Selection of Attenuated Dengue 4 Viruses by Serial Passage in Primary Kidney Cells

Kh, Eckels; Rm, Scott; Wh, Bancroft; Brown, John; Dr, Dubois; Pl, Summers; Pk, Russell; Sb, Halstead

doi:10.4269/ajtmh.1984.33.684

Cited by 54 publications

(2 citation statements)

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“…The most frequent clinical reactions were transient and asymptomatic including a maculopapular rash in 50% of volunteers, increased alanine aminotransferase (ALT) levels in 25% of volunteers and neutropenia in 15% of volunteers. Transient elevations in serum liver enzymes are also seen in vaccinees infected with other live attenuated DENV vaccine candidates (Eckels et al 1984; Edelman et al 1994; Kanesa-thasan et al 2001; Vaughn et al 1996) and the levels observed in vaccinees are generally much lower than those observed in humans experiencing DF or DHF/DSS. Furthermore, the hepatomegaly that is observed in some dengue patients (Kalayanarooj et al 1997; Kuo et al 1992; Mohan et al 2000; Wahid et al 2000) has not been observed in vaccinees.…”

Section: The Rden4δ30 Vaccine Candidatementioning

confidence: 98%

Targeted Mutagenesis as a Rational Approach to Dengue Virus Vaccine Development

Blaney

Durbin

Murphy

et al. 2009

Current Topics in Microbiology and Immunology

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The recombinant dengue virus type 4 (rDEN4) vaccine candidate, rDEN4Δ30, was found to be highly infectious, immunogenic and safe in human volunteers. At the highest dose (105 PFU) evaluated in volunteers, 25% of the vaccinees had mild elevations in liver enzymes that were rarely seen at lower doses. Here, we describe the generation and selection of additional mutations that were introduced into rDEN4Δ30 to further attenuate the virus in animal models and ultimately human vaccinees. Based on the elevated liver enzymes associated with the 105 PFU dose of rDEN4Δ30 and the known involvement of liver infection in dengue virus pathogenesis, a large panel of mutant viruses was screened for level of replication in the HuH-7 human hepatoma cell line, a surrogate for human liver cells and selected viruses were further analyzed for level of viremia in SCID-HuH-7 mice. It was hypothesized that rDEN4Δ30 derivatives with restricted replication in vitro and in vivo in HuH-7 human liver cells would be restricted in replication in the liver of vaccinees. Two mutations identified by. this screen, NS3 4995 and NS5 200,201, were separately introduced into rDEN4Δ30 and found to further attenuate the vaccine candidate for SCID-HuH-7 mice and rhesus monkeys while retaining sufficient immunogenicity in rhesus monkeys to confer protection. In humans, the rDEN4Δ30-200,201 vaccine candidate administered at 105 PFU exhibited greatly reduced viremia, high infectivity and lacked liver toxicity while inducing serum neutralizing antibody at a level comparable to that observed in volunteers immunized with rDEN4Δ30. Clinical studies of rDEN4Δ30-4995 are ongoing.

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Section: The Rden4δ30 Vaccine Candidatementioning

confidence: 98%

Targeted Mutagenesis as a Rational Approach to Dengue Virus Vaccine Development

Blaney

Durbin

Murphy

et al. 2009

Current Topics in Microbiology and Immunology

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“…This vaccine approach was not developed further because extensive virus purification would be required in order to remove contaminating mouse brain antigens. More recently, live candidate dengue vaccines were prepared by repeated passage in cultured cells of nonnatural hosts (9,10,22). Experience from different laboratories indicated that this new approach has not been consistently successful in producing attenuated vaccine strains.…”

mentioning

confidence: 99%

Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys

Men

Bray

Clark

et al. 1996

J Virol

290

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The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3 noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK 2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5 boundary of the 3 noncoding region and whose 3 boundary did not extend beyond the last 113 nt of the 3 end were viable. With the exception of mutant 3d 303-183, which contained a deletion of nt 303 to 183 from the 3 terminus, deletion mutants produced plaques that appeared late on simian LLC-MK 2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK 2 cells than parental wild-type virus. Significantly, mutant 3d 303-183 grew to a high titer and was least restricted in growth. Mutant 3d 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3d 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3 DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK 2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

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Dengue hemorrhagic fever

Sinniah

Igarashi

1995

Reviews in Medical Virology

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Selection of Attenuated Dengue 4 Viruses by Serial Passage in Primary Kidney Cells

Cited by 54 publications

References 0 publications

Targeted Mutagenesis as a Rational Approach to Dengue Virus Vaccine Development

Targeted Mutagenesis as a Rational Approach to Dengue Virus Vaccine Development

Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys

Dengue hemorrhagic fever

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