Antiviral Effect of Bacterially Produced Human Interferon (Hu-IFNα2) Against Experimental Vaccinia Infection in Calves

MaryAnnLiebert, Inc., Publishers

Antiviral

Effect of

_Bacterially

Produced

Human

Interferon

_{(Hu-IFNt*2) Against Experimental}

Vaccinia

Infection

in

Calves

JOHN

WERENNE,1

CATHERINE VANDEN

BROECKE,1

ANNE

SCHWERS,2

ANNE

_GOOSSENS,'

LOUIS

BUGYAKI,3

MARIA

MAENHOUDT,2

and PAUL-PIERRE PASTORET2

ABSTRACT

The

_heterologous

antiviral

efficiency

of

_bacterially

_produced

human interferon

(Hu-IFN«2)

in thebovine

species

was

_studied,

using

vaccinia infectionas

experimental

model.

In adouble blind

experiment,

young calveswere

intramuscularly

injected

daily

forseven

consecutive

days

with different doses of

Hu-IFNa2

or

placebo,

thetreatment

starting

24 h before intradermal inoculation of vaccinia virus. Aclear

_protection

by

interferonwasob¬

served in all the IFN treated

_animals,

_although

individual variations in the

_sensitivity

to

IFNwererecorded. The

efficiency

oftreatmentvaried

according

tothe dose of IFN used:

With the

_highest

dose

(106

_IU/kg),

complete protection

could be obtained.

The

only

side-effect observed was

hyperthermia.

_Circulating

antiviral

activity

appeared

quite early

after each IFN

_injection,

_presented

a more orless

biphasic kinetics,

and was

completely

cleared after 24

_h,

_justifying

the

_daily

treatmentschedule. Thefirst evidence of

an in vivo antiviral effect of humaninterferoninthe bovine

species

opens broad perspec¬ tives for a future use of interferonin

veterinary

medicine.'

INTRODUCTION

In

veterinary medicine, viral diseases cause

important

economic

losses,

especially

in cattle.

Therefore,

abroad_spectrumantiviral

_compound

would be of

high

interestto

_improve

theoverall

efficiency

of bovine

_production.

Interferonseemsa_very

promising

candidate for such

purposes.11'

However, bovine interferon wasnot yet availablein sufficient amounts to testits antiviral

_activity

in vivo.

Since the recombinant DNA

technology

allowed

_large

scale

_production

of different types of

humaninterferon

_(for

review,

seeRef.

2),

numerousclinical studies

_using

these moleculesas

anti-tumororantiviral agentsin manhave been undertaken.

Bacterially

produced

humana2interferon

(HuIFNa2),(3>

oneofthe

subtypes

ofhuman

leukocyte

interferon,

has beenshown forthe firsttimeto exertanin vivo antiviral

activity against experimental

'Departmentof GeneralChemistry, FacultyofSciences,Free_UniversityofBrussels,CP 160,Av. F. Roose¬ velt 50, B.1050 Brussels, Belgium; department of Virology, Faculty of Veterinary Medicine, University of

Liège,Rue des Vétérinaires45, . 1070Brussels, Belgium;^Instituteof_Hygieneand Epidemiology(IHE), Rue A._Delporte27, B.1050Brussels, Belgium.

vaccinia infectionin rhesus

_monkeys.(4>

Furthermore,

different human

interferons,(56>

including

Hu-IFNa2,<7)

are

_highly

active in bovine cell cultures.

Therefore,

the in vivo antiviraleffect ofHu-IFNa2 in thebovine

_species

wastested

using

intra¬

dermal inoculationofvaccinia virus.

Indeed,

this

experimental

system

produces

incalves skin lesions

allowing quantification

of interferon

activity.

MATERIALSAND METHODS

a)

Interferon

Bacterially produced

human

_alpha

interferon Hu-IFNa2 was

kindly

supplied

by

Dr. C. Weiss-mannandDr. M. Fountoulakis from Zürich

University.

Its

specific activity

was 10s

IU/mg

of_pro¬ tein and it

only

contained 5.5ng of

pyrogen/108

IU.

b)

Virus

Vaccinia

_virus,

strain ELSTREE

_(used

for the

_production

of human vaccine in

_Belgium),

was

titratedonchorio-allantoic membrane of

_embryonated

hen's _eggsand

_suspended

ina

_glycerin

con¬

taining

bufferto a final concentration of 108 PFU/ml.

c)

Experimental procedure

A double blind

_experiment

was

performed

(Table 1).

Twelvetwo- tothree-week-oldcalves, from

different

_origins,

weremaintainedintheIHE

_quarantine

stable,

toavoid

spreading

ofvaccinia virus. Hu-IFNa2atdifferent

doses,

or

phosphate

buffer salinefor control

animals,

inavolumeof 0.5

ml,

was

intramuscularly

injected daily

forseven consecutive

days, starting

on

day

-1.

Table 1. DataonExperimentalAnimalsandTheir Treatment.

Calf

number 1 7 8 10 11 12

Weight

(kg)

Breed Sex Treatment 30 43 42 41 25 35 28 35 27 30 BW BW BW BW BW BW BW BW BW BW MFMFMF FF F M 40 40 BB R F F + Vaccinia virus inoculation Hu-IFNa2 0

(IU/kg)

10" 10" 106 0.2 x 106 0.5 X 106 0.2 x 106 0.5 x 106 106 notincluded in the double blindtest

BW: Black and White. BB: _BelgianBlue. R: Red.

F:Female.

M: Male.

At

day

0,all the animalsexcepttwo

₍₁₁

and

12)

were

intradermally

inoculated with vaccinia virus

preparation,

on ashavedareaof the

flank,

using

abifurcated

needle;

thetwouninfectedcalveswere inoculated

by

the same_way, but with

glycerin

buffer

only.

Plasma

_samples

for the determination of

_circulating

antiviral

activity

werecollectedat

0,

30

min,

2

h,

and thereafterevery3 huntil24 h after the firstadministrationof

interferon,

and0,30min,

2,

5,

8,and 24 h after each of the

following injections. Body

temperaturewasmeasured

just

before each blood

sampling.

Skin lesions were

daily

scored for their

size,

number, _appearance, and

degree

of

evolution and

photographed.

Serum

samples

from

days

0 and 15 weretaken for anti-vaccinia anti¬ bodiesdetermination.

d) Interferon

titration

Circulating

antiviral

activity

wasdetermined

by

theclassical

technique

ofreductionof

cytopathic

effect of VSV in human

HEp-2

cells.""

e)

Seroneutralization

test

Sera from

days

0and 15weretested for the_presenceof antibodies

against

vaccinia virus

by

inhibi¬ tion of

pock

formation induced

by

this virusonchorio-allantoicmembrane of

embryonated

hen's eggs.

RESULTS

Twocalves

(5

and

12)

werein poor conditionat the

beginning

of the

experiment.

Both of them died on

day

0; the _necropsyrevealed

cachexia, anemia,

and chronic lesions ofarthritis andpneu¬

monia,

reflecting

a severebacterial

systemic

infection

prior

tointerferontreatment.The

_experiment

was therefore

_performed

with tencalves

_only.

a)

Vaccinia lesions

Thethreeuntreated control calves

(Fig. 1)

showeda_verysimilar evolution ofvaccinialesions. A clear

_protection

by

interferonwasobserved in all the treated animals: lesions

developed

more

slowly

and remained

significantly

smaller.

Figure

2shows theaspectof skin lesionson

_day

_9,intwocontrol andtwoIFN-treatedanimals.

The diameter of skin lesions

_appeared

tobea

good

criteriatoevaluatethe

_efficiency

of IFNtreat¬ ment.Asshownin

_Fig.

1,the inhibition of vaccinia lesions

_{development depended}

onthe IFN dose

injected.

However,

important

individual differences

_appeared

in the

_sensitivity

to IFN treatment.

For

_example,

incalf

3,

noskin lesionsatall could be

observed,

while calf4, treated with thesame

IFN dose

(106 IU/kg) developed

small vesicles

(Fig. 3).

b)

Side

_effects

IFNtreatmentwas well tolerated

by

all the animals.

However,

hyperthermia

wasrecordedin the treated calves

following

each IFN

injection.

c)

Pharmacokinetics

of

plasma

interferon

Figure

4

gives

the evolutionof

_circulating

antiviral

activity

in fivetreatedcalves

during

the 24 h

following

the firstadministration ofIFN. Noantiviral

_activity

wasdetected before the

beginning

of

E o LU O < u. a. z>

INTERFERON DOSE

(

million

_IU/kg

)

FIG.1. Vaccinallesions size. The total surfaceof thevaccinia lesions

₍₃₄

virus inoculationsites in

each

_case)

wasmeasured

_{by planimetry eight}

days

after infection.

FIG. 2. Skinlesions nine

days

after infection.

a,b:

control calves

(a

= calfnumber

7,

b = calf

number

1);

c,d:interferon treated calves

(c

= calf number9,0.2 IO6

U/kg,

d = calf number

3,

IO6

U/kg).

JSHHK

FIG.3. Effect of 106

_U/kg

IFNtreatmentontwodifferent calves,a:calfnumber3,b:calfnumber 4.

TIME AFTER INJECTION (hours)

FIG. 4. Pharmacokinetics of interferon. The

_circulating

antiviral

activity

was titratedin

_HEp2

cells. calf7

_(control),

·calf4

₍₁₀₆

IU

_IFN/kg),

Ocalf 8

_(0.5

106IU

_IFN/kg),

D calf6

_(0.2

106 IU

IFN/kg),

calf12

₍₁₀₆

IU

_IFN/kg),

calf 5

_{(106 IU/kg).}

Thelast animals diedon

day

0 from_pre¬

existing

lesions;

in these cases,

only

aminorfraction ofthe

_injected

interferonwas

_appearing

in the

circulation.

treatment; IFN

_appeared

in

_plasma

ofthe treated animalsas

early

as30 minafterthe first

injection.

The kinetics

_presented

a

biphasic

_aspect:afirst

peak

wasobserved2 h PI

_{(postinjection);}

the level of

IFN

_activity

decreased until 5to8 h PI,increased

_again

toreachasecond

peak

about 11 h PI,and thenreturned

_{progressively}

toundetectable levels after 14to17 h. Untreatedcontrolsnevershowed anydetectable

circulating

antiviral

activity.

The levelof IFNrecovered from

_plasma

wasrelatedtothe dose

_initially

injected,

despite

somein¬

dividual variations.

_{Particularly,}

calves5 and

12,

which died the

following day,

didnotpresentthe

expected

titer of

_circulating

antiviral

_activity.

The

_{pharmacokinetics}

pattern observedafter each of the

following

IFN

_injections

appeared

very

similar,

although

blood

_samples

werecollected

only

for 8 h PI

(data

not

shown).

d)

Seroconversion

None of thetwelve calves

_possessed

anti-vacciniaantibodiesat the

beginning

of the

experiment.

Neutralizing

antibodies

_against

vaccinia virus

appeared

on

day

15 in thesera ofall theinoculated

animals,

exceptcalf3, the

only

one whichshowed noskin lesions.

DISCUSSION

Bacterially

produced

Hu-IFNa2is

clearly

active

against experimental

vaccinia infection in calves

(Figs.

1and

2).

Moreover,the absence of seroconversion in calf 3 confirms the evidence ofastrong inhibition of virus

_{multiplication}

by

interferon. This confirms the results obtained inasimilar_pre¬

liminary experiment performed

ontwocalves."10'

However,

thereare

_important

individualvariations inthe

_sensitivity

toIFNtreatment

_(Figs.

2 and

3).

Although

a

_significant

reduction of skin lesionswas observed foreach dose tested

_(Fig.

1),

106

IU/kg

seemsthe minimal dose abletoconfera

complete protection.

After the end of theseven

_day

treatment, nofurther

_development

of lesionswas seencontrarytowhat is described afterashorter

(two days)

treatmentin the

_previously

reported study.'10 '"

IFNtreatmentis well tolerated

by

all theanimals:the

only

side effect is the classical

hyperthermia

described inmoststudies with exogenous

interferon,'1213)

exceptthat realized in rhesus

monkeys by

Schellekens and coworkers.'4' Asthe interferon

_preparation

usedwas

_highly

purified

and

only

con¬

tained5.5 ngofpyrogen per 108

IU,

this transient

hyperthermia

seemstobe

provided by

interferon

itself.

Circulating

interferon

activity

appeared quite early:

afirst

peak

wasobserved 2to5 h after

injec¬

tion. This_agrees with thenumerous

pharmacokinetics

studiesrealized

following

intramuscular in¬

jection

of human

alpha

interferon in different

species.'14"1"

After a transient

decrease,

a second

peak

is observed 8 to 11 hPI

_(Fig.

4).

Inmost

previous studies,

data are

only reported

for 5to8 h

after

_injection.

However,

Breinig

and coworkers'17' followed the levelof

_circulating

antiviral

_activity

during

24 h inahuman

_{patient repeatedly}

treatedwith

_{conventionally prepared}

humanainterferon:

These authors observed a_verysimilar

_biphasic

kinetics of

circulating

interferon,

with afirst

peak

4-5handasecond one9-11 h after each IM

_injection.

No further

_circulating

antiviral

_activity

is detected24 hPI. This confirmstheresults ofour _pre¬

liminary experiment

in calves'918' and ofmost

_previous

studies.'14"16'

_Breinig

et

al.,'17' however,

still observed low levels of

_plasma

interferon 24 h

_PI,

butno moreat48 h PI.

_Similarly,

Jordanand

co-workers'19' have shown that

_leukocyte

interferon

injected

intramuscularly

in

_patients

with

malig¬

nancy

complicated by

disseminated

herpes

zoster, isnot

completely

eliminated in 24 h.

They

have also shown that

repeated injections

cause a

progressive

rise of theaverage

peak

level indicative ofan accumulation of interferon in tissues. Whether this is indicative ofa slower

degradation

of inter¬ feroninthe

homologous species

orof different

pharmacokinetics

properties

for the differentinter¬ feron

species

is not _yet known. In favor of thelast

_{interpretation}

we should mention the recent

observationmade inmice

by

Sarkar'201

showing

thatHu-IFNa, is cleared from thecirculationmore

rapidly

than the mixture of human interferons

produced

in

leukocytes.

As,inourcase,interferonwas

_completely

clearedfromthe circulationafter 24

h,

the

daily

treat¬ mentschedule

_adopted

seemsconvenient19' andisrecommendedforfurther studies

_using

_exogenous

interferonin the bovine

_species.

Although

the levelof

_circulating

interferon recovered in treated animals varies

according

to the

dose

_injected,

as

already

reported

by

Schellekenset

al.,'4'

someindividual variationswereobserved.

Particularly,

in the two ill animals

(5

and

12),

who died the

_{following day,}

the

_expected

level of

plasma

antiviral

_activity

wasnotrecovered. This raises the

_{interesting possibility}

that

_trapping

of cir¬

culating

interferoncan occuratthesiteof the

_{already developed}

lesions. Further

_{investigations}

are

neededto substantiate such an

interpretation.

In

conclusion,

this is the first

_complete

_reportofanefficient

_{protection against}

aviralinfectionin

cattleobtained

by

awayother than classical vaccination. Interferonappearsthereforevery

promis¬

ing

as abroad spectrum antiviral

compound

in

veterinary

medicine. Some recent

findings

_support this

opinion:

The evidence ofarole of

endogenous

interferon in the control of

experimental

rota¬

virus diarrhea in newborn calves'21·22' and thecurativeeffect of

_orally

administered bovine inter¬

feron incats

suffering

from feline leukemia virus-induced

_{non-regenerative}

anemia.'23' Moreover, Hu-IFNa2is activein vitro

_against

several viruses

_responsible

for

_important

economical lossesinani¬ mal

_production:

infectious bovine rhinotracheitis virus

(BHV1),

pseudorabies

virus

(SHV1),'24'

and

bovine rotavirus

_(unpublished

data).

On the other

hand,

the calfmayrepresentaconvenientmodel for the in vivo

study

of the action of

interferon

against

natural viralinfections.

ACKNOWLEDGMENTS

We wishtothank Dr. C. Weissmann andDr. M. Fountoulakis from Zürich

_University,

Switzer¬

land,

for

giving

us

bacterially

produced

Hu-IFNa2.

We also thank Dr. J. Content from Pasteur

Institute, Brussels,

for

coding

the double blind

study,

and E. Van Kerckhove for

_serological

tests.

We thank Dr. B. Brochier for his occasional but_verykind

_help

during

this

study.

This work was

_partially

_{supported by}

_{grants from the} "Institut pour

_{l'encouragement}

de la Re¬

cherche

_scientifique

dansl'industrieet

_{l'agriculture"}

_(IRSIA)

from which C. VandenBroeckewas a

student fellow.

Part of these datawere

presented

atthe Third Annual International

Congress

for

Interferon,

held in Miami

(USA),

November 1-3, 1982.

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Address

_reprint

requeststo:

Dr. John Werenne

Department

of

General

Chemistry

Faculty of

Sciences Free

University

of

Brussels CP 160, Av. F. Roosevelt 50

B. 1050Brussels,

_Belgium

Received 6August 1984/Accepted4October 1984