IE42043B1 - Influenza type b virus vaccines and preparation thereof - Google Patents

Abstract

1439742 Nasal influenza vaccine RECHERCHE ET INDUSTRIE THERAPEUTIQUES 3 Feb 1975 [4 Feb 1974] 4535/75 Heading A5B A live influenza virus vaccine suitable for nasal administration comprises, as active ingredient an attenuated influenza type B virus strain recombinant obtained from (i) a vaccinal and attenuated influenza type B virus strain and (ii) another influenza type B virus strain, said recombinant having at least one marker property of said attenuated strain which is not shared by said other strain and the antigenic composition of said other strain. The attenuated strain may be Brigit strain. The recombinant may be influenza type BR5, R22 or R22 strain (ATCC VR787, 788 and 789 respectively). The recombinant may be prepared by combining the attenuated strain and the other strain in the allantoic cavity of embryonated chicken eggs, and isolating the recombinant by selective pressure. The isolate may be grown in the allantoic cavity of chicken eggs, admixed with a stabilizer, e.g. peptone or sugar, and freeze-dried. The vaccine may also include an attenuated influenza type A virus vaccine, e.g. Alice strain (ATCC VR776).

Description

This invention relates to influenza virus vaccines for nasal administration comprising as active ingredient an attenuated stable tyjxe B influenza virus strain and, optionally, an attenuated stable type A influenza virus administrable by nasal route; and to a process of preparing said attenuated stable influenza virus vaccines.

Protection against viral'respiratory infections has been shown to be related to the presence of a local immunity in the respiratory mucosa. This aspect has been reviewed recently by Rossen et al. (Progr. Med. Virol. 1971, n, 194).

Several attempts were made in recent years to induce immunity against influenza by the nasal application of inactivated vaccines (see e.g. i Waldman et al. Nature, 1968, 218, 594; JAMA 1969, 207, 520; WHO Bull. 1969, 41, 543). The results were inconsistent, however,, and this was probably due to the insufficient stimulation of the immunity system by the inactivated antigen (Tyrrell et al.

J. Hyg. 1970, 68, 359).

Live influenza type B-virus vaccines are known but are presenting widely varying protection rates and numerous efforts have been made e.g. by Serial passages on eggs (see for instance A.A. Smorodintsev, Proc. Symposium on Acute respiratory Diseases, Zagreb 1969, 391-404) or by Using low temperature mutants (R.M. Davenport et al.

Proc. Symposium on Live Influenza Vaccines, Zagreb 1971, 105-113) to reduce pathogenicity of the virus/' ή Recombination of influenza type B virus strains is also mentioned in the literature (see for instance E.D. Kilbourne .in'Progr. Med. Virol. 5, 79-126, 1963). - 1a42043 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 characteristics of the influenza virus and a property which produces many problems for the control of influenza by vaccination.

The present invention provides a influenza virus vaccine for nasal administration which comprises an attenuated influenza type B virus strain recombinant obtained previously attenuated from a vaccinal and / influenza type B virus strain, and an influenza type B virus strain and 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.

More particularly, the influenza type B attenuated may and vaccinal strain /be a strain herein referred to as the Brigit strain (deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville Md 20852 under the deposit number ATCC VR786) and which has been obtained by passaging the influenza B/Russia/69 strain (ATCC VR79O) in the allantoic cavity of embryonated chicken eggs in the presence of pig serum up to 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 Ξ. Fazekas de St. Groth and D.O. White in J. Hyg. 56: 151-62, 1958) -. - is antigenic with no side-effects on ferrets - shows positive hemagglutinin thermosensitivity at 60° C - shows unstable hemagglutination of sheep red blood cells at 30° C.

In the hereinabove process, the recombination step is carried out in any substrate known to the art for accepting growth of influenza type B virus, e.g. embryonated chicken eggs material or foetal bovine kidney tissue culture, more particularly the allantoic cavity of embryonated chicken eggs. The recombination can be performed with any desired type B virus strain, more particularly a new strain such as the B/Hong Kong/5/72 (ATCC VR791) or < B/Hong Kong/8/73 (ATCC VR792) strain.

Obviously, the so-obtained recombinant strains may be cloned or submitted to several dilution passages without modifying the essence of the invention.

Recombinants obtained according to this invention from the Brigit strain (ATCC VR786) with either the B/HK/5/72 (ATCC VR7 91) or the B/HK/8/73 (ATCC VR792) have been assigned the influenza type B virus R 22 strain (ATCC VR788), R 5 strain (ATCC VR787) and R 75 strain (ATCC VR789) designations respectively.

The virus recombinants obtained by the process of this Invention are non-pathogenic, immunogenic and valuable for influenza type B live virus vaccine production, using therefore any technique known to the art for live influenza virus vaccine production and/or stabilization.

Large amounts of the attenuated influenza virus vaccine of the present invention obtained as hereinabove described may be produced by allowing the virus recombinant to grow in the allantoic cavity of embryonated chicken eggs for a period of time sufficient to permit growth of a large amount of said virus, and harvesting the resulting virus material.

The so-obtained attenuated influenza virus vaccines are administered topically in the nasopharynx at an effective dosage unit (i.e. at least 1O7EID5Q of virus), said administration being eventually repeated if and when necessary.

For vaccinal use, the virus is preferably kept in freeze-dried form and the vaccine is extemporaneously reconstituted by addition of either water or any other pharmaceutical diluent or composition known to the art for the preparation of nasal preparations such as drops or spray. The vaccine formula may obviously include a stabilizer such as for instance peptone, sucrose and other ones known to the art.

According to another embodiment of the present invention the influenza type B virus strain obtained by the hereinabove described process can be combined with influenza type A virus vaccines administrable by nasal route.

The present invention thus relates to a process for preparing a live influenza virus vaccine for nasal adnti.hi'e'tration the active ingredient of which comprises 42()43 an-attenuated influenza ( process type B virus strain recombinant, which/comprises recombining in a substrate known to the art for accepting growth of influenza type B virus -e.g. embryqnated chicken eggs or foetal bovinp kidney tissue culture and preferably the allantci'c' cavity of pmbryonated chicken eggs- an attenuated and vaccinal strain of influenza type B virus -e.g.influenza type B virus Brigit strain(ATCC VR78^>- with an influenza typeB virus strain -e.g. the B/HK/5/72 (ATCC VR791) or the Β/ΗΚ/8/73 (ATCC VR792) strain- isolating by selective pressure a recombinant -e.g. the influenza type B virus R 22 strain (ATCC VR7S8) or R 75 strain (ATCC VR789) 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 and the antigenic composition of the other parent strain, allowing said recombinant to grow in the allantoic cavity of embryoharvesting the virus . . . nated chicken eggs,/and, if desired, adding thereto a stabilizer -e.g. peptone or sucrose- and freeze-drying the mixture.

The following examples illustrate the present invention; they should not be construed as limiting its scope.

EXAMPLE 1 A sample of the influenza B/Russia/69 strain (ATCC VR79O) is submitted to 3 terminal dilution passages on specific pathogen free eggs, for purification. A sample of the last passage is used as seed for the preparation of an experimental lot which is found to be free of avian oayiogenic-·agents and sensitive to serum inhibitors and which shuvs residual pathogenicity for human beings.

Different dilufeijns (i.e. 10~1, 10“^, 10“^ and 10~?) of . · * - 5 42043 the seed viral material in normal saline are mixed, with different concentration of sterile normal pig serum(undiluted, 25 % and 5 %) previously maintained for 15 minutes in a boiling water bath, homogenized and centrifuged at 2,000 rpm for 30 minutes. The supernatant is used for the further step which consists in incubating the virus/serum mixtures at 37° C for one hour before inoculating 0.2 ml. aliquots of said mixture into the allantoic cavity of embryonated chicken eggs previously incubated for 8 to 11 days at 37° C and candled (only the surviving eggs are inoculated).

The eggs are then further 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 hemagglutination method. The harvested virus produced by the inoculum of the highest virus dilution in the presence of the highest serum concentration which shows hemagglutination activity (i.e. virus dilution io6 and serum concentration of 25 %) is used for a second passage performed in the same operative conditions.

A total of 5 passages are performed as described hereinabove.

The virus, harvested after 5 passages, produced by the inoculum of the highest virus dilution (i.e. 10“®) in the presence of the undiluted serum, shows hemagglutination activity and is resistant to serum inhibitors.

Three further passages at terminal dilution are ea?ri^a ’oj»t in 1jhe absence of serum to clone the obtained resistant mutant and to check the stability of the resistant character. The virus harvested at'each of these 3 passages is resistant against the inhibitors of normal serum. The virus at the last passage level, named Brigit and deposited a at the ATCC under the deposit number VR?86, is used as inoculum for the production of a virus seed lot. Therefore, the harvested allantoic fluids are’collected and pooled, sterility and safety tested, mixed'with peptone to reach a final concentration 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 ' (Brigit strain). 1) Inhibitor resistance For testing the resistance against the inhibitors present in normal heated animal serum (previously heated at 75° C for 1 hour), serial twofold dilutions of the heated serums were mixed with 4 hemagglutinating units of the parent strain and the modified virus. After incubation of one hour at room temperature, chicken red blood cells were added and the results recorded. The results show a complete resistance for the seed lot and for experimental lots produced from this seed lot. In the present specification, this character is recorded as positive. 2) Antigenicit^abd^absenc^of^sid^effects A group of 2 ferrets was inoculated nasally with jlcT^IO? EID50 of the Brigit strain (ATCC VR786). The . tempetis^b^e of the animals was recorded daily during 8 days p.i. No significant temperature rise was recer^dQ-(maximum temperature 39.6° C). A second group β of 2 ferrets was inoculated nasally with 10-10 Εΐϋ^θ of the B/Russia/69 parent strain (ATCC VR79O). Two days after the inoculation the animals showed temperatures of 40.2° C. Hemagglutination-inhibition tests demonstrated that 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 EXD5O/(AOS)XD5O (AOS marker) The same inoculum is titrated on embryonated eggs and on the allantoic membrane on shell system (titration on allantoic membrane on shell is described by s.

Pazekas de St. Groth and D.O. white in J. Hyg. 56, 151-62, 1958). The EID5O/(AOS)ID5O ratio for the Brigit strain (ATCC VR786) was found 41°2’In the present specification, a ratio ^.102,is indicated as positive. 4) Hemagglutinin thermosensitivity Hemagglutination test was performed on infective allantoic fluid incubated for one hour at 60° C. After such treatment, Brigit strain (ATCC VR786) has completely lost its hemagglutinating property. This thermosensitivity is herein recorded as positive.

) Hemagglutination test_with sheep_red__blood_cells_at_30°_C An hemagglutination test was performed with sheep red blood cells at 4 and 30° C. The hemagglutination pattern at 4° C was normal whereas at 30° C there was a rapid elution so that no clear cut hemagglutination was observed. When examined 20 hrs. after initiation of the test, the hemagglutination has completely disappeared. This - 8 •λ α\ι «.ι character is herein referred to as unstable. ' ' EXAMPLE 2 A sample of influenza type B virus strain B/HK/5/72 (ATCC VR791) the hemagglutinin of which is different from that of the Brigit strain (ATCC VR786), is inoculated in'the allantoic cavity of 10 to 11 day-old embryonated 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 ό is obtained, the titer of which is 10 ElD^/ml. Aliquots (0.2 ml.) of the undiluted suspension are inoculated in the allantoic cavity of SPF (specific pathogen free) embryonated eggs5' after incubation for 2 hrs. at 33° C, aliquots (0.2 ml.) of a suspension of Brigit strain (ATCC VR786), having a titer of 10?^BID^g/ml., are inoculated in the Same eggs. The eggs are then incubated for 16 hrs· at 33° C and the allantoic fluids are harvested.

I . Aliquots (0.25 ml.) of anti-Brigit strain serotype rabbit serum previously heated for 1 hr. at 56° 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. This mixture is then maintained for 1 hr. at 37° C. Embryonated eggs are inoculated with 0.2 ml. of the incubated mixture(2 eggs for each mixture) and incubated for 48 hrs. at 33° C. The allantoic fluids of each inoculated egg are harvested, passaged in embryonated eggs 9^ (0.2 ml. per egg) and incubated for 72 hrs. at 33° C, using two eggs for each sample. After the 72 hrs. incubation Λ period,„the ppsitive allantoic fluids are harvested and , checked' for : - 9 43043 - hemagglutination of sheep red blood cells - serotype against anti-Brigit serum and against anti-B/HK/ 5/72 serum - resistance to non-specific serum inhibitors - growth in eggs and on AOS (allantoic on shell).

One of the so-obtained viral suspensions which, as indicated in the following Table I, has the serotype of one parent (B/HK/5/72) and the infectivity pattern in the AOS system and the serum inhibitor marker of Brigit strain is assigned the E 22 designation (ATCC VR788) and submitted to 2 passages on embryonated chicken eggs to obtain a substantial amount of E 22 recombinant virus and the harvested allantoic fluid is supplemented with peptone up to a final concentration of 5 % and freeze-dried.

TABLE I Strain Serotype inhi- bitor resis- tance eid50/ (aos)id5O hemagglu- tinin thermosen- sitivity 30° C sheep red blood cells hemagglutination Bri- jit b/hk/ 5/72 Brigit + - + + + unstable B/HK/5/72 - + - - - stable Recoin- binant R 22 (ATCC VR 788) + + stable EXAMPLE 3 Equal volumes of a suspension of the influenza type B Brigit strain (ATCC VR786) and of the B/Hong Kong/ 8/73 .strain .(ATCC VR792) (the titer of each being 10^*^ EID θ/ml.) are'mixed and preincubated for 72 hrs. at 4° C. . -, 1 Tjie mLxtture'is.tlie.n inoculated in the allantoic cavity of two SPP embryoncfted eggs (0.2 ml. per egg). After an - 5¾ 4 2 0 4 3 incubation period of 16 hrs. at 33° C, the allantoic fluids are harvested and sonicated (30 sec.).

The harvested fluids undiluted and diluted to —1 —2 ! and 10 are allowed to react .for one hr. at 37° C with different dilutions of anti-Brigit strain serotype rabbit serum previously 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 of the last positive virus dilution (10 ) are numbered 1 to 7.

Infectivity titers and neutralization by antiBrigit 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 anti15 Brigit serum and which exhibit an (AOS)lD^o titer at least equal to that of Brigit strain (ATCC VR786) are harvested at the highest positive dilution.

These individual harvests are diluted to 10“^ and passaged once in SPF eggs.

One of these viral suspensions showing the serotype and hemagglutinin thermosensitivity Of B/HongKong/ 8/73 strain and the EID^/iAOSjlD^g ratio of Brigit strain is assigned the R 5 designation (ATCC VR787).

In the following Table II the characteristics of recombinant R 5 are summarized and compared to those of the^Brigit (ATCC VR786) and B/HK/8/73 (ATCC VR792) parents. 43043 TABLE II Strain Serotype,.,, eid50/ (aos)id50 hemagglutinin t hermosensitivi ty Brigit 8/73 Brigit + - + + B/HK/8/73 - + - - Recombinant R 5 (ATCC VR787) + + The R 5 recombinant is submitted to two limit dilution passages (in SPE embryonated chicken eggs) to eliminate any eventual avian adventitious agent and to one further passage to obtain a substantial amount of R 5 recombinant virus. The harvested allantoic fluid is supplemented with peptone up to a final concentration of 5 % and freeze-dried.

EXAMPLE 4 Starting from the freeze-dried materials obtained in Example 1 (i.e. the Brigit strain, ATCC VR786) as seed lot for large scale vaccine production, a further passage is carried out in the allantoic fluid of another set of embryonated chicken eggs with are incubated at 35° C for 3 days.

The allantoic fluids are harvested, pooled, sterility and safety tested, mixed with peptone as stabilizer in order to reach a final concentration of 5 % of peptone and distributed into 3 ml. glass vials in order to Qhtain a dosage unit (i.e. at least 10' ΕΙΟ^θ) of virus. •The prddiifct. is then freeze-dried and the vials are iiStob I.V -stop'fjifred.

-Tlflij passage level is used as vaccine batch. For vaccine ^ministration, the contents of one vial is 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 fluids peptone.

, I preparation is distributed in larger glass vials in order to obtain integers of the dosage unit amount of virus to constitute corresponding multi-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 inhibition) titer inferior or equal to 32 against influenza B virus.

Each volunteer received two intranasal administrations with a 8 or 14 day interval. At each administration, subjects were given a virus concentration of 10 ΕΙϋ^θ of Brigi strain (in each nostril 5 drops of the extemporaneously reconsti tuted vaccine in 5 % sucrose solution in water). Seroconversion was observed in 40 subjects (i.e. 87 %); only mild clinical reactions (i.e. rhinorrhea or stuffy nose for 1 to 2 days)were observed. In the trials were control groups were available, no transmission of influenza vaccine virus was observed. Virus isolation was attempted from nasal and throat swabs but the samples remained negative after two blind passages in embryonated eggs.

EXAMPLE.5 Starting from the freeze-dried R 22 strain (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 chicken eggs which are incubated at 35° C for 3 days. - 13 42043 The allantoic fluids are harvested, pooled, sterility and safety tested, mixed with peptone as stabilizer in order to reach a final concentration of 5 % of peptone and distributed into 3 ml. glass vials in order to obtain a dosage unit (i.e. at least W^EXD^q of virus).

The product is freeze-dried and the vials sealed or tightly stoppered.

This passage level is used as vaccine batch. For vaccine administration, the contents of one vial is 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 fluids/peptone preparation is distributed in larger glass vials in order to obtain integers of the dosage unit amount of virus to constitute corresponding multi-doses vaccine preparations. Vaccination with the attenuated type B influenza vaccine R 22 strain.

Six volunteers having an HI antibody titer inferior or equal to 32 were selected for the trial. Two subjects were taken as control. The 6 subjects received, with a 11 to 14 day interval, two administrations of the extemporaneously reconstituted vaccine, R 22 strain. For each administration, all subjects received 5 drops of the vaccine per nostril (io7*5EIDg0).

Blood samples for antibody determination were taken oq/vaccination day and on days 10 to 14 after the first inoculation and on day 14 after the second administration. Nasal washings for local antibody determination were taken on day 14 after the second inoculation. £ii2i£2i_2X!!!££2!!!2A No*-symptoms were recorded.

Virus isolation.

Nasal swabs were collected on day 3 and 6 in the six subjects. All samples remained negative after two blind passages in embryonated eggs.

Serological results and 1θ£έΙ_3ηίϊ^ο^ϊθ5Λ Five' out of six volunteers developed either a serum or nasal response or both.

The results are summarized in the following Table XII.

TABLE III Subject Serum titer (Hl) before vaccination V Serum titer (HI) after 2nd vaccination local antibodies before Vaccination (SN) local antibodies after 2nd vaccination (SN) 1 32 32 <2 24 2 8 8 <2 2-4 3 ’ 32' 64 <2 12 4 8 32 <2 ( 2 5 <8 <8 ND ND 6 . <8. 16 ND ND ND = not determined.

EXAMPLE 6 Starting from the freeze-dried material obtained in Example 3 (R 5 strain,ATCC VR787)as seed lot for large scale vaccine production, a further passage is carried out in the·allantoic fluid of another set of embryonated chicken eggs which are incubated at 35° C for 3 days.

The allantoic fluids are harvested, pooled, sterility and' sSfeJty £h>sted, mixed with peptone as stabilizer in order to reach a final'concentration of 5 % of peptone and distributed into 3 ml. glass vials in order to obtain a dosage unit - 15 42043 (i.e. at least 107 EID,-0) virus. The mixture is then freeze-dried and the vials sealed or tightly stoppeffed.

This passage level is used as vaccine batch. Por vaccine administration, the contents of one vial is reconsti5 tuted by adding 0.5 ml. of water or saline or a 5 % sucrose solution and administered as drops in the nostrils.

Alternatively, the allantoic fluid/ peptone preparation is distributed in larger glass vials in order to obtain integers of the dosage unit amount of virus to constitute corresponding multi-doses vaccine preparations.

EXAMPLE 7 To one dosage unit of the freeze-dried influenza type B virus vaccine (Brigit strain,ATCC VR786)prepared as indicated in example 4·, there is added one dosage unit of live attenuated type A influenza virus (Alice strain,ATCC VR776)in 0.5ml. of a 5% sucrose solution in water. The so-obtained bivalent vaccine is administered as drops in the nostrils and the administration is repeated 14.days later.

Clinical trials were performed in 22 volunteers having a prevaccination HI (hemagglutination inhibition) titer inferior or equal to 64 against influenza A or B virus. Each Volunteer received two nasal administrations (ίθ7*5 ΕΙβ,,θ of the Brigit strain and 107’^ ΕΙϋ^θ of the Alice strain) with a 8 to 14 day interval.

Seroconversion was observed in 80 % of the subjects against influenza type A virus and 73 % against influenza type B virus.

Ohly mild clinical reactions were observed. No transmission of influenza vaccine virus was observed.

EXAMPLE 8 Tq>pne7‘^ ΕΙϋ^θ) of the freeze-dried ~ influenza type B.virus vaccine (R 22 strain,ATCC VR788)prepared as indicated in example 5, there is added one dosage unit *7 (5 * (10'3 ΕΙϋ^θ) of live attenuated type A influenza virus (Alice strain,ATCC VR776)in 0.5 ml. of a 5 % sucrose solution.

The so-obtained bivalent vaccine is administered as drops in the nostrils and the administration is repeated 14 day later. Seroconversion for both components (type B and type a) is observed 16 days after the second administration.

EXAMPLE 9 A sample of influenza type B virus strain. b/HK/5/72 (ATCC VR791) the hemagglutinin of which is different from that of the Brigit strain, is inoculated in the allantoic cavity of 10 tp 11 day-old embryonated chicken 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 0 EID^g/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 33° C, aliquots (0.2 ml.) of a suspension of Brigit strain (obtained in example 1) having a titer of lO^'^ElD^o/ml., are inoculated in the same eggs. The eggs are then incubated for 16 hrs. 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 56° C are mixed serum. Aliquots (0.25 ml) of this serum mixture are added to with the same volume .of normal /’ the harvested allantoic fluid. The mixture is then maintained for one hour at 37° C. SPF embryonated eggs are inoculated with 0.2 ml. of the mixture and - 17 42043 incubated for 48 hrs. at 33° C. The allantoic fluids of each incubated egg are harvested, passaged in SPP embryonated eggs (0.2 ml. per egg) and incubated for 72 hours at 33° C, using two eggs per sanple. After the 72 hours incubation period, the positive allantoic fluids are harvested and checked for : - hemagglutinat ion 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 of the so-obtained viral suspensions showing the serotype of the B/HK/72 parent and the infectivity pattern of the Brigit strain parent in the AOS system is named R 75 (ATCC VR789).

Recombinant R 75 is submitted to two passages in SPP embryonated chicken eggs to obtain a substantial amount of R 75 recombinant.

In the following Table IV, the characteristics of the recombinant are summarized and compared to those of the Brigit and B/HK/5/72 parents.

TABLE IV Strain SerotypeEIV (aos)id5O Hemagglu- tinin thermo sensitivity 30° C sheep red blood cells hemagglutination Bri- git b/hk/ 5/72 Brigit + -. + + unstable B/HK/5/72 - + - - stable Recombinant R 75 - + + stable EXAMPLE 10 The pooled allantoic fluids obtained invexample 9 are sterility and safety tested,mixed with peptone as stabilizer in order to reach a final concentration of 5 % of peptone and distributed into 3 ml. glass vials in order to obtain a dosage - 18 42043 •Ί unit (i.e. at least 1O'EID„_) of virus. The mixture is then JU I J freeze-dried and the vials sealed or tightly stoppered.

This passage level is used as a vaccine batch. For vaccine administration, the contents of one vial is reconstituted.. 5 by adding 0.5 ml. of water or saline or a 5 % sucrose solution and administered as drops in the nostrils.

Clinical trials were performed in 24 volunteers.

Each volunteer received with all to 14 day interval two nasal administrations of the extemporaneously reconstituted vaccine, R 75 strain (ATCC VR789). At each administration, subjects 7 8 were given a virus concentration of ιθ' Είϋ^θ of the reconstituted vaccine in saline solution (5 drops in each nostrils).

The serological results are summarized in the following table V.

TABLE V Subject Serum titer (HI)before vaccination Serum titer (Hl) after 2nd. vaccination 1 256 256 2 16 32 3 16 32 5 <4 32 9 16 32 13 8 32 23 <4 16 25 <4 64 27 16 64 28 4 32 29 16 64 30 4 32 32 16 64 35 8 128 36 8 32 37 128 128 40 8 16 43 64 >256 47 ' · 4 16 49 16 32 59 64 64 63 4 64 65 4 64 66 <4 16 - 19 420 4 3 Seroconversion (fourfold increase of HI titer) was observed in 75 % of the subject showing an prevaccinal HI titer ^16.

' EXAMPLE 11 Suspensions of the influenza type 3 E 75 strain (ATCC VR789) and of the influenza type A Alice strain (ATCC VR 776) are mixed, peptone is added as stabilizer in order to reach a final concentration of 5 % of peptone and the mixture is distributed into 3 ml. glass vials ity order to contain at least lO^EID^g of,each virus. The mixture is freeze-dried and the vials scaled or tightly stoppered. Por vaccine administration, the contents of one vial is reconstituted by adding 0.5 ml. of water or saline or a 5 % sucrose solution and administered as drops in the nostrils.

Claims (10)

CLAIMS.

1. A influenza virus vaccine for nasal administration the active ingredient of which comprises an attenuated influenza type B

2. A vaccine according to claim 1, wherein the attenuated parent is the influenza type B Brigit strain (ATCC VR786).

3. A vaccine according to claim 2, wherein the 15 recombinant is the influenza type B R 22 strain (ATCC VR788).

4. A vaccine according to claim 2, wherein the recombinant is the influenza type B R 5 strain (ATCC VR787). 5. Amount of an attenuated influenza type A virus vaccine which is added after harvesting the influenza type B virus recombinant. I 18. A process according to claim 17, wherein the attenuated influenza type A virus is influenza type A virus Alice strain (ATCC*VR776). 5 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 strain and isolating by selective pressure a recombinant 10 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 and chicken eggs / harvesting the virus. 10. A process according to claitn ? wherein a stabilizer is added 3 to the harvested virus and the mixture is freeze dried. 11. A process according to claim 9 or claim 10 wherein the attenuated and vaccinal strain of influenza type B virus is influenza type B virus Brigit strain (ATCC'VR786). 12. A process according to claim H, wherein recombi20 nation is performed in the allantoic cavity of embryonated chicken eggs· 13. A process according to claim IZ, wherein the isolate is the B/HK/8/73 (ATCC VR792) isolate and the recombinant is influenza type B virus R 5 strain (ATCC VR787). 25 14. A process according to claim 12, wherein the isolate is the b/Hk/5/72 (ATCC VR791) isolate and the recombinant is influenza type B virus R 22 strain (ATCC VR788). . 15. A process according to claim 12, wherein the isolate is the B/HK/5/72 (ATCC VR791) isolate and the 30 recombinant is influenza type Β virus R 75 strain (ATCC VR789) ft α υ α 3 16. A process according to any of claims 10 to 15 wherein the stabilizer is peptone or sugar. 17. A process according to any of claims 9 to 16, wherein the active ingredient also, comprises an effective

5. A vaccine according to claim 2, 20 wherein the recombinant is the influenza type B R 75 strain (ATCC VR7&9). 5 virus strain recombinant obtained from a vaccinal and previously attenuated influenza type B virus strain’and an influenza type B virus strain and 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 10 parent strain.

6. A influenza virus vaccine according to any of claims 1 to 5, wherein the active ingredient also comprises an effective amount of an attenuated influenza. type A virus 25 vaccine administrable by nasal route.

7. A influenza virus Vaccine according to any of claims 1 to 6, wherein the effective amount of attenuated influenza type B virus strain recombinant is at least 10 ΕΧϋ^θ.

8. A influenza virus vaccine according to any 30 of claims 6 and 7, wherein the attenuated influenza type A virus is influenza type A virus Alice strain (ATCC VR776).

9. A process for preparing a influenza virus vaccine for nasal administration the active ingredient of which comprises an attenuated

10. 19, A process according to claim 9 as hereinbefore described in any one of Examples 2,3 or 9.