Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65.

The phosphoprotein 65 (pp65) of human cytomegalovirus is a prominent target of the antiviral CD8 T lymphocyte response. This study focused on investigating the properties of pp65 that render it a privileged antigen. It was found that pp65 was metabolically stable. The tegument protein was introduced into MHC class I presentation following its delivery via non-replicating dense bodies. No ubiquitination was found on particle-associated pp65. Proof was obtained that pp65 was a nucleocytoplasmic shuttle protein, using heterokaryon analyses. Based on this finding, inhibition experiments showed that presentation of particle-derived pp65 by HLA-A2 was sensitive to the impairment of the CRM1-mediated nuclear export pathway. The data support the idea that particle-derived pp65 can serve as a nuclear reservoir for proteasomal processing and MHC class I presentation, following its CRM1-dependent nuclear export. The presentation of pp65-derived peptides was also impaired by CRM1-inhibition following de novo synthesis of the tegument protein. However, pp65 protein levels were also reduced when blocking CRM1-mediated export after transient expression. This indicated that pp65 expression rather than direct interference with its own nuclear export was responsible for its reduced presentation in this case. The functionality of CRM1-mediated nuclear export is thus important for the presentation of pp65-derived peptides in the context of MHC class I on organ cells, both after exogenous uptake and after de novo synthesis of the tegument protein, but different mechanisms may account for either case.

Cytomegalovirus interleukin-10 expression in infected cells does not impair MHC class I restricted peptide presentation on bystanding antigen-presenting cells.

Human cytomegalovirus (HCMV) has evolved strategies to counteract its surveillance by the immune system. Mitigation of antiviral immune responses is considered critical for establishment of viral latency and for spread. Recently, a gene encoding an interleukin-10 homologue (cmvIL-10) has been discovered in the HCMV genome. Using recombinant cmvIL-10, several mostly immunosuppressive functions of the molecule have been described. However, the role of cmvIL-10 in the context of viral infection was not addressed. To be able to analyze this issue, we generated cmvIL- 10-negative viral mutants. Using these mutants, we tested whether the expression of cmvIL-10 by infected cells would render bystander antigen-presenting cells less efficient in their capacity to present antigenic peptides in the context of MHC class I. To test this, CTL clones specific for the viral nonapeptides P65(495-503) and IE1(297-305) were used as tools. Culture supernatant from fibroblasts infected with cmv-IL10-negative viruses was supplemented with increasing concentrations of recombinant cmvIL-10. Treatment of human THP-1 cells with these conditioned media did not impair their capacity to present HCMV-derived nonapeptides in the context of MHC-class I, even when high concentrations of cmvIL-10 were used. To investigate whether close cell contact was important, fibroblasts were infected with either wild-type HCMV or cmvIL-10 null mutants and were cocultured with nonpermissive lymphoblastoid cell lines, serving as target cells. No correlation was found between the ability of HCMV strains to express the cmvIL-10 gene and the capacity of neighboring LCL to present peptides in the context of MHC class I. Consequently, we propose that cmvIL- 10 expressed in the context of HCMV infection has no direct impact on MHC class I-restricted antigen presentation of noninfected bystander cells.

The use of clonal mRNA as an antigenic format for the detection of antigen-specific T lymphocytes in IFN-gamma ELISPOT assays.

Most assay systems for the quantification of antigen-specific T-cell responses in infectious, malignant and autoimmune disease depend on the peptide antigen format and are therefore restricted to known epitopes and their presenting HLA molecules. Here we tested in ELISPOT assays the application of in vitro-transcribed clonal mRNA as an alternative antigen format covering all potential epitopes of a given antigen. As model antigens, we chose pp65 of human cytomegalovirus (HCMV) and human tyrosinase (hTyr). Antigen-presenting cells (APC) were K562 cells stably transfected with single HLA class I alleles and autologous dendritic cells (DC). As effectors, we applied in vitro-generated anti-tyrosinase T-cell populations as well as ex vivo-CD8(+) lymphocytes from HCMV-seropositive donors. APC electroporated with clonal mRNA were efficient inducers of spot formation by antigen-experienced CD8(+) T cells. They were equivalent to peptide-loaded targets. mRNA electroporation did not induce non-specific spot formation. While the use of autologous mRNA-electroporated DC can uncover the complete individual T-cell response towards an antigen, mRNA-electroporated K562 cells stably transfected with single HLA class I alleles help to detect CD8(+) T-cell responses restricted by single HLA class I molecules.

Development of novel vaccine strategies against human cytomegalovirus infection based on subviral particles.

BACKGROUND: Pre- and perinatal human cytomegalovirus (HCMV) infection remains one of the major causes of mental defects and sensineural hearing loss in children. In addition, it is a prominent infectious complication in immunosuppressed individuals such as AIDS patients or transplant recipients. Therefore, the development of an HCMV vaccine has been given top priority by health care institutions. STUDY DESIGN: Defective subviral particles of HCMV, termed Dense Bodies (DB) contain the dominant target antigens for humoral and cellular immune responses elicited during natural infection. These enveloped particles are released from infected culture cells and can be purified by gradient centrifugation. DB were analyzed for their ability to induce virus neutralizing antibodies and cytotoxic T cells (CTL) after immunization of mice. RESULTS: Purified DB entered human and murine hematopoetic and fibroblast cells very efficiently, thereby delivering their protein content into the cytoplasm. The cellular uptake was abrogated after sonication and freeze-thawing of the particles, indicating that the integrity of the viral envelope was important for this process. DB immunization of HLA-A2.K(b) transgenic mice induced significant CTL responses in the absence of viral gene expression and without the use of adjuvant. Induction of cytolytic cells by DB was sensitive to sonication and freeze-thawing as determined by CD3epsilon -redirected lysis analysis. In accordance with that, induction of virus neutralizing antibodies was much more effective when untreated DB were used as immunogen. CONCLUSIONS: DB provide a promising basis for the development of a subunit vaccine against HCMV infection. The ability to genetically engineer HCMV provides a rationale to optimize such a vaccine and to develop concepts for future multicomponent vaccines.

Processing and MHC class I presentation of human cytomegalovirus pp65-derived peptides persist despite gpUS2-11-mediated immune evasion.

Immune control of human cytomegalovirus (HCMV) infection can be mediated by CD8+ cytolytic T lymphocytes (CTL). Adoptive transfer of antiviral CTL confers protection against HCMV reactivation and disease. The tegument protein pp65 and the immediate-early 1 protein (IE1) are recognized to be major CTL targets, even though during productive infection the viral immunoevasion proteins gpUS2-11 act to suppress major histocompatibility complex (MHC) class I-restricted antigen presentation. Thus it was not clear how infected cells could be labelled with antigenic peptides in the face of immunoevasion. We show here that the immunodominant peptide pp65NLV was presented by MHC class I in cells infected with a gpUS2-11-competent virus. Presentation of pp65NLV was still detectable at 96 h post-infection, although at low levels. Partial suppression of pp65NLV presentation was dependent on the ability of the infecting strain to express gpUS2-11. MHC class I-restricted antigen presentation in HCMV-infected cells (encoding gpUS2-11) exhibited specificity for pp65-derived peptides, as infected fibroblasts did not present the IE1-derived nonapeptide IE1TMY. Remarkably, infected cells could restore pp65NLV peptide presentation after acid removal of MHC class I despite gpUS2-11 expression. This recovery was shown to be dependent on proteasome functionality. In contrast to IE1, pp65 peptides are loaded on MHC class I molecules to be transported to the cell surface at early and late times after infection in the face of gpUS2-11-mediated immunoevasion. pp65 is therefore the first example of an HCMV protein only incompletely subjected to gpUS2-11-mediated immunoevasion.

Identification of a conserved HLA-A2-restricted decapeptide from the IE1 protein (pUL123) of human cytomegalovirus.

Control of human cytomegalovirus (HCMV) infection is predominantly mediated by cytolytic CD8+ T lymphocytes (CTL). Among the roughly 200 HCMV-encoded polypeptides, the tegument protein pp65 (ppUL83) and the nonstructural IE1 protein are considered to be dominant CTL targets. Yet the importance of CTL against IE1 for protective immunity against HCMV reactivation and disease has remained elusive. Analyses have been difficult, as all MHC class I presented peptides of IE1 defined so far are located in parts of the protein that are variable between viral strains. In this study a conserved decameric peptide from IE1 (P6, IE1(354-363)) that bound to HLA-A2 was identified. Using peptide-pulsed, HLA-matched stimulator cells, CTL lines which recognized P6 after exogenous loading as well as after endogenous processing could repeatedly be generated. However, memory CTL directed against P6 were not readily detectable by ex vivo ELISPOT analysis in peripheral blood mononuclear cells of healthy seropositive individuals, indicating that this peptide represents a quantitatively subdominant determinant during latent HCMV infection. Using the conserved HLA-A2 presented peptide P6 will enable more detailed studies on the role of IE1-specific CTL in patients suffering from various HCMV-related disease conditions and investigation of the role of such cells for immune control of HCMV. Since IE1 is the first viral protein to be expressed after reactivation from latency, P6 may also serve as an important component of a future recombinant HCMV vaccine.