Success in developing methods for introducing site-specific mutaitons into the dengue type 4 virus (DEN4) genome has provided a new strategy for the construction of safe and effective vaccines against dengue virus infection. A series of cDNA constructs was engineered to contain deletions ranging from 30 to 202 nucleotides in length in the 3' non- coding region of the genome. Full length RNA transcripts of these DNA constructs were tested for infectivity by transfecting permissive tissue culture cells. A panel of viable DEN4 mutants was recovered from mutant RNA transfected cells. Many deletion mutants were stable and produced plaques of reduced size on mosquito C6/36 cells compared to wild type virus. Analysis of the mutants in simian LLC-MK2 cells revealed that most deletion mutants produced plaques that were slow to develop. Furthermore, most mutants that grew slowly attained a lower titer than parental wild type virus. These observations indicate that these DEN4 deletion mutants were growth-restricted in cell culture. Interestingly, deletion mutant 3'd 303-183 produced small plaques on C6/36 cells but grew to a high titer similar to that of parental wild type virus. This deletion mutant plus four other growth-restricted deletion mutants were selected for evaluation of their infectivity and immunogenicity in rhesus monkeys compared to that of the wild type virus. Monkeys infected with those deletion mutants that were restricted in their growth in cultured cells developed fewer days of viremia compared to monkeys infected with the wild type virus. Mutant 3'd 303-183 induced an antibody response equivalent to that of wild type virus as measured by radio- immunoprecipitation and virus neutralization. In contrast, the other deletion mutants induced a low to moderate level of antibodies compared to parental virus. The immunogenicity of these 3' deletion mutants in monkeys appeared to correlate directly with their growth in LLC-MK2 cells. Three deletion mutants of DEN4 which induced a moderate to high level antibody response in monkeys were selected for production of candidate vaccine seed lots that will be evaluated in primates. The results of these studies in primates will determine whether clinical trials will be initiated and which mutants will be studied.
| Anez, German; Men, Ruhe; Eckels, Kenneth H et al. (2009) Passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques. J Virol 83:10384-94 |
