Cytomegalovirus (CMV) Laboratory
Cytomegalovirus (CMV)-specific cellular immune responses in hematopoietic cell transplant (HCT) recipients at the City of Hope. In collaboration with the Hematology department, our laboratory has been observing patients undergoing HCT who are at risk for CMV infection and CMV the question of what elements of immunity are most important for protection from CMV infection in the recipient. A non-therapeutic clinical trial followed patients with such assays as: a) tetramer binding assay (TBA) to locate T-cell recognizing the HLA A2 and B7 presenting CMV peptides; this is done in collaboration with our colleagues Simon Lacey and Don Diamond, b) intra-cellular cytokine (ICC) to locate CD4 and CD8 cells that are activated by CMV peptides and releasing interferon-gamma, c) Q-PCR to detect CMV DNA in plasma, and d) presence of PD-1 expression in T-cells, and effect of KIR genotyping on outcome (this was done in collaboration with our colleagues D. Senitzer and J. Sun.
Preliminary data suggests a prominent role for donor KIR genotype in protection from CMV infection and disease (see Figure 1 below). In addition, once CMV reactivation occurs, it is the expression of PD-1 in CD4 and CD8 T-cells, a measure of T-cell exhaustion, which correlates to the development of CMV disease. This study serves as a platform to other very interesting studies at the COH, which involve vaccine development for protection of patients from CMV.
Figure 1: CMV reactivation and infection relative to donor KIR haplotypes. Group I had minimal donor activating KIR genes, Group II had either aKIR 2DS2 or aKIR2DS4, and Group III had both aKIR 2DS2 and aKIR2DS4 or > 5 aKIR genes. Panel A: Time to first CMV positivity by Q-PCR is shown for subjects in each group (Log-Rank test p<0.001). Panel B: Time to peak Q-PCR is shown for subjects in each group (Log-Rank test p<0.001).
Optimized CMV antigens for induction of immunity. Our laboratory has adopted the approach of using HLAA*0201 transgenic mice immunized with recombinant CMV-DNA vectors followed by a boost with rAAV containing a CMV-transgene. This strategy has been very successful in yielding plenty of CMI for the known CMV genes such as pp65 and IE1. Fusion of shorter constructs are now being developed to remove signals that may interfere with peptide presentation (such as the Nuclear Localization Signal-NLS) and to insert multiple targets into one DNA vaccine (see Figure 2).
Figure 2: CMV- pp65 nuclear localizing signal knock-out
(shown in green) in cytoplasm of cells with
with nuclei stained blue.