Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease
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)
- et al.
The epidemiology of melioidosis in Australia and Papua New Guinea
Acta Trop
(2000) Melioidosis
Lancet
(2003)Treatment and prophylaxis of melioidosis
Int J Antimicrob Agents
(2014)- et al.
Burkholderia pseudomallei: animal models of infection
Trans R Soc Trop Med Hyg
(2008) - et al.
Low-dose exposure of C57BL/6 mice to Burkholderia pseudomallei mimics chronic human melioidosis
Am J Pathol
(2011) - et al.
Crystal structure of IscA, an iron–sulfur cluster assembly protein from Escherichia coli
J Mol Biol
(2004) - et al.
Exploiting the Burkholderia pseudomallei acute phase antigen BPSL2765 for structure-based epitope discovery/design in structural vaccinology
Chem Biol
(2013) - et al.
Melioidosis: epidemiology, pathophysiology and management
Clin Microbiol Rev
(2005) - et al.
Increasing incidence of human melioidosis in Northeast Thailand
Am J Trop Med Hyg
(2010) - et al.
Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei
Nat Rev Microbiol
(2006)
The antimicrobial susceptibility of Pseudomonas pseudomallei—emergence of resistance in vitro and during treatment
J Antimicrob Chemother
Melioidosis
N Engl J Med
Melioidosis: a clinical overview
Br Med Bull
Melioidosis vaccines: a systematic review and appraisal of the potential to exploit biodefense vaccines for public health purposes
PLoS Negl Trop Dis
Development of vaccines against Burkholderia pseudomallei
Front Microbiol
Development of Burkholderia mallei and pseudomallei vaccines
Front Cell Infect Microbiol
Cited by (20)
Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase
2019, Bioorganic and Medicinal Chemistry LettersStructural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain E555:: wbiI (pKnock-KmR) and assessment of the significance of the 2-O-acetyl group in immune protection
2017, Carbohydrate ResearchCitation Excerpt :We have shown that the acetyl group of CPS forms an immunodominant, protective, epitope which is an important finding for chemical efforts to produce synthetic CPS and also places limitations on the methods that can be employed to fragment native CPS polysaccharide. The O-polysaccharide-deficient mutant of B. thailandensis E555 harbouring a kanamycin-resistance marked, in-frame deletion of its wbiI gene (B. thailandensis E555:: wbiI (pKnock-KmR)) [37] was grown in 2 L of LB broth overnight at 37 °C with shaking. Loss of O-polysaccharide expression had previously been confirmed by western immunoblot analysis, with bacterial stocks maintained in 35% (v/v) glycerol suspensions at −80 °C.
Burkholderia pseudomallei and Burkholderia mallei vaccines: Are we close to clinical trials?
2017, VaccineCitation Excerpt :Another approach is to use a combination of proteins. One study has shown that a combination of antigens can provide protection against experimental disease although sterile immunity was still not achieved [49]. An alternative to using combinations of proteins would involve using combinations of epitopes derived from different proteins and there has been some exciting foundation work in this area [50–57].
Protection against experimental melioidosis with a synthetic manno-heptopyranose hexasaccharide glycoconjugate
2016, Bioconjugate ChemistryBurkholderia pseudomallei and melioidosis
2024, Nature Reviews MicrobiologyVaccine Development Against Melioidosis
2023, Vaccines for Neglected Pathogens: Strategies, Achievements and Challenges: Focus on Leprosy, Leishmaniasis, Melioidosis and Tuberculosis