Robert Feldman

Identification of Major Outer Surface Proteins of Streptococcus agalactiae

ABSTRACT To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.

Characterization of Salmonella enterica Derivatives Harboring Defined aroC and Salmonella Pathogenicity Island 2 Type III Secretion System (ssaV) Mutations by Immunization of Healthy Volunteers

ABSTRACT The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.

Signature-tagged mutagenesis in the identification of virulence genes in pathogens

Signature-tagged mutagenesis is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cells. The application of this technique to a range of microbial pathogens has resulted in the identification of novel virulence determinants in each screen performed to date, so that cumulatively several hundred genes have been ascribed a role in virulence.

Vaccination against Group B streptococcus

Streptococcus agalactiae (Group B streptococcus) is an important cause of disease in infants, pregnant women, the elderly and in immunosuppressed adults. An effective vaccine is likely to prevent the majority of infant disease (both early and late onset), as well as Group B streptococcus-related stillbirths and prematurity, to avoid the current real and theoretical limitations of intrapartum antibiotic prophylaxis, and to be cost effective. The optimal time to administer such a vaccine would be in the third trimester of pregnancy. The main limitations on the production of a Group B streptococcus vaccine are not technical or scientific, but regulatory and legal. A number of candidates including capsular conjugate vaccines using traditional carrier proteins such as tetanus toxoid and mutant diphtheria toxin CRM197, as well as Group B streptococcus-specific proteins such as C5a peptidase, protein vaccines using one or more Group B streptococcus surface proteins and mucosal vaccines, have the potential to be successful vaccines. The capsular conjugate vaccines using tetanus and CRM197 carrier proteins are the most advanced candidates, having already completed Phase II human studies including use in the target population of pregnant women (tetanus toxoid conjugate), however, no definitive protein conjugates have yet been trialed. However, unless the regulatory environment is changed specifically to allow the development of a Group B streptococcus vaccine, it is unlikely that one will ever reach the market.

Novel protein vaccine candidates against Group B streptococcal infection identified using alkaline phosphatase fusions

Using an alkaline phosphatase-based genetic screening method, we identified a number of proteins that are potentially located on the outer surface of Group B streptococcus (Streptococcus agalactiae). In an enzyme-linked immunosorbent assay, antisera raised against two of the proteins, the streptococcal yutD homologue and a subunit of an ABC transporter, recognised clinically important serotypes of Group B streptococcus. In a neonatal rat model, purified IgG from the sera conferred significant levels of protection against a lethal challenge infection. The proteins identified show potential as protein subunit candidates for vaccines against Group B streptococcal disease in neonates.

The Group B Streptococcal Capsular Carbohydrate: Immune Response and Molecular Mimicry

The group B streptococcus (GBS) is the commonest cause of lethal bacterial infection in the newborn infant.1 GBS possesses a sialylated carbohydrate capsule which is known to be a major virulence factor.2 Mutant GBS which have a non-sialylated capsule are considerably less virulent, fix complement to a greater degree than the isogenic wild type and are more avidly opsonophagocytosed by polymorhonucleoctes.2 Antibodies to GBS capsule have been shown to be protective but the majority of the population do not possess these antibodies. For example, 90% of the population lack antibodies to the type III capsule of GBS (GBS III).3 Furthermore, 60% of seronegative individuals do not respond to experimental vaccination with purified GBS III.4 There has been considerable interest over the last few years in the development of a vaccine which would prevent neonatal infection with GBS. An appropriate approach would seem to be to conjugate the capsular carbohydrates to proteins as has been successfully achieved for Haemophilus influenzae serotype b. It is envisaged that any such vaccine would be given early in pregnancy and the vaccine induced IgG would subsequently be placentally transported to the fetus. In order to facilitate the development of these new vaccines, it is important to understand the basis of the poor immune response to the native capsular carbohydrate in humans and to document the naturally occurring human antibody response.

Toward a universal multistrain bacterial vaccine

Multigenome screening identifies novel surface-expressed protein vaccine candidates.

Combating Gram-positive pathogens: emerging techniques to identify relevant virulence targets.

Recent progress in microbial genome sequencing, along with functional genomics technologies based on gene expression, proteomics and genetics have facilitated the identification of significant numbers of Gram-positive virulence genes. These genes represent a novel and heterogeneous class of targets for antimicrobial drug development. This review will concentrate of the contribution of two functional genomics technologies, in vivo expression technology (IVET) based on gene expression and signature-tagged mutagenesis (STM), a genetics based technology to the identification of virulence genes in Gram-positive pathogens.

Development of a Group B Streptococcus (GBS) Cloning System

In order to identify genes coding for virulence factors of GBS, a reliable method of incorporating genomic DNA into a cloning system must be developed. Most molecular technologies depend on E. coli based vector-host systems, and we describe here that these methods are unsuitable for use in cloning GBS DNA.