Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections

Abstract

Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

Keywords: FliC; Francisella; OprF; PilA; Pseudomonas; flagellum; pilus; recombinant DNA; vaccine.

Figure 1.

Construction of the plasmids pABST, pBR, pOPRF, and pFLI. The F. tularensis LVS groE promoter was PCR-amplified and the amplicon generated was digested with KpnI and EcoRI, gel-purified, and ligated with pFNLTP8 that had been digested with these same enzymes to generate pABST. Primers were used to PCR-amplify pilA (P. aeruginosa 1244), oprF, or fliC (both of P. aerugionosa PA14). The amplicons generated were digested with EcoRI and NdeI, gel purified, and ligated with pABST that had been digested with these same enzymes to generate pBR, pOPRF, or pFLI respectively.

Figure 2.

Western blotting of recombinant F. tularensis LVS strains expressing proteins of P. aeruginosa. Prior to SDS-PAGE, bacterial cells were normalized to the same density. SDS-PAGE gels were electroblotted onto nitrocellulose paper. After the membrane was blocked, the nitrocellulose paper was probed with mouse monoclonal 5.44 specific for PilA (A), serum specific for OprF (B), or serum specific for FliC (C). Bands were visualized by using alkaline phosphatase-labeled secondary antibodies and naphthol as-mx phosphate with fast red tr salt zinc chloride.

Figure 3.

Construction of the plasmid, pGFLI. Primers were used to amplify fliC of P. aeruginosa PA14. This amplicon was digested with NdeI and BamHI, gel purified, and ligated with pGRP that had been digested with these same enzymes.

Figure 4.

Antibody levels from mice immunized with recombinant F. tularensis LVS expressing P. aeruginosa proteins. Antibody levels from mice immunized with F. tularensis LVS, F. tularensis LVS / pBR, F. tularensis LVS / pGFLI, F. tularensis LVS / pOPRF, or PBS was determined by ELISA. Data points represent antibody titers from individual mice. Serum was extracted from mice on day 42 post-immunization. ELISA plates were coated with P. aeruginosa PA14 (A) or 1244 (B). Lines and error bars represent the medians and quartiles respectively. Antibody levels from mice immunized with LVS / pGFLI produced significant levels of antibodies specific for P. aerugionsa (P < 0.05) compared to mice treated with PBS.