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Featured researches published by Elias Awino.


Infection and Immunity | 2008

Characterization of the Fine Specificity of Bovine CD8 T-Cell Responses to Defined Antigens from the Protozoan Parasite Theileria parva

Simon P. Graham; Roger Pelle; Mat Yamage; Duncan M. Mwangi; Yoshikazu Honda; Ramadhan S. Mwakubambanya; Etienne P. de Villiers; Evelyne Abuya; Elias Awino; James Gachanja; Ferdinand Mbwika; Anthony M. Muthiani; Cecelia Muriuki; John K. Nyanjui; Fredrick O. Onono; Julius Osaso; Victor Riitho; Rosemary Saya; Shirley A. Ellis; Declan J. McKeever; Niall D. MacHugh; Sarah C. Gilbert; Jean-Christophe Audonnet; W. Ivan Morrison; Pierre van der Bruggen; Evans Taracha

ABSTRACT 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.


Veterinary Research | 2011

A minor role of CD4+ T lymphocytes in the control of a primary infection of cattle with Mycoplasma mycoides subsp. mycoides.

Flavio Sacchini; Jan Naessens; Elias Awino; Martin Heller; Andreas Hlinak; Wolfram Haider; Anja Sterner-Kock; Joerg Jores

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.


Veterinary Immunology and Immunopathology | 2012

Cytotoxic T lymphocytes from cattle immunized against Theileria parva exhibit pronounced cross-reactivity among different strain-specific epitopes of the Tp1 antigen

Lucilla Steinaa; R. Saya; Elias Awino; Philip G. Toye

The protozoan parasite Theileria parva causes a usually fatal disease in cattle, known as East Coast fever. Cattle can be vaccinated by injecting live parasites simultaneously with long acting oxytetracycline (the infection and treatment method, ITM). The immunity induced by ITM is believed to be mediated by cytotoxic T lymphocytes (CTL). Although effective, the ITM vaccine has disadvantages such as the need for a liquid nitrogen cold chain and a complex production process, which may be overcome by the development of a subunit vaccine. However, the high level of antigenic polymorphism among different strains of T. parva may hinder the development of a subunit vaccine aimed at induction of a protective CTL response. In this study, the CTL cross-reactivity among T. parva strains was examined. The Tp1(214-224) epitope has previously been shown to be recognized by cattle of the A18 BoLA type. Three different variants of this epitope have been identified from different T. parva strains. Here, bulk CTL and CTL clones were generated from two animals using both the live sporozoite vaccine composed of three different strains and a Muguga strain for immunization. The cross-reactivity of these CTL with the three variant Tp1 epitopes was examined in interferon gamma ELISPOT assays and CTL killing assays. CD8(+) cells from both animals cross-reacted with the three variant CTL epitopes in interferon gamma ELISPOT assays, although the CD8(+) cells from the Muguga-immunized animal showed a more epitope restricted response. Clones from the vaccine immunized animal showed diverse response patterns with clones responding to each variant peptide. Although some variability in the cytotoxic response was observed, overall strong cross-reactivity among the variant Tp1 epitopes was seen in both animals. Such epitope polymorphism does not, in this case, serve as a potential challenge in a putative subunit vaccine as it would be sufficient to only include one of the variant epitopes.


Infection and Immunity | 2012

A Theileria parva Isolate of Low Virulence Infects a Subpopulation of Lymphocytes

Heshborne S. Tindih; Dirk Geysen; Bruno Goddeeris; Elias Awino; Dirk A. E. Dobbelaere; Jan Naessens

ABSTRACT 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.


Veterinary Immunology and Immunopathology | 2015

BoLA-6*01301 and BoLA-6*01302, two allelic variants of the A18 haplotype, present the same epitope from the Tp1 antigen of Theileria parva

Nicholas Svitek; Elias Awino; Vishvanath Nene; Lucilla Steinaa

We have recently shown that the BoLA-A18 variant haplotype (BoLA-6*01302) is more prevalent than the BoLA-A18 haplotype (BoLA-6*01301) in a sample of Holstein/Friesian cattle in Kenya. These MHC class I allelic variants differ by a single amino acid polymorphism (Glu97 to Leu97) in the peptide-binding groove. We have previously mapped an 11-mer peptide epitope from the Theileria parva antigen Tp1 (Tp1214-224) that is presented by BoLA-6*01301. Crystal structure data indicates that Glu97 in the MHC molecule plays a role in epitope binding through electro-static interaction with a lysine residue in position 5 of the epitope, which also functions as an additional anchor residue. In contrast to expectations, we demonstrate that the amino acid substitution in BoLA-6*01302 does not divert the CTL response away from Tp1214-224. The two MHC molecules exhibit similar affinity for the Tp1 epitope and can present the epitope to parasite-specific CTLs derived from either BoLA allelic variants. These data confirm that this BoLA polymorphism does not alter Tp1 epitope specificity and that both allelic variants can be used for Tp1 vaccine studies.


Methods of Molecular Biology | 2016

Analysis of the Cellular Immune Responses to Vaccines

Nicholas Svitek; Evans Taracha; Rosemary Saya; Elias Awino; Vishvanath Nene; Lucilla Steinaa

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.


Immunogenetics | 2016

Sequence diversity between class I MHC loci of African native and introduced Bos taurus cattle in Theileria parva endemic regions: in silico peptide binding prediction identifies distinct functional clusters

Isaiah Obara; Morten Nielsen; Marie Jeschek; Ard M. Nijhof; Camila J. Mazzoni; Nicholas Svitek; Lucilla Steinaa; Elias Awino; Cassandra Olds; Ahmed Jabbar; Peter-Henning Clausen; Richard P. Bishop

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.


npj Vaccines | 2018

An Ad/MVA vectored Theileria parva antigen induces schizont-specific CD8+ central memory T cells and confers partial protection against a lethal challenge

Nicholas Svitek; Rosemary Saya; Elias Awino; Stephen Munyao; Robert Muriuki; Thomas Njoroge; Roger Pelle; Nicholas N. Ndiwa; Jane Poole; Sarah C. Gilbert; Vishvanath Nene; Lucilla Steinaa

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.East Coast fever: Developing an accessible vaccine for African farmersA vaccine expressing parasitic proteins offers more convenient East Coast fever prophylaxis. Current vaccination for the cattle disease, caused by the parasite Theileria parva and a detriment to sub-Saharan African farmers, involves inconvenient injection with live parasites before antibiotic treatment (ITM). A collaboration led by Nicholas Svitek, of the Kenyan International Livestock Research Institute, designed a candidate to provoke cellular immune responses against the parasitic antigen Tp1—an ITM vaccine candidate. In tests on cattle, 93% created Tp1-targeting T cells, and 33% survived a lethal dose of T. parva. The East Coast fever reduction seen in animals in this research outperformed a recent study and was able to generate the same immune memory cells that ITM inspires to provide long-lasting protection. Future research might integrate more antigens with this Tp1 vaccine to provide more comprehensive protection.


Vaccine | 2018

Immune parameters to p67C antigen adjuvanted with ISA206VG correlate with protection against East Coast fever

Anna Lacasta; Stephen Mwalimu; Elisabeth Kibwana; Rosemary Saya; Elias Awino; Thomas Njoroge; Jane Poole; Nicholas N. Ndiwa; Roger Pelle; Vishvanath Nene; Lucilla Steinaa

Highlights • Three doses of p67C antigen generated stronger immune responses than two doses.• Antibody titers and CD4+ T-cell proliferation correlated with protection against ECF.• The number of doses could not be reduced from three to two without compromising the protection.


BMC Veterinary Research | 2018

Immunization with one Theileria parva strain results in similar level of CTL strain-specificity and protection compared to immunization with the three-component Muguga cocktail in MHC-matched animals

Lucilla Steinaa; Nicholas Svitek; Elias Awino; Thomas Njoroge; Rosemary Saya; Ivan Morrison; Philip G. Toye

BackgroundThe 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).ResultsFour 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.ConclusionWe 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.

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Rosemary Saya

International Livestock Research Institute

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Lucilla Steinaa

International Livestock Research Institute

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Nicholas Svitek

International Livestock Research Institute

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Vishvanath Nene

International Livestock Research Institute

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Roger Pelle

International Livestock Research Institute

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Evans Taracha

International Livestock Research Institute

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John K. Nyanjui

International Livestock Research Institute

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