Enhanced immunogenicity in the murine airway mucosa with an attenuated Salmonella live vaccine expressing OprF-OprI from Pseudomonas aeruginosa

Abstract

We constructed an oral live vaccine based on the attenuated aroA mutant Salmonella enterica serovar Typhimurium strain SL3261 expressing outer membrane proteins F and I (OprF-OprI) from Pseudomonas aeruginosa and investigated it in a mouse model. Strains with in vivo inducible protein expression with the PpacC promoter showed good infection rates and immunogenicity but failed to engender detectable antibodies in the lung. However, a systemic booster vaccination following an oral primary immunization yielded high immunoglobulin A (IgA) and IgG antibody levels in both upper and lower airways superior to conventional systemic or mucosal booster vaccination alone. In addition, the proportion of IgG1 and IgG2a antibodies suggested that the systemic booster does not alter the more TH1-like type of response induced by the oral Salmonella primary vaccination. We conclude that an oral primary systemic booster vaccination strategy with an appropriate mucosal vector may be advantageous in diseases with the risk of P. aeruginosa airway infection, such as cystic fibrosis.

FIG. 1.

SDS-PAGE of late log-phase cultures of SL3261(pDB3), SL3261(pDB4), SL3261(pMW151), SL3261(pMW209), SL3261(pMW210), and the SL3261 parent strain (upper panel). Constructs with in vivo inducible protein expression were cultured in standard LB medium and under magnesium-free conditions (left and right lanes, respectively), demonstrating the induction of the desired OprF-OprI antigen as shown by immunoblot (lower panel). For clarity of the figure, vaccine strains are indicated with the name of their plasmid constructs only. Arrows indicate the apparent molecular weight of OprF-OprI.

FIG. 2.

Infection by SL3261 and OprF-OprI vaccine constructs of PP and MLN on day 7 after gastric inoculation of 10⁹ CFU. Bars represent mean CFU ± standard error of the means of 5 (pMW209 and pMW210) to 17 animals. In the spleen, Salmonella was found only occasionally at numbers <100 CFU. In vivo induction of OprF-OprI protein expression resulted in a significantly higher infection rate of SL3261(pMW209) in MLN. The infection level of strain SL3261(pMW210) was indifferent to parent strain SL3261 and higher than all other vaccine strains. Asterisks indicate significantly different mean values as obtained in two-sided Student's t tests. *, P < 0.05; **, P < 0.001. For clarity of the figure, vaccine strains are indicated with the name of their plasmid constructs only.

FIG. 3.

Formation of OprF-OrpI-specific antibodies of the IgG and IgA isotypes in serum, intestine, lung (BALF), and nose (nasal wash) 4 weeks after vaccination with 10⁹ CFU of parent strain SL3261, of a strain with constitutive OprF-OrpI expression [SL3261(pDB3)], and of strains with in vivo inducible OprF-OrpI expression [SL3261(pMW209) and SL3261(pMW210)]. Dots represent individual titer values, bars indicate mean values of ELISA units (EU), error bars indicate the standard errors of the means. Groups comprised 11 to 17 animals from two independent sets of experiments. Asterisks indicate significantly different mean values with a P value of <0.05 (*) and a P value of <0.001 (**) as obtained by a Mann-Whitney test (for comparison of serum titers) or by a two-sided Student's t tests (for comparison of ELISA units). For clarity of the figure, vaccine strains are indicated with the name of their plasmid constructs only.

FIG. 4.

Formation of OprF-OrpI-specific antibodies of IgG and IgA isotypes in serum, intestine, lung (BALF), and nose (nasal wash) after vaccination with 10⁹ CFU of parent strain SL3261, with strain SL3261(pMW209) with in vivo inducible protein expression, with systemic vaccination twice (intramuscularly [i.m.]), and with a combined mucosal primary systemic booster vaccination [SL3261(pMW209)+ i.m.]. Dots represent individual titer values, bars indicate mean values of ELISA units (EU), and error bars indicate the standard errors of the means. Groups comprised 12 to 17 animals from two independent sets of experiments. Significantly different mean values are indicated as follows: *, comparison of SL3261(pMW209) versus i.m.; +, comparison of SL3261(pMW209)+i.m. versus i.m.; and #, comparison of SL3261(pMW209) versus SL3261(pMW209)+i.m. A single sign indicates a P value of <0.05, and a double sign indicates a P value of <0.001 as obtained by a Mann-Whitney test (for comparison of serum titers) or by a two-sided Student's t tests (for comparison of ELISA units). For clarity of the figure, vaccine strains are indicated with the name of their plasmid constructs only.

FIG. 5.

IgG1/IgG2a ratio of OprF-OrpI-specific antibodies in serum, intestine, lung (BALF) and nose (nasal wash). The ratio is considered a surrogate marker for the TH1-TH2 balance, with IgG1 in-dicating a more TH2-type response and IgG2a a more TH1-type response. Mice were vaccinated either twice systemically with an intramuscular application of OprF-I (i.m.), with a single oral application of attenuated recombinant Salmonella with in vivo inducible expression of OprF-OprI [SL3261(pMW209)], or with a combined mucosal primary systemic booster regimen (pMW209+i.m.). Dots represent individual IgG1/IgG2a ratios, and lines indicate the median values of each vaccination group. Groups were compared by one-way analysis of variance for multiple analyses with the Bonferroni and Dunnett post hoc correction for groups with and without equality of variances, respectively. Asterisks indicate a P value of <0.05 (*) and <0.001 (**). In BALF of mice vaccinated with SL3251(pMW209) alone, assessment of the IgG1/IgG2a ratio was not applicable (n.a.) due to the lack of relevant antibody levels. For clarity of the figure, vaccine strains are indicated with the name of their plasmid constructs only.