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Veterinary Immunology and Immunopathology 113 (2006) 357–366

Serum containing ovine IgG2 antibody specific for

maedi visna virus envelope glycoprotein mediates

antibody dependent cellular cytotoxicity

Inderpal Singh a,*, Ian McConnell b, Robert Dalziel c, Barbara A. Blacklaws b

a VaxDesign Corporation, 2721 Discovery Drive, Suite 400, Orlando, FL 32826, USAb Centre for Veterinary Science, University of Cambridge, Madingley Road, Cambridge, UK

c Division of Veterinary Biomedical Sciences, University of Edinburgh, Summerhall, Edinburgh, UK

Received 6 January 2006; received in revised form 12 June 2006; accepted 19 June 2006

Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for maedi visna virus (MVV) has never been described. The

IgG antibody response to MVV is restricted to an IgG1 response whilst MVV specific IgG2 is never seen in persistently infected

sheep. To determine whether the isotypic restriction of the antibody response is responsible for the lack of ADCC, an ADCC assay

was developed using polyclonal serum raised to recombinant MVV ENV protein. Sheep immunised with a recombinant

GST:SUenv fusion protein in complete Freund’s adjuvant produced an antibody response which contained IgG1 and IgG2

antibodies. The activity of this serum in an ADCC assay was compared to serum from persistently infected sheep. Serum from

immunised sheep mediated ADCC reactions whilst no activity was ever seen in persistently infected sheep serum. IgG2 may

therefore be the possible effector isotype for ADCC reactions against MVV. Failure of the IgG2 dependent ADCC system in vivo

may contribute to the persistence of MVV-infected macrophages in vivo.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Maedi visna virus; Ovine progressive pneumonia; Antibody dependent cytotoxicity; Envelope; Lentivirus; ADCC

1. Introduction

Lentiviruses cause slowly progressive disease with

long incubation periods leading to multisystem chronic

inflammatory disease and/or immunodeficiency. All the

lentiviruses infect cells of the monocyte and macro-

phage lineage and all establish persistent infections

(Gendelman et al., 1986; Haase, 1986; McCune, 1991).

Maedi visna virus (MVV) is the prototype lentivirus

which infects sheep. MVV and the similar lentivirus of

* Corresponding author. Tel.: +1 407 249 3671;

fax: +1 407 249 3649.

E-mail address: isingh@vaxdesign.com (I. Singh).

0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2006.06.006

goats caprine arthritis encephalitis virus (CAEV), do

not specifically infect or destroy CD4+ lymphocytes

(Gorrell et al., 1992) unlike the diseases seen with the

human, simian, feline and bovine immunodeficiency

viruses.

Analysis of the immune response to MVV has

revealed that both virus-specific antibody and T cell

immune mechanisms are activated (Bird et al., 1993;

Blacklaws et al., 1994; Blacklaws et al., 1995;

Gudnadottir and Palsson, 1965; Singh et al., 2006).

These responses, however, fail to prevent the

establishment of a persistent infection. It is very rare

to see free infectious virus in the body fluids of MVV

infected sheep (Blacklaws et al., 1994), suggesting

that a major mechanism for virus transmission in vivo

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366358

is by cell-to-cell spread. Therefore immune effector

mechanisms against infected cells rather than free

virus may be of crucial importance in clearing or

controlling virus replication. One such mechanism is

antibody dependent cellular cytotoxicity (ADCC).

Protective activities of antibodies against viruses

include neutralization, complement activation and

ADCC. It is known that anti-MVV neutralizing

antibodies are of lower affinity for the virus than

the virus for its cellular receptor (Kennedy-Stoskopf

and Narayan, 1986), so that neutralization of virus by

antibody may be relatively ineffective in vivo. Anti-

MVV antibodies will also activate complement

(Petursson et al., 1976) but there has been no report

of their mediating ADCC reactions. In the control of

MVV spread therefore, ADCC may be an important

mechanism of antibody protection which is absent in

persistently infected sheep.

In sheep the heavy chain constant region genes of

IgG1 and IgG2 have been sequenced (Clarkson et al.,

1993; Foley and Beh, 1989). Functional studies have

shown that ovine IgG1 activates complement whilst

IgG2 does not (Hein and Dudler, 1998) however which

isotype mediates ADCC reactions is unknown. In MVV

infected sheep, serum IgG responses to viral antigens

are restricted to IgG1, with no detectable IgG2 antibody

(Bird et al., 1995; Mehta and Thormar, 1974). Bird et al.

(1995) showed that in MVV infected sheep this IgG1

restriction was only seen to MVV antigen but not to

other viral infections (orf virus) or proteins adminis-

tered in adjuvant (ovalbumin or keyhole limpet

hemocyanin). Also the restriction in the response to

MVV antigen could be overcome in infected sheep by

boosting animals with MVV GAG p25 antigen in

adjuvant. The absence of an IgG2 antibody response to

MVV may therefore indicate a subtle defect in

immunity to MVV in sheep and could have important

implications for pathogenesis.

Several workers have used the baculovirus system for

the expression of viral glycoproteins, e.g influenza virus

(Kuroda et al., 1986; Possee, 1986), lymphocytic

choriomeningitis virus (LCMV) (Matsuura et al.,

1987), MVV (Huso, 1997; Kwang et al., 1995; Rafnar

et al., 1998), EHV-1 (Stokes et al., 1997) and blue tongue

virus (Roy et al., 1997) so that the recombinant proteins

are glycosylated. The proteins produced are similar in

structure, biological activity, and immunological reac-

tivity to the naturally occuring proteins (O’ Reilly et al.,

1992). Recombinant expression of MVV ENV has been

problematic with few reported successes in prokaryotic

and yeast systems (Carey et al., 1993; Kwang and Cutlip,

1992a). Recently there have been three publications

reporting expression of ENV constructs in baculovirus:

Carter and Dalziel (1997) expressed the gp135 external

surface unit (SU) and Rafnar et al. (1998) expressed the

SU and transmembrane regions (TM), i.e. mature gp155.

Both constructs replaced the viral signal sequence with

heterologous signal sequences. The recombinant proteins

were glycosylated and recognized by immune sera from

MVV infected animals (Carter and Dalziel, 1997; Rafnar

et al., 1998). Similarly, Huso (1997) used the first rev

exon but used env’s own hydrophobic envelope signal

sequence to express gp135 in a baculovirus expression

system to study the specific regions of gp135 which elicit

neutralising and anti-fusion antibody responses.

We have therefore used ENV produced in baculo-

virus (Carter and Dalziel, 1997) to raise anti-ENV IgG2

antibodies in sheep. These antibodies were then

examined for their ability to mediate an ADCC reaction

in vitro.

2. Materials and methods

2.1. Animals

Finnish Landrace crossed sheep (3 years old), kept at

the large animal facilities at the Centre for Veterinary

Science, University of Cambridge were used in the

experiments.

Sheep were experimentally infected with MVV

strain EV1 by subcutaneous injection of 5 � 105 50%

tissue culture infectious doses (TCID50) and had been

infected for longer than 1 year when sera were taken.

Uninfected sheep 0524A was primed by subcutaneous

injection at 2 sites with 130 mg of purified recombinant

GST:SUenv antigen fusion protein (Carter and Dalziel,

1997), emulsified in Freund’s complete adjuvant (1:1).

Secondary immunization was with 40 mg of SUenv

emulsified in Freund’s incomplete adjuvant (1:1),

injected subcutaneously in two sites, 2 weeks later.

Serum was collected at 2 weeks and 6.5 weeks after

initial immunization of sheep.

2.2. Virus

MVV strain EV1 (Sargan et al., 1991) was grown in

sheep skin cell lines as previously described (Bird

et al., 1993) and the infected cell supernatant used as a

virus stock. Virus in the supernatant of infected cells

was also pelleted at 4 8C, 10,000 � g overnight, the

pellet resuspended in PBS and re-pelleted at

120,000 � g for 3 h, 4 8C before finally resuspending

in PBS and storing at �70 8C for use as western

blotting antigen.

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366 359

2.3. Production and purification of recombinant

protein (GST:SUenv)

The construct BacPAK:envl2 containing MVV strain

EV1 nucleotide position 6306–8000 (Carter and

Dalziel, 1997; Sargan et al., 1991) was used. This

does not contain the first 104 amino acids of ENV

(thought to be the signal sequence) which causes low

levels of protein expression in baculovirus (Rafnar

et al., 1998) or the last 323 amino acids of ENV (the TM

domain). The protein was made as a fusion with the

signal sequence of baculovirus gp67 followed by a

GST-tag then the SU unit. The GST-tagged surface unit

of ENV is therefore secreted into the supernatant of

baculovirus infected cells (GST:SUenv). This was

purified using the GST-tag as described by Carter

and Dalziel (1997). Briefly, Sf21 (Spodoptera frugi-

perda) insect cells (Clontech, Basingstoke, UK) were

grown to a cell density of 1 � 106 cells/ml in TC100

with 10% fetal calf serum (FCS). Cells were infected

with baculovirus at a multiplicity of infection (m.o.i.) of

10 plaque forming units/cell in TC100 with 10% FCS

and incubated for 50–60 h. The medium was collected

from the infected cells and clarified by centrifugation at

1000 � g for 5 min at 18 8C before storage at �70 8C.

The presence of GST:SUenv in supernatants was

confirmed by Coomassie staining of SDS–PAGE gel

after purification and elution of the GST:SUenv from

glutathione agarose beads. All the recombinant protein

positive fractions were pooled together and concen-

trated using a centriprep-10 concentrator (Amicon,

Beverly, USA). The quantitation of protein was

determined by Bio-Rad protein assay (Bio-Rad

Laboratories Ltd., Hemel Hempstead, UK). The protein

yield ranged between 0.5–0.8 mg protein per litre of

infected cell.

To separate the surface unit of ENV (SUenv) from

the GST moiety, 0.2 mg of thrombin (Roche Diag-

nostics Ltd., Lewes, UK) was used to cleave 20 mg of

GST:SUenv in thrombin cleavage buffer (Roche

Diagnostics Ltd.) at 25 8C for 2 h. This was then re-

purified on glutathione agarose beads and the unbound,

free SUenv removed from the beads.

2.4. SDS–PAGE and Western blotting

Proteins were separated on 12% or 5–20% poly-

acrylamide gels under reducing conditions using 3%

stacking gels (Laemmli, 1970). Gels were electrophor-

etically blotted to a nitrocellulose membrane (Hybond C,

Amersham) using a semi-dry transblot apparatus (Bio-

Rad Laboratories Ltd.) in blotting buffer (192 mM

glycine, 25 mM Tris in 20% methanol) for 30–45 min at

250 mA. The whole nitrocellulose blot was stained in

Ponceau S (0.1% Ponceau-S in 5% acetic acid) (Sigma–

Aldrich Company Ltd., Poole, UK) and marker tracks

were separated before blocking the blot in 5% low-fat

dried milk powder in PBS (blocking buffer). Blots were

incubated overnight at 4 8C in primary antibody (diluted

in blocking buffer) then washed in PBS, 0.05% Tween 20

before incubation with an optional intermediate layer of

monoclonal antibody specific for sheep IgG1 (McM1) or

IgG2 (McM3) (Beh, 1987), or normal mouse serum for

1 h at room temperature. The immunoblots were washed

as before then incubated in an alkaline phosphatase

conjugated anti-immunoglobulin (species specific,

sheep, rabbit or mouse, 1:1000 diluted in blocking

buffer, Sigma–Aldrich Company Ltd.). The blots were

finally developed with 0.02% nitroblue tetrazolium,

0.01% 5-bromo-4-chloro-3-indolyl phosphate, p-tolui-

dene salt in 20 mM MgCl2, 0.1 M Tris/Cl pH 9.5.

2.5. ADCC

2.5.1. Effector cells

Peripheral blood mononuclear cells (PBMC) and

lymphokine activated killer (LAK) cells, prepared from

peripheral venous blood of uninfected sheep were used

as effector cells. Mononuclear cells were isolated by

overlaying blood buffy coats on Lymphoprep (Nye-

gaard, Oslo, Norway) and then centrifuging at 1400 � g

for 20 min. The PBMC were harvested at the interface

and washed three times with PBS. The PBMC were

finally suspended in RPMI 1640 (with 2 mM L-

glutamine, 25 mM HEPES, 200 U penicillin/ml,

100 mg streptomycin/ml, and 5 � 10�5 M 2-mercap-

toethanol) containing 10% FCS.

To obtain LAK cells, PBMC were cultured at a

concentration of 3 � 106 cells/ml RPMI 1640 with 10%

FCS in the presence of 10 U recombinant human IL-2

(NIBSC, Potters Bar, UK) per ml for 3 days. The

effector cells were overlaid onto Lymphoprep (Nye-

gaard) and viable cells harvested from the interface

(914 � g, 10 min). After two washes with RPMI 1640

with 2% FCS, the viable cells were counted and diluted

to the required concentration in RPMI with 10% FCS.

2.5.2. ADCC assay

Cytolytic activity was measured in a standard 6 h51Cr release assay. Serum samples were heat inactivated

for 30 min at 56 8C prior to assay. Sheep skin fibroblasts

derived from biopsies as detailed in Bird et al. (1993)

were used as target cells. 1 � 104 sheep skin cells were

plated in 96 well flat bottomed microtitre plates and

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366360

incubated overnight at 37 8C, 5% CO2. Cells were either

mock infected or infected with MVV EV1 at a m.o.i. of

0.2 TCID50 per cell in Dulbecco’s Modified Eagle’s

Medium (Gibco, BRL, Paisley, Scotland) containing

2 mM L-glutamine, 200 U penicillin/ml, 100 mg strep-

tomycin/ml and 2% FCS (DME/2%FCS) for 48 h at

37 8C. The target cells were labelled with 1 mCi/well of

Na251CrO4 in 50 ml DME/2% FCS at 37 8C, 5% CO2

overnight (approximately 80% cells express GAG

antigen using this infection regimen). Monolayers were

washed three times with DME/2%FCS and once with

RPMI 1640 with 2% FCS. The cells were pre-incubated

with different dilutions of antibody in 50 ml for an hour

at 37 8C before adding effector cells (50 ml PBMC or

lymphokine activated killer cells (LAK cells), all in

RPMI 1640 with 10% FCS) at an effector–target ratio of

150:1, 100:1 or 50:1. Plates were incubated for 6 h at

37 8C in a humidified atmosphere at 5% CO2. 40 ml of

supernatant were taken from each well, mixed with

120 ml of Optiphase HiSafe 3 scintillant (PerkinElmer

LAS (UK) Ltd., Beaconsfield, UK) then counted in a

Wallac Microbeta 1450 for 1 min. Assays were

performed in triplicate for each dilution of sera tested.

Controls consisted of mock infected target cells, target

cells incubated with medium alone, target cells with

antibody but no effector cells, and target cells with

effector cells alone. The percentage specific lysis or

release was calculated by standard formula:

% specific lysis

¼ mean experimental cpm�mean minimum cpm

mean maximum cpm�mean minimum cpm

� 100

where experimental counts per minute (cpm) is the 51Cr

release by target cells in the presence of effector cells

and antibody, minimum cpm is the 51Cr release by target

cells with medium alone and maximum cpm is 51Cr

release by target cells in the presence of 1% Triton X-

100. Error bars showing the variation in experimental

wells were calculated using the formula [standard

deviation of experimental cpm/(mean maximum

cpm � mean minimum cpm)].

3. Results

3.1. Production of recombinant-ENV (GST:SUenv)

protein

An ENV fusion protein, GST:SUenv, was prepared

from recombinant baculovirus infected cell super-

natants using glutathione agarose columns as previously

described (Carter and Dalziel, 1997). The known

molecular weight of GST:SUenv by SDS–PAGE

analysis is 97–100 kDa (Carter and Dalziel, 1997)

(Fig. 1A). A thrombin cleavage site is situated between

the GST moiety and the ENV protein to allow

purification of ENVas a non-fused recombinant protein.

Cleavage at this site was carried out to further confirm

that the protein made was SUenv. The estimated

molecular weight of thrombin cleaved SUenv without

GST was 70–73 kDa (Fig. 1A). No other reports have

shown the cleavage of affinity tag from the ENV

protein. Theoretically, the cleaved GST moiety may be

removed by binding to glutathione beads leaving the

purified SUenv protein in solution. When this was

attempted, much of the SUenv protein was non-

specifically absorbed onto the agarose beads leading

to losses of more than 50% of the purified SUenv

protein from the solution (data not shown). This

treatment also did not remove all the GST from the

solution (data not shown). Due to the above losses and

inability to remove all GST, many experiments and the

priming immunization for specific sera were performed

with uncleaved GST:SUenv.

3.2. Antigenic authenticity of GST:SUenv and

SUenv protein

Recombinant GST:SUenv and SUenv protein were

used to raise polyclonal anti-ENV serum in a sheep.

When the sheep polyclonal serum was tested on

immunoblots against purified GST: SUenv and SUenv

(Fig. 1A) as well as on pelleted MVV virions (Fig. 1B),

this antiserum showed reactivity against GST:SUenv,

SUenv and GST (Fig. 1A) and viral ENVantigen (smear

at around 50–94 kDa with post immunization serum in

Fig. 1B). Reaction of the sheep antiserum confirmed the

antigenic authenticity of the recombinant GST:SUenv

protein. This was similar to the study of Rafnar et al.

(1998), where serum from sheep immunised with their

recombinant gp135 construct reacted with MVV gp135

and a putative oligomer of gp135.

3.3. Generation of sheep anti-ENV IgG2

There was no detectable IgG2 antibody when

persistently infected sheep serum at a 1/10 dilution

was tested against both GST:SUenv (data not shown)

and thrombin cleaved GST:SUenv (i.e. SUenv) antigen

that had not been purified away from the GST (Fig. 2A).

Serum from a recombinant ENV immunized sheep

(above) was tested for the presence of IgG2 anti-ENV

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366 361

Fig. 1. Specificity of sheep anti-ENV polyclonal antibody. (A.) Recognition of GST:SUenv and SUenv by immunised sheep serum: 1 mg of

GST:SUenv (track 1) or SUenv (track 2) was used per track of a 12% reducing SDS–PAGE gel and blots were probed with pre- and post-

immunization serum from sheep (1/100 dilution) immunized with GST:SUenv and SUenv. Blots were developed using the appropriate anti-species

immunoglobulins conjugated to alkaline phosphatase. The position and size in kDa of molecular weight markers is indicated. The bands seen at

>94 kDa and 70–73 kDa are GST:SUenv and SUenv, respectively, and the GST band is present at 27.5 kDa. (B.) Recognition of virion ENV by

serum from GST:SUenv immunised sheep: pelleted MVV virions were separated on a 5–20% linear gradient SDS–PAGE gel, run under reducing

conditions, and electroblotted onto a nitrocellulose membrane. Strips were made from these blots and incubated overnight with pre-immunization

and post-antigen injection serum (sheep anti-GST:SUenv diluted 1:100). Blots were developed using affinity purified anti-sheep immunoglobulins

conjugated to alkaline phosphatase. Positions of molecular weight standards in kDa are indicated. The smear seen at approximately 70 to>94 kDa is

the outer membrane envelope glycoprotein gp135.

using a similar dilution. Sera collected at 2 weeks (data

not shown) and 6.5 weeks after the initial immunization

showed the presence of both IgG1 and IgG2 isotypes in

the anti-ENV antibody by immunoblot analysis on

Fig. 2. Isotype restriction of the anti-ENV antibody response in immunised

moiety was separated on a 12% reducing SDS–PAGE gel and electroblotted. B

MVV (1/10 dilution) (A) or with sheep anti-GST:SUenv polyclonal antib

indicated) (B and C). After washing, each strip was probed with either M

supernatant), or normal mouse serum (NMS) (1/500) as indicated and develo

alkaline phosphatase. Positions of molecular weight standards in kDa are i

GST:SUenv (data not shown) and SUenv (Fig. 2B and

C). The serum from the recombinant ENV immunized

sheep was titrated by immunoblotting on SUenv

antigen. The titre of the IgG1 anti-ENV antibody in

sheep. Four hundred nanogram per track SUenv cleaved from its GST

lots were incubated with serum from a sheep persistently infected with

ody (collected 6.5 weeks after immunization) (varying dilutions as

cM1 (anti-IgG1) (1/500 ascites fluid), McM3 (anti-IgG2) (saturated

ped using affinity purified anti-mouse immunoglobulin conjugated to

ndicated.

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366362

Fig. 3. Sheep sera activity in ADCC assays. Sera from a MVV

persistently infected (A and B) and a GST:SUenv immunised (C

and D) sheep were tested for activity in ADCC assays at the indicated

dilutions. Effectors cells used from uninfected sheep were PBMC at

effector–target ratios of 100:1 (A and C) or LAK cells at 50:1 (B and

D). Target cells were skin cells, mock infected or infected with MVV

labelled with 51Cr. The cells were incubated with pre- or post-infection

or immunization (immune) serum at the indicated dilutions for 1 h

before the addition of effector cells. Assays were incubated for 6 h and51Cr release was determined. (Open bar) mock infected targets with

immune serum and effectors, (hatched bar) infected targets with pre-

immune serum and effectors, (solid bar) infected targets with immune

serum and effectors, (open square) mock infected targets with immune

serum, no effectors, (open circle) mock infected targets with effectors

but no serum, (solid square) infected targets with immune serum, no

effectors, (solid circle) infected targets with effectors but no serum.

the immunized sheep was 1/1280–1/2560 (Fig. 2B). The

titre of the IgG2 anti-ENV was 1/160–1/320 (Fig. 2C).

A greater IgG1 titre was expected due to the different

concentrations of IgG1 and IgG2 in sheep serum (ca. 20

and 6 mg/ml, respectively) (Hein and Dudler, 1998).

The titre of anti-ENV IgG1 antibody in persistently

infected sheep was 1/2560–1/5120 (data not shown),

when tested by immunoblotting on SUenv antigen.

3.4. ADCC

As the anti-ENV antibody raised to GST: SUenv

contained both IgG1 and IgG2, this polyclonal serum

was examined for its ability to mediate antibody-

dependent cell-mediated cytotoxicity (ADCC) in vitro

using MVV-infected skin fibroblasts as targets. Initially

the serum was analysed to see if it bound to the surface

of infected cells by immunofluorescent staining. Like

serum from persistently infected sheep, this serum

specifically bound to ENV antigen expressed at the

surface of MVV infected skin cells (data not shown).

Infected skin cells and this antiserum could therefore be

used in ADCC reactions.

Experiments were also performed with serum from

MVV-infected sheep. Complement in both serum

samples was inactivated at 56 8C for 30 min before

use in the assay. PBMC and LAK cells were used as

effector cells for the assay. A number of variables,

including effector–target ratios and different concen-

trations of anti-ENV or infected sheep serum were

tested in the assay. The representative results showed

that serum from MVV-infected sheep did not mediate

ADCC activity as the lysis of target cells with

persistently infected sheep serum was always similar

to background controls (Fig. 3A and B). Moreover, no

significant lysis of target cells with MVV-infected

sheep serum was observed even at high effector–target

ratios of PBMC (150:1) or LAK cells (100:1) (data not

shown) and high antibody concentrations (1/5, data not

shown). Anti-ENV polyclonal antibody obtained from

immunized sheep mediated ADCC reactions as

determined by increased levels of specific lysis of

MVV-infected skin cells. Effector–target ratios of

100:1 with PBMC or 50 or 25:1 with LAK cells as

effectors were found to be optimal for the ADCC

reaction (titration not shown) (Fig. 3C and D).

Although pre-immune serum gave background lysis

levels of approximately 12–15%, lysis with immune

serum was more than double at serum concentration of

1/20. No significant increase in ADCC was observed

when high antibody concentrations (1/5 or 1/10) were

used (data not shown). The ADCC activity decreased

with the dilution of antibody to 1/40 (Fig. 3C and D)

and 1/80 (data not shown). Pre-immunization sera and

uninfected target cells were used as background

controls.

4. Discussion

Recombinant GAG (matrix (p17), core (p25) and

nucleocapsid (p14)) and ENV proteins (gp41 and

gp135) of MVV have been generated by several

workers either in bacterial, yeast or baculovirus

expression vectors (Boshoff et al., 1997; Keen et al.,

1995; Kwang and Cutlip, 1992b; Kwang et al., 1995;

Rafnar et al., 1998; Reyburn et al., 1992a) for

analysing humoral (Bird et al., 1993; Boshoff et al.,

1997; Carey et al., 1993; Huso, 1997; Keen et al.,

1996; Keen et al., 1995; Kwang and Cutlip, 1992a,b;

Kwang et al., 1993; Kwang et al., 1995; McConnell

et al., 1998; Power et al., 1995; Reyburn et al., 1992a;

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366 363

Zanoni et al., 1991; Zhang et al., 1997) and cell

mediated (Pinching and Nye, 1990; Rafnar et al.,

1998; Reyburn et al., 1992b) immune responses.

Although antibodies to viral antigens can be elicited in

sheep persistently infected with MVV, their response

has mainly been studied in terms of virus-neutralizing

and complement-fixing antibodies (De Boer, 1970;

Gudnadottir and Kristinsdottir, 1967; Gudnadottir and

Palsson, 1965; Mehta and Thormar, 1974; Petursson

et al., 1976) and no study has directly reported the

correlation of antibodies to ADCC effector function.

This is the first time that ADCC activity has been

examined in MVV infection in vitro.

In this study, ADCC activity of sera was tested

against target cells expressing MVV ENVantigen after

infection with MVV, EV1 strain. Since MVV ENV

(gp135) is a glycosylated surface protein, recombinant

GST:SUenv was expressed in a baculovirus expression

system using a baculovirus signal sequence (Carter

and Dalziel, 1997). Similarly, Rafnar et al. (1998)

expressed gp135 lacking the viral signal sequence but

using an IFN-g signal sequence whilst Huso (1997)

deleted the first rev exon but used the viral signal

sequence to express env SU in baculovirus expression

systems. Baculovirus expressed MVV gp70 has also

been reported by Kwang et al. (1995), but since no

signal sequence was incorporated in the construct

used, glycosylation and secretion of the recombinant

protein would not be expected. The MVV env protein

made in this study was found to be antigenically

authentic when tested by antisera from naturally

infected and experimentally immunized animal,

suggesting that the fusion protein contains epitopes

which are present in MVV (Boshoff et al., 1997; Carey

et al., 1993; Huso, 1997; Kwang and Cutlip, 1992b;

Kwang et al., 1995).

The antibody subclasses produced to infection will

determine which antibody effector mechanisms are

available to help to clear the infection. For example, in

a HIV-infected individual, of the four human IgG

subclasses, IgG1 contained the most ADCC activity

(Khalife et al., 1988; Ljunggren et al., 1988).

Whereas, mice acutely infected with LCMV produced

IgG2a antibodies which fixed complement efficiently

and cleared the virus (Thomsen et al., 1985). The

subclass of antibody produced is dependent on the

cytokine milieu present when B cells are differentiat-

ing: in mice IFN-gamma causes IgG2a and IL-4 IgG1

production; in cattle IL-4 stimulates IgG1 and IFN-

gamma IgG2 class switching (Estes and Brown, 2002;

Mosmann et al., 1986). In MVV infection of sheep,

IgG2 anti-MVV antibodies are not detectable (Bird

et al., 1995; Mehta and Thormar, 1974). Failure to

make an IgG2 MVV specific antibody might result

from the preferential stimulation of subsets of helper T

lymphocytes whose lymphokines influence an IgG1

isotype switch in antibody producing cells, which may

be a defect in the T cell response to viral infection.

Both IgG1 and IgG2 responses can be induced by

immunizing with recombinant GAG (Bird et al., 1995)

or recombinant ENV antigen (above) in complete

Freund’s adjuvant (CFA). We also used Titermax gold

as an adjuvant in sheep with recombinant ENV,

however this did not result in IgG2 production to

GST:SUenv (data not shown). The adjuvant used

therefore also affects which antibody subclass is

produced. It is not known in the sheep which T cell

cytokine is needed to induce IgG2 class switching

although it is most likely to be IFN-gamma from work

in cattle (Estes and Brown, 2002).

The polyclonal sheep anti-ENV antibody was

shown to bind to ENV antigen expressed on the

surface of MVV infected skin cells. These cells

coated with antibody were therefore used as targets in

an ADCC assay. The results showed that serum

containing IgG1 and IgG2 anti-ENV antibody

mediated an ADCC reaction. No viral specific ADCC

reaction was observed with serum from MVV-

infected sheep having only anti-ENV IgG1 antibody

(although the IgG1 anti-MVV titre was similar to that

found in the serum from immunized sheep). The

results shown here are representative of several sera

from persistently infected sheep (data not shown).

Previously, 32 sera from persistently infected sheep

had been tested for ADCC reactivity: none showed

the presence of an anti-viral IgG2 response; the

antibody bound to the surface of infected cells and

directed complement mediated lysis of cells; but none

of the serum samples mediated an ADCC reaction

(Reyburn, 1992). This study therefore suggests that

IgG2 is an effector isotype for ADCC reactions in

sheep. Similarly, no ADCC activity has been detected

in serum of equine infectious anemia virus (EIAV)-

infected horses and this may also allow virus

persistence in carrier horses (Tschetter et al.,

1997). It may be due to the number and the spacing

of EIAV ENV protein molecules on the target cell or

the antibody isotype within the IgG fraction. In

contrast, after CAEV infection of goats, both IgG1

and IgG2 subclasses have been reported to develop in

anti-viral antibodies (Trujillo et al., 2004). In cattle it

has also been reported that IgG2 antibodies are 100

times more efficient than IgG1 immunoglobulins at

mediating antibody-dependent cytotoxicity (Butler,

I. Singh et al. / Veterinary Immunology and Immunopathology 113 (2006) 357–366364

1983) and that resting neutrophils can bind IgG2 or

IgM but not IgG1 (Worku et al., 1994).

In mice and humans variation in the structure of the Fc

portion of the immunoglobulin molecule between

subclasses determines which receptors and other

molecules the immunoglobulin can react with. The

sequence data for ruminant IgG gamma chains indicate

that the ruminant IgG1 isotypes are typically IgG-like,

but the IgG2 isotypes are the most divergent of all known

IgGs, with significant shortening of the lower hinge.

Though ovine g2 has 93% nucleotide identity with ovine

g1, the majority of the differences between them occur in

the hinge-coding region (Clarkson et al., 1993).

Structural models of IgG1 and IgG2 derived from cDNA

sequences suggest the Fab/hinge/Fc interface of IgG1 is

more open than in IgG2 because of the short IgG2 hinge

(Clarkson et al., 1993). The more compact IgG2

molecule is likely to be the molecular basis for

differences in effector functions between the isotypes

mediated by their interaction with Fcg type receptors

(Symons and Clarkson, 1992; Symons et al., 1989).

This study did not specifically examine which

epitopes of the envelope protein were responsible for

binding and subsequently triggering of ADCC activity.

As the construct used in this study was devoid of the

nucleotide sequence to gp41, at least one epitope must

reside in the gpl20 antigen. This study suggested that

IgG2 is an effector antibody isotype required by sheep

effector cells to carry out ADCC reactions. Similarly,

Reyburn (1992) also tested 32 persistently infected

sheep sera for ADCC activity and none of them was

positive for ADCC reaction. The absence of an ADCC

reaction, due to the absence of IgG2 antibody in vivo

may be a major defect in the defense against MVV

infection which could aid the persistence of viral

infection. This finding raises the possibility that

immunization with recombinant ENV may be a way

of ‘rescuing’ the IgG2 response in MVV persistently

infected sheep leading to immune clearance of the

infection in vivo.

Acknowledgements

Recombinant IL-2 was provided by the EU

programme EVA/MRC Centralised Facility for AIDS

Reagents, NIBSC, Potters Bar, UK (Grant numbers

QLK2-CT-1999-00609 and GP828102). Dr. Barbara

Blacklaws was funded by Wellcome Trust Project Grant

Ref: 013786. Dr. Inderpal Singh was supported by a

Nehru Cambridge Scholarship, Overseas Research

Students Award, Cambridge Commonwealth Award

and Jowett Trust Award.

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