Elsevier

Vaccine

Volume 34, Issue 14, 29 March 2016, Pages 1665-1671
Vaccine

Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease

https://doi.org/10.1016/j.vaccine.2016.02.038Get rights and content

Abstract

There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice.

Introduction

Burkholderia pseudomallei is the etiological agent of melioidosis, a serious and often fatal disease of humans and a wide variety of animals [1]. The disease is an important medical problem in many tropical regions of the world. For example, in North-East Thailand, melioidosis is the third most common cause of death from infectious diseases, exceeded only by HIV and tuberculosis [2]. In Darwin, Northern Australia, melioidosis is the most common cause of fatal community-acquired septicemic pneumonia [3]. Melioidosis is difficult to treat as B. pseudomallei is resistant to many antibiotics [4]. The bacterium is susceptible to some newer β-lactam antibiotics including ceftazidime [5], [6] but a protracted course of treatment (typically 20 weeks) is routinely required [7]. Chronic disease is common and disease relapse has been reported [8], [9].

Against this background, there may be an opportunity to use vaccines for the control of naturally occurring disease. Recent work has shown that a vaccine would be a cost-effective intervention in Thailand if used in high-risk populations such as diabetics [10]. However, at present there is no available human vaccine, though a wide range of candidates including live attenuated mutants, killed cells, protein or polysaccharide sub-units and naked DNA vaccines have been tested in mice (for recent reviews see [11], [12], [13]). All of these vaccines are able to induce some protective immunity, protecting against the acute form of the disease. However, all of the candidates fail to provide long-term control of disease of which persistent, chronic melioidosis is a feature.

B. pseudomallei differs from many other pathogens, yet is similar to Mycobacterium tuberculosis, in its capacity for intracellular survival for many years. Chronic melioidosis is often misdiagnosed as tuberculosis and the two diseases have common histological features such as the presence of multinucleated giant cells and granulomas, suggesting that melioidosis and tuberculosis have shared immunological mechanisms. Like melioidosis, it is difficult to devise vaccines that provide sterile immunity against tuberculosis. However, one promising approach is to devise tuberculosis sub-unit vaccines which include antigens expressed during the chronic or latent phase of disease [14], [15], [16]. These vaccines have been shown to be effective in animal models of disease and are currently in human clinical trials [17].

Recently, the B. pseudomallei transcriptional response to a range of in vitro conditions reflecting the diversity of niches occupied by the bacterium was reported [18]. Evaluation of the transcriptional response of B. pseudomallei in conditions mimicking the environment inside the granuloma, including hypoxia and nutrient starvation, has provided clues to the repertoire of antigens expressed by the pathogen as it adapts to long-term persistence in the host. Our hypothesis is that it is possible to prime the host immune system to target B. pseudomallei during the chronic stage of melioidosis by selecting vaccine antigens expressed by the bacterium in the chronic phase of infection. In this study we have identified, constructed and evaluated chronic stage vaccine candidates in the murine model of infection. Our results indicate that, in a murine model of disease, the degree of protection afforded by immunisation with chronic stage vaccine antigens is greater than the protection afforded by two of the leading sub-unit candidates (CPS and LolC).

Section snippets

Bacterial strains

B. pseudomallei strains 576 and K96243 were used in this study. Both are clinical isolates of B. pseudomallei from cases of melioidosis in Thailand [19]. The intraperitoneal (i.p.) median lethal doses of these strains are reported to be 80 and 262 colony forming units (CFU), respectively [19]. Bacteria were cultured in Luria-Bertani (LB) medium at 37 °C.

Identification of chronic stage antigens

B. pseudomallei transcriptional profiles from in vitro conditions thought to reflect the environment inside a granuloma were obtained [18].

Statistical analysis

Survival data was analysed in the program GraphPad PRISM v6.0 using a log rank (Mantel-Cox) test. Significance was assessed at the 95% confidence level.

Identification of chronic stage antigens

We first compared the gene expression profile of B. pseudomallei grown in granuloma mimicking conditions. Nine putative chronic infection stage B. pseudomallei genes that were upregulated in at least three of four in vitro conditions; low oxygen, nutrient deprivation, low pH and peroxide were identified: BPSL0296, BPSL2287, BPSL2289, BPSL3312, BPSS0369, BPSL1899, BPSL3369, BPSL3247 and BPSL1897. The nine genes represented different classes of genes, including hypothetical proteins with membrane

Discussion

A wide range of melioidosis vaccine candidates have been tested in mice over the past decade. None of these candidates consistently provide sterile immunity against disease, but some extend the time to death of mice challenged with B. pseudomallei. It is uncertain whether it will be possible to develop vaccines that provide robust and sterile immunity, and we report here the potential for chronic stage vaccine antigens to contribute to protection.

Recently, tuberculosis vaccines that combine

Authors contributions

This study was conceived by OLC, MB and RWT. LC, CH and GB carried our chronic infection studies and prepared tissues for mRNA isolation and isolated mRNA. OLC carried out the bioinformatics analysis and the analysis of gene expression. LJG, PL and LP cloned, overexpressed and purified the proteins used in this study under the guidance of MB. AS carried out the mouse protection studies under the guidance of JP. OLC, LJG, GB and RWT wrote the manuscript.

Conflict of interest statement

The authors have no competing interests.

Acknowledgements

This work was supported by Fondazione CARIPLO (contract number 2009-3577) “From Genome to Antigen: a Multidisciplinary Approach towards the Development of an Effective Vaccine against Burkholderia pseudomallei, the Etiological Agent of Melioidosis.”

References (33)

  • D.A.B. Dance et al.

    The antimicrobial susceptibility of Pseudomonas pseudomallei—emergence of resistance in vitro and during treatment

    J Antimicrob Chemother

    (1989)
  • W.J. Wiersinga et al.

    Melioidosis

    N Engl J Med

    (2012)
  • D. Limmathurotsakul et al.

    Melioidosis: a clinical overview

    Br Med Bull

    (2011)
  • S.J. Peacock et al.

    Melioidosis vaccines: a systematic review and appraisal of the potential to exploit biodefense vaccines for public health purposes

    PLoS Negl Trop Dis

    (2012)
  • N. Patel et al.

    Development of vaccines against Burkholderia pseudomallei

    Front Microbiol

    (2011)
  • E.B. Silva et al.

    Development of Burkholderia mallei and pseudomallei vaccines

    Front Cell Infect Microbiol

    (2013)
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