PLoS One. 2013;8(2):e56996.

Adenovirus-based vaccine with epitopes incorporated in novel fiber sites to induce protective immunity against Pseudomonas aeruginosa.

Sharma A, Krause A, Xu Y, Sung B, Wu W, Worgall S.

Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, USA.

 

Abstract

Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy.

PMID: 23437292

 

SUPPLEMENT:

Pseudomonas aeruginosa is one of the leading nosocomial bacterial pathogens worldwide and can cause serious infections of the respiratory tract. A vaccine against P. aeruginosa would be useful as treatment is often challenged by antibiotic resistance of the organism. No efficient and marketable vaccine is yet available. P. aeruginosa outer membrane protein F (OprF) is one of the promising vaccine antigens. OprF is surface exposed, antigenically conserved in wild-type strains of P. aeruginosa and elicits cross-reactive, opsonizing and protective antibodies in various animal models and humans. Various immunogenic peptides have been identified in the outer loops of OprF, including the 14-mer peptide Epi8.

Adenovirus (Ad) vectors are attractive delivery vehicles for genetic vaccines due to their ability to act as immune system adjuvants and to rapidly evoke robust immune responses against the transgene product and viral capsid proteins. Ad vectors could also serve as a vaccine platform against P. aeruginosa. We have previously demonstrated that human Ad serotype 5 (Ad5) or non-human primate Ad serotype C7 (AdC7) expressing OprF induced robust protective immunity against pulmonary infections with P. aeruginosa in mice.

One of the limitations of Ad as vaccine carrier is that high prevalence of anti-Ad immunity elicited by the initial immunization usually prevents productive infection with subsequent immunizations, critical to achieve boosting of the anti-transgene immunity. One of the prime-boost strategies for Ad-based vaccines is to incorporate vaccine epitopes into the Ad capsid. Various Ad outer capsid proteins including hexon, fiber knob, penton base and protein IX have been targets for genetic modification. We have also shown that incorporation of influenza hemagglutinin (HA) epitopes into the HI loop of the Ad5 fiber knob elicits stronger humoral and cellular immunity compared to incorporation of the same epitope into the more abundant hexon protein. In this study, we explored different epitope-insertion sites within the Ad fiber knob [CD, DE, FG, HI loops or C-Terminal (CT)] of Ad fiber knob (Figure 1) to enhance the epitope-specific immune response of an Ad-based vaccine. We developed and compared various Ad vectors that display P. aeroginosa Epi8 on the CD [AdZ.F(CD)Epi8], DE [AdZ.F(DE)Epi8], FG [AdZ.F(FG)Epi8], HI [AdZ.F(HI)Epi8] or CT [AdZ.F(CT)Epi8] of Ad fiber knob.

We demonstrated that fiber-modified Ad vectors displaying Epi8 induced anti-P. aeruginosa humoral and cellular immunity that protected against P. aeruginosa infections. FG- and HI- fiber-modified Ad vectors elicited strongest humoral and cellular (both Th1 and Th2 type) protective immunity. In contrast to the Ad vector carrying OprF as transgene (AdOprF), repeated administration of FG- or HI-fiber-modified Ad vectors boosted the anti-OprF humoral immunity to strikingly high levels (Figure 2). Furthermore, this strategy was successful in inducing protective anti-P. aeruginosa immunity even in the presence of high levels of pre-existing anti-Ad immunity, thereby circumventing one of the problems in application of Ad vectors.

Overall, this study suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising strategy for P. aeruginosa vaccine that could also be valuable in the development of Ad-based vaccines against other pathogens.
Figure-1Fig 1. Epitope insertion sites in Ad5 fiber knob. The arrow indicates the location of P. aeruginosa OprF 14-mer epitope Epi8 inserted into loops CD (Green; Gly450/Thr451), DE (Brown; Asn464/Gly465), FG (Red; Gly509/Lys510), HI (Blue; Gly543/Asp544 ) and C terminus (CT) of the Ad5 fiber protein (PDB 1KNB).

 
Figure-2
Fig 2. BALB/c mice were immunized via the intramuscular route with the fiber-modified Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdZ.F(CT)Epi8, the hexon-modified AdZ.HxEpi8 or AdOprF (all 1010 pu/mouse). Mice were boosted with the same vectors after 2 and 5 wk, respectively, and anti-OprF antibodies in serum were analyzed at 2, 5 and 8 wks by ELISA. Data are shown as the mean ± SEM of 5 mice/group. Limit of detection is indicated by the dashed line. * denotes p<0.05

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann