East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8 ؉ cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8 ؉ CTL from immune cattle. CD8 ؉ T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.cattle ͉ East Coast fever ͉ immunoscreening ͉ protozoan parasite ͉ vaccination A single inoculation with a potentially lethal dose of Theileria parva sporozoites and simultaneous treatment with a longacting oxytetracycline induces solid immunity to homologous and, in certain instances, heterologous parasite challenge (1, 2). This methodology has been adopted as a live vaccine for the control of East Coast fever (ECF) (3). The long-lasting immunity to ECF contrasts with the partial immunity to malaria that develops after only several years of exposure to T. parva-related Plasmodium spp. (4). Manufacture and delivery of the live ECF vaccine is difficult to sustain, but it has enabled elucidation of the dominant protective immune response against the disease. Kinetic and adoptive cell transfer studies (5, 6) have demonstrated that protection of cattle is mediated by MHC class I-restricted CD8 ϩ cytotoxic T lymphocytes (CTL) that destroy schizontinfected lymphocytes, the pathogenic life-cycle stage of T. parva. In addition, there is a strong correlation between the specificity of the CTL response and cross-immunity profiles of distinct parasite strains (2). The identification of schizont antigens targeted by CTL from T. parva-immune cattle has been elusive but should pave the way for the development of a subunit vaccine against ECF and provide a long-term solution to a socioeconomically important constraint to livestock agriculture in Africa (7). We adopted two approaches to antigen identification, both dependent on screening of transiently transfected antigenpresenting cells with fully characterized CTL (8, 9) from live vaccine-immunized cattle of diverse bovine leukocyte antigen (BoLA) MHC class I genotypes. First, in a targeted gene approach, we immunoscreened genes that were predicted by using preliminary sequence data from one of the four T. parva chromosomes (10) to contain a secretion signal. The approach was ...

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Influenza virus contains eight single-stranded RNA segments of negative polarity as the genome and an RNA-dependent RNA polymerase as a virion component (26). Influenza virus RNA polymerase catalyzes both transcription [the synthesis of plus-strand mRNA containing the host cell-derived cap I structure at the 5'-terminus and poly (A) tail at the 3'-terminus] and replication [the synthesis of full-length plus-strand complementary RNA (cRNA) and the cRNA-dependent synthesis of minus-strand viral RNA (vRNA)] (14,17,18,24,50). The viral RNA polymerase also catalyzes polyadenylation at the 3'-termini of mRNA in vitro (43). The viral RNA polymerase also performs templatedependent capped RNA cleavage (21, 41) and apparent proofreading of nascent RNA chains (19).The RNA polymerase purified from influenza virus consists of one part each of three subunits, PB1, PB2 and PA (15). In vitro reconstitution studies of enzymatically active RNA polymerase using individual P proteins purified either from baculovirus-infected cells (23), Pichia pastoris cells (16) or by SDS-polyacrylamide gel electrophoresis of virions (49) confirmed the subunit structure. The function of each subunit of influenza virus RNA polymerase has been genetically and biochemically characterized. For instance, PB1 subunit can be cross-linked with nucleotide substrates (3, 4, 6), and nuclear extracts containing PB1 subunit alone or cells expressing both PB1 and NP can catalyze RNA synthesis, depending on the model RNA templates (22,35,52), indicating that PB1 is involved in polymerization of RNA chains. PB1 bound with both negative and positive sense RNAs (10, 28). Cap I analog was cross-linked with PB2 in vitro (6,13,29,53), and RNA synthesized in cells without PB2 lacks the 5'-cap structure (35), suggesting that PB2 is required for cap binding and synthesis of capped mRNAs. The cap-dependent RNase active site has been mapped in PB1 recently (29). However, the information about the role of PA on viral replication remains limited. Temperature-sensitive (ts) mutations in the PA gene affected only vRNA synthesis, but not mRNA synthesis (25,30,33,45,46). Nakagawa et al. demonstrated that PA was essential for cRNA-dependent vRNA synthesis (36). We identified a unique protease activity in purified PA protein (12).The subunit binding sites of the influenza virus RNA polymerase were mapped, which demonstrated that PB1

We investigated the antigenic maturation of rabies virus N protein, for which we used some conformational epitope-specific monoclonal antibodies (MAbs) and an MAb (5-2-26) against a phosphorylation-dependent linear epitope. Infected cells were lysed with a deoxycholate-free lysis buffer and separated by ultracentrifugation into the soluble top and the nucleocapsid fractions. None of the study MAbs recognized N proteins in the top fraction, whereas nucleocapsid-associated N proteins were recognized by all of the MAbs. Immunoprecipitation with polyclonal anti-N antibodies coprecipitated the P proteins from the top fraction, indicating that soluble N proteins are mostly associated with the P protein. The N proteins dissociated from both the N-P complex and nucleocapsids were recognized by none of the study MAbs, whereas the MAb 5-2-6 recognized the SDS-denatured N proteins of the nucleocapsid but not of the top fraction. In addition, the phosphorylation-deficient mutant N proteins were shown to be similarly accumulated as the wild-type N proteins into the viral inclusion bodies, defined as the virus-specific structures composed of viral nucleocapsids, that are produced in the cytoplasm of the infected cells. Based on these results, we believe that newly synthesized N proteins are not immediately phosphorylated at serine-389 (a common phosphorylation site) but are first associated with the P protein. After being used for encapsidation of the viral RNA, the N proteins undergo conformational changes, whereby epitopes for the conformation-specific MAbs are formed and become phosphorylated at serine-389.

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