Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3.

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.

Progress on development of the live BCG recombinant vaccine vehicle for combined vaccine delivery.

BCG, the current vaccine for tuberculosis, has been administered to approximately three billion people. This live vaccine has a low incidence of serious side effects and can be given at birth. Within the past six years, systems for the manipulation and expression of foreign genes in mycobacteria have been developed, allowing the evaluation of rBCG as a vaccine delivery vehicle for heterologous antigens. Recent studies from our group have shown that rBCG expressing outer surface protein A of Borrelia burgdorferi can completely protect mice from an intradermal challenge with this organism. Immune responses protective against Streptococcus pneumoniae challenge have also been achieved by immunization of mice with rBCG expressing PspA. The simplest means of administering multiple vaccine antigens in a rBCG vehicle would be to coexpress these simultaneously in the same BCG recombinant. Currently two general classes of vectors exist for the expression of foreign proteins in BCG: shuttle plasmid vectors, which replicate extrachromosomally in mycobacteria, and shuttle "phasmid" vectors, which integrate as a single copy into the mycobacterial chromosome by means of vector-encoded integration functions of the lysogenic mycobacteriophage L5. The genetic capacity of the multicopy plasmid vectors may be 20 kb or more, while the potential exists for stable integration of much larger amounts of DNA into the mycobacterial genome (L5 itself is 52 kb). Additionally, these two expression systems can have the compatibility to coexist in a single BCG cell. Otitis media is caused by infections of the middle ear chiefly with either S. pneumoniae or H. influenzae. Thus, an effective vaccine would necessarily include antigens from both these pathogens. Our initial attempt at construction of a BCG multivaccine vehicle was to express proteins from each of these pathogens from the same multicopy plasmid. We have recently succeeded in coexpressing the S. pneumoniae PspA and H. influenzae PAL proteins in BCG. Future work will address how the biochemical characterization of and immune responses to the recombinant antigens of the "bivalent" rBCG:PspA/PAL vaccine compare to those of the respective "monovalent" rBCG vaccines.