Efficacies of whole cell and acellular pertussis vaccines against Bordetella parapertussis in a mouse model

Pertussis vaccine development has mainly focused on Bordetella pertussis, and consequently these vaccines contain B. pertussis antigens only. However, the related species Bordetella parapertussis can also cause pertussis, although symptoms associated with the disease are generally considered to be milder. Recent field studies have shown that in some outbreaks B. parapertussis can prevail. Using a mouse model we compared the efficacy against B. parapertussis of two commercially available acellular vaccines and two whole cell vaccines, used in The Netherlands and Finland, respectively. The efficacies of the two whole cell vaccines against B. parapertussis were similar, but much lower compared to the efficacy against B. pertussis. Although, the acellular vaccines conferred some protection against B. parapertussis early in infection, the values were not significant. Later in infection, a highly significant enhancement of colonisation by B. parapertussis was observed in mice vaccinated with acellular vaccines. The whole cell vaccines protected significantly better than the acellular vaccines against B. parapertussis. The possible consequences of a switch from whole cell to acellular vaccines was discussed in the light of our findings.     

Modified intra-cerebral challenge assay for acellular pertussis vaccines: Comparisons among whole cell and acellular vaccines

The modified intra-cerebral challenge assay for acellular pertussis vaccines is used in Japan, Korea, China and possibly other Asian countries as the potency assay for routine release of acellular pertussis (aP) and combination vaccines. National reference standards, typically of whole cell pertussis (Pw) vaccine, are in use in these countries, but there is no agreed international reference standard for acellular pertussis vaccines.     

Comparison of acellular pertussis vaccines-induced immunity against infection due to Bordetella pertussis variant isolates in a mouse model

A significant increase in the incidence of pertussis in adolescents and adults has been observed in vaccinated populations. Concomitantly, emergence of novel pertussis toxin and pertactin types in circulating Bordetella pertussis isolates was noticed. In this study, immunity induced by acellular vaccines against infection due to isolates expressing different pertactin types and fimbriae was monitored in a mouse model. In accordance with previous studies, the effect of a bicomponent DTPa vaccine on bacterial clearance was lower when compared with tri- or pentavalent DTPa vaccines. Whatever the isolates used to infect mice, the tri- or pentavalent DTPa vaccines were both efficacious in inducing immunity that resulted in clearance of infection. These findings suggest that re-emergence of pertussis might not be related to emergence of isolates escaping vaccine protection. The present study reduces potential concerns about acellular vaccine efficacy, but frequent monitoring of protection and surveillance of the evolution of the B. pertussis population remains of particular importance.     

Intranasal murine model of Bordetella pertussis infection. I. Prediction of protection in human infants by acellular vaccines.

Bicomponent, tricomponent and pertactin DTPa vaccines were tested in sublethal aerosol, and lethal and sublethal intranasal murine Bordetella pertussis respiratory challenge models. Pertactin and bicomponent vaccines induced protective immunity against lethality but with little or no bacterial clearance. Intranasal challenge discriminated in a reproducible, statistically significant manner between the efficacies of bicomponent and tricomponent DTPa, in agreement with clinical trial data. This discrimination was not observed in the aerosol challenge. Pertactin had a synergistic effect with bicomponent DTPa. Intranasal challenge may be useful as part of the preclinical evaluation of new acellular pertussis formulations or DTPa-based combinations.     

Lung pathology and immediate hypersensitivity in a mouse model after vaccination with pertussis vaccines and challenge with Bordetella pertussis

While evaluating vaccine efficacy against clinical Bordetella pertussis isolates in mice, after challenge vaccinated mice showed increased lung pathology with eosinophilia, compared to challenged, non-vaccinated animals. This led us to study bacterial clearance, lung pathology, lung TNF-alpha expression, and parameters of immediate hypersensitivity (IH), being serum IgE levels, eosinophil numbers in the bronchoalveolar lavage fluid, and ex vivo IL-4, IL-5, IL-10, IL-13, and IFN-gamma production by the bronchial lymph node cells. BALB/c mice received a combined Diphtheria (D), Tetanus (T), Poliomyelitis, and whole-cell Pertussis vaccine (WCV), a combined D, T, and three-component acellular Pertussis vaccine (ACV), aluminium hydroxide adjuvant, or PBS, 28 and 14 days before B. pertussis infection. Similarly treated non-infected mice were taken as a control. Infection induced pathology; this induction was stronger after (especially WCV) vaccination. WCV but not ACV vaccination induced TNF-alpha expression after challenge. After challenge, IH parameters were strongly increased by (especially ACV) vaccination. Vaccinated IL-4 KO mice showed similar clearance and pathology, in the absence of IgE and with reduced numbers of eosinophils. Vaccinated (Th1-deficient) T-bet KO mice showed reduced clearance and similar pathology. In summary, after challenge vaccination increased lung pathology, TNF-alpha expression (only WCV), and IH parameters. Th1 cells were critical for clearance.     

Analysis of Bordetella pertussis isolates collected in Japan before and after introduction of acellular pertussis vaccines

Because of recent concern that whole-cell pertussis vaccination can drive antigenic divergence of circulating isolates of Bordetella pertussis, we compared 12 clinical isolates of B. pertussis collected in Japan, the first country to introduce acellular pertussis vaccines, with the vaccine strain. We used pulsed-field gel electrophoresis, sequencing of ptx and prn genes and expression of fimbriae. Most of the isolates collected before or after introduction of acellular vaccine possess similar restriction patterns. They contain ptx genes and prn alleles similar to the vaccine strain and to European isolates collected before the introduction of vaccination. Two recently collected isolates exhibiting a different pulsed-field gel electrophoresis pattern possess ptxS1 and prn alleles similar to the alleles harbored by European isolates circulating currently. Our preliminary results suggest that, if acellular pertussis vaccine-induced antigenic divergence exists, it is likely to be a slow or rare process.     

An in vitro assay system as a potential replacement for the histamine sensitisation test for acellular pertussis based combination vaccines

The histamine sensitisation test (HIST) for pertussis toxin is currently an official batch release test for acellular pertussis containing combination vaccines in Europe and North America. However, HIST, being a lethal endpoint assay, often leads to repeated tests due to large variations in test performance. Although a more precise HIST test based on measurement of temperature reduction after the histamine challenge is used in Asian countries, this test still uses animals. An in vitro test system based on a combination of enzyme coupled-HPLC and carbohydrate-binding assays with results analysed by a mathematical formula showed a good agreement with the in vivo HIST results based on measurement of temperature reduction after histamine challenge. The new in vitro test system was shown to be a potential alternative to the current in vivo HIST.     

Efficacy of pertussis components in an acellular vaccine, as assessed in a murine model of respiratory infection and a murine intracerebral challenge model

The efficacy of 10 pertussis vaccines prepared from various concentrations of pertussis toxin (PT) and filamentous hemagglutinin (FHA) was investigated in a murine model of respiratory infection (aerosol challenge model) and a murine intracerebral (ic) challenge model. PT was necessary as a vaccine component for protection against an ic challenge with Bordetella pertussis. FHA appeared to play an important role as a vaccine component in protection against an aerosol challenge with B. pertussis. Vaccines containing a small amount of FHA with a large amount of PT (FHA:PT=1:11 or 2:10, w/w) were strongly protective in both the aerosol challenge and the ic challenge models. Ratios of FHA:PT of 1:11 or 2:10 (w/w) might be suitable for future formulations.     

Potency testing of acellular pertussis vaccines.

The laboratory assessment of the potential of acellular pertussis vaccines to protect against human disease is a major problem. The mouse intracerebral challenge test, which is the accepted potency assay for whole cell pertussis vaccines, is not suitable for testing acellular vaccines, and more recently developed murine respiratory infection assays have methodological drawbacks and doubtful relevance to the human infection. We have found that the ability of several Bordetella pertussis antigens to protect mice against lung colonization correlates with their ability to raise murine antibodies which inhibit the adhesion of the bacteria to Vero cells. In this report we consider the applicability of such in vitro assays, and the in vivo assays, to the potency testing of acellular pertussis vaccines.     

Multiple discontinuity as a remarkable feature of the development of acellular pertussis vaccines.

Development of the Japanese acellular pertussis vaccines (APVs) of the 1980s involved six procedural or conceptual features that were discontinuous with the then-accepted views of how pertussis vaccines should be made and tested. These discontinuities were: modification of the standard intracerebral mouse test for protective potency; use of culture supernates, rather than cells, of Bordetella pertussis as the feedstock for antigen purification; use of haemagglutination as a measure of protective antigen(s); identification of pertussis toxin (PT) as the main protective antigen; complete inactivation of the biological activities of PT by formalin; and the use of a single strain of B. pertussis. Several of these discontinuities had long precedence in the pertussis literature, but the original observations had not been incorporated into the mainstream of pertussis vaccinology and were therefore 'premature'. The APVs, purified from culture-supernates, emerged after a long period of unsuccessful research on the split-cell pertussis vaccines, i.e. those derived from the bacterial cells themselves. There is a brief discussion of why APVs have taken so long to obtain acceptance outside Japan, and of how the listed discontinuities may be explicable in terms of antigen processing by the immune system. General lessions, applicable to vaccines for other infectious diseases, may be learned from this account of how APVs have evolved from whole-cell pertussis vaccines.     

Intranasal murine model of Bordetella pertussis infection: II. Sequence variation and protection induced by a tricomponent acellular vaccine.

When pertussis toxin S1 subunit and pertactin structural genes in Bordetella pertussis clinical isolates from France and Germany were sequenced, 3 previously described S1 subunit types (S1 A, B and E), and 4 pertactin types (PRN A, B, C, A*) were found. PRN A*, present in the WHO reference strain 18323, was not described previously. In a respiratory mouse model, a tricomponent acellular pertussis vaccine (Infanrix) was highly effective in promoting lung clearance of all isolates expressing different S1 subunit and pertactin suggesting that use of acellular vaccine will not increase the risks of pertussis infection by these B. pertussis variants.     

Protection and humoral immune responses against Bordetella pertussis infection in mice immunized with acellular or cellular pertussis immunogens

In the present study, protection against Bordetella pertussis infection and humoral immunological responses in mice has been assessed upon immunization with custom-made acellular pertussis vaccines (ACVs) and whole-cell pertussis vaccine (WCV). Mice were immunized, next intranasally infected with B. pertussis and during 14 days the number of bacteria in the trachea and lungs and the level of serum antibodies were determined. ACV contained five immunogens, filamentous hemagglutinin, pertactin, fimbriae serotypes 2 and 3, and chemically detoxified pertussis toxin (PMC-5), or three immunogens, filamentous hemagglutinin, pertactin, and genetically detoxified (BC-3) or chemically detoxified pertussis toxin (SKB-3). Immunization with a high or low dose of ACV or WCV resulted in significant protection against B. pertussis, with differences in the degree of protection between the vaccines. The lowest protection was found with a low dose of SKB-3 and WCV. The pattern of cytokine production by spleen cells of immunized, non-infected, mice indicated that T-helper 1 cells are activated by vaccination with WCV, and T-helper 1 and T-helper 2 cells are involved in the immune response upon vaccination with ACVs. Each vaccine stimulated the production of IgG, but not IgA, antibodies. In mice immunized with ACV, elimination of B. pertussis from trachea and lungs correlated significantly with the titre of IgG1, but not IgG2a, antibodies.     

Improved protocols for histamine sensitization testing of acellular pertussis vaccines

The histamine sensitization test is a widely used method for measuring the residual toxicity of pertussis toxin in acellular pertussis vaccines. Although it has been used as a routine assay for decades, the current protocols are difficult to standardize because the test results vary considerably and are based on several factors, including mouse strain, age and sex. In this study, we observed that mice of strains CD1, ddY and C57/BL6 were sufficiently sensitive to pertussis toxin among six mice strains tested and that aged male mice were more sensitive to pertussis toxin than younger or female mice. Using this animal model, we showed pertussis toxin dose-dependent responses in the two histamine sensitization test protocols based on either lethal end-point determination or mouse rectal temperature measurement. Sensitivity to pertussis toxin was further enhanced by the addition of lipopolysaccharide in both methods. With these improvements, pertussis toxin activity can be estimated more accurately and reproducibly using a reduced number of animals.     

Effects of adsorption of acellular pertussis antigens onto different aluminium salts on the protective activity in an intranasal murine model of Bordetella pertussis infection

Adsorption of the pertussis antigens, pertussis toxoid (PT), filamentous hemagglutinin (FHA) and pertactin (PRN) onto aluminium phosphate rather than aluminium hydroxide leads to a lower humoral response and poorer protection against intranasal pertussis challenge in mice. These effects could be reversed by inclusion of fimbriae (FIM) 2 and 3 in the formulation. These data emphasis the importance of correct formulation for such vaccines.     

An aerosol challenge mouse model for Moraxella catarrhalis

A simple, reproducible, and non-invasive mouse pulmonary clearance model for Moraxella catarrhalis via aerosol challenge was established. All of eight tested strains could be inoculated into mice at more than 10(5) colony-forming units (CFU)/lung with a challenge concentration of 1x10(9)-6x10(9) CFU/ml in a nebulizer. The number of bacteria retained at 6 h postchallenge was more than 10(4) CFU/lung while at 24 h postchallenge, approximate 10(3) CFU/ml or less remained in the lungs. A maximum of 100 mice could be challenged per aerosol exposure. The number of bacteria inoculated in the lungs could be adjusted by the bacterial challenge concentration, the exposure time, and the negative pressure. Lung tissue sections revealed that bacteria were evenly distributed in the lungs. Passive immunization significantly enhanced pulmonary clearance of the homologous strain in this model. These data indicate that this model will be useful for evaluating M. catarrhalis vaccine candidates and studying roles of immunity against M. catarrhalis.     

Attenuated Bordetella pertussis: new live vaccines for intranasal immunisation

Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious pathogen with a strong capacity to colonize the human respiratory tract. A single infection with virulent B. pertussis induces strong mucosal and systemic humoral and cellular immune responses, as well as long-lasting protection in humans. Therefore, B. pertussis may be a suitable vector for intranasal vaccination against a variety of diseases by a single dose. We showed that intranasally administered recombinant live B. pertussis expressing heterologous antigens induce mucosal immune responses in the respiratory and the genital tracts of mice as well as systemic antibody responses. To consider live B. pertussis for human vaccination, we, therefore, aimed at the development of attenuated strain. Allelic exchange was used to delete the dermonecrotic toxin gene and to replace the pertussis toxin gene by an allele encoding inactive toxin. To reduce the production of tracheal cytotoxin, the ampG gene was over-expressed. After various rounds of homologous recombination, B. pertussis BPZE, affected in the production or activity of three major toxins, was isolated. Histological examinations of infected mice confirmed the strong attenuation of this strain. Nevertheless, it colonized the mouse respiratory tract over a period of roughly 1 month. The level of protection against subsequent challenge with virulent wild-type B. pertussis, induced by a single intranasal administration of BPZE was at least as high as that induced by two injections of commercial acellular vaccine. We conclude that live attenuated B. pertussis may be an attractive vaccine candidate to be administered in a single intranasal dose against whooping cough. Moreover, live recombinant B. pertussis may be used as a vector for intranasal vaccination against a variety of diseases.     

Formulation and characterisation of Bordetella pertussis fimbriae as novel carrier proteins for Hib conjugate vaccines

Haemophilus influenzae type b (Hib) capsular polysaccharide (polyribosylribitol phosphate, PRP) is the active component of conjugate vaccines that have proven successful in preventing invasive Hib disease. Conjugation of PRP to a protein carrier greatly improves its immunogenicity providing protection in infants and subsequent antibody maturation upon boosting. In this study, fimbriae isolated from Bordetella pertussis have been assessed as novel carrier proteins. These proteins are components of some acellular pertussis vaccines and clinical trials have indicated that fimbriae could be important protective antigens against whooping cough. Fimbriae (Fim2 and Fim3) purified from B. pertussis were dissociated in 6 M guanidine hydrochloride, pH 10.5, to produce proteins of defined size and to facilitate the production and characterisation of the conjugates. Both carbodiimide-mediated coupling and reductive amination were used to conjugate PRP to dissociated fimbriae. Efficiency of conjugation was determined by size exclusion chromatography followed by protein and polysaccharide analysis of fractionated components. Immunisation of rabbits with dissociated fimbriae-PRP conjugates (D.fim-PRP) produced high anti-fimbrial and anti-PRP IgG titres. Use of a D.fim-PRP conjugate could protect against Hib disease and may also augment protection against B. pertussis.     

Laboratory assessment problems of the efficacy of acellular pertussis vaccines

Although composition of acellular pertussis vaccines is better defined than whole-cell vaccines, differences in the formulation, content, and detoxification of pertussis vaccine antigens led to a unique character of each of differently produced acellular vaccine. Currently used methods for laboratory evaluation of whole-cell pertussis vaccine efficacy were found not suitable for acellular vaccines. There is a strong need to perform analysis and evaluation of the safety and efficacy profiles of acellular pertussis vaccines combined with other vaccine antigens (e.g. Hib) both before and after conjugation. Mechanisms of interactions seen after conjugation inducing weaker immunogenicity or efficacy are still poorly recognized.