A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.

Discovery of a novel and potent class of FabI-directed antibacterial agents.

Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.

Site-directed mutagenesis of streptococcal plasmin receptor protein (Plr) identifies the C-terminal Lys334 as essential for plasmin binding, but mutation of the plr gene does not reduce plasmin binding to group A streptococci.

Plasmin(ogen) binding is a common property of many pathogenic bacteria including group A streptococci. Previous analysis of a putative plasmin receptor protein, Plr, from the group A streptococcal strain 64/14 revealed that it is a glyceraldehyde-3-phosphate dehydrogenase and that the plr gene is present on the chromosome as a single copy. This study continues the functional characterization of Plr as a plasmin receptor. Attempts at insertional inactivation of the plr gene suggested that this single-copy gene may be essential for cell viability. Therefore, an alternative strategy was applied to manipulate this gene in vivo. Site-directed mutagenesis of Plr revealed that a C-terminal lysyl residue is required for wild-type levels of plasmin binding. Mutated Plr proteins expressed in Escherichia coli demonstrated reduced plasmin-binding activity yet retained glyceraldehyde-3-phosphate dehydrogenase activity. A novel integration vector was constructed to precisely replace the wild-type copy of the plr gene with these mutations. Isogenic streptococcal strains expressing altered Plr bound equivalent amounts of plasmin as wild-type streptococci. These data suggest that Plr does not function as a unique plasmin receptor, and underscore the need to identify other plasmin-binding structures on group A streptococci and to assess the importance of the plasminogen system in pathogenesis by inactivation of plasminogen activators and the use of appropriate animal models.