Human T Cell Responses to Dengue Virus Infections: CD8+CTL and Acute Immunosuppression: a Dissertation

There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses 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+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a 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 PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness