CD8 T cell-evasive functions of human cytomegalovirus display pervasive MHC allele specificity, complementarity, and cooperativity.

Immunoevasive proteins ("evasins") of human CMV (HCMV) modulate stability and localization of MHC class I (MHC I) molecules, and their supply of antigenic peptides. However, it is largely unknown to what extent these evasins interfere with recognition by virus-specific CD8 T cells. We analyzed the recognition of HCMV-infected cells by a panel of CD8 T cells restricted through one of nine different MHC I allotypes. We employed a set of HCMV mutants deleted for three or all four of the MHC I modulatory genes US2, US3, US6, and US11. We found that different HCMV evasins exhibited different allotype-specific patterns of interference with CD8 T cell recognition of infected cells. In contrast, recognition of different epitopes presented by the same given MHC I allotype was uniformly reduced. For some allotypes, single evasins largely abolished T cell recognition; for others, a concerted action of evasins was required to abrogate recognition. In infected cells whose Ag presentation efficiency had been enhanced by IFN-γ pretreatment, HCMV evasins cooperatively impared T cell recognition for several different MHC I allotypes. T cell recognition and MHC I surface expression under influence of evasins were only partially congruent, underscoring the necessity to probe HCMV immunomodulation using specific T cells. We conclude that the CD8 T cell evasins of HCMV display MHC I allotype specificity, complementarity, and cooperativity.

Human cytomegalovirus pp71 stimulates major histocompatibility complex class i presentation of IE1-derived peptides at immediate early times of infection.

Suppression of major histocompatibility complex (MHC) class I-mediated presentation of human cytomegalovirus (HCMV) peptides is an important mechanism to avoid CD8 T lymphocyte recognition and killing of infected cells. Of particular interest is how MHC class I presentation of essential regulatory immediate early (IE) proteins of HCMV can be effectively compromised at times when known viral immunoevasins are not abundantly expressed. The tegument protein pp71 had been suggested to be involved in MHC class I downregulation. Intriguingly, this polypeptide is also critically engaged in the initial derepression of the major IE gene locus, leading to enhanced expression of IE proteins IE1-pp72 and IE2-pp86. Using a set of viral mutants, we addressed the role of pp71 in MHC class I presentation of IE1-pp72-derived peptides. We show that the amount of "incoming" pp71 positively correlates with IE1-pp72 protein levels and with the presentation of IE1-derived peptides. This indicates that the amount of the IE1 protein, induced by pp71, rather than a putative immunoevasive function of the tegument protein, determines MHC class I antigen presentation of IE1-derived peptides. This process proved to be independent of the presence of pp65, which had been reported to interfere with IE1 presentation. It may thus be beneficial for the success of HCMV replication to limit the level of pp71 delivered from infecting particles in order to avoid critical levels of MHC class I presentation of IE protein-derived peptides.

Suppression of CD8+ T-cell recognition in the immediate-early phase of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) interferes with MHC class I-restricted antigen presentation and thereby reduces recognition by CD8(+) T-cells. This interference is mediated primarily by endoplasmic reticulum-resident glycoproteins that are encoded in the US2-11 region of the viral genome. Such a suppression of recognition would be of particular importance immediately after infection, because several immunodominant viral antigens are already present in the cell in this phase. However, which of the evasion proteins gpUS2-11 interfere(s) with antigen presentation to CD8(+) T-cells at this time of infection is not known. Here we address this question, using recombinant viruses (RV) that express only one of the immunoevasins gpUS2, gpUS3 or gpUS11. Infection with RV-US3 had only a limited impact on the presentation of peptides from the CD8(+) T-cell antigens IE1 and pp65 under immediate-early (IE) conditions imposed by cycloheximide/actinomycin D blocking. Unexpectedly, both RV-US2 and RV-US11 considerably impaired the recognition of IE1 and pp65 by CD8(+) T-cells, and both US2 and, to a lesser extent, US11 were transcribed under IE conditions. Thus, gpUS2 and gpUS11 are key effectors of MHC class I immunoevasion immediately after HCMV infection.

Strong and sustained effector function of memory- versus naïve-derived T cells upon T-cell receptor RNA transfer: implications for cellular therapy.

Current protocols used to select CMV-specific T cells for adoptive immunotherapy focus on virus-specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem-cell transplantation (HSCT) from CMV-seronegative donors. Here, we redirected T cells of CMV-seronegative donors with a human genetically engineered TCR recognizing an HLA-A*0201-binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non-viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve-derived T-cell subsets were both efficiently transfected by TCR-RNA, memory-derived T cells showed much stronger levels of HLA-A*0201-restricted cytolytic activity to CMV-infected fibroblasts and maintained acquired function for 5-10 days. In addition to redirection of CD8(+) cytotoxic T cells, TCR-RNA transfection was capable of redirecting CD4(+) T cells into potent Ag-specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve-derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus-specific CD4(+) and CD8(+) T-cell function. CMV TCR-RNA may represent a suitable therapeutic 'off-the-shelf' reagent to be used in severe CMV infections of HSCT patients when endogenous CMV-specific T-cell immunity is insufficient.

Human cytomegalovirus US3 modulates destruction of MHC class I molecules.

Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.

Polyethylenimine is a strong inhibitor of human papillomavirus and cytomegalovirus infection.

Polyethylenimines are cationic polymers with potential as delivery vectors in gene therapy and with proven antimicrobial activity. However, the antiviral activity of polyethylenimines has not been addressed in detail thus far. We have studied the inhibitory effects of a linear 25-kDa polyethylenimine on infections with human papillomaviruses and human cytomegaloviruses. Preincubation of cells with polyethylenimine blocked primary attachment of both viruses to cells, resulting in a significant reduction of infection. In addition, the dissemination of human cytomegalovirus in culture cells was efficiently reduced by recurrent administration of polyethylenimine. Polyethylenimine concentrations required for inhibition of human papillomavirus and cytomegalovirus did not cause any cytotoxic effects. Polyethylenimines and their derivatives may thus be attractive molecules for the development of antiviral microbicides.

Recombinant viruses as tools to study human cytomegalovirus immune modulation.

Infections with cytomegaloviruses are characterized by an intricate balance between the expression of immunomodulatory viral proteins and antiviral immune defence. For human cytomegalovirus (HCMV), several proteins have been described that interfere with the recognition of infected cells by CD8 T lymphocytes. Although the modes of action of these proteins have been elucidated on the molecular level, thus rendering them useful models to understand MHC class I peptide loading and transport, their role during viral infection has remained enigmatic. We exemplify here, how HCMV mutants can help to understand the importance of individual immunomodulatory proteins in the context of viral infection.

Exogenous introduction of an immunodominant peptide from the non-structural IE1 protein of human cytomegalovirus into the MHC class I presentation pathway by recombinant dense bodies.

Exogenous introduction of particle-associated proteins of human cytomegalovirus (HCMV) into the major histocompatibility complex (MHC) class I presentation pathway by subviral dense bodies (DB) is an effective way to sensitize cells against CD8 T-cell (CTL) recognition and killing. Consequently, these particles have been proposed as a platform for vaccine development. We have developed a strategy to refine the antigenic composition of DB. For proof of principle, an HCMV recombinant (RV-VM3) was generated that encoded the immunodominant CTL determinant IE1TMY from the IE1 protein in fusion with the major constituent of DB, the tegument protein pp65. To generate RV-VM3, a bacterial artificial chromosome containing the HCMV genome was modified by applying positive/negative selection based on the expression of the bacterial galactokinase in conjunction with lambda Red-mediated homologous recombination. This method allowed the efficient and seamless insertion of the DNA sequence encoding IE1TMY in frame into the pp65 open reading frame (UL83) of the viral genome. RV-VM3 expressed its fusion protein to high levels. The fusion protein was packaged into DB and into virions. Its delivery into fibroblasts by these viral particles led to the loading of the MHC class I presentation pathway with IE1TMY and to efficient killing by specific CTLs. This demonstrated that a heterologous peptide, not naturally present in HCMV particles, can be processed from a recombinant, DB-derived protein to be subsequently presented by MHC class I. The results presented here provide a rationale for the optimization of a vaccine based on recombinant DB.

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.

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.