Analysis of human T lymphocyte-derived immune responses against <em>Staphylococcus aureus</em> by delivery of <em>in vitro</em> transcribed antigens

Abstract

Staphylococcus aureus is a harmless commensal of the human mucosa and at the same time the main cause of life-threatening nosocomial infections worldwide. In particular, the growing number of methicillin-resistant S. aureus strains, which are resistant to a broad spectrum of β lactam antibiotics defines the need for alternative treatments such as vaccination. However, so far, all vaccination trials have failed to induce protectivity in patients. Next to successful manipulation of the human immune system by a broad spectrum of pathogen-derived virulence factors, one major issue could lie in insufficient induction of protective T lymphocyte responses. <br /> To analyze T cell immunity against S. aureus, the virulence factor protein A (SpA) was selected because of its surface expression and known immunogenicity. SpA is a highly variable Ig-binding protein that allows S. aureus to manipulate and evade the human immune response. It consists of a homologous region that harbors the Ig-binding sites and a variable region that determines the spa type. To characterize T cell responses to SpA, an mRNA-based approach was chosen because it circumvents the immune response attributable to the Ig-binding properties of SpA. Thus, the gene of interest was cloned into a specialized plasmid, which is suitable for in vitro transcription (ivt) and monocyte-derived dendritic cells (MoDC) were transfected with ivt mRNA. Following translation in the cytoplasm, the cells process the antigen and present the peptides via MHC molecules to autologous T cells in co-culture. <br /> Analysis of T cell activation by IFN-γ ELISpot assay revealed the presence of antigen-specific CD8⁺ and CD45RO⁺ T cells activated by spa mRNA-transfected MoDC, whereas activation of CD4⁺ T cells was rather weak. Furthermore, donor- and spa type-dependent T cell activation was observed, indicating differences in preformed immune memory among donors and in the response to spa variants. In average, approximately 0.04% of peripheral CD8⁺ T cells were activated by spa mRNA in healthy donors. This high frequency was comparable to the number of T cells activated by an Influenza control antigen. In order to better define the T cell-activating SpA epitopes, mRNA of a SpA Ig-binding mutant and truncated versions of SpA, either lacking the homologous or the variable domain were generated. Interestingly, ELISpot assays demonstrated higher IFN-γ production with the Ig-binding mutant and the truncated spa compared to wildtype spa mRNA. <br /> Moreover, the T cell response to mRNA-encoded antigens (spa, mecA and sitc) differed profoundly from that induced by the corresponding protein antigens (SpA, PBP2a and SitC). In contrast to mRNA, protein-based antigen presentation elicited only very low amounts of IFN-γ after overnight incubation. Co-culture assays of mRNA and protein antigens suggested that the mRNA’s adjuvant effect is necessary to trigger IFN-γ secretion in S. aureus specific T cells. Analysis of cytokine secretion patterns in MoDC:T cell co-cultures after five days of stimulation further revealed that mRNA-encoded antigens elicited a high Th1-biased immune response characterized by production of IFN-γ and TNF. In contrast, stimulation with staphylococcal proteins led to secretion of low levels of IL-13 and IL-5, indicators for a T_h2-biased immune response. On the contrary to mRNA, SpA protein and, to a minor account, PBP2a induced secretion of IL-2, IL-10 and G-CSF, cytokines that play a role in the differentiation of T regulatory cells. Furthermore, activation of CD8⁺ T lymphocytes by spa mRNA but not by SpA protein triggered degranulation, as suggested by flow cytometric detection of LAMP1. <br /> In conclusion, this study presents mRNA as a novel tool for the analysis and quantification of T cell responses specific for staphylococcal antigens. The results further suggest that this technique represents a promising approach to overcome the T_h2/Treg-dominated skewing of T cell responses to this pathogen.