Justin T. LaBrooy

Burkholderia pseudomallei virulence: definition, stability and association with clonality

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.

Oral delivery of foreign antigens by attenuated Salmonella: Consequences of prior exposure to the vector strain

Several strains of Salmonella have been used as vectors for the delivery of Escherichia coli fimbrial proteins to the gut-associated lymphoid tissue (GALT) of the mouse. Plasmids carrying a complementing thyA+ gene, together with genes specifying synthesis of K88 or K99, were introduced into non-reverting thyA Salmonella mutants. The resulting constructs expressed the foreign pilin protein on their surfaces and, provided the vector was able to colonize the GALT, elicited strong serum responses to K88 or K99. These responses were dramatically impaired however, in recipients with pre-existing immunity to the vector strain. Mice initially infected with Salmonella stanley ca 4, 10 or 20 weeks prior to oral administration of S. stanley-K88 showed greatly reduced serum responses to K88 as determined by ELISA. The hypo-responsiveness seen in vector-primed mice could be largely overcome by changing the serotype of the strain subsequently used to deliver the foreign protein.

Specific immune response in humans following rectal delivery of live typhoid vaccine

The specific immune responses to the live vaccine Salmonella typhi Ty21a following rectal administration were determined in serum, peripheral blood lymphocytes, saliva and in jejunal fluid of adult human subjects. Following vaccination, all seven subjects had a detectable anti-typhoid IgA antibody response using their peripheral blood lymphocytes (p = 0.009). Significant rises in postvaccination anti-typhoid IgA antibody were observed in the jejunal fluid (p = 0.033), serum (p = 0.010) and saliva (p = 0.050) of these subjects. This study confirms that the normal rectal mucosa is an efficient route of entry to the systemic immune system for microbial agents, and therefore may provide a further possible route of immunization with attenuated bacterial vaccines.

Demonstration of a Cell-Mediated Immune Response in Melioidosis

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei. The aim of this study was to determine whether a cell-mediated adaptive immune response against B. pseudomallei developed in patients who had recovered from melioidosis. Lymphocyte proliferation assays were done on peripheral blood mononuclear cells from patients (n=13) and control subjects (n=10) to determine the lymphocyte response to B. pseudomallei antigens. Production of interferon-gamma and interleukin-10 was also determined. Activation of T cell subsets was assessed by fluorescence-activated cell sorter analysis, using antibodies to CD4, CD8, and CD69 antigens. Lymphocyte proliferation and interferon-gamma production in response to B. pseudomallei antigens were significantly higher (P<.001 for both) in patients than in control subjects. There was also an increase in the percentage of activated CD4+ (P<.004) and activated CD8+ T cells (P<.035) in cell cultures from patients. The development of such a cell-mediated immune response in patients may be essential for their survival.

Induction of autoimmune valvulitis in lewis rats following immunization with peptides from the conserved region of group A streptococcal M protein

Rheumatic heart disease (RHD) is considered to be an autoimmune disorder mediated by group A streptococcal (GAS) M protein-specific T cells and antibodies that cross-react with cardiac antigens and epitopes of the GAS M protein. In this study, Lewis rats were immunized with a pool of overlapping peptides spanning the conserved region of the GAS M protein in Complete Freunds Adjuvant, followed by immunization with Bordetella pertussis. Controls received adjuvants alone. Spleen-derived lymphocytes from rats immunized with the conserved region peptides proliferated in response to the immunogen and to cardiac myosin. Moreover, histological examination of cardiac tissue from rats immunized with conserved region peptides revealed the presence of inflammatory lesions in both the myocardium and valve tissue indicating a role for GAS M protein-specific autoreactive T cells in the development of cardiac lesions. This study may support the use of the rat model of autoimmune valvulitis to investigate the immunopathogenesis of RHD and possible preventive strategies.

Recovery of the small intestine in coeliac disease on a gluten‐free diet: Changes in intestinal permeability, small bowel morphology and T‐cell activity

Abstract Intestinal permeability was assessed before and 1, 2, 4, 8 and 12 weeks after commencing a gluten‐free diet (GFD) in eight coeliac subjects. Intestinal morphology was quantified in six coeliac subjects on a normal diet, six coeliac subjects on a GFD, and 21 normal subjects. T‐cell activity was measured in the eight coeliac subjects by soluble interleukin‐2 receptor (sIL‐2R) concentration (normal < 477 U/mL). Intestinal permeability was increased 10‐fold with a geometric mean value of 0.72 on a normal diet, and decreased to 0.17 at 4 weeks (P=0.04), to 0.07 at 8 weeks (P=0.010), and to 0.20 at 12 weeks (P=0.015) of a GFD. Two of the eight subjects showed a poor response to gluten withdrawal. Quantitative intestinal morphology showed no significant improvement after 3 to 6 months of a GFD. Mean ± s.d. sIL‐2R concentrations in the eight subjects were increased 5‐fold higher than control values at 1400 ± 530 U/mL on a normal diet and decreased to 750 ± 200 U/mL after 12 weeks of a GFD (P=0.004). We conclude that intestinal permeability improves rapidly in the majority of coeliac subjects after commencing a GFD, although some abnormal permeability and increased T‐cell activity persists. This may be due to varying degrees of gluten ingestion resulting in continued immune activation.

SPECIFIC IMMUNE RESPONSE IN THE HUMAN RESPIRATORY TRACT FOLLOWING ORAL IMMUNIZATION WITH LIVE TYPHOID VACCINE

Specific antibody responses in the lower respiratory tract of human subjects to orally administered Salmonella typhi Ty21a are reported. These responses, predominantly of the immunoglobulin G class, were determined to be a transudate from serum. These results were supported by the similarity in responses to parenteral administration of heat-killed typhoid vaccine. Specific immunoglobulin A antibody was a poor contributor to the respiratory antibody response to either vaccine.

Gut immunity to typhoid: The immune response to a live oral typhoid vaccine Ty21a

Abstract The oral typhoid vaccine, Ty21a, generated an intestinal antibody response that was consistent and long‐lasting in contrast to the irregular response it generated in serum. Studies with different doses of the vaccine showed that 109 organisms appeared to be around the threshold dose required for a response and there was an increase in the consistency and magnitude of the response without side effects up to the maximum dose used of 1011 organisms. Revaccination after 6 months generated a further response though this response did not have the features of a memory response.

In vivo evidence of immunological masking of the Vibrio cholerae O antigen of a hybrid Salmonella typhi Ty21a-Vibrio cholerae oral vaccine in humans

The immunogenicity of the live oral hybrid vaccine organism Salmonella typhi Ty21a/V. cholerae Inaba (EX210) following its growth in media containing variable concentrations of supplemental galactose was examined in human volunteer subjects. The local intestinal IgA-specific antibody responses to both typhoid and cholera lipopolysaccharide (LPS) preparations were determined. It was observed that the immunogenicity of the galactose-independent Vibrio cholerae O antigen in vivo was dependent upon the variation in galactose-dependent long chain S. typhi O antigen production which was directly proportional to the media galactose concentration. It is likely that this observation was a result of steric hindrance of the presentation of the V. cholerae O antigen by S. typhi Ty21a in the presence of the longer, immunodominant S. typhi Ty21a O antigen. This observation may have relevance to the use of S. typhi vectors in vaccine development involving the presentation of LPS-associated heterologous antigens.

Antibody responses reveal differences in oral tolerance to wheatand maize grain protein fractions

The influence of diet on humoral immune responses to gluten‐ and maize‐derived proteins wasexamined using ELISA and protein blotting techniques. Mice raised on the maize‐based (gluten‐free)diet responded well to parenteral immunization with each of six gluten‐derived protein preparations(whole gliadin, two omega‐gliadin fractions, wheat salt‐soluble proteins, a peptic‐tryptic digest and asubtilisin digest of gluten), as serum antibody levels increased at least 300‐fold in each case. Incontrast, mice raised on the wheat‐based diet responded poorly to immunization with either wholegliadin or omega‐gliadin and were virtually non‐responsive to enzymic digest of gluten. Diet had littleeffect on the magnitude of the antibody response to wheat salt‐soluble proteins, with both groupsshowing a 300‐fold increase in titre. Similarly, tolerance to alpha‐zeins, the alcohol‐soluble proteinsof maize, did not occur on either diet. However, some oral tolerance was observed to maize glutelin. The specificity of the various antibody responses was then analysed by immunoblotting. Followingimmunization with gluten proteins or digests, antibodies from the maize‐fed mice bound more or lessequally to each of the main gliadin bands and to the glutenins while the mice on the wheat‐based diethad antibody specific for omega‐gliadin proteins. Serum antibodies from the maize‐fed mice, immunized with cither alpha‐zein or maize glutelin, showed even labelling of the major maizeendosperm proteins while antibodies from mice on the wheat diet showed strong labelling of the M,27000 and 58000 bands. These results show that diet influenced the specificity, as well as themagnitude of serum antibody responses to cereal proteins. In addition, oral tolerance appeared toaffect the humoral response to some cereal proteins more than others. Both of these findings haveimportant implications for our understanding of coeliac disease.