Helen S. Atkins

Towards a Brucella vaccine for humans

There is currently no licensed vaccine for brucellosis in humans. Available animal vaccines may cause disease and are considered unsuitable for use in humans. However, the causative pathogen, Brucella, is among the most common causes of laboratory-acquired infections and is a Center for Disease Control category B select agent. Thus, human vaccines for brucellosis are required. This review highlights the considerations that are needed in the journey to develop a human vaccine, including animal models, and includes an assessment of the current status of novel vaccine candidates.

The ABC Transporter Protein OppA Provides Protection against Experimental Yersinia pestis Infection

ABSTRACT The identification of Yersinia pestis as a potential bioterrorism agent and the emergence of antibiotic-resistant strains have highlighted the need for improved vaccines and treatments for plague. The aim of this study was to evaluate the potential for ATP-binding cassette (ABC) transporter proteins to be exploited as novel vaccines against plague. Western blotting of ABC transporter proteins using sera from rabbits immunized with killed whole Y. pestis cells or human convalescent-phase sera identified four immunologically reactive proteins: OppA, PstS, YrbD, and PiuA. Mice immunized with these proteins developed antibody to the immunogen. When the immunized mice were challenged with Y. pestis, the OppA-immunized mice showed an increased time to death compared to other groups, and protection appeared to correlate with the level of immunoglobulin G antibody to OppA.

Identification of a LolC Homologue in Burkholderia pseudomallei, a Novel Protective Antigen for Melioidosis

ABSTRACT Melioidosis is an emerging disease of humans in Southeast Asia and tropical Australia. The bacterium causing this disease, Burkholderia pseudomallei, is also considered a bioterrorism agent, and as yet there is no licensed vaccine for preventing B. pseudomallei infection. In this study, we evaluated selected proteins (LolC, PotF, and OppA) of the ATP-binding cassette systems of B. pseudomallei as candidate vaccine antigens. Nonmembrane regions of the B. pseudomallei proteins were expressed and purified from Escherichia coli and then evaluated as vaccine candidates in an established mouse model of B. pseudomallei infection. When delivered with the monophosphoryl lipid A-trehalose dicorynomycolate adjuvant, the proteins stimulated antigen-specific humoral and cellular immune responses. Immunization with LolC or PotF protein domains afforded significant protection against a subsequent challenge with B. pseudomallei. The most promising vaccine candidate, LolC, provided a greater level of protection when it was administered with immune-stimulating complexes complexed with CpG oligodeoxynucleotide 10103. Immunization with LolC also protected against a subsequent challenge with a heterologous strain of B. pseudomallei, demonstrating the potential utility of this protein as a vaccine antigen for melioidosis.

Phenotypic and Functional Characterization of Human Memory T Cell Responses to Burkholderia pseudomallei

Background Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-γ) is critical for resistance, but in humans the characteristics of IFN-γ production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined. Methods Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-γ responses were analyzed by ELISPOT and flow cytometry. Findings B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-γ. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4+ (and CD8+) T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector–memory (TEMRA) phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum. Conclusions Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-γ that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide–conjugate subunit vaccines against this important but neglected disease.

Microbial TIR domains: not necessarily agents of subversion?

The Toll/interleukin-1 receptor (TIR) domain plays a crucial role in the mammalian innate immune response. Recently, proteins containing TIR domains have been described in bacteria and it has been suggested that these bacterial proteins are involved in subversion of the vertebrate immune system. Here we describe the distribution of TIR-domain proteins among bacteria, fungi, archaea and viruses and evaluate the subversion hypothesis in the light of our findings. We suggest that most TIR domains in bacteria have nothing to do with subverting eukaryotic cells; instead, TIR domains function simply as general purpose protein-protein interaction domains put to diverse uses.

An anthrax subunit vaccine candidate based on protective regions of Bacillus anthracis protective antigen and lethal factor

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.

Towards a human oral vaccine for anthrax: the utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy

We previously demonstrated the ability of an orally administered attenuated Salmonella enterica serovar Typhimurium strain expressing the protective antigen (PA) of Bacillus anthracis to confer protection against lethal anthrax aerosol spore challenge [Stokes MG, Titball RW, Neeson BN, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75(April (4)):1827-34]. To extend the utility of this approach to humans we constructed variants of S. enterica serovar Typhi Ty21a, an attenuated typhoid vaccine strain licensed for human use, which expressed and exported PA via two distinct plasmid-based transport systems: the Escherichia coli HlyA haemolysin and the S. Typhi ClyA export apparatus. Murine immunogenicity studies confirmed the ability of these constructs, especially Ty21a expressing the ClyA-PA fusion protein, to stimulate strong PA-specific immune responses following intranasal immunization. These responses were further enhanced by a subsequent boost with either parenterally delivered recombinant PA or the licensed US human alum-adsorbed anthrax vaccine (AVA). Anthrax toxin neutralizing antibody responses using this prime-boost regimen were rapid, vigorous and broad in nature. The results of this study demonstrate the feasibility of employing a mucosal prime with a licensed Salmonella Typhi vaccine strain followed by a parenteral protein boost to stimulate rapid protective immunity against anthrax.

A carpet-based mechanism for direct antimicrobial peptide activity against vaccinia virus membranes

Antimicrobial peptides have activity against a wide variety of biological membranes and are an important component of innate immunity in vertebrate as well as invertebrate systems. The mechanisms of action of these peptides are incompletely understood and a number of competing but not necessarily mutually exclusive models exist. In this study we examined the virucidal activity of four peptides, the human cathelicidin derived LL37, Xenopus alanine-substituted Magainin-2 amide, uperin-3.1, and a cecropin-LL37 hybrid against vaccinia virus. The peptides were shown to be differentially virucidal but all were shown to attack the viral envelope, with LL37 being the most effective and uperin-3.1 the least. Density gradient analysis of the treated virions indicated the virus outer membrane was efficiently removed by peptide action and suggests a mechanism of direct virus inactivation that is consistent with the carpet model for peptide-mediated membrane disruption. Interestingly, the least effective peptide uperin-3.1 was equally effective as the others at inducing susceptibility to neutralizing antibody. This suggests that in addition to direct killing by a carpet-based mechanism, the peptides may simultaneously operate a different mechanism that exposes sequestered antigen without membrane removal.

ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei

BackgroundATP binding cassette (ABC) systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243 and Burkholderia mallei strain ATCC 23344 has been compiled using bioinformatic techniques.ResultsThe ABC systems in the genomes of B. pseudomallei and B. mallei have been reannotated and subsequently compared. Differences in the number and types of encoded ABC systems in belonging to these organisms have been identified. For example, ABC systems involved in iron acquisition appear to be correlated with differences in genome size and lifestyles between these two closely related organisms.ConclusionThe availability of complete inventories of the ABC systems in B. pseudomallei and B. mallei has enabled a more detailed comparison of the encoded proteins in this family. This has resulted in the identification of ABC systems which may play key roles in the different lifestyles and pathogenic properties of these two bacteria. This information has the potential to be exploited for improved clinical identification of these organisms as well as in the development of new vaccines and therapeutics targeted against the diseases caused by these organisms.

Bacterial TIR-containing proteins and host innate immune system evasion

The innate immune system provides the first line of host defence against invading pathogens. Key to upregulation of the innate immune response are Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) and trigger a signaling pathway culminating in the production of inflammatory mediators. Central to this TLR signaling pathway are heterotypic protein–protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms. Recent work has identified a potentially novel evasion mechanism involving bacterial TIR domain proteins. Such domains have been identified in a wide range of pathogenic bacteria, and there is evidence to suggest that they interfere directly with the TLR signaling pathway and thus inhibit the activation of NF-κB. The individual TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis, Brucella sp, uropathogenic E. coli and Yersinia pestis have been analyzed in detail. The individual bacterial TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of the bacterial TIR domains.