Immunological surveys of antibodies against B. pertussis and B. parapertussis in some African and Asian countries.

Immunological surveys in African and Asian countries showed a different degree of herd immunity in the respective countries and the circulation of various types of B. pertussis. Antibodies against B. parapertussis, the second aertiological agent of whooping cough, were found in all countries in all age groups. Before planning any vaccination program, attention should be paid to the vaccine concerning the content of all types of B. pertussis, as was shown in the results of the testing of different vaccines used in Mongolia and Algeria. It is also possible to estimate the age limit for vaccination and thus economic use of the vaccine. The contemporary state of reporting whooping cough cases is very unsatisfactory.     

Enhancement of Bordetella parapertussis infection by Bordetella pertussis in mixed infection of the respiratory tract

The epidemiological and pathogenic relationship between Bordetella pertussis and Bordetella parapertussis, the two causes of whooping cough (pertussis), is unclear. We hypothesized that B. pertussis, due to its immunosuppressive activities, might enhance B. parapertussis infection when the two species were present in a coinfection of the respiratory tract. The dynamics of this relationship were examined using the mouse intranasal inoculation model. Infection of the mouse respiratory tract by B. parapertussis was not only enhanced by the presence of B. pertussis, but B. parapertussis significantly outcompeted B. pertussis in this model. Staggered inoculation of the two organisms revealed that the advantage for B. parapertussis is established at an early stage of infection. Coadministration of PT enhanced B. parapertussis single infection, but had no effect on mixed infections. Mixed infection with a PT-deficient B. pertussis strain did not enhance B. parapertussis infection. Interestingly, the depletion of airway macrophages reversed the competitive relationship between these two organisms, but the depletion of neutrophils had no effect on mixed infection or B. parapertussis infection. We conclude that B. pertussis, through the action of PT, can enhance a B. parapertussis infection, possibly by an inhibitory effect on innate immunity.     

Review of the biology of Bordetella pertussis.

Bordetella pertussis produces a complex array of adhesins, aggressins and toxins that are presumed to be important in the colonisation of its human host and in ensuring its survival and propagation. The organism also has highly sophisticated mechanisms for regulating virulence factor expression, in response to environmental signals or by reversible mutations. Despite the rapidly increasing knowledge of these aspects of the biology of B. pertussis, our understanding of the pathogenesis of whooping cough is still far from clear. In defining the role of individual factors, reliance has to be placed on in vitro assays or animal models of the human infection, particularly in the mouse, where different conditions may prevail. Some clues to pathogenic mechanisms may be provided by considering other bordetellae, especially B. parapertussis, B. bronchiseptica and B. avium, their similar, but not identical, range of virulence factors and the common features of the diseases caused by these species in their respective hosts. The bordetellae are usually defined as obligate, non-invasive parasites of the respiratory tracts of warm-blooded animals, including birds, with a predilection for the respiratory ciliated epithelium. This definition has been challenged by a number of recent observations. For example, the ability of Bordetella spp. to regulate virulence factor expression in response to external signals strongly suggests that they have alternative habitats where such regulation would be an advantage. These habitats may be intracellular, since it has been shown that B. pertussis, B. parapertussis and B. bronchiseptica can invade and survive within host cells, or they may be in other sites within the same or different hosts. Recent DNA fingerprinting studies of B. pertussis have revealed hitherto unsuspected heterogeneity amongst isolates which could be reflected in antigenic differences between strains. Some of these new perspectives on Bordetella pathogenicity may have implications for pertussis vaccine development.     

Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth.     

Impact of vaccination on the infectious diseases epidemiology: example of pertussis

Several vaccines are now routinely used since fifty years in different developed countries. Their principal impact has been to decrease morbidity and mortality of the infectious diseases they are targeting. One disease, smallpox, is eradicated, poliomyelitis will be soon, diphteria is controlled in several countries but pertussis is still endemic although an efficacious vaccine was used. Why? Pertussis is an example of an infection for which the immunity of the population has changed after the introduction of generalized vaccination with killed whole cell pertussis vaccines, from a natural immunity due to infection to different types of vaccine-induced immunity. These different types of immunity have changed the protection against infection, disease and transmission. The impact of the generalized vaccination in a human population has been an important change in the epidemiology of the disease. In fact, a child-to-child transmission observed before the introduction of vaccination is now replaced by an adolescent-adult to infant transmission. The major consequence is an increase in the mortality and morbidity in non vaccinated infants mostly contaminated by their parents. Researches undertaken on the agent of the disease, the bacterium, Bordetella pertussis, conducted to the development of subunits vaccines, efficacious and better tolerated by infants than whole-cell vaccines. Many developed countries decided to change vaccines but also to add vaccine boosters for adolescents and adults in order to stop the transmission of the disease to infants. However, even after 15 years of studies in many countries, pertussis is still underestimated in adults and generalized adult vaccination remains difficult. The new goal now is to give information to medical students and health care workers in general in order to increase adolescent and adult's vaccination coverage.     

Humoral imunity to pertussis in children with diabetes of type 1

To determine the state of humoral immunity to pertussis in children with insulin-dependent diabetes, IgG antibodies to pertussis toxin (PT) were determined in blood serum samples by means of EIA. In a group of children aged up to 6 years the highest percentage (100%) received the complete course of vaccination against pertussis with Russian adsorbed DPT vaccine, containing whole-cell pertussis monovaccine, while in a group over 6 years the complete vaccination course (3 vaccinations and 1 revaccination) had 53.4% of children. Pertussis morbidity was considerably higher in nonvaccinated subjects than in children with 4-fold vaccination (p < 0.001). The coefficient of association (Q) was 0.84. Children of all age groups were found to have low and average titers of antibodies to PT. The regressive analysis showed a decrease in antibodies in persons completely immunized against pertussis by the age of 6 years old. The presence of antibodies in nonimmunized persons showed that cases of pertussis or carrier state took place among the population. High titers of antibodies, indicative of recent cases of pertussis, were registered in all age groups, but high titers of antibodies were registered mostly in the group of children over 13 years old (p < 0.05), which confirmed an increase in pertussis morbidity in adolescents. Thus, vaccination against pertussis effectively protected children with diabetes of type 1, aged up to 6 years. For more prolonged protection the vaccination and revaccination of children aged over 4 years old is necessary.     

Molecular typing of the strains of Bordetella pertussis circulating in St. Petersburg during an outbreak of the disease

Growth of the whooping-cough morbidity during the last years in Russia and other countries with 40-year-long history of immunization gave rise to significant interest of researchers to variability of the Bordetella pertussis population. Comparative assay of the genomes of the B. pertussis strains circulating in St. Petersburg in 1998-2000 and strains used to produce domestic vaccines AKDS was performed using the pulse-field electrophoresis and sequencing. It was found that most strains of B. pertussis circulating during this period were distinguished from the vaccine strains by the DNA-profile and structure of genes involved in encoding of biosynthesis of the S1 subunit of the whooping-cough toxin (ptxS1) and pertactin (prn). It was shown that 62% of wild-type strains had electrophoretic profiles IV alpha and IV beta, whereas vaccine strains had electrophoretic profiles II and III. Circulation of strains with profiles IV alpha and IV beta was found to correlate with the whooping-cough morbidity rate in vaccinated children. Our results and data of other researchers were compared and analyzed.     

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis . It was found that this homolog, named AfuA_Bpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis , showed no influence on antibody recognition of AfuA_Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

     

Molecular typing of Bordetella parapertussis isolates circulating in Pakistan

Although a whole-cell pertussis vaccine was introduced in Pakistan in 1980, little is known about the pertussis prevalence and circulating strains in Pakistan. The aim of this study was to analyze Bordetella parapertussis isolates circulating between 2005 and 2009 in Pakistan and to compare them with those found in other countries during different periods. A total of 59 (7.35%) B.?parapertussis isolates from 802 subjects (median age, 3?years) from Pakistan, with pertussis-like symptoms were investigated. We carried out genotyping and DNA microarray analyses on these isolates and compared them with some international isolates of B.?parapertussis. We found that the allele for pertactin (prn) found in strains studied from Pakistan was identical to the predominant type found in Europe. We showed that B.?parapertussis isolates circulating in Pakistan are part of the same pulsed-field gel electrophoresis group to those circulating in Finland during the period of 1982-2007. Finally, microarray analysis confirmed that the isolates collected in Pakistan, were quite similar to international strains. Overall, these results confirm that B.?parapertussis is extremely monomorphic. The high isolation rate of B.?parapertussis (7.35%) compared to Bordetella pertussis (0.5%) may suggest that the whole-cell vaccine used in Pakistan is effective against B.?pertussis (0.5% infections detected), but much less so against B.?parapertussis.     

Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis.

The ptl locus of Bordetella pertussis contains eight open reading frames which are predicted to encode proteins (PtlA to PtlH) that are essential for secretion of pertussis toxin from the bacterium and which are members of a family of transport proteins found in other types of bacteria. We have detected PtlE, PtlF, and PtlG in immunoblots of extracts of B. pertussis by using antibodies raised to fusion proteins consisting of maltose-binding protein and the individual Ptl proteins. These proteins have apparent molecular weights similar to those predicted by DNA sequence analysis. Cell fractionation studies indicated that all three Ptl proteins are associated with the membranes of B. pertussis, suggesting that the Ptl proteins form a gate or channel which facilitates transport of pertussis toxin. Cell extracts of other Bordetella spp. were probed with antibodies to Ptl proteins for the presence of these transport proteins. Neither Bordetella parapertussis nor Bordetella bronchiseptica contained detectable levels of PtlE or PtlF. This lack of detectable Ptl protein may provide an explanation for previous observations which indicated that introduction of the genes encoding pertussis toxin subunits from B. pertussis into other Bordetella spp. results in production of the toxin but not secretion of the toxin.     

Differentiation between B. pertussis and B. parapertussis according to their fatty acid composition

The fatty acid composition of 3 B. pertussis strains and 2 B. parapertussis strains grown on casein-carbon agar (CCA) with 8% of sheep blood added and without blood, as well as B. parapertussis strain grown on beef-extract agar (BEA) has been studied by gas chromatography. The fatty acid profiles characteristic of B. pertussis and B. parapertussis were greatly different, as B. parapertussis has a considerable amount of methylene-hexadenocanoic acid, while containing less hexadecenoic and octadecanoic acids and more tetradecanoic acid. The fatty acid composition of 2- to 5-day Bordetella cultures grown on CCA with and without blood has no essential differences. Differences in the content of various fatty acids in B. parapertussis grown on CCA and BEA had no essential influence on the fatty acid profile. The specificity of the fatty acid composition of B. pertussis and B. parapertussis allows to use this characteristics for their differentiation.     

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

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.     

Development of acellular pertussis vaccines.

In 1974, the authors reported the isolation and characterization of protective antigens of Bordetella pertussis in mice. With this information, an acellular pertussis vaccine was developed, composed mainly of pertussis toxin (PT) and filamentous haemagglutinin (FHA). Substances causing side effects, especially lipopoly sacahoride (LPS) or endotoxin that cause fever, were removed, and detoxification of the PT by formaldehyde with retention of potency was achieved. In 1981, an acellular pertussis vaccine called the "Adsorbed Purified Pertussis Vaccine" was approved in Japan, in place of the whole-cell pertussis vaccine. The acellular pertussis vaccine has been widely accepted as safer and more efficacious in Japan. Since 1981, intense surveillance has shown that there are only rare adverse reactions and that pertussis has virtually been eliminated in Japan. Evaluation of active immunization with highly purified and pharmacologically inert PT and FHA and passive immunization with polyclonal and monoclonal antibodies, provide quantitative data about the vaccine-induced immunity in mice. Finally, it was discovered that the PT toxoid in the vaccine is the major and essential protective antigen. The toxoid of PT should be sufficient for protection against pertussis.     

Lipopolysaccharides from Bordetella pertussis and Bordetella parapertussis differently modulate human dendritic cell functions resulting in divergent prevalence of Th17-polarized responses

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1beta production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1beta, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IkappaBalpha and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

Composition of acellular pertussis and combination vaccines: a general review.

Since the development and introduction of the acellular pertussis vaccine in Japan in the early eighties, we have come a long way in using this component in combination with other vaccines. However, the basic problem in development of an effective and safe pertussis vaccine is that the antigens to induce complete protection against clinical pertussis and the precise mechanism by which pertussis vaccine confers immunity is yet unknown. Hence, the composition of future acellular pertussis vaccine remains an open issue. Recently, acellular pertussis vaccine has been licensed for the booster doses in the U.S.A. and for primary immunization of infants in Italy and Germany. A multicentric trial has been carried out to compare the serological response and adverse reactions of 13 acellular pertussis vaccines. These vaccines contained one or more of the four components, i.e. FHA, PT, 69 kDa OMP and fimbriae. All vaccines were associated with substantially fewer and less adverse reactions and were more immunogenic with respect to antibodies against the added antigens. DTP vaccines in the near future will have combinations of other components and the key antigen for combination will be acellular pertussis component which is going to replace whole cell pertussis component in DTP vaccines. In view of this, manufacturers like ourselves from the developing countries are still groping in the dark, uncertain whether we should have a single component acellular pertussis vaccine or multicomponent one. This will have a major impact on the cost of production, the final cost of the combination vaccines and the regulatory issues that we will have to tackle in view of the recent thinking on harmonization in the pharmaceutical industry.     

Epidemic process of pertussis in Moscow

Materials reflecting the dynamics of pertussis morbidity during the period of 1958 - 2003 under the conditions of prolonged mass immunization of the child population with adsorbed DPT vaccine are presented. The planned vaccination of children led to the decrease of pertussis morbidity during the first 10 years, but groundless abstentions from vaccination during the 1980s - 1990s contributed to a sharp rise in morbidity among children of younger age groups. During the recent four years a rise in pertussis morbidity was registered in 2000 (71.79 per 100,000 of the population), followed by the most significant for the last 20 years drop in morbidity in 2002--down to 9.89. But in 2003 the growth of morbidity was again registered (38.67). Recently periodic rises and drops in morbidity occurred simultaneously with the increased coverage of children of younger age groups with vaccination. In recent years changes in the age structure of patients were observed: the specific proportion of school children increased (in 2003 morbidity rates in children aged 6 - 10 years were 288.6 - 270.7), simultaneously high morbidity among children aged up to one year (274.9) was registered. The specific proportion of pertussis-affected children aged above 7 years reached 65%. From the late 1990s until present in 87.1% of cases strains of serotype 1.0.3 prevailed in the population of B. pertussis strains. But in recent years the circulation of strains 1.2.3, spread in the prevaccination period and having toxicity similar to that of strains of serotype 1.0.3, while exceeding them in virulence, in sufficiently high proportion (7.0% in 2002) was noted. This was indicative of the possibility of the unfavorable development of the epidemic process of pertussis infection.     

Identification of a Bordetella pertussis bvg-regulated porin-like protein.

Bordetella pertussis 18323 produces a bvg-regulated 39.1-kDa porin-like protein, OmpQ. OmpQ had 61% similarity to the major porin of B. pertussis and contains conserved regions common to both the neisserial and enteric porin families. The results of Southern blot analysis indicate that strains of Bordetella parapertussis and Bordetella bronchiseptica but not Bordetella avium contain this gene.     

Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.     

Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes

Pertussis toxin, the major virulence factor of Bordetella pertussis, is not produced by the closely related species Bordetella parapertussis and Bordetella bronchiseptica. It is shown here that these two species possess but do not express the complete toxin operon. Nucleotide sequencing of an EcoRI fragment of 5 kilobases comprising the regions homologous to the pertussis toxin genes shows that in this region, B. parapertussis and B. bronchiseptica are 98.5% and 96% homologous, respectively, to B. pertussis. The changes (mostly base pair substitutions) in many cases are identical in B. parapertussis and B. bronchiseptica, suggesting that these two species derive from a common ancestor. Many of the mutations common to B. parapertussis and B. bronchiseptica involve the promoter region, which becomes very inefficient. The S1 subunits of both species, when expressed in Escherichia coli, have the same ADP-ribosylating activity as the S1 subunit from B. pertussis, indicating that the mutations in the S1 gene described here do not affect its function.