Efficacy of a Pseudomonas aeruginosa Serogroup O9 Vaccine

Abstract

There are currently no approved vaccines against the opportunistic pathogen Pseudomonas aeruginosa. Among vaccine targets, the lipopolysaccharide (LPS) O antigen of P. aeruginosa is the most immunodominant protective candidate. There are twenty different O antigens composed of different repeat sugars structures conferring serogroup specificity, and ten are found most frequently in infection. Thus, one approach to combat infection by P. aeruginosa could be to generate immunity with a vaccine cocktail that includes all these serogroups. Serogroup O9 is one of the ten serogroups commonly found in infection, but it has never been developed into a vaccine, likely due, in part, to the acid labile nature of the O9 polysaccharide. Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa serogroup O11 LPS O antigen was effective in clearing and preventing mortality in mice following intranasal challenge with serogroup O11 P. aeruginosa. Consequently, we set out to develop a P. aeruginosa serogroup O9 vaccine using a similar approach. Here we show that Salmonella expressing serogroup O9 triggered an antibody-mediated immune response following intranasal administration to mice and that it conferred protection from P. aeruginosa serogroup O9 in a murine model of acute pneumonia.

Figure 1.. Expression of P. aeruginosa serogroup O9 in S. typhimurium.

LPS was extracted, applied to SDS-PAGE, and visualized with silver stain (A) or by (B) immunoblot analysis using Pseudomonas serotype O9-specific rabbit polyclonal antibody, followed by incubation with anti-rabbit secondary antibody conjugated to alkaline-phosphatase (Sigma). P. aeruginosa serogroup O9 strain PAO9 (Lane 1), S. typhimurium SL321 (lane 2), and from S. typhimurium SL3261 containing the plasmid expressing the P. aeruginosa serogroup O9 antigen (pLAFRO9) (Lane 3). Molecular weight (MW).

Figure 2.. Serum antibody response of BALB/c mice following intranasal immunization with S. typhimurium SL3261 expressing P. aeruginosa serogroup O9 and bacterial loads in organs of intranasally immunized BALB/c mice after intranasal lethal challenge with P. aeruginosa PAO9.

Mice were immunized intranasally on day 0 and day 14 with 10⁷ CFU/mouse of the vector control (SL3261 (pLAFR376)) or the vaccine (SL3261 (pLAFRO9)). Sera were collected 2- and 4-weeks post-vaccination, and the data were analyzed by the Mann-Whitney U test. (A) Serum IgM and (B) IgG response to P. aeruginosa PAO9 whole antigen. (C) Survival rates for naïve mice after intranasal challenge with various doses of P. aeruginosa PAO9 (n=4 mice/group). (D) Bacterial load in the nasal wash and lungs, (E) liver and spleens of intranasally immunized mice 24 hours post-challenge with 2 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on Pseudomonas isolation agar (PIA). Each point represents a single mouse.

Figure 2.. Serum antibody response of BALB/c mice following intranasal immunization with S. typhimurium SL3261 expressing P. aeruginosa serogroup O9 and bacterial loads in organs of intranasally immunized BALB/c mice after intranasal lethal challenge with P. aeruginosa PAO9.

Mice were immunized intranasally on day 0 and day 14 with 10⁷ CFU/mouse of the vector control (SL3261 (pLAFR376)) or the vaccine (SL3261 (pLAFRO9)). Sera were collected 2- and 4-weeks post-vaccination, and the data were analyzed by the Mann-Whitney U test. (A) Serum IgM and (B) IgG response to P. aeruginosa PAO9 whole antigen. (C) Survival rates for naïve mice after intranasal challenge with various doses of P. aeruginosa PAO9 (n=4 mice/group). (D) Bacterial load in the nasal wash and lungs, (E) liver and spleens of intranasally immunized mice 24 hours post-challenge with 2 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on Pseudomonas isolation agar (PIA). Each point represents a single mouse.

Figure 2.. Serum antibody response of BALB/c mice following intranasal immunization with S. typhimurium SL3261 expressing P. aeruginosa serogroup O9 and bacterial loads in organs of intranasally immunized BALB/c mice after intranasal lethal challenge with P. aeruginosa PAO9.

Mice were immunized intranasally on day 0 and day 14 with 10⁷ CFU/mouse of the vector control (SL3261 (pLAFR376)) or the vaccine (SL3261 (pLAFRO9)). Sera were collected 2- and 4-weeks post-vaccination, and the data were analyzed by the Mann-Whitney U test. (A) Serum IgM and (B) IgG response to P. aeruginosa PAO9 whole antigen. (C) Survival rates for naïve mice after intranasal challenge with various doses of P. aeruginosa PAO9 (n=4 mice/group). (D) Bacterial load in the nasal wash and lungs, (E) liver and spleens of intranasally immunized mice 24 hours post-challenge with 2 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on Pseudomonas isolation agar (PIA). Each point represents a single mouse.

Figure 3.. Immune response and bacterial burden in response to immunizing BALB/c mice with various doses of S. typhimurium SL3261 expressing P. aeruginosa serogroup O9.

Anti-P. aeruginosa serogroup O9 serum (A) IgM and (B) IgG antibody response after intranasal vaccination of BALB/c mice to various doses of vector or vaccine. (C) Bacterial load in the nasal wash and lungs of intranasally immunized mice 24 hours post-challenge with 4.5 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on PIA. Each point represents a single mouse. Data were analyzed by one-way ANOVA, * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars represent the mean and SEM.

Figure 3.. Immune response and bacterial burden in response to immunizing BALB/c mice with various doses of S. typhimurium SL3261 expressing P. aeruginosa serogroup O9.

Anti-P. aeruginosa serogroup O9 serum (A) IgM and (B) IgG antibody response after intranasal vaccination of BALB/c mice to various doses of vector or vaccine. (C) Bacterial load in the nasal wash and lungs of intranasally immunized mice 24 hours post-challenge with 4.5 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on PIA. Each point represents a single mouse. Data were analyzed by one-way ANOVA, * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars represent the mean and SEM.

Figure 4.. Immune response and bacterial burden in response to immunizing BALB/c mice with S. typhimurium SL3261 expressing P. aeruginosa serogroup O9 at 10⁹ CFU/mouse.

Anti-P. aeruginosa serogroup O9 serum (A) IgM, (B) IgG antibody, and (C) IgG isotype response after intranasal vaccination of BALB/c mice with 10⁹ CFU/mouse of SL3261 (pLAFR376) (vector) and SL3261 (pLAFRO9) (vaccine). (D) Bacterial load in the nasal wash and lungs, and (E) liver and spleens of intranasally immunized mice 24 hours post-challenge with 8.6 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on PIA. Each point represents a single mouse. Data were analyzed by one-way ANOVA. * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars represent the mean and SEM.

Figure 4.. Immune response and bacterial burden in response to immunizing BALB/c mice with S. typhimurium SL3261 expressing P. aeruginosa serogroup O9 at 10⁹ CFU/mouse.

Anti-P. aeruginosa serogroup O9 serum (A) IgM, (B) IgG antibody, and (C) IgG isotype response after intranasal vaccination of BALB/c mice with 10⁹ CFU/mouse of SL3261 (pLAFR376) (vector) and SL3261 (pLAFRO9) (vaccine). (D) Bacterial load in the nasal wash and lungs, and (E) liver and spleens of intranasally immunized mice 24 hours post-challenge with 8.6 × 10⁷ CFU of PAO9. All samples were plated for viable CFU on PIA. Each point represents a single mouse. Data were analyzed by one-way ANOVA. * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars represent the mean and SEM.

Figure 5.. Antibody response induced by intranasal vaccination mediates efficient opsonic killing of P. aeruginosa PAO9 in vitro.

Opsonophagocytic killing of P. aeruginosa PAO9 P1-lux using dilutions of pooled antisera collected from intranasally PBS-, SL3261 (pLAFR376) (vector)-, and SL3261 (pLAFRO9) (vaccine)-immunized BALB/c mice. Plates were read at 120 minutes following the co-incubation of the opsonophagocytosis assay components.

Figure 6.. Passive antisera transfer from immunized animals provides protection to acute P. aeruginosa pneumonia in naïve mice.

Bacterial loads in (A) nasal wash and lungs, (B) liver and spleens of BALB/c mice after passive intranasally transferred antisera immediately followed by challenge with 1 × 10⁷ CFU of PAO9. Mice were euthanized 24 hours post-infection. All samples were plated for viable CFU on PIA. Each point represents a single mouse. Data were analyzed by one-way ANOVA. *P<0.05, **P<0.01, ****P<0.001. Each point represents a single mouse. Error bars represent the mean and SEM.