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We analyzed the CD4+ T-lymphocyte responses to dengue, West Nile, and yellow fever viruses 4 months after immunization of a volunteer with an experimental live-attenuated dengue virus type 1 vaccine (DEN-1 45AZ5). We examined bulk culture proliferation to noninfectious antigens, determined the precursor frequency of specific CD4+ T cells by limiting dilution, and established and analyzed CD4+ T-cell clones. Bulk culture proliferation was predominantly dengue virus type 1 specific with a lesser degree of cross-reactive responses to other dengue virus serotypes, West Nile virus, and yellow fever virus. Precursor frequency determination by limiting dilution in the presence of noninfectious dengue virus antigens revealed a frequency of antigen-reactive cells of 1 in 1,686 peripheral blood mononuclear cells (PBMC) for dengue virus type 1, 1 in 9,870 PBMC for dengue virus type 3, 1 in 14,053 PBMC for dengue virus type 2, and 1 in 17,690 PBMC for dengue virus type 4. Seventeen CD4+ T-cell clones were then established by using infectious dengue virus type 1 as antigen. Two patterns of dengue virus specificity were found in these clones. Thirteen clones were dengue virus type 1 specific, and four clones recognized both dengue virus types 1 and 3. Analysis of human leukocyte antigen (HLA) restriction revealed that five clones are HLA-DRw52 restricted, one clone is HLA-DP3 restricted, and one clone is HLA-DP4 restricted. These results indicate that in this individual, the CD4+ T-lymphocyte responses to immunization with live-attenuated dengue virus type 1 vaccine are predominantly serotype specific and suggest that a multivalent vaccine may be necessary to elicit strong serotype-cross-reactive CD4+ T-lymphocyte responses in such individuals.

journal of virology

Dengue virus-specific human CD4+ T-lymphocyte responses in a recipient of an experimental live-attenuated dengue virus type 1 vaccine: bulk culture proliferation, clonal analysis, and precursor frequency determination