A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immunologically mediated and virus-specific cytotoxic T lymphocytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from dengue virus infections. There is little available information on the human CD8 ϩ T cell responses to dengue viruses. Memory CD8ϩ CTL responses were analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenuated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8 ϩ CTL activity was demonstrated. The nonstructural (NS3 and NS1.2a) and envelope (E) proteins were recognized by CD8 ϩ CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using the PBMC of two donors were serotype specific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35.These results indicate that the CD8 ϩ CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vaccines for dengue. ( J. Clin. Invest. 1996. 98:1684-1691.)

Full-length simian hemorrhagic fever virus (SHFV) genome RNA (about 15 kb in length) and six subgenomic RNAs, ranging in size from 0.65 to 4.7 kb, were detected by Northern blot hybridization in MA104 cytoplasmic extracts with a 3' genomic antisense probe. The 5' regions of the two smallest subgenomic RNAs (RNAs 6 and 7) were cloned and sequenced. Sequence analysis indicated that these two RNAs contained a common 5' leader sequence joined to the subgenomic RNA bodies via a highly conserved junction sequence; the junction sequence of RNA 7 was 5'-TTAACC-3', while that of RNA 6 was 5'-TCAACC-3'. The complete 5' leader sequence (208 nt) was obtained from genomic RNA. The genomic 5' junction sequence is identical to that of RNA 7. Northern blot hybridization with an antisense 5' leader probe confirmed the presence of the complete leader sequence in all six species of subgenomic RNA. In its virion morphology, genome size, gene order, and replication strategy, SHFV is most similar to viruses such as equine arteritis virus, lactate dehydrogenase-elevating virus, and Lelystad virus/porcine respiratory and reproductive syndrome virus.

The role of flavivirus-cross-reactive T lymphocytes in recovery from and pathogenesis of flavivirus infections is not known. In the present paper, we have defined a flavivirus-cross-reactive epitope recognized by two CD4 + CD8 cytotoxic T lymphocyte (CTL) clones, JK4 and JK43. The T cell clones were established from the peripheral blood T lymphocytes of a dengue-4-immune donor, using a limiting-dilution method with dengue-4 antigen. These two T cell clones were cross-reactive for dengue virus types 1, 2, 3 and 4, yellow fever virus and West Nile virus, and recognized NS3 protein. The smallest synthetic peptide recognized by these T cell clones was an identical 9 amino acid peptide which contains amino acids 146 to 154 (VIGLYGNGV) of dengue-4 NS3. HLA-DR15 was the restriction allele for recognition of this epitope by JK4 and JK43. JK4 and JK43 both used T cell receptor V~8, but JK4 used Vfl8 and JK43 used Vfl2. This result indicates that this epitope is recognized by two flavivirus-cross-reactive CD4 ÷ T cell clones which originated from different T cells in vivo.

SllmmaryIt is generally accepted that virus-specific CD8 + cytotoxic T lymphocytes (CTLs) recognize nine-amino acid peptides in conjunction with HLA class I molecules. We recently reported that dengue virus-specific CD8 + CTLs of two different serotype specifidties, which were established by stimulation with dengue virus, recognize a single nine-amino acid peptide of the nonstructural protein NS3 of dengue virus type 4 (D4V) in an HLA-B35-restricted fashion. To further analyze the relationships between the serotype specificities ofT cells and the amino acid sequence of the recognized peptides, we examined the ability of this viral peptide D4.NS3.500-508 (TPEGIIFTL) to stimulate T lymphocytes of an HLA-B35-positive, dengue virus type 4-immune donor. Peptide stimulation of the PBMC generated dengue virus-specific, HLA-B35-restricted CD8 § CTL clones. These clones lysed dengue virus-infected autologous cells, as well as autologous target cells pulsed with this peptide. Four patterns of dengue virus serotype specificities were demonstrated on target cells infected with dengue-vaccinia recombinant viruses or pulsed with synthetic peptides corresponding to amino acid sequences of four dengue virus serotypes. Two serotype-specific clones recognized only D4V. Three dengue virus subcomplex-specific clones recognized D1V, D3V, and D4V, and one subcomplex-spedfic clone recognized D2V and D4V. Three dengue virus serotype-cross-reactive clones recognized D1V-D4V. Thus, a single nine-amino acid peptide induces proliferation of a heterogeneous panel of dengue virus-specific CD8 + CTL clones that are all restricted by HLA-B35 but have a variety of serotype specificities. Peptides that contain a single amino acid substitution at each position of D4.NS3.500-508 were recognized differently by the T cell clones. These results indicate that a single epitope can be recognized by multiple CD8 + CTLs that have a variety of serotype specificities, but the manner of recognition by these multiple CTLs is heterogeneous.H uman T lymphocytes recognize antigenic peptides that are bound to HLA (1, 2). TCRs consist of a heterodimer of oe and 3 chains expressed on the cell surface and are responsible for recognition of the peptide--HLA complexes (3, 4). The specificity of T lymphocytes is, therefore, determined by the interaction of TCRs with peptide-HLA complexes. It is generally accepted that the V(D)Jjunction (complementarity determining region [CDR] t 3 portion) of 1 Abbreviations used in tkis~r: CD, complimentarity determining region; DHF, dengue hemorrhagic fever; D1V-D4V, dengue virus types 1-4; LCL, B lymphoblastoid cell lines; TCGF, T cell growth factor; VV, vaccinia virus; VV-DV, vaccinia dengue recombinant virus.TCR is mainly responsible for recognition of peptide, and that the CDR1 and CDR2 portions recognize HLA (5); however, how the TCR interacts with peptide and HLA is not completely understood (6).Virus-specific CTLs play an important role in recovery from infection (7,8) and in immunopathological mechanisms that may occur in vir...