Elsevier

Vaccine

Volume 33, Issue 46, 17 November 2015, Pages 6277-6281
Vaccine

Pertactin negative Bordetella pertussis demonstrates higher fitness under vaccine selection pressure in a mixed infection model

https://doi.org/10.1016/j.vaccine.2015.09.064Get rights and content

Abstract

Whooping cough or pertussis is a highly infectious respiratory disease in humans caused by Bordetella pertussis. The use of acellular vaccines (ACV) has been associated with the recent resurgence of pertussis in developed countries including Australia despite high vaccination coverage where B. pertussis strains that do not express pertactin (Prn), a key antigenic component of the ACV, have emerged and become prevalent. In this study, we used an in vivo competition assay in mice immunised with ACV and in naïve (control) mice to compare the proportion of colonisation with recent clinical Prn positive and Prn negative B. pertussis strains from Australia. The Prn negative strain colonised the respiratory tract more effectively than the Prn positive strain in immunised mice, out-competing the Prn positive strain by day 3 of infection. However, in control mice, the Prn positive strain out-competed the Prn negative strain. Our findings of greater ability of Prn negative strains to colonise ACV-immunised mice are consistent with reports of selective advantage for these strains in ACV-immunised humans.

Introduction

Pertussis, also known as whooping cough, is a life-threatening, highly contagious respiratory tract disease in unvaccinated infants and a cause of significant morbidity in children and adults [1]. The causal organism, Bordetella pertussis, a Gram-negative aerobic bacterium, is transmitted via air droplets and has tropism for ciliated respiratory epithelial cells. In the 1950s, whole cell vaccine (WCV) was introduced and the pertussis notification rate was dramatically reduced. However, due to side effects, a less reactogenic acellular vaccines (ACV) was developed in the 1980s and found to be efficacious by a series of randomised trials in the 1990s [2]. A variety of ACVs containing one to five components have been used in different countries [3], [4]. In Australia, ACV replaced the WCV by 2000 for both primary and booster doses. Since that time, a 3-component ACV-containing detoxified pertussis toxin (Ptx), pertactin (Prn), and filamentous haemagglutinin (FHA) has been used almost exclusively, singly or in combination [5], [6].

In recent years, many developed countries have reported resurgence of pertussis despite high vaccine coverage [1], [7]. Several causes have been suggested for the re-emergence of pertussis including bacterial adaptation, waning immunity and more sensitive molecular diagnostic methods [1], [7], [8], [9], [10]. A clear example of bacterial evolution is the emergence and global spread of ptxP3 strains, which carry a novel ptx promoter ptxP3 allele [10], [11] linked with increased production of Ptx [11]. Our genotyping studies using single nucleotide polymorphisms (SNPs) as molecular markers have shown that the predominant strains currently circulating in Australia belonged to Cluster I, which is associated with the ptxP3 allele [12], [13]. In Australia, a prolonged pertussis epidemic occurred in different regions from 2008 to 2012 [14], [15]. We demonstrated that strains that did not produce Prn emerged and expanded during the 2008−2012 epidemic [16], providing evidence of vaccine-driven evolution. Prn negative isolates have also been reported in many countries with high vaccine coverage including the European Union, Japan, the USA and Canada [17], [18], [19], [20], [21]. Martin et al. [21] reported that vaccinated individuals have a 2-fold higher probability of infection by Prn negative B. pertussis, suggesting that Prn negative B. pertussis strains have an advantage in the immunised host compared with Prn positive strains.

Prn is an autotransporter protein [22] that promotes adhesion to tracheal epithelial cells [23]. A recent study by van Gent et al. showed that the prn knock-out mutant had decreased ability to colonise the trachea and lungs in mice, but complementation of the prn knock-out mutant restored this ability [24]. In B. bronchiseptica, Prn also plays a role in resistance to neutrophil-mediated clearance, promoting persistence in the lower respiratory tract [25]. The emergence of B. pertussis strains with inactivation of Prn raises questions of the effect of this on virulence and the possibility that the presence of other virulence factors may have rendered Prn dispensable under selection pressure.

In this study, we employed an established mouse model of B. pertussis infection to investigate the effect of the ACV vaccine on the fitness (selective advantage) of Prn negative strains. We hypothesised that Prn negative B. pertussis could survive better in the host immunised with the ACV and carried out a competition assay in the mouse model using a mixed infection of recently isolated Prn positive and Prn negative B. pertussis strains from Australia.

Section snippets

B. pertussis clinical strains

A Prn negative strain (L1756) and a Prn positive strain (L1423) isolated from patients in Australia during 2008−2012 pertussis epidemic were selected for this study. The non-production of Prn in L1756 was due to an IS481 insertion in the prn gene [16] and our genome data showed that these two strains differ by 18 SNPs (Safarchi et al. unpublished data) (Table S1). L1423 and L1756 were isolated in 2010 and 2011, respectively. Both strains shared the same genotype, SP13, prn2, fim3A and ptxP3.

Statistical analysis

The significance of ACV-induced bacterial clearance from lungs and trachea were determined by T-test at each time point between the log10 (CFUs/lungs or trachea) of the control group of mice against the immunised one. The significant differences in Prn negative proportion in lungs and trachea of immunised mice against the control group were determined by t-test of the average percentage of Prn negative strain calculated based on the number of reads for each set of primers. P < 0.05 was considered

In vitro growth rate of the isolates used in this study

As intrinsic differences in growth rate may affect the in vivo competition outcome, we determined the doubling time separately for the Prn negative strain (L1756) and positive strain (L1423) based on CFU counts done in three separate experiments. The doubling time of L1756 and L1423 was 5.64 ± 0.51 and 6.55 ± 0.43 h, respectively, which was not statistically significantly different (p = 0.79).

Bacterial clearance in immunised mice infected with Prn positive and negative strains

Groups of 3 mice for each time point were infected with a mix of L1756 and L1423 in 1:1 ratio. Bacterial

Discussion

Increasing numbers of Prn negative strains were identified in the 2008−2012 pertussis epidemic in Australia [16]. In this study, we investigated the effect of ACV in a mouse model by examining the comparative fitness of the Prn negative strain in immunised and unimmunised mice. We carried out a competition assay by inducing mixed infection in mice with Prn negative and Prn positive strains which demonstrated that the Prn negative strain was relatively fitter in ACV-immunised mice and the Prn

Conflict of Interest Statement

Investigators on vaccine trials funded by GSK (Helen Marshall, Nicholas Wood) or partly supported by GSK (Peter McIntyre). All other co-authors have no conflicts of interest to declare.

Acknowledgement

The funding for this project was provided by the National Health and Medical Research Council of Australia (NHMRC) (APP1011942) and Helen Marshall was supported by an NHMRC Career Development Fellowship (APP1016272). Azadeh Safarchi was supported by a UNSW international postgraduate research award. We thank Dr Jani O’Rourke for technical assistance in the mouse experiments.

References (34)

  • J. Kurniawan et al.

    Bordetella pertussis clones identified by multilocus variable-number tandem-repeat analysis

    Emerg Infect Dis

    (2010)
  • H.E. Quinn et al.

    Duration of protection after first dose of acellular pertussis vaccine in infants

    Pediatrics

    (2014)
  • J.D. Cherry

    Pertussis: challenges today and for the future

    PLoS pathog

    (2013)
  • F.R. Mooi et al.

    Pertussis resurgence: waning immunity and pathogen adaptation - two sides of the same coin

    Epidemiol Infect

    (2014)
  • M.J. Bart et al.

    Global population structure and evolution of Bordetella pertussis and their relationship with vaccination

    mBio

    (2014)
  • F.R. Mooi et al.

    Bordetella pertussis strains with increased toxin production associated with pertussis resurgence

    Emerg Infect Dis

    (2009)
  • S. Octavia et al.

    Insight into evolution of Bordetella pertussis from comparative genomic analysis: evidence of vaccine-driven selection

    Mol Biol Evol

    (2011)
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