LATENCY AND REACTIVATION OF CYTOMEGALOVIRUS AFTER BONE MARROW TRANSPLANTATION

Stanford University, Stanford CA

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Abstract

This project will investigate the role of cytomegalovirus (CMV) reactivation as a contributor to disease in allogeneic hematopoietic cell transplant recipients, with a focus on quantitative and qualitative detection of indicators that precedes active infection. Reactivation of latent virus following transplantation contributes significantly to the incidence of disease in this patient population. Reactivation is believed to depend upon latent viral load, the level to which persistent infection or reactivation is present at the time of transplantation and the quality and pace of reconstitution of antiviral immunity following transplantation. Despite being widely used in a pre-emptive manner to control viral replication, anti-viral drugs exhibit toxicity that decreases success of engraftment and opens the way for other microbial pathogens. Studies on patients will focus on evaluation of levels of viral DNA and latency-associated mRNA transcripts in peripheral blood leukocytes as quantitative measures of reactivation and active replication that indicate progression to virus disease. The First Aim will seek to assemble a molecular definition of the latent state of the virus. This aim will identify the complete set of viral genes expressed by human CMV during latency in hematopoietic progenitor cells. Sets of viral open reading frame specific primers will be employed for both conventional and real time RT-PCR analyses of experimental and natural latent infection to dissect viral gene expression and establish criteria for latent infection, reactivation and active replication. This aim will focus on the natural processes of latency and reactivation. The Second Aim will assess viral DNA levels and patterns of viral gene expression in peripheral blood leukocyte populations. Real time PCR and RT-PCR methods, as well as in situ antibody staining and hybridization will be used to evaluate the levels and cell type distribution of viral infections in patients following transplantation. This study design will differentiate latent from active infection by defining viral reactivation in the transplanted host. The Third Aim will study lymphocyte responses to murine CMV in mice transplanted with allogeneic versus congenic hematopoietic cell transplants with pluripotent stem cells plus purified lymphoid subsets. The study will investigate host immune correlates of protection from viral infection and identify lymphocyte populations that can contribute to protection from viral disease. This aim will employ virus-specific MHC class I-tetramer positive cells for transfer. Together, the proposed studies will form a composite of information that will be of central importance to the transplanted patient and will enable a more informed and complete understanding of the pathogen and mechanisms of host immune control during the engraftment process.