Safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers

Abstract

A hybrid protein [Met-Ala-(His)6OprF190-342-OprI21-83] consisting of the mature outer membrane protein I (OprI) and amino acids 190 to 342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography. After safety and pyrogenicity evaluations in animals, four groups of eight adult human volunteers were vaccinated intramuscularly three times at 4-week intervals and revaccinated 6 months later with either 500, 100, 50, or 20 microg of OprF-OprI adsorbed onto A1(OH)3. All vaccinations were well tolerated. After the first vaccination, a significant rise of antibody titers against P. aeruginosa OprF and OprI was measured in volunteers receiving the 100- or the 500-microg dose. After the second vaccination, significant antibody titers were measured for all groups. Elevated antibody titers against OprF and OprI could still be measured 6 months after the third vaccination. The capacity of the elicited antibodies to promote complement binding and opsonization could be demonstrated by a C1q-binding assay and by the in vitro opsonophagocytic uptake of P. aeruginosa bacteria. These data support the continued development of an OprF-OprI vaccine for use in humans.

FIG. 1

Lanes 2 to 7: SDS-polyacrylamide gel electrophoresis under reducing conditions of 8 μl (8 μg) of OprF-OprI, purified from E. coli. Lanes: 1, 8 μl marker (Novex; blue); 6, silver staining; 7, Coomassie blue staining; 2, Western blotting with antibody 11179 against E. coli 1:500; 3, Western blotting with antibody 944/5 against OprF epitope D5 1:7,500; 4, Western blotting with antibody 948-12 against OprF epitope D1 1:10,000; 5, Western blotting with antibody 2-A1 against Opr-I 1:10,000.

FIG. 2

Serum anti-OprF-OprI IgG ELISA titers measured in sera from volunteers. Groups of volunteers were vaccinated intramuscularly three times with 20 μg (a), 50 μg (b), 100 μg (c), or 500 μg (d) of OprF-OprI at 4-week intervals and 6 months after the third vaccination. Blood was taken before each vaccination (day 0) and 2 weeks after each vaccination. Key: , day 0; , day 14; , day 42; , day 70; , day 240; , day 254.

FIG. 3

Western immunoblot analysis of antibodies against P. aeruginosa serogroup 1. Cell extracts were prepared from P. aeruginosa ATCC 33348. Blots were developed with alkaline phosphatase-conjugated monoclonal antihuman antibodies (Sigma A-2064) or with alkaline-phosphatase-conjugated rabbit anti-mouse antibodies (Zymed no. 61-6522). Lanes: 1, marker (Sigma wide range, B-2787); 2 and 3, preimmune serum and serum obtained from volunteer 4 in group 2 after the third vaccination; 4 and 5, preimmune serum and serum obtained from volunteer 4 in group 3 after the third vaccination; 6 and 7, preimmune serum and serum obtained from volunteer 3 in group 4 after the third vaccination; 8 and 9, preimmune serum and serum obtained from volunteer 5 in group 4 after the third vaccination; 10 and 11, mouse monoclonal antibodies against OprF (epitope D5); 12, mouse monoclonal antibodies against OprI (2-A1).

FIG. 4

IgG1-specific antibody response in sera of volunteers as shown in Fig. 2 measured by ELISA with 1:100 diluted mouse human IgG1 as described in Materials and Methods. Key: □, day 0; ▨, day 14; , day 42; , day 70; , day 240; , day 254.

FIG. 5

The binding of C1q, a subcomponent of the complement classical pathway component C1, was measured by ELISA as reported previously (11, 54). The sera were tested before ■ and after  the third vaccination. Plates were coated with OprF-OprI and incubated with 50 μl of the respective 1:4-diluted serum and 50 μl of complement source. Binding was measured with peroxidase-linked anti-C1q antibodies, and ortho-phenylen-diamine was used as the substrate.