Daniela Bottero

Global Population Structure and Evolution of Bordetella pertussis and Their Relationship with Vaccination

ABSTRACT Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape. Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

Outer membrane vesicles as acellular vaccine against pertussis

In this study the development and evaluation of outer membrane vesicles (OMVs) obtained from Bordetella pertussis as vaccines against pertussis disease is described. SDS-PAGE, immunoblot techniques and gel electrophoresis associated to tandem mass spectrometry were used to describe the composition of the OMVs obtained from B. pertussis Tohama CIP 8132 strain. These techniques revealed the presence of the main well-known pertussis surface immunogens in the OMVs such as pertactin, adenylate cyclase-haemolysin, pertussis toxin, as well as the lipo-oligosaccharide (LOS). A total of 43 proteins were identified by mass spectrometry. Some of them were predicted to have outer membrane or periplasmic location and the others with cytoplasmic or unknown location. The characterized pertussis OMVs were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by different routes. Killed detoxified whole-cell B. pertussis bacteria were used as reference. For intraperitoneal (i.p.) immunization, aluminum hydroxide was used as adjuvant. Since i.n. treatment with OMVs as well as killed whole-cell bacteria enhanced markers of innate immune response such as TNFalpha, IL-6 and CCL20, i.n. immunizations were performed with no adjuvant added. Immunized BALB/c mice were intranasally challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p<0.001). Adequate elimination rates (p<0.005) were observed in mice immunized either with OMV or whole-cell bacteria. Comparable results were obtained with both types of immunization route. In view to their capacity to induce airways innate and protective immunity in the mouse model, OMVs obtained from B pertussis are candidates to be used to protect against pertussis.

Pulsed-field gel electrophoresis, pertactin, pertussis toxin S1 subunit polymorphisms, and surfaceome analysis of vaccine and clinical Bordetella pertussis strains.

ABSTRACT To add new insight to our previous work on the molecular epidemiology of Bordetella pertussis in Argentina, the prn and ptxS1 gene sequences and pulsed-field gel electrophoresis (PFGE) profiles of 57 clinical isolates obtained during two periods, 1969 to 1989 and 1997 to 2006, were analyzed. Non-vaccine-type ptxS1A was detected in isolates obtained since 1969. From 1989 on, a shift of predominance from the vaccine prn1 type to the nonvaccine prn2 type was observed. This was also reflected in a transition of PFGE group IV to group VI. These results show that nonvaccine B. pertussis strains are currently circulating. To analyze whether the observed genomic divergences between vaccine strains and clinical isolates have functional implications, protection assays using the intranasal mouse challenge model were performed. For such experiments, the clinical isolate B. pertussis 106 was selected as representative of circulating bacteria, since it came from the major group of the PFGE dendrogram (PFGE group VI). Groups of mice were immunized either with diphtheria-tetanus-whole-cell pertussis vaccine (ptxS1B prn1) or a vaccine prepared by us containing B. pertussis 106. Immunized mice were then challenged with a B. pertussis vaccine strain (Tohama, harboring ptxS1B and prn1) or the clinical isolate B. pertussis 106 (ptxS1A prn2). An adequate bacterial-elimination rate was observed only when mice were immunized and challenged with the same kind of strain. For further characterization, comparative proteomic profiling of enriched membrane proteins was done using three vaccine strains and the selected B. pertussis 106 clinical isolate. By matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, a total of 54 proteins were identified. This methodology allowed us to detect differing proteins among the four strains studied and, in particular, to distinguish the three vaccine strains from each other, as well as the vaccine strains from the clinical isolate. The differing proteins observed have cellular roles associated with amino acid and carbohydrate transport and metabolism. Some of them have been proposed as novel vaccine candidate proteins for other pathogens. Overall, the global strategy described here is presented as a good tool for the development of next-generation acellular vaccines.

Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate.

In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles (OMVs) were engineered to decrease their endotoxicity. The pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-deacylase, was expressed in Bordetella pertussis strain Tohama I. The resulting OMVs, designated OMVs(BpPagL), contain tetra- instead of penta-acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin, as the wild type OMVs. The characterized pertussis OMVs(BpPagL) were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by i.n. routes. Immunized BALB/c mice were challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p<0.001). Adequate elimination rates (p<0.005) were observed in mice immunized either with OMVs(BpPagL) and wild type OMVs. All OMV preparations tested were non toxic according to WHO criteria; however, OMVs(BpPagL) displayed almost no weight loss at 3 days post administration, indicating less toxicity when compared with wild type OMVs. Induction of IL6- and IL1-expression in lung after i.n. delivery as well as neutrophil recruitment to airways showed coincident results, with a lower induction of the proinflammatory cytokines and lower recruitment in the case of OMVs(BpPagL) compared to wild type OMVs. Given their lower endotoxic activity and retained protective capacity in the mouse model, OMVs(BpPagL) obtained from B. pertussis seem as interesting candidates to be considered for the development of novel multi-antigen vaccine.

Pertussis epidemiology in Argentina: trends over 2004–2007

OBJECTIVES Pertussis continues causing significant morbidity and mortality worldwide. Although its epidemiology has been studied in many developed countries, the current pertussis situation in South America is scarcely known. This review summarizes the most important recent data concerning pertussis in a country of South America, Argentina. METHODS CDC criteria were used for pertussis diagnosis. Proportion of pertussis cases by age, immunization status, and immunization coverage rate evaluated at the Argentinean National Pertussis Reference Centers was reported. Bordetella pertussis isolates were characterized and compared with vaccine strains. RESULTS From 2002 to nowadays, a steady increase of pertussis cases was observed. Most of these cases correspond to patients younger than six months old that received less than three doses of vaccine. However, cases in adolescent and adults have also been detected. For this situation, which is not peculiar to Argentina, several explanations have been proposed. Among them, the inability of current vaccines to induce long-lasting immunity is the most widely accepted as a cause of pertussis resurgence. Furthermore, antigenic divergence between local clinical isolates and vaccine strains may have aggravated the effect of waning immunity. CONCLUSIONS Pertussis is an important problem for public health in Argentina. Divergence between vaccine strains and local isolates could contribute to the described pertussis epidemiology.

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.

GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF BORDETELLA PERTUSSIS STRAINS USED IN DIFFERENT VACCINE FORMULATIONS IN LATIN AMERICA

Aim:  To characterize Bordetella pertussis vaccine strains in comparison with current circulating bacteria.

Outer membrane vesicles derived from Bordetella parapertussis as an acellular vaccine against Bordetella parapertussis and Bordetella pertussis infection

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.

Laboratory Adaptation of Bordetella pertussis Is Associated with the Loss of Type Three Secretion System Functionality

ABSTRACT Although Bordetella pertussis contains and transcribes loci encoding type III secretion system (TTSS) homologues, expression of TTSS-associated proteins has been reported only for non-laboratory-adapted Irish clinical isolates. Here we confirm such a result for clinical isolates obtained from patients treated in Argentinean hospitals. Moreover, we demonstrate that the expression of TTSS-associated proteins is independent both of the year in which the isolate was obtained and of the types of polymorphic alleles for other virulence factors but is dependent on environmental growth conditions. Interestingly, we observed that TTSS-associated protein expression is lost after successive in vitro passages but becomes operative again when bacteria come into contact with the host. This in vivo activation of TTSS expression was observed not only for clinical isolates previously adapted to the laboratory after successive in vitro passages but also for vaccine strains that did not express the system in vitro. The reversibility of TTSS expression, demonstrated by its switching off-on when the bacterium comes into contact with the host, appears to be an adaptive response of this pathogen.

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.