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Ticks and Tick-borne Diseases 7 (2016) 549–564

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Ticks and Tick-borne Diseases

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eview Article

he biology of Theileria parva and control of East Coast fever – Currenttatus and future trends

ishvanath Nenea,b,∗, Henry Kiaraa, Anna Lacastaa, Roger Pellea, Nicholas Sviteka,ucilla Steinaaa

International Livestock Research Institute, Old Naivasha Road, P.O. Box 30709, Nairobi 00100, KenyaAdjunct Faculty, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, United States

r t i c l e i n f o

rticle history:eceived 16 September 2015eceived in revised form 1 February 2016ccepted 2 February 2016vailable online 26 February 2016

eywords:heileria parvaast Coast feveraccines

a b s t r a c t

Tremendous progress has been made over the last ten years on East Coast fever (ECF) research. Publi-cation of a reference genome sequence of Theileria parva, the causative agent of ECF, has led to a morethorough characterization of the genotypic and antigenic diversity of the pathogen. It also facilitatedidentification of antigens that are targets of bovine major histocompatibility complex class I restrictedcytotoxic T-lymphocytes (CTLs), induced by a live parasite-based infection and treatment method (ITM)vaccine. This has led to improved knowledge of epitope-specific T-cell responses to ITM that most likelycontribute to the phenomenon of strain-specific immunity. The Muguga cocktail ITM vaccine, which pro-vides broad-spectrum immunity to ECF is now a registered product in three countries in eastern Africa.Effort is directed at improving and scaling up the production process to make this vaccine more widely

nfection and treatmentntigens

available on a commercial basis in the region. Meanwhile, research to develop a subunit vaccine based onparasite neutralizing antibodies and CTLs has been revived through convening of a research consortiumto develop proof-of-concept for a next generation vaccine. Many new scientific and technical advancesare facilitating this objective. Hence, the next decade promises even more progress toward an improvedcontrol of ECF.

© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5502. A background to East Coast fever, a lethal bovine disease present in sub-Saharan Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5503. Theileria parva exhibits a complex life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5514. Host cell transformation and morbidity caused by the schizont life-cycle stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5515. Genotypic diversity is a hallmark of Theileria parva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5516. A live sporozoite-based vaccine for the control of ECF via infection and treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5537. Chemotherapy of East Coast fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5548. A role for humoral immune responses in mediating immunity to East Coast fever. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .554

8.1. Identification and characterization of antigens that are targets of neutralizing antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5568.2. Immunization with recombinant p67 can protect against East Coast fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556

9. A role for cellular immune responses in mediating immunity to East Coast fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

9.1. Identification and characterization of CD8+ T-cell antigens . . . . . . .

9.2. A role for NetMHCpan in the identification and characterization o9.3. Epitope specificity of the CTL response in cattle immunized by th

Abbreviations: BoLA, bovine leukocyte antigen; CD, cluster of differentiation; cM, cemmunospot assay; IFN-�, interferon-�; ITM, infection and treatment method; mAb, monf tandem repeat.∗ Corresponding author at: International Livestock Research Institute, Old Naivasha Ro

E-mail address: v.nene@cgiar.org (V. Nene).

ttp://dx.doi.org/10.1016/j.ttbdis.2016.02.001877-959X/© 2016 The Authors. Published by Elsevier GmbH. This is an open access artic.0/).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557f CTL epitopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558e infection and treatment method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

ntiMorgan; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linkedoclonal antibody; MHC, major histocompatibility complex; VNTR, variable number

ad, P.O. Box 30709, Nairobi 00100, Kenya.

le under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

550 V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564

9.4. The challenge of priming CD8+ CTLs in cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55810. Desirable qualities in a subunit vaccine for the control of East Coast fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55911. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .559

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560. . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Theileriosis caused by the hemoprotozoan pathogen Theileriaarva still ranks first among the tick-borne diseases of cattle inub-Saharan Africa (Minjauw and McLeod, 2003). Organisms inhe genus Theileria are found in many wild and domesticatednimals (reviewed in Norval et al., 1992). They are phylogeneti-ally most closely related to members of the Babesia genus andall in the order Piroplasmida under the phylum ApicomplexaGou et al., 2012; Kuo et al., 2008; Lack et al., 2012; Levine,985; Morrison, 2009a; Schnittger et al., 2012). Theileria parva, T.nnulata and T. lestoquardi feature prominently among pathogenicpecies of Theileria that affect domesticated ruminants (reviewedn Norval et al., 1992). They share a unique biology of beingble to transform a subset of infected mononuclear host cells,hich can be cultured in vitro as persistently infected cell lines.ot all species of Theileria can transform mononuclear host cells,.g., T. orientalis, T. mutans and T. velifera. If such species causeisease it is mainly due to multiplication of the parasite life-ycle stage within red blood cells (reviewed in Norval et al.,992).

It is believed that T. parva co-evolved with the African Capeuffalo, in which infection does not cause disease, and has under-one a “host jump” to cattle, where it causes the disease referredo as East Coast fever (ECF) (reviewed in Norval et al., 1992). Differ-nces in the epidemiology, clinical symptoms, and parasitologicalarameters in cattle associated with different T. parva infectionsave led, in the past, to sub-speciation of T. parva. This nomencla-ure was abandoned (Perry and Young, 1993), and parasite isolatesre now described as cattle- or buffalo-derived (Anon, 1988a,b).owever, in southern Africa, cattle-derived T. parva is associatedith a less virulent disease than ECF, called January disease in

imbabwe (reviewed in Latif and Hove, 2011). The disease causedy buffalo-derived T. parva is called Corridor disease, and it issually more acute than ECF (reviewed in Norval et al., 1992).nother characteristic of the latter is that it is extremely diffi-ult to maintain buffalo-derived parasites through serial passageetween ticks and cattle, whereas this is relatively easy with cattle-erived parasite isolates (Young and Purnell, 1973). This scenario

s complicated by the presence of multiple strains in cattle- anduffalo-derived parasite populations, with possible co-infectionsetween them in individual animals (Conrad et al., 1987; Manst al., 2015; Oura et al., 2003, 2004, 2005, 2011). In evolutionaryerms, the change in host-species is likely to have occurred rel-tively recently as cattle were introduced to Africa ∼5000 yearsgo (Epstein, 1971). Although molecular data do not currently sup-ort sub-species status of T. parva (Allsopp et al., 1993; Conradt al., 1989), the biological observations suggest the presence ofwo segregating populations of the parasite (see also Mans et al.,015). The importance of the parasite buffalo reservoir in eval-ating approaches to ECF control is variable depending on thearming system, geographical region and wildlife-cattle interfaceGachohi et al., 2012; Norval et al., 1992). The remainder of thiseview concentrates on aspects of the biology of T. parva and

ecent advances in vaccine-related research, which is helping tohape the development of a subunit vaccine for the control ofCF.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

2. A background to East Coast fever, a lethal bovine diseasepresent in sub-Saharan Africa

In the early 1900s, Dr. Arnold Theiler recognized that Theileriaparva was the causative agent of ECF in South Africa (Theiler, 1912).He distinguished ECF from Redwater caused by Babesia, and identi-fied the principal tick vector that transmits T. parva as Rhipicephalusappendiculatus. The disease was associated with cattle importedfrom East Africa, and caused high levels of morbidity and mortality(reviewed in Norval et al., 1992). South Africa eradicated the dis-ease through a strict control of cattle movement, surveillance withslaughter of infected cattle, and fencing to restrict buffalo to gameparks. Occasional outbreaks of buffalo-derived T. parva disease stilloccur, but appear to be self-limiting in nature (Mbizeni et al., 2013;Thompson et al., 2008). It has not been possible to implement suchmeasures in other countries as a sustainable method of diseasecontrol. Hence, ECF remains an acute and usually lethal diseasepresent in 12 countries in eastern, central and southern Africa,including Burundi, Democratic Republic of Congo, Kenya, Malawi,Mozambique, Rwanda, South Sudan, Tanzania, Uganda, Zambia andZimbabwe (reviewed in Norval et al., 1992; Malak et al., 2012), and,more recently, in the Comoro Islands (De Deken et al., 2007).

On a regional basis ECF kills ∼1 million cattle/year with annualeconomic losses of ∼USD300 million (McLeod and Kristjanson,1999). These estimates were derived many years ago and currentimpacts are likely to be much larger. ECF can establish in new areasdue to a wider distribution of the tick vector and/or suitable tickhabitats (Norval et al., 1992), as occurred in the Comoro Islandsin 2004 (De Deken et al., 2007). Recent political changes in SouthSudan has led to a growing incidence of ECF in a naïve popula-tion of cattle with mortality rates close to 80% (Malak et al., 2012;Marcellino et al., 2012). This spread poses a direct threat to cat-tle in the Central African Republic and regions to the west. To theeast, although ECF has not been described in Ethiopia, this coun-try houses the largest population of cattle in Africa, and predictivemodels indicate that the tick vector could thrive in the Ethiopianhighlands (Norval et al., 1991).

There is some evidence that Bos taurus cattle are more sus-ceptible to ECF than Bos indicus animals, and there may be breeddifferences in susceptibility to disease (Norval et al., 1992). Theseverity of ECF caused by T. parva is parasite dose-dependent andthere also appear to be differences in the threshold of infectivityin individual cattle (Cunningham et al., 1974; Dolan et al., 1984).These observations could obscure true host genetic differences insusceptibility to ECF. Cattle that exhibit mild or moderate ECF clin-ical symptoms are considered resistant to disease, while those thatexhibit severe clinical reactions are judged to be susceptible (Anon,1988a). Cattle can spontaneously recover from infection, usuallyafter a mild or moderate reaction, and are solidly immune to re-infection. This phenomenon contributes to endemic stability ofthe disease in indigenous cattle in regions where there is contin-ual transmission of the parasite (Medley et al., 1993; Norval et al.,1992). Animals that recover from ECF do not usually eliminate theinfection, they remain as carriers of the parasite and a source of

infections to ticks (Kariuki et al., 1995). The recent observation ofan apparent protective effect in young cattle due to co-infectionswith non-pathogenic Theileria raises interesting new questions on

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he epidemiology of ECF (Thumbi et al., 2014; Woolhouse et al.,015).

. Theileria parva exhibits a complex life cycle

Understanding the life cycle of the parasite is important as itrovides information that may be used to improve disease con-rol strategies, and for identifying novel targets of intervention.heileria parva undergoes a series of sequential developmentalhanges in the tick and bovine host (Fig. 1). These changes haveeen primarily characterized at the morphological level throughlegant electron microscopy studies (Fawcett et al., 1981, 1982a,987; Shaw and Tilney, 1992; Shaw, 1996b). Unfortunately, notuch is known about the molecular details of parasite-host cell

nteraction or pathways that control parasite differentiation intoifferent life-cycle stages. Rhipicephalus appendiculatus is a three-ost tick and the principal vector for transmission of T. parva,hich occurs trans-stadially (reviewed in Norval et al., 1992).

hus, tick larvae and nymphs acquire an infection from the piro-lasm stage present in red blood cells (RBCs) in infected cattle.

nfected nymphs and adults transmit sporozoites, the life-cycletage infective to cattle, during feeding. In vitro, sporozoites canind and rapidly enter lymphocytes via a passive “zippering pro-ess” of sporozoite and host cell membranes (Fawcett et al., 1982c;awcett and Stagg, 1986; Shaw, 1996a, 1997). Unlike other api-omplexan organisms, T. parva sporozoites are non-motile andpherical/ovoid in shape with a diameter of 0.75–1.5 �m (Fawcettt al., 1982a,b). They do not have well developed apical com-lexes and host cell entry is not orientation specific (reviewed inhaw, 2003). Discharge from sporozoite secretory organelles, rhop-ries/microspheres, is seen after host-cell entry, and coincides withapid escape from the surrounding host membrane. Sporozoitesifferentiate into the schizont stage, which is a multinucleatedtage that resides free in the host cell cytoplasm surrounded by

basket of host cell microtubules that seem to be nucleatedy parasite molecules (reviewed in Shaw, 2003). These infectedells acquire a metastatic, cancer-like phenotype and are the pri-ary cause of pathology (reviewed in Dobbelaere and Heussler,

999). Sporozoites also enter macrophages and dendritic cells,ut only develop to an early stage of the schizont (Shaw et al.,993).

Schizonts undergo a process of merogony to produce mero-oites, which are released by host cell rupture. Merozoites invadeBCs, where they develop into the piroplasm stage. The mecha-ism of merozoite entry into RBCs is similar to that of sporozoitentry into lymphocytes and merozoites develop into the piro-lasm stage, free in the RBC cytoplasm (Shaw and Tilney, 1995).iroplasms undergo a limited number of cell divisions (Conradt al., 1986) and anemia due to destruction of infected RBCs isot a prominent feature of ECF (reviewed in Norval et al., 1992).he piroplasm stage is infective to ticks and differentiation toametes seems to occur post-ingestion in the tick gut (reviewed inorval et al., 1992). Following fusion of macro- and micro-gametes,ygotes enter cells of the tick gut epithelium and develop intootile kinetes, which are released into the tick hemocel. Sporo-

ony occurs in the e-cells in type III acini (Fawcett et al., 1982b).elease of Theileria sporozoites from the salivary glands occurss a trickle between day 4–8 post-attachment (Shaw and Young,995). Approximately 30,000–50,000 sporozoites develop in an

nfected acinus (Fawcett et al., 1982a,b). All life cycle stages of. parva, except the zygote, are haploid in nature and zygotes

ndergo a two-step meiotic division (Gauer et al., 1995). Theexual cycle of T. parva in the tick vector results in genera-ion of extensive parasite genotypic and antigenic diversity (seeelow).

Diseases 7 (2016) 549–564 551

4. Host cell transformation and morbidity caused by theschizont life-cycle stage

ECF is described as an acute, lymphoproliferative disease associ-ated with pathology induced by schizont-infected cells, and deathis usually caused by pulmonary edema (reviewed in Norval et al.,1992). Full schizogony of T. parva only occurs in B- and T-cells(Baldwin et al., 1988), where the parasite causes the host cell toproliferate. The schizont divides in synchrony with the host cell sothat daughter host cells remain infected (Irvin et al., 1982). In vivoand in vitro most transformed cell lines are of the T-cell lineage,and higher levels of pathogenicity are associated with infected T-cells rather than infected B-cells (Morrison et al., 1996). Growthof infected cells is rapid, independent of exogenous growth fac-tors, and completely dependent on the presence of live parasitesas treatment with anti-theilerial drugs, e.g., buparvaquone, resultsin cells reverting to a non-proliferative state (Brown et al., 1989a;Dobbelaere et al., 1991; Heussler et al., 1992).

In contrast to T. parva, T. annulata transformsmacrophages/dendritic cells and B-cells, but not T-cells(Dobbelaere and Heussler, 1999; Spooner et al., 1989). Theseobservations indicate that a two-way communication betweenparasite and host cell type plays a role in the process of hostcell transformation. However, the situation appears to be morecomplex as not all infected lymphocytes become transformed(Morzaria et al., 1995; Rocchi et al., 2006). It is beyond thescope of this review to cover excellent research on the topic ofmolecular mechanisms that contribute to host cell transformationand the reader is directed to several publications (Chaussepiedand Langsley, 1996; Chaussepied et al., 2010; Cheeseman andWeitzman, 2015; Dobbelaere and Rottenberg, 2003; Heussleret al., 2002; Marsolier et al., 2015; Medjkane et al., 2014; Shielset al., 2006; Tretina et al., 2015; von Schubert et al., 2010).Understanding the mechanisms by which Theileria organismscause mammalian cells to transform has an obvious relevance tocancer biology. Disruption of these mechanisms may offer noveldrug-targets for the control of ECF (Marsolier et al., 2015).

5. Genotypic diversity is a hallmark of Theileria parva

A reference genome sequence for T. parva and T. annulata waspublished in 2005 (Gardner et al., 2005; Pain et al., 2005). Areference sequence for other Theileria species (Hayashida et al.,2012; Kappmeyer et al., 2012) and whole genome sequence datafrom other strains/isolates of T. parva have also been published(Hayashida et al., 2013; Henson et al., 2012; Norling et al., 2015).These resources provide essential baseline data to study T. parvapopulation structure, antigenic diversity, and facilitate reverse vac-cinology approaches for vaccine development. In contrast to otherApicomplexa, e.g., Plasmodium and Babesia (Jackson et al., 2014;Singh et al., 2014), a prominent role for members of the major T.parva multi-gene families (Gardner et al., 2005) as antigens remainsto be established.

In general, buffalo-derived parasites are more diverse thancattle-derived parasites. A number of loci (Allsopp and Allsopp,1988; Bishop et al., 1995, 1998; Conrad et al., 1987; Katzer et al.,2010; Pelle et al., 2011) and variable number of tandem repeat(VNTR) markers (mini- and micro-satellites) derived from the T.parva genome sequence data have been used as markers to deter-mine parasite diversity (Oura et al., 2003, 2004, 2005, 2011; Patelet al., 2011). Such studies are influenced by the distribution, den-

sity, and type of markers across the genome, and current resultsare preliminary in nature. A study of parasite population struc-ture using VNTRs (Oura et al., 2003, 2004, 2005, 2011) indicatedthat there was no obvious relationship between geographical origin

552 V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564

Fig. 1. Life-cycle of Theileria parva. The figure illustrates the different life cycle stages of the parasite as it cycles through the mammalian and tick host. The figure wasinspired by fluorescence and electron micrograph images of the parasite life cycle (Fawcett et al., 1982a; Norval et al., 1992; von Schubert et al., 2010). Stages of the lifecycles are not drawn to scale. Sporozoites enter lymphocytes where they develop to the schizont stage, a process that results in host cell transformation resulting in clonalexpansion of schizont-infected cells. The schizont stage is polyploid and multiple schizont nuclei are observed during host cell interphase. It is during the metaphase periodof the cell cycle that schizont division occurs (Irvin et al., 1982). Some of the schizonts undergo merogony, giving rise to merozoites, which mature into the piroplasm stage inRBCs, and are infective to ticks. Tick larvae and nymphs acquire an infection by feeding on infected cattle or buffalo, and transmit the parasite as the next tick instar, nymphs and

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nd level of genetic similarity between parasite isolates, such thatifferent parasite isolates from the same farm exhibited distinctenotypes. Older cattle tended to have a larger number of differ-nt parasite genotypes than younger ones, which tended to have

pre-dominating genotype (Oura et al., 2005). Some geographi-al sites exhibited a sub-structure in parasite populations, othersid not, and some exhibited an epidemic structure characteristicf recent predominating infections (Oura et al., 2005). Thus, it haseen suggested that cattle movement, their interface with buffalo,nd parasite transmission rates are likely to play a major role inetermining T. parva parasite population structures (Oura et al.,003).

VNTR mapping the progeny from two cattle-derived parasiteloned stocks after transit through the tick vector has led to aenetic linkage map for T. parva (Katzer et al., 2011), and identi-cation of regions with higher (hot spots) and lower (cold spots)ates of recombination than a genome-wide average. Using a phys-cal size of ∼8.3 Mbp of the T. parva genome (Gardner et al., 2005)he total genetic size was calculated at 1683.8 cM (Katzer et al.,011). This map provides an important reference for placement ofenes into regions with different rates of recombination. Wholeenome sequence data from a few different cattle- and buffalo-erived parasite isolates mapped to the reference genome has

ed to identification in excess of 500,000 single-nucleotide poly-orphisms (SNPs) (Hayashida et al., 2013; Henson et al., 2012).

NP density/kbp was 2–4-fold higher in buffalo-derived parasites,ore prevalent in coding than non-coding regions, and has led

o identification of SNP-rich and SNP-poor chromosomal regionsHayashida et al., 2013). Sequence data have also identified recom-ination events, and a large number of crossover events lead toene conversion (Henson et al., 2012). Interestingly, phylogeneticelationships as measured by SNPs detected in the coding regionsf 200 random selected genes place the sequences derived fromuffalo-derived parasites into a separate cluster from the sequencesf cattle-derived parasites (Norling et al., 2015). These data begino reveal the genetic complexity of the parasite nuclear genome,vents leading to diversity between different strains, and a list ofrotein coding genes that are under different rates of evolutionaryressure (Hayashida et al., 2013; Henson et al., 2012). Unravelingow such observations relate to genes that play a role in medi-ting host and vector interactions, the differences in the biologyetween cattle- and buffalo-derived parasites, and to immunity toCF, remains a fascinating prospect.

. A live sporozoite-based vaccine for the control of ECF vianfection and treatment

There is solid evidence from both field observations as well asrom laboratory-based studies that cattle acquire robust immu-ity to ECF following recovery from infection (reviewed in Norvalt al., 1992). As the infection and treatment method (ITM) namemplies, the process involves deliberate infection of cattle with con-omitant drug treatment. The latter controls but does not kill thearasite permitting generation of a protective acquired immuneesponse. The ITM process has evolved over several decades start-

ng in South Africa. Initial studies were based on injecting cattle

ith heavily parasitized spleen and lymph node homogenatesbtained from infected cattle slaughtered in the last stages of theisease (Theiler, 1912). This gave way to the finding that prolonged

dults. There is no transovarial transmission of the parasite (reviewed in Norval et al., 19here sporogony occurs. Mature merozoites (Mz) and sporozoites (Sz) originate from a m

espectively. See main text for additional details. The vertebrate host cell nucleus is colnd micronemes in merozoites are depicted as small dark green dots inside the parasiterawings and artistic creation by N. Svitek.

Diseases 7 (2016) 549–564 553

intra-venous tetracycline cover during natural T. parva infectionsresulted in immunity to ECF (Neitz, 1953). The process of usingtriturated infected tick suspensions, a method for cryopreservingthem, and titration of the infectivity of frozen sporozoite stabilates(Cunningham et al., 1973, 1974) provided a source of standardizedparasite material for vaccination. Development and optimizationof first short, and subsequently long-acting formulations of oxy-tetracyclines to control the infections played an important role indevelopment of the ITM protocol (Radley, 1981). It has been demon-strated that at low dilutions the Boleni isolate of T. parva fromZimbabwe can be used to immunize cattle in the absence of tetra-cycline (reviewed in Latif and Hove, 2011), but it has been difficultto replicate this in other parasite isolates.

It was discovered early on that exposure of immune cattle toparasite isolates from different geographical sites could result inbreak-through infection and disease in some animals, giving rise tothe concept of strain/isolate specific immunity (Young et al., 1973).Although individual parasite isolates can be placed in differentcross-immunity groups, there is usually 20–30% cross-protectionbetween them (Morzaria, 1996). Combining different isolatesbroadens the vaccine coverage of ITM. Hence, the Muguga cocktail,which consists of three T. parva isolates (called Muguga, Serengeti-transformed and Kiambu-5) was shown to provide broad-spectrumimmunity to ECF (Radley et al., 1975a,b). Developed in principlealmost 50 years ago, the Muguga cocktail ITM vaccine has hada checkered history in its translation to a commercially availableproduct as it is time consuming and difficult to produce, requiresa liquid nitrogen cold chain for its storage, is potentially a lethalproduct if not given with adequate antibiotic cover, and is expen-sive.

The hurdles in producing and delivery of the Muguga cock-tail can be overcome, and the vaccine has proved to be highlyeffective in controlling ECF (Di Giulio et al., 2009). In response toa FAO/multi-donor regional program, the International LivestockResearch Institute (ILRI) produced ∼600,000 doses of the cocktailin two batches, called FAO1 and FAO2 (Morzaria et al., 1997). Use ofa higher oxytetracycline dose than before reduced the number ofclinical reactors to the vaccine, eliminating need for a second doseof antibiotic treatment and visit by the service provider after immu-nization. This, together with more extensive use of the vaccine inpastoral areas of Tanzania has played a major role in increasingthe demand for the cocktail vaccine (Di Giulio et al., 2009). Thenext commercial batch of the cocktail of ∼1 million doses, calledILRI08, was made by ILRI (Patel et al., 2015) in recognition of deplet-ing stocks of the FAO vaccine batches, and in response to a majorinitiative to invest in commercialization of the Muguga cocktail(GALVmed, 2010). An indication of the success of this revitaliza-tion is that the ILRI08 batch was sold out in just five years, andre-investments in the Center for Ticks and Tick-Borne Diseases(CTTBD) in Malawi to become the site of production and distri-bution of future batches of the cocktail as a commercial enterpriseare near to fruition (GALVmed, 2014). The Muguga cocktail has beenregistered as a vaccine in Malawi, Kenya and Tanzania, and no doubtmore lessons will be learnt as vaccine production is scaled up anddelivered through public and private veterinary services.

It is puzzling how three T. parva isolates in the Muguga cocktail,selected from a limited geographical area, have shown protec-tion across such a wide geographical area (Di Giulio et al., 2009;Norval et al., 1992; Radley et al., 1975a,b). Using a very limited

92). Kinetes, the final products of the sexual cycle, invade the tick salivary glandsultinucleated residual body (Rb) during the process of merogony and sporogony,

ored in purple and the parasite nucleus is in orange. Microspheres in sporozoites. Host cell microtubules in the dividing schizont-infected cell are drawn in green.

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anel of five VNTR markers on schizont-infected cell lines estab-ished from the components of the Muguga cocktail revealed theresence of 17 genotypes in the cocktail (Patel et al., 2011). Theaccine strains present in the ILRI08 cocktail batch also exhibitimited diversity of genes encoding the known candidate vaccinentigens (Hemmink, Weir, MacHugh, Graham, Shiels, Toye, Mor-ison and Pelle, unpublished manuscript). Recent whole genomeequence data derived from the piroplasm stage of parasite compo-ents of the Muguga cocktail revealed only 957 SNPs between theerengeti-transformed and Muguga sequence, but close to 40,000NPs between the Kiambu-5 and Muguga sequence (Norling et al.,015). The sequence differences between Serengeti-transformednd Muguga were primarily restricted to sub-telomeric regions,hile those between Kiambi-5 and Muguga were located across the

enome. The latter altered the sequences of almost half of the par-site protein coding genes. A curious feature of the sequence dataas an absence of mixed sequences, suggesting that the Serengeti-

ransformed and Kiambu-5 piroplasm DNA sequenced were clonaln nature (Norling et al., 2015). Although the true level of hetero-eneity in the Muguga cocktail remains to be determined, the veryigh degree of sequence similarity between Serengeti-transformednd Muguga questions the origin of the former, and the value of itsrotective role in the Muguga cocktail (Norling et al., 2015). Unfor-unately, our current tools for typing parasite genotypic diversityo not predict cross-immunity profiles. Nevertheless, moleculararkers can be used to determine identity, and as quality con-

rol tools to ensure that the composition of strains present in theuguga cocktail remain relatively constant during the complex

roduction process of the vaccine.As mentioned previously, parasite isolates placed in different

ross-immunity groups usually exhibit 20–30% cross-protectionetween them (Morzaria, 1996). Sequential immunization by ITMith single isolates in different cross-immunity groups provides

dditive immunity (Taracha et al., 1995b). This suggests that somerotective antigens are shared between different parasite isolates,nd the breadth of the immune response can be expanded inesponse to new infections. Novel genotypes can be found in cat-le vaccinated with the Muguga cocktail (Oura et al., 2004). This

ay help to further broaden the spectrum of immunity to strainsf the parasite not present in the cocktail. Currently, there is noimple answer as to whether buffalo-derived parasites will break-hrough the Muguga cocktail. For example, the cocktail has provedighly effective in pastoral areas of Tanzania where cattle are likelyo be exposed to buffalo-derived parasites (Di Giulio et al., 2009).ecent experiments in Kenya demonstrate that this immunity isusceptible to buffalo-derived parasites on the Ol Pejeta ranch inaikipia district (Sitt et al., 2015) but not in the Narok County areaMukholi, Kitala, Gathuma, Kidula, Turasha, Mbwira, Mugambi,iara, in preparation). Hence, buffalo-derived parasites may remain

confounding factor in some areas where the Muguga cocktail iseployed and will have to be evaluated on an empirical basis.

There still remains lingering concern about the long-termmpact that exotic T. parva strains might have on local parasiteopulations when used outside the region of origin of vaccinetrains leading to a debate on the use of autogenous ITM vac-ines versus cocktail vaccines (Geysen, 2008; McKeever, 2007). Forxample, following some apparent complications due to use of theuguga cocktail, Zambia has chosen to use local isolates from the

astern and southern provinces to control ECF (Nambota et al.,994). Fortunately, in Zimbabwe, a single parasite stock (Boleni)rovides broad-spectrum immunity within the country (reviewed

n Latif and Hove, 2011). The concern of using exotic parasites

evolves around the observation that cattle can remain as carriersf vaccine stocks, and that these may recombine with local parasitetrains during tick transmission giving rise to new more viru-ent parasite variants and upset the balance of endemic stability.

Diseases 7 (2016) 549–564

However, the Muguga cocktail ITM vaccine has been used in differ-ent livestock production systems and geographical areas over manyyears (Uilenberg, 1999), without any major reports of a changein the epidemiology of ECF or large-scale failure of the vaccine,although it has been shown that components of the cocktail canestablish itself locally (Oura et al., 2007). What is required is toeither dispel or confirm the concerns by establishing monitoringexperiments where a good baseline study of the clinical natureof the disease and a comprehensive study of the existing para-site genotypes are studied prior to vaccination, and longitudinalmonitoring is carried out for several years. In this way the riskfrom the use of live parasites in exotic locations could be betterassessed. For the moment ITM remains the only vaccine based solu-tion available for the control of ECF. Efforts are underway to developnext generation subunit vaccines but these are several yearsaway.

7. Chemotherapy of East Coast fever

Thirty years later buparvaquone still remains the front line com-mercial drug of choice for the treatment of T. parva (McHardy et al.,1985) and T. annulata infections (Dhar et al., 1986). However, tobe completely effective the drug needs to be administered earlyin infection (Martins et al., 2010). As with other hydroxynaptho-quinones the drug most likely functions by inhibiting electrontransport through the mitochondrion (Birth et al., 2014). Resis-tance to buparvaquone has been described in T. annulata (Mhadhbiet al., 2010) and correlates with mutations in the cytb gene(Mhadhbi et al., 2015; Sharifiyazdi et al., 2012). Hence, the recentidentification of a T. annulata nuclear gene encoding a secretedpeptidyl-prolyl isomerase, as an additional target of buparvaquoneresistance was unexpected (Marsolier et al., 2015). These findingsare of concern for the future control of T. annulata as drug resis-tance could spread. Although buparvaquone resistance in T. parvaremains to be documented it may just be a question of time beforethis occurs. Effort should be made to identify new anti-theilerialdrugs, leveraging on the drive to develop new anti-malarial drugs(Burrows et al., 2014).

8. A role for humoral immune responses in mediatingimmunity to East Coast fever

Cattle exposed to T. parva infection develop an antibodyresponse to several parasite proteins. Among these specificities,antibodies to the polymorphic immunodominant molecule (PIM)have proved to be the most reliable in measuring exposure to T.parva. PIM can be polymorphic in size between different T. parvastrains (Shapiro et al., 1987; Toye et al., 1995a), but there is suf-ficient conservation of sequences between the variants for PIM tofunction as a diagnostic antigen (Katende et al., 1998; Toye et al.,1996). Hence, an ELISA assay based on recombinant PIM from theMuguga stock of the parasite is routinely used to determine preva-lence (Gitau et al., 2000), and sero-conversion to the ITM vaccine(Patel et al., 2015).

Early studies on passive transfer of sera from immune cat-tle failed to protect against challenge (Muhammed et al., 1975).In addition, immunization with parasite antigen failed to protectagainst ECF (Wagner et al., 1974), leading to a consensus in theECF literature that antibodies did not play a major role in mediat-ing immunity to the disease. However, indirect evidence for a rolefor antibodies in mediating immunity to ECF was derived from the

observation that cattle can develop sporozoite neutralizing anti-bodies (Musoke et al., 1982). However, this activity is seen aftermultiple sporozoite exposure, suggesting weak immunogenicity ofthe relevant target/s.

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555Table 1Experimental vaccine trials in cattle using different p67 constructs and antigen delivery systems.

Immunogen p67 antigensequence

Dose Route Adjuvant Animal numbers Control Challenge dose andstabilate number

Outcome (SRreduction)

References

NS1-p67 (E. coli) 1–709 5 × 1 mg s.c. 3% SA (Merck) 9 immunized10 controls

Non immunizedandNS1 in saponin

LD68, Muguga 3087 66% Musoke et al. (1992)

BEV-p67 (insect) 1–709 5 × 300 �g CFA/IFA or3% SA (Merck)

6 immunized3 controls

Non immunized LD68, Muguga 3087 66% Nene et al. (1995)

NS1-p67 (E. coli) 1–709 5 × 600 �g 3% SA (Merck)12 immunized10 controls Non immunized

LD68, Muguga 3087 58% Neneet al.(1996)11 immunized

10 controls1:8 dilutionMarikebuni 3014

55%

p67 in attenuated Salmonella Mix of:1–70920–70920–622

3–4 × 1–2 × 109

c.f.ui.m. None 10 immunized

15 controlsNon immunizedand Salmonellaimmunized

Equivalent to oneinfected acinus,Muguga 3087 or 4133

25% Heussler et al.(1998)

p67 in attenuatedSalmonella

21–699 3 × 109–1011 c.f.u.secreting p67

i.m.None

3 immunized3 controls Non immunized LD80 Muguga 4133

66% Gentschev et al.(1998)

21–699 3 × 109–1011 c.f.u.secreting p67

oral 3 immunized3 controls

33%

Mix of:1–70920–70920–622

3 × 109–1011 c.f.u.p67

i.m. 3 immunized3 controls

33%

V-67-GPI in recombinantvaccinia virus

1–643 2 × 5 × 108 p.f.ui.d. None

7 immunized7 controls

Non immunizedLD70, Muguga 4133

0% Honda et al. (1998)

V583-IL2 in recombinantvaccinia virus

1–583 2 × 5 × 108 p.f.u 10 immunized20 controls

Non immunizedand V-IL2

20%

V-67-GPI + V-IL2 inrecombinant vaccinia virus

1–643 2 × 5 × 108 p.f.u 7 immunized7 controls

Non-immunized 51%

p67C (E. coli) 572–651 3 × 450 �gs.c.

RWL (Pfizer AnimalHealth)

7 immunized7 controls RWL immunized LD70, Muguga 4133

70% Bishop et al. (2003)

P67N (E. coli) 21–225 3 × 450 �g 7 immunized7 controls

13%

P67635 (E. coli) 17–635 3 × 450 �g 7 immunized7 controls

40%

GFP:p67DSS 21–651 2 × 100 �gs.c.

SA or WOE (bothIntervetInternational)

26 immunized (13SA and 13 WOE)15 controls

GFP in SA or WOELD70, Muguga 4133

51% in SA and43% in WOE

Kaba et al. (2005)

GP64:p67C (cell extract) 572–651 2 × 10 �g SA (IntervetInternational)

13 immunized15 controls

GFP in SA 36%

p67635 (E. coli) 17–635 3 × 450 �gs.c.

RWL (PfizerAnimal Health)and ISA206(SEPPIC)

42 immunized35 controls

Ovalbumin in RWLor ISA206 LD70, Muguga 4133

40% Musoke et al. (2005)

p67C (E. coli) 572–651 3 × 450 �g 14 immunized35 controls

69%

p67635 and p67C (E. coli)field experiment

17–635 and572–651respectively

3 × 450 �g RWL (PfizerAnimal Health)

83 immunized50 controls

Ovalbumin in RWL 26%

Results from published p67 vaccine trials in Bos indicus cattle are presented. The outcome of challenge is expressed as a percentage of cattle that were immune to challenge and was based on the severity of clinical reactions tochallenge, e.g., severe reactors are classified as susceptible to ECF. This was calculated by subtracting the percentage of non-severe reactors in the control group from that in the immunized group. Abbreviations: c.f.u. (colonyforming units); CFA (complete Freund’s adjuvant); GFP (green fluorescent protein); i.d. (intradermal); IFA (incomplete Freund’s adjuvant); i.m. (intramuscular); LD (lethal dose); p.f.u. (plaque forming units); SA (saponin); s.c.(subcutaneous); SR (severe reactor); WOE (water-in-oil emulsion).

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.1. Identification and characterization of antigens that areargets of neutralizing antibodies

Characterization of a series of murine mAbs that neutralizedporozoite infectivity in in vitro assays identified a ∼67 kDa protein,alled p67, as a major surface antigen of sporozoites, and as the pri-ary target of neutralizing mAbs (nmAbs) (Dobbelaere et al., 1984,

985; Musoke et al., 1984). The p67 protein probably contributeso the process of host cell recognition and entry, during which it ished (Webster et al., 1985). It is encoded by a single copy gene, and isredicted to contain an N-terminal signal peptide and a membranenchor domain (Nene et al., 1992). No sequence variation was foundn this gene in a set of cattle-derived T. parva isolates (Nene et al.,996). Allelic polymorphisms in p67 sequences are observed inuffalo-derived T. parva and in samples directly collected from cat-le (Sibeko et al., 2010). However, it is not entirely clear if the latterarasite types can be maintained through serial passage betweenicks and cattle. Sequence identity between full-length p67 alleles isreater than 90% (Nene et al., 1996). This suggests that the recentlyescribed diversity in a few p67 B-cell epitopes (Obara et al., 2015)

s unlikely to negatively influence the ability of p67 to functions a cross-protective immunogen, a prediction that remains to bealidated.

Host MHC class I molecules appear to facilitate sporozoite entrys mAbs to them can neutralize sporozoite infectivity, but they arenlikely to function as unique host cell receptors as sporozoiteso not bind or enter all nucleated cells (Shaw et al., 1995). Moreecently the function of p67 as a primary ligand has been broughtnto question. This is based on the observation that T. parva Chi-ongo sporozoites only binds to CD8+ T-cells yet the gene codingor p67 is identical in sequence to that of T. parva Muguga sporo-oites, which bind B-cells and all subsets of T-cells (Tindih et al.,010, 2012). Comparative molecular studies between sporozoitesf these two T. parva isolates should help to resolve their differencesn host cell tropism.

The PIM antigen was identified as a second target of nmAbshrough serendipity. A series of mAbs raised to the schizont stageas found to be specific for PIM, and has been used to type differ-

nt parasite strains (Minami et al., 1983; Shapiro et al., 1987). The T.arva PIM gene sequence is highly polymorphic in both cattle- anduffalo-derived parasites (Baylis et al., 1993; Geysen et al., 2004;oye et al., 1995a). Some of the mAbs were subsequently foundo neutralize sporozoite infectivity (Toye et al., 1995a), and lin-ar epitopes they bound were mapped by Pepscan analysis (Toyet al., 1996). PIM is localized to sporozoite rhoptries/micronemesToye et al., 1996). In contrast, PIM is located on the schizont sur-ace (Baylis et al., 1993; Shapiro et al., 1987), most likely as a

olecule that spans the surface membrane several times. The cDNAequence of sporozoite and schizont PIM is identical, so it is notlear how the molecule is targeted to different cellular compart-ents in the different life cycle stages. It is possible that nmAbs

ain access to PIM in a transient manner during sporozoite inva-ion to inhibit its function. However, the immunobiology of theIM antigen is quite complex as rats immunized with recombinantIM make neutralizing antibodies (Toye et al., 1995b) but cattleo not, although the bovine antibodies to PIM also bind to peptideequences bound by nmAbs (Toye et al., 1996). A role for PIM asn anti-sporozoite vaccine antigen remains to be explored, as it isossible that immunization of cattle with different constructs ofIM may induce neutralizing antibodies.

A third candidate target of neutralizing antibodies, a 32 kDa pro-ein, was identified through immunoprecipitation of biotin labeled

porozoites with the mAb 4C9. This protein proved to be a homologf the Tams1 antigen of T. annulata, a major surface antigen oferozoites (Shiels et al., 1995). Bovine antisera to recombinant

32 did not induce neutralizing antibodies and immunized cattle

Diseases 7 (2016) 549–564

were susceptible to challenge (Skilton et al., 2000). Hence, work onthis antigen was discontinued. The role of two other immunogenicsporozoite antigens (p104 and p150) as candidate vaccine antigenshas not been evaluated (Iams et al., 1990; Skilton et al., 1998).Based on the large number of apicomplexan parasite moleculesthat generally play a role in host cell entry (Counihan et al., 2013),it is possible that additional T. parva candidate sporozoite vaccinetargets remain to be discovered.

8.2. Immunization with recombinant p67 can protect againstEast Coast fever

Full-length and smaller regions of the p67 protein from theMuguga parasite isolate have been expressed in a recombinantform using a variety of different expression systems (Table 1).The high degree of sequence conservation of this protein makesit an intrinsically more attractive candidate vaccine antigen than,e.g., the PIM antigen. Unfortunately, the full-length p67 antigenhas proved to be an extremely difficult molecule to express in itsnative format as a recombinant protein in E. coli (Bishop et al.,2003; Musoke et al., 1992), Pichia pastoris (unpublished data), insectcells (Kaba et al., 2002, 2005; Nene et al., 1995) or mammaliancells (Honda et al., 1998, and unpublished data). Under labora-tory conditions, recombinant p67 protein with adjuvants given viathe sub-cutaneous route has been found to consistently induceimmunity to ECF. Table 1 also provides a summary of results fromexperimental vaccine trials carried out over several years, includ-ing those derived from a small number of studies with bacterial orviral antigen delivery systems.

Experimental laboratory trials have mainly used a needle chal-lenge of sporozoites from the Muguga isolate. The dose was titratedto provide a lethal dose 70% (LD70) challenge, which in controlanimals represented a 100% infectivity with ∼70% of the cattleundergoing a severe ECF reaction (Musoke et al., 1992). Relative tocontrol groups, cattle immunized with recombinant antigen havegiven consistent results exhibiting a range of immunity to ECF from13 to 70% (Table 1). Immunization with p67 protects against a chal-lenge with sporozoites from the Marikebuni isolate (see below),which in ITM studies falls in a different cross-immunity group tothe Muguga isolate (Irvin et al., 1983). Immunization with recom-binant p67 protein even protects against a cross-species challengewith T. annulata sporozoites (Hall et al., 2000). Surprisingly, p67C,an 80 amino acid section toward the C-terminal end of the protein,induces equivalent levels of immunity to full-length p67 (Bishopet al., 2003). Cattle exhibit a range of ECF clinical symptoms fromnon-reactors to severe symptoms, with only the latter classified assusceptible to disease (Anon, 1988a). All cattle generate high levelsof antibody titers to p67, but in most experiments there has beenno consistent correlate of various antibody assays with immunity(Musoke et al., 1992; Nene et al., 1996, 1999).

Field trials exposing p67 immunized cattle to naturaltick/parasite challenge have been carried out at three sites in Kenya.In these experiments, about 50% of the immunized cattle and 25%of the control animals exhibited immunity to ECF (Musoke et al.,2005). While there was a statistically significant level of immunitythat could be ascribed to the vaccine, the overall level of immunityin the field relative to the laboratory challenge data was low, asunder the field conditions some control cattle naturally recoveredfrom infection. These results are qualitatively different to thosereported from a field trial in Zambia, where immunized and con-trol cattle were equally susceptible to ECF (Schetters et al., 2014).

However, immunized cattle were immune to ECF when given asporozoite needle challenge. It is difficult to reconcile the differ-ences in field results between the experiments in Kenya and Zambiaas different antigen doses, adjuvants, and cattle types were used.

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We firmly believe that p67 remains a strong candidate vac-ine antigen, but research is required to increase the efficacy ofhe current p67 antigen formulation/immunization scheme. Underaboratory conditions, it is presumed that sterilizing immunity

as induced in a small number of cattle immunized with p67,s they exhibited no reaction on challenge (Musoke et al., 1992).his is not a desirable outcome of a vaccine as this is likely toisrupt endemic stability of the disease. The lack of an in vitro cor-elate with immunity hampers efforts to improve the efficacy of67, as immunogenicity studies alone do not predict the outcomef challenge data. Efforts are underway to determine if differentypes of correlates of immunity (Plotkin, 2010) can be devel-ped.

. A role for cellular immune responses in mediatingmmunity to East Coast fever

Development of the ITM vaccine gave rise to a robust and repro-ucible research model. Summarizing studies initiated in the late970s, there is now very good evidence that classical CD8+ MHClass I restricted cytotoxic T-lymphocytes (CTLs) that kill schizont-nfected cells play a major role in mediating immunity primed byTM (Morrison and Goddeeris, 1990; Morrison, 2009b). The in vivomportance of cellular responses in mediating immunity to ECF wasstablished in passive transfer experiments of thoracic duct leuco-ytes from immune animals to their naïve twins, which conferredesistance to challenge (Emery, 1981). These studies were refinedy demonstrating that immunity was associated with transfer ofD8+ enriched cells from efferent lymph following mAb-mediatedomplement lysis of CD4+ cells, �� T-cells and B-cells (McKeevert al., 1994).

As mentioned previously, the ITM vaccine induces strain specificmmunity. Most of what we know about the nature of this responseerives from studies using the T. parva Muguga and Marikebuni par-site isolates, which fall in different cross-immunity groups (Irvint al., 1983). In brief, these studies demonstrated that there was aarked correlation between the in vitro CTL strain specificity to kill

he different parasite isolates and cross-protection (Taracha et al.,995a). There was immuno-dominance in the antigen specificityf the CTL response that was influenced by the parasite antigenicype (Taracha et al., 1995a,b). In addition, the majority of the CTLesponse was often restricted by one BoLA class-I allele, with a ten-ency of some alleles to dominate the immune response over othersreviewed in Morrison et al., 2015).

The contribution of other cellular immune responses or innateesponses to immunity induced by ITM has not been extensivelyharacterized (reviewed in Morrison et al., 2015). CD4+ T-cellsBaldwin et al., 1987; Brown et al., 1989b,c, 1990) and �� T-cellsDaubenberger et al., 1999) show marked responses post-ITM vac-ination, but their roles in directly mediating immunity to ECFemain to be fully characterized. The former are likely to play aole in priming the CTL response (Taracha et al., 1997) and the lat-er can lyse schizont-infected cells (Daubenberger et al., 1999). Theheer numbers of �� T-cells in cattle leads to speculation that theseells could contribute to immunity via cross reactivity toward dif-erent T. parva strains, as MHC does not restrict the lytic responsef these cells (Daubenberger et al., 1999). This is consistent withhat is known about �� T-cells in other mammals (Adams et al.,

015). Efforts are underway by others to identify CD4+ T-cell anti-ens, but little is known about the �� antigens, which in otherystems have been identified as lipids or phosphoantigens (Adams

t al., 2015). Interestingly, T. annulata schizonts express a largeumber of surface molecules that are phosphorylated (Wiens et al.,014). The role of other innate cellular immune responses to infec-ion (reviewed in Morrison et al., 2015), including a novel subset

Diseases 7 (2016) 549–564 557

of NKp46+CD3+ cells (Connelley et al., 2014), remains to be fullycharacterized.

9.1. Identification and characterization of CD8+ T-cell antigens

The antigen specificity of CD8+ T-cells is mediated by their T-cell receptor (TCR), which bind to peptide-MHC class I complexesat the surface of target cells (reviewed by Rossjohn et al., 2015).These peptides, usually from 8 to 9 amino acid residues in length,are derived from proteosomal degradation of antigens and bind to apeptide-binding groove in the MHC molecule (reviewed by Neefjeset al., 2011). Methods for the identification of T. parva CTL anti-gens in a high-throughput manner were established fairly recently(Graham et al., 2007a). Briefly, this involved transfection of sch-izont cDNA made from candidate gene sequences mined from thereference T. parva Muguga genome sequence, or the transfectionof random pools of clones from a Muguga schizont cDNA libraryinto antigen presenting cells. Transfected cells were co-culturedwith CTL lines generated from cattle immunized by ITM with theMuguga parasite isolate. De-convoluting the components of pooledcDNA that were positive in an IFN-� ELISpot assay led to the identi-fication of individual parasite cDNA encoding a CTL antigen. TheBoLA class-I heavy chain allele restricting the CTLs was definedby co-transfection of parasite cDNA with different BoLA class-IcDNA. Finally, overlapping synthetic peptides were used to mapthe location of CTL epitopes. This first round of screening defined 8antigens, labeled Tp1 to Tp8, and mapped 11 CTL epitopes restrictedby one of seven different BoLA class I alleles (Table 2) (Akooloet al., 2008; Graham et al., 2006, 2007a, 2008). Since then threemore CTL antigens have been characterized, Tp9, Tp10 and Tp12(cited in Morrison et al., 2015). Some of the Tp antigens have apredicted function, including housekeeping functions. Not all ofthe Tp antigens encode a predicted signal peptide raising ques-tions on how these antigens gain access to the host cell MHC class Iantigen processing pathway. Interestingly, in T. annulata a memberof the subtelomeric variable secreted protein (SVSP) gene family,labeled Ta11, has been identified as a CTL antigen (MacHugh et al.,2011). This provides the first evidence that members of a multi-gene family in T. parva may play a role in the immune response toinfection.

The immunoscreens for CTL antigens were carried out using lessthan a dozen BoLA haplotypes, as defined by serology (Oliver et al.,1989) Classical BoLA class I genes are located on bovine chromo-some 23 (Brinkmeyer-Langford et al., 2009) and expressed fromsix different loci (Codner et al., 2012). There is no co-dominantexpression of the six alleles and usually no more than three will beexpressed in a given haplotype (Codner et al., 2012). Some of theCTL lines used in the screens did not yield positive data (Grahamet al., 2006, 2007a). The Immuno-Polymorphism-Database (IPD-MHC) houses ∼100 BoLA class I sequences (Robinson et al., 2010),and there is little doubt that the repertoire of bovine BoLA class Ialleles is much higher in number. This indicates that many moreT. parva antigens/epitopes, from both cattle- and buffalo-derivedparasites, remain to be characterized. The few antigens identi-fied so far have helped to unravel some of the complexity of theimmunobiology of T. parva CTL antigens (see below). The recentavailability of recombinant BoLA class I molecules (Hansen et al.,2014) has allowed the “on demand” production of peptide-MHCclass-I tetramers and ELISA-based peptide-MHC binding assays(Svitek et al., 2014). These permit in vitro and ex vivo epitope specificT-cell studies using flow cytometry, where co-staining with various

markers of interest can be used to describe the phenotype of epi-tope specific CD8+ T-cells (Svitek et al., 2014; Wendoh et al., 2014).Such data will provide important information on CTLs induced byITM and experimental vaccines.

558 V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564

Table 2A list of known T. parva CTL antigens and restricting BoLA class-I alleles.

Antigen Antigen annotation a Antigen length Signal peptide b Restricting BoLAclass-I allele

CTL epitope References

Tp1 Hypothetical 543 NO 6*01301 and6*01302

214VGYPKVKEEML224 Graham et al. (2008)

Tp2 Hypothetical 174 YES

6*04101 29EELKKLGML37 Svitek et al. (2014)

Unknown 40DGFDRDALF48 Nene et al. (2012)

2*01201 49KSSHGMGKVGK59 Graham et al. (2008)

BoLA-T2c c 96FAQSLVCVL104 Graham et al. (2008)

2*01201 98QSLVCVLMK106 Graham et al. (2008)

Unknown 138KTSIPNPCKW147 Akoolo et al. (2008)Tp4 �-TCP1 579 NO 3*00101 328TGASIQTTL336 Graham et al. (2008)Tp5 eIF-1A 155 NO BoLA-T5 d 87SKADVIAKY95 Graham et al. (2008)Tp7 Hsp90 721 NO BoLA-T7 c 206EFISFPISL214 Graham et al. (2008)Tp8 Cysteine proteinase 440 NO 3*00101 379CGAELNHFL387 Graham et al. (2008)Tp9 Hypothetical 334 YES 1*02301 67AKFPGMKKSK76

e Nene et al. (2012)

The antigens and CTL epitopes were identified from the Muguga strain of T. parva as briefly reviewed in the text. Data presented include antigen name, antigen annotation,the length of each antigen in amino acid residues and presence of a predicted signal sequence for secretion. If known, the BoLA class-I allele (Robinson et al., 2010) thatpresents the CTL epitope and the sequence of the CTL epitope is also listed.

a �-TCP1 (eta subunit of T complex protein 1), eIF-1A (translation elongation initiation factor 1A), Hsp90 (heat shock protein 90), hypothetical is of unknown function.b Predicted using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/).

alleleer, AK

9c

rretTmcamrttbetrta

9t

atlshworiTer

c Unassigned BoLA class-I allele.d Unassigned BoLA class-I allele, but the �1-�2 domain sequence of the BoLA-T5e Initially reported as a 10-mer, more recent data indicate the epitope is the 9-m

.2. A role for NetMHCpan in the identification andharacterization of CTL epitopes

The NetMHCpan algorithm (Hoof et al., 2009) can play a usefulole in the prediction of bovine CTL epitopes (Nene et al., 2012). Weecently reported on optimization of NetMHCpan using data gen-rated by measuring the binding of all possible nonameric peptideso nine recombinant BoLA class I molecules (Hansen et al., 2014).he data generated a motif for peptides that bind each BoLA class Iolecules and identified anchor residues, which play a more criti-

al role in peptide binding. Based on such parameters NetMHCpanssigns a score to peptide sequences that can bind to a given MHColecule (Hoof et al., 2009). NetMHCpan predicted that Tp229–37

ather than Tp227–37 (Graham et al., 2006) is likely to be the epitopehat binds to BoLA-6*04101 (Hansen et al., 2014). It also predictedhat the Tp587–95 epitope bound by BoLA-T5 could also be boundy BoLA-1*02301, an allele originally identified as presenting anpitope on Tp9 (Table 2). We experimentally verified these predic-ions (Hansen et al., 2014; Svitek et al., 2014). In the context of CTLesponses to T. annulata, the BoLA-1*02301 allele has been showno present epitopes on Ta9 and Ta5, which are homologs of the Tp9nd Tp5 antigens of T. parva (MacHugh et al., 2011).

.3. Epitope specificity of the CTL response in cattle immunized byhe infection and treatment method

With the identification of CTL antigens it has been possible to re-ssess the phenomenon of immuno-dominance in cattle immuneo ECF. Almost 70% of the CTL response to the Muguga parasite iso-ate in cattle homozygous for BoLA-A18 haplotype is directed to aingle epitope on the Tp1 antigen, while the response in BoLA-A10omozygous cattle is directed to one epitope on the Tp2 antigen,ith no detectable response to Tp2 in the BoLA-A18 cattle type

r to Tp1 in the BoLA-A10 animals (MacHugh et al., 2009). T-celleceptor analysis has revealed that the CTL response is polyclonal

n nature with predominant clonotypes responding to either thep1214–224 or Tp249–59 epitope (Connelley et al., 2011; Macdonaldt al., 2010). The Tp2 antigen contains a second epitope, Tp298–106,estricted by BoLA-2*01201 (Table 2), yet the CTL were directed

is identical to the �1-�2 domain sequence of 1*00902.FPGMKKS (Svitek et al., in preparation).

mainly to the N-terminal epitope. The specificities of the remainingCTL in these cattle are yet to be defined, and the relative role of thedominant versus less-dominant specificities in mediating immunityto ECF remains to be determined. These studies highlight extremeskewing of CTL responses in these cattle, which presumably con-tributes to the phenomenon of strain specific immunity induced byITM. Unfortunately, there has not been extensive characterizationof the influence of BoLA-type on the specificity of CTL responses incattle that are heterozygous at the BoLA locus, or on the specificityof CTL responses to infection with mixed parasite isolates, e.g., theMuguga cocktail.

Alanine scanning experiments of the peptide Tp1214–224 andTp249–59 epitopes with different CTL clones highlight the impor-tance of different anchor residues in the peptide binding to BoLA,and the influence of peptide-BoLA recognition by different TCRs(Connelley et al., 2011; Macdonald et al., 2010). It has also beendemonstrated that animals possessing BoLA-6*01302, which dif-fers from BoLA-6*1301 by a single amino acid substitution in thepeptide binding groove still presents the Tp1214–224 epitope (Sviteket al., 2015). Sequencing of Tp genes from different cattle- andbuffalo-derived parasites has revealed extensive antigenic diver-sity in the Tp antigens (Morrison et al., 2015; Pelle et al., 2011). Theability of variant sequences to function as cross-reactive epitopeswill depend on the molecular flexibility of the peptide-MHC-TCRinteraction. For example, in the context of the respective BoLA classI allele, variants of the Tp249–59 epitope seem to be less toleratedthan variants of Tp1214–224 (Connelley et al., 2011; Macdonald et al.,2010; Steinaa et al., 2012). In some cases amino acid substitutionsmay influence the proteolytic processing of antigens to generatethe relevant epitopes (Morrison et al., 2015). These data indicatethat effort is needed to produce a denser map of T. parva CTL anti-gens that contribute to strain specific and cross-reactive responses.This should aid the selection of antigens required to prime broad-spectrum immunity to ECF.

9.4. The challenge of priming CD8+ CTLs in cattle

Attempts to induce CTL in cattle that kill autologous schizont-infected cells using the Tp antigens have, in general, not been

V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564 559

Table 3Experimental vaccine trials in cattle using schizont antigens.

Delivery system Immunogen Dose Route Animalnumbers

Control Challenge Outcome References

CP/MVA andDNA/MVA

Tp1, Tp2, Tp4,Tp5 and Tp8

0.5 mg DNA or108 p.f.u. CPprime, boost5 × 108 p.f.u.MVA

DNA i.d./CP andMVA s.c.

12 DNA/MVAimmunized12 CP/MVAimmunized4 controls

Immunizedwith vaccinediluents

LD100 Muguga4133

17% survivors.Presence of CTLcorrelated withsurvival.

Graham et al.(2006)

DNA (PcDNA4//V5-HIS)

PIM 3 × 1 mg i.d. 2 immunized1 control

Plasmid only LD100 Katete 1 PIM immunizedanimal with CTLsurvived. The otherPIM animal hadMHC class Iunrestricted CTLand wassusceptible, as wasthe control.

Ververken et al.(2008)

Flt3 + GM-SCFDNA/MVA

Tp1, Tp2, Tp4,Tp5 and Tp8

0.5 mg of FLT3andGM-CSF/0.5 mgDNA(each)/5 × 108

p.f.u. MVA

i.d. 12 immunized8 controls

Immunizedwith vaccinediluents

LD100 Muguga4133

All animals died,but the controlgroup died fasterindicating someeffect ofimmunization.

Mwangi et al.(2011)

The results derived from three published experimental trials with schizont antigens are presented. Abbreviations: CP(canary pox); i.d. (intradermal); Flt3L (FMS-like tyrosinek (modD ot und

sb(e(afctsttAmplAIFpocwfhsttt(�Rttsteiro

inase ligand); GM-CSF (granulocyte-macrophage colony-stimulating factor); MVANA experiment, although the control animal was susceptible to challenge, it did n

uccessful. One study using CpG oligodeoxynucleotides and recom-inant PIM antigen resulted in priming of cytolytic CD4+ T-cellsGraham et al., 2007b). A study using immunization with DNAncoding the PIM antigen in two animals gave equivocal resultsVerverken et al., 2008). The most successful approaches have usedt least one viral vectored antigen delivery system in a prime-boostormat (Table 3). In order to facilitate immunogenicity studies,attle carrying BoLA class I alleles that present the known Tp epi-opes were used. For example, cattle with the BoLA-A18 haplotypehould respond to the Tp1214–224 epitope (Table 2). The antigensested so far derive from the Muguga parasite isolate and cat-le were given a homologous LD100 sporozoite needle challenge.

prime-boost regimen in 24 cattle primed with a mix of plas-ids (DNA prime) or a mix of recombinant canary pox virus (CP

rime) expressing five antigens (Tp1, Tp2, Tp4, Tp5 and Tp8), fol-owed by a boost with a mix of recombinant modified vacciniankara (MVA) virus expressing the same 5 antigens, resulted in

FN-� ELISpot positive results in 19 cattle (Graham et al., 2006).our cattle exhibited CTL activity and an additional three wereositive 2 weeks post-challenge. These seven animals were thenly ones that were immune to challenge. When compared to theontrol group the overall survival rate in the experimental groupas 17%, as one animal out of four in the control group recovered

rom challenge. Although cattle were pre-selected for their BoLAaplotype not all cattle responded with the expected Tp epitopepecificities. In addition, very few animals mounted a responseo more than one immunogen (Graham et al., 2008). Approacheso augment the immune response to recombinant MVA by usinghe cytokines granulocyte-macrophage colony-stimulating factorGM-CSF) and FMS-like tyrosine kinase ligand (Flt3L) elicited IFN-

secreting CD4+ and CD8+ cells, but no CTL (Mwangi et al., 2011).ecent unpublished data using AdHu5/MVA as a prime-boost sys-em with the Tp1 antigen confirmed an ability to prime CD8+ CTLhat will kill peptide-pulsed targets. However, these CTL do not killchizont-infected cells, indicating that CTL generated by this sys-em are qualitatively different to those generated by ITM (Svitek

t al, in preparation). Resolution of these differences will be anmportant step forward. Currently, as in other large mammals, theoutine priming of CTLs remains a technical challenge. Our collab-rators and we are exploring other antigen delivery systems. Until

ified vaccinia Ankara); s.c. (subcutaneous); p.f.u. (plaque forming units). In the PIMergo a typical response (Ververken et al., 2008).

one is found, the number of CTL antigens that will be required inan anti-schizont vaccine remains unknown.

10. Desirable qualities in a subunit vaccine for the controlof East Coast fever

One component of a target product profile of a subunit vaccineis to prime immunity against a broad-spectrum parasite chal-lenge, e.g., that of the Muguga cocktail. The intensity, parasitecomposition, and time to first challenge post-vaccination will varydepending on the livestock production system in which a vaccine isdeployed. Whatever the composition of the vaccine, it is desirablethat cattle undergo a mild to moderate disease reaction on chal-lenge. For an anti-sporozoite based vaccine, this will also resultin priming a parasite-induced CTL response thereby mimicking thelive parasite method of vaccination. For an anti-schizont based vac-cine, this should boost vaccine induced CTL specificities, and mayprime additional CTL specificities not present in the subunit vac-cine, as is thought to occur in animals vaccinated by ITM. If theseantigens can function in a synergistic fashion, a vaccine targetingboth life cycle stages is conceptually appealing and may be of spe-cial value in reducing the dose of sporozoite strains not covered bythe vaccine CTL specificities. Maintaining an adequate level of pro-tective antibody and CTL responses in a combination vaccine maybe less challenging than for either one alone. One concern that willneed to be addressed is whether recovery from an infection inter-feres with the subsequent quality and longevity of vaccine inducedimmune responses.

11. Conclusions

Substantial advances have been made in the identification andcharacterization of candidate vaccine antigens of T. parva. Althoughtime consuming, the technical challenge of identifying T. parva CTLantigens has been largely overcome. What is not clear at this stageis the identity and number of sporozoite and/or schizont antigens

that will be required to prime broad-spectrum immunity, and howto induce a robust CTL response. The application of more modernapproaches to vaccine-related research (Koff et al., 2013; Nakayaand Pulendran, 2015) should lead to improved understanding

5 -borne

avfTabacIds

A

puIfiIvgCCttd

R

A

A

A

A

A

A

B

B

B

B

B

B

B

B

60 V. Nene et al. / Ticks and Tick

nd profiling of the immune response to ITM and experimentalaccines. Hence, the prospects for developing a subunit vaccineor the control of ECF are good. The buffalo wildlife reservoir of. parva remains a confounding factor. However, we anticipate

greater understanding of the differences between cattle- anduffalo-derived T. parva, as more whole genome sequence dataccumulate. In the interim, commercialization of the Mugugaocktail ITM vaccine is good news for the cattle industry in Africa.t is reasonable to expect improvements in the manufacture andelivery of this vaccine as experience in its use in different farmingystems accumulates.

cknowledgements

We wish to thank the many years of excellent contribution ofast and present scientists and laboratory personnel, farm and ticknit staff in support of East Coast fever research at ILRI. We thank

van Morrison for a critical review of the manuscript. Previous corenancial support from ILRI donors is gratefully acknowledged and

FAD for funding activities related to commercialization of the ITMaccine. More recent research at ILRI has been funded throughrants from the USA National Science Foundation (0965346), theGIAR Research Program on Livestock and Fish, the Norman Borlaugommemorative Research Initiative, an initiative between the Feedhe Future program of USAID and USDA-ARS (58-5348-2-117F) andhe Department for International Development of the United King-om and the Bill and Melinda Gates Foundation (OPP1078791).

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-borne

F

F

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GGG

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