Therapeutic Vaccination of Hematopoietic Cell Transplantation Recipients Improves Protective CD8 T-Cell Immunotherapy of Cytomegalovirus Infection.

Reactivation of latent cytomegalovirus (CMV) endangers the therapeutic success of hematopoietic cell transplantation (HCT) in tumor patients due to cytopathogenic virus spread that leads to organ manifestations of CMV disease, to interstitial pneumonia in particular. In cases of virus variants that are refractory to standard antiviral pharmacotherapy, immunotherapy by adoptive cell transfer (ACT) of virus-specific CD8+ T cells is the last resort to bridge the "protection gap" between hematoablative conditioning for HCT and endogenous reconstitution of antiviral immunity. We have used the well-established mouse model of CD8+ T-cell immunotherapy by ACT in a setting of experimental HCT and murine CMV (mCMV) infection to pursue the concept of improving the efficacy of ACT by therapeutic vaccination (TherVac) post-HCT. TherVac aims at restimulation and expansion of limited numbers of transferred antiviral CD8+ T cells within the recipient. Syngeneic HCT was performed with C57BL/6 mice as donors and recipients. Recipients were infected with recombinant mCMV (mCMV-SIINFEKL) that expresses antigenic peptide SIINFEKL presented to CD8+ T cells by the MHC class-I molecule Kb. ACT was performed with transgenic OT-I CD8+ T cells expressing a T-cell receptor specific for SIINFEKL-Kb. Recombinant human CMV dense bodies (DB-SIINFEKL), engineered to contain SIINFEKL within tegument protein pUL83/pp65, served for vaccination. DBs were chosen as they represent non-infectious, enveloped, and thus fusion-competent subviral particles capable of activating dendritic cells and delivering antigens directly into the cytosol for processing and presentation in the MHC class-I pathway. One set of our experiments documents the power of vaccination with DBs in protecting the immunocompetent host against a challenge infection. A further set of experiments revealed a significant improvement of antiviral control in HCT recipients by combining ACT with TherVac. In both settings, the benefit from vaccination with DBs proved to be strictly epitope-specific. The capacity to protect was lost when DBs included the peptide sequence SIINFEKA lacking immunogenicity and antigenicity due to C-terminal residue point mutation L8A, which prevents efficient proteasomal peptide processing and binding to Kb. Our preclinical research data thus provide an argument for using pre-emptive TherVac to enhance antiviral protection by ACT in HCT recipients with diagnosed CMV reactivation.

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.

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.