The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL

Abstract

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8 ؉ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple shortterm CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor V␤ gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8 ؉ T cells utilizing a single V␤ gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Specific activation of CMV-primed human T lymphocytes by cytomegalovirus pp65 expressed in fission yeast

“…Importantly, CTL speci¢c for pp65 will recognise autologous HCMV-infected cells without the requirement for de novo viral gene expression resulting in the lysis of infected cells before viral assembly [39]. The immune response induced by pp65 in vitro is similar to that of whole viral antigen considering T cell proliferation as well as interleukin 2 and interferon production [22,27,28]. In this study, the latter could also be con¢rmed for pp65 expressed in ¢ssion yeast.…”

An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

“…Other, perhaps minor, phosphoprotein components of the tegument have been reported, including ppUL97 (51). Genetic or functional analysis of two of the most-studied phosphorylated tegument proteins has indicated the following: (i) pp71, encoded by UL82, is a transcriptional transactivator, acting early in the infectious cycle, is highly beneficial for viral growth in culture, and stimulates progression through the cell cycle (5,6,24,30) and (ii) pp65, encoded by UL83, is a prominent target of the cellular immune system, is nonessential for growth in culture, is required for the production of dense bodies, and mediates accumulation and degradation of HLA class II in lysosomes (18,25,37,44,54). Functional analysis of the UL32 gene product, pp150, has not been reported.…”

Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines

“…route. Spleens were removed three weeks post-immunization and were stimulated in vitro for one week using a simplified protocol with HLA-matched human EBV-LCL [35] as antigen presenting cells (APC), loaded either with the relevant CMV-CTL epitope HLA-A*0201 IE-1 316-324 (IE1-A2), pp65 495-503 (pp65-A2) [36][37][38] or, IE2 CMV-peptide library (4 g/ml) made in our laboratory [29].…”