Construction and characterization of a Pseudomonas aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine

Abstract

Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P. aeruginosa infection. When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies. To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit. Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity. Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P. aeruginosa. Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies. Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate. However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate. Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P. aeruginosa in vitro. These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer.

FIG. 1.

Sephacryl S-1000 gel filtration profile of MEP conjugated to KLH. Fractions were assayed for protein by the Bradford assay (595 nm) and for uronic acid by the meta-hydroxybiphenyl assay (525 nm).

FIG. 2.

Sandwich ELISA with rabbit anti-KLH IgG for antigen capture and mouse anti-MEP serum for detection of the binding of the polysaccharide component of the MEP-KLH conjugate. A mixture of MEP and KLH did not bind anti-MEP IgG. In contrast, wells incubated with the MEP-KLH conjugate reacted with the anti-MEP antibody.

FIG. 3.

Titers of anti-MEP IgG in C3H-HeN mice over a period of 84 days after the beginning of immunization. Lines represent the geometric mean of ELISA titers, and error bars indicate the range. Arrows indicate the time points of immunization. For numerical values of titers for days 0 to 21, see Table 1. Titers of anti-MEP antibodies peaked at day 35 and declined gradually until day 63 (P < 0.001 [as determined by repeated measures ANOVA and Bonferroni test for pairwise comparisons of the time points]). A booster dose given on day 63 elicited a significant rise in titers by day 84 (P < 0.05).

FIG. 4.

Opsonophagocytic killing of P. aeruginosa strain 2192 (vaccine strain) by serum from C3H-HeN mice after immunization with either MEP-KLH conjugate or native MEP mixed with KLH and by serum from a rabbit hyperimmunized with MEP-KLH. Bars represent the mean percent killing of four replicates relative to the respective preimmune serum, and error bars represent the standard error of the mean. At serum dilutions ranging from 1:5 to 1:80, sera of mice immunized with MEP-KLH generated significantly higher killing activity than did the sera of mice immunized with the nonconjugated polysaccharide (✽, P < 0.001; ○, P < 0.01 [as determined by ANOVA with the Bonferroni test for pairwise comparison]). At a serum dilution of 1:80, mouse serum maintained a significantly higher opsonic activity than rabbit serum (#, P < 0.01).

FIG. 5.

Cross-reactive binding and opsonic killing activity of rabbit and mouse antisera raised to MEP-KLH conjugate vaccine. (A) IgG titers against the vaccine strain (strain 2192) and multiple mucoid, LPS rough P. aeruginosa isolates from CF patients. (B) Opsonic activity of a serum dilution of 1:10 of the rabbit and mouse sera after immunization with MEP-KLH conjugate against the same target strains. No significant difference was found in binding to MEP isolated from the vaccine strain or heterologous strains of mouse and rabbit antisera. Also, the opsonic activity of the antisera against heterologous mucoid strains of P. aeruginosa did not differ significantly from their activity against strain 2192, except for strain 258, for which the killing activity was significantly lower than that against the vaccine strain (P < 0.001 [as determined by ANOVA with the Dunnet test for comparison with strain 2192]). The sera tested were obtained from a hyperimmunized rabbit and from five C3H-HeN mice 35 days after the first immunization. The mouse sera were pooled for analysis. MEP from strain 258 was not tested by ELISA for cross-reactive IgG, since this strain reverts to the nonmucoid phenotype when grown in the Mian’s medium used to produce MEP. The bars represent the mean of titers of two replicates determined by linear regression (A) and the means of four replicates (B); error bars indicate the standard error of the mean in panel B.

FIG. 6.

(A) MEP-specific IgG in mice preimmunized with 50 μg of native MEP given i.p., 50 μg of seaweed alginate given i.p., or seaweed alginate given in the drinking water (1 mg/ml) compared to that in naive mice. Weekly starting at 190 days after preimmunization, the mice were immunized with three 1-μg doses of MEP-KLH, and serum was collected on day 225. Mice with preexisting antibodies to MEP produced significantly lower titers than those in naive mice (○, P < 0.01; #, P < 0.05 [as determined by ANOVA with the Dunnet test for comparison to naive mice]). Bars represent the geometric mean of individual titers of five mice, and error bars represent the standard error of the mean. (B) Opsonic activity of sera pooled from mice with preexisting antibodies to MEP after vaccination with three 1-μg doses of MEP-KLH conjugate or a mixture of MEP and KLH. At a serum dilution of 1:5, the opsonic activity in sera from mice immunized with the conjugate vaccine was significantly higher than in sera from mice that had received nonconjugated polysaccharide, regardless of the presence of preexisting antibodies (○, P < 0.001 [as determined by ANOVA and Bonferroni test for pairwise comparison]). There was no significant difference in the opsonic activity at this serum dilution among any of the groups given the MEP-KLH conjugate regardless of the preimmunization regimen. At a serum dilution of 1:10, the sera of mice initially immunized i.p. with MEP showed no difference in opsonic killing activity regardless of whether the animals were immunized with MEP-KLH conjugate or the mixture. The sera of mice preimmunized with seaweed alginate and boosted with the MEP-KLH conjugate vaccine had a significantly higher killing activity at a 1:10 serum dilution than did sera from mice immunized with the mixture of MEP and KLH (○, P < 0.001). The opsonic killing activity of sera of naive mice immunized with the MEP-KLH conjugate was significantly higher than that of sera from animals vaccinated with MEP-KLH conjugate that were preimmunized with MEP (P < 0.001 [as determined by ANOVA by using the Dunnet test for comparison with naive mice]). Preimmunization with alginic acid either i.p. or perorally did not reduce the opsonic killing activity of sera compared to that in sera from nonpreimmunized mice given the conjugate vaccine. Bars represent mean percent killing of four replicates compared to preimmune serum, and the error bars represent the standard error of the mean.

FIG. 7.

Effect of acetate substituents on the binding of conjugate-induced antisera to MEP. (A) Binding of rabbit IgG after immunization with the MEP-KLH conjugate vaccine to MEP and de-O-acetylated MEP as the coating antigen in an ELISA. (B) Competitive inhibition ELISA with native MEP and de-O-acetylated MEP to inhibit binding of conjugate-induced antibody to native MEP as the solid-phase antigen. The percent inhibition was determined by comparison of the absorbance at 405 nm in the presence or absence of inhibitor. Native and de-O-acetylated MEP used for coating and inhibition were derived from P. aeruginosa strain 2192.

FIG. 8.

Inhibition of opsonic killing of rabbit serum raised against MEP-KLH by various inhibitors. All inhibitors were tested in concentrations up to 100 μg/ml, without any increase in inhibition at concentrations higher than 1.25 μg/ml (data not shown). (A) Comparative inhibition with native or deacetylated MEP. In concentrations of ≥0.625 μg/ml, native MEP inhibited opsonic killing activity significantly better than did de-O-acetylated MEP (✽, P < 0.001). (B) Comparative inhibition with native, non-O-acetylated, or chemically O-acetylated seaweed alginate. O-acetylated seaweed alginate inhibited opsonophagocytic killing activity significantly better than did the native polysaccharide (✽, P < 0.001 for inhibitor concentrations ≥0.125 μg/ml). The target strain in the opsonophagocytic killing assay was 2192. Native and de-O-acetylated MEP used for inhibition were also derived from strain 2192. Bars represent the mean of four replicates, and the error bars represent the standard error of the mean. Inhibition of opsonophagocytic killing was compared by ANOVA and Bonferroni test for pairwise comparison of O-acetylated and non-O-acetylated polysaccharide.