- INTRODUCTION
- MATERIALS AND METHODS
- YFE design, cloning, and expression in plants.
- YFE protein purification.
- YFE protein characterization.
- Immunogenicity assessments of YFE proteins in mice.
- Virus challenge studies in mice and NHPs.
- Assessment of VN antibody responses in mouse and NHP sera from challenge studies.
- Assessment of viremia in NHP sera after challenge infection.
- Evaluation of antibody avidity.
- IFNγ ELISPOT assay.
- RESULTS
- YFE subunit vaccine engineering, expression in N. benthamiana, purification, and characterization.
- Immunogenicity of four YFE subunit vaccines in mice.
- VN antibody responses in mice immunized with YFE-1 or YFE-2E using a two-immunization regimen.
- Immunization with YFE subunit vaccines partially protected mice against lethal viral challenge.
- Immunization with YFE subunit vaccines elicited VN antibody responses in NHPs and reduced viremia after viral challenge.
- YFE-specific IgG avidity in NHPs immunized with YFE subunit vaccines.
- IFNγ production by PBMCs from NHPs immunized with YFE subunit vaccines.
- DISCUSSION
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Plant-Produced Subunit Vaccine Candidates against Yellow Fever Induce Virus Neutralizing Antibodies and Confer Protection against Viral Challenge in Animal Models
Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20–50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens’ eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.
Author Notes
Financial support: The study was supported by Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos (Rio de Janeiro, Brazil).
Authors’ addresses: Stephen Tottey, Yoko Shoji, R. Mark Jones, Jessica A. Chichester, Brian J. Green, Konstantin Musiychuk, Huaxin Si, Slobodanka D. Manceva, Moneim Shamloul, Joey Norikane, Stephen J. Streatfield, and Vidadi Yusibov, Fraunhofer USA Center for Molecular Biotechnology, Newark, DE, E-mails: stephen.tottey@fhcmb.org, yoko.shoji@fhcmb.org, mark.jones@fhcmb.org, jessica.chichester@fhcmb.org, brian.green@fhcmb.org, konstantin.musiychuk@fhcmb.org, huaxin.si@fhcmb.org, slobodanka.manceva@fhcmb.org, moneim.shamloul@fhcmb.org, joey.norikane@fhcmb.org, stephen.streatfield@fhcmb.org, and vidadi.yusibov@fhcmb.org. Rosane C. Guimarães, Elena Caride, Marisol Simões, Patricia C. C. Neves, Renato Marchevsky, and Marcos S. Freire, Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos, Fiocruz, Manguinhos, Rio de Janeiro, Brazil, E-mails: rosane@bio.fiocruz.br, elena@bio.fiocruz.br, marisol.simoes@bio.fiocruz.br, pcristina@bio.fiocruz.br, march@bio.fiocruz.br, and freire@bio.fiocruz.br. Amy Rhee, Amgen, Thousand Oaks, CA, E-mail: amyc.rhee@yahoo.com. Andrea N. M. R. Silva, Universidade Federal do Pará, Belém, Pará, Brazil, E-mail: andrearangel@ufpa.br.