mechanism(s) of attenuation of theileria annulata vaccine cell lines

Tropical Medicine and International Health

volume 4 no 9 pp a78–a84 september 1999

© 1999 Blackwell Science LtdA78

Mechanism(s) of attenuation of Theileria annulata vaccine celllines

Roger Hall1, Tulin Ilhan2, Erol Kirvar2, Gwen Wilkie2, Patricia M. Preston3, Mohamed Darghouth4,Robert Somerville1 and Rachel Adamson1

1 Department of Biology, University of York, UK,

2 Centre for Tropical Veterinary Medicine, University of Edinburgh, UK,

3 Institute of Cell & Population Biology, University of Edinburgh UK

4 Ecole Nationale de Médecine Vétérinaire, Tunisia

Summary Attenuated vaccines are an important means of controlling Theileria annulata infection of cattle. Production

is by prolonged cultivation of macroschizont-infected cells. The mechanism of attenuation remains unclear.

There are three general nonmutually exclusive possibilities: Selection of avirulent subpopulations, genome

rearrangements and alterations in gene expression. Several groups, including ours, have provided evidence

that the population structure usually tends to simplify during attenuation. Our data on the T. annulata (Ta)

Ankara cell line show that attenuation is not necessarily accompanied by the population becoming clonal.

We have been unable to detect large DNA rearrangements. Evidence for alterations in host and parasite gene

expression during attenuation is available. With respect to the host we have shown that attenuation is accom-

panied by loss of expression of parasite induced matrix metalloproteinases (MMPs). However, in different

lines different protease activities are involved. In the T. annulata Ode line we have shown that 8 activities

(including MMP9) are downregulated and that this correlates with a loss of metastatic behaviour. This has

previously been shown in vitro using reconstituted basement membrane (MatrigelTM) and is demonstrated in

vivo using scid mice in this study. Thus part of the pathology, namely the ability to disseminate, mediated by

host MMPs, is lost upon attenuation. Re-isolation experiments have shown that the reduction/loss of MMP

is a stable transferable trait. A logical extension is that loss of MMP activity (and virulence in general) must

be at the most fundamental level a genetic trait of the parasite. Evidence for loss of parasite gene expression

is implied by the loss of the ability to differentiate into merozoites on attenuation. Specific evidence for loss

of parasite gene expression has been obtained using differential RNA display. We view virulence as a multi-

factorial phenomenon involving interacting subpopulations of cells and attenuation is a threshold effect

whereby the number of virulence factors is reduced below a critical level. On this basis there will be many

different ways to achieve attenuation.

keywords Theileria annulata, attenuation, virulence, vaccine, matrix metalloproteinase, metastasis,

microschizont

correspondence Roger Hall, Department of Biology, PO Box 373, University of York, YO10 5YW, UK,

Fax: 144 (0)1904 432860; E-mail: [email protected]

Introduction

Control of tropical theileriosis is mainly with attenuated cell-

line vaccines (Pipano & Tsur 1966; Gill et al. 1976; Ozkoc &

Pipano 1981; Pipano 1981; Singh 1990). These vaccines, pro-

duced by prolonged in vitro cultivation of the macroschizont

stage of the life cycle, have an efficacy approaching 95–100%

(Brown 1990). A subunit vaccine is being developed, but much

research is still required before it will rival the live vaccines

(Boulter et al. 1995). Despite the widespread use of live vac-

cines, the process of attenuation is only poorly understood.

However, the primary event(s) resulting in the attenuated

phenotype must occur in the parasite and not in the host cell.

This assertion relies on the fact that vaccination requires the

TMIH454

Tropical Medicine and International Health volume 4 no 9 pp a78–a84 september 1999

R. Hall et al. Epidemiology and control of bovine theileriosis

© 1999 Blackwell Science Ltd A79

transfer of the macroschizont from the donor cell, in which it

was attenuated, to the cells of the new host (Pipano & Israel

1971).

A number of phenomena, of both host and parasite origin,

correlated with the acquisition of the attenuated phenotype,

have been described. Thus attenuation is associated with a

reduction in host matrix metalloproteinase (MMP) activity,

reduced ability to differentiate into merozoites, reduction in

specific parasite gene expression and alteration of the popu-

lation structure (Baylis et al. 1992, 1995; Adamson & Hall

1996, 1997; Darghouth et al. 1996; Sutherland et al. 1996;

Somerville et al. 1998ab). In this paper we present data based

on parasitized vaccine cell lines derived from 6 stocks from

India, Turkey and Tunisia (Table 1). The combined data show

that these phenomena are general. Furthermore we show that

the loss/reduction of MMP activity is a transferable trait in

several re-isolated lines. Our results are discussed in the con-

text of a multifactorial model for the virulent phenotype.

Materials and methods

T. annulata cell lines, cell culture, in vitro differentiation

The cell lines used in this study, and their origin, are listed in

Table 1 alongside the appropriate references. The cells were

cultured according to the method of Brown (1987). The

capacity to differentiate into microschizonts was estimated by

placing the cultures at 41 °C for 11 or 12 days as described

(Glascodine et al. 1990 Shiels et al. 1992; Baylis et al. 1992;

Somerville et al. 1998a).

Southern blotting and gelatin substrate gels

DNA was extracted from macroschizont-infected cells,

digested with Eco RI, blotted and probed with the 39 SPAG-1

gene probe as previously described (Katzer et al. 1994; Somer-

ville et al. 1998a), metalloproteinase profiles were assayed on

gelatin substrate gels exactly as detailed by Baylis et al. (1992).

Metastasis of macroschizont-infected cells in scid mice

This was performed as described by Somerville et al. (1998b).

Briefly, female C.B.-17 scid mice (Harlan, UK) aged

3–4 months were used. They were maintained in sterile cabi-

nets (Kytota, Muurame, Finland). Groups of 10 mice were

inoculated i.p with 2 3 106 Ta-Ode cells at passage levels 58

(p58) or 158 (p158) in PBS. The mice were sacrificed on day

11 and impression smears of the organs prepared and stained

with Giemsa. Macroschizonts were counted in 100 random

fields for each organ from each mouse and the data are pre-

sented as the mean number of macroschizonts per field per

tissue.

Results

Attenuation is accompanied by fluctuations in the parasite

population structure but clonality is not essential

Restriction fragment length polymorphism (RFLP) analysis,

using the 39 end of the SPAG-1 gene as a probe (Katzer et al.

1994), shows that the genetic composition of 3 of 4 vaccine

lines alters during attenuation (Table 2). The population in

the early (p4) Ta-Diyarbakir line carries only the 3.4 kb RFLP

Table 1 Details of T. annulata cell lines

Vaccine Country of Early passage Attenuated passage

cell line origin level level Reference

Ankara Turkey 14 387 Ozkoc & Pipano (1981); Somerville et al. (1998a)

Diyarbakir Turkey 04 457 Pipano & Shkap (1979)

Hissar India 15 096 Gill et al. (1976); Sutherland et al. (1996)

Ode India 58 158 & 300 Baylis et al. (1992)

Jedeida Tunisia 11 201 & 302 Darghouth et al. (1996)

Batan Tunisia 09 199 & 302 Darghouth et al. (1996)

Table 2 Distribution of allelic SPAG-1 RFLP in T. annulata cell

lines

SPAG-1 RFLP Alleles

Cell line 3.4 kb 4.8 kb 6.0 kb

Ankara p14 1 1 1 1 no 1 1 1 1

Ankara p387 1 1 1 1 no 1

Diyarbakir p4 1 1 1 1 no no

Diyarbakir p457 no 1 1 1 1 no

Hissar p15 1 1 1 1 1 1 1 1 1 1

Hissar p96 1 1 1 1 no no

Ode p58 no no 1 1 1 1

Ode p158 no no 1 1 1 1

1 1 1 1 very strong band; 1 1 1 strong band; 1 weak band

no, no band.




allelic marker, whereas by p457 this has been completely

replaced by the 4.8 kb allele. In the Ta-Hissar and Ta-Ankara

lines, the SPAG-1 allele pattern simplifies upon long-term cul-

ture (Table 2). The Ta-Hissar line initially (at p15) has all 3

allelic RFLP markers of 3.4, 4.8 and 6.4 kb, but at p96 only

the 3.4 kb allele is present. In the Ta-Ankara line both the

early (p14) and late (p387) passages have two SPAG-1 alleles

of 3.4 and 6.0 kb but the late (p387) attenuated cell popu-

lation however, has a much lower frequency of the 6.0 kb

allelic marker. Since the attenuated Ta-Ankara cell line still

contains two SPAG-1 alleles, and since this parasite is

haploid, we conclude that attenuation is not necessarily a

clonal phenomenon.

Matrix metalloproteinase profiles and attenuation

In all 7 lines examined the early passage levels had MMP

activity which was lost or substantially diminished upon

attenuation (Table 3). However there were quantitative and

qualitative differences between lines. Thus a major activity in

5 of the cell lines was MMP9. Two lines (Ta-Ankara & Ta-

Diyarbakir), which are adherent in character, however,

Metalloproteinase Metalloproteinase profile

Cell line activity score*

Ankara p14 1 1 1 Major band at 72 kD, MMP2?

p387 2 72 kD activity lost

Diyarbakir p4 1 1 1 Major band at 72 kD, MMP2?

p457 1 72 kD activity lost

Hissar S45 p5 1 1 1 1 8 bands of activity including MMP9

RI1 1 1 1 8 bands of activity including MMP9

RI2 1 1 1 1 8 bands of activity including MMP9


p50 1 1 1 8 bands of activity including MMP9

p130 1 8 bands of activity including MMP9

RI1 1 8 bands of activity including MMP9



Ode p58 1 1 1 1 8 bands of activity including MMP9

RI1 2 8 bands of activity including MMP9 lost


p158 1





p300 2 8 bands of activity including MMP9 lost



Ankara (Pendik) p4 1 1 1 8 bands of activity including MMP9







Jedeida p11 1 2 bands of activity including MMP9




Batan p9 1 1 2 bands of activity including MMP9



RI, re-isolate of line after passage through a calf. *The score is presented on a sliding arbitrary

scale with 1 1 1 1 highest, 1 lowest and 2 no activity

Table 3 Metalloproteinase activity of

T. annulata cell lines




expressed a different MMP at 72 kd which is probably

MMP2. Of substantial interest is the observation that, in all

cases, parasitized cells re-isolated (labelled RI in Table 3)

from cattle infected with low or null producing cells were also

poor MMP expressors. This suggests that the MMP pheno-

type is a result of a parasite influence on the host cell and that

loss of MMP production is due to a genetic component of

the parasite.

Metastatic properties are related to passage level and are

mediated by MMPs in scid mice

The ability of the Ta-Ode line to disseminate in scid mice was

investigated. The p58 level, which produces abundant MMP

activity, was found in all 5 organs examined. In contrast the

p158 cells, which produce a much diminished level of MMP

activity, were not found in any tissue except for a low level in

the lungs (Table 4). This metastatic phenotype is MMP

dependent since it can be inhibited by a highly specific MMP

inhibitor called BB94 (own observations, Somerville et al.

1998b).

Attenuation is accompanied by a reduction in the ability to

differentiate from macroschizont to microschizont

In 4 lines the late passage cells showed a marked reduction in

the production of microschizonts when placed at 41 °C for

11–12 days (Table 5). Thus the early Ta-Ankara, Ta-

Diyarbakir, Ta-Hissar and Ta-Ode lines produced 33, 26, 38

and 45% microschizonts, respectively, whereas the corre-

sponding values for the late lines are 3, 2.5. 6 and 9%, respec-

tively.

Discussion

The work summarized here demonstrates that the attenuated

phenotype is associated with several phenomena, namely

changes in the parasite population structure, reduction in

MMP activity with concomitant reduction in metastatic

potential and reduction in microschizont production (Baylis

et al. 1992; Adamson & Hall 1996, 1997; Darghouth et al.

1996; Sutherland et al. 1996; Somerville et al. 1998ab). The

alterations in parasite population structure we observed are

consistent with an overall simplification of the genetic com-

position and a trend towards clonality. However, the fact that

the attenuated Ta-Ankara line retains two SPAG-1 alleles

demonstrates that loss of virulence is not necessarily exclu-

sively a clonal phenomenon.

Perhaps the most striking aspect and generic feature of the

attenuated lines is their loss of host MMP activity relative to

their progenitors. This phenomenon has been observed in 7

different lines from 3 geographical locations (Table 3). There

are quantitative and qualitative differences between the lines

with respect to this phenomenon but the trend is most per-

suasive. The fact that the same enzymes are characteristic of

metastatic tumours (Stetler-Stevenson et al. 1993) leads to the

obvious suggestion that they contribute to the dissemination

of the schizont-infected cells throughout the body of the

bovine host (Adamson & Hall 1997). We provided evidence

in support of this notion based on the ability of infected cells

to invade reconstituted basement membranes as well as

spread throughout the bodies of scid mice (Adamson & Hall

1996; Somerville et al. 1998b). Thus the highly secreting p58

Ta-Ode line spreads through scid mice whereas the low

secretor p158 does not (Table 4). Furthermore this ability of

Ta-Ode p58 to metastasize is specifically inhibited by an ex-

quisite MMP inhibitor BB94 (unpublished observations;

Somerville et al. 1998b). Loss of the ability to express MMPs

upon attenuation could go some way to explaining the reduc-

tion in virulence.

Table 4 Metastasis of the T. annulata Ode cell line in scid mice

Numbers of macroschizonts per field

———————————————Tissue ODE p58 ODE p158

Kidney 1.09 6 0.4 0

Liver 1.32 6 0.45 0

Mesentery 1.44 6 0.65 0

Tumour 2.00 6 1.2 0

Spleen 0.57 6 0.55 0

Lung 1.00 6 0.2 0.175 6 0.4

The figures in the table are the average of 100 random fields

Table 5 Comparison of the ability of early and late passage T.

annulata cell lines to differentiate from macroschizonts into

microschizonts

Cell line % microschizonts

Ankara*

p14 33

p387 03

Diyarbakir*

p4 26

p457 02.5

Hissar*

p15 38

p96 06

Ode†

p58 45

p158 09

Cells were left for *11 or †12 days at 41 °C.




Virulent Less virulent Attenuated

RFLP = 1, 2, 3, 4 RFLP = 1, 3, 4 RFLP = 3, 4

MMP+

MMP+

MMP+

MMP+

MMP+

MMPÐ

MMPÐMMPÐ

CKÐCKÐ

CKÐ

CKÐCKÐ

CKÐ

CK+

CK+

CK+

M

M

M

M

M

M

M

M

M

B

B

B

B

B

B

B

2

2

1

3

4

1

3

4

3

4

1

3

4

1

3

4

3

Figure 1 Multifactorial model of virulence and attenuation in Theileria annulata. The virulent population is composed of phenotypically dis-

tinct host cells (i.e M 5 macrophage/monocyte and B 5 B cells) infected with genotypically distinct macroschizonts (i.e genotypes 1, 2, 3 and 4

which are defined by a specific allelic RFLP marker). Some genotypes interact with the host to produce virulence factors (i.e genotype 2 interacts

with B cells to produce MMPs and macrophages to produce various cytokines (CK), e.g. TNF a or IL1-a (etc). The mixture of host factors pro-

duced leads to virulent disease. Some genotypes do not stimulate the production of virulence factors in certain host cells (i.e 3 does not stimulate

B cells to secrete MMPs or macrophages to secrete cytokines). Over time in culture the genotypic complexity of the parasite reduces and poss-

ibly less virulent genotypes prevail although a general reduction in the numbers of parasite genotypes will probably result in production of fewer

types of virulence factor. Eventually the number/activity of virulence determining factors will fall below a threshold even before the parasite has

gone clonal. At this point the culture is attenuated since the loss in diversity of parasite genotypes means that the threshold spectrum of viru-

lence factors cannot be induced in the new host after macroschizont transfer.

B



One of the most remarkable aspects of the mechanics of

the attenuated vaccine is the necessity for the macroschizont

to transfer from bovine donor cells to the cells of the recipi-

ent. This means that fundamentally the attenuated phenotype

is determined by the parasite and any host effects on the

degree of virulence must be due to effects exerted by the par-

asite. Thus if loss of virulence is related to loss of MMP

activity, the inability to induce MMPs must be a stable feature

of the attenuated parasite. In fact this prediction is shown to

be true in all 12 re-isolates of low/null MMP expressors

examined covering 3 vaccine lines (Table 3). That is to say

reduction/loss of MMP activity is a stably inherited parasite

determined character as judged by the fact that re-isolates

after transfer to the new host cell show the same phenotype

(Table 3).

Evidence that genetic changes in the parasite itself are

responsible for attenuation is provided by the loss of ability

to differentiate to microschizonts upon cultivation at 41 °C,

shown in the 4 lines examined (Table 5). This could underlie

the lack of appearance of piroplasms in vivo characteristic of

attenuated vaccine-induced infections. Specific examples of

parasite genes downregulated upon attenuation which may

influence virulence are provided by the loss of expression of

the antigen recognized by monoclonal antibody EU106 by the

S45 Ta-Hissar line and a gene called deg1.10 cloned from the

Ta-Ankara line (Sutherland et al. 1996; Somerville et al.

1998a).

Taken together, we would interpret virulence as being a

multifactorial phenomenon and suggest that the model

shown in Fig. 1 may be used to explain attenuation. In

essence we envisage the virulent population consisting of

multiple genetically distinct parasites residing in several

classes of host mononuclear cell. Each combination of para-

site and host will have different outcomes, some of which

include secretion of mediators of virulence such as MMPs

and cytokines such as TNF a or IL1-a. (Brown et al. 1995).

The virulent features of the infection will be determined by

this cocktail of factors, which must be present above a quan-

titative or qualitative threshold. During long-term culture the

population structure simplifies such that the number of para-

site genotypes reduces and the complexity of virulence fac-

tors diminishes below a threshold. The result is a parasite

population genetically incapable of inducing sufficient quan-

tity and/or diversity of mediators of virulence upon transfer

to a new host. Whilst this model leaves out much detail and

complexity it broadly describes the known characteristics of

the phenomenon of attenuation in Theileria annulata vaccine

cell lines.

Acknowledgements

We are particularly grateful to Duncan Brown for provision

of parasite material, critically reading the manuscript and

keeping us all in line as only he knows how. Thanks to DK

Singh for the Ode line and Eugene Pipano and Varda Shkap

for the Ankara and Diyarbakir lines. Work in this paper was

supported by grant number 050220 from the Wellcome Trust.

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