RESUMEN
Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.
Asunto(s)
Vacunas Antiprotozoos/administración & dosificación , Theileria parva/inmunología , Theileriosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos de Protozoos/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Bovinos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Interferón gamma/biosíntesis , Activación de Linfocitos , Proteínas de la Membrana/administración & dosificación , Proteínas Recombinantes , Linfocitos T Citotóxicos/inmunología , Theileria parva/patogenicidad , Theileriosis/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.
Asunto(s)
Bovinos/inmunología , Inmunización/veterinaria , Vacunas Antiprotozoos/inmunología , Linfocitos T Citotóxicos/inmunología , Theileria parva/inmunología , Theileriosis/inmunología , Animales , Antígenos de Protozoos/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunización/métodos , Interferón gamma/sangre , Masculino , Biblioteca de Péptidos , Vacunas Antiprotozoos/uso terapéutico , Theileriosis/parasitología , Theileriosis/prevención & control , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéuticoRESUMEN
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.
Asunto(s)
Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , Theileria parva/inmunología , Animales , Bovinos , Mapeo Epitopo , Epítopos de Linfocito T/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Epítopos Inmunodominantes/inmunología , Masculino , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Enhancement of the induction of cytotoxic T-cell responses by immunostimulatory CpG oligodeoxynucleotides has been described in humans and mouse models. The present study attempted to address whether CpG has a similar effect in cattle. Immunisation of cattle with a recombinant form of the polymorphic immunodominant molecule from Theileria parva emulsified with immunostimulatory CpG oligodeoxynucleotides in adjuvant had no effect on the induction of antibody responses including the isotype profile, but significantly enhanced the induction of cytolytic responses that were mediated by CD4+CD3+ T cells utilizing the perforin-granzyme pathway.
Asunto(s)
Antígenos de Protozoos/inmunología , Linfocitos T CD4-Positivos/inmunología , Oligodesoxirribonucleótidos/farmacología , Proteínas Protozoarias/inmunología , Theileria parva/inmunología , Theileriosis/inmunología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Antígenos de Protozoos/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Bovinos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Citometría de Flujo/veterinaria , Inmunización/veterinaria , Interferón gamma/inmunología , Activación de Linfocitos , Masculino , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
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.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas Antiprotozoos/inmunología , Linfocitos T Citotóxicos/inmunología , Theileria parva/inmunología , Theileriosis/inmunología , Animales , Bovinos , Línea Celular , Theileriosis/parasitología , Theileriosis/patología , VacunaciónRESUMEN
Two recombinant vaccines against Theileriaparva, based on a near full-length version of the sporozoite surface antigen p67 (p67(635)), or an 80 amino acid C-terminal section (p67C), were evaluated by exposure of immunized cattle to natural tick challenge in two sites at the Kenya Coast and one in Central Kenya. Vaccination reduced severe ECF by 47% at the coast and by 52% in central Kenya from an average incidence of 0.53+/-0.07 (S.E.) in 50 non-immunised controls to an average of 0.27+/-0.05 in 83 immunised animals. The reduction in severe East Coast fever was similar to that observed in laboratory experiments with p67(635) and p67C. The p67 coding sequence from thirteen T. parva field isolates including seven from vaccinated cattle that were not protected, was 100% identical to the gene on which the recombinant vaccine is based, suggesting a predominantly homologous p67 antigenic challenge. The same parasite isolates were however genetically heterogeneous at several loci other than p67.
Asunto(s)
Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Garrapatas/parasitología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Inmunización , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Análisis de Secuencia de ADN , Vacunas de Subunidad/inmunologíaRESUMEN
Full-length recombinant versions of p67, the 709 amino acid major surface protein of Theileria parva sporozoites, induce immunity to East Coast fever (ECF) in cattle. We show that a soluble Escherichia coli recombinant version of p67 (p67(635)), in which a prokaryotic signal peptide replaces the eukaryotic one, confers protection comparable to that induced by the full-length molecule, but is unstable. Peptides encoding 80 (p67C) and 205 (p67N) amino acid fragments of p67, containing epitopes recognised by sporozoite neutralising monoclonal antibodies, exhibit improved stability in E. coli. Antibodies raised against the central region of p67 (p67M) neutralise sporozoite infectivity in vitro. The p67C peptide induced immunity against ECF in cattle, at a level equivalent to p67(635), suggesting that a synthetic peptide vaccine might be achievable.
Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Bovinos/prevención & control , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Theileria parva/inmunología , Theileriosis/prevención & control , Vacunación/veterinaria , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/química , Bovinos , Enfermedades de los Bovinos/inmunología , Epítopos/inmunología , Pruebas de Neutralización , Fragmentos de Péptidos/inmunología , Proteínas Protozoarias/química , Ratas , Proteínas Recombinantes de Fusión/inmunología , Theileriosis/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
There is growing evidence that immunity of cattle to Cowdria ruminantium infection is mediated by T lymphocytes. C. ruminantium antigens that stimulate these responses are therefore of considerable importance to the development of a sub-unit vaccine against the disease. We have examined T cell responses against recombinant analogues of the surface-exposed C. ruminantium major antigen 1 (MAP1) a 28.8 kDa protein and MAP2 (21 kDa) antigen in cattle immunised by infection and treatment. Vigorous and sustained proliferative responses to both antigens were observed in peripheral blood mononuclear cells from immune cattle. MAP1-specific responses were predominantly restricted to cluster of differentiation four antigen positive T cells (CD4+ T cells). Reverse transcription polymerase chain reaction (RT-PCR) analysis of cytokine expression by T cell lines derived from this population revealed strong expression of interferon gamma (IFN-gamma), interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), tumour necrosis factor beta (TNF-beta), interleukin-2 receptor alpha (IL-2Ralpha) transcripts, and weak expression of IL-2 and IL-4. Supernatants from these T cell cultures contained IFN-gamma protein. CD4+ T cell clones specific for MAP1 were generated. Two of these clones proliferated in the presence of autologous infected endothelial cells. In contrast, the response to MAP2 was characterised largely by proliferation of gamma delta (gammadelta) T cells. RT-PCR analysis of cytokine expression by T cell lines which were dominated by gammadelta T cells revealed expression of IFN-gamma, TNF-alpha, TNF-beta, IL-2Ralpha transcripts. Supernatants of these T cell cultures also contained IFN-gamma protein. Our findings indicate that immunisation of cattle by infection with C. ruminantium results in generation of MAP1- and MAP2-specific T cell responses that may play a role in protection against the pathogen.