Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

ABSTRACT A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met→Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.

[1]  S. Harrison,et al.  The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution , 1995, Nature.

[2]  F. Guirakhoo,et al.  Construction, Safety, and Immunogenicity in Nonhuman Primates of a Chimeric Yellow Fever-Dengue Virus Tetravalent Vaccine , 2001, Journal of Virology.

[3]  S. Kuhara,et al.  Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. , 1987, Virology.

[4]  L. Dalgarno,et al.  Neurovirulence and neuroinvasiveness of Murray Valley encephalitis virus mutants selected by passage in a monkey kidney cell line. , 1995, The Journal of general virology.

[5]  C. Huang,et al.  Chimeric Dengue Type 2 (Vaccine Strain PDK-53)/Dengue Type 1 Virus as a Potential Candidate Dengue Type 1 Virus Vaccine , 2000, Journal of Virology.

[6]  I. Tokimatsu,et al.  Genetic Determinants Responsible for Acquisition of Dengue Type 2 Virus Mouse Neurovirulence , 1998, Journal of Virology.

[7]  A S Kolaskar,et al.  Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus. , 1999, Virology.

[8]  A. Nakajima,et al.  PPARγ and inflammatory bowel disease: a new therapeutic target for ulcerative colitis and Crohn's disease , 2001 .

[9]  J. Grainger.  The Virus , 1940, Nature.

[10]  J. Lewis,et al.  Probit Analysis (3rd ed). , 1972 .

[11]  L. Reed,et al.  A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS , 1938 .

[12]  R. Marchevsky,et al.  Phenotypic analysis of yellow fever virus derived from complementary DNA. , 1995, The American journal of tropical medicine and hygiene.

[13]  C. Rice,et al.  Yellow Fever/Japanese Encephalitis Chimeric Viruses: Construction and Biological Properties , 1999, Journal of Virology.

[14]  J. Fox,et al.  BEHAVIOR OF 17D YELLOW FEVER VIRUS IN RHESUS MONKEYS RELATION TO SUBSTRAIN, DOSE, AND NEURAL OR EXTRANEURAL INOCULATION , 1943 .

[15]  P. Desprès,et al.  Determinants in the envelope E protein and viral RNA helicase NS3 that influence the induction of apoptosis in response to infection with dengue type 1 virus. , 2000, Virology.

[16]  C. Mandl,et al.  Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH , 1995, Journal of virology.

[17]  M. Bray,et al.  Construction of intertypic chimeric dengue viruses by substitution of structural protein genes. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Shope,et al.  Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA. , 1995, The Journal of infectious diseases.

[19]  C. B. Cropp,et al.  Variation in virulence for mice and rhesus monkeys among St. Louis encephalitis virus strains of different origin. , 1980, The American journal of tropical medicine and hygiene.

[20]  A Marchler-Bauer,et al.  Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus , 1996, Journal of virology.

[21]  A. Barrett,et al.  Comparison of the genomes of the wild-type French viscerotropic strain of yellow fever virus with its vaccine derivative French neurotropic vaccine. , 1995, The Journal of general virology.

[22]  G. Chang,et al.  Nucleotide sequence of the virulent SA-14 strain of Japanese encephalitis virus and its attenuated vaccine derivative, SA-14-14-2. , 1990, Virology.

[23]  Koji Watanabe,et al.  Identification of mutations that occurred on the genome of Japanese encephalitis virus during the attenuation process , 1991, Virus Genes.

[24]  P. Mason,et al.  Partial nucleotide sequence of the Japanese encephalitis virus genome. , 1987, Virology.

[25]  E. Gould,et al.  Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants. , 1991, Virology.

[26]  P. McMinn,et al.  Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein , 2001, Journal of Virology.

[27]  I. Levenbook,et al.  The monkey safety test for neurovirulence of yellow fever vaccines: the utility of quantitative clinical evaluation and histological examination. , 1987, Journal of biological standardization.

[28]  A. Barrett,et al.  Immunogenicity, genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate against Japanese encephalitis. , 1999, Virology.

[29]  Y. L. Lin,et al.  Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro. , 1996, Virology.

[30]  T. Monath.  Pathobiology of the Flaviviruses , 1986 .

[31]  T. Monath,et al.  Yellow fever vector live-virus vaccines: West Nile virus vaccine development , 2001, Trends in Molecular Medicine.

[32]  A. Barrett,et al.  Chimeric Yellow Fever Virus 17D-Japanese Encephalitis Virus Vaccine: Dose-Response Effectiveness and Extended Safety Testing in Rhesus Monkeys , 2000, Journal of Virology.

[33]  G. Atkins,et al.  A recombinant Semliki Forest virus particle vaccine encoding the prME and NS1 proteins of louping ill virus is effective in a sheep challenge model. , 2001, Vaccine.

[34]  C. Mandl,et al.  Adaptation of Tick-Borne Encephalitis Virus to BHK-21 Cells Results in the Formation of Multiple Heparan Sulfate Binding Sites in the Envelope Protein and Attenuation In Vivo , 2001, Journal of Virology.

[35]  F. Guirakhoo,et al.  Molecular Basis for Attenuation of Neurovirulence of a Yellow Fever Virus/Japanese Encephalitis Virus Chimera Vaccine (ChimeriVax-JE) , 2001, Journal of Virology.

[36]  M. Cetron,et al.  Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases , 2001, The Lancet.

[37]  J. Roehrig,et al.  Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. , 1994, The Journal of infectious diseases.

[38]  Y. Kobayashi,et al.  Mutations in the envelope protein of Japanese encephalitis virus affect entry into cultured cells and virulence in mice. , 1992, Virology.

[39]  C. Rice,et al.  Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. , 1989, Science.

[40]  H. Guzmán,et al.  Experimental yellow fever virus infection in the Golden hamster (Mesocricetus auratus). II. Pathology. , 2001, The Journal of infectious diseases.

[41]  T. Monath,et al.  Serious adverse events associated with yellow fever 17DD vaccine in Brazil: a report of two cases , 2001, The Lancet.

[42]  A. Davidson,et al.  Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA. , 1998, The Journal of general virology.

[43]  S. Schlesinger,et al.  The Togaviridae and Flaviviridae , 1986, The Viruses.

[44]  H. Guzmán,et al.  Experimental yellow fever virus infection in the Golden Hamster (Mesocricetus auratus). I. Virologic, biochemical, and immunologic studies. , 2001, The Journal of infectious diseases.

[45]  A. Barrett,et al.  Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructural genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primates. , 1999, Vaccine.