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227 Improved Adenovirus-Based Gene Delivery Technology for Vaccination
R. Vogels, M. Havenga, D. J. Opstelten, B. Bout, M. Mehtali, and T. Logtenberg*
Crucell NV, Leiden, The Netherlands
Background: Recombinant adenoviruses derived from Ad5 constitute attractive vectors for vaccination against AIDS since they stimulate in vivo potent cellular and humoral immune responses against the encoded HIV1 antigens. However, their use for human vaccination is still hampered by: (i) the generation of replication competent adenoviruses (RCA) when using current helper cells for manufacturing; (ii) the poor efficiency of transduction of human antigen-presenting cells (APC) due to the absence or low expression of the coxsackie-adenovirus receptor (CAR); and (iii) the high prevalence of anti-Ad5 neutralizing antibodies in most human sera. To specifically overcome these limitations, we have developed AdVac, a new gene delivery system for vaccination
Methods: Fifty-one human adenovirus serotypes were collected, produced, and tested for their sensitivity to neutralization by 560 human serum samples collected from 6 different locations in Europe, Japan, and the US. In parallel, the fiber-encoding sequences were isolated from these serotypes and used to generate chimeric Ad5 vectors. The ability of the chimeric viruses to infect human immature and mature APC was investigated in vitro. Finally, to eliminate the risks of generation of RCA during manufacturing, a novel human helper cell line was generated by transfection into primary human retinoblasts of Ad5 E1 sequences not overlapping with the recombinant vectors.
Results: Although most adenovirus serotypes were neutralised with high frequencies, adenovirus serotype 35 (Ad35) was neutralised in only 7% (range 0(13%) of the sera. In addition, titers found in such patients were lower than the anti-Ad5 titers. E1-deleted vectors derived from Ad35 were shown not to be neutralised by human sera. Interestingly, Ad5 vectors carrying the fiber from Ad35 were found to better transduce human antigen-presenting cells (immature and mature dendritic cells) and skeletal muscle cells. As a consequence, a 100-fold lower dosage of the new vector is needed to elicit in vitro a potent cellular immune response against candidate antigens. Finally, a new human helper cell line (PER.C6) was developed that prevents the generation of RCA during manufacturing of recombinant vectors. Such cells can be grown in suspension in serum-free medium allowing the large-scale production of RCA-free viruses. Fully characterized Master Cell Banks were established and a Biologics Master File was filed at the FDA.
Conclusions: The AdVac gene delivery vectors transduce very effectively human antigen presenting cells and skeletal muscle cells and are not sensitive to neutralization by human sera. They can be produced in absence of serum on complementation cells derived from PER.C6, a well-characterized human cell line that grows in suspension. This vector technology combines features required for an optimal vaccination.
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