Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

Figure 1

Schematic of the P. aeruginosa outer membrane protein OprF. The amino acid sequences of OprF epitopes Epi1, Epi6, and Epi8, which were incorporated into the Ad hexon protein of the Ad vectors used in the present study, are shown in bold (figure modified with permission from ref. 21).

Figure 2

Humoral response to P. aeruginosa after immunization with Ad vectors with OprF epitopes in the hexon protein of the Ad capsid. C57BL/6 mice were either not immunized (Naive) or immunized via the subcutaneous route with AdZ.Epi1, AdZ.Epi6, AdZ.Epi8, or AdZ at a dose of 1 × 10⁹ pu/animal. Total IgG antibodies against P. aeruginosa were determined by ELISA at 0, 2, and 4 weeks after administration, using the laboratory P. aeruginosa strain PAO1 as the antigen. Data are shown as a single measurement of pooled sera obtained from 5 individual mice per group.

Figure 3

Anti–P. aeruginosa and anti-OprF after immunization with AdZ.Epi8. Sera from C57BL/6 mice immunized subcutaneously with AdZ.Epi8 (lanes 4–6) or AdNull (lanes 1–3) at a dose of 1 × 10¹⁰ pu/mouse were analyzed by Western blot for the presence of antibodies against whole PAO1 (lanes 1 and 4), the outer membrane of PAO1 (OM; lanes 2 and 5), or purified OprF (OprF; lanes 3 and 6). The bands representing OprF in the PAO1 preparation and recombinant OprF (rOprF) are shown.

Figure 4

MHC haplotype influences the humoral response to OprF after immunization with AdZ.Epi8. (A and B) C57BL/6 (H2-Ab), BALB/c (H2-Ed), or CBA (H2-Ek) mice were immunized via the subcutaneous route with AdZ.Epi8 or AdZ at a dose of 1 × 10¹⁰ pu/animal. (A) Total anti-OprF IgG titers determined at 2 weeks by ELISA. (B) IgG subclasses of antibodies against Epi8. Data are shown as single measurement of pooled sera obtained from 5 individual mice per group. The dashed lines indicate the limit of detection.

Figure 5

CD4 and CD8 T cell IFN-γ response after immunization with AdZ.Epi8. (A and B) C57BL/6 mice were immunized with AdZ.Epi8 or AdZ at a dose of 1 × 10¹⁰ pu/animal via the subcutaneous route. Ten days after immunization, CD4 and CD8 cells were isolated from spleens and the IFN-γ response after in vitro stimulation with Epi8 or β-galactosidase was determined by intracellular cytokine staining. Shown are data for CD4 (A) and CD8 (B) cells after immunization with nothing (Control), AdZ, or AdZ.Epi8 and in vitro stimulation with DCs alone (DC), DCs plus β-galactosidase (DC + β-gal) or DCs plus Epi8 (DC + Epi8). Data represent the mean of pooled cells from 5 individual mice per group from 3 separate experiments.

Figure 6

Ability of immunization with AdZ.Epi8 to protect against pulmonary challenge with P. aeruginosa. (A–D) C57BL/6 mice were immunized via the subcutaneous route with AdZ.Epi8 at a dose of 1 × 10¹⁰ pu/animal. Immunization with AdZ or heat-inactivated PAO1 (1 × 10⁸ CFU/mouse) served as the control. Five weeks after immunization, the mice were challenged with a lethal intratracheal dose of agar-encapsulated P. aeruginosa and survival was monitored for 2 weeks (A, C, and D) or lungs were assessed by histology (B). (A) Challenge with laboratory strain PAO1. (B) Lung morphology 48 hours after challenge with agar-encapsulated PAO1 compared with lung morphology of an uninfected mouse. Shown are hematoxylin and eosin–stained sections of formalin-fixed lungs. Scale bar: 0.5 μm. (C) Challenge with the clinical isolate MI6. (D) Challenge with the clinical isolate PA514.

Figure 7

Repeated administration of AdZ.Epi8 results in boosting of the anti-OprF immune response. (A and B) C57BL/6 mice were immunized via the subcutaneous route with AdZ.Epi8 (Epi8), AdNull (Null) or nothing (–) at a dose of 1 × 10¹⁰ pu/animal followed by a second immunization 2 weeks later. Total IgG antibodies against OprF (Anti-OprF; A) or β-galactosidase (Anti–β-gal; B) were assessed by ELISA at 4 weeks after the first immunization. Data are shown as mean ± SEM of 5 mice per group of 1 representative of 3 independent experiments.

Figure 8

Anti-OprF humoral response after administration of Epi8 peptide compared with AdZ.Epi8. C57BL/6 mice were immunized via the subcutaneous route with AdZ.Epi8 or AdZ (1 × 10¹⁰ pu/animal) or with an equimolar amount of Epi8 peptide (12 pmol = 3.6 ng/mouse) in complete Freund’s adjuvant, followed by a second immunization 2 weeks later. Total IgG antibodies against OprF were assessed by ELISA at 4 weeks after the first immunization. Data are shown as mean ± SEM of 4 mice per group.

Figure 9

Repeated administration of AdZ.Epi8 results in boosting of the Epi8-specific cellular immune response. (A and B) C57BL/6 mice were immunized via the subcutaneous route with AdZ.Epi8 or AdZ at a dose of 1 × 10¹⁰ pu/animal. Some mice were not immunized further (×1); others received a second immunization 2 weeks later (×2). Ten days after the second immunization, CD4 and CD8 cells were isolated from spleens and the IL-4 and IFN-γ responses after in vitro stimulation with Epi8 or β-galactosidase were assessed by ELISPOT assay. Shown are data for CD4 IL-4 (A), CD4 IFN-γ (B), and CD8 IFN-γ (C) after in vitro stimulation with DCs plus β-galactosidase or DCs plus Epi8. For each condition, data were normalized to the total number of polyclonal cells stimulated with PMA/ionomycin.