Lazzaroni, Sharon (2009) Immune responses in Burkholderia pseudomallei infection, relapse, reactivation and protection. Masters (Research) thesis, James Cook University.

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Abstract

Burkholderia pseudomallei is the bacterium that causes the potentially fatal disease melioidosis. Endemic regions include South-east Asia and northern Australia. The immunopathogenesis of melioidosis is not fully understood as the bacterium can evade the host immune responses surviving within cells becoming apparent when the host is immunocompromised. Since B. pseudomallei is an intracellular organism, cell-mediated immune (CMI) responses are important in clearance and protection. The processes involved in the induction of a protective immune response is necessary to understand how the disease would progress and for developing vaccine strategies against B. pseudomallei. Therefore, the focus of the research in this thesis is to determine the extent of exposure to B. pseudomallei in northern Queensland and to characterise the CMI responses of seropositive healthy individuals from this endemic region. The immune responses involved in relapse and latent B. pseudomallei infection will also be determined by analysing the clonality of isolates from patients with recurrent melioidosis and developing a murine model of latency. The protective effect of DNA constructs of putative virulence factors will be studied in an experimental model of melioidosis.

Using the indirect haemagglutination assay, 1500 blood donors from northern Queensland were assessed for antibodies to B. pseudomallei. The results demonstrated 2.47% (n=37) of the population had an antibody response to B. pseudomallei with a titre of ≥1:40. Using these seropositive donors, the Melioidosis IgG Rapid Cassette test was evaluated as a potential screening test for melioidosis. The results found only 32% were positive using this rapid test. This study demonstrated that individuals living in endemic regions may have been exposed to B. pseudomallei and consequently produced an antibody response. By characterising the CMI response of these seropositive individuals it will be possible to determine whether these individuals develop protective immunity against the bacterium.

Lymphocyte proliferation assays and flow cytometry were used to characterise the CMI response in seropositive individuals (n=8). Lymphocytes were isolated and stimulated with B. pseudomallei antigen (BpLy1) the proliferative response was assessed, as well as activation of T cell subsets and cytokine production. Results demonstrated no significant differences in the proliferative response of lymphocytes in seropositive individuals when compared to seronegative individuals. There were no significant differences observed in CD4+ and CD8+ populations, interferon (IFN)-γ or interleukin (IL)-4 production. These results did not demonstrate an induction of a CMI response following stimulation with BpLy1. Although these seropositive individuals may have produced an antibody response to B. pseudomallei, the exposure was not sufficient to produce a CMI response.

Clonality of B. pseudomallei isolates from patients who had relapsed with melioidosis was determined using pulsed-field gel electrophoresis (PFGE). Fragment patterns produced following PFGE demonstrated clonal isolates were responsible for each relapse episode in four patients. One patient who had relapsed with melioidosis nine times over a period of eight years was due to the same B. pseudomallei isolate recrudescing rather than infection with a different isolate. These results suggest that the bacterium is able to evade the immune responses and cause continual episodes of melioidosis, therefore, treatment of the disease may need to be reassessed.

The development of a latent model of melioidosis involved C57BL/6 mice being infected with a low dose of a low virulence strain of B. pseudomallei (NCTC13179). The mice were allowed to clear the infection over a period of 30 days with bacterial load, delayed type hypersensitivity (DTH) and lymphocyte proliferation responses assessed at days 10, 20 and 30. These results demonstrated that a CMI response had been produced with significant differences seen in the DTH response and proliferative response of lymphocytes following stimulation with BpLy1. There was also an apparent clearance of B. pseudomallei, with negative culture from spleen and liver of infected animals. Mice were then immunosuppressesed with dexamethasone and monitored for survival, DTH and lymphocyte proliferation responses and bacterial load. Following immunosuppression, there was a significantly reduced immune response demonstrated by the DTH response. The proliferative response of lymphocytes in infected, immunosuppressed mice was not reflective of observations seen in vivo with no differences observed in proliferation of lymphocytes, following stimulation with BpLy1, in infected, immunosuppressed mice compared to infected, non-immunosuppressed mice. Interestingly, results from bacterial loads demonstrated that by bacterial culture, B. pseudomallei was not present, however using real-time PCR (qPCR) B. pseudomallei DNA was detected in the spleens of both groups of mice. There was apparent reactivation of B. pseudomallei infection with a significant decrease in survival of infected mice that had been immunosuppressed and infected mice that had not been immunosuppressed. This aspect of the investigation demonstrates the initial development of an animal model of latent melioidosis.

Potential DNA vaccine constructs were evaluated using a BALB/c mouse model of experimental B. pseudomallei infection to determine their protective potential. The DNA vaccine constructs were developed against selective putative virulence factors of B. pseudomallei. Mice were vaccinated at three time points using a gene gun and subsequently challenged with a low virulence strain of B. pseudomallei (NCTC13179). Mice were monitored for survival and bacterial load. There were no significant differences seen in survival of mice vaccinated with different DNA constructs. When a combination of DNA constructs or CpG, an immunostimulator, was added to the vaccination regime, no significant differences were seen in survival of mice in these groups when compared to control mice. Splenic bacterial loads were not reduced in vaccinated groups when compared to control mice.

The results of this present study have provided further knowledge into host-pathogen interactions of B. pseudomallei. There is evidence that although there is exposure to B. pseudomallei in northern Queensland, detected by assessing antibody production, this exposure does not produce a CMI response which is essential in protection against the bacterium. Initial studies in determining whether B. pseudomallei persists within the host has been modelled in a murine model of latency. To produce a successful vaccine to protect against B. pseudomallei infection, further understanding of the immune responses and interaction of the bacterium is required.

Item ID:	11605
Item Type:	Thesis (Masters (Research))
Keywords:	meliodosis infections, immunology, immune responses, cell mediated immune responses, protective immunities, meliodosis exposure, latent meliodosis, infection relapses, Burkholderia pseudomallei
Copyright Information:	Copyright © 2009 Sharon Lazzaroni
Date Deposited:	25 Jun 2010 01:19
FoR Codes:	06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060502 Infectious Agents @ 100%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50% 97 EXPANDING KNOWLEDGE > 970111 Expanding Knowledge in the Medical and Health Sciences @ 50%
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