Note: Descriptions are shown in the official language in which they were submitted.
a73
INFECTIOUS BRONCHITIS V~CCINES FOR P3ULTRY AND PRCCESS FOR THE
PREPARATION OF SUCH VPCCINES.
The invention is relating to infectious bronchitis (IB) vaccines
for poultry and a process for the preparation of such vaccines and
more particularly to improve infectious bronchitis vaccines, derived
from at least one in~ectious bronchitis virus strain of a no~el
serotype as to those of the virusses, which are known up to now
for vaccination purposes.
The application of live infectious bronchitis vaccines for
poultry is already known for many years.
Infectious bronchitis is an important affection of the
respiratory system, the kidneys and oviduct of poultry. The cause
of this syndrome is a corona virus. The poultry is severely
affected by epizootics of this disease.
~he infectious bronchitis still causes a high mortality,
especially in young poultry. Besides the mortality and more or
less skrong respiratory symptoms, lesions to the oviducts and
as a result thereof egg production drops occur caused by an IB
infection.
Moreover infections with IB virus may stimulate latent vir~ls-
or bacterial infections and may give rise in ~is way to severe
economical losses, especially in the broiler field.
~or the combat~ent of infectious hronchitis as well vaccines,
derived from inactivated virus as tho9e ones der:ived ~rom live
virus, are applied. However, it was ~ound that a loss of immunogenic
pr.ope~ties o~urxed after inactivation of these viruses with e.g.
~.ormaline cmd ultra violet light (M.S. ~lo~stad, Diseases of Poultry,
Biester and Schwarte, Iowa State University Press. Ames. (1565), 615).
As also normal, sound chickens can be killed or diseased by
primary vaccination with a live, non or less attenuated virus vaccine,
whereby an especial danger is existing for animals of less than
2 to 3 weeks old or for chickens short before the start of or during
laying, pecple skilled in this art have a clear preference for the
app.llc~lon of d~ad vaccin~ or of live vaccines, whereby ~a~ tried
to ~ncre~e th~ h~rmlessness of such vaccines by means o~
~1
,.
attenuation of the original IB field virus isolate.
For exa~ple the H-strain, which is presently applied on world
wide scale on account of his broad immunization spectre against
am~ng others Massachusetts and Connecticut type IB-virus and which
was isolated and attenuated by sijlenga, Hoekstra and Rispens, is
disclosed in Tijdschr. Diergeneesk. 81:43, "Infectious bronchitis
in ehicks in the Netherlands" (1956), Tijdschr. Diergeneesk, 85:320
(1960), Tijdschr. Diergeneesk. 85-279 (1960) and Tijdschr. Diergeneesk.
85:398 (1960).
E'or such m~dified vaccines, viruses, having undergone 25 or more
embryo passages to reduce their pathogenicit~ and their disseminating
ability, are applied up to ncw, such as e.g. the Massachusetts type
and more particularly the IsV W 48, M 41, 82828 besides the H52
and H120 strains thereof, these two having been passaged approximately
52 and 120 times respectively on ehieken embryonated eggs, a
Connectieut isolate, e.g. A 5968 or the Beaudette IBV type (I~V-42).
'I~e imm~nizing eapaeity of these viruses is very specifie against
Massachusetts or Cbnnecticut types of IB virusses.
Although ~he use of vaeeines of these modified strains has
presently appeared t~ be safe and effeetive, these vaeeines have
appeared to be still unable to prevent outbreaks of infeetious
bronehitis in a suffieient way under certain conditions, as appears
from ~vian diseases vol. 20, no. 1, p. 42 and 177 and Avian Diseases
vol. 19, no. 2, p. 323 and 583.
q~his shorteommg o the present IB vaeeines is attributed to
c~eurrLng antlc~enie variations of the virus in an important degree,
a~ al~o a~pp~ax~ e.g. from ~rehiv f~r die Ge.s,lmte Virusforschung 3~,
p. 32 ~ 71) arld Cunningham C.EI., Develop. Biol. Standard, 33, 311 (1976).
Efforts were made therefore to reaeh an adequate vaeeination of
poultry by means of preparation and application of co~bined vaeeines,
derived from several IBV strains of different serotypes.
Elowever, hereby a elearly eneountered difficulty appeared to form
the deerease of immunogenie properties of the respeet:ive start~lg
viruse~, eaused by mutual ~Iterae-tion, as appears from ~n. J. Vet. Res.
36, ~, 52~ an~ 525 ~1965) a~ ~vian Diseases 12, 577 (1~68).
'rhexefore ~here :is ~ill existing a larqe need
~!
~!33~3
-- 3 --
for IB vaccines with adequate immunizing properties.
It will be appreciated that the pursued improve-
ment of these vaccines are still severely hampered, caused
by changing immunogenic and other properties of
the presently avai]able Is viruses after a large nuT~er of
passages in embryonated chicken eggs, the appearance of new
serotypes and the lack of sufficiently effectively a~plicable
serological and immunological test procedures respecti~ely.
In this connection reference may be made to ~vian
Diseases, 19, 2, 323 and 324 (1975).
As a result of extensive research and experimenta-
tion, a numher of novel IB viruses could surprisingly be
obtained, which can be regarded as deviating from the up to
now most frequently applied IB viruses of the H type
(e.g. IB H120 and IB H52) in cross neutralization tests
(virus neutralization tests) according to e.g. the method as
described in American Association of Avian Pathologist~,
"Isolation and Iclentification of Avian Pathogens", page 184
(1975)~in the understanding that antisera diluted in a ratio
of 1:5 are applied~and in challenge experiments with subse-
quent vlrusreisolation tests. In other words at an 1nocula-
tion with a virus o~ the H-type the concerning animals are
not protected against virusreplication in the mucosa o~ the
re~piratory sy~tem after a ch~llenge with the beforementioned
cleviatlng novel IB vlru~cs.
Ilumoral antihodles against the IB H strain equally
~ppellrcd not to be able to neutralize ~ignificant amounts of
IB vlru~ oE the mentioned devlating ~ypes.
O special irnportance ~or the practice is that
the~ rlovel ~B vlru~e~ cause respiratory symp~om3 wLth ani-
mal~ ~how:ln~ hL~h all~ibo(ly ~:iters a~ainst ihe ~B H sl:rain,
and wi-th stlll l~ying anlmals, ec3cJ produ~tion clrons.
~ xamples of such novel viruses are those ones,
identifi.~3d by tlle internal notation ~trecht.101 (U.101),
Utrecht 102 (U.102), Drente.201 (Dv201), Lirnburg.501 (L.501),
Llmburg.502 (L.502), Brabant.801 (B.801),
I,;lmbu~c3.$86 ~L. 536), Overij~sel 728 ~0.728) ancl Utrecht.121
(U~ 12L) a~ CIQPO~:It~d at the Czeclloslovak National Collectlon
o~ Typ~ Cul~uros o~ the Institute oE ~Iyg:Lene and ~p:Ldem:Lolo~Jy
in Prague under the respective nos. CNCTC A07/80 ~ CNCTC A08t80,
CNCTC A09/80, CNCTC A010/80, CNCTC A011~80,
(on March 6, 1980), CNCTC A016/80, CNCTC A014/80, CNCTC A015/80
and CNCTC A013/80 (on September 9, 1980), and with the Col-
5 lection Nationale de Cultures de11icroorganismes d'Institut Pasteur,Paris,
under the resp.
nos. I-111, I- 112, I-113~ I-109, I-110 (on November 14, 1979)
and I-132, I-134, I-135 and I-133 (on October 3, 1980) with
10 the Collection Nationale de Cultures de Microorganismes
Institut Pasteur, Paris.
These viruses were isolated by means of the trachea
swab method with flocks of laying chickens, which had been
vaccinated two and three times respectively with IB H120 and
15 IB ~152 vaccine and which showed high humoral antibody titers
as -to the Massachusetts type of the IB virus at the beginning
of the occurrence of respiratory symptoms and/or egg production
drops and with broilers, which sho~Jed in the second half of
the ~attening period, respiratory symptoms a~ter previous vaccina-
20 tlons with IB vaccine of the I1120 type, whereby both kindsof animals found themselves at the moment of isolation in
districts, which have been indicated in the hereinbefore
usecl internal notatlons.
There ~7a5 now found that by attenuation to SPF-
~5 chitkt~n~mbryos, the isolrlted virusstrains have lost theirpakh~gqnicLk~ for SPF chlckens in a major degree, in spite
o~ the fact that lheir immunising ability has rernained s-till
present.
For example the viruss-traLn with lnternal notation
30 xr~v U~ c~ht.l01 ~howet~ these beEorementioned characterlst:lcs
aEker 85 SPE' ~ype I chlckenelnhryo passages and the virus-
strain IBV Limburg.536 ~howed these characteristics after 56
SPF type I chickenembryo passages.
There ~s surprisingly found during a comparing
35 test, using conven-tional ~1120 or H52 vaccines, ~hat the
protectton against LB viru~;es of thc Massachusetts type
~ m~ur:~d a~ ~he amoullt oE virus neu~raliæ~ng antiboclles -
h~cl n~t ~.o be dllninl.shet~ 1n a ~i~3ni~lcant d~yrce, i~ a l-l-
7;~
type vaccine combined with e.g. the novel IBV isolate Utrecht
101 was administered instead of the H-ty~e vaccine. During
this experimentation one star-ted from intran~sal a~lication
of the concerning vaccines.
For instance the following neutrali7ation indices
were determined ~ weeks a.~ter adminlstration of the respective
vacclnes and vaccine combinations.
~Table 1. I~nunisin~ ability of dif~erent vaccines measured
on the humoral immune res~onse (neutralization-
index)
_ _ _ .. , ., . _ _ .. , _ _ _ _ .. _ _ _ _ _ .. ~ _ _ _ _ .. , .. .. _, .. _ _ _ _ . _ _ _ _ _ _ _ _ . _ .. _ _ . _ _ _ . _, _ _
vaccine testvirus/ IBV IBV
N.I.H (B222) U.101 L.536
1. H120 6.5 1.7 0.8
2. :[BV U.101 2.1 6.8 N.D.
15 3. H120 ~ IBV U.101 6.2 6.5 N.D.
(53~d ~gg pas~ags~) 0 9 N.D. 5.8
5. H120 ~ IBV L,536 6.7 N.D. 5.7
... _~.~ .. ._. ,_ _____ ~ .. _ ._.. , ___,._._._ _ __ . ._ ___ ____
In addition -to the immuno response af-ter administra-
t.i.on of different vaccines and vaccine cornbinations respec-
20 tive:ly, the res.istance agains!t virusreplication and ~ersis-
tence was also d~termi.1lesl in and on the mucosa of the trachea,
cls3termlned by means Oe the v.irus rei.solation technique, as
de~s~.lbed .in "Speci~:l.cat.ions ~or the pxoduct.ion and control
O.e ~v:l.an li.ve v.lrus vaccines" of the Mini~try of Agriculture,
25 Fi.sherls~ ~nd Food of the United Kin~dom Central Veterlnary
~Jahoratory of F~ioloc~:i.c~l Products alld Stallsla:rds ~e~artm~ntr
N~W ll~w, W~br.~dsJe, SuLxey KT 153 MB, 2nd Edlt.l~n (1977), p.12
Cxoss ncutralizati.on ~ests in SPF chicken embryos
and cross inEection tests on SPF chickens were carried ou-t
30 accord.i.ng to the hereunder followiny tables:
- 6 ~ 73
Table 2
Cross neutrali2ation test in SPF chickenembryos
Antiserum
vlruslsolate
against virustype H U.101 o. 728 L. 536
5 H ~,7,2 neg. neg. n~g.
U.101 neg. 5,3 neg. neg. x
0.728 neg. neg. 5,8 neg.N.I.(10 )
L.5~6 neg. neg. neg, ~,6,2
In this connection with the expression "neg." i~
10 meant, that no significant positive neutralization indexes
(N.I.), i.e. N.I. ~ 102' are obtained.
Table 3.
Cross challenge tests car~ied out o~ SPF chickens;
results of virusresolationtests.
15 vacc~ne virus challenge virus
"VOET" U.101 0.728 L.536
type Massachusetts
__ ___
H neg. + + +
120th egg-
passage
U.101 + neg,
50th egg-
passage
0,~.827 + ~ neg. +
pas sage
20 ~.S36 + + + neg.
80th ecJ~3,
p~ ge ~
With the term "neg." is meant in this connection
that none of the SPF eggs, injected wlth the tracheaswab
material, ~howed symptorns which rnay be attributed to an
infection with IB virus.
--7--
From the results of tables 2 and 3 (neutralization and cross-
challenge tests) it appears that the four applied serotypes of the
IB virus, namely those of the H-, U.101-, 0.728- and L.536-type,
not only differ antigenitically from each other, but additionally
- ha~7ing in mind the results of the challenge experiments with
additional virusreisolation - show attractive im~nogenic properties
as to the hom~logeous virus.
Field experiments shcwed that in sera of broilers, reproduction
chickens and laying hens, antibodies were frequently occurring
against the virustypes Utrecht.101, Limburg.536 and ~7erijssel.728.
It will be appreciated therefore that the novel IB virustypes
are not only differing antigenitically in a significant degree from
the usually applied H-virus, but moreover are shcwing significantly
different properties as to each other.
The novel isolated virusstrains could be characteri~ed by the
following tests:
Treat~ent of the infectious amnion-allantoic fluid obtained
by cultivation of original virus containing samples from infected
homDgenized organ and tracheaswabmaterial in the allantoic hole
of 10 days prebrooded SPF eggs with chloroform according to Mayr,
A. et al, Virologische Arbeitsmethoden G. Fischer Verlag, Jena,
1977, p. 2B5. resulted, in comparison with the non treated ~terial,
un a reduction o the repeatedly measured viruscontent from 107 5
to 101'5 EID~o. ~his experience may point to the presence of a virus
agent, which is containing in his envelope a lip:lde, which is
nece~sary Eor the infectivity.
~ e in~ctious amnion-allantoic ~luid caused no agglutination
with erythrocytes derived rom SPF chickens.
Addition of 5-fluordesoxyuridine (Ft~) to the culture medium
of chicken kidney cell culturest serving for replication of the agent,
did not influence the intracellular synthesis of the virus agent in
a significank degree.
The EID50 content of the cellmaterial and culture medium appeared
k~ xe~ide on comparable levels 2, ~ and 7 days after -the inoculation
oE the vixu~ a~nt, l.e. the
'73
--8--
nucleic acid to be replicated belonged to the group of the
ribonucleic acid.
Exam mation with electron microscope showed, that the virus
agent, present in the amnion allantoic fluid which was harvested
within 30 hours after the artificial infection, possessed a
diameter of about 100 nm. About 15 nm long projections were present
on the surface of this virus. The virus has the size and shape of
a corona virus, to which also the avian bronchitisviruses are
regarded to belong.
It will be appreciated that the properties of the novel
serotypes of IB-viruses as described hereinbefore make the novel
virusstra~ns expecially suitable for the preparation of as well
inactivated as live poultry vaccines on behalf of a more
efficient protection against infectious bronchitis, especially in
areas or countries, wherein the described deviating serotypes
according to the present invention occur besides the IB viruses
of the socalled H-tyee.
More particularly virusstrains of the serotypes of the
hereinbefore mentioned novel virusstrains may successfully be
applied for the preparation of mixed live and inactivated vaccines,
derived as well ~rom the novel IB strains as from the H~strains
with one or more o the novel IB-strains~
The nov~l IBV vaccines o~ the present invention may be
obtained by propagation of one or more novel virusstrains by
methods kncwn in the ar~ in principle and optionally follc~ d by
inaativation by methods known in the art in principle.
For instance the virus may b~ propayated in embryonated
SPF chicken egg~ or in suitable cell cultures such as chicken
lcidney cell cultures. However wit,h such a process there has to
be checked whether the antigenic properties do not significantly
change.
Hereafter the cultivatJed virus mat,erial is collected and
puxi~ied. ~t last one or more stabilizers ancl possihly
an~lbio~,ic~, ~uch as sodium penicillin Gl streptomycin or
r
- 9 ~
natamycin, may be added and the mix-ture is lyophilized.
~ore particularly, the concerning seed virus is
lnoculated under sterile conditions into the allantoic cavity
in 10-ll days prebrooded chicken egc3s of type I SPF
After incubation for 28 to 34 hours at 37C, the
amnion-allantoic fluid of the then still living and of the
specifically died (i.e. after 24 hours after the seed virus
inoculation) embryos is harvested, purified and lyophilized
after optional addition of stabili~ers and/or antibiotics.
According to this process, single vaccines could
be prepared, which contained the virus after lyonhilisa~ion
in an amount of ~lO4- ~ID50 per dose, while e.g. so
prepared combined vaccines of a novel virusstrain and a
known ~1-strain or of more novel virusstrains showed a virus
15 content of ~ 2 x 109- EID50 per dose and preerably a
content of each of the virus cornponents of ~ 104 EID50
pcr dose.
It will ~e appreciated that the present invention
.is also relating to novel, as well inactivated as live,
~o IBV vaccines, which have been at least derived fror~ one of the
novel. IB virus~trains and to the application of such vaccines.
Preferably live vaccines, derived from one or more
Oe t:he nove] vlruses or Erom the 11-tyne virus with one or ~ore
of thr. novel IB-virllses, are applied.
More preferahly live vaccines, derived from ~120 or
1152 vlrusstrain and frorn one or rnore ~B viruses, selected
Erom the Jrol1p cons:i6l:irlc3 of U.101, L.536 and ~728, are
appL:Ied. ~rlle vclccl.ncs rnay also he applied with youny anim~ls
an~ rnore preferab].y with broilers.
'rhe v~lccines may he ~dministered by the soccl1led
eyerirop- or nose(1rop, t:l)e drLr1k;r)c3-water- or spraylneli1od
~or 1Ive vacc:lne~.
Vacc:ination hy means of novel live vaccines accord-
ing to the present invention has preferably to be carried out
35 with poul~ry of an age of l day to 18 weeks. ~ovel inactivated
vaccines are subcutaneously or intra1n1:lsc-1larly
aclm:Lnil,tered to arlirnals. .,
It wlll br3 a~ rec:i.cll.ed that a].so combLncd live (~r
Lnacllvat:ed vacc:l1le~, r1erlvr~ rom at icast onr of the novel
- 1 o - ~ 3
IB virustypes and one or more completelv other vlrusty~es, such
as e.~. the Newcastle disease virus, adeno- or reo viruses,
form a feature of the present invention too.
For the preparation of inactivated IBV vaccines
according to the present invention there may be started from
e.g. an amnion-allantoic fluid, to which a suitable carrier
is added, after inactivation by methods known in the art,
e.g. by means of beta-propiolactone or formaline.
Preferably the virus liquid of a suitable titer is
processed to a water in oil emulsion vaccine, derived from
a mineral or plant oil and one or more emulsifiers, such as
non-ionic surface-active cornpounds derived from alkylene
oxide and/or hexahydric alcohols and/or higher natural fatty
acids (Clo-C20) such as esters or ester-ethers.
Examples of the lastmentioned emulsifiers are
rn~nnide rnonooleate (Span ~ 80, Arlacel 80 ~ , Arlacel A ~
and polyoxyethylene (20) sorbita~ monooleate ~e.g. Tween 80 ~ )
The volurne ratio between the aqueous phase (virus
1uid) and the oily phase may vary from 3:7 to l:l and lies
20 preferably at a ratio of about 7:13.
~ rhe invention is illustrated by the following exam-
plcs, however without any restriction o the object of the
invention to these specific embodiments.
Example 1
25 Pre~Axation of CB virus vaccine of the strain U.101.
A. Cultiv~tion of virus~
'rype ~ SE'F chicken eyyci, which were preincubal:ed
10 to 11 days, are inoculated with 103- to 104~ E~D50
C~V U.101 seed vixus ~0.2 rnl pro eyy) in the allantoic
vlt:y.
The ecJy~ ~re inspected for the first t;me after
20 to 24 hollrs from the virusinoculAtion and all a3pecific
died embryos are removed.
After an incubationperiod of in total 28 hours
35 at-~37C, the arnnion-al]antoic fluid (AAF) i3 harves~ed.
B. Treat.men~ of virus su.~pension.
Aeter purl~ic~ion of the A~E' b~ rnean.s of centr1~u-
gation at 2000 r.p.m. in a cooling oe ntrifuge and/or filtration,5 x 105 units of sodium penicillin G and 800 mg of streptomycin
per liter are added to this AAF.
The virusmaterial is subsequently stabilized by a~dition of
at least 3% by weight of albumin and/or mannitol.
The stabilized buLk virusmaterial is frozen to at least
-35C and stored at such temperature until the further processing
phase.
Samples of this material are meanwhile tested on their virus
content by means of the EID50 (Egg Infection Dose 50%) assay method.
After the test results have become available, the virus
material is thawn up again and filled out into lyophilization flasks.
~ he virus content (volume) is adjusted hereby in such a way,
that at the end of the subsequent lyophilization there are still
at least 10 5 EID50 of the concerning virus per dose present
in the vaccine.
m e flasks are sealed under vacuurn at the end of the
lyophilization.
With the preparation of the multivalent (mixed) vaccine care
ha to be taken that the minimal virus contents for all virus-
components are reached.
Example 2
Preparation of IB virus vaccine of the strain L.536.
~. Cultivation of virus.
Type I SPF chicken eggs, which have been preincubated during
10 to 11 day~, are inoculated with 103- to 104- EID50 IgV L.536
seed virus (0.2 ml pro egg) into the allantoic cavity.
The egy~ are inspected for the firsk time and all aspecifically
died ~nb~y~ a~ remov~d after 20 to 24 hours ~rom the virus
inoculation. A~ter an incubation period of in total 32 ho~s
at ~37C, the A~F is harvested.
B. Treatment of the virus suspension.
After purification of the AAF by means of centrifugation at
2000 r.p.m. in a cooling centrifuge and/or
-- 12 -- 131.~?9L73
filtration, 6 x 105 units of sodium penicillin G and 900 mg
of streptomycin per liter are added to this AAF.
The virus material is subsequently stabilized by
addition of about 5~ by ~eight of albumin and/or mannitol.
As albumin e.g. bovine albumin may be applied. The stabilized
bulk virus material is subsequently f~ozen to at least -35C
and kept at such temperatu~e until the further processing ,_
phase.
Meanwhile samples of this material are tested on
their virus content by means of the EID50 (Egg Infectious
Dose 50%) assay method. The virus material is thawn u~
again after the testresults have become available and filled
out into lyophilisationflasks.
The virus content (volume) is adjusted hereby in
~uch a way that at the end of the lyophilisation process at
least 10 '5EID50 of the concerning virus per dose are ~resent
in the vaccine. The flasks are sealed under vacuum at the end
o~ the lyoph:ilisation.
During the preparation of the multivalent (mixed)
vaccine care has to be taken that the minimum virus content
for all vLrus components is reached.
Example _
Pre~arati,on of IB-virus vacc~ne of th~? strain 0.728.
A. Cultivation of viru.s.
Type I SPF chicken eggs, Which have been preincubated
2~ durin~ 10 to 11 day~, are inoculated with 103- to 104'0EID50
I~V 0.728 seed viru~ (0,2 ml per egg).
'rhe c?gcJs are inspec-ted or the first tirne and all
aspe?ciflcally 11ed embryos are rernoved after 20 t:o 24 hours
~m ~he ViX'll~ inoc~ .ation. ~t~?r an 1ncuhation period of
3~ ln l,otal 3~ hours at ~37C, I:h~ ~AF is haL-vested.
B. Treatment of lhe virus suspension.
After purification of the ~AF by means of centrifu~
yation at 2000 r.p.m in a cooling cen-trifuc3e and/or iltratlon,
8 x 105 wnits of sodiurrl penicillln G and 1100 mg of strepto-
35 rrly~Ln~? I1~?r lit~?r a~? ac~cled to th~? ~F.
'rh? viru~ malerial ls ~ub~ec,~u~?ntly stabilizecl by
~3~73
- 13 -
the addition of about 7% by weiyht of albumine and/or manni-
tol.
The stabilized hulk virus material is subsequently
frozen to at least -35C and kept at this temperature until
the further processing phase.
Meanwhile samples of this material are tested on
their virus content by means of the EID50 assay method.
The virus material is thawn un again and filled
out into lyophilisationflasks after the testresults have be-
come available.
The virus content (volume) is adjusted hereby in
such a way tha-t at the end of the lyophilisation process at
least 10 EID50 of the concerning virus per dosis are present
ln the vaccine. The flasks are sealed under vacuurn at the end
of the lyophilisation.
During the preparation of the multivalent (mixed)
vaccine care has to be taken that the minimum virus content
for all virus components is reached.
~ le 4
Preparation of a combined IB-virus vaccine of the strains
U.lOl, L.536 and 0.728.
.. . .. . _ _ ... . , . . . . _ . . . ............ . _
A. Cultivation of virus.
Type I SPF chicke~ eggs, which have been preincubated
during 10 to ll days, are inoculated wi-th 103- to 104-0EID50
LBV U.lOl, 1..536 and 0.728 seed virus (0,2 ml in total per egg)
inko the allantoi.c cavLty.
T}le egc3~ are in~pected for the fJrst tlrne and all
aspeci~lcally died embryos are removed after 20 to 24 hours
~rom the virus inoculation. After an incubatLon period of in
total 32 hours a~ 37C, the ~AF :Ls harve~tecl.
B. rrreatment oE the virus suspension.
After purification of the A~F by rneans of centrifu-
gation at 2000 r.p.m in a cooling centrifuge and/or filtration,
8 x 105 units of sodium penicillin G and 1000 rng of strepto-
lnycine per liter are aclded to this AAF.
3S '['he vi~us Inater:ial is subs4quently stabilizecl by
~clclLt-Lon o~ at ~e~ 3~ by weicJht o;E albllnlLne and/or mannltol.
~ he stabilized bulk v,irus material is subsequently
frozen to at least -35C and kept at this temperature until
the further processing phase.
Meanwhile samples of this material are tested on
their virus content by means of the EID50 assay method.
The virus material is thawn u~' again and filled
c,ut into lyophilisationflasks after the teqtresults have be-
come available.
The virus content (volume) is adjusted hereby in
10' such a way that at the end of the lyophilisation Process at
least 10 ' EID50 of each concernincJ virus per dosis is present
in the vaccine. The flasks are sealed under vacuo at the end
of the lyophilisation.
During the preparation of the multivalent (mi~ed)
vaccine care has to be taken that the minimum virus content
for all virus components is reached.
Preparation of inactivated combined IB-virus vaccine of the
strains H.120 U.101, I,.'536 and 0.7~8
In a similar way as described in example 4.A., the
' 20 virus was cultiva-ted in SPF eggs and the obtained virus sus-
pension was treated in a similar way as in example 4.s., un-
til the frozen phase is rea~hed, however, without addition of
antibiotics and stabilizers.
The ~rozen ~AF is defrosted and inactivated in a
water halh by 0.l~ o~ propiolactone during 90 minutes at
37~C.
The virus suspension Ls kept overnight a-t ~4C, The
inactlvation ls checked by ino~ulation of prebrooded embryo-
nakecl SPF chicken e~s wi-th thc lnactlvated vlru~ material and
~0 ~uhsQc,luQnk Lncuba~Lon.
rrhe Lnactivated A~F is diluted if necessary, w,ith
PBS t 0,3% of formaline, dependent on t,he E:LD50 of each
virus t~rpe, determined in the non-inacti~atecl A~F (at least
107' for all virusstrains).
To the virus suspension of the four st:rains 3.5% of Tween 80
are ac1dcd.
- 15 -
The inactivated virus suspension is mixed with an
oil phase in the ratio of 6.5 parts of oil / 3.5 parts of
virus fluid and emulsified, so that the average particle
size of the aqueous phase is about 0.5 u.
The emulsifying .is carried out by means of an
Ultra Turrax homogeniser or by means of passing the starting
mixture through a colloid+mill.
The applied oily phase has the following composi-
tion:
10 Marcol 5 2~ (white paraffinic Esso oil) 93,5
Arlacel A~ Arlacel 8 ~ or Span 8 ~
(mannide monooleate) 6,5 ~,
The components of the oily phase are separately
heated to l10C in an autoclave or the mixture is sterily
filtrated.
