No abstract

Pertussis is a respiratory infectious disease that has been resurged during the last decades. The change from the traditional multi-antigen whole-cell pertussis (wP) vaccines to acellular pertussis (aP) vaccines that consist of a few antigens formulated with alum, appears to be a key factor in the resurgence of pertussis in many countries. Though current aP vaccines have helped to reduce the morbidity and mortality associated with pertussis, they do not provide durable immunity or adequate protection against the disease caused by the current circulating strains of Bordetella pertussis , which have evolved in the face of the selection pressure induced by the vaccines. Based on the hypothesis that a new vaccine containing multiple antigens could overcome deficiencies in the current aP vaccines, we have designed and characterized a vaccine candidate based on outer membrane vesicle (OMVs). Here we show that the OMVs vaccine, but not an aP vaccine, protected mice against lung infection with a circulating pertactin (PRN)-deficient isolate. Using isogenic bacteria that in principle only differ in PRN expression, we found that deficiency in PRN appears to be largely responsible for the failure of the aP vaccine to protect against this circulating clinical isolates. Regarding the durability of induced immunity, we have already reported that the OMV vaccine is able to induce long-lasting immune responses that effectively prevent infection with B. pertussis . Consistent with this, here we found that CD4 T cells with a tissue-resident memory (T RM ) cell phenotype (CD44 + CD62L low CD69 + and/or CD103 + ) accumulated in the lungs of mice 14 days after immunization with 2 doses of the OMVs vaccine. CD4 T RM cells, which have previously been shown to play a critical role sustained protective immunity against B. pertussis , were also detected in mice immunized with wP vaccine, but not in the animals immunized with a commercial aP vaccine. The CD4 T RM cells secreted IFN-γ and IL-17 and were significantly expanded through local proliferation following respiratory challenge of mice with B. pertussis . Our findings that the OMVs vaccine induce respiratory CD4 T RM cells may explain the ability of this vaccine to induce long-term protection and is therefore an ideal candidate for a third generation vaccine against B. pertussis .

Pertussis resurgence had been attributed to waning vaccine immunity and Bordetella pertussis adaptation to escape vaccine-induced immunity. Circulating bacteria differ genotypically from strains used in production of pertussis vaccine. Pertactin-deficient strains are highly prevalent in countries that use acellular vaccine (aP), suggesting strong aP-imposed selection of circulating bacteria. To corroborate this hypothesis, systematic studies on pertactin prevalence of infection in countries using whole-cell vaccine are needed. We provide pertussis epidemiologic data and molecular characterization of B. pertussis isolates from Buenos Aires, Argentina, during 2000–2017. This area used primary vaccination with whole-cell vaccine. Since 2002, pertussis case incidences increased at regular 4-year outbreaks; most cases were in infants <1 year of age. Of the B. pertussis isolates analyzed, 90.6% (317/350) contained the ptxP3-ptxA1-prn2-fim3-2 allelic profile. Immunoblotting and sequencing techniques detected only the 2 pertactin-deficient isolates. The low prevalence of pertactin-deficient strains in Argentina suggests that loss of pertactin gene expression might be driven by aP vaccine.

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.

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.

En este trabajo de tesis hemos abordado el diseño de diferentes formulaciones vacunales con el objetivo de hacer frente a una enfermedad respiratoria inmunoprevenible que ha resurgido en las últimas décadas en muchos países [1,2] a pesar de la introducción de vacunación masiva desde los años 60 con calendarios de vacunación que contienen al menos 4 dosis de vacunas (https://apps.who.int/immunization_monitoring/globalsummary/diseases). Esta enfermedad denominada pertussis afecta a todas las edades siendo el grupo de los lactantes menores de 6 meses los más vulnerables y en los que se registran las tasas más altas de letalidad [3,4]. Trabajamos con dos enfoques que, aunque diferentes, ambos buscaron superar debilidades de las actuales formulaciones vacunales contra pertussis. En particular, buscamos superar las debilidades de las modernas vacunas acelulares (aP) que: 1- ejercen una presión de selección sobre la población bacteriana del agente causal (Bordetella pertussis) más fuerte que las tradicionales vacunas celulares favoreciendo la prevalencia de bacterias más resistentes a la inmunidad conferida por vacunación [5,6]; y 2- inducen una respuesta inmune más debil de corta duración y con un perfil mayormente del tipo Th2 [7,8]. Uno de nuestro enfoques se refirió a combinar a nuestro candidato vacunal multiepitope basado en vesículas de membrana externa (del inglés OMVs, cuya patente fue aceptada en Estados Unidos) [9-16] con la vacuna aP comercial de diferentes farmaceuticas que están formuladas a partir de pocos inmunógenos del agente causal de la enfermedad. Esta combinación buscó no sólo direccionar la respuesta inmune inducida por la aP hacia el perfil mixto Th1/Th17 recomendado para alcanzar una protección robusta de más largo plazo sino también posicionar a nuestro candidato vacunal basado en OMVs de cara a los ensayos clínicos que hoy resultan engorrosos o impracticables de realizar. Esta dificultad en los ensayos clínicos reside en el hecho que no existe un correlato de protección para pertussis sencillo de medir y porque muchos diseños incluirían grupos placebo que atentan contra el derecho del hombre a la vacunación actualmente vigente. La combinación de vacunas que incluye una vacuna en uso y una formulación novel permitiría diseñar ensayos clínicos de no inferioridad. Estos ensayos no sólo evitan trabajar con un grupo placebo que conllevaría a la negación de un derecho, sino que permiten realizar de mediciones sencillas de respuesta inmune (título de anticuerpos específico) en forma comparativa con un grupo vacunado con la vacuna en uso. El otro enfoque buscó fundamentalmente superar el efecto negativo en la protección de la divergencia entre la población bacteriana circulante y las cepas vacunales tratando de no incrementar y en lo posible disminuir, la presión de selección que ejercen las actuales vacunas aP por estar consitutidas por pocos inmunógenos en altas concentraciones. Decidimos para ello obtener a nuestro candidato vacunal basado en OMVs de cultivos B. pertussis en biofilm. Esta decisión se basó en: 1- que los aislamientos de B. pertussis se diferencian de las cepas vacunales en el hecho que estas últimas se adaptaron a las condiciones de cultivo de laboratorio dejando de expresar componentes propios del desarrollo in vivo del patógeno [17,18], 2- la detección de la formación de biofilm in vivo [19,20]; y 3- expresión de proteínas con potencial poder protector en condiciones de cultivo en biofilm [21,22]. Estos dos diseños vacunales fueron evaluados en el modelo murino de desafío subletal intranasal ya puesto a punto en nuestro laboratorio. La seguridad de estas vacunas se evaluó mediante ensayos de liberación de IL-6 en sangre entera murina. Los resultados de los ensayos demostraron que ambas formulaciones que contienen a las OMVs de B. pertussis exhiben menor capacidad endotoxica que las vacunas celulares comerciales y muy próxima a la de las vacunas aP. Respecto de la capacidad protectora detectamos que ambas vacunas son efectivas contra las cepas a partir de las cuales se formularon las vacunas aquí testeadas pero también contra los aislamientos circulantes, incluidos los que no expresan el antígeno vacunal pertactina cuya prevalencia ha aumentado en los últimos años en países que solo usan aP en sus calendarios [23,24]. La vacuna aP en uso frente a estos aislamientos que fueron seleccionados luego de realizar la caracterización genotípica y fenotípica de ellos [25] se mostró muy poco efectiva (p<0,05). Nuestro candidato vacunal obtenido de cultivos planctónicos, aunque mejora la protección contra aquellos aislamientos respecto de la vacuna aP comercial, el mismo presenta alguna debilidad que no se detectó en las formulaciones aquí ensayadas. Más aún, ambos candidatos vacunales en contraposición a la vacuna aP comercial indujeron un perfil mixto de respuesta celular con una robusta respuesta humoral. En resumen, nuestros diseños que contienen a OMVs derivados de B. pertussis conteniendo un mayor número de inmunógenos en conformaciones cercanas a las encontradas al agente causal se presentan como superadoras de las actuales vacunas acelulares y con potencialidad de transformarse en la tercera generación de vacunas contra pertussis.

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.