Novel Chlamydia pneumoniae vaccine candidates confirmed by Th1-enhanced genetic immunization

Abstract

Identification of highly immunogenic antigens is critical for the construction of an efficacious subunit vaccine against Chlamydia pneumoniae infections. A previous project used a genome-wide screen to identify 12 protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al. [14]). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the Escherichia coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these 12 genes, in parallel with six genes identified by other investigators. Vaccine candidate genes cutE and Cpn0420 conferred significant protection by all criteria evaluated (prevention of C. pneumoniae-induced death, reduction of lung disease, elimination of C. pneumoniae). Gene oppA_2 was protective by disease reduction and C. pneumoniae elimination. Four other genes were protective by a single criterion. None of the six genes reported elsewhere protected by reduction of lung disease or elimination of C. pneumoniae, but three protected by increasing survival.

Fig. 1

Use of E. coli heat-labile enterotoxin (LT) as genetic adjuvant boosts total and Th1 immune response. Groups of 5 mice were immunized with C. abortus dnaX2 in presence (white bars) or absence (shaded bars) of the LT adjuvant. Mice (n = 5/group) were vaccinated 3 times in 3-week intervals, and antibody levels at 4 weeks after the last vaccination were evaluated by ELISA using recombinant DnaX2 protein as antigen, goat-anti mouse horseradish peroxidase conjugates, and TMB substrate. Data are shown as means of absorbance of 1:2,000 diluted sera or the ratio of the IgG2a/IgG1 OD values ± 95% confidence intervals. Asterisks indicate significant differences between adjacent groups (*, P < 0.05; ***, P < 0.001; Student’s t-test).

Fig. 2

Disease protection efficacy of final vaccine candidates. After testing of 46 individual candidates in Round 2, 12 of these genes (Table 1) were cloned as full-length genes (except ide_ab and Cpn0095_a) into genetic immunization plasmid CMVi-UB and used for vaccination together with genetic vaccine adjuvant LT A+B in Round 3. Cpn0095_a was not included in the round-3 high-dose challenge. Vaccinated mice (n = 10/group) were intranasally challenged with an LD₅₀ of 5×10⁸ C. pneumoniae elementary bodies. Surviving mice (Table 2) were sacrificed on day 10 p.i., lungs were weighed, and the lung weight increase over the average lung weight of unchallenged age-matched female A/J mice was calculated. The lung weight increase is a reliable measure of disease intensity, and high increases reflect severe disease. A. Lung weight increase data were linearly transformed into protection scores by setting the increase for unprotected mock-vaccinated mice at 0 and for optimally protected live-vaccinated mice at 1. B. For vaccination Rounds 2 and 3 of the final vaccine candidate genes, protection scores were calculated based on the logarithm of the total C. pneumoniae lung load on day 10. Protection scores from Round 2 with the use of LEE constructs and from Round 3 with plasmid-cloned genes (full-length except for partial genes ide_ab and Cpn0095_a) were pooled and analyzed by one-way ANOVA. Data are shown as means ± 95% confidence intervals (mock-vaccinated, live vaccine groups n = 60; genetic vaccine groups n = 13–20). Asterisks indicate protected groups that are significantly different from mock-vaccinated mice (*, P < 0.05; **, P < 0.01; ***, P < 0.001; Dunnett’s test).

Fig. 3

Vaccine candidates reported as protective do not confer protection against C. pneumoniae disease. Genes previously reported as protective [8,9,10,11,12,17] against C. pneumoniae or C. abortus challenge were tested for direct comparison to the vaccine candidates found in this study. A. Lung weight increase data were converted into protection scores as described and analyzed by one-way ANOVA. B. Reported protective genes were compared to the vaccine candidates found in this study. The logarithm of the total C. pneumoniae lung load was converted into protection scores as described. One-way ANOVA results are shown as means ± 95% confidence intervals (**, P < 0.01; Dunnett’s test).