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As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles., Competing Interests: The authors have declared that no competing interests exist.

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.

Synapse elimination in the adult central nervous system can be modelled by axotomy of spinal motoneurons which triggers removal of synapses from the cell surface of lesioned motoneurons by processes that remain elusive. Proposed candidate mechanisms are removal of synapses by reactive microglia and astrocytes, based on the remarkable activation of these cell types in the vicinity of motoneurons following axon lesion, and/or decreased expression of synaptic adhesion molecules in lesioned motoneurons. In the present study, we investigated glia activation and adhesion molecule expression in motoneurons in two mouse strains with deviant patterns of synapse elimination following axotomy. Mice deficient in complement protein C3 display a markedly reduced loss of synapses from axotomized motoneurons, whereas mice with impaired function of major histocompatibility complex (MHC) class Ia display an augmented degree of stripping after axotomy. Activation of microglia and astrocytes was assessed by semiquantative immunohistochemistry for Iba 1 (microglia) and GFAP (astrocytes), while expression of synaptic adhesion molecules was determined by in situ hybridization. In spite of the fact that the two mouse strains display very different degrees of synapse elimination, no differences in terms of glial activation or in the downregulation of the studied adhesion molecules (SynCAM1, neuroligin-2,-3 and netrin G-2 ligand) could be detected. We conclude that neither glia activation nor downregulation of synaptic adhesion molecules are correlated to the different extent of the synaptic stripping in the two studied strains. Instead the magnitude of the stripping event is most likely a consequence of a precise molecular signaling, which at least in part is mediated by immune molecules.