Genome-Based Approach Delivers Vaccine Candidates Against Pseudomonas aeruginosa

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

Environmental Acidification Drives S. pyogenes Pilus Expression and Microcolony Formation on Epithelial Cells in a FCT-Dependent Manner

Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin- Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection

Investigating the Role of Antigen Orientation on the Immune Response Elicited by Neisseria meningitidis Factor H Binding Protein on GMMA

GMMA are outer membrane vesicles (OMVs) released from Gram-negative bacteria genetically modified to enhance OMVs formation that have been shown to be optimal systems to enhance immunogenicity of protein antigens. Here, we selected Neisseria meningitidis factor H binding protein (fHbp) and used the conjugation chemistry as a tool to alter antigen orientation on GMMA. Indeed, fHbp was randomly linked to GMMA or selectively attached via the N-terminus to mimic native presentation of the protein on the bacterial surface. Interestingly, protein and peptide array analyses confirmed that antibodies induced by the selective and the random conjugates showed a pattern very similar to fHbp natively expressed on bacterial surfaces or to the recombinant protein mixed with GMMA, respectively. However, the two conjugates elicited antibodies with similar serum bactericidal activity against meningococcal strains, superior to the protein alone or physically mixed with GMMA. Presentation of fHbp on GMMA strongly enhances the functional immune response elicited by the protein but its orientation on the bacterial surface does not have an impact. This study demonstrates the flexibility of the GMMA platform as a display and delivery system for enhancing antigen immunogenicity and further supports the use of such promising technology for the development of effective vaccines

Preventing bacterial infections with pilus-based vaccines: The group B streptococcus paradigm

We recently described the presence of 3 pilus variants in the human pathogen group B streptococcus (GBS; also known as Streptococcus agalactiae), each encoded by a distinct pathogenicity island, as well as the ability of pilus components to elicit protection in mice against homologous challenge. To determine whether a vaccine containing a combination of proteins from the 3 pilus types could provide broad protection, we analyzed pili distribution and conservation in 289 clinical isolates. We found that pilus sequences in each island are conserved, all strains carried at least 1 of the 3 islands, and a combination of the 3 pilus components conferred protection against all tested GBS challenge strains. These data are the first to indicate that a vaccine exclusively constituted by pilus components can be effective in preventing infections caused by GBS, and they pave the way for the use of a similar approach against other pathogenic streptococci. \ua9 2008 by the Infectious Diseases Society of America. All rights reserved

Immunology: Identification of a universal group B Streptococcus vaccine by multiple genome screen

Group B Streptococcus (GBS) is a multiserotype bacterial pathogen representing a major cause of life-threatening infections in newborns. To develop a broadly protective vaccine, we analyzed the genome sequences of eight GBS isolates and cloned and tested 312 surface proteins as vaccines. Four proteins elicited protection in mice, and their combination proved highly protective against a large panel of strains, including all circulating serotypes. Protection also correlated with antigen accessibility on the bacterial surface and with the induction of opsonophagocytic antibodies. Multigenome analysis and screening described here represent a powerful strategy for identifying potential vaccine candidates against highly variable pathogens

SslE Elicits Functional Antibodies That Impair <i>In Vitro</i> Mucinase Activity and <i>In Vivo</i> Colonization by Both Intestinal and Extraintestinal <i>Escherichia coli</i> Strains

<div><p>SslE, the <u>S</u>ecreted and <u>s</u>urface-associated <u>l</u>ipoprotein from <i><u>E</u>scherichia coli</i>, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of <i>in vitro</i> bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired <i>E. coli</i> mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslE_IHE3034), are able to inhibit translocation of <i>E. coli</i> strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslE_IHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in <i>E. coli</i> colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic <i>E. coli</i> species.</p></div

SslE_IHE3034 induces cross-protection in intestinal colonization, UTI and sepsis models.

<p>(A) Thirty CD1 mice were intranasally immunized with 30 µg of SslE_IHE3034 at days 1, 21 and 35. Saline was used in the negative control groups. Challenge was done by oral gavage with 5×10⁷ CFU of strain GL53 at day 49. Serial dilutions of the homogenized intestinal caecum tract were plated and the CFU number was enumerated. Statistical significance of protection was obtained using the Mann Whitney test. (B) SslE_IHE3034 prevents the spread of the UPEC strain 536 into the kidneys and spleen in an ascending model of urinary tract infection. Thirty mice were immunized intranasally with 10 µg cholera toxin (CT) alone or with 100 µg of SslE_IHE3034 at a 10∶1 ratio of antigen:CT (day 1). After two boosts of 25 µg antigen (10∶1 ratio of antigen to CT) or CT alone (day 7 and 14), mice were transurethrally challenged with 10⁸ CFU of strain 536 at day 21. After 48 h, bladder, kidneys and spleen were harvested and homogenized. Bacteria in urine and in the tissue homogenates were enumerated by plating serial dilutions. Symbols represent CFU/g tissue or CFU/ml urine of individual mice, and bars indicate median values. P values were determined using the nonparametric Mann-Whitney significance test. (C) SslE_IHE3034 protects against the SEPEC strain IN1S in a sepsis mouse model. CD1 out-bred mice were immunized by subcutaneous injections at day 1, 21, and 35 with 20 µg of recombinant SslE_IHE3034 formulated with alum or alum alone. Immunized animals were challenged at day 49 with a sublethal dose of heterologous strain IN1S and survival was monitored for up to 4 days. The results are indicated as percentage of survival out of a total number of 40 mice. P values were determined using the nonparametric Mann-Whitney significance test.</p