Delivery of Cytomegalovirus T cell antigens for diagnostic and vaccination purposes

Human Cytomegalovirus (CMV) is a highly prevalent β-herpesvirus that establishes life-long latency after primary infection. Congenital CMV infection is the most common viral compli-cation in newborns, causing a number of late sequelae that range from impaired hearing to mental retardation. At the same time, managing CMV reactivation during immunosup-pression remains a major hurdle in post-transplant care. Since CMV-specific T cells are critical for controlling viral reactivation, monitoring of T cell responses is a promising strategy to identify patients at risk for symptomatic CMV disease. T cell responses are mostly quantified after antigen-specific restimulation, which requires presentation of pathogen-derived peptides by antigen-presenting cells (APCs). Here, it was tested if protein carbamoylation, a posttranslational modification of amino groups occur-ring in vivo during renal dysfunction or inflammation, impacts the uptake of recombinant proteins into APCs and, as a result, also T cell restimulation. The CMV proteins immediate-early 1 (IE 1) and pp65, which are the main targets of T cell-mediated immunity during CMV infection, as well as the Epstein-Barr virus (EBV) protein BZLF1 were chosen as model proteins and recombinantly produced for addressing this question. As a result of protein carbamoylation, antigen-specific restimulation of T cells was increased for pp65 (mean in-crease 39%, ranging from 6-112% for different blood donors) and BZLF1 (mean increase 80%, ranging from 5-161%), but decreased for IE 1. Mass spectrometry analysis revealed that lysine residues were almost completely modified (>90%), while arginine car-bamoylation was negligible under the chosen conditions (0.3%). The removal of positive charges from amino groups as a result of carbamoylation was found to be important for the enhanced restimulation of pp65-specific T cells, since this effect could be reverted by addition of the polyanionic competitor fucoidan. In addition, protein maleylation, another posttranslational modification removing positive charges from amino groups, mediated a similar increase in the restimulation of pp65-specific T cells. T cell responses to car-bamoylated pp65 were increased compared to the unmodified protein, irrespective of whether monocytes, macrophages or dendritic cells acted as APCs. Improved restimulation of pp65-specific T cells was shown to be the result of enhanced protein uptake into APCs, which increased both MHC I and MHC II presentation. In summary, protein charges appear to have a major influence on uptake into antigen-presenting cells, which could be harnessed for improving current methods for T cell im-munomonitoring and the design of novel vaccines. In the second part of this thesis, new viral vectors delivering the T cell immunogens IE 1 and pp65 were evaluated with regard to suitability as future CMV vaccine candidates. The vectors were based on Adenovirus 19a/64 (Ad19a/64) or Sendai virus (SeV) and compared side-by-side to the well-established vector platforms Ad5 and Modified Vaccinia Ankara (MVA). All vectors were characterized virologically and immunologically in a series of ex vivo assays with a focus on dendritic cells (DCs), which are the main population priming T cell responses in vivo. It was found that unlike Ad5, Ad19a/64 vectors readily transduce a broad panel of im-mune cells, including monocytes, T cells, NK cells and monocyte-derived dendritic cells (moDCs). Both Ad19a/64- and MVA-transduced moDCs efficiently restimulated IE 1 or pp65-specific T cells, but MVA induced a higher amount of cytotoxicity in this cell type. Ad5 and Ad19 induced upregulation of the maturation markers CD86 and HLA-DR in moDCs whereas expression of CD80 and CD83 was largely unaltered. By contrast, MVA transduction led to downregulation of all markers. To enhance the safety of SeV vectors, a replication-deficient strain (rdSeV) that infects tar-get cells in a non-productive manner while retaining viral gene expression was used in this thesis. rdSeV was compared to the parental, replication-competent Sendai virus strain (rcSeV) as well as MVA. rcSeV was capable of replicating to high titers in DCs while rdSeV infected cells abortively. Due to the higher degree of attenuation, IE 1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to the parental rcSeV strains, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of Interferon α and Interleukin 6. Taken together, these data demonstrate that both rdSeV and Ad19a/64 have great po-tential as novel vector systems for the delivery of CMV immunogens