Th2 immune response. In contrast, commercial Tdap induced Th2 but weak Th1 responses. All results presented here showed that Tdap(OMVsBpPagL) is an interesting formulation to be considered for the development of novel acellular multi-antigen vaccine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bordetella parapertussis, a close related species of B. pertussis, can also cause the disease named pertussis or whooping cough. The number of cases caused by this related pathogen has risen sustained in the last years. The widely used cellular (wP) or acellular (aP) pertussis vaccines have little or no efficacy against B. parapertussis. In an effort to devise an effective acellular vaccine against B. parapertussis infection, outer membrane vesicles (OMVs) were obtained from B. parapertussis. Proteomic analysis of the resulting OMVs, designated OMVsBpp, evidenced the presence of several surface immunogens including pertactin. The characterized OMVsBpp were used in murine B. parapertussis intranasal challenge model to examine their protective capacity when administered by systemic route. Immunized BALB/c mice were challenged with sublethal doses of B. parapertussis. Significant differences between immunized animals and the negative control group were observed (p<0.001). OMVsBpp protected against B. parapertussis infection, whereas current commercial aP vaccine showed little protection against such pathogen. More interestingly, protection induced by OMVsBpp against B. pertussis was comparable to our previously designed vaccine consisting in OMVs derived from B. pertussis (OMVsBp). For these experiments we used as a positive control the current commercial aP vaccine in high dose. As expected aP offered protection against B. pertussis in mice. Altogether the results presented here showed that the OMVs from B. parapertussis are an attractive vaccine candidate to protect against whooping cough induced by B. parapertussis but also by B. pertussis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)