Pertussis epidemiology in Argentina: TRENDS after the introduction of maternal immunisation.

Data on the impact of the recently recommended maternal pertussis vaccination are promising, but still insufficient to universalise this approach. We thus compared the epidemiological data prior to the implementation of this vaccination strategy in Argentina (2012) with the figures reported after 2012. During that 2010-2016 period, two outbreaks occurred, one in 2011 and another in 2016. In the former, the incidence was 6.9/100 000 inhabitants and the case-fatality rate 2.6%. Thereafter, a decline in incidence was detected until 2014. During 2015 and 2016 an increase in the incidence transpired, but this rise was fortunately not accompanied by one in the case fatality ratio. Indeed, in 2016 the case fatality ratio was the lowest (0.6%). Moreover, during the 2016 outbreak, the incidence (3.9/100 000 inhabitants) and the case severity detected in the most vulnerable population (infants 0-2 months) were both lower than those in 2011. Consistent with this pattern, in 2016, in the most populated province of Argentina (Buenos Aires), the case percentage with laboratory-positive results indicating a high number of symptoms (59.1% of the total cases) diminished compared with that detected in the 2011 outbreak without maternal immunisation (71.9%). Using the mathematical model of pertussis transmission we previously designed, we assessed the effect of vaccination during pregnancy on infant incidence. From comparisons between the epidemiological data made through calculations, emerged the possibility that vaccinating women during pregnancy would benefit the infants beyond age 2 months, specifically in the 2-12-month cohort.

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.

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.

Bordetella holmesii in children suspected of pertussis in Argentina.

We describe nine patients (eight aged <1 year) clinically diagnosed with pertussis yet laboratory-confirmed with Bordetella holmesii infections, a human pathogen normally isolated from blood. Most patients reported cough and cold symptoms. No death was reported. We report B. holmesii isolation in infants with respiratory symptoms in Argentina.

Desarrollo y puesta a punto de una metodología diagnóstica serológica para coqueluche, una enfermedad resurgente / Development and commissioning of a serological diagnostic method for pertussis, a resurgent disease

INTRODUCCIÓN Coqueluche es una enfermedad respiratoria aguda que presenta 16 millones de casos anuales, aunque la vacunación masiva los ha reducido considerablemente. La alta comunicabilidad de la enfermedad marca la necesidad de realizar una detección temprana del caso índice y los contactos cercanos. OBJETIVOS Realizar la puesta a punto de una nueva metodología de ELISA, que emplea como antígeno a la subunidad S1 y requiere un único suero. MÉTODOS Se analizaron sueros de título conocido, titulados mediante la metodología de ELISA a células enteras de Bordetella pertussis, especialmente de adolescentes y adultos que habían recibido la última dosis de vacuna anti-pertussis más de tres años antes. Además, se escaló la obtención de la subunidad S1 de la toxina pertussis PTXS1. RESULTADOS La mejor condición de ensayo fue sensibilizando la placa de ELISA con 10 µg/ ml de PTXS1, tres horas de bloqueo, dilución de 1/1500 del anticuerpo secundario y 150 µl del sustrato de la enzima con 50 µl de solución stop para revelar la reacción. En esas condiciones se obtuvieron, para los sueros con títulos de ELISA considerados como positivos por la metodología de ELISA a células enteras (10), títulos de 1/320, mientras que para los sueros considerados como negativos (10), los títulos obtenidos a partir del ensayo de ELISA utilizando PTXS1 recombinante purificada fueron de 1/20-1/40. El rendimiento promedio obtenido con las purificaciones automatizadas fue tres veces mayor que con purificación manual. DISCUSIÓN El desarrollo de un test de ELISA que emplee la subunidad S1 de la toxina pertussis PTXS1, obtenida de forma recombinante a nivel local, y que pueda ser validado contra otros ensayos de ELISA disponibles, redundaría en una herramienta útil para el diagnóstico de la enfermedad en la población adolescente y adulta.

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. METHODS AND RESULTS: Genomic and proteomic analyses of Bp137 were performed in comparison with other vaccine strains used in Latin America (Bp509 and Bp10536) and with the clinical Argentinean isolate Bp106. Tohama I strain was used as reference strain. Pulse-field gel electrophoresis (PFGE) and pertussis toxin promoter (ptxP) sequence analysis revealed that Bp137 groups with Bp509 in PFGE group III and contains ptxP2 sequence. Tohama I (group II) and Bp10536 (group I) contain ptxP1 sequence, while Bp106 belongs to a different PFGE cluster and contains ptxP3. Surface protein profiles diverged in at least 24 peptide subunits among the studied strains. From these 24 differential proteins, Bp10536 shared the expression of ten proteins with Tohama I and Bp509, but only three with Bp137. In contrast, seven proteins were detected exclusively in Bp137 and Bp106. CONCLUSIONS: Bp137 showed more features in common with the clinical isolate Bp106 than the other vaccine strains here included. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented show that the old strains included in vaccines are not all equal among them. These findings together with the data of circulating bacteria should be taken into account to select the best vaccine to be included in a national immunization programme.

Laboratory adaptation of Bordetella pertussis is associated with the loss of type three secretion system functionality.

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.

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)

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.

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.

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.

Differences of circulating Bordetella pertussis population in Argentina from the strain used in vaccine production.

In Argentina, as in other countries, the number of pertussis cases has been increasing, even in highly vaccinated zones. Many reports suggest that the decline of vaccine efficacy due to antigenic shifts in the circulating Bordetella pertussis might be among the factors that contribute to pertussis re-emergence in different parts of the world. To evaluate the incidence of this factor in Argentina, we decided to characterize the circulating bacteria of an important demographic area of this country in comparison with the strain used for vaccine production. From 1997 to 2003 we collected nasopharyngeal samples from pediatric patients with signs of Bordetella infection hospitalized in the metropolitan area of Buenos Aires and La Plata, Argentina. From these samples we identified 28 B. pertussis, which were characterized by biochemical techniques, PCR, DNA fingerprint, prn and ptx genes sequencing, and lipopolysaccharides (LPS) pattern. BOX-PCR from B. pertussis isolates yielded one cluster containing 13 isolates and some smaller ones, being all fingerprints different from the vaccine strain. Differences between Argentinean circulating bacteria and the vaccine strain were also observed for the Prn and Ptx variants as well as for the LPS pattern. Moreover, this last pattern seemed to change over the years. In addition, we identified two B. bronchiseptica. The presence of this Bordetella species together with the observed differences between circulating B. pertussis and the strain used in vaccine production should be considered for the development of an improved vaccine.

Sex steroid hormones act as growth factors.

We observed that sex steroid hormones, like growth factors, stimulate the Src/Ras/erk pathway of cell lines derived from human mammary or prostate cancers. In addition, hormone-dependent pathway activation can be induced in Cos cells, upon transfection of classic steroid receptors. Cross-talks between sex steroid receptors regulate their association with Src and consequent pathway activation. Oestradiol treatment of MCF-7 cells triggers simultaneous association of ER with Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase (PI3-kinase) and activation of Src- and PI3-K-dependent pathways. Activation of the latter pathway triggers cyclin D1 transcription, that is unaffected by Mek-1 activation. This suggests that simultaneous activation of different signalling effectors is required to target different cell cycle components. Thus, a novel reciprocal cross-talk between the two pathways appears to be mediated by the ER. In all tested cells, activation of the signalling pathways has a proliferative role. Transcriptionally inactive ER expressed in NIH 3T3 cells responds to hormone causing Src/Ras/Erk pathway activation and DNA synthesis. This suggests that in these cells genomic activity is required for later events of cell growth.