Colonization of Bordetella pertussis clinical isolates that differ by pulsed field gel electrophoresis types in the lungs of naïve mice or mice immunized with the whole-cell pertussis vaccine used in Poland.

The goal of our study was to compare the elimination of Bordetella pertussis clinical isolates that differ according to pulsed field gel electrophoresis (PFGE), serotypes and genes encoding virulence factors from the lungs of naïve mice or mice immunized with commercial diphtheria-tetanus-whole-cell pertussis vaccine used in Poland. When a mixture of four isolates, given in equal proportions and harboring different PFGE profiles, serotypes, and alleles encoding virulence factors, was used to infect non-immunized mice, a single isolate, characterized by PFGE type IVγ, Fim2 phenotype and ptxA1-prn2-tcfA2-fim2-1-ptxP1-ptxC1-fim3-1 alleles, was found to be significantly predominant compared to the others. This PFGE profile is commonly found in B. pertussis isolates circulating in some European countries since the late 1990s, confirming its high fitness. The Polish commercial whole-cell pertussis vaccine induced an immunity effective at eliminating the B. pertussis isolates from the lungs. However, the elimination of the isolate harboring PFGE type C profile, Fim2,3 phenotype and ptxA1-prn1-tcfA2-fim2-1-ptxP1-ptxC1-fim3-1 alleles was delayed as compared to the others, suggesting phenotypic differences with the other isolates and vaccine strains. Nevertheless, the same isolate, when challenged into mice in the defined mixture of strains, lost the competition with the others, as measured by lung colonization efficiency. This PFGE profile represents 15 % of the isolates circulating in Poland between 2001 and 2012.

Effectiveness of experimental whole-cell pertussis vaccines in murine model.

INTRODUCTION: Since the 1990s pertussis re-emergence has been observed in many highly immunized countries. Genetic divergence between circulating B. pertussis isolates and vaccine strains has been suggested as one of the reasons responsible for the resurgence of pertussis. This divergence was observed in some studies to affect the effectiveness of pertussis vaccine when tested in murine model. In the study, using the murine intranasal challenge model we evaluated the effectiveness of four experimental wP vaccines, prepared with B. pertussis isolates belonging to different PFGE groups, in the elimination of the bacterial infection induced with mixture of the four B. pertussis isolates. METHODS: The experimental wP vaccines were prepared with clinical isolates belonging to PFGE groups V, IVγ and C, used individually or together. The mixture of four isolates classified to PFGE groups V, IVγ, III and C was used as intranasal mice challenge. The chosen strains represent PFGE groups characteristic for isolates currently circulating in Europe (PFGE groups IV and V), specific for Poland (PFGE group C) and vaccine strains of Polish wP vaccine (PFGE group III). Additionally, to study bacterial fitness, changes in the proportions of four isolates used as the challenge within the course of infection in mice lungs were monitored. RESULTS AND CONCLUSIONS: All experimental wP vaccines were found to be equally effective in eliminating B. pertussis from mice lungs. Their effectiveness was independent on PFGE group of vaccine strain. The results on bacterial fitness during mixed infections induced in the non-immunized mice found the isolate of PFGE group IVγ dominating among the other isolates used in the mixture belonging to PFGE group III, V, and C. This data might suggest that the isolates belonging to PFGE group IV, so commonly seen in Europe, might be more fitted to explore in conditions of waning immunity.

Consistency of Bordetella pertussis vaccine seed strains and potency of whole-cell pertussis vaccine still in use in Poland.

In Poland, where the wP vaccine has been used since 1960, pertussis rates increased in the mid-1990s. In 2012, the rate of pertussis recognised by surveillance was unexpectedly found to be two-fold higher than in the previous decade. Quality measures on potency and vaccine working seeds were introduced, to confirm the possible impact of manufacturing inconsistency or potency lowering on the observed increase in pertussis. Shewhart charts on potency values for lots released between 2001 and 2013 did not reveal any significant fluctuations. Working seeds of three vaccine strains used within last decade for wP manufacturing belong to the PFGE group III and were highly related. According to PFGE and SDS-PAGE data, all vaccine strains were found consistent according profiling on the genomic and protein levels. According to the sequencing data, they harboured ptxA2, ptxC1, prn1, fim2-1, fim3-1, tcfA2, ptxP1 and were assigned as MLST-2 type. Other factors apart from vaccine manufacturing inconsistency might be responsible for the increase in pertussis noted in 2012 in Poland.

[Evaluation of Bordetella pertussis strains toxicity in vitro using CHO cell lines]. / Ocena toksycznosci szczepów Bordetella pertussis z zastosowaniem linii komórkowych CHO in vitro.

INTRODUCTION: Whooping cough is still a significant disease with regular outbreaks despite the decades of mass vaccination and good immunization coverage. The aim of this study was to evaluate the capacity of Bordetella pertussis toxicity testing among strains harbouring different alleles of the pertussis toxin promoter ptxP using hamster ovary cell line CHO (Hamster Ovary). METHODS: The study assessed the limits of detection of high and low Ptx levels producing strains using a reference preparation ofpertussis toxin and B. pertussis strains that increased toxicity in vitro has been previously correlated with ptxP3 allele presence. RESULTS: The presence of the strong agglomerates on CHO cell line confirmed the higher toxicity of B. pertussis strains isolated in France. Preliminary toxicity study with use of selected strains of B. pertussis differing by ptxP1 and ptxP3 promdter alleles with respect to relevant reference preparation indicate lower toxicity of strains B. pertussis isolated in Poland. CONCLUSIONS: The toxicity measured on CHO line will be used to assess the virulence of all available B. pertussis strains isolated in Poland.

[The studies of elimination of nontypeable Haemophilus influenzae strains using the animal model of infection]. / Badania eliminacji nietypowalnych szczepów Haemophilus influenzae na modelu zwierzecym.

INTRODUCTION: The intranasal immunization is considered the effective method to induce immunological response in the mucosa and the model useful to develop the vaccine against otitis media due to nontypaeble Haemophilus influenzae infections. In the study, elimination of NTHi strains isolated from healthy and carrier individuals, varying with several genetic determinants, from mice nasopharynx, lungs and ears tissues and the interactions among strains during mixed infections were evaluated. METHODS: Mice were infected with single and mixture of NTHi strains previously identified as non or potentially invasive. The curves of infections were drown from the averages of log cfu/mg values obtained for nasopharynx or ears leavages or lungs homogenates coupled with standard deviation at each time point using 5 mice. The significance of the differences was confirmed using ANOVA method. RESULTS: The course of infections induced with a single noninvasive or potentially invasive strains in the mice nasopharynx, ear or lung tissues were not found to differ in respect to the level and duration. Regardless the strain used, higher elimination rates in the ear and lung tissues in comparison with nasopharynx were fund. Different rate of elimination was found in the case of mixture infections where the strain, determined in the previous study as potentially invasive, was dominating. CONCLUSION: The concurrence model among not-invasive and potentially invasive NTHi strains in the host niche was thus confirmed using in vivo model of infection.

[Reproducibility of Fim2 and Fim3 antigens determination in Bordetella pertussis by serotyping method]. / Ocena porównywalnosci oznaczen obecnosci antygenów Fim2 i Fim3 Bordetella pertussis metoda serotypowania.

INTRODUCTION: Serotyping is a commonly used method to characterize the presence of Fimbriae 2 and 3 in Bordetella pertussis strains for epidemiological purposes and optimal choice of strain composition of the pertussis whole-cell vaccine. Monoclonal antisera against Fim2 and Fim3 are recommended to be used for microplate serotyping instead ofpolyclonal. Reliable evaluation offimbriae expressed by B. pertussis strains influence interpretation of vaccine-driven strain evolution. METHODS: To evaluate the impact of tests conditions on the reproducibility of serotyping, results of serotyping based on a standardized protocol for microplate agglutination with monoclonal antisera performed in three different accredited laboratories were compared. For the study isolates of three vaccine strains of B. pertussis deposited within seed lot system originating from different liofilization lots were compared. RESULTS: Lack of the complete agreement on serotyping results among three labs might relates to the differences of media used, subjective reading, test conditions, and specificity of the reagents. CONCLUSIONS: Serotyping results should be interpreted with caution and the type of media and culture conditions used should be precisely recommended after validation studies. Inconsistent results should be confirmed using an alternative technique, eg. ELISA or by reference laboratory.

[Immunogenicity and reactogenicity of acellular pertussis vaccines intended for adolescent and adults]. / Immunogennosc i odczynowosc bezkomórkowych szczepionek przeciw krztuscowi przeznaczonych dla mlodziezy i osób doroslych.

Although high immunization status against pertussis maintained in neonates and children, the increase in pertussis cases has been observed in Poland since 1990-ies, especially in adolescent and adults. Wide use of pertussis acellular vaccines intended for adolescents and adult's immunization might prolong immunity against pertussis. The goal of the work was to present current view of immunogenicity and reactogenicity of those pertussis vaccines in different age groups.

[Immunization of adolescents and adults as the strategy of improvement of epidemiology of pertussis]. / Szczepienia mlodzieizy i osób doroslych jako strategia poprawy sytuacji epidemiologicznej zachorowan na krztusiec.

Increase of pertussis incidence has been recognised mainly among adolescents and adults since 90. As adolescents and adults sustain the reservoir of infection for non immunized and not completely immunized newborns and neonates, increased rates of pertussis are dangerous. The improvement of pertussis epidemiology might have been obtained through routine immunization of adolescents and adults able to interrupt of B. pertussis circulation in the population. The improvement of surveillance and diagnostics might have result in better detection of the disease in children at the age up to first year and in older age groups.

Effectiveness of the whole-cell pertussis vaccine produced in Poland against different Bordetella parapertussis isolates in the mouse intranasal challenge model.

The aim of the study was to evaluate the effectiveness of the whole-cell pertussis vaccine produced locally and routinely used in Poland in the elimination of Bordetella parapertussis strains from the lungs and trachea of a mouse model. We found that the average protective effect against B. parapertussis in the lungs of mice immunized with the whole-cell pertussis vaccine (DTwP) was significantly higher than in animals immunized with the acellular pertussis vaccine (DTaP). The effectiveness of B. parapertussis elimination rates from the lungs of DTwP-immunized mice, depending on the strain used as a challenge, was found to be 1.2-3.0 times or 3.1-7.0 times lower than against Bordetella. pertussis Tohama I or vaccine B. pertussis 606/67 isolates, respectively. Our results show that the locally produced DTwP vaccine is able to protect against B. parapertussis isolates; however, the level of protection and course of B. parapertussis infection in the lungs and trachea seems to be strain specific.

Strain variation among Bordetella pertussis isolates circulating in Poland after 50 years of whole-cell pertussis vaccine use.

In the present study, clinical isolates of Bordetella pertussis collected in Poland from 1960 to 2005 were analyzed by pulsed-field gel electrophoresis (PFGE) according to protocols recommended in previous studies. Among the 110 isolates from 1995 to 2005, 59 PFGE patterns were found, most of which were different from those currently circulating in other European Union (EU) countries for which data are available. The PFGE patterns of currently disseminating B. pertussis clones were found within PFGE groups III and IV, as elsewhere in the EU, and in newly identified clusters A and C. Up to 70, 26, and 4%, respectively, of the currently isolated strains in Poland harbored ptxA1-prn1, ptxA1-prn2, and ptxA1-prn3 allele combinations, and most (82%) were found to be of the Fim2 phenotype. Differences in the extent of heterogeneity estimated by PFGE typing in B. pertussis populations circulating in Poland in comparison to other EU countries may be due to the different vaccine composition strategy, since routine pertussis vaccination was initiated in Poland in 1960.

[Evaluation of whole-cell and acellular pertussis vaccines effectiveness in clearance of experimental B. pertussis infection in mice]. / Ocena efektywnosci pelnokomórkowych i bezkomórkowych szczepionek przeciw krztuscowi w eliminacji eksperymentalnego zakazenia myszy Bordetella pertussis.

The study is based on assumption that B. pertussis strains harbouring different allele variants of genes encoding subunit S1 of pertussis toxin and pertactin might be eliminated with different efficiency from lung tissue of mice which were immunized with whole-cell and acellular pertussis vaccines. It has been assumed that strains containing combinations of genes alleles which were not prevalent since 1990-ties are consisting of mutated strains in respect to pertussis toxin subunit S1 and pertactin, and are capable to decrease efficiency of pertussis vaccines. Experiments performed in vivo dealt with activity of tested vaccines against B. pertussis strains of different combinations of ptxS1/prn. The study indicated for lowered efficiency of whole-cell and acellular pertussis vaccines in elimination of mutated strains of B. pertussis from animal lung tissue in comparison with strains currently used for vaccine production.

[Analysis of chosen parameters of immuno response in mice immunized with whole-cell or acellular pertussis vaccines and challenged with B. pertussis strains harbouring different ptxS1/prn allele genes combinations]. / Analiza wybranych parametrów immmunologiczej odpowiedzi myszy szczepionych pelno- lub bezkomórkowa szczepionka przeciw krztuscowi i zakazanych szczepami B. pertussis o róznych kombinacjach alleli genów ptxS1/prn.

Studies concerned evaluation of differences between parameters of cell-mediated immunity in mice, induced with whole-cell and acellular pertussis vaccines with subsequent challenge with B. pertussis strains harbouring different ptxS1/prn allele genes. In the study, concentrations of IFN-gamma/Il-2 and 1l-4/Il-5 in supernatants of cultured mice splenocytes have been determined to evaluate differences in Th1 or Th2 lymphocytes subpopulation response. Simultaneously, studies of intracellular expression of genes encoding of Il-2, Il-12, IFN-gamma and Il-4, Il-5, Il-10, Il-13 in mice splenocytes, and genes encoding factors involved in inflammatory process in the lung tissue (GM-CSF, TNF-alpha, Il-1beta, Il-6 i TGF-beta) have been performed on RNA level. The obtained results, confirmed high polarization of immunological response toward Th1 in mice immunized with DTP vaccine with whole-cell pertussis component, and toward Th2 in mice immunized with acellular pertussis vaccine. Inflammatory process in the lung tissue was more pronounced in animals immunized with whole-cell pertussis vaccine. There were no quantitative differences of analysed factors involved in the immune response among mice challenged B. pertussis strains containing different ptxS1/prn composition.

Amplified fragment length polymorphism (AFLP) versus randomly amplified polymorphic DNA (RAPD) as new tools for inter- and intra-species differentiation within Bordetella.

Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intra- and inter-species differentiation of isolates of the Bordetella genus. The most precise level of inter-species discrimination and the highest level of intra-species discrimination of the Bordetella isolates of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both techniques might provide alternative tools for the identification of Bordetella at the genomic species and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in epidemiology. By applying the AFLP technique presented in this article, more precise data on the emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the Bordetella genus in the human and animal environments might be obtained.

[Increase in the incidence of pertussis and quality of whole-cell pertussis component of the DTP vaccine produced in Poland. Part I. Potency of DTP vaccine]. / Wzrost zachorowan na krztusiec a wyniki analizy jakosci komponentu krztuscowego polskiej szczepionki DTP. Czesc I: Moc szczepionki DTP.

This study aimed to identify and to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine lots produced within 1972-2001 due to the changes having occurred in production and potency testing procedures. The study confirms that higher potency values were obtained for vaccine lots produced since 80-ties, e.g. after changes of: references lots (1975), vaccine strains (1978) and source of animals used in Kendrick tests (1979). Additionally, the comparisons performed revealed a down trend in potency levels within 1992-1999 correlating to the lowering of the number of IOU/dose.

[Increase in the incidence of pertussis and quality of whole-cell pertussis component of the DTP vaccine produced in Poland. Part II. Consistency of control and production]. / Wzrost zachorowan na krztusiec a wyniki analizy jakosci komponentu krztuscowego polskiej szczepionki DTP. Czesc II: Jednorodnosc badan i produkcji.

This study aimed to identify and to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine lots produced within 1972-2001 due to the changes having occurred in production and potency testing procedures. Obtained results showed high consistency of the production and control tests confirming relevant quality of country-produced DTP vaccine.