RESUMO
East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Nanotecnologia/métodos , Vacinas Protozoárias/imunologia , Theileria parva/fisiologia , Theileriose/imunologia , Doenças Transmitidas por Carrapatos/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Camundongos , Óleo Mineral/administração & dosagem , Nanopartículas/química , Proteínas de Protozoários/genética , Vacinas Protozoárias/genética , Células RAW 264.7 , Dióxido de Silício/química , Carrapatos , Vacinação , Vacinas de Subunidades Antigênicas , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
BACKGROUND: The tick-borne protozoan parasite Theileria parva causes a usually fatal cattle disease known as East Coast fever in sub-Saharan Africa, with devastating consequences for poor small-holder farmers. Immunity to T. parva, believed to be mediated by a cytotoxic T lymphocyte (CTL) response, is induced following natural infection and after vaccination with a live vaccine, known as the Infection and Treatment Method (ITM). The most commonly used version of ITM is a combination of parasites derived from three isolates (Muguga, Kiambu 5 and Serengeti-transformed), known as the "Muguga cocktail". The use of a vaccine comprising several strains is believed to be required to induce a broad immune response effective against field challenge. In this study we investigated whether immunization with the Muguga cocktail induces a broader CTL response than immunization with a single strain (Muguga). RESULTS: Four MHC haplotype-matched pairs of cattle were immunized with either the trivalent Muguga cocktail or the single Muguga strain. CTL specificity was assessed on a panel of five different strains, and clonal responses to these strains were also assessed in one of the MHC-matched pairs. We did not find evidence for a broader CTL response in animals immunized with the Muguga cocktail compared to those immunized with the Muguga strain alone, in either the bulk or clonal CTL analyses. This was supported by an in vivo trial in which all vaccinated animals survived challenge with a lethal dose of the Muguga cocktail vaccine stabilate. CONCLUSION: We did not observe any substantial differences in the immunity generated from animals immunized with either Muguga alone or the Muguga cocktail in the animals tested here, corroborating earlier results showing limited antigenic diversity in the Muguga cocktail. These results may warrant further field studies using single T. parva strains as future vaccine candidates.
Assuntos
Vacinas Protozoárias/farmacologia , Linfócitos T Citotóxicos/imunologia , Theileria parva/imunologia , Theileriose/prevenção & controle , Animais , Bovinos , Genes MHC Classe I/imunologia , Haplótipos , Complexo Principal de Histocompatibilidade/imunologia , Vacinas Protozoárias/imunologia , Especificidade da Espécie , Linfócitos T Citotóxicos/efeitos dos fármacos , Theileriose/imunologiaRESUMO
There is strong evidence that the immunity induced by live vaccination for control of the protozoan parasite Theileria parva is mediated by class I MHC-restricted CD8(+) T cells directed against the schizont stage of the parasite that infects bovine lymphocytes. The functional competency of class I MHC genes is dependent on the presence of codons specifying certain critical amino acid residues that line the peptide binding groove. Compared with European Bos taurus in which class I MHC allelic polymorphisms have been examined extensively, published data on class I MHC transcripts in African taurines in T. parva endemic areas is very limited. We utilized the multiplexing capabilities of 454 pyrosequencing to make an initial assessment of class I MHC allelic diversity in a population of Ankole cattle. We also typed a population of exotic Holstein cattle from an African ranch for class I MHC and investigated the extent, if any, that their peptide-binding motifs overlapped with those of Ankole cattle. We report the identification of 18 novel allelic sequences in Ankole cattle and provide evidence of positive selection for sequence diversity, including in residues that predominantly interact with peptides. In silico functional analysis resulted in peptide binding specificities that were largely distinct between the two breeds. We also demonstrate that CD8(+) T cells derived from Ankole cattle that are seropositive for T. parva do not recognize vaccine candidate antigens originally identified in Holstein and Boran (Bos indicus) cattle breeds.
Assuntos
Linfócitos T CD8-Positivos/parasitologia , Epitopos de Linfócito T/imunologia , Genes MHC Classe I/genética , Fragmentos de Peptídeos/imunologia , Theileria parva/genética , Theileriose/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/citologia , Bovinos , Simulação por Computador , Doenças Endêmicas , Epitopos de Linfócito T/metabolismo , Genes MHC Classe I/imunologia , Imunidade Celular/imunologia , Fragmentos de Peptídeos/metabolismo , Homologia de Sequência de Aminoácidos , Software , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/parasitologia , Theileria parva/imunologia , Theileriose/genética , Theileriose/parasitologiaRESUMO
Peptide-major histocompatibility complex (p-MHC) class I tetramer complexes have facilitated the early detection and functional characterisation of epitope specific CD8+ cytotoxic T lymphocytes (CTL). Here, we report on the generation of seven recombinant bovine leukocyte antigens (BoLA) and recombinant bovine ß2-microglobulin from which p-MHC class I tetramers can be derived in ~48 h. We validated a set of p-MHC class I tetramers against a panel of CTL lines specific to seven epitopes on five different antigens of Theileria parva, a protozoan pathogen causing the lethal bovine disease East Coast fever. One of the p-MHC class I tetramers was tested in ex vivo assays and we detected T. parva specific CTL in peripheral blood of cattle at day 15-17 post-immunization with a live parasite vaccine. The algorithm NetMHCpan predicted alternative epitope sequences for some of the T. parva CTL epitopes. Using an ELISA assay to measure peptide-BoLA monomer formation and p-MHC class I tetramers of new specificity, we demonstrate that a predicted alternative epitope Tp229-37 rather than the previously reported Tp227-37 epitope is the correct Tp2 epitope presented by BoLA-6*04101. We also verified the prediction by NetMHCpan that the Tp587-95 epitope reported as BoLA-T5 restricted can also be presented by BoLA-1*02301, a molecule similar in sequence to BoLA-T5. In addition, Tp587-95 specific bovine CTL were simultaneously stained by Tp5-BoLA-1*02301 and Tp5-BoLA-T5 tetramers suggesting that one T cell receptor can bind to two different BoLA MHC class I molecules presenting the Tp587-95 epitope and that these BoLA molecules fall into a single functional supertype.
Assuntos
Algoritmos , Epitopos de Linfócito T/imunologia , Complexo Principal de Histocompatibilidade , Linfócitos T Citotóxicos/imunologia , Theileria parva/imunologia , Microglobulina beta-2/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática/veterinária , Antígenos de Histocompatibilidade , Dados de Sequência Molecular , Alinhamento de Sequência/veterináriaRESUMO
Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1(+) γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4(+), CD8(+), and WC1(+) T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8(+) T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+), or WC1(+) T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8(+) lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.
Assuntos
Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/parasitologia , Theileria parva/imunologia , Theileria parva/patogenicidade , Animais , Antígenos CD4/análise , Antígenos CD8/análise , Bovinos , Células Cultivadas , Citometria de Fluxo , Glicoproteínas de Membrana/análise , Subpopulações de Linfócitos T/química , VirulênciaRESUMO
Flow cytometry, enzyme-linked immunospot (ELISpot), and cellular cytotoxicity assays are powerful tools for studying the cellular immune response toward intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity toward a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific toward cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium (51Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included, and a newly developed and simple assay to measure TCR avidity.
Assuntos
Epitopos de Linfócito T , Vacinas , Animais , Bovinos , Imunidade Celular , Peptídeos , Linfócitos T CitotóxicosRESUMO
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known.We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.
Assuntos
Imunidade Adaptativa , Linfócitos T CD4-Positivos/imunologia , Doenças dos Bovinos/imunologia , Citocinas/sangue , Mycoplasma mycoides/imunologia , Pleuropneumonia Contagiosa/imunologia , Animais , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais Murinos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/microbiologia , Testes de Fixação de Complemento/veterinária , Citocinas/imunologia , Citometria de Fluxo/veterinária , Masculino , Pleuropneumonia Contagiosa/sangue , Pleuropneumonia Contagiosa/microbiologiaRESUMO
African swine fever virus (ASFV) is the etiological agent of a contagious and fatal disease of domestic pigs that has significant economic consequences for the global swine industry. Due to the lack of effective treatment and vaccines against African swine fever, there is an urgent need to leverage cutting-edge technologies and cost-effective approaches for generating and purifying recombinant virus to fast-track the development of live-attenuated ASFV vaccines. Here, we describe the use of the CRISPR/Cas9 gene editing and a cost-effective cloning system to produce recombinant ASFVs. Combining these approaches, we developed a recombinant virus lacking the non-essential gene A238L (5EL) in the highly virulent genotype IX ASFV (ASFV-Kenya-IX-1033) genome in less than 2 months as opposed to the standard homologous recombination with conventional purification techniques which takes up to 6 months on average. Our approach could therefore be a method of choice for less resourced laboratories in developing nations.
RESUMO
Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright's fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite.
Assuntos
Búfalos/parasitologia , DNA de Protozoário/genética , Variação Genética , Theileria parva/genética , Theileriose/parasitologia , Animais , Bovinos , Genoma de Protozoário , Genótipo , Especificidade da Espécie , Theileria parva/classificação , Theileria parva/isolamento & purificaçãoRESUMO
Chemotherapy of East Coast fever, a lymphoproliferative cancer-like disease of cattle causing significant economic losses in Africa, is largely dependent on the use of buparvaquone, a drug that was developed in the late 1980's. The disease is caused by the tick-borne protozoan pathogen Theileria parva. Buparvaquone can be used prophylactically and it is also active against tropical theileriosis, caused by the related parasite Theileria annulata. Recently, drug resistance was reported in T. annulata, and could occur in T. parva. Using a 3H-thymidine incorporation assay we screened 796 open source compounds from the Medicines for Malaria Venture (MMV) to discover novel chemicals with potential inhibitory activity to T. parva. We identified nine malaria box compounds and eight pathogen box compounds that inhibited the proliferation of F100TpM, a T. parva infected lymphocyte cell line. However, only two compounds, MMV008212 and MMV688372 represent promising leads with IC50 values of 0.78 and 0.61⯵M, respectively, and CC50 valuesâ¯>â¯5⯵M. The remaining compounds exhibited a high degree of toxicity (CC50 valuesâ¯<â¯1.09⯵M) on the proliferation of bovine peripheral blood mononuclear cells stimulated with concanavalin A. We also tested the anti-cancer drug, dasatinib, used in the chemotherapy of some leukemias. Dasatinib was as active and safe as buparvaquone in vitro, with an IC50 of 5 and 4.2â¯nM, respectively, and CC50â¯>â¯10⯵M. Our preliminary data suggest that it may be possible to repurpose compounds from the cancer field as well as MMV as novel anti-T. parva molecules.
Assuntos
Antimaláricos/farmacologia , Reposicionamento de Medicamentos , Theileria parva/efeitos dos fármacos , Animais , Antiprotozoários/farmacologia , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dasatinibe/farmacologia , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Naftoquinonas/farmacologia , Bibliotecas de Moléculas Pequenas , Theileriose/tratamento farmacológicoRESUMO
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.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Theileria parva/imunologia , Animais , Bovinos , Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Epitopos Imunodominantes/imunologia , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
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.
Assuntos
Bovinos/imunologia , Imunização/veterinária , Vacinas Protozoárias/imunologia , Linfócitos T Citotóxicos/imunologia , Theileria parva/imunologia , Theileriose/imunologia , Animais , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Imunização/métodos , Interferon gama/sangue , Masculino , Biblioteca de Peptídeos , Vacinas Protozoárias/uso terapêutico , Theileriose/parasitologia , Theileriose/prevenção & controle , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
OBJECTIVES: The objective of this study was to assess whether cytotoxic T cells (CTL) generated by the live vaccine, known as "ITM Muguga cocktail", which is used for the cattle disease East Cost fever (ECF) in Sub-Saharan Africa, showed a broad reactivity against many different strains of the causative parasite Theileria parva. We also assessed whether immune responses were similar in cattle expressing the same MHC class I haplotypes. RESULTS: The antigenic specificity of CTL from MHC class I-matched cattle vaccinated with the Muguga cocktail were different. Three cattle of MHC class I haplotype A18, one A18/A19 and two haploidentical (A18v/A12) animals, showed differential recognition of autologous cells infected with a panel of T. parva isolates. This could have implications in the field where certain strains could break through the vaccine. Furthermore, neither of the haploidentical cattle recognized the CTL epitope (Tp1214-224), presented by the A18 haplotype, in contrast to the third animal, showing differences in immunodominance in animals of the same haplotype A18. This suggests that the CTL specificities following immunization with the Muguga cocktail can vary even between haploidentical individuals and that some parasite strains may break through immunity generated by the Muguga cocktail.
Assuntos
Antígenos de Protozoários/imunologia , Doenças dos Bovinos/prevenção & controle , Bovinos , Epitopos/imunologia , Genes MHC Classe I/genética , Linfócitos T Citotóxicos/imunologia , Theileria parva/imunologia , Theileriose/prevenção & controle , Animais , Bovinos/sangue , Bovinos/genética , Bovinos/imunologia , Haplótipos , Quênia , MasculinoRESUMO
The parasite Theileria parva is the causative agent of East Coast fever (ECF), one of the most serious cattle diseases in sub-Saharan Africa, and directly impacts smallholder farmers' livelihoods. There is an efficient live-parasite vaccine, but issues with transmission of vaccine strains, need of a cold chain, and antibiotics limit its utilization. This has fostered research towards subunit vaccination. Cytotoxic T lymphocytes (CTL) are crucial in combating the infection by lysing T. parva-infected cells. Tp1 is an immunodominant CTL antigen, which induces Tp1-specific responses in 70-80% of cattle of the A18 or A18v haplotype during vaccination with the live vaccine. In this study, human adenovirus serotype 5 (HAd5) and modified vaccinia Ankara (MVA) were assessed for their ability to induce Tp1-specific immunity. Both viral vectors expressing the Tp1 antigen were inoculated in cattle by a heterologous prime-boost vaccination regimen. All 15 animals responded to Tp1 as determined by ELISpot. Of these, 14 reacted to the known Tp1 epitope, assayed by ELISpot and tetramer analyses, with CTL peaking 1-week post-MVA boost. Eleven animals developed CTL with specific cytotoxic activity towards peripheral blood mononuclear cells (PBMC) pulsed with the Tp1 epitope. Moreover, 36% of the animals with a Tp1 epitope-specific response survived a lethal challenge with T. parva 5 weeks post-MVA boost. Reduction of the parasitemia correlated with increased percentages of central memory lymphocytes in the Tp1 epitope-specific CD8+ populations. These results indicate that Tp1 is a promising antigen to include in a subunit vaccine and central memory cells are crucial for clearing the parasite.
RESUMO
East Coast fever (ECF) is a lymphoproliferative disease caused by the tick-transmitted protozoan parasite Theileria parva. ECF is one of the most serious cattle tick-borne diseases in Sub-Saharan Africa. We have previously demonstrated that three doses of the C-terminal part of the sporozoite protein p67 (p67C) adjuvanted with ISA206VG confers partial protection against ECF at a herd level. We have tested the efficacy of two doses of this experimental vaccine, as reducing the vaccination regimen would facilitate its deployment in the field. We reconfirm that three antigen doses gave a significant level of protection to severe disease (46%, ECF scoreâ¯<â¯6) when compared with the control group, while two doses did not (23%). Animals receiving three doses of p67C developed higher antibody titers and CD4+ T-cell proliferation indices, than those which received two doses. A new panel of immune parameters were tested in order to identify factors correlating with protection: CD4+ proliferation index, total IgG, IgG1, IgG2 and IgM half maximal titers and neutralization capacity of the sera with and without complement. We show that some of the cellular and humoral immune responses provide preliminary correlates of protection.
Assuntos
Adjuvantes Imunológicos , Antígenos de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Theileria/imunologia , Theileriose/prevenção & controle , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos/imunologia , Bovinos , Imunização , Imunização Secundária , Vacinas Protozoárias/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
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.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Proteínas de Protozoários/imunologia , Theileria parva/imunologia , Theileriose/imunologia , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Bovinos , Ensaio de Imunoadsorção Enzimática/veterinária , Citometria de Fluxo/veterinária , Imunização/veterinária , Interferon gama/imunologia , Ativação Linfocitária , Masculino , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Flow cytometry, enzyme-linked immunospot (ELISpot) and cellular cytotoxicity assays are powerful tools for studying the cellular immune response towards intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity towards a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific towards cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium ((51)Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included.
Assuntos
ELISPOT/métodos , Citometria de Fluxo/métodos , Imunidade Celular/imunologia , Vacinas/imunologia , Sequência de Aminoácidos/genética , Animais , Bovinos , Epitopos de Linfócito T/imunologia , Imunidade Celular/genética , Gado/virologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
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.