Inmunodominancia y procesamiento de epítopos de la glicoproteína F del virus respiratorio sincitial humano reconocidos por linfocitos T citotóxicos CD8+ murinos

Abstract

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-06-2006Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T lymphocyte (CTL) and antibody responses. Two murine H-2Kd-restricted CTL epitopes (F85-93 and F92-106) are known in the F protein of the A2 strain of RSV. We generated F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators and found that in this strain only the F85-93 epitope is conserved. Motif based epitope prediction programmes and an F2 chain deleted F protein encoded in a recombinant vaccinia virus (rVV) enabled us to identify a new epitope in the Long strain, F249-258, which is presented by Kd as a 9mer (TYMLTNSEL) or a 10mer (TYMLTNSELL) peptide. F249-258-specific CTL are induced in vivo during the immune response to RSV or to vvF, a rVV encoding the entire F protein. The immunodominance pattern between epitopes F85-93 and F249-258 was assessed by quantifying CD8+ T-lymphocyte responses to epitopes F85-93 and F249-258. Our results show CD8+ T-lymphocyte responses are strongly skewed to F85-93 in in vitro multispecific CTL lines and in vivo during a secondary response to vvF. However, no hierarchy in CD8+ T-lymphocyte responses to F85-93 and F249-258 epitopes was observed in vivo during a primary response. Processing and presentation of epitopes F85-93 and F249-258 was studied and major differences in presentation pathways were found depending on the epitope and on the viral context of the antigen. Epitope F85-93 is presented through an endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein is in either RSV or rVV context. The proteasome mediates this pathway in cells infected with rVV encoding either native F protein, a cytosolic form, or a form that is retained before the mid-Golgi. Moreover, in rVV-infected cells, an additional endogenous TAP-independent presentation pathway is also functioning for this epitope. In contrast, epitope F249-258 is mainly presented through two TAP-independent pathways when the F protein is expressed from RSV or rVV, that differ in that presentation is endogenous or mostly exogenous, respectively. Our results contribute to the study in murine models of the role of CTL in the immune response to RSV F protein, and underscore the diversity of MHC class I processing pathways available for presentation of epitopes to CTL