Fusion of a cell penetrating peptide from HIV-1 TAT to the Theileria parva antigen Tp2 enhances the stimulation of bovine CD8+ T cell responses.

Immunity to the bovine apicomplexan parasite Theileria parva is associated with MHC-I restricted CD8+ T cell responses directed against the intralymphocytic schizont stage of the parasite. A number of schizont-stage antigens that are targets of CD8+ T cell responses from immune animals have been identified but an effective delivery strategy that consistently induces protective CD8+ T cell responses remains to be developed. This study aimed to determine whether fusing Tat, a cell penetrating peptide (CPP) from HIV-1 TAT, to a CD8+ T cell target antigen from T. parva (Tp2) enhances the cytosolic delivery and subsequent stimulation of bovine CD8+ T cell responses in vitro. Using IFN-gamma ELISpot and cytotoxicity assays, it was demonstrated that recombinant Tat-Tp2 fusion protein possessed a superior ability to access MHC-I processing and presentation pathway and to stimulate CD8+ T cell responses compared to recombinant Tp2 protein. Exposure of APC to Tat-Tp2 protein for only 30 min was sufficient for protein uptake and stimulation of CD8+ T cells. This work describes for the first time the utility of a CPP to enhance MHC-I presentation in a veterinary species and supports the evaluation of CPP fusion proteins in the induction of CD8+ T cell responses in vivo.

Expression analysis of the Theileria parva subtelomere-encoded variable secreted protein gene family.

BACKGROUND: The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. METHODOLOGY/PRINCIPAL FINDINGS: We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. CONCLUSIONS: Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.

Comparative genomics of the neglected human malaria parasite Plasmodium vivax.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Plasmodium vivax in India.

Four Plasmodium species cause malaria in humans: Plasmodium vivax is the most widespread and results in pronounced morbidity. India (population >1 billion) is a major contributor to the burden of vivax malaria. With a resurgence in interest concerning the neglected burden of vivax malaria and the completion of the P. vivax genome, it is timely to review what is known concerning P. vivax in India. The P. vivax population is highly diverse in terms of relapse patterns, drug response and clinical profiles, and highly genetically variable according to studies of antigen genes, isoenzyme markers and microsatellites. The unique epidemiology of malaria in India, where P. vivax predominates over Plasmodium falciparum, renders this location ideal for studying the dynamics of co-infection.

Genes transcribed in the salivary glands of female Rhipicephalus appendiculatus ticks infected with Theileria parva.

We describe the generation of an auto-annotated index of genes that are expressed in the salivary glands of four-day fed female adult Rhipicephalus appendiculatus ticks. A total of 9162 EST sequences were derived from an uninfected tick cDNA library and 9844 ESTs were from a cDNA library from ticks infected with Theileria parva, which develop in type III salivary gland acini. There were no major differences between abundantly expressed ESTs from the two cDNA libraries, although there was evidence for an up-regulation in the expression of some glycine-rich proteins in infected salivary glands. Gene ontology terms were also assigned to sequences in the index and those with potential enzyme function were linked to the Kyoto encyclopedia of genes and genomes database, allowing reconstruction of metabolic pathways. Several genes code for previously characterized tick proteins such as receptors for myokinin or ecdysteroid and an immunosuppressive protein. cDNAs coding for homologs of heme-lipoproteins which are major components of tick hemolymph were identified by searching the database with published N-terminal peptide sequence data derived from biochemically purified Boophilus microplus proteins. The EST data will be a useful resource for construction of microarrays to probe vector biology, vector-host and vector-pathogen interactions and to underpin gene identification via proteomics approaches.