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Journal of Virology, September 2004, p. 9998-10008, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9998-10008.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Single Amino Acid Substitution in the Envelope Protein of Chimeric Yellow Fever-Dengue 1 Vaccine Virus Reduces Neurovirulence for Suckling Mice and Viremia/Viscerotropism for Monkeys

F. Guirakhoo,1* Z. Zhang,1 G. Myers,1 B. W. Johnson,2 K. Pugachev,1 R. Nichols,1 N. Brown,1 I. Levenbook,3 K. Draper,4 S. Cyrek,4 J. Lang,5 C. Fournier,5 B. Barrere,5 S. Delagrave,6 and T. P. Monath1

Acambis, Inc., Cambridge, Massachusetts,1 Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado,2 99 Riverchase Court, Rensselaer, New York,3 Sierra Division, Discovery and Development Services, Charles River Laboratories, Inc., Sparks, Nevada,4 Aventis Pasteur, Campus Merieux, Marcy-L'Etoile, France,5 BioTech Studio LLC, Newark, Delaware6

Received 26 February 2004/ Accepted 13 April 2004

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78:4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

* Corresponding author. Mailing address: Acambis, Inc., 38 Sidney St., Cambridge, MA 02139. Phone: (617) 761-4323. Fax: (617) 494-1741. E-mail: farshad.guirakhoo{at}acambis.com.

Journal of Virology, September 2004, p. 9998-10008, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9998-10008.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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