Kate F. Griffin

Passive protection against Burkholderia pseudomallei infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is endemic in south-east Asia and northern Australia, where it is an important cause of human disease. There is no vaccine available and antibiotic therapy is associated with high relapse rates. A panel of seven monoclonal antibodies (MAbs) that recognise capsular polysaccharide, lipopolysaccharide or proteins was produced and their ability to protect mice passively against experimental melioidosis was evaluated. The MAbs were capable of protecting mice against intra-peritoneal challenge with 10(4) cfu/250 MLD of a virulent strain of B. pseudomallei (NCTC 4845), when pooled, and four of the MAbs were individually protective. However, at a higher B. pseudomallei challenge level of 10(6) cfu none of the MAbs afforded protection and only the anti-exopolysaccharide MAbs produced a significantly delayed time to death.

The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens.

ABSTRACT We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.

The Use of Live Attenuated Bacteria as a Delivery System for Heterologous Antigens

Live attenuated mutants of several pathogenic bacteria have been exploited as potential vaccine vectors for heterologous antigen delivery by the mucosal route. Such live vectors offer the advantage of potential delivery in a single oral, intranasal or inhalational dose, stimulating both systemic and mucosal immune responses. Over the years, a range of strategies have been developed to allow controlled and stable delivery of antigens and improved immunogenicity where required. Most of these approaches have been evaluated in Salmonella vaccine vectors and, as a result, several live attenuated recombinant Salmonella vaccines are now in human clinical trials. In this review, these strategies and their use in the development of a delivery system for the Yersinia pestis V antigen are described.

Probing Molecular Interactions in Intact Antibody: Antigen Complexes, an Electrospray Time-of-Flight Mass Spectrometry Approach

Using a combination of nanoflow-electrospray ionization and time-of-flight mass spectrometry we have analyzed the oligomeric state of the recombinant V antigen from Yersinia pestis, the causative agent of plague. The mass spectrometry results show that at pH 6.8 the V antigen in solution exists predominantly as a dimer and a weakly associated tetramer. A monoclonal antibody 7.3, raised against the V antigen, gave rise to mass spectra containing a series of well-resolved charge states at m/z 6000. After addition of aliquots of solution containing V antigen in substoichiometric and molar equivalents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the antibody-antigen complex results in the selective release of the dimer from the complex supporting the proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the antibody is specific for the V antigen because no complex with F1 was detected even in the presence of a 10-fold molar excess of F1 antigen. More generally this work demonstrates a rapid means of assessing antigen subunit interactions as well as the stoichiometry and specificity of binding in antibody-antigen complexes.

Protective efficacy of a fully recombinant plague vaccine in the guinea pig

A fully recombinant sub-unit vaccine comprising the protein antigens rF1 + rV has been demonstrated to protect immunised guinea pigs against exposure to 10(5) colony-forming units (CFU) of virulent Yersinia pestis. Additionally, IgG purified from rF1 + rV-immunised guinea pig serum, protected the mouse by passive immunisation against challenge with Y. pestis whereas IgG purified from the serum of guinea pigs immunised with a licensed killed whole cell (KWC) vaccine for plague, protected less well. Guinea pigs immunised with the licensed killed whole cell vaccine developed an IgG titre for fraction 1 (F1) but not for V antigen. The differential in protection conferred on the mouse by passive immunisation with guinea pig IgG, was abrogated by the use of IgG purified from guinea pigs immunised with killed whole cell vaccine supplemented with V antigen. These findings indicate that the reduced efficacy of the licensed killed whole cell vaccine formulation previously observed in the mouse can be attributed to lack of the V antigen. Cross-protection of the mouse with guinea pig IgG suggests that the recognition of neutralising epitopes in the F1 and V proteins is conserved between these two species.

Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague

Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine.

Antibiotic-Free Plasmid Stabilization by Operator-Repressor Titration for Vaccine Delivery by Using Live Salmonella enterica Serovar Typhimurium

ABSTRACT Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyers patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.

Salmonella enterica serovar Typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge

ABSTRACT Protective immunity against infection with Bacillus anthracis is almost entirely based on a response to the protective antigen (PA), the binding moiety for the two other toxin components. We cloned the PA gene into an auxotrophic mutant of Salmonella enterica serovar Typhimurium as a fusion with the signal sequence of the hemolysin (Hly) A gene of Escherichia coli to allow the export of PA via the Hly export system. To stabilize the export cassette, it was also integrated into the chromosome of the live Salmonella carrier. When S. enterica serovar Typhimurium with the chromosomally integrated PA gene was given intravenously to A/J mice, they developed high levels of antibody to PA. These mice were protected against intraperitoneal challenge with 100 or 1,000 50% lethal doses of B. anthracis strain STI. This work contributes to the development of a Salmonella-based orally delivered anthrax vaccine.

Antibody responses to Yersinia pestis F1-antigen expressed in Salmonella typhimurium aroA from in vivo-inducible promoters.

Attenuated mutants of Salmonella typhimurium are being evaluated as delivery systems for a variety of heterologous vaccine antigens. Gene promoters which are induced in vivo can direct the stable expression of genes encoding these antigens. We have investigated the utility of the phoP, ompC, pagC and lacZ gene promoters for expression of the Y. pestis F1-antigen in S. typhimurium SL3261 (aroA). After i.g. (intragastric) dosing the highest level of spleen colonisation was found with recombinant Salmonella expressing F1-antigen from the phoP gene promoter, and this recombinant was most effective in inducing serum and mucosal antibody responses. The use of the pagC gene promoter to direct expression of F1-antigen resulted in the induction of serum and mucosal antibody responses even though the recombinant Salmonella were unable to colonise spleen tissues suggesting that colonisation of these tissues is not essential for the induction of antibody responses.

Critical role of type 1 cytokines in controlling initial infection with Burkholderia mallei

ABSTRACT Burkholderia mallei is a gram-negative bacterium which causes the potentially fatal disease glanders in humans; however, there is little information concerning cell-mediated immunity to this pathogen. The role of gamma interferon (IFN-γ) during B. mallei infection was investigated using a disease model in which infected BALB/c mice normally die between 40 and 60 days postinfection. IFN-γ knockout mice infected with B. mallei died within 2 to 3 days after infection, and there was uncontrolled bacterial replication in several organs, demonstrating the essential role of IFN-γ in the innate immune response to this pathogen. Increased levels of IFN-γ, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 were detected in the sera of immunocompetent mice in response to infection, and splenic mRNA expression of IFN-γ, IL-6, IL-12p35, and IL-27 was elevated 24 h postinfection. The effects of IL-18, IL-27, and IL-12 on stimulation of the rapid IFN-γ production were investigated in vitro by analyzing IFN-γ production in the presence of heat-killed B. mallei. IL-12 was essential for IFN-γ production in vitro; IL-18 was also involved in induction of IFN-γ, but IL-27 was not required for IFN-γ production in response to heat-killed B. mallei. The main cellular sources of IFN-γ were identified in vitro as NK cells, CD8+ T cells, and TCRγδ T cells. Our data show that B. mallei is susceptible to cell-mediated immune responses which promote expression of type 1 cytokines. This suggests that development of effective vaccines against glanders should target the production of IFN-γ.