The Biology of Theileria Parva and Control of East Coast Fever – Current

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

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

j ournal homepage: www.elsevier.com/locate/ttbdis

Review Article

The biology of Theileria parva and control of East Coast fever – Current

status and future trends

a,b,∗ a a a a

Vishvanath Nene , Henry Kiara , Anna Lacasta , Roger Pelle , Nicholas Svitek , a

Lucilla Steinaa

International Livestock Research Institute, Old Naivasha Road, P.O. Box 30709, Nairobi 00100, Kenya

Adjunct 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 a b s t r a c t

Article history: Tremendous progress has been made over the last ten years on East Coast fever (ECF) research. Publi-

Received 16 September 2015

cation of a reference genome sequence of Theileria parva, the causative agent of ECF, has led to a more

Received in revised form 1 February 2016

thorough characterization of the genotypic and antigenic diversity of the pathogen. It also facilitated

Accepted 2 February 2016

identiﬁcation of antigens that are targets of bovine major histocompatibility complex class I restricted

Available online 26 February 2016

cytotoxic 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 likely

Keywords:

contribute to the phenomenon of strain-speciﬁc immunity. The Muguga cocktail ITM vaccine, which pro-

Theileria parva

vides broad-spectrum immunity to ECF is now a registered product in three countries in eastern Africa.

East Coast fever

Vaccines Effort is directed at improving and scaling up the production process to make this vaccine more widely

Infection and treatment available on a commercial basis in the region. Meanwhile, research to develop a subunit vaccine based on

Antigens parasite neutralizing antibodies and CTLs has been revived through convening of a research consortium

to develop proof-of-concept for a next generation vaccine. Many new scientiﬁc and technical advances

are facilitating this objective. Hence, the next decade promises even more progress toward an improved

control of ECF.

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

Contents

1. Introduction ...... 550

2. A background to East Coast fever, a lethal bovine disease present in sub-Saharan Africa ...... 550

3. Theileria parva exhibits a complex life cycle ...... 551

4. Host cell transformation and morbidity caused by the schizont life-cycle stage ...... 551

5. Genotypic diversity is a hallmark of Theileria parva ...... 551

6. A live sporozoite-based vaccine for the control of ECF via infection and treatment ...... 553

7. Chemotherapy of East Coast fever ...... 554

8. 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 ...... 556

8.2. 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 ...... 557

9.2. A role for NetMHCpan in the identiﬁcation and characterization of CTL epitopes ...... 558

9.3. Epitope speciﬁcity of the CTL response in cattle immunized by the infection and treatment method ...... 558

Abbreviations: BoLA, bovine leukocyte antigen; CD, cluster of differentiation; cM, centiMorgan; ELISA, enzyme-linked immunosorbent assay; ELISpot, enzyme-linked

immunospot assay; IFN-␥, interferon-␥; ITM, infection and treatment method; mAb, monoclonal antibody; MHC, major histocompatibility complex; VNTR, variable number of tandem repeat.

∗

Corresponding author at: International Livestock Research Institute, Old Naivasha Road, P.O. Box 30709, Nairobi 00100, Kenya.

E-mail address: [email protected] (V. Nene).

http://dx.doi.org/10.1016/j.ttbdis.2016.02.001

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

9.4. The challenge of priming CD8 CTLs in cattle ...... 558

10. Desirable qualities in a subunit vaccine for the control of East Coast fever ...... 559

11. Conclusions...... 559

Acknowledgements ...... 560

References ...... 560

1. Introduction 2. A background to East Coast fever, a lethal bovine disease

present in sub-Saharan Africa

Theileriosis caused by the hemoprotozoan pathogen Theileria

parva still ranks ﬁrst among the tick-borne diseases of cattle in In the early 1900s, Dr. Arnold Theiler recognized that Theileria

sub-Saharan Africa (Minjauw and McLeod, 2003). Organisms in parva was the causative agent of ECF in South Africa (Theiler, 1912).

the genus Theileria are found in many wild and domesticated He distinguished ECF from Redwater caused by Babesia, and identi-

animals (reviewed in Norval et al., 1992). They are phylogeneti- ﬁed the principal tick vector that transmits T. parva as Rhipicephalus

cally most closely related to members of the Babesia genus and appendiculatus. The disease was associated with cattle imported

fall in the order Piroplasmida under the phylum Apicomplexa from East Africa, and caused high levels of morbidity and mortality

(Gou et al., 2012; Kuo et al., 2008; Lack et al., 2012; Levine, (reviewed in Norval et al., 1992). South Africa eradicated the dis-

1985; Morrison, 2009a; Schnittger et al., 2012). Theileria parva, T. ease through a strict control of cattle movement, surveillance with

annulata and T. lestoquardi feature prominently among pathogenic slaughter of infected cattle, and fencing to restrict buffalo to game

species of Theileria that affect domesticated ruminants (reviewed parks. Occasional outbreaks of buffalo-derived T. parva disease still

in Norval et al., 1992). They share a unique biology of being occur, but appear to be self-limiting in nature (Mbizeni et al., 2013;

able to transform a subset of infected mononuclear host cells, Thompson et al., 2008). It has not been possible to implement such

which can be cultured in vitro as persistently infected cell lines. measures in other countries as a sustainable method of disease

Not all species of Theileria can transform mononuclear host cells, control. Hence, ECF remains an acute and usually lethal disease

e.g., T. orientalis, T. mutans and T. velifera. If such species cause present in 12 countries in eastern, central and southern Africa,

disease it is mainly due to multiplication of the parasite life- including Burundi, Democratic Republic of Congo, Kenya, Malawi,

cycle stage within red blood cells (reviewed in Norval et al., Mozambique, Rwanda, South Sudan, Tanzania, Uganda, Zambia and

1992). Zimbabwe (reviewed in Norval et al., 1992; Malak et al., 2012), and,

It is believed that T. parva co-evolved with the African Cape more recently, in the Comoro Islands (De Deken et al., 2007).

buffalo, in which infection does not cause disease, and has under- On a regional basis ECF kills ∼1 million cattle/year with annual

gone a “host jump” to cattle, where it causes the disease referred economic losses of ∼USD300 million (McLeod and Kristjanson,

to as East Coast fever (ECF) (reviewed in Norval et al., 1992). Differ- 1999). These estimates were derived many years ago and current

ences in the epidemiology, clinical symptoms, and parasitological impacts are likely to be much larger. ECF can establish in new areas

parameters in cattle associated with different T. parva infections due to a wider distribution of the tick vector and/or suitable tick

have led, in the past, to sub-speciation of T. parva. This nomencla- habitats (Norval et al., 1992), as occurred in the Comoro Islands

ture was abandoned (Perry and Young, 1993), and parasite isolates in 2004 (De Deken et al., 2007). Recent political changes in South

are now described as cattle- or buffalo-derived (Anon, 1988a,b). Sudan has led to a growing incidence of ECF in a naïve popula-

However, in southern Africa, cattle-derived T. parva is associated tion of cattle with mortality rates close to 80% (Malak et al., 2012;

with a less virulent disease than ECF, called January disease in Marcellino et al., 2012). This spread poses a direct threat to cat-

Zimbabwe (reviewed in Latif and Hove, 2011). The disease caused tle in the Central African Republic and regions to the west. To the

by buffalo-derived T. parva is called Corridor disease, and it is east, although ECF has not been described in Ethiopia, this coun-

usually more acute than ECF (reviewed in Norval et al., 1992). try houses the largest population of cattle in Africa, and predictive

Another characteristic of the latter is that it is extremely difﬁ- models indicate that the tick vector could thrive in the Ethiopian

cult to maintain buffalo-derived parasites through serial passage highlands (Norval et al., 1991).

between ticks and cattle, whereas this is relatively easy with cattle- There is some evidence that Bos taurus cattle are more sus-

derived parasite isolates (Young and Purnell, 1973). This scenario ceptible to ECF than Bos indicus animals, and there may be breed

is complicated by the presence of multiple strains in cattle- and differences in susceptibility to disease (Norval et al., 1992). The

buffalo-derived parasite populations, with possible co-infections severity of ECF caused by T. parva is parasite dose-dependent and

between them in individual animals (Conrad et al., 1987; Mans there also appear to be differences in the threshold of infectivity

et al., 2015; Oura et al., 2003, 2004, 2005, 2011). In evolutionary in individual cattle (Cunningham et al., 1974; Dolan et al., 1984).

terms, the change in host-species is likely to have occurred rel- These observations could obscure true host genetic differences in

atively recently as cattle were introduced to Africa ∼5000 years susceptibility to ECF. Cattle that exhibit mild or moderate ECF clin-

ago (Epstein, 1971). Although molecular data do not currently sup- ical symptoms are considered resistant to disease, while those that

port sub-species status of T. parva (Allsopp et al., 1993; Conrad exhibit severe clinical reactions are judged to be susceptible (Anon,

et al., 1989), the biological observations suggest the presence of 1988a). Cattle can spontaneously recover from infection, usually

two segregating populations of the parasite (see also Mans et al., after a mild or moderate reaction, and are solidly immune to re-

2015). The importance of the parasite buffalo reservoir in eval- infection. This phenomenon contributes to endemic stability of

uating approaches to ECF control is variable depending on the the disease in indigenous cattle in regions where there is contin-

farming system, geographical region and wildlife-cattle interface ual transmission of the parasite (Medley et al., 1993; Norval et al.,

(Gachohi et al., 2012; Norval et al., 1992). The remainder of this 1992). Animals that recover from ECF do not usually eliminate the

review concentrates on aspects of the biology of T. parva and infection, they remain as carriers of the parasite and a source of

recent advances in vaccine-related research, which is helping to infections to ticks (Kariuki et al., 1995). The recent observation of

shape the development of a subunit vaccine for the control of an apparent protective effect in young cattle due to co-infections

ECF. with non-pathogenic Theileria raises interesting new questions on

V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564 551

the epidemiology of ECF (Thumbi et al., 2014; Woolhouse et al., 4. Host cell transformation and morbidity caused by the

2015). schizont life-cycle stage

ECF is described as an acute, lymphoproliferative disease associ-

3. Theileria parva exhibits a complex life cycle

ated with pathology induced by schizont-infected cells, and death

is usually caused by pulmonary edema (reviewed in Norval et al.,

Understanding the life cycle of the parasite is important as it

1992). Full schizogony of T. parva only occurs in B- and T-cells

provides information that may be used to improve disease con-

(Baldwin et al., 1988), where the parasite causes the host cell to

trol strategies, and for identifying novel targets of intervention.

proliferate. The schizont divides in synchrony with the host cell so

Theileria parva undergoes a series of sequential developmental

that daughter host cells remain infected (Irvin et al., 1982). In vivo

changes in the tick and bovine host (Fig. 1). These changes have

and in vitro most transformed cell lines are of the T-cell lineage,

been primarily characterized at the morphological level through

and higher levels of pathogenicity are associated with infected T-

elegant electron microscopy studies (Fawcett et al., 1981, 1982a,

cells rather than infected B-cells (Morrison et al., 1996). Growth

1987; Shaw and Tilney, 1992; Shaw, 1996b). Unfortunately, not

of infected cells is rapid, independent of exogenous growth fac-

much is known about the molecular details of parasite-host cell

tors, and completely dependent on the presence of live parasites

interaction or pathways that control parasite differentiation into

as treatment with anti-theilerial drugs, e.g., buparvaquone, results

different life-cycle stages. Rhipicephalus appendiculatus is a three-

in cells reverting to a non-proliferative state (Brown et al., 1989a;

host tick and the principal vector for transmission of T. parva,

Dobbelaere et al., 1991; Heussler et al., 1992).

which occurs trans-stadially (reviewed in Norval et al., 1992).

In contrast to T. parva, T. annulata transforms

Thus, tick larvae and nymphs acquire an infection from the piro-

macrophages/dendritic cells and B-cells, but not T-cells

plasm stage present in red blood cells (RBCs) in infected cattle.

(Dobbelaere and Heussler, 1999; Spooner et al., 1989). These

Infected nymphs and adults transmit sporozoites, the life-cycle

observations indicate that a two-way communication between

stage infective to cattle, during feeding. In vitro, sporozoites can

parasite and host cell type plays a role in the process of host

bind and rapidly enter lymphocytes via a passive “zippering pro-

cell transformation. However, the situation appears to be more

cess” of sporozoite and host cell membranes (Fawcett et al., 1982c;

complex as not all infected lymphocytes become transformed

Fawcett and Stagg, 1986; Shaw, 1996a, 1997). Unlike other api-

(Morzaria et al., 1995; Rocchi et al., 2006). It is beyond the

complexan organisms, T. parva sporozoites are non-motile and

scope of this review to cover excellent research on the topic of

spherical/ovoid in shape with a diameter of 0.75–1.5 ␮m (Fawcett

molecular mechanisms that contribute to host cell transformation

et al., 1982a,b). They do not have well developed apical com-

and the reader is directed to several publications (Chaussepied

plexes and host cell entry is not orientation speciﬁc (reviewed in

and Langsley, 1996; Chaussepied et al., 2010; Cheeseman and

Shaw, 2003). Discharge from sporozoite secretory organelles, rhop-

Weitzman, 2015; Dobbelaere and Rottenberg, 2003; Heussler

tries/microspheres, is seen after host-cell entry, and coincides with

et al., 2002; Marsolier et al., 2015; Medjkane et al., 2014; Shiels

rapid escape from the surrounding host membrane. Sporozoites

et al., 2006; Tretina et al., 2015; von Schubert et al., 2010).

differentiate into the schizont stage, which is a multinucleated

Understanding the mechanisms by which Theileria organisms

stage that resides free in the host cell cytoplasm surrounded by

cause mammalian cells to transform has an obvious relevance to

a basket of host cell microtubules that seem to be nucleated

cancer biology. Disruption of these mechanisms may offer novel

by parasite molecules (reviewed in Shaw, 2003). These infected

drug-targets for the control of ECF (Marsolier et al., 2015).

cells acquire a metastatic, cancer-like phenotype and are the pri-

mary cause of pathology (reviewed in Dobbelaere and Heussler,

1999). Sporozoites also enter macrophages and dendritic cells,

5. Genotypic diversity is a hallmark of Theileria parva

but only develop to an early stage of the schizont (Shaw et al.,

1993).

A reference genome sequence for T. parva and T. annulata was

Schizonts undergo a process of merogony to produce mero-

published in 2005 (Gardner et al., 2005; Pain et al., 2005). A

zoites, which are released by host cell rupture. Merozoites invade

reference sequence for other Theileria species (Hayashida et al.,

RBCs, where they develop into the piroplasm stage. The mecha-

2012; Kappmeyer et al., 2012) and whole genome sequence data

nism of merozoite entry into RBCs is similar to that of sporozoite

from other strains/isolates of T. parva have also been published

entry into lymphocytes and merozoites develop into the piro-

(Hayashida et al., 2013; Henson et al., 2012; Norling et al., 2015).

plasm stage, free in the RBC cytoplasm (Shaw and Tilney, 1995).

These resources provide essential baseline data to study T. parva

Piroplasms undergo a limited number of cell divisions (Conrad

population structure, antigenic diversity, and facilitate reverse vac-

et al., 1986) and anemia due to destruction of infected RBCs is

cinology approaches for vaccine development. In contrast to other

not a prominent feature of ECF (reviewed in Norval et al., 1992).

Apicomplexa, e.g., Plasmodium and Babesia (Jackson et al., 2014;

The piroplasm stage is infective to ticks and differentiation to

Singh et al., 2014), a prominent role for members of the major T.

gametes seems to occur post-ingestion in the tick gut (reviewed in

parva multi-gene families (Gardner et al., 2005) as antigens remains

Norval et al., 1992). Following fusion of macro- and micro-gametes,

to be established.

zygotes enter cells of the tick gut epithelium and develop into

In general, buffalo-derived parasites are more diverse than

motile kinetes, which are released into the tick hemocel. Sporo-

cattle-derived parasites. A number of loci (Allsopp and Allsopp,

gony occurs in the e-cells in type III acini (Fawcett et al., 1982b).

1988; Bishop et al., 1995, 1998; Conrad et al., 1987; Katzer et al.,

Release of Theileria sporozoites from the salivary glands occurs

2010; Pelle et al., 2011) and variable number of tandem repeat

as a trickle between day 4–8 post-attachment (Shaw and Young,

(VNTR) markers (mini- and micro-satellites) derived from the T.

1995). Approximately 30,000–50,000 sporozoites develop in an

parva genome sequence data have been used as markers to deter-

infected acinus (Fawcett et al., 1982a,b). All life cycle stages of

mine parasite diversity (Oura et al., 2003, 2004, 2005, 2011; Patel

T. parva, except the zygote, are haploid in nature and zygotes

et al., 2011). Such studies are inﬂuenced by the distribution, den-

undergo a two-step meiotic division (Gauer et al., 1995). The

sity, and type of markers across the genome, and current results

sexual cycle of T. parva in the tick vector results in genera-

are preliminary in nature. A study of parasite population struc-

tion of extensive parasite genotypic and antigenic diversity (see

ture using VNTRs (Oura et al., 2003, 2004, 2005, 2011) indicated

below).

that 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 was

inspired 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 life

cycles 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 clonal

expansion of schizont-infected cells. The schizont stage is polyploid and multiple schizont nuclei are observed during host cell interphase. It is during the metaphase period

of 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 in

RBCs, 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

V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564 553

and level of genetic similarity between parasite isolates, such that intra-venous tetracycline cover during natural T. parva infections

different parasite isolates from the same farm exhibited distinct resulted in immunity to ECF (Neitz, 1953). The process of using

genotypes. Older cattle tended to have a larger number of differ- triturated infected tick suspensions, a method for cryopreserving

ent parasite genotypes than younger ones, which tended to have them, and titration of the infectivity of frozen sporozoite stabilates

a pre-dominating genotype (Oura et al., 2005). Some geographi- (Cunningham et al., 1973, 1974) provided a source of standardized

cal sites exhibited a sub-structure in parasite populations, others parasite material for vaccination. Development and optimization

did not, and some exhibited an epidemic structure characteristic of ﬁrst short, and subsequently long-acting formulations of oxy-

of recent predominating infections (Oura et al., 2005). Thus, it has tetracyclines to control the infections played an important role in

been suggested that cattle movement, their interface with buffalo, development of the ITM protocol (Radley, 1981). It has been demon-

and parasite transmission rates are likely to play a major role in strated that at low dilutions the Boleni isolate of T. parva from

determining T. parva parasite population structures (Oura et al., Zimbabwe can be used to immunize cattle in the absence of tetra-

2003). cycline (reviewed in Latif and Hove, 2011), but it has been difﬁcult

VNTR mapping the progeny from two cattle-derived parasite to replicate this in other parasite isolates.

cloned stocks after transit through the tick vector has led to a It was discovered early on that exposure of immune cattle to

genetic linkage map for T. parva (Katzer et al., 2011), and identi- parasite isolates from different geographical sites could result in

ﬁcation of regions with higher (hot spots) and lower (cold spots) break-through infection and disease in some animals, giving rise to

rates of recombination than a genome-wide average. Using a phys- the concept of strain/isolate speciﬁc immunity (Young et al., 1973).

∼

ical size of 8.3 Mbp of the T. parva genome (Gardner et al., 2005) Although individual parasite isolates can be placed in different

the total genetic size was calculated at 1683.8 cM (Katzer et al., cross-immunity groups, there is usually 20–30% cross-protection

2011). This map provides an important reference for placement of between them (Morzaria, 1996). Combining different isolates

genes into regions with different rates of recombination. Whole broadens the vaccine coverage of ITM. Hence, the Muguga cocktail,

genome sequence data from a few different cattle- and buffalo- which consists of three T. parva isolates (called Muguga, Serengeti-

derived parasite isolates mapped to the reference genome has transformed and Kiambu-5) was shown to provide broad-spectrum

led to identiﬁcation in excess of 500,000 single-nucleotide poly- immunity to ECF (Radley et al., 1975a,b). Developed in principle

morphisms (SNPs) (Hayashida et al., 2013; Henson et al., 2012). almost 50 years ago, the Muguga cocktail ITM vaccine has had

SNP density/kbp was 2–4-fold higher in buffalo-derived parasites, a checkered history in its translation to a commercially available

more prevalent in coding than non-coding regions, and has led product as it is time consuming and difﬁcult to produce, requires

to identiﬁcation of SNP-rich and SNP-poor chromosomal regions a liquid nitrogen cold chain for its storage, is potentially a lethal

(Hayashida et al., 2013). Sequence data have also identiﬁed recom- product if not given with adequate antibiotic cover, and is expen-

bination events, and a large number of crossover events lead to sive.

gene conversion (Henson et al., 2012). Interestingly, phylogenetic The hurdles in producing and delivery of the Muguga cock-

relationships as measured by SNPs detected in the coding regions tail can be overcome, and the vaccine has proved to be highly

of 200 random selected genes place the sequences derived from effective in controlling ECF (Di Giulio et al., 2009). In response to

buffalo-derived parasites into a separate cluster from the sequences a FAO/multi-donor regional program, the International Livestock

of cattle-derived parasites (Norling et al., 2015). These data begin Research Institute (ILRI) produced ∼600,000 doses of the cocktail

to reveal the genetic complexity of the parasite nuclear genome, in two batches, called FAO1 and FAO2 (Morzaria et al., 1997). Use of

events leading to diversity between different strains, and a list of a higher oxytetracycline dose than before reduced the number of

protein coding genes that are under different rates of evolutionary clinical reactors to the vaccine, eliminating need for a second dose

pressure (Hayashida et al., 2013; Henson et al., 2012). Unraveling of antibiotic treatment and visit by the service provider after immu-

how such observations relate to genes that play a role in medi- nization. This, together with more extensive use of the vaccine in

ating host and vector interactions, the differences in the biology pastoral areas of Tanzania has played a major role in increasing

between cattle- and buffalo-derived parasites, and to immunity to the demand for the cocktail vaccine (Di Giulio et al., 2009). The

ECF, remains a fascinating prospect. next commercial batch of the cocktail of ∼1 million doses, called

ILRI08, was made by ILRI (Patel et al., 2015) in recognition of deplet-

ing stocks of the FAO vaccine batches, and in response to a major

6. A live sporozoite-based vaccine for the control of ECF via

initiative to invest in commercialization of the Muguga cocktail

infection and treatment

(GALVmed, 2010). An indication of the success of this revitaliza-

tion is that the ILRI08 batch was sold out in just ﬁve years, and

There is solid evidence from both ﬁeld observations as well as

re-investments in the Center for Ticks and Tick-Borne Diseases

from laboratory-based studies that cattle acquire robust immu-

(CTTBD) in Malawi to become the site of production and distri-

nity to ECF following recovery from infection (reviewed in Norval

bution of future batches of the cocktail as a commercial enterprise

et al., 1992). As the infection and treatment method (ITM) name

are near to fruition (GALVmed, 2014). The Muguga cocktail has been

implies, the process involves deliberate infection of cattle with con-

registered as a vaccine in Malawi, Kenya and Tanzania, and no doubt

comitant drug treatment. The latter controls but does not kill the

more lessons will be learnt as vaccine production is scaled up and

parasite permitting generation of a protective acquired immune

delivered through public and private veterinary services.

response. The ITM process has evolved over several decades start-

It is puzzling how three T. parva isolates in the Muguga cocktail,

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

selected from a limited geographical area, have shown protec-

with heavily parasitized spleen and lymph node homogenates

tion across such a wide geographical area (Di Giulio et al., 2009;

obtained from infected cattle slaughtered in the last stages of the

Norval et al., 1992; Radley et al., 1975a,b). Using a very limited

disease (Theiler, 1912). This gave way to the ﬁnding that prolonged

adults. There is no transovarial transmission of the parasite (reviewed in Norval et al., 1992). Kinetes, the ﬁnal products of the sexual cycle, invade the tick salivary glands

where sporogony occurs. Mature merozoites (Mz) and sporozoites (Sz) originate from a multinucleated residual body (Rb) during the process of merogony and sporogony,

respectively. See main text for additional details. The vertebrate host cell nucleus is colored in purple and the parasite nucleus is in orange. Microspheres in sporozoites

and micronemes in merozoites are depicted as small dark green dots inside the parasite. Host cell microtubules in the dividing schizont-infected cell are drawn in green.

Drawings and artistic creation by N. Svitek.

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

panel of ﬁve VNTR markers on schizont-infected cell lines estab- However, the Muguga cocktail ITM vaccine has been used in differ-

lished from the components of the Muguga cocktail revealed the ent livestock production systems and geographical areas over many

presence of 17 genotypes in the cocktail (Patel et al., 2011). The years (Uilenberg, 1999), without any major reports of a change

vaccine strains present in the ILRI08 cocktail batch also exhibit in the epidemiology of ECF or large-scale failure of the vaccine,

limited diversity of genes encoding the known candidate vaccine although it has been shown that components of the cocktail can

antigens (Hemmink, Weir, MacHugh, Graham, Shiels, Toye, Mor- establish itself locally (Oura et al., 2007). What is required is to

rison and Pelle, unpublished manuscript). Recent whole genome either dispel or conﬁrm the concerns by establishing monitoring

sequence data derived from the piroplasm stage of parasite compo- experiments where a good baseline study of the clinical nature

nents of the Muguga cocktail revealed only 957 SNPs between the of the disease and a comprehensive study of the existing para-

Serengeti-transformed and Muguga sequence, but close to 40,000 site genotypes are studied prior to vaccination, and longitudinal

SNPs between the Kiambu-5 and Muguga sequence (Norling et al., monitoring is carried out for several years. In this way the risk

2015). The sequence differences between Serengeti-transformed from the use of live parasites in exotic locations could be better

and Muguga were primarily restricted to sub-telomeric regions, assessed. For the moment ITM remains the only vaccine based solu-

while those between Kiambi-5 and Muguga were located across the tion available for the control of ECF. Efforts are underway to develop

genome. The latter altered the sequences of almost half of the par- next generation subunit vaccines but these are several years

asite protein coding genes. A curious feature of the sequence data away.

was an absence of mixed sequences, suggesting that the Serengeti-

transformed and Kiambu-5 piroplasm DNA sequenced were clonal

7. Chemotherapy of East Coast fever

in nature (Norling et al., 2015). Although the true level of hetero-

geneity in the Muguga cocktail remains to be determined, the very

Thirty years later buparvaquone still remains the front line com-

high degree of sequence similarity between Serengeti-transformed

mercial drug of choice for the treatment of T. parva (McHardy et al.,

and Muguga questions the origin of the former, and the value of its

1985) and T. annulata infections (Dhar et al., 1986). However, to

protective role in the Muguga cocktail (Norling et al., 2015). Unfor-

be completely effective the drug needs to be administered early

tunately, our current tools for typing parasite genotypic diversity

in infection (Martins et al., 2010). As with other hydroxynaptho-

do not predict cross-immunity proﬁles. Nevertheless, molecular

quinones the drug most likely functions by inhibiting electron

markers can be used to determine identity, and as quality con-

transport through the mitochondrion (Birth et al., 2014). Resis-

trol tools to ensure that the composition of strains present in the

tance to buparvaquone has been described in T. annulata (Mhadhbi

Muguga cocktail remain relatively constant during the complex

et al., 2010) and correlates with mutations in the cytb gene

production process of the vaccine.

(Mhadhbi et al., 2015; Shariﬁyazdi et al., 2012). Hence, the recent

As mentioned previously, parasite isolates placed in different

identiﬁcation of a T. annulata nuclear gene encoding a secreted

cross-immunity groups usually exhibit 20–30% cross-protection

peptidyl-prolyl isomerase, as an additional target of buparvaquone

between them (Morzaria, 1996). Sequential immunization by ITM

resistance was unexpected (Marsolier et al., 2015). These ﬁndings

with single isolates in different cross-immunity groups provides

are of concern for the future control of T. annulata as drug resis-

additive immunity (Taracha et al., 1995b). This suggests that some

tance could spread. Although buparvaquone resistance in T. parva

protective antigens are shared between different parasite isolates,

remains to be documented it may just be a question of time before

and the breadth of the immune response can be expanded in

this occurs. Effort should be made to identify new anti-theilerial

response to new infections. Novel genotypes can be found in cat-

drugs, leveraging on the drive to develop new anti-malarial drugs

tle vaccinated with the Muguga cocktail (Oura et al., 2004). This

(Burrows et al., 2014).

may help to further broaden the spectrum of immunity to strains

of the parasite not present in the cocktail. Currently, there is no

8. A role for humoral immune responses in mediating

simple answer as to whether buffalo-derived parasites will break-

immunity to East Coast fever

through the Muguga cocktail. For example, the cocktail has proved

highly effective in pastoral areas of Tanzania where cattle are likely

Cattle exposed to T. parva infection develop an antibody

to be exposed to buffalo-derived parasites (Di Giulio et al., 2009).

response to several parasite proteins. Among these speciﬁcities,

Recent experiments in Kenya demonstrate that this immunity is

antibodies to the polymorphic immunodominant molecule (PIM)

susceptible to buffalo-derived parasites on the Ol Pejeta ranch in

have proved to be the most reliable in measuring exposure to T.

Laikipia district (Sitt et al., 2015) but not in the Narok County area

parva. PIM can be polymorphic in size between different T. parva

(Mukholi, Kitala, Gathuma, Kidula, Turasha, Mbwira, Mugambi,

strains (Shapiro et al., 1987; Toye et al., 1995a), but there is suf-

Kiara, in preparation). Hence, buffalo-derived parasites may remain

ﬁcient conservation of sequences between the variants for PIM to

a confounding factor in some areas where the Muguga cocktail is

function as a diagnostic antigen (Katende et al., 1998; Toye et al.,

deployed and will have to be evaluated on an empirical basis.

1996). Hence, an ELISA assay based on recombinant PIM from the

There still remains lingering concern about the long-term

Muguga stock of the parasite is routinely used to determine preva-

impact that exotic T. parva strains might have on local parasite

lence (Gitau et al., 2000), and sero-conversion to the ITM vaccine

populations when used outside the region of origin of vaccine

(Patel et al., 2015).

strains leading to a debate on the use of autogenous ITM vac-

Early studies on passive transfer of sera from immune cat-

cines versus cocktail vaccines (Geysen, 2008; McKeever, 2007). For

tle failed to protect against challenge (Muhammed et al., 1975).

example, following some apparent complications due to use of the

In addition, immunization with parasite antigen failed to protect

Muguga cocktail, Zambia has chosen to use local isolates from the

against ECF (Wagner et al., 1974), leading to a consensus in the

eastern and southern provinces to control ECF (Nambota et al.,

ECF literature that antibodies did not play a major role in mediat-

1994). Fortunately, in Zimbabwe, a single parasite stock (Boleni)

ing immunity to the disease. However, indirect evidence for a role

provides broad-spectrum immunity within the country (reviewed

for antibodies in mediating immunity to ECF was derived from the

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

observation that cattle can develop sporozoite neutralizing anti-

revolves around the observation that cattle can remain as carriers

bodies (Musoke et al., 1982). However, this activity is seen after

of vaccine stocks, and that these may recombine with local parasite

multiple sporozoite exposure, suggesting weak immunogenicity of

strains during tick transmission giving rise to new more viru-

the relevant target/s.

lent parasite variants and upset the balance of endemic stability.

V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564 555 to

s.c.

(colony

(1992) (2005) al.

(2003) (1998)

al.

(1995) et al. al. (2005)

reactions

al.

al. c.f.u.

et et al. (saponin); al.

et et

al. clinical

References Musoke Nene Nene et Heussler Gentschev Honda Bishop Kaba Musoke (1996) (1998) (1998) of

units);

Abbreviations:

and severity (SR

forming SA WOE

the

group.

in in

33% 33% 13% 40% 69% 20% 25% 43% Outcome reduction) 66% 66% 58% 51% 36% 26% 55% 0% 70% 51% 40% 66% (plaque

based

p.f.u. immunized

was

4133

the and

3087 3087 3087 4133 4133 4133 4133 4133

or and

one

dose); in

3014

dose

number 3087 acinus,

that

Muguga Muguga Muguga Muguga Muguga Muguga Muguga Muguga

(lethal

dilution challenge

from LD

to LD68, 1:8 Muguga LD70, LD70, Equivalent Challenge LD68, LD70, LD68, LD80 LD70, Marikebuni stabilate infected

group

immune

RWL RWL

WOE

in in

control

were

(intramuscular);

saponin

SA SA the

immunized immunized immunized immunized immunized immunized immunized immunized in in in Salmonella V-IL2

that

i.m. ISA206

Non Non Non Non-immunized RWL Control Non Non Non Ovalbumin Non GFP GFP Ovalbumin or immunized and NS1 and and cattle

reactors of

adjuvant);

(13

WOE)

percentage numbers non-severe 13 Freund’s

as and immunized immunized controls immunized controls immunized controls immunized controls immunized controls immunized controls controls controls immunized immunized controls controls

immunized immunized controls immunized controls immunized controls immunized controls immunized controls immunized controls immunized controls immunized controls immunized controls

3 7 83 26 15 Animal 9 11 10 3 3 10 7 7 7 14 7 10 3 3 3 3 7 7 7 7 7 13 15 20 10 10 15 35 35 50 42 6 12 SA (incomplete

percentage expressed

IFA is Animal

the (both

Health) Health)

(Merck) (Merck) (Merck) WOE

(Pfizer (Pfizer (Pfizer

ISA206

SA SA SA or (Intervet

challenge

SA 3% RWL RWL None None and 3% Adjuvant 3% CFA/IFA None SA RWL Intervet International) Health) International) Animal Animal (SEPPIC) of subtracting

(intradermal); systems.

i.d.

outcome

delivery

The

calculated

protein); s.c. s.c. s.c. i.m. oral i.m. i.d. i.m. Route s.c.

was antigen

and This

presented. c.f.u. c.f.u. c.f.u.

p.f.u p.f.u p.f.u ﬂuorescent 11 11 11

× are ECF.

8 8 8 p67 p67

g g g g g g g g g

g ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮

–10 –10 –10 10 10 10 to

␮ 1–2 9 9 9

mg × × ×

× constructs emulsion). (green

1 300 600 10 10 10 5 5 5 450 450 450 100 10 450 450 450 cattle

× × × × × × × × × × × × × × × × ×

5 3 secreting secreting p67 5 5 c.f.u 3 3 2 2 2 3 3 3 2 2 3 3 3–4 3 Dose p67 GFP

indicus

susceptible

as Bos

(water-in-oil different

and

adjuvant);

of: of: antigen

WOE using

trials

classiﬁed

1–709 17–635 21–699 1–643 1–583 572–651 respectively 1–643 p67 1–709 1–709 Mix 21–699 Mix 572–651 21–225 17–635 21–651 572–651 17–635 572–651 1–709 20–709 20–622 1–709 20–709 20–622 sequence Freund’s are

cattle

vaccine in reactor);

virus )

p67

reactors trials

coli

(complete

(severe E.

Salmonella

(

extract)

SR ) ) vaccinia

CFA

) ) severe

vaccine

coli coli ) recombinant

recombinant ) ) published p67C

(cell

V-IL2 virus virus

coli coli

E. E. in in + e.g. ( ( (insect)

coli

coli coli

E. E.

( and ( units); experiment attenuated attenuated E.

E. E.

from ( ( (

1 in in

635 635

635 vaccinia vaccinia recombinant ﬁeld Salmonella Immunogen NS1-p67 BEV-p67 NS1-p67 p67 V-67-GPI V583-IL2 V-67-GPI P67N P67 GP64:p67C p67C p67 GFP:p67DSS p67C p67 p67 Experimental Table Results forming challenge, (subcutaneous);

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

8.1. Identiﬁcation and characterization of antigens that are were susceptible to challenge (Skilton et al., 2000). Hence, work on

targets of neutralizing antibodies this antigen was discontinued. The role of two other immunogenic

sporozoite antigens (p104 and p150) as candidate vaccine antigens

Characterization of a series of murine mAbs that neutralized has not been evaluated (Iams et al., 1990; Skilton et al., 1998).

sporozoite infectivity in in vitro assays identiﬁed a ∼67 kDa protein, Based on the large number of apicomplexan parasite molecules

called p67, as a major surface antigen of sporozoites, and as the pri- that generally play a role in host cell entry (Counihan et al., 2013),

mary target of neutralizing mAbs (nmAbs) (Dobbelaere et al., 1984, it is possible that additional T. parva candidate sporozoite vaccine

1985; Musoke et al., 1984). The p67 protein probably contributes targets remain to be discovered.

to the process of host cell recognition and entry, during which it is

shed (Webster et al., 1985). It is encoded by a single copy gene, and is

predicted to contain an N-terminal signal peptide and a membrane 8.2. Immunization with recombinant p67 can protect against

anchor domain (Nene et al., 1992). No sequence variation was found East Coast fever

in this gene in a set of cattle-derived T. parva isolates (Nene et al.,

1996). Allelic polymorphisms in p67 sequences are observed in Full-length and smaller regions of the p67 protein from the

buffalo-derived T. parva and in samples directly collected from cat- Muguga parasite isolate have been expressed in a recombinant

tle (Sibeko et al., 2010). However, it is not entirely clear if the latter form using a variety of different expression systems (Table 1).

parasite types can be maintained through serial passage between The high degree of sequence conservation of this protein makes

ticks and cattle. Sequence identity between full-length p67 alleles is it an intrinsically more attractive candidate vaccine antigen than,

greater than 90% (Nene et al., 1996). This suggests that the recently e.g., the PIM antigen. Unfortunately, the full-length p67 antigen

described diversity in a few p67 B-cell epitopes (Obara et al., 2015) has proved to be an extremely difﬁcult molecule to express in its

is unlikely to negatively inﬂuence the ability of p67 to function native format as a recombinant protein in E. coli (Bishop et al.,

as a cross-protective immunogen, a prediction that remains to be 2003; Musoke et al., 1992), Pichia pastoris (unpublished data), insect

validated. cells (Kaba et al., 2002, 2005; Nene et al., 1995) or mammalian

Host MHC class I molecules appear to facilitate sporozoite entry cells (Honda et al., 1998, and unpublished data). Under labora-

as mAbs to them can neutralize sporozoite infectivity, but they are tory conditions, recombinant p67 protein with adjuvants given via

unlikely to function as unique host cell receptors as sporozoites the sub-cutaneous route has been found to consistently induce

do not bind or enter all nucleated cells (Shaw et al., 1995). More immunity to ECF. Table 1 also provides a summary of results from

recently the function of p67 as a primary ligand has been brought experimental vaccine trials carried out over several years, includ-

into question. This is based on the observation that T. parva Chi- ing those derived from a small number of studies with bacterial or

tongo sporozoites only binds to CD8 T-cells yet the gene coding viral antigen delivery systems.

for p67 is identical in sequence to that of T. parva Muguga sporo- Experimental laboratory trials have mainly used a needle chal-

zoites, which bind B-cells and all subsets of T-cells (Tindih et al., lenge of sporozoites from the Muguga isolate. The dose was titrated

2010, 2012). Comparative molecular studies between sporozoites to provide a lethal dose 70% (LD70) challenge, which in control

of these two T. parva isolates should help to resolve their differences animals represented a 100% infectivity with ∼70% of the cattle

in host cell tropism. undergoing a severe ECF reaction (Musoke et al., 1992). Relative to

The PIM antigen was identiﬁed as a second target of nmAbs control groups, cattle immunized with recombinant antigen have

through serendipity. A series of mAbs raised to the schizont stage given consistent results exhibiting a range of immunity to ECF from

was found to be speciﬁc for PIM, and has been used to type differ- 13 to 70% (Table 1). Immunization with p67 protects against a chal-

ent parasite strains (Minami et al., 1983; Shapiro et al., 1987). The T. lenge with sporozoites from the Marikebuni isolate (see below),

parva PIM gene sequence is highly polymorphic in both cattle- and which in ITM studies falls in a different cross-immunity group to

buffalo-derived parasites (Baylis et al., 1993; Geysen et al., 2004; the Muguga isolate (Irvin et al., 1983). Immunization with recom-

Toye et al., 1995a). Some of the mAbs were subsequently found binant p67 protein even protects against a cross-species challenge

to neutralize sporozoite infectivity (Toye et al., 1995a), and lin- with T. annulata sporozoites (Hall et al., 2000). Surprisingly, p67C,

ear epitopes they bound were mapped by Pepscan analysis (Toye an 80 amino acid section toward the C-terminal end of the protein,

et al., 1996). PIM is localized to sporozoite rhoptries/micronemes induces equivalent levels of immunity to full-length p67 (Bishop

(Toye et al., 1996). In contrast, PIM is located on the schizont sur- et al., 2003). Cattle exhibit a range of ECF clinical symptoms from

face (Baylis et al., 1993; Shapiro et al., 1987), most likely as a non-reactors to severe symptoms, with only the latter classiﬁed as

molecule that spans the surface membrane several times. The cDNA susceptible to disease (Anon, 1988a). All cattle generate high levels

sequence of sporozoite and schizont PIM is identical, so it is not of antibody titers to p67, but in most experiments there has been

clear how the molecule is targeted to different cellular compart- no consistent correlate of various antibody assays with immunity

ments in the different life cycle stages. It is possible that nmAbs (Musoke et al., 1992; Nene et al., 1996, 1999).

gain access to PIM in a transient manner during sporozoite inva- Field trials exposing p67 immunized cattle to natural

sion to inhibit its function. However, the immunobiology of the tick/parasite challenge have been carried out at three sites in Kenya.

PIM antigen is quite complex as rats immunized with recombinant In these experiments, about 50% of the immunized cattle and 25%

PIM make neutralizing antibodies (Toye et al., 1995b) but cattle of the control animals exhibited immunity to ECF (Musoke et al.,

do not, although the bovine antibodies to PIM also bind to peptide 2005). While there was a statistically signiﬁcant level of immunity

sequences bound by nmAbs (Toye et al., 1996). A role for PIM as that could be ascribed to the vaccine, the overall level of immunity

an anti-sporozoite vaccine antigen remains to be explored, as it is in the ﬁeld relative to the laboratory challenge data was low, as

possible that immunization of cattle with different constructs of under the ﬁeld conditions some control cattle naturally recovered

PIM may induce neutralizing antibodies. from infection. These results are qualitatively different to those

A third candidate target of neutralizing antibodies, a 32 kDa pro- reported from a ﬁeld trial in Zambia, where immunized and con-

tein, was identiﬁed through immunoprecipitation of biotin labeled trol cattle were equally susceptible to ECF (Schetters et al., 2014).

sporozoites with the mAb 4C9. This protein proved to be a homolog However, immunized cattle were immune to ECF when given a

of the Tams1 antigen of T. annulata, a major surface antigen of sporozoite needle challenge. It is difﬁcult to reconcile the differ-

merozoites (Shiels et al., 1995). Bovine antisera to recombinant ences in ﬁeld results between the experiments in Kenya and Zambia

p32 did not induce neutralizing antibodies and immunized cattle as different antigen doses, adjuvants, and cattle types were used.

V. Nene et al. / Ticks and Tick-borne Diseases 7 (2016) 549–564 557

+ +

We ﬁrmly believe that p67 remains a strong candidate vac- of NKp46 CD3 cells (Connelley et al., 2014), remains to be fully

cine antigen, but research is required to increase the efﬁcacy of characterized.

the current p67 antigen formulation/immunization scheme. Under

laboratory conditions, it is presumed that sterilizing immunity

9.1. Identiﬁcation and characterization of CD8 T-cell antigens

was induced in a small number of cattle immunized with p67,

as they exhibited no reaction on challenge (Musoke et al., 1992).

The antigen speciﬁcity of CD8 T-cells is mediated by their T-

This is not a desirable outcome of a vaccine as this is likely to

cell receptor (TCR), which bind to peptide-MHC class I complexes

disrupt endemic stability of the disease. The lack of an in vitro cor-

at the surface of target cells (reviewed by Rossjohn et al., 2015).

relate with immunity hampers efforts to improve the efﬁcacy of

These peptides, usually from 8 to 9 amino acid residues in length,

p67, as immunogenicity studies alone do not predict the outcome

are derived from proteosomal degradation of antigens and bind to a

of challenge data. Efforts are underway to determine if different

peptide-binding groove in the MHC molecule (reviewed by Neefjes

types of correlates of immunity (Plotkin, 2010) can be devel-

oped. et 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 the

9. A role for cellular immune responses in mediating

reference T. parva Muguga genome sequence, or the transfection

immunity to East Coast fever

of random pools of clones from a Muguga schizont cDNA library

into antigen presenting cells. Transfected cells were co-cultured

Development of the ITM vaccine gave rise to a robust and repro-

with CTL lines generated from cattle immunized by ITM with the

ducible research model. Summarizing studies initiated in the late

+ Muguga parasite isolate. De-convoluting the components of pooled

1970s, there is now very good evidence that classical CD8 MHC

cDNA that were positive in an IFN-␥ ELISpot assay led to the identi-

class I restricted cytotoxic T-lymphocytes (CTLs) that kill schizont-

ﬁcation of individual parasite cDNA encoding a CTL antigen. The

infected cells play a major role in mediating immunity primed by

BoLA class-I heavy chain allele restricting the CTLs was deﬁned

ITM (Morrison and Goddeeris, 1990; Morrison, 2009b). The in vivo

by co-transfection of parasite cDNA with different BoLA class-I

importance of cellular responses in mediating immunity to ECF was

cDNA. Finally, overlapping synthetic peptides were used to map

established in passive transfer experiments of thoracic duct leuco-

the location of CTL epitopes. This ﬁrst round of screening deﬁned 8

cytes from immune animals to their naïve twins, which conferred

antigens, labeled Tp1 to Tp8, and mapped 11 CTL epitopes restricted

resistance to challenge (Emery, 1981). These studies were reﬁned

by one of seven different BoLA class I alleles (Table 2) (Akoolo

by demonstrating that immunity was associated with transfer of

+ et al., 2008; Graham et al., 2006, 2007a, 2008). Since then three

CD8 enriched cells from efferent lymph following mAb-mediated

+ more CTL antigens have been characterized, Tp9, Tp10 and Tp12

complement lysis of CD4 cells, ␥␦ T-cells and B-cells (McKeever

(cited in Morrison et al., 2015). Some of the Tp antigens have a

et al., 1994).

predicted function, including housekeeping functions. Not all of

As mentioned previously, the ITM vaccine induces strain speciﬁc

the Tp antigens encode a predicted signal peptide raising ques-

immunity. Most of what we know about the nature of this response

tions on how these antigens gain access to the host cell MHC class I

derives from studies using the T. parva Muguga and Marikebuni par-

antigen processing pathway. Interestingly, in T. annulata a member

asite isolates, which fall in different cross-immunity groups (Irvin

of the subtelomeric variable secreted protein (SVSP) gene family,

et al., 1983). In brief, these studies demonstrated that there was a

labeled Ta11, has been identiﬁed as a CTL antigen (MacHugh et al.,

marked correlation between the in vitro CTL strain speciﬁcity to kill

2011). This provides the ﬁrst evidence that members of a multi-

the different parasite isolates and cross-protection (Taracha et al.,

gene family in T. parva may play a role in the immune response to

1995a). There was immuno-dominance in the antigen speciﬁcity

infection.

of the CTL response that was inﬂuenced by the parasite antigenic

The immunoscreens for CTL antigens were carried out using less

type (Taracha et al., 1995a,b). In addition, the majority of the CTL

than a dozen BoLA haplotypes, as deﬁned by serology (Oliver et al.,

response was often restricted by one BoLA class-I allele, with a ten-

1989) Classical BoLA class I genes are located on bovine chromo-

dency of some alleles to dominate the immune response over others

some 23 (Brinkmeyer-Langford et al., 2009) and expressed from

(reviewed in Morrison et al., 2015).

six different loci (Codner et al., 2012). There is no co-dominant

The contribution of other cellular immune responses or innate

expression of the six alleles and usually no more than three will be

responses to immunity induced by ITM has not been extensively

+ expressed in a given haplotype (Codner et al., 2012). Some of the

characterized (reviewed in Morrison et al., 2015). CD4 T-cells

CTL lines used in the screens did not yield positive data (Graham

(Baldwin et al., 1987; Brown et al., 1989b,c, 1990) and ␥␦ T-cells

et al., 2006, 2007a). The Immuno-Polymorphism-Database (IPD-

(Daubenberger et al., 1999) show marked responses post-ITM vac-

MHC) houses ∼100 BoLA class I sequences (Robinson et al., 2010),

cination, but their roles in directly mediating immunity to ECF

and there is little doubt that the repertoire of bovine BoLA class I

remain to be fully characterized. The former are likely to play a

alleles is much higher in number. This indicates that many more

role in priming the CTL response (Taracha et al., 1997) and the lat-

T. parva antigens/epitopes, from both cattle- and buffalo-derived

ter can lyse schizont-infected cells (Daubenberger et al., 1999). The

parasites, remain to be characterized. The few antigens identi-

sheer numbers of ␥␦ T-cells in cattle leads to speculation that these

ﬁed so far have helped to unravel some of the complexity of the

cells could contribute to immunity via cross reactivity toward dif-

immunobiology of T. parva CTL antigens (see below). The recent

ferent T. parva strains, as MHC does not restrict the lytic response

availability of recombinant BoLA class I molecules (Hansen et al.,

of these cells (Daubenberger et al., 1999). This is consistent with

2014) has allowed the “on demand” production of peptide-MHC

what is known about ␥␦ T-cells in other mammals (Adams et al.,

+ class-I tetramers and ELISA-based peptide-MHC binding assays

2015). Efforts are underway by others to identify CD4 T-cell anti-

(Svitek et al., 2014). These permit in vitro and ex vivo epitope speciﬁc

gens, but little is known about the ␥␦ antigens, which in other

T-cell studies using ﬂow cytometry, where co-staining with various

systems have been identiﬁed as lipids or phosphoantigens (Adams

markers of interest can be used to describe the phenotype of epi-

et al., 2015). Interestingly, T. annulata schizonts express a large

tope speciﬁc CD8 T-cells (Svitek et al., 2014; Wendoh et al., 2014).

number of surface molecules that are phosphorylated (Wiens et al.,

Such data will provide important information on CTLs induced by

2014). The role of other innate cellular immune responses to infec-

ITM and experimental vaccines.

tion (reviewed in Morrison et al., 2015), including a novel subset

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

Table 2

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

a b

Antigen Antigen annotation Antigen length Signal peptide Restricting BoLA CTL epitope References

class-I allele

214

Tp1 Hypothetical 543 NO 6*01301 and VGYPKVKEEML224 Graham et al. (2008) 6*01302

6*04101 EELKKLGML37 Svitek et al. (2014)

Tp2 Hypothetical 174 YES Unknown DGFDRDALF48 Nene et al. (2012)

2*01201 KSSHGMGKVGK59 Graham et al. (2008)

c 96

BoLA-T2c FAQSLVCVL104 Graham et al. (2008)

2*01201 QSLVCVLMK106 Graham et al. (2008)

138

Unknown KTSIPNPCKW147 Akoolo et al. (2008)

328

Tp4 ␧-TCP1 579 NO 3*00101 TGASIQTTL336 Graham et al. (2008)

d 87

Tp5 eIF-1A 155 NO BoLA-T5 SKADVIAKY95 Graham et al. (2008)

c 206

Tp7 Hsp90 721 NO BoLA-T7 EFISFPISL214 Graham et al. (2008)

379

Tp8 Cysteine proteinase 440 NO 3*00101 CGAELNHFL387 Graham et al. (2008)

67 e

Tp9 Hypothetical 334 YES 1*02301 AKFPGMKKSK76 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) that

presents 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.

Predicted using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/).

Unassigned BoLA class-I allele.

Unassigned BoLA class-I allele, but the ␣1-␣2 domain sequence of the BoLA-T5 allele is identical to the ␣1-␣2 domain sequence of 1*00902.

Initially reported as a 10-mer, more recent data indicate the epitope is the 9-mer, AKFPGMKKS (Svitek et al., in preparation).

9.2. A role for NetMHCpan in the identiﬁcation and mainly to the N-terminal epitope. The speciﬁcities of the remaining

characterization of CTL epitopes CTL in these cattle are yet to be deﬁned, and the relative role of the

dominant versus less-dominant speciﬁcities in mediating immunity

The NetMHCpan algorithm (Hoof et al., 2009) can play a useful to ECF remains to be determined. These studies highlight extreme

role in the prediction of bovine CTL epitopes (Nene et al., 2012). We skewing of CTL responses in these cattle, which presumably con-

recently reported on optimization of NetMHCpan using data gen- tributes to the phenomenon of strain speciﬁc immunity induced by

erated by measuring the binding of all possible nonameric peptides ITM. Unfortunately, there has not been extensive characterization

to nine recombinant BoLA class I molecules (Hansen et al., 2014). of the inﬂuence of BoLA-type on the speciﬁcity of CTL responses in

The data generated a motif for peptides that bind each BoLA class I cattle that are heterozygous at the BoLA locus, or on the speciﬁcity

molecules and identiﬁed anchor residues, which play a more criti- of CTL responses to infection with mixed parasite isolates, e.g., the

cal role in peptide binding. Based on such parameters NetMHCpan Muguga cocktail.

assigns a score to peptide sequences that can bind to a given MHC Alanine scanning experiments of the peptide Tp1214–224 and

molecule (Hoof et al., 2009). NetMHCpan predicted that Tp229–37 Tp249–59 epitopes with different CTL clones highlight the impor-

rather than Tp227–37 (Graham et al., 2006) is likely to be the epitope tance of different anchor residues in the peptide binding to BoLA,

that binds to BoLA-6*04101 (Hansen et al., 2014). It also predicted and the inﬂuence of peptide-BoLA recognition by different TCRs

that the Tp587–95 epitope bound by BoLA-T5 could also be bound (Connelley et al., 2011; Macdonald et al., 2010). It has also been

by BoLA-1*02301, an allele originally identiﬁed as presenting an demonstrated that animals possessing BoLA-6*01302, which dif-

epitope on Tp9 (Table 2). We experimentally veriﬁed these predic- fers from BoLA-6*1301 by a single amino acid substitution in the

tions (Hansen et al., 2014; Svitek et al., 2014). In the context of CTL peptide binding groove still presents the Tp1214–224 epitope (Svitek

responses to T. annulata, the BoLA-1*02301 allele has been shown et al., 2015). Sequencing of Tp genes from different cattle- and

to present epitopes on Ta9 and Ta5, which are homologs of the Tp9 buffalo-derived parasites has revealed extensive antigenic diver-

and Tp5 antigens of T. parva (MacHugh et al., 2011). sity in the Tp antigens (Morrison et al., 2015; Pelle et al., 2011). The

ability of variant sequences to function as cross-reactive epitopes

will depend on the molecular ﬂexibility of the peptide-MHC-TCR

9.3. Epitope speciﬁcity of the CTL response in cattle immunized by

interaction. For example, in the context of the respective BoLA class

the infection and treatment method

I allele, variants of the Tp249–59 epitope seem to be less tolerated

than variants of Tp1214–224 (Connelley et al., 2011; Macdonald et al.,

With the identiﬁcation of CTL antigens it has been possible to re-

2010; Steinaa et al., 2012). In some cases amino acid substitutions

assess the phenomenon of immuno-dominance in cattle immune

may inﬂuence the proteolytic processing of antigens to generate

to ECF. Almost 70% of the CTL response to the Muguga parasite iso-

the relevant epitopes (Morrison et al., 2015). These data indicate

late in cattle homozygous for BoLA-A18 haplotype is directed to a

that effort is needed to produce a denser map of T. parva CTL anti-

single epitope on the Tp1 antigen, while the response in BoLA-A10

gens that contribute to strain speciﬁc and cross-reactive responses.

homozygous cattle is directed to one epitope on the Tp2 antigen,

This should aid the selection of antigens required to prime broad-

with no detectable response to Tp2 in the BoLA-A18 cattle type

spectrum immunity to ECF.

or to Tp1 in the BoLA-A10 animals (MacHugh et al., 2009). T-cell

receptor analysis has revealed that the CTL response is polyclonal

in nature with predominant clonotypes responding to either the 9.4. The challenge of priming CD8 CTLs in cattle

Tp1214–224 or Tp249–59 epitope (Connelley et al., 2011; Macdonald

et al., 2010). The Tp2 antigen contains a second epitope, Tp298–106, Attempts to induce CTL in cattle that kill autologous schizont-

restricted by BoLA-2*01201 (Table 2), yet the CTL were directed 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 3

Experimental vaccine trials in cattle using schizont antigens.

Delivery system Immunogen Dose Route Animal Control Challenge Outcome References

numbers

CP/MVA and Tp1, Tp2, Tp4, 0.5 mg DNA or DNA i.d./CP and 12 DNA/MVA Immunized LD100 Muguga 17% survivors. Graham et al.

DNA/MVA Tp5 and Tp8 10 p.f.u. CP MVA s.c. immunized with vaccine 4133 Presence of CTL (2006)

prime, boost 12 CP/MVA diluents correlated with

5 × 10 p.f.u. immunized survival.

MVA 4 controls

DNA (PcDNA4// PIM 3 × 1 mg i.d. 2 immunized Plasmid only LD100 Katete 1 PIM immunized Ververken et al.

V5-HIS) 1 control animal with CTL (2008)

survived. The other

PIM animal had

MHC class I

unrestricted CTL

and was

susceptible, as was

the control.

Flt3 + GM-SCF Tp1, Tp2, Tp4, 0.5 mg of FLT3 i.d. 12 immunized Immunized LD100 Muguga All animals died, Mwangi et al.

DNA/MVA Tp5 and Tp8 and 8 controls with vaccine 4133 but the control (2011)

GM-CSF/0.5 mg diluents group died faster

DNA indicating some

(each)/5 × 10 effect of

p.f.u. MVA immunization.

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

kinase ligand); GM-CSF (granulocyte-macrophage colony-stimulating factor); MVA (modiﬁed vaccinia Ankara); s.c. (subcutaneous); p.f.u. (plaque forming units). In the PIM

DNA experiment, although the control animal was susceptible to challenge, it did not undergo a typical response (Ververken et al., 2008).

successful. One study using CpG oligodeoxynucleotides and recom- one is found, the number of CTL antigens that will be required in

binant PIM antigen resulted in priming of cytolytic CD4 T-cells an anti-schizont vaccine remains unknown.

(Graham et al., 2007b). A study using immunization with DNA

encoding the PIM antigen in two animals gave equivocal results 10. Desirable qualities in a subunit vaccine for the control

(Ververken et al., 2008). The most successful approaches have used of East Coast fever

at least one viral vectored antigen delivery system in a prime-boost

format (Table 3). In order to facilitate immunogenicity studies, One component of a target product proﬁle of a subunit vaccine

cattle carrying BoLA class I alleles that present the known Tp epi- is to prime immunity against a broad-spectrum parasite chal-

topes were used. For example, cattle with the BoLA-A18 haplotype lenge, e.g., that of the Muguga cocktail. The intensity, parasite

should respond to the Tp1214–224 epitope (Table 2). The antigens composition, and time to ﬁrst challenge post-vaccination will vary

tested so far derive from the Muguga parasite isolate and cat- depending on the livestock production system in which a vaccine is

tle were given a homologous LD100 sporozoite needle challenge. deployed. Whatever the composition of the vaccine, it is desirable

A prime-boost regimen in 24 cattle primed with a mix of plas- that cattle undergo a mild to moderate disease reaction on chal-

mids (DNA prime) or a mix of recombinant canary pox virus (CP lenge. For an anti-sporozoite based vaccine, this will also result

prime) expressing ﬁve antigens (Tp1, Tp2, Tp4, Tp5 and Tp8), fol- in priming a parasite-induced CTL response thereby mimicking the

lowed by a boost with a mix of recombinant modiﬁed vaccinia live parasite method of vaccination. For an anti-schizont based vac-

Ankara (MVA) virus expressing the same 5 antigens, resulted in cine, this should boost vaccine induced CTL speciﬁcities, and may

␥

IFN- ELISpot positive results in 19 cattle (Graham et al., 2006). prime additional CTL speciﬁcities not present in the subunit vac-

Four cattle exhibited CTL activity and an additional three were cine, as is thought to occur in animals vaccinated by ITM. If these

positive 2 weeks post-challenge. These seven animals were the antigens can function in a synergistic fashion, a vaccine targeting

only ones that were immune to challenge. When compared to the both life cycle stages is conceptually appealing and may be of spe-

control group the overall survival rate in the experimental group cial value in reducing the dose of sporozoite strains not covered by

was 17%, as one animal out of four in the control group recovered the vaccine CTL speciﬁcities. Maintaining an adequate level of pro-

from challenge. Although cattle were pre-selected for their BoLA tective antibody and CTL responses in a combination vaccine may

haplotype not all cattle responded with the expected Tp epitope be less challenging than for either one alone. One concern that will

speciﬁcities. In addition, very few animals mounted a response need to be addressed is whether recovery from an infection inter-

to more than one immunogen (Graham et al., 2008). Approaches feres with the subsequent quality and longevity of vaccine induced

to augment the immune response to recombinant MVA by using immune responses.

the cytokines granulocyte-macrophage colony-stimulating factor

(GM-CSF) and FMS-like tyrosine kinase ligand (Flt3L) elicited IFN- 11. Conclusions

+ +

␥ secreting CD4 and CD8 cells, but no CTL (Mwangi et al., 2011).

Recent unpublished data using AdHu5/MVA as a prime-boost sys- Substantial advances have been made in the identiﬁcation and

tem with the Tp1 antigen conﬁrmed an ability to prime CD8 CTL characterization of candidate vaccine antigens of T. parva. Although

that will kill peptide-pulsed targets. However, these CTL do not kill time consuming, the technical challenge of identifying T. parva CTL

schizont-infected cells, indicating that CTL generated by this sys- antigens has been largely overcome. What is not clear at this stage

tem are qualitatively different to those generated by ITM (Svitek is the identity and number of sporozoite and/or schizont antigens

et al, in preparation). Resolution of these differences will be an that will be required to prime broad-spectrum immunity, and how

important step forward. Currently, as in other large mammals, the to induce a robust CTL response. The application of more modern

routine priming of CTLs remains a technical challenge. Our collab- approaches to vaccine-related research (Koff et al., 2013; Nakaya

orators and we are exploring other antigen delivery systems. Until and Pulendran, 2015) should lead to improved understanding

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

and proﬁling of the immune response to ITM and experimental Brown, W.C., Shaw, M.K., Conrad, P.A., Dolan, T.T., 1989a. Theileria parva:

reappearance of schizonts in infected lymphoblastoid cells treated with

vaccines. Hence, the prospects for developing a subunit vaccine

parvaquone is dependent on interleukin 2-like growth factors. Exp. Parasitol.

for the control of ECF are good. The buffalo wildlife reservoir of

68, 308–325.

T. parva remains a confounding factor. However, we anticipate Brown, W.C., Sugimoto, C., Conrad, P.A., Grab, D.J., 1989b. Differential response of

bovine T-cell lines to membrane and soluble antigens of Theileria parva

a greater understanding of the differences between cattle- and

schizont-infected cells. Parasite Immunol. 11, 567–583.

buffalo-derived T. parva, as more whole genome sequence data

Brown, W.C., Sugimoto, C., Grab, D.J., 1989c. Theileria parva: bovine helper T cell

accumulate. In the interim, commercialization of the Muguga clones speciﬁc for both infected lymphocytes and schizont membrane

antigens. Exp. Parasitol. 69, 234–248.

cocktail ITM vaccine is good news for the cattle industry in Africa.

Brown, W.C., Lonsdale-Eccles, J.D., DeMartini, J.C., Grab, D.J., 1990. Recognition of

It is reasonable to expect improvements in the manufacture and

soluble Theileria parva antigen by bovine helper T cell clones: characterization

delivery of this vaccine as experience in its use in different farming and partial puriﬁcation of the antigen. J. Immunol. 144, 271–277.

systems accumulates. Burrows, J.N., Burlot, E., Campo, B., Cherbuin, S., Jeanneret, S., Leroy, D.,

Spangenberg, T., Waterson, D., Wells, T.N., Willis, P., 2014. Antimalarial drug

discovery – the path towards eradication. Parasitology 141, 128–139.

Acknowledgements Chaussepied, M., Langsley, G., 1996. Theileria transformation of bovine leukocytes:

a parasite model for the study of lymphoproliferation. Res. Immunol. 147,

127–138.

We wish to thank the many years of excellent contribution of Chaussepied, M., Janski, N., Baumgartner, M., Lizundia, R., Jensen, K., Weir, W.,

Shiels, B.R., Weitzman, J.B., Glass, E.J., Werling, D., Langsley, G., 2010. TGF-␤2

past and present scientists and laboratory personnel, farm and tick

induction regulates invasiveness of Theileria-transformed leukocytes and

unit staff in support of East Coast fever research at ILRI. We thank

disease susceptibility. PLoS Pathog. 6, e1001197.

Ivan Morrison for a critical review of the manuscript. Previous core Cheeseman, K., Weitzman, J.B., 2015. Host-parasite interactions: an intimate

epigenetic relationship. Cell. Microbiol. 17, 1121–1132.

ﬁnancial support from ILRI donors is gratefully acknowledged and

Codner, G.F., Birch, J., Hammond, J.A., Ellis, S.A., 2012. Constraints on haplotype

IFAD for funding activities related to commercialization of the ITM

structure and variable gene frequencies suggest a functional hierarchy within

vaccine. More recent research at ILRI has been funded through cattle MHC class I. Immunogenetics 64, 435–445.

Connelley, T.K., Machugh, N.D., Pelle, R., Weir, W., Morrison, W.I., 2011. Escape

grants from the USA National Science Foundation (0965346), the

from CD8 T cell response by natural variants of an immunodominant epitope

CGIAR Research Program on Livestock and Fish, the Norman Borlaug

from Theileria parva is predominantly due to loss of TCR recognition. J.

Commemorative Research Initiative, an initiative between the Feed Immunol. 187, 5910–5920.

the Future program of USAID and USDA-ARS (58-5348-2-117F) and Connelley, T.K., Longhi, C., Burrells, A., Degnan, K., Hope, J., Allan, A.J., Hammond,

+ +

J.A., Storset, A.K., Morrison, W.I., 2014. NKp46 CD3 cells: a novel

the Department for International Development of the United King-

nonconventional T cell subset in cattle exhibiting both NK cell and T cell

dom and the Bill and Melinda Gates Foundation (OPP1078791).

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Conrad, P.A., Denham, D., Brown, C.G., 1986. Intraerythrocytic multiplication of

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