Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a threecomponent vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the highlevel antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Tolllike receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.

The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.

Summary CsrRS (or CovRS) is a two-component system implicated in the control of multiple virulence determinants in the important human pathogen, group A Streptococcus (GAS). We observed CsrS-dependent regulation of 72 of the 73 genes whose expression changed in response to elevated extracellular Mg 2+ in wild-type bacteria, a result that identifies CsrS as the principal, if not exclusive, sensor for extracellular Mg 2+ in GAS. To our knowledge, this study is the first to characterize global gene regulation by a GAS two-component system in response to a specific environmental stimulus.

The peroxide response transcriptional regulator, PerR, is thought to contribute to virulence of group A Streptococcus (GAS); however, the specific mechanism through which it enhances adaptation for survival in the human host remains unknown. Here, we identify a critical role of PerR-regulated gene expression in GAS phagocytosis resistance and in virulence during pharyngeal infection. Deletion of perR in M-type 3 strain 003Sm was associated with reduced resistance to phagocytic killing in human blood and by murine macrophages in vitro. The increased phagocytic killing of the perR mutant was abrogated in the presence of the general oxidative burst inhibitor diphenyleneiodonium chloride (DPI), a result that suggests PerR-dependent gene expression counteracts the phagocyte oxidative burst. Moreover, an isogenic perR mutant was severely attenuated in a baboon model of GAS pharyngitis. In competitive infection experiments, the perR mutant was cleared from two animals at 24 h and from four of five animals by day 14, in sharp contrast to wild-type bacteria that persisted in the same five animals for 28 to 42 d. GAS genomic microarrays were used to compare wild-type and perR mutant transcriptomes in order to characterize the PerR regulon of GAS. These studies identified 42 PerR-dependent loci, the majority of which had not been previously recognized. Surprisingly, a large proportion of these loci are involved in sugar utilization and transport, in addition to oxidative stress adaptive responses and virulence. This finding suggests a novel role for PerR in mediating sugar uptake and utilization that, together with phagocytic killing resistance, may contribute to GAS fitness in the infected host. We conclude that PerR controls expression of a diverse regulon that enhances GAS resistance to phagocytic killing and allows adaptation for survival in the pharynx.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.MenB vaccine | selected reaction monitoring mass spectrometry | SRM-MS | fHbp antigen | antigen density

We have characterized group A Streptococcus (GAS) genome-wide responses to hydrogen peroxide and assessed the role of the peroxide response regulator (PerR) in GAS under oxidative stress. Comparison of transcriptome changes elicited by peroxide in wild-type bacteria with those in a perR deletion mutant showed that 76 out of 237 peroxide-regulated genes are PerR dependent. Unlike the PerR-mediated upregulation of peroxidases and other peroxide stress defense mechanisms previously reported in Gram-positive species, PerR-dependent genes in GAS were almost exclusively downregulated and encoded proteins involved in purine and deoxyribonucleotide biosynthesis, heme uptake, and amino acid/peptide transport, but they also included a strongly activated putative transcriptional regulator (SPy1198). Of the 161 PerR-independent loci, repressed genes (86 of 161) encoded proteins with functions similar to those coordinated by PerR, in contrast to upregulated loci that encoded proteins that function in DNA damage repair, cofactor metabolism, reactive oxygen species detoxification, pilus biosynthesis, and hypothetical proteins. Complementation of the perR deletion mutant with wild-type PerR restored PerR-dependent regulation, whereas complementation with either one of two PerR variants carrying single mutations in two predicted metal-binding sites did not rescue the mutant phenotype. Metal content analyses of the recombinant wild type and respective PerR mutants, in addition to regulation studies in metal-supplemented and iron-depleted media, showed binding of zinc and iron by PerR and an iron requirement for optimal responses to peroxide. Our findings reveal a novel physiological contribution of PerR in coordinating DNA and protein metabolic functions in peroxide and identify GAS adaptive responses that may serve to enhance oxidative stress resistance and virulence in the host.

High incidence, severity and increasing antibiotic resistance characterize Pseudomonas aeruginosa infections, highlighting the need for new therapeutic options. Vaccination strategies to prevent or limit P. aeruginosa infections represent a rational approach to positively impact the clinical outcome of risk patients; nevertheless this bacterium remains a challenging vaccine target. To identify novel vaccine candidates, we started from the genome sequence analysis of the P. aeruginosa reference strain PAO1 exploring the reverse vaccinology approach integrated with additional bioinformatic tools. The bioinformatic approaches resulted in the selection of 52 potential antigens. These vaccine candidates were conserved in P. aeruginosa genomes from different origin and among strains isolated longitudinally from cystic fibrosis patients. To assess the immune-protection of single or antigens combination against P. aeruginosa infection, a vaccination protocol was established in murine model of acute respiratory infection. Combinations of selected candidates, rather than single antigens, effectively controlled P. aeruginosa infection in the in vivo model of murine pneumonia. Five combinations were capable of significantly increase survival rate among challenged mice and all included PA5340, a hypothetical protein exclusively present in P. aeruginosa. PA5340 combined with PA3526-MotY gave the maximum protection. Both proteins were surface exposed by immunofluorescence and triggered a specific immune response. Combination of these two protein antigens could represent a potential vaccine to prevent P. aeruginosa infection.

We previously investigated immunogenicity of meningococcal native outer membrane vesicle (NOMV) vaccines prepared from recombinant strains with attenuated endotoxin (ΔLpxL1) and over-expressed factor H binding protein (fHbp) in a mouse model. The vaccines elicited broad serum bactericidal antibody responses. While human toll-like receptor 4 (TLR-4) is mainly stimulated by wildtype meningococcal endotoxin, mouse TLR-4 is stimulated by both the wildtype and mutant endotoxin. An adjuvant effect in mice of the mutant endotoxin would be expected to be much less in humans, and may have contributed to the broad mouse bactericidal responses. Here we show that as previously reported for humans, rhesus primate peripheral blood mononuclear cells incubated with a NOMV vaccine from ΔLpxL1 recombinant strains had lower proinflammatory cytokine responses than with a control wildtype NOMV vaccine. The cytokine responses to the mutant vaccine were similar to those elicited by a detergent-treated, wildtype outer membrane vesicle vaccine that had been safely administered to humans. Monkeys (N=4) were immunized beginning at ages 2 to 3 months with three doses of a NOMV vaccine prepared from ΔLpxL1 recombinant strains with over-expressed fHbp in the variant 1 and 2 groups. The mutant NOMV vaccine elicited serum bactericidal titers ≥1:4 against all 10 genetically diverse strains tested, including 9 with heterologous PorA to those in the vaccine. Negative-control animals had serum bactericidal titers <1:4. Thus, the mutant NOMV vaccine elicited broadly protective serum antibodies in a non-human infant primate model that is more relevant for predicting human antibody responses than mice.