Simian adenoviruses as vaccine vectors

Abstract

Replication incompetent human adenovirus serotype 5 (HAdV-C5) has been extensively used as a delivery vehicle for gene therapy proteins and infectious disease antigens. These vectors infect replicating and nonreplicating cells, have a broad tissue tropism, elicit high immune responses and are easily purified to high titers. However, the utility of HAdV-C5 vectors as potential vaccines is limited due to pre-existing immunity within the human population that significantly reduces the immunogenicity of HAdV-C5 vaccines. In recent years, adenovirus vaccine development has focused on simian-derived adenoviral vectors, which have the desirable vector characteristics of HAdV-C5 but with negligible seroprevalence in the human population. Here, we discuss recent advances in simian adenovirus vaccine vector development and evaluate current research specifically focusing on clinical trial data.

Keywords: BAC recombineering; chimpanzee adenoviruses; clinical trials; simian adenoviruses; vector vaccine.

Figure 1.. Generation of a molecular clone of ChAd68.

(A) Insertion of ChAd68 genomic DNA into the pBAC ‘rescue vector’ by gap repair. The E1 LF1 and 2 and terminal right hand side region (RF) are amplified from ChAd68 genomic DNA and cloned into pBACe3.6 to produce a BAC adenovirus rescue clone. Recombination occurs between LF1 and LF2 of the isolated ChAd68 genome and the BAC rescue clone and the RF of ChAd68 genome and the BAC rescue clone. The resulting product is a BAC containing an E1 deleted ChAd68 genome. (B) Excision of the E3 region of ChAd68 by recombineering. First, the galactokinase gene (GalK) is amplified from pGalK using primers containing sequences homologous to the flanking region of E3 (E3LF and E3RF). The E3 region is replaced by the GalK gene using λ red recombination. The GalK gene is subsequently replaced by a PCR product consisting of E3LF and E3RF, again using λ red recombination. The resulting product is a BAC containing an E1E3-deleted ChAd68 genome. (C) Insertion of an antigen cassette at the E1 locus. First, the GalK gene is amplified from pGalK using primers containing sequences homologous to the flanking region of E1 (LF1 and LF2). The E1 region is replaced by the GalK gene using λ red recombination. The GalK gene is subsequently replaced by a PCR product consisting of LF1-antigen-expression cassette-LF2 using λ red recombination. The resulting product is a BAC containing an E1E3-deleted ChAd68 genome with an antigen-expression cassette at the E1 locus. BAC: Bacterial artificial chromosome; ChAd68: Chimpanzee adenovirus 68; LF: Left flanking region; RF: Right flanking region.

Figure 2.. Insertion of an antigen-expression cassette into adenovirus vector using att recombination sites.

A universal cassette expressing a bacteria antibiotic resistance gene and ccdB suicide gene flanked by the specific recombination sequences, attR1 and attR2 is located at the E1 locus and/or the E3 locus of the BAC-adenovirus genome clone. Shuttle plasmids containing an antigen-expression cassette flanked by specific recombination sites paired with those present in the adenovirus genome (attL1/L2) allow site-specific recombination in the presence of an enzyme mixture containing bacteriophage λ integrase, integration host factor and excisionase. BAC: Bacterial artificial chromosome; ChAdOx2: E1/E3 deleted adenovirus vector derived from ChAd68 with a modified E4 region; CMV: Cytomegalovirus.

Figure 3.. Growth of ChAdOx2 compared with ChAd68.

E1-complementing HEK293 cells were infected with one multiple of infection of viral vectors ChAdOx2 (black line) or ChAd68 (gray line) each expressing GFP from the E1 locus. Samples were taken at 48 and 96 h postinfection. Virus yield was determined by titration in triplicate on HEK293 cells and GFP-positive cells counted 48-h postinfection. Results are expressed as log₁₀ fluorescent units per milliliter from two separate experiments with triplicate titrations for each sample. Student's unpaired t-test was used to statistically analyze the results and the mean with standard deviation is depicted. FU: Fluorescent unit; GFP: Green fluorescent protein.

Figure 4.. Immunogenicity of ChAdOx1-eGFP compared with ChAdOx2-eGFP.

Female BALB/c mice (four per group) were injected intramuscularly with 10⁸ infectious units of vector and spleens harvested 2 weeks later to measure the response to GFP by IFN-γ ELISPOT. Results are expressed as SFUs per million splenocytes. Mann–Whitney test was used to statistically analyze the results and the mean with SEM is depicted. GFP: Green fluorescent protein; SFU: Spot-forming unit.