Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

Epstein-Barr virus-encoded LMP1 induces a hyperproliferative and inflammatory gene expression programme in cultured keratinocytes.

SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell-cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1beta and IL-1alpha was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.

Deletions within the epstein-barr virus latent membrane protein-1 oncogene in adult ordinary, HIV-associated and paediatric Hodgkin's disease.

The aims of this study were the following: a) to perform Epstein-Barr virus (EBV) strain type assignment in three groups of Hodgkin's disease(HD): adult ordinary (39 cases), paediatric (24 cases), and HIV-associated (30 cases) and to compare the prevalence of type 1 and type 2 in each of the groups with that existing in two reference populations made up of 50 adults and 39 children; b) to assess the frequency of latent membrane protein-1 (LMP-1) 30-base pair (bp) deletions in the HD groups and in the healthy controls; and c) to relate the presence of LMP-1 deletions with EBV type. Type 2 EBV was observed in 12.8% of ordinary HD, in 26.7% of HIV-associated HD, in 25% of paediatric HD, in 4% of adult controls, and in none of the healthy children. The existence of double infections by type 1 and 2 EBV was also observed in 5.1% of ordinary HD, in 6.7% of HIV-associated HD, and in 10% of adult controls. The 30-bp deletion was identified overall in 33.3% of ordinary HD, in 83.3% of HIV-positive HD, 79.2% of paediatric HD, 34.7% of adult controls, and 36.4% of healthy children. Statistical analysis showed a significant association of the deleted strains with HD occurring in HIV-positive patients (P= 0.00003) and childhood HD (P= 0.006). On the other hand, the prevalence of the 30-bp deletion in the adult ordinary HD group reflects the prevalence of the deletion in the general population. Co-infections by deleted and non-deleted EBV strains were detected in 12.8% of ordinary HD, in 33.3% of HIV-associated HD, in 50% of paediatric HD, in 26.5% of adult controls, and in 27.3% of healthy children. Concerning the relationship between the deletion and the EBV typing, 26% of type 1 specimens carried the 30-bp deletion in an isolated manner compared with 64.7% of type 2. The statistical analysis showed that the deletion was associated with type 2 strains when coinfections were excluded and only the cases in which the deletion appeared alone were considered (P=0.003).