The rate of acquisition of human cytomegalovirus (HCMV) infection is very high during the first two years of life. Most infections are asymptomatic in young children, but the virus can be transmitted to susceptible pregnant women, causing severe neurologic damage to the fetus. Very little is known about virus-specific CD8 T cell responses in young children. HCMV infection provides an excellent context in which to examine the functional maturation of this component of antiviral immunity in healthy children because virus excretion in urine can be quantitated and monitored in parallel with immunologic studies. We propose to examine whether HCMV-specific CD8 T cells contribute to resolution of active primary HCMV infection in early childhood. Highly sensitive flow cytometry techniques requiring small amounts of peripheral blood afford an unprecedented opportunity to assess the capacity of the developing immune system to respond to this systemic viral infection. CD8 T cell responses will be correlated with changes in viral 'load', measured by virus titration of urine. Intracellular cytokine (ICC) assays will be carried out using HCMV-infected cell lysate and pp65 protein as antigens, with CD69 as the marker of T cell activation and IFN-gamma as the marker of the antigen-specific response. HLA-A2 positive infants will also be tested for acquisition for HCMV-specific CD8 T cells using a class I-HCMV pp65/495-503-A2 tetramer. In order to evaluate the prevalence of 'effector' versus 'memory' phenotypes and the functional characteristics of HCMV-specific CD8 T cells, HCMV- specific CD8 T cells that are detected in infected infants will be characterized for cell surface phenotypes using antibodies to RA and CD27. These experiments will determine whether 'effector' or 'memory' CD8 T cell subpopulations predominate at early or late times during the course of primary HCMV infection. The same infants will be tested for HCMV-specific CD4 T cell responses in Project 1 while viral tropism for peripheral blood cells and maturation of antigen presentation will be studied in Project 3. Defining whether the HCMV-specific CD8 T cell response is a correlate of reduce viral replication in the healthy host is important to HCMV vaccine design, providing criteria for immunogenicity based upon similarities to natural immunity. More generally, investigation of CD8 T cell responses to HCMV antigens will help to elucidate the contribution of this arm of the immune system to the control of viral infections in early childhood. This information is relevant for the design of vaccines against other viral disease acquired in infancy of early childhood.
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