Immunization with Pseudomonas aeruginosa outer membrane vesicles stimulates protective immunity in mice

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of severe nosocomial and community acquired infections, these infections are major health problems for cystic fibrosis patients and immune-compromised individuals. The emergence of multidrug-resistant isolates highlights the need to develop alternative strategies for treatment of P. aeruginosa infections. Outer membrane vesicles (OMVs) are spherical nanometer-sized proteolipids that are secreted from numerous of pathogenic Gram-negative bacteria, and a number of studies have confirmed the protective efficacy for use of OMVs as candidate vaccines. In this study, OMVs from P. aeruginosa (PA_OMVs) were isolated, formulated with aluminum phosphate adjuvant and used as a vaccine in a mouse model of acute lung infection. The results confirmed that active immunization with PA_OMVs was able to reduce bacterial colonization, cytokine secretion and tissue damage in the lung tissue, thus protecting mice from lethal challenge of P. aeruginosa. Cytokines assay validated that immunization with PA_OMVs was efficient to induce a mixed cellular immune response in mice. Further, high level of specific antibodies was detected in mice immunized with PA_OMVs, and results from opsonophagocytic killing assay and passive immunization suggested that humoral immune response may be critical for PA_OMVs mediated protection. These findings demonstrated that PA_OMVs may be served as a novel candidate vaccine for the prevention of P. aeruginosa infection.     

Identification and characterization of OmpL as a potential vaccine candidate for immune-protection against salmonellosis in mice

Salmonella is gram-negative flagellated bacteria that can cause food and waterborne gastroenteritis and typhoid fever in humans. Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined, the effectiveness of the currently available vaccines is also limited. Outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against salmonellosis. OMPs interface the cell with the environment, thus representing important potential vaccine candidate for pathogen infection. We showed that the outer membrane porin L (OmpL) is a transmembrane β barrel (TMBB) protein, which forms 12 transmembrane β-strands. OmpL of S. Typhimurium is highly immunogenic, OmpL could evoke humoral and cell-mediated immune responses, and confer 100% protection to immunized mice against challenge with very high doses of S. Typhimurium. Besides, very efficient clearance of bacteria from the reticuloendothelial systems of immunized mice was seen. The homology search further revealed that OmpL is widely distributed and conserved, homologous proteins were identified in S. Typhi and Paratyphi by RT-PCR and western blot. We also found that anti-rOmpL serum harber a high bactericidal activity for Salmonella serovars tested in this study. Therefore, OmpL provide a promising target for the development of a candidate vaccine against Salmonella infection.     

Vaccination of attenuated EIS-producing Salmonella induces protective immunity against enterohemorrhagic Escherichia coli in mice

There is an urgent need for vaccine against enterohemorrhagic Escherichia coli (EHEC), which causes a wide range of life-threatening diseases in human and animals. E. coli secreted protein A (EspA), intimin and shiga toxin (Stx) are important pathogenic factors and protective antigens of EHEC. In our previous study, we found that recombinant trivalent protein EIS, which is composed of EspA (E), the 300 amino acids of the carboxyl terminus of intimin (I) and the B subunit of Stx2 (S), was able to efficiently elicit protective immunity against EHEC. The application of live attenuated Salmonella as a carrier for vaccine against mucosal pathogens provided unparalleled merits. Therefore, in this study we constructed live attenuated EIS-producing Salmonella vaccine and tested it as vaccine in mice model. We found that the vaccination of EIS-producing recombinant Salmonella was able to induce significant increases of EspA, intimin and Stx2 specific IgG in serum and secretory IgA in feces. Antigen specific T cell proliferation was also observed in the mice immunized with recombinant EIS-producing Salmonella. In addition, this immunity was able to protect mice from a challenge of a lethal dose of EHEC, even after a period of 70 days. Moreover, the EIS-producing Salmonella induced immunity can be boosted by a single subcutaneous injection of purified EIS protein, even after an interval of 70 days. This EIS-producing Salmonella vaccine provides an alternative approach for the prevention of EHEC infection.     

Development of a bivalent conjugate vaccine candidate against malaria transmission and typhoid fever

Immune responses to poorly immunogenic antigens, such as polysaccharides, can be enhanced by conjugation to carriers. Our previous studies indicate that conjugation to Vi polysaccharide of Salmonella Typhi may also enhance immunogenicity of some protein carriers. We therefore explored the possibility of generating a bivalent vaccine against Plasmodium falciparum malaria and typhoid fever, which are co-endemic in many parts of the world, by conjugating Vi polysaccharide, an approved antigen in typhoid vaccine, to Pfs25, a malaria transmission blocking vaccine antigen in clinical trials. Vi-Pfs25 conjugates induced strong immune responses against both Vi and Pfs25 in mice, whereas the unconjugated antigens are poorly immunogenic. Functional assays of immune sera revealed potent transmission blocking activity mediated by anti-Pfs25 antibody and serum bactericidal activity due to anti-Vi antibody. Pfs25 conjugation to Vi modified the IgG isotype distribution of antisera, inducing a Th2 polarized immune response against Vi antigen. This conjugate may be further developed as a bivalent vaccine to concurrently target malaria and typhoid fever.     

Nontyphoidal salmonella disease: Current status of vaccine research and development

Among more than 2500 nontyphoidal Salmonella enterica (NTS) serovars, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis account for approximately fifty percent of all human isolates of NTS reported globally. The global incidence of NTS gastroenteritis in 2010 was estimated to be 93 million cases, approximately 80 million of which were contracted via food-borne transmission. It is estimated that 155,000 deaths resulted from NTS in 2010. NTS also causes severe, extra-intestinal, invasive bacteremia, referred to as invasive nontyphoidal Salmonella (iNTS) disease. iNTS disease usually presents as a febrile illness, frequently without gastrointestinal symptoms, in both adults and children. Symptoms of iNTS are similar to malaria, often including fever (>90%) and splenomegaly (>40%). The underlying reasons for the high rates of iNTS disease in Africa are still being elucidated. Evidence from animal and human studies supports the feasibility of developing a safe and effective vaccine against iNTS. Both antibodies and complement can kill Salmonella species in vitro. Proof-of-principle studies in animal models have demonstrated efficacy for live attenuated and subunit vaccines that target the O-antigens, flagellin proteins, and other outer membrane proteins of serovars Typhimurium and Enteritidis. More recently, a novel delivery strategy for NTS vaccines has been developed: the Generalized Modules for Membrane Antigens (GMMA) technology which presents surface polysaccharides and outer membrane proteins in their native conformation. GMMA technology is self-adjuvanting, as it delivers multiple pathogen-associated molecular pattern molecules. GMMA may be particularly relevant for low- and middle-income countries as it has the potential for high immunologic potency at a low cost and involves a relatively simple production process without the need for complex conjugation. Several vaccines for the predominant NTS serovars Typhimurium and Enteritidis, are currently under development.     

A combination vaccine comprising of inactivated enterovirus 71 and coxsackievirus A16 elicits balanced protective immunity against both viruses

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand, foot and mouth disease (HFMD), which is an infectious disease frequently occurring in children. A bivalent vaccine against both EV71 and CA16 is highly desirable. In the present study, we compare monovalent inactivated EV71, monovalent inactivated CA16, and a combination vaccine candidate comprising of both inactivated EV71 and CA16, for their immunogenicity and in vivo protective efficacy. The two monovalent vaccines were found to elicit serum antibodies that potently neutralized the homologous virus but had no or weak neutralization activity against the heterologous one; in contrast, the bivalent vaccine immunized sera efficiently neutralized both EV71 and CA16. More importantly, passive immunization with the bivalent vaccine protected mice against either EV71 or CA16 lethal infections, whereas the monovalent vaccines only prevented the homologous but not the heterologous challenges. Together, our results demonstrate that the experimental bivalent vaccine comprising of inactivated EV71 and CA16 induces a balanced protective immunity against both EV71 and CA16, and thus provide proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.     

A bivalent outer membrane vesicle-based intranasal vaccine to prevent infection of periodontopathic bacteria

Periodontal disease has become a serious public health problem, not only causing tooth loss, but also inducing chronic disorders of extra-oral organs. The present study assessed an intranasal vaccine strategy to prevent periodontal disease using outer membrane vesicles (OMVs) of two major periodontopathic bacteria, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). We compared the morphology, composition, and immune activity between OMVs of Pg strain ATCC 33277 and Aa strain Y4. Aa OMVs had a smoother surface and stronger lipid A activity compared to Pg OMVs. The in vitro immune activity elicited by Aa OMVs in macrophage-like cells was remarkably stronger than that of Pg OMVs. Intranasal immunization of mice with Aa OMVs alone resulted in robust, humoral immune responses in blood and saliva. Despites the intrinsically low mucosal immunogenicity of Pg OMVs alone, using Aa OMVs as a mucosal adjuvant strongly enhanced Pg-specific immune responses, resulting in both serum IgG and salivary IgA, both of which aggregated Pg and Aa cells. Furthermore, Aa OMVs were found to be a more potent mucosal adjuvant than Poly(I:C) in the context of enhancing the production of Pg-specific IgG (especially IgG2a) and IgA. In addition, in a randomized, blinded study, mice oral challenged with Pg and Aa after intranasal immunization with Pg OMVs and Aa OMVs had significantly decreased numbers of both microorganisms compared to mock-immunized mice. Furthermore, in an intracerebral injection mouse model, there were no serious adverse effects on the brain even after administrating a dose of OMVs as same as that used for intranasal administration. Taken together, the bivalent OMV intranasal vaccine may be effective in preventing colonization of periodontopathic bacteria in the oral cavity and related systemic disorders associated with periodontal diseases.     

Zebrafish as a novel model for non-typhoidal Salmonella pathogenesis,transmission and vaccine efficacy

Salmonella-induced gastroenteritis causes massive morbidity and mortality in both adults and children of developing countries. However, it is difficult to study the mode of infection and vaccine efficacy due to inadequacies of current animal models. For this reason, we have explored using zebrafish as an improved model for non-typhoidal Salmonella (NTS) infection, including Salmonella enterica Typhimurium, Salmonella enterica Enteritidis and Salmonella enterica Weltevreden. In this study, we found that after infection of zebrafish with NTS, severe diarrhea like symptoms were observed and NTS significantly colonized the zebrafish intestine without any manipulation of the normal intestinal microbiota of the fish. Furthermore, these strains can colonize for longer than 72 h and induce severe inflammation in the intestine, which may induce fish death. We also found that infected fish can transmit the pathogen into naïve fish. Moreover, we have established that zebrafish is an excellent model for vaccine study. Successive triple bath vaccination with heat-killed single serotype S. Typhimurium and S. Enteritidis immunogen induced protective efficacy against a high dose (10⁸ CFU/ml) of infection with these pathogens. This study provides a natural infection model for the study of NTS infection, transmission and vaccine efficacy.     

Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice

Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunately, there is still no safe and efficacious vaccine available. We investigated the protection conferred by a new vaccine containing outer membrane vesicles (OMVs) from Shigella flexneri with an adjuvant based on nanoparticles in an experimental model of shigellosis in mice. OMVs were encapsulated in poly(anhydride) nanoparticles prepared by a solvent displacement method with the copolymer PMV/MA. OMVs loaded into NPs (NP-OMVs) were homogeneous and spherical in shape, with a size of 197 nm (PdI = 0.06). BALB/c mice (females, 9-week-old, 20 ± 1 g) were immunized by intradermal, nasal, ocular (20 μg) or oral route (100 μg) with free or encapsulated OMV. Thirty-five days after administration, mice were infected intranasally with a lethal dose of S. flexneri (1 × 10⁷ CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intradermal route the NP-OMVs formulation increased the protection from 20%, obtained with free extract, to 100%. Interestingly, both OMVs and OMV-NP induced full protection when administered by the nasal and conjuntival route. A strong association between the ratio of IL-12p40/IL-10 and protection was found. Moreover, low levels of IFN-γ correlate with protection. Under the experimental conditions used, the adjuvant did not induce any adverse effects. These results place OMVs among promising candidates to be used for vaccination against Shigellosis.     

rOmpF and OMVs as efficient subunit vaccines against Salmonella enterica serovar Enteritidis infections in poultry farms

Salmonella enterica serovar Enteritidis remains the most prevalent serotype causing human salmonellosis through the consumption of contaminated foods, especially poultry products. The development of a subunit vaccine against S. Enteritidis can not only protect chickens against Salmonella infection in the poultry industry but also cut the transmission sources. In this study, both the expressed recombinant outer membrane protein F (rOmpF) and extracted outer membrane vesicles (OMVs) were developed as subunit vaccines against S. Enteritidis challenge in chickens. Immunization with the subunit vaccine could induce not only antibody production but also strong cell-mediated immune response. Both rOmpF plus QuilA adjuvant and OMVs alone had highly protective efficacy against S. Enteritidis challenge and rapidly decreased the colonization of bacteria in chicken. These findings revealed the potential application of rOmpF and OMVs as subunit vaccines in the poultry industry.     

A bivalent vaccine derived from attenuated Salmonella expressing O-antigen polysaccharide provides protection against avian pathogenic Escherichia coli O1 and O2 infection

Avian pathogenic Escherichia coli (APEC), a leading cause of avian airsacculitis and colibacillosis, is responsible for significant economic loss in the poultry industry. APEC serogroups O1, O2, and O78 are predominantly associated with disease. Lipopolysaccharide (LPS) O-antigen has been shown to be a potent antigen for inducing specific protective immune responses. Therefore, we sought to develop a multivalent polysaccharide vaccine to prevent most APEC infections. We previously reported the stable expression of plasmid pSS27 encoding the APEC O1 O-antigen gene cluster (10.8 kb) in attenuated Salmonella enterica serovar Typhimurium S740 provided excellent protection against APEC O1 challenge. In this study, the plasmid pSS28 harboring the APEC O2 O-antigen polysaccharide gene cluster (15.5 kb) was constructed. Biosynthesis of pSS28-encoded APEC O2 O-antigen in Salmonella vaccine strain S740 was validated by Western blot. The recombinant Salmonella vaccine strain S740 (pSS28) elicited homologous protection against virulent wild-type APEC O2 challenge in a chicken model. Furthermore, through equal-volume mixing the two monovalent vaccine strains S740 (pSS27) and S740 (pSS28), a bivalent vaccine candidate against both APEC O1 and O2 was developed. Immunization of chickens with the bivalent vaccine elicited production of serum IgG and mucosal sIgA antibodies against the LPS of both APEC O1 and O2. Moreover, antibodies induced by the bivalent vaccine promoted opsonization, provoked complement-mediated bactericidal activity, and elicited protection against lethal challenge with both virulent APEC O1 and O2 strains. These results demonstrate that the bivalent vaccine comprised of S740 (pSS27) and S740 (pSS28) is a promising vaccine candidate against APEC O1 and O2 infection.     

Expression of human CEACAM1 in transgenic mice limits the Opa-specific immune response against meningococcal outer membrane vesicles

Outer membrane vesicles (OMVs) have been extensively investigated as meningococcal vaccine candidates. Among their major components are the opacity (Opa) proteins, a family of surface-exposed outer membrane proteins important for bacterial adherence and entry into host cells. Many Opa-dependent interactions are mediated through the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of receptors. Importantly, binding of Opa to CEACAM1 has been reported to suppress human CD4 T cell proliferation in vitro in response to OMV preparations. This raises the question whether OMV vaccines should contain Opa proteins at all. Until now it has been difficult to answer this question, as the proposed immunosuppressive effect was only demonstrated with human cells in vitro, while immunization experiments in mice are not informative because the Opa interaction is specific for human CEACAM1. In the present study we have used Opa+ and Opa− OMVs for immunization experiments in a human CEACAM1 transgenic mouse model. OMVs were prepared from a meningococcal strain H44/76 variant expressing the CEACAM1-binding OpaJ protein, and from an isogenic variant in which all opa genes have been inactivated. Both the CEACAM1 expressing transgenic mice and their congenic littermates lacking it were immunized twice with the OMV preparations, and the sera were analyzed for bactericidal activity and ELISA antibody titres. Total IgG antibodies against the OMVs were similar in both mouse strains. Yet the titres for IgG antibodies specific for purified OpaJ protein were significantly lower in the mice expressing human CEACAM1 than in the nontransgenic mice. No significant differences were found in bactericidal titres among the four groups. Overall, these data indicate that expression of human CEACAM1 confers a reduced Opa-specific antibody response in vivo without affecting the overall immune response against other OMV antigens.     

Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice

Shigellosis is one of the leading causes of diarrhea worldwide with more than 130 million cases annually. Hence, the research of an effective vaccine is still a priority. Unfortunately, a safe and efficacious vaccine is not available yet. We have previously demonstrated the capacity of outer membrane vesicles (OMVs) to protect mice against an experimental infection with Shigella flexneri. Now, we present results on the capacity of this antigenic complex to confer a longer-term protection by oral or nasal routes when encapsulated into nanoparticles. OMVs were encapsulated in poly(anhydride) nanoparticles (NP) prepared by a solvent displacement method with the copolymer poly methyl vinyl ether/maleic anhydride. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 148 nm (PdI = 0.2). BALB/c mice were immunized with OMVs either free or encapsulated in nanoparticles by nasal (20 μg or 10 μg of OMVs) or oral route (100 μg or 50 μg of OMVs). All immunized animals remained in good health after administration. Challenge infection was performed intranasally on week 8th with a lethal dose of 5 × 10⁷ CFU/mouse of S. flexneri 2a. The number of dead mice after challenge was recorded daily. Results confirmed the value of OMVs as a vaccine. By oral route, the OMV-vaccine was able to protect independently either the dose or the formulation. When vaccine was delivered by nasal route, encapsulation into NPs resulted beneficial in increasing protection from 40% up to 100% when low dose was administered. These results are extraordinary promising and put in relevance the positive effect of nanoencapsulation of the OMV subcellular vaccine.     

Characterization of an inactivated whole-virus bivalent vaccine that induces balanced protective immunity against coxsackievirus A6 and A10 in mice

Coxsackievirus A6 (CVA6) and CVA10 are two of the major pathogens associated with hand, foot and mouth disease (HFMD) in children. The majority of CVA6 and CVA10 infections result in mild, self-limiting episodes (fever and herpangina) in pediatric populations; however, in some cases, can proceed to severe neurological disease and death. Efforts to mitigate viral transmission to decrease the morbidity and mortality associated with infection would be greatly strengthened by the availability of an efficacious CVA6 and CVA10 bivalent vaccine. Here we report the immunogenicity and protective efficacy of a bivalent combination vaccine comprised of formaldehyde-inactivated, whole-virus CVA6 and CVA10. We demonstrate that subcutaneous delivery of the bivalent vaccine can induce antigen-specific systemic immune responses, particularly the induction of polyfunctional T cells, which elicit active immunization to achieve a protection rate of >80% in the infected neonatal mice. Furthermore, passive transfer of the antisera from vaccinated mice potently protected recipient mice against CVA6 and CVA10 challenge. Importantly, the bivalent vaccine could induce high levels of IgG and neutralizing antibodies in adult female mice and the maternal antibody transmitted to the recipient mice played an important role in controlling homotypic and heterotypic CVA6 and CVA10 infections and viral replication in vivo. Collectively, these findings indicate that there is no immunological interference between the two antigens with respect to their ability to induce virus-specific immune responses, and thus provides proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.     

Multi-serotype outer membrane vesicles of Shigellae confer passive protection to the neonatal mice against shigellosis

Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 Δstx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50 μg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3–4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future.     

Immunogenicity and protective effects of recombinant bivalent COVID-19 vaccine in mice and rhesus macaques

Coronavirus disease (COVID-19) is still spreading rapidly worldwide, and a safe, effective, and cheap vaccine is still required to combat the COVID-19 pandemic. Here, we report a recombinant bivalent COVID-19 vaccine containing the RBD proteins of the prototype strain and beta variant. Immunization studies in mice demonstrated that this bivalent vaccine had far greater immunogenicity than the ZF2001, a marketed monovalent recombinant protein COVID-19 vaccine, and exhibited good immunization effects against the original COVID-19 strain and various variants. Rhesus macaque challenge experiments showed that this bivalent vaccine drastically decreased the lung viral load and reduced lung lesions in SARS-CoV-2 (the causative virus of COVID-19)-infected rhesus macaques. In summary, this bivalent vaccine showed immunogenicity and protective efficacy that was far superior to the monovalent recombinant protein vaccine against the prototype strain and provided an important basis for developing broad-spectrum COVID-19 vaccines.     

A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection

Burkholderia pseudomallei, and other members of the Burkholderia, are among the most antibiotic-resistant bacterial species encountered in human infection. Mortality rates associated with severe B. pseudomallei infection approach 50% despite therapeutic treatment. A protective vaccine against B. pseudomallei would dramatically reduce morbidity and mortality in endemic areas and provide a safeguard for the U.S. and other countries against biological attack with this organism. In this study, we investigated the immunogenicity and protective efficacy of B. pseudomallei-derived outer membrane vesicles (OMVs). Vesicles are produced by Gram-negative and Gram-positive bacteria and contain many of the bacterial products recognized by the host immune system during infection. We demonstrate that subcutaneous (SC) immunization with OMVs provides significant protection against an otherwise lethal B. pseudomallei aerosol challenge in BALB/c mice. Mice immunized with B. pseudomallei OMVs displayed OMV-specific serum antibody and T-cell memory responses. Furthermore, OMV-mediated immunity appears species-specific as cross-reactive antibody and T cells were not generated in mice immunized with Escherichia coli-derived OMVs. These results provide the first compelling evidence that OMVs represent a non-living vaccine formulation that is able to produce protective humoral and cellular immunity against an aerosolized intracellular bacterium. This vaccine platform constitutes a safe and inexpensive immunization strategy against B. pseudomallei that can be exploited for other intracellular respiratory pathogens, including other Burkholderia and bacteria capable of establishing persistent infection.     

Vaccination with meningococcal outer membrane vesicles carrying Borrelia OspA protects against experimental Lyme borreliosis

Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines, as such a vaccine has been proven to be successful in the past. The expression of recombinant antigens in meningococcal Outer Membrane Vesicles (OMVs), with the OMV functioning both as adjuvant and delivery vehicle, greatly enhances their potential. Immunization studies in mice have shown that OMV-based vaccines can protect against various pathogens and an OMV-based meningococcal vaccine is approved and available for human use. Because of its surface localization in Borrelia and the detailed knowledge regarding its immunogenicity and structure, OspA was chosen as a suitable lipoprotein to be tested as an OMV-based vaccine against Lyme borreliosis. We have previously shown that the OMV-OspA vaccine was immunogenic in mice and here we assessed the efficacy of OMV-OspA.We generated a second-generation OMV-OspA vaccine and vaccinated C3H/HeN mice with (EDTA extracted) meningococcal OMVs expressing OspA from B. burgdorferi strain B31. The adjuvant effect of empty OMVs on recombinant OspA was tested as well. We subsequently challenged mice with a subcutaneous injection of B. burgdorferi.Average antibody end-point titers against the OspA-OMV construct were high, although lower compared to the antibodies raised against recombinant OspA. Interestingly, antibody titers between recombinant OspA adjuvanted with aluminum hydroxide and recombinant OspA with OMV as adjuvant were comparable. Finally, qPCR and culture data show that both the OspA-OMV and the vaccine based on recombinant OspA with OMV as adjuvant provided significant, yet partial protection, against Borrelia infection.OMV-based vaccines using Borrelia (lipo)proteins are an easy and feasible vaccination method protecting against B. burgdorferi infection and could be a promising strategy in humans.     

Characterization of the immune response induced by pertussis OMVs-based vaccine

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 (Tdap_OMVsBp,) 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 Tdap_OMVsBp-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.     

Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles

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.