Abstract
The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors.
Highlights
Recombinant adenoviruses have attracted tremendous interest as gene delivery vectors due to their ability to efficiently infect a variety of cells and to be generated to high titers in vitro[1,2,3,4]
We analyzed the structural stability of chimeric hexon proteins and their interactions with L4-100K proteins in order to determine the reason for the failure to be rescued and change in growth of hexon-chimeric recombinant adenovirus serotype 5 (rAd5) vectors
Afterwards, the infectious particles/physical particles (IFU/PP) of the HVRchimeric rAd5 vectors were detected. These vector yields were determined at indicated time points after infection at the multiplicity of infection (MOI) of 10 infectious titers (IFU)/cell, and observations of the cell morphology showed that viral CPE varied markedly between Ad5-37(5, 7) and Ad5-43 (5, 7) after 48 h post infection (h p.i.) (Fig. 1c)
Summary
Recombinant adenoviruses have attracted tremendous interest as gene delivery vectors due to their ability to efficiently infect a variety of cells and to be generated to high titers in vitro[1,2,3,4] Because of these and other features, many studies focus on the construction of novel genetically capsid-modified adenovirus vectors using genetic engineering for gene therapy and vaccine at present. The L4-100K protein was found to interact with both hexon monomers and trimers within the cytoplasm, whereas it interacts predominantly with hexon trimers in the nucleus[17,18] Based on these prior observations, we hypothesized that the L4-100K protein can bind chimeric hexon monomers but fail to assist in the proper folding of hexon trimers, thereby causing some hexon-chimeric adenoviruses to fail to rescue virus and others to grow efficiently to high titers. We analyzed the structural stability of chimeric hexon proteins and their interactions with L4-100K proteins in order to determine the reason for the failure to be rescued and change in growth of hexon-chimeric rAd5 vectors
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