We conducted a phase 1 study of 9 pediatric patients with recurrent brain tumors using monocyte-derived dendritic cells pulsed with tumor RNA to produce antitumor vaccine (DCRNA) preparations. The objectives of this study included (1) establishing safety and feasibility and (2) measuring changes in general, antigen-specific, and tumor-specific immune responses after DCRNA. Dendritic cells were derived from freshly isolated monocytes after 7 days of culture with IL-4 and granulocyte-macrophage colony-stimulating factor, pulsed with autologous tumor RNA, and then cryopreserved. Patients received at least 3 vaccines, each consisting of an intravenous and an intradermal administration at biweekly intervals. The study showed that this method for producing and administering DCRNA from a single leukapheresis product was both feasible and safe in this pediatric brain tumor population. Immune function at the time of enrollment into the study was impaired in all patients tested. While humoral responses to recall antigens (diphtheria and tetanus) were intact in all patients, cellular responses to mitogen and recall antigens were below normal. Following DCRNA vaccine, 2 of 7 patients showed stable clinical disease and 1 of 7 showed a partial response. Two of 7 patients who were tested showed a tumor-specific immune response to DCRNA. This study showed that DCRNA vaccines are both safe and feasible in children with tumors of the central nervous system with a single leukapheresis.

More effective antibiotics and a protective vaccine are desperately needed to combat the ‘superbug’ Staphylococcus aureus. While in vivo pathogenicity studies routinely involve infection of mice with human S. aureus isolates, recent genetic studies have demonstrated that S. aureus lineages are largely host-specific. The use of such animal-adapted S. aureus strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted S. aureus strain (JSNZ) which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an hlb-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization.

Arginase of the Helicobacter pylori urea cycle hydrolyzes l-arginine to l-ornithine and urea.H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved inH. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori genehp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 androcF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was ∼1,000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of l-arginine, in contrast to the WT, and yielded a ∼10,000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylorimouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log10 CFU) were 6.1, 5.5, and 4.1, respectively. Thus,H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.

The efficacy of an orogastric vaccine comprised of purified Helicobacter pylori catalase plus the mucosal adjuvant cholera toxin (CT) was examined with both the Helicobacter felis and H. pylori mouse models with BALB/c mice. Native H. pylori catalase (200 g) plus CT was initially used as a vaccine antigen in the H. felis mouse model and protected 80% (8 of 10) of the challenged animals, while all control animals were infected (20 of 20). In a follow-up experiment, recombinant H. pylori catalase plus CT was used for immunization, and groups of mice were challenged with the Sydney strain of H. pylori. Immunization with recombinant catalase protected a significant proportion (9 of 10) of the mice from H. pylori challenge, indicating that this enzyme should be considered as a candidate for a future vaccine. This study provides the first available data on the efficacy of protective immunization with the new Sydney strain of H. pylori in a mouse model. These data also provide indirect evidence that proteins which are normally intracellular, such as catalase, may be present on the surface of H. pylori and thus may provide targets for immunization.

Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H. pylori, we generated low-infectivity variants of a mouse-colonizing H. pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. The gene expression levels of five open reading frames (ORFs) were significantly reduced in low-infectivity variants, with the most significant changes observed for ORFs HP1583 and HP1582. These ORFs were annotated as encoding homologs of the Escherichia coli vitamin B6 biosynthesis enzymes PdxA and PdxJ. Functional complementation studies with E. coli confirmed H. pylori PdxA and PdxJ to be bona fide homologs of vitamin B6 biosynthesis enzymes. Importantly, H. pylori PdxA was required for optimal growth in vitro and was shown to be essential for chronic colonization in mice. In addition to having a well-known metabolic role, vitamin B6 is necessary for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. Thus, for the first time, we identify vitamin B6 biosynthesis enzymes as novel virulence factors in bacteria. Interestingly, pdxA and pdxJ orthologs are present in a number of human pathogens, but not in mammalian cells. We therefore propose that PdxA/J enzymes may represent ideal candidates for therapeutic targets against bacterial pathogens.