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Expression, Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin

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Abstract

Ubiquitin–proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8+ T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies. The antigens were solubilized using urea and purified by nickel affinity chromatography in denatured condition. Fusion of ubiquitin to E6E7 resulted in marked polyubiquitination in vitro mainly when fused to the E6E7 N-terminal. When tested in a therapeutic scenario, the fusion of ubiquitin to E6E7 reinforced the anti-tumor protection and increased the E6/E7-specific cellular immune responses. Present results encourage the investigation of the adjuvant potential of the ubiquitin fusion to recombinant vaccines requiring CD8+ T cells.

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Abbreviations

Ub:

Ubiquitin

MHC:

Major histocompatibility complex

APC:

Antigen-presenting cells

LB:

Luria–Bertani

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered saline

DTT:

1,4-Dithiothreitol

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Acknowledgements

The authors would like to thank Fapesp (2010/04490-4 and 2007/51698-7), CNPq (304467/2010-3 and 306992/2014-0), and Fundação Butantan for financial support.

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Authors and Affiliations

  1. Laboratório de Biotecnologia Molecular I, Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP, Brazil

    Liliane M. Fernandes de Oliveira, Mirian G. Morale, Agtha A. M. Chaves & Paulo L. Ho

  2. Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, SP, Brazil

    Marilene Demasi

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  1. Liliane M. Fernandes de Oliveira
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  2. Mirian G. Morale
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  3. Agtha A. M. Chaves
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Correspondence to Paulo L. Ho.

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de Oliveira, L.M.F., Morale, M.G., Chaves, A.A.M. et al. Expression, Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin. Mol Biotechnol 59, 46–56 (2017). https://doi.org/10.1007/s12033-016-9990-6

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