Membrane Vesicles Derived from Bordetella bronchiseptica: Active Constituent of a New Vaccine against Infections Caused by This Pathogen.

Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.

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 (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.

Acid tolerance response of Bordetella bronchiseptica in avirulent phase.

Bordetella bronchiseptica is a Gram-negative bacterium responsible for respiratory diseases in many mammalian hosts, including humans. This pathogen has been shown as able to persist inside the host cells, even in the phagosomes that are acidified to pH 4.5-5.0 after bacterial infection. Here we evaluated the resistance of B. bronchiseptica to survive under acidic conditions. In particular we analyzed the bacterial capacity to develop the mechanism known as acid tolerance response (ATR). Our studies were mainly focused on the avirulent phase of the bacteria since this phenotypic phase was reported to be more resistant to environmental stress conditions than the virulent phase. Results from B. bronchiseptica in virulent phase were also included for comparison purposes. In fact, for B. bronchiseptica 9.73 bacteria in virulent phase we observed that the viability of bacteria does not decrease significantly when grown at pH as low as 4.5, but it is affected when the pH of the medium was equal to or less than 4.0. After acid-adaptation at pH 5.5 for several hours, the survival rate of B. bronchiseptica 9.73 at lethal pH 4.0 for 6h was increased. Interestingly, the avirulent phase mediated by the two-component BvgAS system conferred further resistance to lethal acid challenge and a marked increase in the magnitude of the expressed ATR. The ATR for this avirulent phase seems to be associated with changes in LPS and surface protein profiles. 2D-gel electrophoresis revealed at least 25 polypeptides differentially expressed, 17 of which were only expressed or over-expressed under acid conditions. Using MALDI-TOF mass spectrometry, 10 of these differentially expressed polypeptides were identified.

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production.

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 µg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

Vigilancia nacional de tos convulsa, coqueluche o pertussis: experiencia del hospital Garrahan y de los laboratorios nacionales de referencia / National vigilance program for whooping cough or pertussis: Experience of the Garrahan Hospital and national reference laboratories

Pertussis es una enfermedad particularmente grave en menores de 1 año. No sólo no se ha podido erradicar pese al uso de vacunas por más de cincuenta años, sino que en la actualidad ha reemergido. En junio del 2010 se registró uno de los mayores brotes de coqueluche de los Estados Unidos, con 910 casos confirmados. En la Argentina se ha venido registrando un aumento significativo de casos de pertussis. En este trabajo se presentan datos nacionales y de nuestro hospital confirmados por cultivo y/o métodos moleculares (PCR). Durante el período 2008-2010 se registraron anualmente en nuestro país entre 600 y 1.000 casos con sintomatología compatible y en el Hospital Garrahan entre 110 y 150. La confirmación se concretó en alrededor de 24% de los casos nacionales y entre un 7% y un 36,4% en el hospital. Los datos obtenidos en los laboratorios nacionales de referencia muestran un registro actual que vuelve a alcanzar los valores del 2008, luego de un descenso en el 2009. En el Hospital Garrahan, un aumento relativo en el número de casos sospechosos no confirmados podría estar vinculados a cuadros respiratorios compatibles con pertussis producidos por otros agentes etiológicos. Más allá de las variaciones anuales se puede observar la vigencia de esta enfermedad en la Argentina. Desde el punto de vista de la prevención es importante destacar que muchos de estos niños no habían recibido el esquema de vacunación completo (la mayoría de los casos se registró en menores de 6 meses). (AU)

Pertussis is a especially severe illness in infants. The use of vaccines during more than 50 years could not eradicate this illness, that now it has reemerged. In June 2010 one of the greatest outbreaks of coqueluche was recorded in the United States, with 910 confirmed cases. In Argentina, a significant increase of pertussis cases was recorded. In the present study both national and hospital data is presented, including cases confirmed by culture and/or molecular methods (PCR). During 2008-2010 between 600 and 1,000 cases were recorded in our country, and between 110 and 150 at the Hospital Garrahan. Confirmation was done in almost 24% of national cases and between 7% and 36.4% in the hospital. Data obtained by national reference laboratories showed that cases decreased from 2007 to 2008, to regain similar numbers in 2009. In the Hospital Garrahan a relative increase of non-confirmed suspected cases could be related to respiratory syndromes due to other agents but compatible with pertussis. Beyond annual fluctuations the prevalence of this illness in Argentina can be observed. From the prevention point of view it is very important to highlight that many of these children have not received the complete vaccination scheme (most of cases have been recorded in less than 6-month-old children) (AU)

Bordetella Bronchiseptica aislada en paciente con fibrosis quística / Isolation Bordetella bronchiseptica in a patients with cystic fibrosis

Presentamos el caso de una paciente de 15 años con Fibrosis Quística (FQ) en la cual, en dos oportunidades y con un intervalo de 2 años; se aisló Bordetella Bronchiseptica con idéntico perfil genético estudiado por electroforesis de campo pulsado. El mecanismo lesional de B. Bronchiseptica en el árbol bronquial de pacientes con FQ no esta claramente establecido, pero la habilidad de esta bacteria para inhibir la función de los leucocitos y su capacidad de adherirse a las células del epitelio bronquial explicaría su capacidad infectiva y su persistencia en el tracto respiratorio.

Release of outer membrane vesicles from Bordetella pertussis.

The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly- showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.