Pertussis Vaccine Candidate Based on Outer Membrane Vesicles Derived From Biofilm Culture.

Outer membrane vesicles (OMV) derived from Bordetella pertussis-the etiologic agent of the resurgent disease called pertussis-are safe and effective in preventing bacterial colonization in the lungs of immunized mice. Vaccine formulations containing those OMV are capable of inducing a mixed Th1/Th2/Th17 profile, but even more interestingly, they may induce a tissue-resident memory immune response. This immune response is recommended for the new generation of pertussis-vaccines that must be developed to overcome the weaknesses of current commercial acellular vaccines (second-generation of pertussis vaccine). The third-generation of pertussis vaccine should also deal with infections caused by bacteria that currently circulate in the population and are phenotypically and genotypically different [in particular those deficient in the expression of pertactin antigen, PRN(-)] from those that circulated in the past. Here we evaluated the protective capacity of OMV derived from bacteria grown in biofilm, since it was observed that, by difference with older culture collection vaccine strains, circulating clinical B. pertussis isolates possess higher capacity for this lifestyle. Therefore, we performed studies with a clinical isolate with good biofilm-forming capacity. Biofilm lifestyle was confirmed by both scanning electron microscopy and proteomics. While scanning electron microscopy revealed typical biofilm structures in these cultures, BipA, fimbria, and other adhesins described as typical of the biofilm lifestyle were overexpressed in the biofilm culture in comparison with planktonic culture. OMV derived from biofilm (OMVbiof) or planktonic lifestyle (OMVplank) were used to formulate vaccines to compare their immunogenicity and protective capacities against infection with PRN(+) or PRN(-) B. pertussis clinical isolates. Using the mouse protection model, we detected that OMVbiof-vaccine was more immunogenic than OMVplank-vaccine in terms of both specific antibody titers and quality, since OMVbiof-vaccine induced antibodies with higher avidity. Moreover, when OMV were administered at suboptimal quantity for protection, OMVbiof-vaccine exhibited a significantly adequate and higher protective capacity against PRN(+) or PRN(-) than OMVplank-vaccine. Our findings indicate that the vaccine based on B. pertussis biofilm-derived OMV induces high protection also against pertactin-deficient strains, with a robust immune response.

Outer-Membrane-Vesicle-Associated O Antigen, a Crucial Component for Protecting Against Bordetella parapertussis Infection.

Bordetella parapertussis is a respiratory-disease pathogen producing symptomatology similar to that of pertussis but of underestimated incidence and with no specific vaccine existing. We recently designed a vaccine candidate from B. parapertussis outer-membrane vesicles (OMVs) that proved to be safe and protective in a murine-infection model. Based on protection recently reported for the B. parapertussis O antigen in aqueous solution, we assessed here whether the B. parapertussis O-antigen-containing lipopolysaccharide (BppLPS-O+) embedded in the membranes, as present in B. parapertussis-derived OMVs (OMVs(Bpp-LPS-O+)), was the component responsible for that previously observed protection by OMVs. By performing a comparative study with OMVs from a human strain with undetectable O antigen (OMVs(Bpp-LPS-O-)), we demonstrated that the OMVs(Bpp-LPS-O+), but not the OMVs(Bpp-LPS-O-), protected mice against sublethal B. parapertussis infections. Indeed, the B. parapertussis loads were significantly reduced in the lungs of OMVs(Bpp-LPS-O+) -vaccinated animals, with the CFUs recovered being decreased by 4 log units below those detected in the non-immunized animals or in the animals treated with the OMVs(Bpp-LPS-O-), (p < 0.001). We detected that the OMVs(Bpp-LPS-O+) induced IgG antibodies against B. parapertussis whole-cell lysates, which immunocomponents recognized, among others, the O antigen and accordingly conferred protection against B. parapertussis infection, as observed in in-vivo-passive-transfer experiments. Of interest was that the OMVs(Bpp-LPS-O+) -generated sera had opsonophagocytic and bactericidal capabilities that were not detected with the OMVs(Bpp-LPS-O-)-induced sera, suggesting that those activities were involved in the clearance of B. parapertussis. Though stimulation of cultured spleen cells from immunized mice with formulations containing the O antigen resulted in gamma interferon (IFN-γ) and interleukin-17 production, spleen cells from OMVs(Bpp-LPS-O+) -immunized mice did not significantly contribute to the observed protection against B. parapertussis infection. The protective capability of the B. parapertussis O antigen was also detected in formulations containing both the OMVs derived from B. pertussis and purified BppLPS-O+. This combined formulation protected mice against B. pertussis along with B. parapertussis.

Pertussis Maternal Immunization: Narrowing the Knowledge Gaps on the Duration of Transferred Protective Immunity and on Vaccination Frequency.

Maternal safety through pertussis vaccination and subsequent maternal-fetal-antibody transfer are well documented, but information on infant protection from pertussis by such antibodies and by subsequent vaccinations is scarce. Since mice are used extensively for maternal-vaccination studies, we adopted that model to narrow those gaps in our understanding of maternal pertussis immunization. Accordingly, we vaccinated female mice with commercial acellular pertussis (aP) vaccine and measured offspring protection against Bordetella pertussis challenge and specific-antibody levels with or without revaccination. Maternal immunization protected the offspring against pertussis, with that immune protection transferred to the offspring lasting for several weeks, as evidenced by a reduction (4-5 logs, p < 0.001) in the colony-forming-units recovered from the lungs of 16-week-old offspring. Moreover, maternal-vaccination-acquired immunity from the first pregnancy still conferred protection to offspring up to the fourth pregnancy. Under the conditions of our experimental protocol, protection to offspring from the aP-induced immunity is transferred both transplacentally and through breastfeeding. Adoptive-transfer experiments demonstrated that transferred antibodies were more responsible for the protection detected in offspring than transferred whole spleen cells. In contrast to reported findings, the protection transferred was not lost after the vaccination of infant mice with the same or other vaccine preparations, and conversely, the immunity transferred from mothers did not interfere with the protection conferred by infant vaccination with the same or different vaccines. These results indicated that aP-vaccine immunization of pregnant female mice conferred protective immunity that is transferred both transplacentally and via offspring breastfeeding without compromising the protection boostered by subsequent infant vaccination. These results-though admittedly not necessarily immediately extrapolatable to humans-nevertheless enabled us to test hypotheses under controlled conditions through detailed sampling and data collection. These findings will hopefully refine hypotheses that can then be validated in subsequent human studies.

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.

Immunization with the recombinant Cholera toxin B fused to Fimbria 2 protein protects against Bordetella pertussis infection.

This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2)-cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 µg of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P < 0.01 or P < 0.001 , resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.

Acellular pertussis vaccine based on outer membrane vesicles capable of conferring both long-lasting immunity and protection against different strain genotypes.

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.

Fatty acid composition and dynamics of phospholipids from fresh water fish Prochilodus Lineatus brain and spinal cord

Se estudió la composición de fosfolípidos y ácidos grasos de lípidos de cerebro y médula del pez de de agua dulce Prochilodus lineatus (sábalo). También hemos investigado la anisotropía de fluorescencia de fosfolípidos marcados con 1.6-difenil-1.3.5 hexatrieno. Se halló que la fosfatidilcolina era el fosfolípido más abundante, seguido por la fosfatidil etanolamina, fosfatidil-serina, fosfatidil-inositol y la esfingomielina. La composición de ácidos grasos de todos los fosfolípidos, exceptuando la esfingomielina, mostró la presencia de ácidos no saturados de la series n-3, n-6 y n-9. El ácido araquidónico evidenció la presencia de ácidos grasos polinosaturados de la serie n-6, y se lo encontró preferentemente en el fosfatidil-inositol. Los ácidos grasos n-3 fueron representados por los ícidos 20:5n-3, (araquidónico) y 3 en peces de agua dulce contrasta con la ausencia de los n-6 en el tejido nervioso de peces marinos. La fosfatidilcolina mostró la mayor fluidez de todos los fosfolípidos de cerebro y médula

Fatty acid composition and dynamics of phospholipids from fresh water fish Prochilodus Lineatus brain and spinal cord

Se estudió la composición de fosfolípidos y ácidos grasos de lípidos de cerebro y médula del pez de de agua dulce Prochilodus lineatus (sábalo). También hemos investigado la anisotropía de fluorescencia de fosfolípidos marcados con 1.6-difenil-1.3.5 hexatrieno. Se halló que la fosfatidilcolina era el fosfolípido más abundante, seguido por la fosfatidil etanolamina, fosfatidil-serina, fosfatidil-inositol y la esfingomielina. La composición de ácidos grasos de todos los fosfolípidos, exceptuando la esfingomielina, mostró la presencia de ácidos no saturados de la series n-3, n-6 y n-9. El ácido araquidónico evidenció la presencia de ácidos grasos polinosaturados de la serie n-6, y se lo encontró preferentemente en el fosfatidil-inositol. Los ácidos grasos n-3 fueron representados por los ícidos 20:5n-3, (araquidónico) y 3 en peces de agua dulce contrasta con la ausencia de los n-6 en el tejido nervioso de peces marinos. La fosfatidilcolina mostró la mayor fluidez de todos los fosfolípidos de cerebro y médula (AU)