Pamela S. Fink

Heterogeneous in vivo expression of clumping factor A and capsular polysaccharide by Staphylococcus aureus: Implications for vaccine design

There is a clear unmet medical need for a vaccine that would prevent infections from Staphylococcus aureus (S. aureus). To validate antigens as potential vaccine targets it has to be demonstrated that the antigens are expressed in vivo. Using murine bacteremia and wound infection models, we demonstrate that the expression of clumping factor A (ClfA) and capsular polysaccharide antigens are heterogeneous and dependent on the challenge strains examined and the in vivo microenvironment. We also demonstrate opsonophagocitic activity mediated by either antigen is not impeded by the presence of the other antigen. The data presented in this report support a multiantigen approach for the development of a prophylactic S. aureus vaccine to ensure broad coverage against this versatile pathogen.

On the role of subunit III in proton translocation in cytochromec oxidase

Mammalian mitochondrial cytochromec oxidase catalyzes the transfer of electrons from ferrocytochromec to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III,Mr 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.

Challenges for the evaluation of Staphylococcus aureus protein based vaccines: Monitoring antigenic diversity

Clumping factors A (ClfA) and B (ClfB) are Staphylococcus aureus virulence proteins that are displayed on the cell surface of the organism and have potential as vaccine antigens for the prevention of S. aureus disease. Here we evaluate the phylogeny of S. aureus in the context of antigenic variation of these two surface proteins. ClfA and ClfB gene sequences, along with epidemiological markers (MLST, spa and capsule genotype) were obtained for 224 S. aureus isolates including both historical strains and a collection representative of current MRSA isolates from the United States. Variation within ClfA and ClfB was consistent with the established population biology of S. aureus, namely, that S. aureus strains belong to a relatively small number of clonal lineages, with evolution proceeding mainly by mutation and with little to no recombination between clades. Thus most variation in ClfA and ClfB occurs between but not within lineages, and particular groups of ClfA and ClfB variants are closely linked. This has important implications for vaccine development and assessment as it suggests that a relatively small survey of strains will be representative of the total population variation, whereas for species that evolve mainly by recombination, such as Neisseria meningitidis, analysis of a much larger number of strains is needed to accomplish the same purpose. Our study also revealed evidence for the de-evolution of ClfB and therefore its reduced suitability as a target for vaccine development compared to ClfA.

NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.

Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD50 value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D.

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.

Murine response to DNA encoding herpes simplex virus type-1 glycoprotein D targeted to the liver

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3′‐end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa 3‐type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.