Susceptibility to macrolide antibiotics of Bordetella pertussis and Bordetella parapertussis strains isolated from whooping cough patients in 1968 and in 1997-99]

The activity of erythromycin, azithromycin, clarithromycin, dirithromycin, oleandomycin, roxithromycin, spiramycin and josamycin against 21 and 34 B. pertussis strains and against 6 and 8 B. parapertussis strains isolated respectively in the years 1968 and 1997-99 was examined. The antibiotic agar dilution method was used. The minimum concentration of macrolides which inhibited growth of B. pertussis and B. parapertussis was calculated for 50% (MIC50) and 90% (MIC90) of isolates. The susceptibility to macrolides of B. pertussis and B. parapertussis strains isolated in the years 1968 and 1997-99 did not differ significantly. The MIC90 values of erythromycin were the same for B. pertussis (MIC90 = 0.125 mg/l) and B. parapertussis strains (MIC90 = 0.25 mg/l) recovered in 1968 as for those recovered in the years 1997-99. The most active antibiotic against all strains was azithromycin (MIC90 = 0.06 mg/l). The least active antibiotics were oleandomycin (MIC90 = 2-4 mg/l) and spiramycin (MIC90 = 8 mg/l). The study showed that erythromycin remains the antibiotic of choice for treatment of whooping cough and in case of emergence of B. pertussis and/or B. parapertussis strains erythromycin resistant, can be replaced by azithromycin.     

Efficacy of Whooping-cough Vaccines used in the United Kingdom before 1968: Final Report to the Director of the Public Health Laboratory Service by the Public Health Laboratory Service Whooping-Cough Committee and Working Party

The efficacy of pertussis vaccines was investigated in 33 areas in the United Kingdom from November 1966 until April 1968 inclusive. Bordetella pertussis was isolated from 1,293 persons, but there were only six isolations of B. papapertussis. Among vaccinated contacts under 5 years in homes in which B. pertussis was isolated 52% developed paroxysmal cough. The corresponding attack rate among unvaccinated contacts was 69%. These findings suggest that much of the pertussis vaccine in use for five or six years before 1968 was not very effective. However, vaccine from one producer was more effective than vaccine from another. Of the cultures of B. pertussis identified 89% were serotype 1, 3 and only about 9% were serotype 1, 2, 3. Serotype 1, 2, 3 was isolated much more frequently from unvaccinated than from vaccinated children, but some cultures identified as type 1, 2, 3 were found on re-examination to contain colonies of type 1, 3. Virological investigations were made in some areas during the first year of the study. Of the wide variety of viruses identified adenovirus and parainfluenza virus were the most common groups. Virus isolation rates were similar in patients and symptomless contacts, but B. pertussis was isolated far more often from patients than from symptomless contacts. The evidence suggests that B. pertussis remained the major cause of whooping cough in the U.K.     

Bordetella pertussis serotypes in Canada.

A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.9%) of the pertussis strains examined were serotype 1,3.All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich in each of the three main pertussis agglutinogens.     

Surface-exposed antibody-accessible outer membrane proteins of Bordetella pertussis

The convalescent sera from a patient recovered from whooping cough were used to identify the surface-exposed antibody-accessible outer membrane proteins (OMPs) of Bordetella pertussis. The results indicated that the 69,000 OMP, the 40,000 porin, agglutinogens 2 and 3, and a number of presently unknown OMPs were exposed on the surface. The importance of these surface-exposed antigens in the protection against whooping cough is discussed.     

Duration of effectiveness of pertussis vaccine: evidence from a 10 year community study

A 10 year study of whooping cough in a discrete general practice community was performed to assess longitudinally the efficacy of pertussis vaccine from one to seven years after immunisation. Of the 436 cases of whooping cough over 10 years, 326 occurred in children aged 1-7 years. The rate of immunisation was known for each cohort of children born during each year, and the attack rate of whooping cough was thus calculated for those immunised and unimmunised. The attack rates were highest in those cohorts exposed to the epidemics of 1977-9, 1981-3, and 1985-7. The efficacy of the vaccine was calculated as a percentage as (attack rate in unimmunised group—attack rate in immunised group) × 100/attack rate in unimmunised group. It fell from 100% in the first year to 46% in the seventh, being 84% in the fourth and only 52% in the fifth.Thus the pertussis vaccine or its schedule of use does not seem to provide sufficient herd immunity to prevent outbreaks of whooping cough. Matters might be improved if vaccination against pertussis were included in the preschool immunisation programme.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Persistence of antibody after accelerated immunisation with diphtheria/tetanus/pertussis vaccine.

OBJECTIVE--To determine the persistence of antibody to diphtheria, tetanus, and pertussis in children receiving an accelerated schedule of primary immunisation. DESIGN--Controlled study of antibody testing of blood samples from children immunised according to various schedules: three doses of triple vaccine completed at 8-13 calendar months, 6-7 calendar months, before 6 calendar months, or three doses followed by diphtheria/tetanus before age 2. SETTING--Plymouth Health Authority. SUBJECTS--129 children aged 4 years who had received three doses of diphtheria/tetanus/pertussis vaccine with or without a diphtheria/tetanus booster. MAIN OUTCOME MEASURES--Diphtheria and tetanus antitoxin concentrations and antibody titres to pertussis toxin, filamentous haemagglutinin, and agglutinogens 2 and 3. RESULTS--All children had protective concentrations of antitoxin to diphtheria and tetanus (greater than or equal to 0.01 IU/ml). There was no evidence of a significant difference in diphtheria or tetanus antitoxin concentrations and pertussis antibody titres in children immunised with an accelerated course (third dose of triple vaccine before 6 months) compared with those who received a longer course (third dose at 8-13 months) with no booster (geometric mean antitoxin concentration 0.411 (95% confidence interval 0.273 to 0.618) v 0.426 (0.294 to 0.616) for diphtheria and 0.358 (0.231 to 0.556) v 0.299 (0.197 to 0.453) for tetanus; geometric mean antibody titres 903 (500 to 1631) v 1386 (848 to 2266) for pertussis filamentous haemagglutinin, 179 (130 to 248) v 232 (167 to 322) for pertussis toxin, and 2002 (1276 to 3142) v 3591 (2220 to 5809) for agglutinogens 2 and 3). CONCLUSION--Immunisation with three doses of triple vaccine at monthly intervals completed before 6 months of age probably provides adequate protection against diphtheria, tetanus, and whooping cough which will persist until the age of the preschool booster.     

Molecular cloning of pertussis toxin genes.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.     

Protective effects of in vivo‐expressed autotransporters against Bordetella pertussis infection

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re‐emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

     

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.     

Pathogenic properties of freshly isolated strains of the pertussis microbe

The serovar composition, toxicity, virulence and lymphocytosis stimulating activity (LSA) of B. pertussis strains circulating in the 1980-ies were studied in comparison with the strains circulating in previous years. The study revealed changes in the toxic properties of B. pertussis: their decrease in the years of the intensive immunization of children against whooping cough and rise at the period when the number of immunized children was reduced. The toxic properties and LSA in most B. pertussis strains were less pronounced in the 1980-ies than in the 1960-ies. The serovar composition of the circulating strains remained stable for 15 years with the prevalence of serovar 1.0.3.     

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.     

Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis

The filamentous hemagglutinin (FHA) of Bordetella pertussis is a principal adhesin, which plays a key role in the colonization of the upper respiratory tract. FHA is also a protective antigen, which has been incorporated in the new generation of acellular vaccines against whooping cough. The protein is synthesized as a large 367-kDa precursor, which is then processed into a 220-kDa secreted polypeptide. To optimize the use of this protein for vaccine purposes it would be helpful to define the regions encompassing immunodominant epitopes. Twelve recombinant plasmids have been generated encoding fusion proteins between fragments of the matured-secreted 220-kDa form of FHA and the vector-encoded phage MS2 polymerase. Protein extracts of the resulting recombinant clones have been tested for reactivity with sera from 20 patients convalescent from whooping cough, and two human standard sera. The results indicate the presence of an immunodominant B cell epitope in the polypeptide coded by a 1-kb DNA fragment encompassing positions 5781-6800 of the published sequence. These results suggest that the identified fragment should be conserved in the formulation of vaccines against pertussis.     

Consequences of the expression of lipopolysaccharide-modifying enzymes for the efficacy and reactogenicity of whole-cell pertussis vaccines

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is, due to its endotoxic activity, responsible for the relatively high reactogenicity of whole-cell vaccines. In addition, lipopolysaccharide has strong immune stimulating properties, which makes it, potentially, an interesting vaccine component. In a previous study, we have shown that expression of two lipopolysaccharide-modifying enzymes, i.e., PagP and PagL, modulates the endotoxic activity of the Gram-negative bacterium Bordetella pertussis, the causative agent of whooping cough. To assess the consequences of PagP and PagL expression on the efficacy and reactogenicity of whole-cell pertussis vaccines, we have immunised mice and challenged them intranasally with wild-type B. pertussis. Vaccine efficacy, B. pertussis-specific antibody responses, and cytokine profiles were evaluated. The results show that expression of PagL, but not of PagP, significantly increases vaccine efficacy without altering vaccine reactogenicity. Therefore, PagL-expressing B. pertussis strains may form a basis for the development of a new and safer whole-cell pertussis vaccine, as higher vaccine efficacies may allow a reduced vaccine dosage. These data show, for the first time, that lipopolysaccharide composition is an important determinant for the efficacy of whole-cell pertussis vaccines.     

Modern strains of Bordetella pertussis: immunobiological properties and vaccine improvement

Strains of B. pertussis isolated from patients in Moscow in 2001-2005 as well as strains included in locally produced diphtheria-tetanus-whole cell pertussis (DTP) vaccine were studied. Nucleotide sequences in genes of pertactin and S1-subunit of pertussis toxin of isolated strains, their immunobiological properties and opportunity to use for producing of the acellular pertussis vaccine were determined. Genes of pertactin and S1-subunit of pertussis toxin in the isolated wild strains differed from the same genes in strains included in the local DTP vaccine. Majority of the isolated strains belonged to serotype 1.0.3 and were markedly virulent.     

Use of an immunoblotting method for identifying the individual proteins in the antigenic complexes of Bordetella pertussis

The composition of antigenic complexes isolated from the supernatant fluid of B. pertussis culture has been studied by means of immunoblotting techniques. In preparations obtained from B. pertussis strains 305 and 475 fragments of the molecule of fimbrial hemagglutinin, three subunits of B. pertussis toxin and agglutinogens 2 and 3 have been detected with the use of antisera to B. pertussis protective substances.     

Temporal analysis of French Bordetella pertussis isolates by comparative whole-genome hybridization

Bordetella pertussis, a gram-negative beta-proteobacterium, is the agent of whooping cough in humans. Whooping cough remains a public health problem worldwide, despite well-implemented infant/child vaccination programs. It continues to be endemic and is observed cyclically in vaccinated populations. Classical molecular subtyping methods indicate that genome diversity among B. pertussis isolates is limited. Although the whole bacterial genome has been studied by pulsed-field gel electrophoresis, the genes implicated in the diversity have not been identified. We developed a B. pertussis whole-genome DNA microarray representing over 91% of the predicted coding sequences of the sequenced strain Tohama I. Genomic DNA from clinical isolates with various pulsed-field gel electrophoresis profile patterns was competitively hybridized with the DNA microarray and coding sequences were classified as present, absent or duplicated. Our data strongly suggest that the B. pertussis population is dynamic. In France, with highly vaccinated population, the genetic diversity is low and decreasing with time, and clonal expansion correlates with cycles of the disease. This decrease in diversity is essentially due to loss of genes and pseudogenes. The genes deleted are most of the time flanked by insertion sequences.     

Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. II. Occurence and level of B. pertussis antigens in children with suspected whooping cough

The aim of this study was to determine and evaluate IgG, IgM and IgA levels to pertussis toxin (PT), filamentous hemagglutinin (FHA) and endotoxin (LPS) of B. pertussis in children with clinical symptoms of whooping cough. The serum samples obtained from 265 children (age range: 2 months-16 years) suspected of pertussis were examined by indirect haemagglutination (IH) and ELISA tests. Higher antibody level was most frequently observed in IgA class to PT, FHA and LPS in 45.3%, 35.1% and 66% of pertussis patients sera respectively. The least positive results were obtained in IgM class to PT and FHA (in 9.8% and 2.6% of children sera respectively) but in the case of LPS applied as the antigen in ELISA, higher IgM level was determined in 46.8% of pertussis patients sera. The four times increase of antibody level to LPS determined by IH was observed in 86.7% of children suspected of pertussis. Humoral response to B. pertussis infection is mainly connected with higher IgA level to PT, FHA, LPS and IgM to LPS in children with clinical symptoms of whooping cough.     

Neonatal immunization with a single dose of recombinant BCG expressing subunit S1 from pertussis toxin induces complete protection against Bordetella pertussis intracerebral challenge

The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-gamma production and an increase in both IFN-gamma(+) and TNF-alpha(+)-CD4(+)-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.