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Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach

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Abstract

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

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Abbreviations

BEVS:

Baculovirus expression vector system

rBVG:

Recombinant baculovirus expressing rabies lyssavirus glycoprotein

rBVM:

Recombinant baculovirus expressing rabies lyssavirus matrix protein

RABV:

Rabies lyssavirus

rBV:

Recombinant baculoviruses

RVGP:

Rabies lyssavirus glycoprotein

RVM:

Rabies lyssavirus matrix protein

VLPs:

Virus-Like Particles

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Acknowledgements

The authors are grateful to Dr. Ralf F. Hopkins and Dr. Dominic Esposito for providing the Sf9-ET cells used in this study, to Prof. Bergmann Ribeiro and Lorena Chaves for useful discussions.

Funding

This study was financially supported by the National Council of Technological and Scientific Development (CNPq) (grant no. 152538/2012–7), the São Paulo Research Foundation (FAPESP) (grant no. 2009/08038–1), CAPES, and the Butantan Foundation. C.A. Pereira is the recipient of a CNPq 1A senior fellowship.

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Authors and Affiliations

  1. Laboratório de Biotecnologia Viral, Instituto Butantan, Av Vital Brasil 1500, São Paulo, CEP, 05503-900, Brazil

    Thaissa Consoni Bernardino, Renato Mancini Astray, Carlos Augusto Pereira, Vera Lucia Boldorini & Soraia Attie Calil Jorge

  2. Laboratório de Biologia Estrutural, Instituto Butantan, São Paulo-SP, Brazil

    Marta Maria Antoniazzi & Simone Gonçalves Silva Jared

  3. Grupo de Engenharia de Bioprocessos. Escola de Artes, Ciências E Humanidades (EACH), Universidade de São Paulo, São Paulo, SP, Brazil

    Eutimio Gustavo Fernández Núñez

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  1. Thaissa Consoni Bernardino
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Bernardino, T.C., Astray, R.M., Pereira, C.A. et al. Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach. Mol Biotechnol 63, 1068–1080 (2021). https://doi.org/10.1007/s12033-021-00366-z

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