Self-adjuvanting bacterial vectors expressing pre-erythrocytic antigens induce sterile protection against malaria.

Genetically inactivated, Gram-negative bacteria that express malaria vaccine candidates represent a promising novel self-adjuvanting vaccine approach. Antigens expressed on particulate bacterial carriers not only target directly to antigen-presenting cells but also provide a strong danger signal thus circumventing the requirement for potent extraneous adjuvants. E. coli expressing malarial antigens resulted in the induction of either Th1 or Th2 biased responses that were dependent on both antigen and sub-cellular localization. Some of these constructs induced higher quality humoral responses compared to recombinant protein and most importantly they were able to induce sterile protection against sporozoite challenge in a murine model of malaria. In light of these encouraging results, two major Plasmodium falciparum pre-erythrocytic malaria vaccine targets, the Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS) fused to the Maltose-binding protein in the periplasmic space and the Circumsporozoite Protein (CSP) fused to the Outer membrane (OM) protein A in the OM were expressed in a clinically relevant, attenuated Shigella strain (Shigella flexneri 2a). This type of live-attenuated vector has previously undergone clinical investigations as a vaccine against shigellosis. Using this novel delivery platform for malaria, we find that vaccination with the whole-organism represents an effective vaccination alternative that induces protective efficacy against sporozoite challenge. Shigella GeMI-Vax expressing malaria targets warrant further evaluation to determine their full potential as a dual disease, multivalent, self-adjuvanting vaccine system, against both shigellosis, and malaria.

Proteomics for the development of vaccines and therapeutics.

Proteomics permits the large-scale and high-throughput analysis of proteins and has become a powerful tool with which to study the pathogenic mechanisms of bacteria. It not only provides a metabolic snapshot at a particular moment in the life of a pathogen, but can also determine where a protein resides, its function, whether it is secreted, and its interactions with other proteins, including those of the host. Comparative proteomics can yield important information on the differences between attenuated and pathogenic organisms and whether a protein is conserved among various strains. Our laboratory has utilized traditional and novel techniques to investigate the global and subproteomes of Bacillus anthracis as they relate to vaccine and therapeutic development. Recently, our efforts have focused on the use of mass spectrometry for B-cell epitope discovery and identification of components of a pathogen that interact with host proteins. Development of vaccines and therapeutics based on proteomic data in combination with novel adjuvants and delivery systems will be presented.

Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.

Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans.

A rapid and sensitive magnetic bead-based immunoassay for the detection of staphylococcal enterotoxin B for high-through put screening.

Staphylococcal enterotoxin B (SEB) is one of many toxins produced by the Gram-positive bacterium Staphylococcal aureus. While SEB is known as the causative agent of certain food poisonings it is also considered a biological Select Agent. Thus, rapid and accurate identification of SEB during either surveillance or in response to a biothreat is critical to the mitigation of the suspect agent. This report presents an improved method for the detection of SEB based on a SEB-specific, two-antibody system where one antibody was bound to a magnetic bead particle while the other was labeled with Alexa fluor 647. The assay consisted of one incubation period for 30 minutes where all reagents necessary to detect SEB were included. Using this assay 100 pg of recombinant purified SEB, as well as SEB from the culture supernatant of several strains of methicillin-resistant S. aureus were detected with fidelity. This assay presents improvements over current assays in terms of a combination of the reduction in assay time length, assay sensitivity, ease of use, and application to automated high-throughput analysis. Additionally, this assay can be easily modified to detect a wide range of proteins and whole organisms.