Derivation and Characterization of a Dengue Type 1 Host Range-Restricted Mutant Virus That Is Attenuated and Highly Immunogenic in Monkeys

Abstract

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3 stem-loop structure (3-SL) that is conserved among all flavivirus species. The 3-SL constitutes the downstream terminal ϳ95 nucleotides of the 3 noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a humanvirulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.The four serotypes of dengue viruses (DEN1, DEN2, DEN3, and DEN4) cause dengue fever (DF) and dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS). The incidences of both DF and DHF/DSS are rising worldwide (10), and there is currently no vaccine available to prevent the spread of dengue or reduce the incidence of disease. A live virus vaccine is desirable for a variety of reasons, including reduced cost in comparison to that of subunit vaccines, the possibility for single-dose vaccination, and the need to elicit long-term immunity. Vaccine development has been hampered thus far by the lack of any animal model for DF or DHF/DSS and the perceived need to elicit a protective response to all four serotypes of dengue virus concurrently (10,16,27).The dengue viruses are members of the Flavivirus genus. Flaviviruses contain a positive-strand RNA genome that is ϳ11 kb long and includes a single open reading frame (ORF) encoding a polyprotein which is processed co-and posttranslationally to yield three structural and at least seven nonstructural (NS) proteins (6). The ORF is flanked at the 5Ј and 3Ј termini of the genome by noncoding region...

A Live, Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3′ Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys

“…Although monkeys inoculated with rDEN1mutF showed a decreased level of viremia compared to those inoculated with wt rDEN1, this difference was not statistically significant. Previously published results for studies with rhesus monkeys have shown a similar level of attenuation for rDEN1mutF (11). In addition, Markoff et al have shown that there was a difference in the mean number of days of viremia (determined by reverse transcription-PCR) between monkeys that received DEN1mutF virus and those that received wt DEN1 (11).…”

“…1A). The use of wt DEN1 virus as the parent for the introduction of the ⌬30 mutation also permitted a comparison of the level of attenuation of rDEN1⌬30 with that of the previously described rDEN1mutF virus, which also contains mutations in the 3Ј UTR (11). The mutF mutation consists of a pair of deleted nucleotides and a two-nucleotide substitution in the terminal 3Ј stem-loop structure conserved among all flavivirus species (22).…”

Dengue virus type 3 vaccine candidates generated by introduction of deletions in the 3′ untranslated region (3′-UTR) or by exchange of the DENV-3 3′-UTR with that of DENV-4

“…Specifically, modification of the 3'-UTR as a means to attenuate DENV has resulted in the development of several promising live attenuated vaccine candidates [19,22,32,33]. The introduction of deletion mutations into untranslated regions is an attractive strategy for development of live attenuated DENV vaccines for two reasons.…”

Size Heterogeneity in the 3′ Noncoding Region of South American Isolates of Yellow Fever Virus

“…Experimental studies indicate that variations in the 3Ј NCR are well tolerated but may sometimes lead to altered growth characteristics in cell culture (3,6,19), changes in virulence and immunogenicity in vertebrate animals (10,17,21,22), or reduced infectivity in mosquitoes (1,44). Molecular epidemiological studies of naturally occurring flaviviruses also suggest that 3Ј NCR structural differences correlate with patterns of virus distribution and transmission (39,50).…”