Target Structures of the CD8⁺-T-Cell Response to Human Cytomegalovirus: the 72-Kilodalton Major Immediate-Early Protein Revisited

Abstract

Cell-mediated immunity plays an essential role in the control of infection with the human cytomegalovirus (HCMV). However, only a few CD8+-T-cell epitopes are known, with the majority being contained in the pp65 phosphoprotein, which is believed to dominate the CD8+-T-cell response to HCMV. Here, we have readdressed the issue of CD8+ T cells specific for the 72-kDa major immediate-early protein (IE-1), which is nonstructural but is found very early and throughout the replicative cycle. Using a novel flow-cytometric assay, we were able to identify CD8+-T-cell epitopes (by IE-1 peptide-specific induction of cytokine synthesis) and simultaneously measure the frequency of cells directed against them. For this purpose, 81 pentadecamer peptides covering the complete 491-amino-acid sequence of IE-1 were tested on peripheral blood mononuclear cells of anti-HCMV immunoglobulin G-seropositive donors. At least 10 new epitopes were identified, and the fine specificity and presenting HLA molecule of the first of them was determined. The frequencies of CD8+ T cells directed against IE-1 were similar to those directed against pp65 in donors tested with known pp65-derived peptides. Importantly, additional testing of a corresponding set of peptides covering the complete sequence of pp65 on 10 of these donors identified individuals whose CD8+ T cells recognized IE-1 but not pp65 and vice versa, clearly illustrating that either protein may be a major target. In summary, our results suggest that IE-1 is far more important as a CD8+-T-cell target than current opinion suggests.

DNA vaccine prime followed by boost with live attenuated virus significantly improves antigen-specific T cell responses against human cytomegalovirus

“…49 We also presume that weaker cellular responses against IE1 may be explained by the following facts: (1) pp65 protein, as a highly immunodominant antigen, could suppress the T cell response to IE1.4 antigen; (2) IE1 is a less abundant virion component (< 0.1% of total virion proteins) as opposed to pp65 (15.4%); (3) in vaccine development, application of replication deficient virus (injections of mice with HCMV) excludes the possibility of production of proteins that are usually highly immunogenic in the host after natural infection; (4) we decided to utilize only part of the IE1 protein, which, even though it contains highly immunogenic epitopes, does not include all other epitopes that can also stimulate the immune system. 9,50 The protective efficacy of a pp65-specific response is still unknown, however, it was previously shown that strong CD8 T cell responses, specific to IE1, correlated with protection from CMV disease in solid organ transplant recipients. 31 Therefore, it is critical to include this antigen in future CMV vaccine development.…”

“…Microneutralization assay. Microneutralization assay was performed as previously described 49,50 . Rabbit sera collected at 1 week after the fourth immunizations were serially diluted in this assay to block the infection of AD169 strain of HCMV to FSK cells.…”

Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection

“…The size of these peptides allows them to be processed and presented by HLA class I molecules (which typically bind peptides 8 to 11 amino acids long) and by HLA class II molecules (whose binding groove accommodates peptides ranging from about 10 to 25 amino acids long). In theory, because the overlapping 15mer peptides contain every possible T-cell epitope, they can be used to quantify total T-cell responses independently of the patient's MHC haplotype, as shown recently for Mycobacterium tuberculosis, 23 cytomegalovirus, 24 and HIV infection. 25 The complete amino acid sequence of the HCV NS3 protein derived from HCV-1 (genotype 1a) was covered by 150 overlapping peptides, divided evenly into 3 peptide pools of 50: pool NS3-A, amino acids (aa) 1007-1218; NS3B, aa 1207-1418; and NS3C, aa 1407-1618.…”

Immune Regulation of Human Herpesviruses and Its Implications for Human Transplantation

“…The complexity of CMV has led to a significant amount of work investigating the source of antigenic peptides recognized by CMV-specific T cells during the lytic cycle of infection. Early studies indicated that immunodominant T cells responses are predominantly directed against phosphoprotein (pp) 65 and the immediate early (IE)-1 protein (47)(48)(49). These two proteins remain the immunodominant antigens recognized by CMV-specific T cells, however, more thorough analysis of the CMV-specific T cell repertoire has revealed that immunodominant T cell responses are also directed against an array of other antigens, including pp28, pp50, pp150 and the surface glycoproteins, gH and gB (50).…”