Production of Monoclonal Antibodies Against Equine Flu Virus

Production of monoclonal antibodies against equine influenza : application to a comparative study of various

strains of the virus

C. Cruci` ere, M.C. Guillemin, A. Roseto, A. Wirbel, E. PLATEAU

To cite this version:

C. Cruci` ere, M.C. Guillemin, A. Roseto, A. Wirbel, E. PLATEAU. Production of monoclonal antibodies against equine influenza : application to a comparative study of various strains of the virus. Annales de Recherches V´ et´ erinaires, 1989, 20 (3), pp.243-250. <hal-00901886>

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Original ^article

Production of monoclonal antibodies against

equine influenza : application ^{to a} comparative study

of various strains of the virus

C. Crucière M.C. Guillemin ^A. Roseto ^A. Wirbel E. Plateau

1

Ministère de

I Agriculture,

^Direction

générale

^de

l’alimentation,

services vétérinaires de la santé et de la

protection animale,

laboratoire central de recherches

vétérinaires, 22,

^rue

Pierre-Curie,

^B.P.

67,

94703 Maisons-Alfort

Cedex;

^and

2

Hôpital Saint-Louis,

Institut de recherches sur les maladies du sang, ^{Unité 197} INSERM-LOI 101

CNRS,

² Place du Docteur

Fournier,

^{75470 Paris}

Cedex,

^France

(accepted

¹³

September 1988)

Summary &horbar;

Monoclonal antibodies

(Mo Abs)

^were

prepared against influenza/A/equine/Prague/1/

^l

56

(H7N7)

^and

influenza/A/equine/Miami/l/63 (H3N8)

^reference strains of

equine

influenza virus.

These monoclonals were tested

against

^{the 2 reference}

strains,

^{8 field} strains of

equine

^influenza

virus,

3 human influenza viruses

possessing

^{the H3}

hemagglutinin,

^{and one} virus of human

origin possessing

^{the H1}

hemagglutinin.

Two antibodies were obtained in one fusion

against

^the

Prague/1/56

strain and reacted

only

with this strain. Four

anti/A/equine/Miami/1/63

^{Mo Abs were}

obtained in one fusion.

They

differentiated 8 strains of

equine origin

^{from all 3} strains of human

origin

^{and from one strain of}

equine origin (Joinville/l/78)

isolated in 1978. The

specificity

^{of this}

difference was confirmed

by

cross-seroneutralization between

A/equine/Miami/1/63

^{strain and}

A/equine/Joinville/1/78

^strain.

monoclonal antibodees-

equine

^{influenza -} virus&horbar;

production

^-test

Résumé &horbar; Production

d’anticorps

monoclonaux

antigrippe équine : application

^{à une étude}

comparative

de diverses souches de virus. Des

anticorps

monoclonaux ont été

préparés

^contre

les souches de référence de la

grippe équine Influenzal!4/equinelPraguelll56 (H7N7)

^et

lnfluenzalAlequinelMiamilll63 (H3N8).

^Ces

anticorps

^ont été testés contre les deux souches de

référence,

^contre huit souches du terrain de virus de la

grippe équine,

^{trois souches de virus}

grippal

humain

possédant l’hémagglutinine

^{H3 et une souche}

d’origine

^humaine

possédant l’hémagglutinine

^{H1. Deux}

anticorps

^ont été obtenus contre la souche

AlequinelPraguelll56

^au

cours d’une fusion et se sont montrés totalement

spécifiques

^{de cette souche. Quatre}

anticorps

^anti

AlequinelMiamilll63

^{obtenus en une fusion ont} pu différencier huit souches

d’origine équine

^des

trois souches

d’origine

^humaine

possédant l’hémagglutinine

^{H3 et} d’une souche

d’origine équine (Joinvillelll78)

^{isolée en 1978. La}

spécificité

^{de cette} différence a été confirmée par neutralisation croisée entre la souche

AlequinelMiamilll63

^{et la souche}

AlequinelJoinvillelll78.

anticorps

monoclonaux &horbar;

grippe équipe

^{- virus -}

production

^{- test}

Introduction

The differentiation of

subtypes ^and

^va-

riants

among human and animal strains

^of

influenza virus

is a

continuing

preoc-

cupation ^of diagnostic

laboratories and

vaccine producers. ^Studies

^are

usually

carried

out

by

^means of different serolo-

gical

^{tests :}

hemagglutination

^inhibition

(HAI); complement ^fixation (CF);

^inhibition

of neuraminidase activity (INA); agar gel

immunodiffusion

(AGID); ^and

seroneutra-

lization (SN). Monospecific antibodies

are

traditionally prepared

^on

laboratory

^ani-

mals (ferrets

^or

guinea pigs); the strains

are

compared by titrations in parallel against homologous

^and

heterologous antigens (Archetti

^and

Horsfall, 1950).

^In

the

horse, the serological

response

is low, and the poor efficiency of vaccination led

to

investigations

^{into the}

antigenicity

^and

immunogenicity

of the virus.

In recent

years, the monoclonal antibodies (Mo Abs) technique has pro-

vided

highly specific reagents ^and serological ^reactions

^have

changed,

^from

quantitative

^to

qualitative, with each strain

of virus

being ^tested against

^a

panel ^of

Mo Abs with which the reaction is either

positive

^or

negative.

With influenza virus

the

most

widely used reaction

for

such

studies is HAI. This reaction can diffe-

rentiate

numerous variants in each

subtype,

and it has been

proved

^{that a}

correlation

exists between the HAI titers

of the

sera

of

animals

and their

resistance to

infection (Lucam et al., 1974). Thus it is

possible simultaneously

^{to measure}

^the antigenic

^drifts

(Kendal

^et

al., 1981;

Underwood, 1982), the relations between the strains, ^and

^to

^have indications

on

the level of protection

^{that can}

^be expected.

In

this study

^we

prepared ^Mo

^Abs

against

²

reference strains of equine

Influenza/A/virus : Prague/1/56 (H7N7) strain, and Miami/1/63 (H3N8) ^strain.

These Mo Abs

were

tested against

various strains

of

equine ^{influenza virus,}

against

⁴

^strains

^{of human}

origin ^also possessing

^{the H3}

hemagglutinin,

^and

against

^one strain of human

origin possessing ^{the H1} hemagglutinin.

Materials

and Methods

Virus

Fourteen strains of virus were used in this

study.

Reference strain

Influenza/A/equine/Miami/

1/63

(H3N8)

obtained from the American

Type

Culture Collection

(ATCC, VR-317).

Eight

^other strains of

equine

Influenza/A/

possessing

^{the H3}

hemagglutinin :

Russe/1/65, Roubaix/1/67,

Alfort/1/67 and Fontainebleau/1/79 isolated at the National

Veterinary

School of Alfort

(Department

^of

Medicine); 595/l/73,

2409/1/80, ^81229/1/81 isolated at the

Laboratory

^of

Equine Virology,

Pasteur

Institute,

Paris. Strain Joinville/l/78 was isolated in 1978 at the

Department

^of

Equine Pathology,

^Central

Veterinary

^Research

Laboratory (Plateau et al., 1983).

Three human influenza/A/viruses posses-

sing

^{the H3}

hemagglutinin : Texas/1/77;

Bangkok/1/79;

and Port Chalmers/l/73.

One human influenza/A/virus

possessing

the H1

hemagglutinin :

USSR/2161/80.

Reference strain

Influenza/A/equine/Pra- gue/1/56 (H7N7) possessing

^{the H7}

hemag- glutinin

and obtained from the ATCC

(VR, 297).

All strains were grown ^on 11-d

embryonated

eggs inoculated via the allantoic ^route. After 2d of incubation at 35 °C the allantoic fluids were

harvested and tested

by hemagglutination (HA).

Each strain was

pooled

and stock viruses

were

kept

ⁱⁿ

aliquots

^{at -80 °C.}

Immunization of animals

BALB/c mice were inoculated

intraperitoneally (I.P.)

with 1 200

hemagglutinating

^units

(HAU)

of virus emulsified in Freund’s

complete adjuvant (200 !i/200 jll).

Three weeks after

inoculation,

^{animals were} tested

by hemagglutination

inhibition

(HAI)

^for

the detection of anti-influenza

antibodies;

positive

animals received a booster

injection

^of

antigen (I.P.)

^without

adjuvant

3d before fusion.

Guinea

pigs weighing

^{= 200} g ^were inoculated with 4 000 HAU of virus mixed with

complete

^Freund’s

adjuvant.

^{After a booster}

injection

^at

d30,

the animals were sacrificed and bled.

Hemagglutination inhibition

reaction

This test was

performed according

^{to the}

recommendations of the U.S.

Department

^of

Health Education and Welfare

(1975).

Before

titration,

^{the sera were} treated

by

different

procedures

^{in order to} eliminate the

non

specific hemagglutinins

^{and the non-}

specific

inhibitors of

hemagglutination (Belyavin

and

Cohen, 1962;

^Terzin

et al., 1979).

^{Mo Abs}

were not treated.

Treatment of

sera

Sera were heated at 56 °C for 30

min,

^and

placed

^{in contact}

(vol/vol)

^{with a 20% chick red}

blood cell

(CRBC) suspension

ⁱⁿ

phosphate

buffer solution

(PBS)

for 30 min. After

centrifugation

for 10 min at 2 000 g, the

supernatant

^was collected and mixed with a

suspension

^{of kaolin}

(1

^{vol of}

supernatant

^{for 4} vol of

kaolin).

After 30 min of contact, ^the

suspension

^was

centrifuged (10 min,

^{1 500}

g),

and the

supernatant

collected and titrated. After treatment the initial dilution of the sera was 1:10.

Titration of antigen

Twofold serial dilutions of

antigen

^were

performed

in 96-well

microplates (Becton Dickinson, Grenoble)

ⁱⁿ

50-pl

^{vol of a 0.5%}

CRBC

suspension.

^{After 30 min at}

laboratory temperature,

^the

antigen

^{titer was}

given by

^the

highest

dilution where

hemagglutination

^was

complete (HAU

^{in 50}

!I).

Titration

of

antibodies

Twofold serial dilutions of sera were

performed

in 96-well

microplates

^{in a 25}

jll

vol. To each dilution was added

25 pl

^{of a}

suspension

^of

antigen containing

4 HAU. After 30 min of contact at

laboratory temperature,

⁵⁰

pl

^{of a}

0.5%

suspension

^{of CRBC was} added. The

hemagglutination

^{titer was} recorded after 30 min at

laboratory temperature.

The titer of antibodies is

given by

^the

reciprocal

of dilution

inhibiting

100% of the

hemagglutination

^reaction.

Seroneutralization

reaction

The seroneutralization reaction was

performed

as described

by

Rouse and Ditchfield

(1970) :

^a

given quantity

^of

antigen

^was

placed

^{in contact}

with different dilutions of antibodies and the mixture was inoculated into

embryonated

eggs.

The virus was titrated

by

inoculation of serial 10-fold dilutions of virus into 4

embryonated

eggs. After 48 h allantoic fluids ^were harvested and tested

by

HA. The infectious dose 50

(ID 50

)

^was calculated

by

the method of Reed and Muench

(1938).

One hundred

ID 50

^{of virus}

was incubated with 2-fold serial dilutions of antibodies. After 1 h of contact at

laboratory temperature,

^0.2 ml of each of the different dilutions was inoculated into 4

embryonated

eggs. After 48 h of

incubation,

the allantoic fluids were harvested and the presence of virus

was tested

by

HA. The titer of the antibodies

was

given by

^the

highest

^dilution

completely inhibiting

^viral

development.

Preparation of monoclonal

antibodies

Monoclonal antibodies were

prepared

^accor-

ding

to the

procedure

^described

by

^{Wictor et}

al. (1980).

Cell

fusion

The

spleen

^{of a}

hyperimmunized

^BALB/c

mouse was

aseptically

removed and

ground

ⁱⁿ

3 ml of RPMI 1640 medium. The cellular

suspension

^was

centrifuged

^{at 1}

500 g

^{for 10 0}

min and washed twice in RPMI.

Lymphocytes

were

resuspended

^{in 10} ml of RPMI without

serum and counted. SP2/0 AG14

myeloma

cells were

prepared by

cultivation in RPMI 1640

supplemented

^with

glucose

^{2 x}

10- 3 M, pyruvate

¹

M, glutamine

²

M,

10% fetal calf

serum

(FCS),

^{and 8}

azaguanine

^{2 x}

10- 5

M.

Before

fusion,

several flasks of SP2/0 cells

were

collected, centrifuged

for 10 min at 1 500 g, and washed twice. The cells ^were

resuspended

ⁱⁿ 10 ml of RPMI without FCS.

The 2 cell

populations

^were mixed in the

proportion

^{of 5}

splenocytes

^{to 1 SP2/0}

myeloma

^cell.

The mixture was

centrifuged

for 10 min at 2 000 g and the

supernatant

discarded. Fusion

was

performed

^with

polyethylene-glycol (PEG)

4 000 diluted 1:2 in RPMI. One ml of PEG ^was added

dropwise

^{to the cell}

pellet.

^{After 1 min of}

contact,

^the PEG was diluted with 5 ml of RPMI under

gentle agitation.

^{The cells were}

resuspended

in RPMI with 20% FCS at the final concentration of

10 6

cells per 1.5 ml.

Each well of a 24-well

microplate

^{was filled}

with 1.5 ml of cell

suspension.

After 24 h at 37

°C,

^the culture medium was

replaced

^{with a}

RPMI selective medium

containing

^{20% of}

FCS, hvpoxanthine

^{5 x}

^{10- 5 M,}

^{and asazerine}

2 x

10 !

^{M. Cells were} incubated for 2

wk,

and the medium was

replaced

every 5 d

by

^an asazerine-free medium.

Cloning

The presence of antibodies in the wells was

checked

by

HAI when the

hybrid

cells covered 50% of the bottom of the well.

Producing

^cells

were cloned

by limiting

^{dilution on 96-well}

microplates

^with

Mv 1

^Lu

^(NBL-7)

irradiated cells

(American Type

Culture Collection

CCL64;

Mink

Lung)

^{at 2 500 rads as feeder}

layer.

Hybridoma

^{cells were further}

multiplied

ⁱⁿ

large

volume flasks in order to obtain sufficient amounts of cells to be

kept by freezing

ⁱⁿ

liquid nitrogen.

Results

Two clones of

hybridomas secreting

monoclonal antibodies

were

selected after

mouse

immunization with

the

Prague/1/56 strain and

4 clones after

immunization

with

the Miami/1/63 strain.

After

testing by

^agar

gel immunodiffusion

against IgG (IgGi, IgG2a, IgG2ab), IgA, IgM

^{it was}

concluded that

all the Mo Abs

were of

the IgGl ^subclass.

The

Mo

Abs

were

tested by ^HAI against different strains of

virus

(Tables

^I

and 111). ^Control polyclonal ^antibodies

were

tested

in

parallel against

^{the same}

strains (Table 1).

Discussion

Four Mo

Abs

were

obtained against

^the

A/equine/Miami/1/63 virus and

2

against

the A/equine/Prague/1/56

virus. These Mo Abs were

specific

^for

^each subtype

^{as no}

cross-reaction

^was

observed by ^{HAI. One} anti-A/equine/Miami/l/63

^{Mo Ab and one}

anti-A/equine/Prague/1/56

^Mo

^Ab

^were

also

tested by ^SN against

²

strains of virus possessing ^the

^H3

hemagglutinin.

The

anti-A/equine/Prague/1/56 ^Mo

^Ab

^did

not react

with either of the

2

viruses, ^and

the

anti-A/equine/Miami/1/63

^{Mo Ab reac-}

ted only

^with

^the homologous strain and

not

with

the

other.

Although

^the

monoclonal antibodies

were

obtained

from

different clones, ^it

^was

not

established

if

each

of the 4

anti-

A/equine/ Miami/1/63

Mo

Abs and each of the

2

anti-A/equine/Prague/1/56 ^Mo

^Abs

were

different,

^as

they ^all belonged

^to

^the IgG1

¹

subtype ^and

^no

^{reaction of} identification against

^viral

proteins

^was

performed. Meanwhile each monoclonal

antibody

^was

denominated separately M1, M2, M3, M4, ^{and P1,}

^P2.

The 4

anti-A/equine/Miami/1/63 ^Mo

Abs

reacted with

all

the isolates of equine origin excepted ^the A/equine/Joinville/l/78 virus, ^and

^{did not react with any}

^of

^the

isolates of

human

origin possessing

^the

H3 hemagglutinin,

^from

^which

^the

^Join-

ville/1/78 virus

could

not

be distinguished.

The

epitope recognized by ^these

^{Mo Abs}

is

therefore specific

^{for most}

^{of the}

influenza viruses

of equine origin ^posses- sing

^{the H3}

hemagglutinin.

^As

expected,

the

USSR/2161/80

virus

possessing ^the

H1

hemagglutinin ^did

^{not react}

with any of

the

monoclonal

or

polyclonal ^antibodies

tested.

The biological significance ^{of the}

difference between A/equine/Joinville/l/78

and

the

other isolates of equine origin

^was

confirmed by seroneutralization.

The Mo

Abs reacted

only

^with

^the homologous A/equine/Miami/l/63

virus and not with

the A/equine/Joinville/l/78

^virus.

^This

difference between A/equine/Joinville/l/78

and the other

viruses may

^{be of some}

importance ⁱⁿ

^the

protection ^{of the} horse, when

one

considers that the antigenic

sites of the

A/equine/Joinville/l/78 ^virus

are not

completely ^covered by ^the

antibodies induced by ^the A/equine/

Miami/l/63

strain

present

^{in most of}

^the

vaccines.

This

risk is

confirmed by ^the

observation

that HAI titers

of guinea pig

sera

after immunization with

the

A/equine/

Miami/1/63 virus

were much lower

against

the

A/equine/Joinville/l/78

^strain

^than against ^all

^the

^other

strains tested.

In

fact the epidemiological

^conse-

quences

of the difference between

A/equine/Joinville/l/78 isolate and the reference A/equine/Miami/l/63 ^virus

^must

be minimized because this difference did

not

persist

and the 3 strains

isolated

after 1978

tested in this study

^were

recognized by ^all

^of

^the anti-A/equine/Miami/1/63

monoclonals. Either the epitope missing

ⁱⁿ

the

A/equine/Joinville/l/78

^{virus was}

recovered very

quickly

^{or, more}

probably,

the circulation

of

viruses possessing ^this

epitope

^{was never}

interrupted.

The

conservation of

^the

epitope

indicates

great stability

^of

the overall

antigenic

^{structure of}

^the equine

^influenza

viruses and sustains the

possibility

^of

natural

or

vaccinal immunization.

^Mean-

while, polyclonal antibodies obtained by

immunization of guinea pigs ^{with the} A/equine/Joinville/l/78

virus confirm a

partial difference between the strains isolated before

1978

and those isolated

later.

There

is a

4-fold dilution difference

between

the

titers

against

^{the 5}

^strains

isolated before

1978

and the

4

strains isolated post-1978.

Even if the

A/equine/

Miami/1/63 virus induces

cross-protection against

^recent

^isolated (Burrows

^et

^al., 1981; Burrows and Denyer, 1982), ^the

introduction of these isolates into the vaccinal strains, ^such

^as

A/equine/Fontai-

nebleau/1/79

or

A/equine/ Joinville/1/78, contributes

to

reinforce the potency ^{of the} vaccines.

In

1983,

^{Hinshaw et}

al., established that

Mo

Abs against A/equine/Fontaine-

bleau/1/79 do

not react

by ^HAI against

any of the strains isolated before 1971. In

parallel,

^one

^of

^the

anti-A/equine/Miami/

1/63

Mo Abs does

not react

with any of the strains isolated after

1979.

Moreover, Daniels

et al.

(1985) demonstrated, by comparison ^of

^the

amino acid sequences, the existence of

a

modification

^between the

A/equine/Miami/1/63

strain and

the

A/equine/Fontainebleau/1/79

strain. Some modifications concern

residues

which are

recognized by ^{Mo Abs} against ^{the human} X,

³¹

(H3N2) variant. These residues

are

located

on the

external part ^{of the}

molecule and could play a part ⁱⁿ

^the

antigen-antibody

reaction and the anti-

genic ^drift.

Recently,

^a

modification of the anti-

genic pattern

in the strains isolated after 1976 was demonstrated

by Appleton

^{e t}

al. (1987). ^When analyzed by

^HI

^and

ELISA

the viruses

fell

into one

of the

2

groups :

⁶

A/equine ^isolates

^-

^Miami/

1/63, Kentucky/1/63, Milwaukee/2/63, Sao Paolo/2/69, Sachiyama/1/71, ^and Algiers/

1/72 ^-

reacted with

4

Mo Abs, ^whereas

⁸

A/equine

^{strains -} California/

1/76,

^New-

market/76, Newmarket/79, California/1/80, Kentucky/1/81, Finger Lakes/1/83, Finger Lakes/2/83, Syracuse/

^{1/85 -}

^did

^not

react with these monoclonals. In

contrast,

4

other monoclonals reacted

with

almost all the strains.

Continuing evolution of the antigenic

characters of the

equine

influenza virus

has

now

been demonstrated,

^but

ⁱⁿ practice

the appearance of ^{new or} unmasked

epitopes

^{seems to} be balanced

by ^the persistence ^{of stable} epitopes.

^The

number and

immunogenicity ^{of these} epitopes

have been sufficient until now to ensure a

minimal natural

or vaccinial

protection

^of

^the

^overall

population,

^even

though frequent individual failures still

occur.

References

U.S.

Department

^of

^Health,

Education and Welfare

(1975)

^Advanced

Laboratory

^Tech-

niques

for Influenza

Diagnosis. Immunology

series no. 6

procedural guide.

Center for Disease

Control,

Atlanta GA.

Appleton J.A.,

Antczak D.E. &

Lopes

^A.D.

(1987)

Characterisation of

equine

^influenza

virus H3 with monoclonal antibodies. Arch.

Virol. 94, ^339-346

Archetti I. & Horsfall F.L.

(1950)

^Persistent

antigenic

^variation of influenza virus after

incomplete

neutralisation in ovo. J.

Exp.

^Med.

92, 441-446

Belyavin

^{G. & Cohen A.}

(1962)

^Chromato-

graphy

^of influenza virus

hemagglutination

inhibitors.

Virology 18,

11-145

Burrows R. &

Denyer

^M.

(1982) Antigenic properties

^{of some}

equine

influenza viruses.

Arch.

Virol. 73, 15-24

Burrows

R., Denyer M., Goodridge

^{D. &}

Hamilton F.

(1981)

^{Field and}

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