CA1047922A - Live influenza virus vaccines and preparation thereof - Google Patents
Live influenza virus vaccines and preparation thereofInfo
- Publication number
- CA1047922A CA1047922A CA218,508A CA218508A CA1047922A CA 1047922 A CA1047922 A CA 1047922A CA 218508 A CA218508 A CA 218508A CA 1047922 A CA1047922 A CA 1047922A
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- Prior art keywords
- virus
- strain
- atcc
- influenza type
- recombinant
- Prior art date
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pulmonology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Attenuated stable influenza type B virus strains are obtained by recombining a vaccinal and previously attenuated influenza type B virus strain with an influenza type B virus isolate having the desired serotype and iso-lating a recombinant having at least one marker property of the attenuated parent strain which is not shared by the other parent strain and the antigenic composition of the other parent strain. The obtained strains are useful for the production of nasal influenza vaccines.
Attenuated stable influenza type B virus strains are obtained by recombining a vaccinal and previously attenuated influenza type B virus strain with an influenza type B virus isolate having the desired serotype and iso-lating a recombinant having at least one marker property of the attenuated parent strain which is not shared by the other parent strain and the antigenic composition of the other parent strain. The obtained strains are useful for the production of nasal influenza vaccines.
Description
~ 7~2;~
This invention relates to live influenza virus vaccines ~or nasal administration comprising as active ~
ingredient an attenuated stable type B in~luenza virus ~ -strain and, eventually, an attenuated stable type A in~luenza virus administrable by nasal route, and to a process o~
preparing said attenuated stable in~luenza virus vaccines.
Protection against viral respiratory in~ections has been shown to be related to the presence o~ a local immunity in the respiratory mucosa. This aspect has been reviewed recently by Rossen et al. (Progr. Med. Virol.
1971, 13, 194). `
Several attempts were made in recent years to ~
induce immunity against in~luenza by the nasal application `
of inactivated vaccines (see e.g. : Waldman et al. Nature, !~ `
1968, 218, 594; JAMA 1969, 207, 520; WH0 Bull~ 1969, 41, .~;
543). The results were inconsistent, however, and this was probably due to the insu~icient stimulation o~ the immunity system by the inactivated antigen (Tyrrell et al. ~`~
J. Hyg. 1970, 68, 359).
Live inPluenza type B virus vaccines are known ~ ~ `
b~t are presenting widely v`arying protection rates and ~ ;
numerous e~orts have been made e.g. by serial passages on eggs (see ~or instance A.A. Smorodintsev, Proc. Symposium on Acute respiratory Diseases, Zagreb 1969, 391-404) or by using low temperature mutants (F.M. Davenport et al.
Proc. Symposium on Llve In~luenza Vaccines, Zagreb 1971, 105-113) to reduce pathogenicity o~ the virus.
Recombination oP in~luenza~type B ~irus strains is also mentioned in the literature (see ~or instance E.D. `~
Kilbourne in Progr. Med. Virol. 5, 79-126, 1963).
:
10~7~;22 As indicated for instance by G.C. Schild et al.
in Brit. Med. J. 4, 127-31, 1973, antigenic variations in its surface antigens is one of the most important character-istics of the influenza~virus and a property which produces many problems for the control of influenza by vaccination.
The present invention provides a~tenuated stable in~luenza type B virus strains valuable for vaccinal use or production by a process which comprises attenuating a virulent ~ ;
influenza type B virus strain up to getting an attenuated and vaccinal influenza type B virus strain, recombining said attenuated and vaccinal strain with an influenza type B virus isolate and isolating by selective pressure a recombinant having at least one marker property of the attenuated parent strain which is not shared by the other parent strain and the antigenic composition of the other parent strain. ~-Thus, in accordance with the present teachlngs, `
a process is provided for preparing a live influenza virus ;
vaccine for nasal administration the active ingredient of which comprises at least an effective amount of an attenuated inEluenza `~
type B virus strain recombinant. The process comprises xecombining a substrate known to the art for accepting growth ~ , o~ influenza type B virus an attenuated and vaccinal strain of ln~luenæa type B virus with an influenza type B virus isolate -and isolating by selective pressure a recombinant having at least on~ ~arker property of the attenuated parent strain which is no-t shared by the other parent strain and the antigenic com- ~ -position of the other parent strain and allowing the recombinant to grow in the allantoic cavity of embryonated chicken eggs and harvesting the virus and adding thereto a stabilizer 3n and subsequently freeze-drying the mixture. ~;
More particularly, the influenza type B attenuated Ll ,
This invention relates to live influenza virus vaccines ~or nasal administration comprising as active ~
ingredient an attenuated stable type B in~luenza virus ~ -strain and, eventually, an attenuated stable type A in~luenza virus administrable by nasal route, and to a process o~
preparing said attenuated stable in~luenza virus vaccines.
Protection against viral respiratory in~ections has been shown to be related to the presence o~ a local immunity in the respiratory mucosa. This aspect has been reviewed recently by Rossen et al. (Progr. Med. Virol.
1971, 13, 194). `
Several attempts were made in recent years to ~
induce immunity against in~luenza by the nasal application `
of inactivated vaccines (see e.g. : Waldman et al. Nature, !~ `
1968, 218, 594; JAMA 1969, 207, 520; WH0 Bull~ 1969, 41, .~;
543). The results were inconsistent, however, and this was probably due to the insu~icient stimulation o~ the immunity system by the inactivated antigen (Tyrrell et al. ~`~
J. Hyg. 1970, 68, 359).
Live inPluenza type B virus vaccines are known ~ ~ `
b~t are presenting widely v`arying protection rates and ~ ;
numerous e~orts have been made e.g. by serial passages on eggs (see ~or instance A.A. Smorodintsev, Proc. Symposium on Acute respiratory Diseases, Zagreb 1969, 391-404) or by using low temperature mutants (F.M. Davenport et al.
Proc. Symposium on Llve In~luenza Vaccines, Zagreb 1971, 105-113) to reduce pathogenicity o~ the virus.
Recombination oP in~luenza~type B ~irus strains is also mentioned in the literature (see ~or instance E.D. `~
Kilbourne in Progr. Med. Virol. 5, 79-126, 1963).
:
10~7~;22 As indicated for instance by G.C. Schild et al.
in Brit. Med. J. 4, 127-31, 1973, antigenic variations in its surface antigens is one of the most important character-istics of the influenza~virus and a property which produces many problems for the control of influenza by vaccination.
The present invention provides a~tenuated stable in~luenza type B virus strains valuable for vaccinal use or production by a process which comprises attenuating a virulent ~ ;
influenza type B virus strain up to getting an attenuated and vaccinal influenza type B virus strain, recombining said attenuated and vaccinal strain with an influenza type B virus isolate and isolating by selective pressure a recombinant having at least one marker property of the attenuated parent strain which is not shared by the other parent strain and the antigenic composition of the other parent strain. ~-Thus, in accordance with the present teachlngs, `
a process is provided for preparing a live influenza virus ;
vaccine for nasal administration the active ingredient of which comprises at least an effective amount of an attenuated inEluenza `~
type B virus strain recombinant. The process comprises xecombining a substrate known to the art for accepting growth ~ , o~ influenza type B virus an attenuated and vaccinal strain of ln~luenæa type B virus with an influenza type B virus isolate -and isolating by selective pressure a recombinant having at least on~ ~arker property of the attenuated parent strain which is no-t shared by the other parent strain and the antigenic com- ~ -position of the other parent strain and allowing the recombinant to grow in the allantoic cavity of embryonated chicken eggs and harvesting the virus and adding thereto a stabilizer 3n and subsequently freeze-drying the mixture. ~;
More particularly, the influenza type B attenuated Ll ,
- 2 -I?, '''~ ;;
:~479Z~ ~
and vaccinal strain is a strain herein referred to as the :
srigit strain (deposited in the ~-merican ~ype Culture Collection, . ~:
12301 Parklawn Drive, Roc~ville Md 20852 under the deposit number ATCC VR786) and which has been obtained by passaging the -influenza s/Russia/69 strain (ATCC VR790) in the allantoic cavity of embryonated chicken eggs in the presence of pig serum up to i-isolating therefrom an attenuated and vaccinal influenza type -B virus strain which~
- is completely resistant to serum inhibitors ;;~
- has a positive AOS (allantoic on shell) marker (titration ~ :
on "allantoic membrane on shell" is described by S. Fazekas :.
de St. Groth and D. O. White in J~ Hyg. 56: 151-62, 1958) ,: ~-' :.' ' ` :
~'` ,``: ''' 2~ ' ~.
;
'~
- 2a -10479Z;~ ~ ~
- i5 antigenic with no side-e~ects on ferrets - shows positive hemagglutinin the~mosensitivity at 60 C
- shows unstable hemagglutination ~f sheep red blood cells at 30 C.
In the hereinabove process, the recombination step is carried out in any substrate known to the art ~or accepting growth o~ in~luenza type B vlrus, e.g. -~
embryonated chicken eggs material or foetal bovine kidney tissue culture, more particularly the allantoic cavity of ~ .
embryonated chicken eggs. The recombina~ion can be per~ormed with any desired type B virus isolate, more particularly a ;
new isolate such as the B/Hong Kong/5/72 (ATCC VR791) or ~
~/Hong Kong/~/73 (ATCC VR792) isolate. ~ ;
Obviously, the so-obtained recomblnant strains ;~;
may be cloned or submitted to several dilution passages without-modi~ying the essence o~ the lnvention.
Recombinants obtained accor~iny ~to this invention from the Brigit strain (ATCC VR786) with either the B/H~/5/72 (ATCC VR791)or the B/HK/R/73 (ATCC VR792) isolate have been assigned the in~luenza type B virus R 22 strain - (ATCC VR7~8), R 5 strain (ATCC VR787) and 1~ 75 strain ~ATCC V~7~9) designations respectively.
., The virus recombinants obtained by the process o~ this invention are non-pathogenic, immunogenic and valuable for in~luenza type B live virus vaccine production, ;~
using therefore any technique known to the art for live influenza virus vaccine production and/or stabilization.
~, .
ConsequentIy, the present invention also relates to X
attenuated in~luenza type B virus vaccine5 containing at least one said in~luenza type B recombinant and to the , .;~;"' :.' -. 3- -; `:
~L0479;;~
process o~ preparin~ said vaccines therefrom.
According to -this embodiment, the invention - ,~
relates to a method oP preparing an attenuated in~luenza virus vaccine comprising allowing ~m inPluenza type B virus strain as obtained by the hereinabove described process ,~
to grow in the allantolc cavity oP embryonated chicken ~ '~
eggs Por a period of time suPficient to permit growth oP a large amount of said virus, and harvesting the ' ~
resulting virus material. - ~''~,! , The so-obtained attenuated in~luenza vïrus vaccines are administered topically in the nasopharynx at an ePPective dosage unit (i.e~ at least 107EID50 ~
virus), said administration being eventually repeated iP -and when necessary. ;
For vaccinal use, the virus is preferably kept ~in Preeze-dried Porm and the vaccine is extemporane,ously ' ~, reconstituted by addition oP either water or any other ' ~''' pharmaceutical diluent or composition known to the art ~, ~ .
Por the prçparation oP nasal preparations such as drops ~.
or spray.' The vaccine Pormula may obviously include a stabilizer such as Por instance pep~one, sucrose and other ones known to the art.
~ccording to another embodiment,of the present invention the inPluenza type B virUs strain obtained by the hereinabove described process can be combined with inPluenza type A virus vaccines administrable by nasal '~
route. ~ "
The present invention thus relates to a process Eor preparing a live inPluenza vlrus vaccine Por nasal
:~479Z~ ~
and vaccinal strain is a strain herein referred to as the :
srigit strain (deposited in the ~-merican ~ype Culture Collection, . ~:
12301 Parklawn Drive, Roc~ville Md 20852 under the deposit number ATCC VR786) and which has been obtained by passaging the -influenza s/Russia/69 strain (ATCC VR790) in the allantoic cavity of embryonated chicken eggs in the presence of pig serum up to i-isolating therefrom an attenuated and vaccinal influenza type -B virus strain which~
- is completely resistant to serum inhibitors ;;~
- has a positive AOS (allantoic on shell) marker (titration ~ :
on "allantoic membrane on shell" is described by S. Fazekas :.
de St. Groth and D. O. White in J~ Hyg. 56: 151-62, 1958) ,: ~-' :.' ' ` :
~'` ,``: ''' 2~ ' ~.
;
'~
- 2a -10479Z;~ ~ ~
- i5 antigenic with no side-e~ects on ferrets - shows positive hemagglutinin the~mosensitivity at 60 C
- shows unstable hemagglutination ~f sheep red blood cells at 30 C.
In the hereinabove process, the recombination step is carried out in any substrate known to the art ~or accepting growth o~ in~luenza type B vlrus, e.g. -~
embryonated chicken eggs material or foetal bovine kidney tissue culture, more particularly the allantoic cavity of ~ .
embryonated chicken eggs. The recombina~ion can be per~ormed with any desired type B virus isolate, more particularly a ;
new isolate such as the B/Hong Kong/5/72 (ATCC VR791) or ~
~/Hong Kong/~/73 (ATCC VR792) isolate. ~ ;
Obviously, the so-obtained recomblnant strains ;~;
may be cloned or submitted to several dilution passages without-modi~ying the essence o~ the lnvention.
Recombinants obtained accor~iny ~to this invention from the Brigit strain (ATCC VR786) with either the B/H~/5/72 (ATCC VR791)or the B/HK/R/73 (ATCC VR792) isolate have been assigned the in~luenza type B virus R 22 strain - (ATCC VR7~8), R 5 strain (ATCC VR787) and 1~ 75 strain ~ATCC V~7~9) designations respectively.
., The virus recombinants obtained by the process o~ this invention are non-pathogenic, immunogenic and valuable for in~luenza type B live virus vaccine production, ;~
using therefore any technique known to the art for live influenza virus vaccine production and/or stabilization.
~, .
ConsequentIy, the present invention also relates to X
attenuated in~luenza type B virus vaccine5 containing at least one said in~luenza type B recombinant and to the , .;~;"' :.' -. 3- -; `:
~L0479;;~
process o~ preparin~ said vaccines therefrom.
According to -this embodiment, the invention - ,~
relates to a method oP preparing an attenuated in~luenza virus vaccine comprising allowing ~m inPluenza type B virus strain as obtained by the hereinabove described process ,~
to grow in the allantolc cavity oP embryonated chicken ~ '~
eggs Por a period of time suPficient to permit growth oP a large amount of said virus, and harvesting the ' ~
resulting virus material. - ~''~,! , The so-obtained attenuated in~luenza vïrus vaccines are administered topically in the nasopharynx at an ePPective dosage unit (i.e~ at least 107EID50 ~
virus), said administration being eventually repeated iP -and when necessary. ;
For vaccinal use, the virus is preferably kept ~in Preeze-dried Porm and the vaccine is extemporane,ously ' ~, reconstituted by addition oP either water or any other ' ~''' pharmaceutical diluent or composition known to the art ~, ~ .
Por the prçparation oP nasal preparations such as drops ~.
or spray.' The vaccine Pormula may obviously include a stabilizer such as Por instance pep~one, sucrose and other ones known to the art.
~ccording to another embodiment,of the present invention the inPluenza type B virUs strain obtained by the hereinabove described process can be combined with inPluenza type A virus vaccines administrable by nasal '~
route. ~ "
The present invention thus relates to a process Eor preparing a live inPluenza vlrus vaccine Por nasal
3~ administration the active lngFedient o~ whlch comprises ~ , '~ ..
_ 4 ~
-` ~o~
at least an effective amount oE an attenuated influenza -type B virus s-train recombinant, which comprises recom-bining in a substrate known to -the art for accep-ting growth o~ influenza type B virus -e.g, embryonated chicken eggs or foetal bovine kidney tissue culture and pre~erably the all antoic cavity of embryonated chicken eggs- an attenuated and vaccinal strain of influexa ;~
type B virus -e.g. influenza type B virus Brigit s-train (ATCC VR786)- with an influenza type B virus iso-late -e.g. the B/HK/5/72 (ATCC VR791) or the B/HK/8/73 ~
~ATCC VR792 isolate- isolating by selective pressure -on a recombinant -e.g. the influenza type B virus R 22 s-train (ATCC Vr788) or R 5 strain (ATCC VR787) -having at least one marker property of the attenuated parent ;
strain which is not shared by the other parent strain 1 and the antigenic composition of the other parent strain, Rllowing said recombinant to grow in -the allantoic cav-i-~y of embryona~ed chicken eggs and, if desired, adding thereto a stabilizer -e.g. peptone or sucrose- and freeæe-drying the mixture.
The -~ollowing examples illustrate -the present inven--tlo; they should not be cons-trued as limiting its s~pe.
~LX~MPLE 1 A sample of -the in-flueza B/Russis/69 s-train (AT'~C
VR 790) is submitted -to 3 terminal dilution passages on specific pathogen free eggs, for purification. A
sample of the last passage is used as seed ~or the ~;
preparatiOn of an experiméntal lot which is fuund to be free of avian pathogenic agents and sensitive to serum inhibitors and which shows residual pathogenicity ~or human beings. Dif-ferent dilutions (i.e. 10 1, 10 3, 10 5, and 10-7) o~ ~
792~
the seed virRl material in normal saline are mixed wi-th different concentration of sterile normal pig serum (undiluted, 25% and 5%) previously maintained for 15 minutes in a boiling wa-ter bath, homogenized and centrl-fuged at 2,000 rpm for 30 minutes. The supernatant is used for the further step which consists in incubating the virus/serum mixtures at 37C for one hour before inoculating 0.2 ml. aliquots of said mixture into the ~ ~`
allantoic cavity of embryonated chicken eggs previollsly incubated 8 to ll days at 37C and candled (only the surviving eggs are inoculated).
The eggs are then fur-ther incubated for a period of time varying between 24 and 96 hours.
At the end of this incubation period, the eggs~ ~
are candled and the surviving eggs are chilled at .
_ 4 ~
-` ~o~
at least an effective amount oE an attenuated influenza -type B virus s-train recombinant, which comprises recom-bining in a substrate known to -the art for accep-ting growth o~ influenza type B virus -e.g, embryonated chicken eggs or foetal bovine kidney tissue culture and pre~erably the all antoic cavity of embryonated chicken eggs- an attenuated and vaccinal strain of influexa ;~
type B virus -e.g. influenza type B virus Brigit s-train (ATCC VR786)- with an influenza type B virus iso-late -e.g. the B/HK/5/72 (ATCC VR791) or the B/HK/8/73 ~
~ATCC VR792 isolate- isolating by selective pressure -on a recombinant -e.g. the influenza type B virus R 22 s-train (ATCC Vr788) or R 5 strain (ATCC VR787) -having at least one marker property of the attenuated parent ;
strain which is not shared by the other parent strain 1 and the antigenic composition of the other parent strain, Rllowing said recombinant to grow in -the allantoic cav-i-~y of embryona~ed chicken eggs and, if desired, adding thereto a stabilizer -e.g. peptone or sucrose- and freeæe-drying the mixture.
The -~ollowing examples illustrate -the present inven--tlo; they should not be cons-trued as limiting its s~pe.
~LX~MPLE 1 A sample of -the in-flueza B/Russis/69 s-train (AT'~C
VR 790) is submitted -to 3 terminal dilution passages on specific pathogen free eggs, for purification. A
sample of the last passage is used as seed ~or the ~;
preparatiOn of an experiméntal lot which is fuund to be free of avian pathogenic agents and sensitive to serum inhibitors and which shows residual pathogenicity ~or human beings. Dif-ferent dilutions (i.e. 10 1, 10 3, 10 5, and 10-7) o~ ~
792~
the seed virRl material in normal saline are mixed wi-th different concentration of sterile normal pig serum (undiluted, 25% and 5%) previously maintained for 15 minutes in a boiling wa-ter bath, homogenized and centrl-fuged at 2,000 rpm for 30 minutes. The supernatant is used for the further step which consists in incubating the virus/serum mixtures at 37C for one hour before inoculating 0.2 ml. aliquots of said mixture into the ~ ~`
allantoic cavity of embryonated chicken eggs previollsly incubated 8 to ll days at 37C and candled (only the surviving eggs are inoculated).
The eggs are then fur-ther incubated for a period of time varying between 24 and 96 hours.
At the end of this incubation period, the eggs~ ~
are candled and the surviving eggs are chilled at .
4 C. The allantoic fluid of each series of surviving eggs is harvested separately and tested for the presence of influenza virus by the hemagglu-tination me-thod. The ~ `
harvested virus produced by the inoculum of the highest vlrus dilution in the preserce of the highest serum concentration which shows hemagglutination activity (i.e. virus d`ilution 10 and serum concentration of 25Yo ) is used for a second passage performed in the -~
same operative conditions.
A total o~ S passages are performed as described hereinabove.
The virus, harvested after 5 passages, produced by the inoculum of the highest virus dilution (i.e. ;~
) in the presence of the undiluted serum, shows hemagglutination activi-ty and is resistant to serum ~ ;
inhibitors.
Three further passages at terminal dilutlon are ;
carried out in the absence of serum to clone the ob--tained 75~2Z
resis-tant mutant and to check the stability of the .
resistant character. The virus harvested at each of these 3 passages is resistant against the inhibi-tors of normal serum. The virus at the last passage level, named "Brigit" and deposited a-t the ATCC under the deposit number Vr786, is used as inoculum for the pro-duction of a virus seed lot. ~herefore, the harves-ted ~;~
allantoic fluids are collected and pooled, sterility and safety tested, mixed with peptone to reach a ~;
final concentra-tion of 5% of peptone. A volume~ of 0.5 ml. of the viral suspension is distributed into :
3 ml. vials and freeze-dried to yield the Brigit strain - .
(ATCC VR786) lot. IN VITRO AND IN VIVO CHARACTERISTICS ~.
OF THE MODIFIED VIRUS (Bri~it strain).
1) Inhibitor resistance ..
For testing the resistance against the inhibi-tors present in normal heated animal serum (previously :. ;`
heated at 75 C for 1 hour), serial twofold dilu-tlons of the heated serums were mixed with ~ hema- .
gglutinating units of the paren-t strain and the ;.
modi~ied virus. A~ter incubation of one hour at room tempera-ture, chicken red blood cells were added ~ :
~nd -thq results recorded. ~he results show a com- .
pl~-t~ resistance ~or the seed lot and for experi-men-tal lots produced from this seed lot. In -the present specification, this character is recorded as "positive".
. :~
2) Antigenicity and absence of side e~fects ~ :
A group of 2 ferrets was inoculated nasally wi-th - 10 EID50 of the Brigit strain (ATCC VR786).
~ _ 7 ~
~ 79ZZ
The tempera-ture of the animals was recorded daily during 8 days p.i. No signif`icant temperature rise was recorded (maximum temperature 39.6 C).
A second group . . ~
, ~` :
.
, ~
' , :, ''' ,: '`
~":
- 7a -.. . , . . ~ . . . . . . . . . . .
~9L7922 of 2 ~errets wa~ inoculated nasally with 10 - 10 EID50 ~ the B/Russia/69 parent strain (AT¢C vR790).
Two days after the inocula-t:ion -the animals showed temperatures oE 40.2C. Hemagglutination-inhibition tests demostrated tha-t the modified strain induced an antigenic response in ferrets : 14 days after .
the nasal inoculation, serum antibody titers were very high among the inoculated animals (titer : . ~ :
1/256) opposed to the uninoculated control group (titer : 1/8). ~ -~
3) Ratio EIDsQ/ (AOS) IDsQ _AOS _arker) The same inoculum is titrated on embryonated eggs ..
and on the "allantoic membrane on shell" systern (titration on "allantoic membrane on shell" is described by S. Faekas de St. Groth and D.O. White in J. Hyg. 56, 151-62, 1958). The EIDso/AOS)ID
ratio for the Brigit strain (ATCC V~786):was ~ound 102~5. In the present specification, a ratio 102 5 is indicated as "positive". . .:`~ ~, 4) He~agglutination tes-t was performed on infec-tive allantoic fluid incubated ~or one hour at 60 C. .
A~-ter such treatment, Brigit strain (ATCC VR786) has ~`.
completelylost its hemagglutinating property. This I , ~hermosensitivity is herein recorded as "posi-tive". . .;
harvested virus produced by the inoculum of the highest vlrus dilution in the preserce of the highest serum concentration which shows hemagglutination activity (i.e. virus d`ilution 10 and serum concentration of 25Yo ) is used for a second passage performed in the -~
same operative conditions.
A total o~ S passages are performed as described hereinabove.
The virus, harvested after 5 passages, produced by the inoculum of the highest virus dilution (i.e. ;~
) in the presence of the undiluted serum, shows hemagglutination activi-ty and is resistant to serum ~ ;
inhibitors.
Three further passages at terminal dilutlon are ;
carried out in the absence of serum to clone the ob--tained 75~2Z
resis-tant mutant and to check the stability of the .
resistant character. The virus harvested at each of these 3 passages is resistant against the inhibi-tors of normal serum. The virus at the last passage level, named "Brigit" and deposited a-t the ATCC under the deposit number Vr786, is used as inoculum for the pro-duction of a virus seed lot. ~herefore, the harves-ted ~;~
allantoic fluids are collected and pooled, sterility and safety tested, mixed with peptone to reach a ~;
final concentra-tion of 5% of peptone. A volume~ of 0.5 ml. of the viral suspension is distributed into :
3 ml. vials and freeze-dried to yield the Brigit strain - .
(ATCC VR786) lot. IN VITRO AND IN VIVO CHARACTERISTICS ~.
OF THE MODIFIED VIRUS (Bri~it strain).
1) Inhibitor resistance ..
For testing the resistance against the inhibi-tors present in normal heated animal serum (previously :. ;`
heated at 75 C for 1 hour), serial twofold dilu-tlons of the heated serums were mixed with ~ hema- .
gglutinating units of the paren-t strain and the ;.
modi~ied virus. A~ter incubation of one hour at room tempera-ture, chicken red blood cells were added ~ :
~nd -thq results recorded. ~he results show a com- .
pl~-t~ resistance ~or the seed lot and for experi-men-tal lots produced from this seed lot. In -the present specification, this character is recorded as "positive".
. :~
2) Antigenicity and absence of side e~fects ~ :
A group of 2 ferrets was inoculated nasally wi-th - 10 EID50 of the Brigit strain (ATCC VR786).
~ _ 7 ~
~ 79ZZ
The tempera-ture of the animals was recorded daily during 8 days p.i. No signif`icant temperature rise was recorded (maximum temperature 39.6 C).
A second group . . ~
, ~` :
.
, ~
' , :, ''' ,: '`
~":
- 7a -.. . , . . ~ . . . . . . . . . . .
~9L7922 of 2 ~errets wa~ inoculated nasally with 10 - 10 EID50 ~ the B/Russia/69 parent strain (AT¢C vR790).
Two days after the inocula-t:ion -the animals showed temperatures oE 40.2C. Hemagglutination-inhibition tests demostrated tha-t the modified strain induced an antigenic response in ferrets : 14 days after .
the nasal inoculation, serum antibody titers were very high among the inoculated animals (titer : . ~ :
1/256) opposed to the uninoculated control group (titer : 1/8). ~ -~
3) Ratio EIDsQ/ (AOS) IDsQ _AOS _arker) The same inoculum is titrated on embryonated eggs ..
and on the "allantoic membrane on shell" systern (titration on "allantoic membrane on shell" is described by S. Faekas de St. Groth and D.O. White in J. Hyg. 56, 151-62, 1958). The EIDso/AOS)ID
ratio for the Brigit strain (ATCC V~786):was ~ound 102~5. In the present specification, a ratio 102 5 is indicated as "positive". . .:`~ ~, 4) He~agglutination tes-t was performed on infec-tive allantoic fluid incubated ~or one hour at 60 C. .
A~-ter such treatment, Brigit strain (ATCC VR786) has ~`.
completelylost its hemagglutinating property. This I , ~hermosensitivity is herein recorded as "posi-tive". . .;
5) Hemagglutination test wi_h sheep red blood cells a-t _ _ _ ' ~;' ~ .:
An hemagglutination test was performed,with sheep red blood cells at 4 and 30 C. The hemagglutination ..
pattern at 4 C eas normal whereas at 30 C there was .
a rapid elution so -that no clear cut hemagglu-tina- ~
-tion was observed. When examined 20 hrs. after .~.
initia-tion of the test, the hemagglutination has , completely disappeared. This .~ .
~4~92;2 :
character is herein referred to as "unstable".
A sample of influenza type B virus strain B/HK/5/72 (ATCC VR791) the hemagglutinin of which is difEerent from that of the Brigit strain (~TCC VR786) is inocula-ted in the allantoic cavity of lO to ll day-old embryo nated eggs (inoculum : 0.2 ml./egg), and submitted to three passages, in order to obtain a substantial amount of virus. At the end of the third passage, a viral suspension is obtained, the titer of which is lO EID50 ;
/ml. Aliquots (0.2 ml.) of the undiluted suspension are inoculated in the allantoic cavity of SPF (specific pathogen free) embryonated eggs; after incubation for 2 hrs. at 33C, aliquots (0.2 ml.) of a suspension of Brigit strain (ATCC VR 786), having a titer of 10 ~
EID50/ml., are inoculated in the same eggs. The eggs ~ ~-are then incubated for 16 hrs. at 33 C and the allan-toic fluids are harves-ted.
Aliquots (o.25 ml.) of anit-Brigit strain sero-type rabbit serum pre~iously heated for 1 hr. at 56 C, ~:
are mixed with the same volume of normal serum. Ali-quots (0.25 ml.~ o~ -this serum mixture are added to the hArves-ted allantoic ~luids. This mixture is then main-tained for 1 hr. at 37C. Embryonated eggs are ln~6ula-ted wi-th 0.2 ml. of the incuba-ted mixture (2 eggs :~or each mix-ture) and incubated for 48 hrs. at 33 C. The allantoic fluids of each inoculated egg are harvested, passaged in embryonated eggs (0.2 ml. per egg) and incubated for 72 hrs. at 33 C, using two eggs for each sample. After the 72 hrs. incubation period, the positive allantoic fluids are harvested checked for _ g _ ~47922 - hemagglutination o~ sheep red blood cells - serotype against anti-Brigit serum and against anti-B/HK/
5/72 serum - resis~ance to non-speci~ic serum inhibitors - growth in eggs and on AOS (allantolc on shell).
One o~ the so~obtained viral suspensions which, ~`
as indicated in the Pollowing Table I, has the serotype oP
one parent (B/HK/5/72) and the infectivity pattern in the AOS system and the serum inhibitor marker oP Brigit strain is assigned the "R 22" designation (ATCC VR788) and submitted to 2 passayes on embryonated chicken eggs to obtain a substantial amount o~ R 22 recombinant virus and the harvested ailantoïc Pluid is supplemented with peptone up to a Pinal concentration oP 5 % and ~reeze-dried.
TABLE I ;
Serotype inhi- EID hemagglu- 30 C sheep Strain ~ri- B/HK/ rbeisti5r 50/ tinin cells hemag-glt 5/72 tance (AoS~ID50 sitivity glutination _ _ _ . ~ ,.
Brigit + _ + + +unstable . B/HK/5/72 _ + _ . _stable ~ ;~
20 Recom-R 22 , + + ~ _stable (ATCC VR _ . _ ~
' ~ .: ~' . ;
. '. ,' ~ ' Equal volumes oP a suspension oP the inPluenza type B "Brigit" strain (ATCC VR786) ànd oP the B/Hong Kong/
8j73 strain (ATCC VR792) ( the titer o~ each being 107 5 EID50/ml.) are mixed and preincubated Por 72 hrs. at 4 C.
The mixture is then inoculated in the allantoic cavity oP
two SPF embryonated eggs (0.2 ml. per egg). After an ~
- 10 - ~;
' `' ~, ~' .. .. -. -.. , . - .. , . - .. ,.. - , .. .. , i~ - . . , ~, . .
~4~47~Z
incubation period of 16 hrs. at 33 C, the allantoic fluids are harvested and sonicated (30 sec.).
The harves-ted fluids undiluted and diluted to 10 and 10 2 are allowed to react for one hr. at 37 C with different dilutions of an-ti-Brigit strain serotype , rabbit serum perviously heated at 56 C for 30 min. ~
The resulting mixtures are inoculated on allantoic on ~ -shell system and incubated for 72 hrs. at 37 C. The samples o-f the last positive virus dilu-tion (lO ) are numbered 1 to 7.
Infectivity titers and neutralization by anit-Brigit serum in the AOS system are then compared for the 7 samples and for the two parental strains. ;;
The samples which are not neutralized by anti-Brigit serum and which exhibit an (AOS)ID50 titers at least equal to that of Brigit strain (ATCC VR786) are harvested at the highest positive dilution.
Theee individual harvests are duluted to 10 and passaged once in SPF eggs.
One oE these viral suspensions showing the sero- ,~
type and hemagglutinin thermosensitivity of B/Hong ~ong/ 8/73 strain and the EID50/(AOS)ID50 ratio of arlgi-t strain is assigned the "R 5" designation (ATCC
VR787,) In the ~ollowing Table II -the characteris-tias of recombinan-t R 5 are summarized and compared to those o-f the Brigit (ATCC VR786) and B/HK/8/73 (A~CC VE792) parents.
;
:'; ' .. , . : : :. , ~
~ID4792;~ ~
TABLE II
_ . _ _ Seroty~flK EID50/ hemagglutinin Strain Brigit 8/73 (AOS)ID50 thermosensitivity . __ _ . _ Brigit + _ B/HK/8/73 ~ + _ Recombinant R 5 + _ (ATGC VR787) _ ,' , The R 5 recombinant is submitted to two limit dilution passages (in SPF embryonated chicken eggs) to eliminate any eventual avian adventitious agent and to one further passage to obtain a substantial amount o~ R 5 recombinant virus. The harvested allantoïc fluid is supple-mented with peptone up to a inal concentration o~ 5 /0 and freeze-dried.
Starting ~rom the ~reeze-dried materials obtained in Example 1 (i.e. the Brigit strain, ATCC VR786) as seed lot or large scale vaccine production, a Purther passage is carried out in the allantoïc ~luid o~ another set o~
embryonated chicken eggs with are incubated at 35 C
or 3 days.
The allantoïc Pluids are harvested, pooled, sterility and safety tested, mixed with peptone as stabi-lizer in order to reach a inal concentration o 5 % o peptone and dis-tributed into 3 ml. glass vials in order to obtain a dosage unit (i~e. at least 107 EID50) ~ virus. ;~
The product is then freeze-dried and the vials are tightly stoppered.
This passage level is used as vaccine batch. For vaccine administration, the contents of one vial is ,' "~
~479~Z
reconstituted by adding 0.5 ml. of water or saline or a 5% sucrose solution and administered as drops in the nostrils.
Alternatively, the allantoic ~luids peptone prepar-ation i5 dis-tributed in larger glass vials in order to obtain integers of the dosage unit amount of virus to constitute corresponding mult-doses vaccine preparations.
Vaccination with the attenuated type B (Brigit strain) `~
influenza virus.
Clinical trials were performed in 46 volunteers having a prevaccination HI (hemagglutination inhibi-tion) titer inferior or equal to 32 against influenza B virus. Each volunteer received two in-tranasal admin-istrations with a 8 or 14 day interval. At each admin~
istration, subjects were given a virus concentration of EID of Brigit strain (in each nostril 5 drops of the extemporaneously reconstituted vaccine in 5% sucrose solution inl:water). Seroconversion was observed in 40 sub~ects (i.e. 87%); only mild clinical r-eactions (i.e. ~;
rhinorrhea or stuffy nose for l to 2 days~ were observed In the trials were control groups were available, not: ~ `
~ransmission of influenza vaccine virus was observed.
Vlrua isolatlon was attempted from nasal and throat awabs but the samples remained negative a~-ter -two blind pasaages in embryonated eggs.
EX~MPLE-5 Starting from the freeze-dried R 22 s-train (ATCC
VR788 obtained in example 2 as seed lot for large scale vaccine production, a further passage is carried out in the allantoic fluid of another set of embryonated chic-lcen eggs which are incubated at 35 C for 3 days.
`~' _ 13 -3L0gL73;Z2 The allantoïc fluids are harvested, pooled, sterility and safety tested, mixed with peptone as stabi-lizer in order to reach a ~inal concentration oP 5 % oP
peptone and distributed into 3 ml. glass vials in order to ~
obtain a dosage unit (i.e. at least 107F,ID50 ~ virus)~ ~ `
The product is Preeze-dried and the vials sealed or tightly stoppered.
This passage 1evel is used as vaccine batch. For vaccine administration, the contents of one vial is recons-tituted by adding 0.5 ml. of water or saline or a 5 /0 sucrose ~ ;
solution and administered as drops in the nostrils.
Alternatively, the allantoic fluids/peptone prepa-ration is distributed in larger glass vials in order to obtain integers of the dosage unit amount o~ virus to constitute corresponding multi-doses vaccine preparations. "
Vaccination with the attenuated type B influ za vaccine R 22 strain. " ;
Material and methods. ~ ~
__ _________________ , ~ .
Six volunteers haviny an HI antibody titer inferior or equal to 32 were selected ~or the trial. Two subjects were taken as control. The 6 subjects received, with a ~ .
11 to 14 day interval, two administrations of the extempo- `
raneously reconstituted vaccine, R 22 strain~ ~or each adminis-tra-tion, all subjects received 5 drops o~ the vaccine per nostril (107'5EID50).
Blood samples for antibody determinatlon were ta`ken on vaccination day and on days 10 to 14 af-ter the first inoculation and on day 14 after the second administration. ;~
Nasal washings for local antibody determination were taken ~;~
on day 14 a~ter the second inoculation.
''' ` ~
- 14 - ~
, 7~
Clinlcal sym~toms.
No symp~omS were recorded.
Virus isolation.
________________ Nasal swabs were collected on day 3 and 6 in the six subjects. All s~lples remained nega-tive a~ter two blind passages in embryonated eggs.
Serological results and local antibodies.
______ __________________________________ Five out of 5iX volunteers developéd either a serum or nasal response or both.
The ~;esultsare summarized ln the following Table III.
TABLE III
., ~ . ~
Ser~lm titer Serum titer local local Sub ect (HI) before (HI) after antibodies antibodies J vacc:ination 2nd bePore after 2nd vaccination vaccination vaccination (SN) (SN) , __ . _ ~
1 32 32 ~2 24 2 8 8 <2 2-4 3 32 64 ~2 12 4 8 32 <2 ~2 <8 <8 ND ND
An hemagglutination test was performed,with sheep red blood cells at 4 and 30 C. The hemagglutination ..
pattern at 4 C eas normal whereas at 30 C there was .
a rapid elution so -that no clear cut hemagglu-tina- ~
-tion was observed. When examined 20 hrs. after .~.
initia-tion of the test, the hemagglutination has , completely disappeared. This .~ .
~4~92;2 :
character is herein referred to as "unstable".
A sample of influenza type B virus strain B/HK/5/72 (ATCC VR791) the hemagglutinin of which is difEerent from that of the Brigit strain (~TCC VR786) is inocula-ted in the allantoic cavity of lO to ll day-old embryo nated eggs (inoculum : 0.2 ml./egg), and submitted to three passages, in order to obtain a substantial amount of virus. At the end of the third passage, a viral suspension is obtained, the titer of which is lO EID50 ;
/ml. Aliquots (0.2 ml.) of the undiluted suspension are inoculated in the allantoic cavity of SPF (specific pathogen free) embryonated eggs; after incubation for 2 hrs. at 33C, aliquots (0.2 ml.) of a suspension of Brigit strain (ATCC VR 786), having a titer of 10 ~
EID50/ml., are inoculated in the same eggs. The eggs ~ ~-are then incubated for 16 hrs. at 33 C and the allan-toic fluids are harves-ted.
Aliquots (o.25 ml.) of anit-Brigit strain sero-type rabbit serum pre~iously heated for 1 hr. at 56 C, ~:
are mixed with the same volume of normal serum. Ali-quots (0.25 ml.~ o~ -this serum mixture are added to the hArves-ted allantoic ~luids. This mixture is then main-tained for 1 hr. at 37C. Embryonated eggs are ln~6ula-ted wi-th 0.2 ml. of the incuba-ted mixture (2 eggs :~or each mix-ture) and incubated for 48 hrs. at 33 C. The allantoic fluids of each inoculated egg are harvested, passaged in embryonated eggs (0.2 ml. per egg) and incubated for 72 hrs. at 33 C, using two eggs for each sample. After the 72 hrs. incubation period, the positive allantoic fluids are harvested checked for _ g _ ~47922 - hemagglutination o~ sheep red blood cells - serotype against anti-Brigit serum and against anti-B/HK/
5/72 serum - resis~ance to non-speci~ic serum inhibitors - growth in eggs and on AOS (allantolc on shell).
One o~ the so~obtained viral suspensions which, ~`
as indicated in the Pollowing Table I, has the serotype oP
one parent (B/HK/5/72) and the infectivity pattern in the AOS system and the serum inhibitor marker oP Brigit strain is assigned the "R 22" designation (ATCC VR788) and submitted to 2 passayes on embryonated chicken eggs to obtain a substantial amount o~ R 22 recombinant virus and the harvested ailantoïc Pluid is supplemented with peptone up to a Pinal concentration oP 5 % and ~reeze-dried.
TABLE I ;
Serotype inhi- EID hemagglu- 30 C sheep Strain ~ri- B/HK/ rbeisti5r 50/ tinin cells hemag-glt 5/72 tance (AoS~ID50 sitivity glutination _ _ _ . ~ ,.
Brigit + _ + + +unstable . B/HK/5/72 _ + _ . _stable ~ ;~
20 Recom-R 22 , + + ~ _stable (ATCC VR _ . _ ~
' ~ .: ~' . ;
. '. ,' ~ ' Equal volumes oP a suspension oP the inPluenza type B "Brigit" strain (ATCC VR786) ànd oP the B/Hong Kong/
8j73 strain (ATCC VR792) ( the titer o~ each being 107 5 EID50/ml.) are mixed and preincubated Por 72 hrs. at 4 C.
The mixture is then inoculated in the allantoic cavity oP
two SPF embryonated eggs (0.2 ml. per egg). After an ~
- 10 - ~;
' `' ~, ~' .. .. -. -.. , . - .. , . - .. ,.. - , .. .. , i~ - . . , ~, . .
~4~47~Z
incubation period of 16 hrs. at 33 C, the allantoic fluids are harvested and sonicated (30 sec.).
The harves-ted fluids undiluted and diluted to 10 and 10 2 are allowed to react for one hr. at 37 C with different dilutions of an-ti-Brigit strain serotype , rabbit serum perviously heated at 56 C for 30 min. ~
The resulting mixtures are inoculated on allantoic on ~ -shell system and incubated for 72 hrs. at 37 C. The samples o-f the last positive virus dilu-tion (lO ) are numbered 1 to 7.
Infectivity titers and neutralization by anit-Brigit serum in the AOS system are then compared for the 7 samples and for the two parental strains. ;;
The samples which are not neutralized by anti-Brigit serum and which exhibit an (AOS)ID50 titers at least equal to that of Brigit strain (ATCC VR786) are harvested at the highest positive dilution.
Theee individual harvests are duluted to 10 and passaged once in SPF eggs.
One oE these viral suspensions showing the sero- ,~
type and hemagglutinin thermosensitivity of B/Hong ~ong/ 8/73 strain and the EID50/(AOS)ID50 ratio of arlgi-t strain is assigned the "R 5" designation (ATCC
VR787,) In the ~ollowing Table II -the characteris-tias of recombinan-t R 5 are summarized and compared to those o-f the Brigit (ATCC VR786) and B/HK/8/73 (A~CC VE792) parents.
;
:'; ' .. , . : : :. , ~
~ID4792;~ ~
TABLE II
_ . _ _ Seroty~flK EID50/ hemagglutinin Strain Brigit 8/73 (AOS)ID50 thermosensitivity . __ _ . _ Brigit + _ B/HK/8/73 ~ + _ Recombinant R 5 + _ (ATGC VR787) _ ,' , The R 5 recombinant is submitted to two limit dilution passages (in SPF embryonated chicken eggs) to eliminate any eventual avian adventitious agent and to one further passage to obtain a substantial amount o~ R 5 recombinant virus. The harvested allantoïc fluid is supple-mented with peptone up to a inal concentration o~ 5 /0 and freeze-dried.
Starting ~rom the ~reeze-dried materials obtained in Example 1 (i.e. the Brigit strain, ATCC VR786) as seed lot or large scale vaccine production, a Purther passage is carried out in the allantoïc ~luid o~ another set o~
embryonated chicken eggs with are incubated at 35 C
or 3 days.
The allantoïc Pluids are harvested, pooled, sterility and safety tested, mixed with peptone as stabi-lizer in order to reach a inal concentration o 5 % o peptone and dis-tributed into 3 ml. glass vials in order to obtain a dosage unit (i~e. at least 107 EID50) ~ virus. ;~
The product is then freeze-dried and the vials are tightly stoppered.
This passage level is used as vaccine batch. For vaccine administration, the contents of one vial is ,' "~
~479~Z
reconstituted by adding 0.5 ml. of water or saline or a 5% sucrose solution and administered as drops in the nostrils.
Alternatively, the allantoic ~luids peptone prepar-ation i5 dis-tributed in larger glass vials in order to obtain integers of the dosage unit amount of virus to constitute corresponding mult-doses vaccine preparations.
Vaccination with the attenuated type B (Brigit strain) `~
influenza virus.
Clinical trials were performed in 46 volunteers having a prevaccination HI (hemagglutination inhibi-tion) titer inferior or equal to 32 against influenza B virus. Each volunteer received two in-tranasal admin-istrations with a 8 or 14 day interval. At each admin~
istration, subjects were given a virus concentration of EID of Brigit strain (in each nostril 5 drops of the extemporaneously reconstituted vaccine in 5% sucrose solution inl:water). Seroconversion was observed in 40 sub~ects (i.e. 87%); only mild clinical r-eactions (i.e. ~;
rhinorrhea or stuffy nose for l to 2 days~ were observed In the trials were control groups were available, not: ~ `
~ransmission of influenza vaccine virus was observed.
Vlrua isolatlon was attempted from nasal and throat awabs but the samples remained negative a~-ter -two blind pasaages in embryonated eggs.
EX~MPLE-5 Starting from the freeze-dried R 22 s-train (ATCC
VR788 obtained in example 2 as seed lot for large scale vaccine production, a further passage is carried out in the allantoic fluid of another set of embryonated chic-lcen eggs which are incubated at 35 C for 3 days.
`~' _ 13 -3L0gL73;Z2 The allantoïc fluids are harvested, pooled, sterility and safety tested, mixed with peptone as stabi-lizer in order to reach a ~inal concentration oP 5 % oP
peptone and distributed into 3 ml. glass vials in order to ~
obtain a dosage unit (i.e. at least 107F,ID50 ~ virus)~ ~ `
The product is Preeze-dried and the vials sealed or tightly stoppered.
This passage 1evel is used as vaccine batch. For vaccine administration, the contents of one vial is recons-tituted by adding 0.5 ml. of water or saline or a 5 /0 sucrose ~ ;
solution and administered as drops in the nostrils.
Alternatively, the allantoic fluids/peptone prepa-ration is distributed in larger glass vials in order to obtain integers of the dosage unit amount o~ virus to constitute corresponding multi-doses vaccine preparations. "
Vaccination with the attenuated type B influ za vaccine R 22 strain. " ;
Material and methods. ~ ~
__ _________________ , ~ .
Six volunteers haviny an HI antibody titer inferior or equal to 32 were selected ~or the trial. Two subjects were taken as control. The 6 subjects received, with a ~ .
11 to 14 day interval, two administrations of the extempo- `
raneously reconstituted vaccine, R 22 strain~ ~or each adminis-tra-tion, all subjects received 5 drops o~ the vaccine per nostril (107'5EID50).
Blood samples for antibody determinatlon were ta`ken on vaccination day and on days 10 to 14 af-ter the first inoculation and on day 14 after the second administration. ;~
Nasal washings for local antibody determination were taken ~;~
on day 14 a~ter the second inoculation.
''' ` ~
- 14 - ~
, 7~
Clinlcal sym~toms.
No symp~omS were recorded.
Virus isolation.
________________ Nasal swabs were collected on day 3 and 6 in the six subjects. All s~lples remained nega-tive a~ter two blind passages in embryonated eggs.
Serological results and local antibodies.
______ __________________________________ Five out of 5iX volunteers developéd either a serum or nasal response or both.
The ~;esultsare summarized ln the following Table III.
TABLE III
., ~ . ~
Ser~lm titer Serum titer local local Sub ect (HI) before (HI) after antibodies antibodies J vacc:ination 2nd bePore after 2nd vaccination vaccination vaccination (SN) (SN) , __ . _ ~
1 32 32 ~2 24 2 8 8 <2 2-4 3 32 64 ~2 12 4 8 32 <2 ~2 <8 <8 ND ND
6 <8 16 ND ND
. _ _ ~0 ND = not determined.
Starting Prom the Preeze-dried material obtained in ~x~mple 3 (R 5 strain,ATCC VR787)as seed lot Por large scale vaccine~
production, a ~urther passage is carried out in the allantoic ~lUid oE anothèr set o~ embryonated chlcken eggs which are incubated at 35 C ~or 3 days.
The allantoic ~luids are harvested, pooled, sterility and saPety tested, mixed with peptone as stabilizer in order to reach a final concentration~oP 5 ~0 o~ peptone and distributed into 3 ml. glass vials in order to obtain a dosage unit ' ~L047922 (i.e. at least 107 EID50) Ç virus. The mixture is then ;~
Preeze-dried and the vials sealed or tightly stoppered.
This passage level is used as vaccine batch. For `
vaccine administration, the contents oP one vial is reconsti- ~ ;
tuted by adding 0.5 ml. oP water or saline or a 5 % sucrose solution and administered as drops in the nostrils.
Alternatively, the allantolc fluid / peptone prepa-ration is distributed in larger glass vials in order to obtain integers oP the dosage unit amount oP virus to constitute corresponding multi-doses vaccine preparations.
EXAMPLE 7 -~
: .
To one dosage unit of the ~reeze-dried inPluenza type B
virus vaccine (Brigit strain,ATCC VR786)prepared as indi~ated in example 4, there is added one dosage unit of live atbenuated type A influenza virus (Alice strain,ATCC VR776)in 0.5ml. oP a 5%
sucrose solutlon in water. The so-obtained bivalent vaccine is administered as drops in the nostrils and the administration is repeated 14 days later.
Clin-ical trials were perPormed in 22 volunteers ~`
having a prevaccination HI (hemagglutination inhibition) titer inPerior or equal to 64 against inPluenza A or B virus. Each volunteer received two nasal administrations (107-5 EID50 oP the Brigit strain and 107-5 EID50 P the Alice strain) with a ~ to 14 day interval.
Seroconversion was obsèrved in 80 % oP the subjects against in~luenza type A virus and 73 % against inPluenza type B virus.
- Only mild clinical reactions were observed. No transmission oP ~nPluenza vaccine virus was observed.
EXAMR~E 8 To one dosag~ unit ClQ7 5 EID~ Qf the freeze-dried influenza type B virus vaccine CR 22 strain, ATCC VR7881 prepared as indicated in axample 5, there i~ added one dosage unit (lQ7-5 EID50~ of li~e attenuated type A influenza virus (Alice strain, ATCC V~7761 in Q.5 ml. of a 5~ sucrose solution.
The so-obtained hivalen~ vaccine is administered as drops in the nostrils and the administration is repeated 14 days later. Seroconversion for both oomponents (type B and type A~
1~ is observed 16 days after the second administration.
A sample of influenza type B viru~ ~train B/HK~5~72 (ATCC VR 791~ the hemagglutinin of which is different from that of the Brigit strain, is inoculated in the allantoic cavity of 10 to 11 day-old embryonated chicken eggs (inoculum : ~.2 ml.~egg~, and submitted to three passages, in order to obtain a substantial amount of virus. At the end of the third passage, a viral suspension is obtained, the titer of which is 10~ EID5~/ml. Aliquots (0.2 ml.) of the undiluted suspension 24 are inoculated in the allantoic cavity of SPF (specific pathogen free) embryonated eggs; after incubation for 2 hrs. at 33 C, aliquots (a.2 ml.~ of a suspension of Brigit strain (o~tained i~ exa~ple 1I havin~ a titer of la7-5 EID 5~/ml., are inoculated in the same eggs. The eggs are then incu~ated for 16hrs. at 33 C and the allantoic fluids are harvested.
Aliquots (0.25 ml.~ of anti-Brigit strain serotype rabbit serum previously heated for one hour at 5G C are mixed with the same volume of normal serum. Aliquots (0.25 ml.) of this serum mixture are added to the harvested allantoic fluids. ;
3a The mixture is then maintained for one hour at 37C. SPF
embryonated eggs are inao~lated ~ith Q.2 ml. of the mixture and :
-- . . . . ~ . .
~ 4792Z
incubated for 4~ hrs. at 33 C. The allantoïc fIuids of each incubated egg are harvested, passaged in SPF embryonated eggs (0.2 ml. per egg) and incubated ~or 72 hours at 33 C, using :
two eggs per sample. A.~ter the 72 hours incubation period, the S positive allantoic .~luids are harvested and checked ~or : ~
- hemagglutination of sheep red blood cells ~.
- serotype against anti-Brigit serum and against anti-B/HK/5/72 ~ :
serum - growth in eggs and~on AOS (allantoic on shell) One o~ t~e so-obtained viral suspensio~s showing thesero- ~
type of the B/HK/72 parent and the infectivity pattern oP the . .
Brigit strain parent in the AOS system is named R 75 (ATCC VR789).
Recombinant R 75 is submitted to two passages in ::~
SPF embryonated chicken eggs to obtain a substantial amount 15 o~ R 75 recombinant. ~`
In the following Table IV, the characteristics of the recombinant are summarized and compared to those of the Bri.git .
and B/HK/5/72 parents.
TABLE IV
_ . _ . ' ' : i Serotype EID / Hemagglu- 30 C sheep .
Strain Brit- B~HK/ ( ) thermosen- red blood ~;
gl 5 7 sitivity gl.utination .: .
, ~___ ,.. .
Brigit .+ _ + ` + unstable ~/HK/5/72 _ + ._ _ stable .
25 Rccombinant _ _ . ` stable -:
~, The pooled allantoic ~luids obtained in example 9 .:
are sterility and safety tested,mixed with peptone as stabilizer in order to reach a ~inal concentration of 5 % of peptone and distributed into 3 ml. glass vials in order to obtain a dosage ~ ;
- 18 - : `
;. ,.
, '' , ~479Z;~ ~ .
unit (i.e. at least 1'07EID50) ~ virus.The mixture is then Preeze-dried and the~vials sealed or tightly stoppered.
This passage level is used as a vaccine batch. For vaccine administration, the conten-ts o~ one vial is reconstituted, by adding 0~5 ml. oP water or saline or a 5 % sucrose solution and administered as drops in the nostrils. ~ , Clinical trials were perPormed in 24 volunteers.
Each volunteer received with a 11 to 14 day interval two nasal ~, administrations of the extemporaneously reconstituted vaccine, R 75 strain (Al'CC VR789}. At each administration, subjects were given a~virus concentration oP 107-8EID50 of the recons~
tituted vaccine in saline solution (5 drops in each nostrils).
The serological results are summari~ed in the following table V.
TABLE V
_ ~
Serum titer (HI)bePore Serum titer (HI) aPter Subject vaccination 2nd. vaccination I ~ 256 256 27 ¦ < I ~ C
~5 ~I A :
, 79Z;~
Seroconversion (fourfold increase o~ ~I titer) was observed in 75 % of the subject showing an prevaccinal HI
titer ~16.
Suspensions o~ the inPluenza type B R 75 strain (ATCC VR789) and o~ the in~luenza type A ~Alice~ strain ~
(ATCC VR 776) are mixed, peptone is added as ~-stabilizer in order to reach a Pinal concentration oE 5 %
o~ peptone and the mixture is distributed into 3 ml.
glass vials in order to contain at least 107EID50 o~ each virus. The mixture is ~reeze-dried and the vials sealed or tightly stoppered. ~or vaccine administration, the contents o~ one vial is reconstituted by adding 0.5 ml. o~ water or saline or a 5 % sucrose solution ~ ~
15 and administered as drops in the nostrils. `
~ `~
, .'.
'. ,,;'~'..~.'.
'' ":-, ,' - 20 ~
~ :''
. _ _ ~0 ND = not determined.
Starting Prom the Preeze-dried material obtained in ~x~mple 3 (R 5 strain,ATCC VR787)as seed lot Por large scale vaccine~
production, a ~urther passage is carried out in the allantoic ~lUid oE anothèr set o~ embryonated chlcken eggs which are incubated at 35 C ~or 3 days.
The allantoic ~luids are harvested, pooled, sterility and saPety tested, mixed with peptone as stabilizer in order to reach a final concentration~oP 5 ~0 o~ peptone and distributed into 3 ml. glass vials in order to obtain a dosage unit ' ~L047922 (i.e. at least 107 EID50) Ç virus. The mixture is then ;~
Preeze-dried and the vials sealed or tightly stoppered.
This passage level is used as vaccine batch. For `
vaccine administration, the contents oP one vial is reconsti- ~ ;
tuted by adding 0.5 ml. oP water or saline or a 5 % sucrose solution and administered as drops in the nostrils.
Alternatively, the allantolc fluid / peptone prepa-ration is distributed in larger glass vials in order to obtain integers oP the dosage unit amount oP virus to constitute corresponding multi-doses vaccine preparations.
EXAMPLE 7 -~
: .
To one dosage unit of the ~reeze-dried inPluenza type B
virus vaccine (Brigit strain,ATCC VR786)prepared as indi~ated in example 4, there is added one dosage unit of live atbenuated type A influenza virus (Alice strain,ATCC VR776)in 0.5ml. oP a 5%
sucrose solutlon in water. The so-obtained bivalent vaccine is administered as drops in the nostrils and the administration is repeated 14 days later.
Clin-ical trials were perPormed in 22 volunteers ~`
having a prevaccination HI (hemagglutination inhibition) titer inPerior or equal to 64 against inPluenza A or B virus. Each volunteer received two nasal administrations (107-5 EID50 oP the Brigit strain and 107-5 EID50 P the Alice strain) with a ~ to 14 day interval.
Seroconversion was obsèrved in 80 % oP the subjects against in~luenza type A virus and 73 % against inPluenza type B virus.
- Only mild clinical reactions were observed. No transmission oP ~nPluenza vaccine virus was observed.
EXAMR~E 8 To one dosag~ unit ClQ7 5 EID~ Qf the freeze-dried influenza type B virus vaccine CR 22 strain, ATCC VR7881 prepared as indicated in axample 5, there i~ added one dosage unit (lQ7-5 EID50~ of li~e attenuated type A influenza virus (Alice strain, ATCC V~7761 in Q.5 ml. of a 5~ sucrose solution.
The so-obtained hivalen~ vaccine is administered as drops in the nostrils and the administration is repeated 14 days later. Seroconversion for both oomponents (type B and type A~
1~ is observed 16 days after the second administration.
A sample of influenza type B viru~ ~train B/HK~5~72 (ATCC VR 791~ the hemagglutinin of which is different from that of the Brigit strain, is inoculated in the allantoic cavity of 10 to 11 day-old embryonated chicken eggs (inoculum : ~.2 ml.~egg~, and submitted to three passages, in order to obtain a substantial amount of virus. At the end of the third passage, a viral suspension is obtained, the titer of which is 10~ EID5~/ml. Aliquots (0.2 ml.) of the undiluted suspension 24 are inoculated in the allantoic cavity of SPF (specific pathogen free) embryonated eggs; after incubation for 2 hrs. at 33 C, aliquots (a.2 ml.~ of a suspension of Brigit strain (o~tained i~ exa~ple 1I havin~ a titer of la7-5 EID 5~/ml., are inoculated in the same eggs. The eggs are then incu~ated for 16hrs. at 33 C and the allantoic fluids are harvested.
Aliquots (0.25 ml.~ of anti-Brigit strain serotype rabbit serum previously heated for one hour at 5G C are mixed with the same volume of normal serum. Aliquots (0.25 ml.) of this serum mixture are added to the harvested allantoic fluids. ;
3a The mixture is then maintained for one hour at 37C. SPF
embryonated eggs are inao~lated ~ith Q.2 ml. of the mixture and :
-- . . . . ~ . .
~ 4792Z
incubated for 4~ hrs. at 33 C. The allantoïc fIuids of each incubated egg are harvested, passaged in SPF embryonated eggs (0.2 ml. per egg) and incubated ~or 72 hours at 33 C, using :
two eggs per sample. A.~ter the 72 hours incubation period, the S positive allantoic .~luids are harvested and checked ~or : ~
- hemagglutination of sheep red blood cells ~.
- serotype against anti-Brigit serum and against anti-B/HK/5/72 ~ :
serum - growth in eggs and~on AOS (allantoic on shell) One o~ t~e so-obtained viral suspensio~s showing thesero- ~
type of the B/HK/72 parent and the infectivity pattern oP the . .
Brigit strain parent in the AOS system is named R 75 (ATCC VR789).
Recombinant R 75 is submitted to two passages in ::~
SPF embryonated chicken eggs to obtain a substantial amount 15 o~ R 75 recombinant. ~`
In the following Table IV, the characteristics of the recombinant are summarized and compared to those of the Bri.git .
and B/HK/5/72 parents.
TABLE IV
_ . _ . ' ' : i Serotype EID / Hemagglu- 30 C sheep .
Strain Brit- B~HK/ ( ) thermosen- red blood ~;
gl 5 7 sitivity gl.utination .: .
, ~___ ,.. .
Brigit .+ _ + ` + unstable ~/HK/5/72 _ + ._ _ stable .
25 Rccombinant _ _ . ` stable -:
~, The pooled allantoic ~luids obtained in example 9 .:
are sterility and safety tested,mixed with peptone as stabilizer in order to reach a ~inal concentration of 5 % of peptone and distributed into 3 ml. glass vials in order to obtain a dosage ~ ;
- 18 - : `
;. ,.
, '' , ~479Z;~ ~ .
unit (i.e. at least 1'07EID50) ~ virus.The mixture is then Preeze-dried and the~vials sealed or tightly stoppered.
This passage level is used as a vaccine batch. For vaccine administration, the conten-ts o~ one vial is reconstituted, by adding 0~5 ml. oP water or saline or a 5 % sucrose solution and administered as drops in the nostrils. ~ , Clinical trials were perPormed in 24 volunteers.
Each volunteer received with a 11 to 14 day interval two nasal ~, administrations of the extemporaneously reconstituted vaccine, R 75 strain (Al'CC VR789}. At each administration, subjects were given a~virus concentration oP 107-8EID50 of the recons~
tituted vaccine in saline solution (5 drops in each nostrils).
The serological results are summari~ed in the following table V.
TABLE V
_ ~
Serum titer (HI)bePore Serum titer (HI) aPter Subject vaccination 2nd. vaccination I ~ 256 256 27 ¦ < I ~ C
~5 ~I A :
, 79Z;~
Seroconversion (fourfold increase o~ ~I titer) was observed in 75 % of the subject showing an prevaccinal HI
titer ~16.
Suspensions o~ the inPluenza type B R 75 strain (ATCC VR789) and o~ the in~luenza type A ~Alice~ strain ~
(ATCC VR 776) are mixed, peptone is added as ~-stabilizer in order to reach a Pinal concentration oE 5 %
o~ peptone and the mixture is distributed into 3 ml.
glass vials in order to contain at least 107EID50 o~ each virus. The mixture is ~reeze-dried and the vials sealed or tightly stoppered. ~or vaccine administration, the contents o~ one vial is reconstituted by adding 0.5 ml. o~ water or saline or a 5 % sucrose solution ~ ~
15 and administered as drops in the nostrils. `
~ `~
, .'.
'. ,,;'~'..~.'.
'' ":-, ,' - 20 ~
~ :''
Claims (8)
1. A process for preparing a live influenza virus vaccine for nasal administration the active ingredient of which comprises at least an effective amount of an attenuated influenza type B virus strain recombinant, which comprises recombining in a substrate known to the art for accepting growth of influenza type B virus an attenuated and vaccinal strain of influenza type B virus with an influenza type B virus isolate and isolating by selective pressure a recombinant having at least one marker property of the attenuated parent strain which is not shared by the other parent strain and the antigenic composition of the other parent strain, allowing said recombinant to grow in the allantoic cavity of embryonated chicken eggs, harvesting the virus, adding thereto a stabilizer and freeze-drying the mixture.
2. A process according to claim 1, wherein the attenuated and vaccinal strain of influenza type B virus is influenza type B virus Brigit strain (ATCC VR 786).
3. A process according to claim 2, wherein recombination is performed in the allantoic cavity of embryonated chicken eggs.
4. A process according to claim 2, wherein the isolate is the B/HK/8/73 (ATCC VR 792) isolate and the recombinant is influenza type B virus R 5 strain (ATCC VR 787).
5. A process according to claim 2, wherein the isolate is the B/HK/5/72 (ATCC VR 791) isolate and the recombinant is influenza type B virus R 22 strain (ATCC VR 788).
6. A process according to claim 2, wherein the isolate is the B/HK/5/72 (ATCC VR 791) isolate and the recombinant is influenza type B virus R 75 strain (ATCC VR 789).
7. A process according to any of claims 1 to 3, wherein the stabilizer is peptone or sugar.
8. A process according to any of claims 1 to 3, wherein the active ingredient also comprises an effective amount of an attenutated influenza type A virus vaccine which is added after harvesting the influenza type B virus recombinant.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US43917674A | 1974-02-04 | 1974-02-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1047922A true CA1047922A (en) | 1979-02-06 |
Family
ID=23743623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA218,508A Expired CA1047922A (en) | 1974-02-04 | 1975-01-23 | Live influenza virus vaccines and preparation thereof |
Country Status (21)
| Country | Link |
|---|---|
| JP (1) | JPS50107122A (en) |
| AT (1) | AT338416B (en) |
| BE (1) | BE824970A (en) |
| CA (1) | CA1047922A (en) |
| CH (1) | CH596316A5 (en) |
| CS (1) | CS202545B2 (en) |
| DD (1) | DD116474A5 (en) |
| DE (1) | DE2504373A1 (en) |
| DK (1) | DK141926B (en) |
| ES (1) | ES434384A1 (en) |
| FI (1) | FI750260A (en) |
| FR (1) | FR2259616B1 (en) |
| GB (1) | GB1439742A (en) |
| HU (1) | HU170522B (en) |
| IE (1) | IE42043B1 (en) |
| IL (1) | IL46446A (en) |
| LU (1) | LU71784A1 (en) |
| NL (1) | NL7501028A (en) |
| NO (1) | NO750295L (en) |
| SE (1) | SE424503B (en) |
| ZA (1) | ZA75248B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2588918A (en) * | 1947-11-07 | 1952-03-11 | William T Graham | Mechanism for raising and lowering plow frames relative to the ground wheels |
| JPS6042209B2 (en) * | 1974-05-31 | 1985-09-20 | 北里研究所（社団法人） | A method for acclimating fresh influenza virus isolates to embryonated chicken eggs in a short period of time |
-
1975
- 1975-01-14 ZA ZA00750248A patent/ZA75248B/en unknown
- 1975-01-16 DK DK9775AA patent/DK141926B/en not_active IP Right Cessation
- 1975-01-16 IL IL46446A patent/IL46446A/en unknown
- 1975-01-20 SE SE7500549A patent/SE424503B/en unknown
- 1975-01-23 CA CA218,508A patent/CA1047922A/en not_active Expired
- 1975-01-24 FR FR7502331A patent/FR2259616B1/fr not_active Expired
- 1975-01-27 IE IE160/75A patent/IE42043B1/en unknown
- 1975-01-29 NL NL7501028A patent/NL7501028A/en not_active Application Discontinuation
- 1975-01-30 NO NO750295A patent/NO750295L/no unknown
- 1975-01-30 BE BE152864A patent/BE824970A/en not_active IP Right Cessation
- 1975-01-31 FI FI750260A patent/FI750260A/fi not_active Application Discontinuation
- 1975-02-01 ES ES434384A patent/ES434384A1/en not_active Expired
- 1975-02-03 AT AT76675A patent/AT338416B/en not_active IP Right Cessation
- 1975-02-03 JP JP50014790A patent/JPS50107122A/ja active Pending
- 1975-02-03 DD DD183973A patent/DD116474A5/xx unknown
- 1975-02-03 HU HUTE805A patent/HU170522B/hu unknown
- 1975-02-03 DE DE19752504373 patent/DE2504373A1/en not_active Withdrawn
- 1975-02-03 CS CS75669A patent/CS202545B2/en unknown
- 1975-02-03 GB GB453575A patent/GB1439742A/en not_active Expired
- 1975-02-03 CH CH124275A patent/CH596316A5/xx not_active IP Right Cessation
- 1975-02-03 LU LU71784A patent/LU71784A1/xx unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IE42043B1 (en) | 1980-05-21 |
| AT338416B (en) | 1977-08-25 |
| DK141926B (en) | 1980-07-21 |
| FR2259616A1 (en) | 1975-08-29 |
| LU71784A1 (en) | 1975-06-24 |
| GB1439742A (en) | 1976-06-16 |
| CS202545B2 (en) | 1981-01-30 |
| SE7500549L (en) | 1975-08-05 |
| BE824970A (en) | 1975-07-30 |
| IE42043L (en) | 1975-08-04 |
| ZA75248B (en) | 1976-01-28 |
| FI750260A (en) | 1975-08-05 |
| JPS50107122A (en) | 1975-08-23 |
| SE424503B (en) | 1982-07-26 |
| DK141926C (en) | 1981-01-19 |
| NO750295L (en) | 1975-09-01 |
| NL7501028A (en) | 1975-08-06 |
| DE2504373A1 (en) | 1975-08-07 |
| IL46446A0 (en) | 1975-04-25 |
| IL46446A (en) | 1978-01-31 |
| CH596316A5 (en) | 1978-03-15 |
| FR2259616B1 (en) | 1980-01-04 |
| DK9775A (en) | 1975-09-29 |
| ATA76675A (en) | 1976-12-15 |
| HU170522B (en) | 1977-06-28 |
| ES434384A1 (en) | 1976-12-01 |
| AU7734675A (en) | 1976-07-15 |
| DD116474A5 (en) | 1975-11-20 |
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