Decreased accumulation of subgenomic RNA in human cells infected with vaccine candidate DEN4Δ30 increases viral susceptibility to type I interferon.

The NIH has developed live attenuated dengue virus (DENV) vaccine candidates by deletion of 30 nucleotides (Δ30) from the untranslated region of the viral genome. Although this attenuation strategy has proven to be effective in generating safe and immunogenic vaccine strains, the molecular mechanism of attenuation is largely unknown. To examine the mediators of the observed attenuation phenotype, differences in translation efficiency, genome replication, cytotoxicity, and type I interferon susceptibility were compared between wild type parental DENV and DENVΔ30 attenuated vaccine candidates.

Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice.

Unlabelled: The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome.

Genetic and phenotypic properties of vero cell-adapted Japanese encephalitis virus SA14-14-2 vaccine strain variants and a recombinant clone, which demonstrates attenuation and immunogenicity in mice.

The live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine, produced in primary hamster kidney cells, is safe and effective. Past attempts to adapt this virus to replicate in cells that are more favorable for vaccine production resulted in mutations that significantly reduced immunogenicity. In this study, 10 genetically distinct Vero cell-adapted JEV SA14-14-2 variants were isolated and a recombinant wild-type JEV clone, modified to contain the JEV SA14-14-2 polyprotein amino acid sequence, was recovered in Vero cells.

Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78.

La Crosse virus infectivity, pathogenesis, and immunogenicity in mice and monkeys.

Background: La Crosse virus (LACV), family Bunyaviridae, was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl with fatal encephalitis in La Crosse, Wisconsin. LACV is a major cause of pediatric encephalitis in North America and infects up to 300,000 persons each year of which 70-130 result in severe disease of the central nervous system (CNS). As an initial step in the establishment of useful animal models to support vaccine development, we examined LACV infectivity, pathogenesis, and immunogenicity in both weanling mice and rhesus monkeys.

Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the structural genes of rDEN4 and rDEN4Delta30 with those of DEN1.

Background: Antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Delta30(ME) and rDEN3/4Delta30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Delta30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Delta30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME structural genes of DEN1 Puerto Rico/94 strain.

Genetically modified, live attenuated dengue virus type 3 vaccine candidates.

Three novel recombinant dengue type 3 (DEN3) virus vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in the Sleman area of central Java in Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope (ME) proteins of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4Delta30(ME)) and the absence (rDEN3/4(ME)) of the Delta30 mutation, a previously described 30-nucleotide deletion in the 3' untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate rDEN3Delta30.

Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells.

Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 virus in Vero cells. DEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice.

Genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in SCID mice transplanted with human liver cells.

Mutations that restrict replication of dengue virus have been sought for the generation of recombinant live-attenuated dengue virus vaccines. Dengue virus type 4 (DEN4) was previously grown in Vero cells in the presence of 5-fluorouracil, and the characterization of 1248 mutagenized, Vero cell passaged clones identified 20 temperature-sensitive (ts) mutant viruses that were attenuated (att) in suckling mouse brain (J. E.