DK141926B - Process for producing an attenuated influenza virus strain. - Google Patents

Description

(11) PUBLICATION MANUAL 1926 DENMARK <6 ”lntcl · 3 CA" "" "" i; "§ (21) Application No. 97/75 (22) Filed on 1 6 · Jan. 1975 (23) Running Day 16 Jan. 1975 (44) The application submitted and "the publication of the publication published on 21 · JU1. I you DIRECTORATE OF T ^ Λ u.

PATENT AND TRADE MARKET <30> Priority requested from it

Feb 4 1974, 439176, US

(71) INQUIRY AND INDUSTRY THERAPEUTIQUES R.I.T., rue du Tilleul, 13, B-T320 Genval, BE.

(72) Inventor: Julien Peetermans, avenue Herman, 6, 1330 Rixensart, BE: Mi5 * 1 chele Lobmann, rue de Morimont, 30, 1341 Ceroux-Mousty, BE: Jean-Marie PrevosT, avenue Paul Hymans, 6, 1200 Brussels , BE: Emil Vascoboi = nlc, avenue des Eperviers, 90, 1150 Brussels, BE.

(74) Plenipotentiary in the proceedings:

The company Chas. Hude.

(54) Process for producing an attenuated influenza virus strain.

The present invention relates to a method of producing an attenuated influenza virus strain valuable for vaccine use by nasal administration, wherein an attenuated influenza virus strain is recombined with an influenza virus isolate and is characterized by the characterizing portion of the claim.

Protection against viral respiratory infections has been shown to be associated with the presence of a local immunity in the respiratory mucosa. This relationship has Rossen et al. recently reported on 141926 2 (Progr. Med. Virol. 1971, 13, 194).

In recent years, several attempts have been made to induce immunity to influenza by nasal application of inactivated vaccines (see, for example: Waldman et al. Nature, 1968, 218, 594; Jama 1969, 207, 520; WHO Bull (1969, _41, 543). However, the results were unsustainable and this may be due to the insufficient stimulation of the immunity system by the inactivated antigen (Tyrrell et al. J. Hyg. 1970, 6j3, 359).

Live influenza type B virus vaccines are known, but these provide widely varying degrees of protection and numerous attempts have been made to reduce the pathogenicity of the virus, e.g. by serial transfers to eggs (see, e.g., AA Smorodintsev, Proc. Symposium on Acute Respiratory Diseases, Zagreb 1969, 391-404) or by using low temperature mutants (FM Davenport et al. Proc. Symposium on Live Influenza Vaccines, Zagreb 1971, 105-113).

Recombination of influenza type B virus strains has also been mentioned in the literature (see, e.g., E. D. Kilbourne in Progr. Med. Virol. 5, 79-126, 1963).

As indicated by e.g. G.C. Schild et al. in Brit. With. J. _4, 127-131, 1973, antigenic variations in its surface antigens are one of the most important traits of the influenza virus and a feature that causes many problems in influenza control during vaccination.

The object of the invention is to improve the preparation of attenuated influenza virus strain valuable for vaccine use by nasal administration, which can be obtained by recombining a attenuated virus strain with a virulent viral isolate by improving the step of isolating an appropriate recombinant.

When two viruses are allowed to recombine, the medium finally contains a mixture of different viruses, each of the parent viruses plus not just a single recombinant but a series of recombinants. According to the prior art, the parent viruses are eliminated from the medium one (i.e., the attenuated) by the addition of a specific antiserum and the other, (i.e., the pathogen) because of its poor growth ability, but the recombinants are still present. present, and they are isolated (i.e., dones) by the final dilution method. The question then is to choose a re-combinator, which is a suitable subject for vaccine preparation.

According to the prior art, the recombinants are examined for one or more properties and selection is made on the basis of the results of these studies. In contrast, according to the invention, the recombinants are placed under conditions (i.e., selective pressure) selected such that only the recombinants exhibiting appropriate properties will evolve: The selection conditions are determined by the specific characteristics of each parent virus relative to the other parent virus. .

The advantage of the method of the invention is to substantially limit the number of recombinants to be further investigated and to isolate the most suitable recombinants directly.

The present invention provides recombination of an attenuated vaccine influenza type B virus strain with a influenza type B virus isolate and selective pressure isolation of a recombinant having at least one label property of the attenuated parent strain characteristics not shared by the other parent strain and antigenic material of the other parent tribe.

The term "virus isolate" used above means any wild pathogenic strain variant found in the world and then isolated.

More specifically, the attenuated type B influenza strain and vaccine strain is a strain referred to herein as the Brigit strain (deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville Md 20852 under the landfill number ATCC VR786) and obtained by transmitting influenza B / Russia / 69 strain (ATCC VR790) to the allantoic cavity of embryonated eggs in the presence of swine serum until there is isolated from this attenuated vaccine influenza type B virus strain, which: - is completely resistant to serum inhibitors, - has a positive allantoic AOS on shell) marking property (titration on "allantoic membrane on shell" is described by S. Fazekas de St. Groth and DO White in J. Hyg. 56; 151-162, 1958), said AOS marking property herein being defined as the ratio of the egg-infecting dose in 50% of the inoculated eggs to the infecting dose in 50% of the inoculated allantoic membranes on shell, viz. EIDgg / (AOS) ID ™, said AOS brand property being considered positive when said -3 ° 2 5 ratio is at least 10 '- is antigenic without side effects on ferrets

- exhibits positive hemagglutinin thermosensitivity at 60 ° C

- exhibits unstable hemagglutination of sheep red blood cells at 30 ° C.

Influenza virus recombination and selection of recombinants can be summarized as follows, the scheme being applicable to any recombination and recombinant selection. From the art, it is known that the surface antigens (i.e., neuraminidase and hemagglutinin) determine the serotype of the strain.

Reactants Intestine I Stanrne II

I_I

Step recarbination i-i-1-1-1-1

Yield Virus I X recombinants Y recombinants Z recombinants Virus II

with surface down surface with one surface antigens of antigens of the antigen of I

virus I virus II and the other surface antigen I _, __ l_951135? __ 1

Step Treatment with Anti-I Serum

i

Yield Y recombinants with virus II

the surface antigens of strain ΓΕ

It is clear from this scheme that it is always necessary to have an antiserum treatment to simultaneously eliminate the parent strain and recombinants that are antigenically related to I.

In the method according to the invention, strain I is ATCC VR-786, strain II is the strain isolate and the above scheme must be supplemented with the following.

5 U1926

Reactants Y Recombinants with Virus Strain II (i.e.

the surface antigens the stem isolate) of strain II (i.e., the domestic isolate) 1-1--1

Step Selective tap to allow

Brigit virus type culture virus (examples are vsdcst on AOS, hemag-glutin intimose sensitivity, treatment in the presence of normal serum)

Yield Y recombinants with the surface antigen genes of the stem isolate and at least one mark of Brigit (ie, growth on AOS and / or growth in the presence of normal serum, which means resistance to normal serum inhibitors).

In the method described above, the recombination step is carried out in any substrate known in the art to accept the growth of influenza type B virus, e.g. embryonated chicken egg material or fetal bovine kidney tissue culture, especially the allantoic cavity of embryonic hen eggs. The recombination can be carried out with any desired type B virus isolate, especially a novel isolate such as the B / Hong Kong / 5/72 (ATGC VR791) isolate or the B / Hong Kong / 8/73 (ATCC VR792) isolate.

The recombinant strains thus obtained can, of course, be cloned or subjected to numerous dilution transfers, without thereby substantially modifying the present invention.

The recombinants obtained according to this invention from the Brigit strain (ATCC VR786) with either B / HK / 5 / 72- (ATCC VR791) or B / HK / 8 / 73- (ATCC VR792) The isolate have been assigned to influenza type B virus R 22, respectively. strain (ATCC VR788), R 5 strain (ATCC VR787) and R 75 strain (ATCC VR789) designation. The strains R 1, R 22 and R 1 are novel.

The virus recombinants obtained by the method of the present invention are non-pathogenic, immunogenic and valuable for the preparation of influenza type B live virus vaccine, using any technique known to produce and / or stabilize live influenza virus vaccine.

The method of producing such an attenuated influenza virus vaccine is obtained by allowing a influenza type B virus strain obtained by the above-described method to develop in the allantoic cavity of embryonic hen eggs for a period sufficient to allow growth of a large amount of said virus, and recover the resulting virus material.

The attenuated influenza virus vaccines thus obtained are administered topically in the nasal pharynx in an effective dosage unit (ie, at least 10 EIDj-q virus), said administration being optionally repeated if and where necessary.

For vaccination use, the virus is preferably kept in lyophilized form and the vaccine is prepared for use by the addition of either water or another pharmaceutical diluent or product which can be used to prepare nasal preparations such as drops or syringes. Of course, the vaccine may include a stabilizer such as e.g. peptone, sucrose or other known stabilizers.

According to another embodiment of the vaccine, the influenza type B virus strain obtained by the method described above can be combined with influenza type A virus vaccines which can be administered by nasal route.

A method for preparing a live influenza virus vaccine for nasal administration, wherein the active ingredient comprises at least an effective amount of an attenuated influenza type B virus strain recombinantly, is obtained by growing in a substrate known to accept growth of influenza type B virus, f. eg. embryonated chicken eggs or fetal bovine kidney tissue culture and preferably embryonated chicken eggs allantoic cavity. To prepare the recombinant used for the vaccine, an attenuated vaccine influenza type B virus strain ATCC VR786 (Brigit strain) is recombined with a influenza type B virus isolate, e.g. The B / HK / 5/72 (ATCC VR791) isolate or the B / HK / 8/73 (ATCC VR792) isolate, at selective pressure, isolates a recombinant, e.g. influenza type B virus R 22 strain (ATCC VR788) or R 75 strain (ATCC VR789) or R 5 strain (ATCC VR787), with at least one of the attenuated parent strain's brand characteristics not shared by the other parent strain and the other parent strain's antigenic composition, allowing said recombinant to be grown on the allantoic cavity of embryonic hen eggs, and if desired, a stabilizer, e.g. peptone or sucrose, and freeze-dry the mixture.

The following examples illustrate, but in no way limit the present invention.

Example 1

A sample of influenza strain B / Russia / 69 (AICC VR790) is subjected to three terminal dilution transfers on specific pathogen-free eggs for purification. A sample from the last transfer is used as the basis for the preparation of a test portion which is found to be free of avian or poultry pathogenic agents and sensitive to serum inhibitors and which exhibits residual pathogenicity to humans. Various dilutions (i.e., ΙΟΙΟ, 10 ”, 10- ^, and 10“) of the viral product or viral base material in normal saline are mixed with different concentrations of sterile normal pig serum (undiluted, $ 25 and $ 5). "Preceded for 15 minutes in a boiling water bath, homogenized and centrifuged at 2000 rpm for 30 minutes. The supernatant is used for the additional step which consists of incubating the virus / serum mixtures at 37 ° C for 1 hour before grafting 0.2 ml aliquots of said mixture into the allantoic cavity of embryonic hen eggs, the eggs being pre-incubated for 8-11 days at 37 ° C and illuminated (live eggs inoculated only).

The eggs are then further incubated for a time ranging from 20-96 hours.

After the end of this incubation period, the eggs are illuminated and live eggs are cooled to 4 ° C. The allantoic fluid from each series of live eggs is isolated separately and tested for the presence of influenza virus by the hemagglutination method. The recovered virus formed by the inoculum from the largest virus dilution in the presence of the highest serum concentration, spm shows hemagglutination activity (i.e. virus dilution 10 6 and serum concentration of 25 $), is used for a second transfer performed under the same conditions.

A total of 5 transfers are performed as described above.

The virus harvested after 5 transfers produced by the inoculation from the largest virus dilution (i.e., 10 “) in the presence of the undiluted serum exhibited hemagglutination activity and is resistant to serum inhibitors.

141926 8

Three additional transfers at terminal dilution are performed in the absence of serum to clone the obtained resilient mutant and check the stability of the resilience character. The virus harvested at each of these three transfers is resistant to the normal serum inhibitors. The virus at the last transfer concentration referred to as "Brigit" and deposited at ATCC under the deposit number VR786 is used as an inoculant in the preparation of a virus graft portion. The harvested allantoic fluids are then collected and combined, sterility and safety determined, then mixed with peptone to give a final concentration of 5 $ peptone. 0.5 ml of the virus suspension is partitioned into 3 ml vials and freeze-dried to obtain the Brigit strain portion (ATCC VR786).

In vitro and in vivo properties of the modified virus (Brigit strain).

1) Inhibitor Resistance

To determine the resistance to the inhibitors found in normal heated animal serum (pre-heated to 75 ° C for 1 hour), serial dilutions of the heated sera were mixed with 4 hemagglutination units of the parent strain and the modified virus. After 1 hour of incubation at room temperature, red blood cells from chickens were added and the results noted. The results show a complete resistance to the graft portion and to test portions prepared from this graft portion. In the present description, this property is referred to as "positive".

2) Antigenicity and absence of side effects

A group of 2 ferrets was seeded nasally with 10-10 EID ^ Q of the Brigit strain (ATCC VR786). The animals' temperature was taken daily for 8 days after grafting. No significant temperature increase (highest temperature 39.6 ° C) was observed. Another group of 2 ferrets was seeded nasally with 10 ^ -10® EID ^ q of the B / Russia / 69 parent strain (ATCC YR790). Two days after inoculation, the animals had temperatures of 40.2 ° C. Hemagglutination inhibition assays demonstrated that the modified strain induced an antigenic response in ferrets: 14 days after nasal inoculation, serum antibody titers were very high among the inoculated animals (titer: 1/256) as opposed to the inoculated control group (titer: <1/8).

3) Ratio EID ^ q / (AOS) ID ^ q (AOS brand property)

The same inoculum is applied to embryonated eggs and to the "allantoic membrane on shell" system (titration on "allantoic membrane on shell" is described by S. Fazekas de St. Groth and D. P. White in J. Hyg.

56, 151-162, 1958). The EID ^ 0 / (AOS) 1D ^ 0 ratio of the Brlgit strain (ATCC VR786) was found to be ^ 10 ^ ** '. In the present description, a ratio of 10 is indicated as "positive".

4) The hemagglutinin thermosensitized Hemagglutination assay was performed on infected allantoic fluid incubated for 1 hour at 60 ° C. Following such treatment, the Brigit strain (ATCC VR786) had completely lost its haemagglutination property. This thermosensitivity is herein termed "positive".

52__Hemagglutinate lon sample of red blood cells from sheep at 30 ° C

A hemagglutinatone test was performed with sheep red blood cells at 4 ° C and 30 ° C. The hemagglutination pattern at 4 ° C was normal, whereas at 30 ° C there was a rapid elution, so that no defined hemagglutination was observed. By examination 20 hours after the start of the experiment, hemagglutination had completely disappeared. This property is herein referred to as "unstable".

Example 2

A specimen type B influenza virus strain B / HK / 5/72 (A! ECC VR791), whose hemagglutination is different from that of the Brigit strain (ATCC VR786), is inoculated into the allantoic cavity of 10-11 day old embryos (inoculum: 0.2 ml / eggs) and subjected to 3 transfers to obtain a significant amount of virus. Upon completion of the third transfer, a

Q

Russian suspension, whose titer is 10 EID ^ / ml. Portions (0.2 ml) of the undiluted suspension are inoculated into specific pathogen-free (SPF) embryonic eggs of the allantoic cavity. After incubation for 2 hours at 33 ° C, portions (0.2 ml) of a Brigit strain (ATCC VR786) suspension are seeded with a titer of ΙΟ ^^ ΕΙΏ ^ / πΟ. in the same eggs. The eggs are then incubated for 16 hours at 33 ° C and the allantoic fluids are harvested.

141926 ίο

Portions (0.25 ml) of anti-Brigit strain serum type rabbit serum, preheated to 56 ° C for 1 hour, are mixed with the same volume of normal serum. Portions (0.25 ml) of this serum mixture are added to the harvested allantoic fluids. This mixture is then kept for 1 hour at 57 ° C. Embryonated eggs are seeded with 0.2 ml of the incubated mixture (2 eggs for each mixture) and incubated for 48 hours at 33 ° C. The allantoic fluids from each inoculated egg are harvested, transferred to embryonated eggs (0.2 ml per egg) and incubated for 72 hours at 33 ° C, using two eggs for each sample. After 72 hours of incubation, the positive allantoic fluids are harvested and tested for: - Hemagglutination of sheep red blood cells - Serotype against anti-Brigit serum and anti-B / HK / 5/72 serum - Resistance to non-specific serum inhibitors - growth in eggs and on AOS (allantoic on shell).

One of the virus suspensions thus obtained which, as given in the following Table I, has the serotype of one parent strain (B / HK / 5/72) and the infection pattern of the AOS system and the serum inhibitor label property of the Brigit strain, is attributed to the "R 22" designation ( ATCC YR788) and subjected to 2 transfers on embryonated chicken eggs to obtain a substantial amount of R22 recombinant virus, and the harvested allantoic fluid is supplemented with peptone until a final concentration of 5i and lyophilized.

Table I

Strain Serotype Irihibi- EID, -n / Hemagglu- 30 ° C red

Bri- B / HK / tormod- (δos) fininther- sheep blood cells git 5/72 stops- J mosensiti- haemagglutinin-

Brigit + - + 4- + unstable B / HK / 5/72 - + - - - stable

Recombinant + + + - stable R 22

(ATCC

VR788) ___ _ _

Example 3

Equal volumes of a suspension of the influenza virus strain type B "Brigit" (ATCC VR 786) and the strain B / Hong Eong / 8/73 (ATCC VR792) (each titre of 10 µl EID ^ / ml) are mixed and preincubated for 72 hours at 4 ° C. The mixture is then seeded in the allantoic cavity of two SPE embryonic eggs (0.2 ml per egg). After an incubation period of 16 hours at 33 ° C, are allantoic fluids harvested? and audio processing (30 seconds).

The harvested liquids in undiluted state and diluted to ΙΟ- · * · and 10 "2 are allowed to react for 1 hour at 37 ° C with various solutions of anti-Brigit strain serotype rabbit serum, preheated to 56 ° C for 30 min. The resulting mixtures are seeded on the "allantoic on shell" system and incubated for 72 hours at 37 ° C. The last positive virus dilution (ICT) samples numbered 1 to 7.

Infection titers and neutralization using anti-Brigit serum in the AOS system are then compared for the 7 samples and for the two parent strains.

The samples, which are not neutralized by anti-Brigit serum, and which exhibit an (AOS) ID ^ q titer which is at least equal to that of the Brigit strain (ATCC VR786), are harvested at the largest positive dilution.

These different portions are diluted to 10 “and transferred once in SPE eggs.

One of these virus suspensions, showing the serotype and B / Hong Kong / 8/72 strain's hemagglutinin thermosensitivity and Brigit strain's EID ^^ / (AOS) IDfjQ ratio, is designated "R 5" (ATCC VR787).

The following Table II summarizes the properties of recombinant R 5 and compares with the properties of Brigit (ATCC VR786) and B / HK / 8/73 (ATCC VR792) parent strains.

fork II

12 U1926

Strain Serotype EID, -0 / Hemagglutinin

Brigit B ^ HK / (A0S) ID ^ q thermosensitivity

Brigit + - + + B / HK / 8/73 - +

Recombinant R 5. - + + (AICC VR787) The R 5 recombinant is subjected to two limited dilution transfers (in SPE embryonated chicken eggs) to eventually eliminate any avian or poultry adventitious agent and one additional transfer to obtain a substantial amount of R 5 recombinant virus. The harvested allantoic liquid is supplemented with peptone until a final concentration of 5 $ and freeze-dried.

Example 4

With the freeze-dried R 22 strain (ATCC VR788) obtained in Example 2 'as a graft portion for large-scale vaccine production, a further transfer into the allantoic fluid of a second set of embryonated chicken eggs incubated at 35 ° C for 3 days is performed.

The allantoic fluids are harvested, combined, sterility and safety tested, mixed with peptone as a stabilizer to reach a final concentration of 5 µg peptone and distributed in 3 ml vials to obtain a dosage unit (i.e., at least 10 µ EID ^ q virus). The product is freeze-dried and the vials are closed or clogged tightly.

This transfer level is used as a vaccine charge. For vaccine administration, the contents of 1 vial are prepared by adding 0.5 ml of water or saline or a $ 5 sucrose solution and the administration takes place as drops in the nostrils.

Alternatively, the peptone preparation is distributed with the allantoic fluids in larger vials to obtain the viral dosage unit amount several times to produce corresponding multidose vaccine preparations.

141926 13

Vaccination with the attenuated type B Influenza Vaccine R 22 strain Material_and methods

Six volunteers with an HI antibody titer of less than or equal to 32 were selected for the trial. Two persons were selected for control. At six to 14 day intervals, the six individuals received 2 administrations of the on-line vaccine R 22 strain. For each administration, all subjects received 5 drops of the vaccine per nostril (io7'5eid50).

Blood samples for antibody determination were taken on the day of vaccination and on the tenth to fourteenth day after the first inoculation and on the fourteenth day after the second administration. Nasal wash fluids for local antibody determination were taken on the fourteenth day after the second inoculation.

Eliniske_symgtomer

No symptoms were noted.

T ± ri_isisols; irrigated nasal samples were collected on days 3 and 6 of the six individuals. All samples remained negative after two blind transfers in embryonic eggs.

Serological results and local (ie, nasal) antibodies.

Five out of six volunteers developed either a serum or nasal response or both (namely 2 for serum, 2 for local, and 1 for both, cf. those with x marked numbers).

The results are summarized in the following Table III.

The numbers are the reciprocal values of the last active dilution of the serum and of the nasal wash fluids, respectively.

Table III

14 141926

Serumanti- Sertmanti-

Person Dust Titer (HI) Dust Titer (HI) Local Anti-Local Antibody Before Vaccination After Second Dust Titer Before Titer After Second Vaccination Vaccination Vaccination _ (SN) _ (SN) _ 1 32 32 <2 24 * 2 8 8 <2 2- 4 * 3 32 64 * <2 12 * 4 8 3235 <2 <2

5 <8 <8 ND ND

6 <8 16 * ND 'ND

ND = not determined HI = hemagglutination inhibition SN = serum neutralization test.

Example 5

With the freeze-dried material obtained in Example 3 (R5 strain, ATCC VR787) as a graft portion for large-scale vaccine production, a further transfer was made into the allantoic fluid from a second batch of embryonated chicken eggs, incubated at 35 ° C for 3 days. .

The allantoic fluids are harvested, combined, sterility and safety tested, mixed with peptone as a stabilizer to achieve a final concentration of 5µ peptone and distributed in 3 ml vials to obtain a dosage unit (i.e., at least 10? EID ^ q) of virus. The mixture is then freeze-dried and the vials closed or clogged tightly.

This transfer level is used as a vaccine charge. For vaccine administration, the contents of one vial are diluted by adding 0.5 ml of water or saline or a 5% sucrose solution, ag administration as drops in the nostrils.

Alternatively, the allantoic liquid / peptone preparation is distributed in larger vials to obtain the viral dosage unit amount several times to produce corresponding multidose vaccine preparations.

IS 141926

Example 6

To one dosing unit (107.5 EID ^ 0) of the freeze-dried influenza type B virus vaccine (R 22 strain, ATCC VR788) prepared as set forth in Example 5 is added one dosing unit (10'EID ^ q) live attenuated type A influenza virus ( Alice strain, ATCC YR776) in 0.5 ml of a 5 $ sucrose solution.

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

Example 7

A sample of influenza type B virus strain B / HK / 5/72 (ATCC VR791), whose hemagglutination is different from that of the Brigit strain, is inoculated into the allantoic cavity of the 10 to 11 day old embryonic hen egg (inoculum: 0.2 ml / egg), and subjected to three transfers to obtain a substantial amount of virus. After the third transmission, a virus is obtained.

Q

suspension whose titer is 10 EID ^ 0 / ml. Aliquots (0.2 ml) of the undiluted suspension are seeded into the allantoic cavity of SPE (specific pathogen-free) embryonic eggs. After incubation for 2 hours at 33 ° C, in the same egg portions (0.2 ml) of a suspension of Brigit strain (obtained in Example 1) were seeded with a titer of 10 7 The eggs are then incubated for 16 hours at 33 ° C and the allantoic fluids are harvested.

Aliquots (0.25 ml) of anti-Brigit strain serotype rabbit serum preheated for 1 hour to 56 ° C are mixed with aliquots (0.25 ml) of the recovered allantoic fluid. The mixture is then kept at 37 ° C for 1 hour. Specific pathogen-free embryonic eggs are seeded with 0.2 ml of the mixture and incubated for 48 hours at 33 ° C. The allantoic fluids from each inoculated egg are recovered, transferred into specific pathogen-free embryonic eggs (0.2 ml per egg) and incubated for 72 hours at 33 ° C using two eggs per egg. sample. After the 72 hour incubation period, the positive allantoic fluids are recovered and examined for: 16 1419-28 hemagglutination of sheep red blood cells - serotype against anti-Brigit serum and anti-B / HK / 5/72 serum - growth in eggs and on AOS (allantoic on shell).

One of the virus suspensions thus obtained, showing the serotype of the B / HK / 5/72 parent strain and the infection pattern of the Brigit parent strain in the AOS system, is called R75 (ATCC VR789).

The above procedure for preparing R 75 is the same as used for preparing R 22. Both strains are similar in that the hemagglutination, serotype and AOS markers are used. They are nonetheless different regarding additional properties, e.g. their serum response in hamsters as given in the following table.

Antigenicity of R 22 strain (ATCC VR788) and R 75 strain (ATCC VR789) R 22 (ATCC VR788) R 75 (ATCC VR789)

Serum anti- (<8 '<8' Dust (HI)) (<8 16 in 5 hamsters (<8 8) <8 <8 (<8 <8

Geometric mean titer ~ "~" "~~ ~" "" serum reaction in% "0 40

The figures in this table show that serum response is not induced in hamsters by the ATCC VR788 strain, while a serum reaction is induced in 40% of the hamsters inoculated with ATCC VR789.

The geometric mean titer of serum antibody titers HI is the antilogarithm of the arithmetic mean of the logarithmic values of the various serum antibody titers. In this case, the logarithmic values were calculated on base 2 and the number "<8" was taken as "0".

Recombinant R75 is subjected to two transfers in specific pathogen-free embryonic chicken eggs to obtain a substantial amount of R75 recombinant.

In the following Table IV, the properties of the recombinant are summarized and compared with the Brigit and Β / ΗΚ / 5/72 parent strains.

Table IV

Strain Serotype EID-Q / Hemagglutinin 30 ° C Red Sheep

Bri- B / HK / ('Λης'ίτπ thermosensitic blood cells elevated 5/72 ^' 50 white_magglutination

Brigit + - + + unstable B / HK / 5/72 + - stable

Recombinant + + _ stable R 75

Example 8

The united allantoic fluids obtained in Example 7 are sterility and safety tested, mixed with peptone as a stabilizer to achieve a final concentration of 5% peptone and distributed in 3 ml vials to obtain a dosage unit (i.e., at least 10 µ EID ^ q) of virus. The mixture is then freeze-dried and the vials closed or clogged tightly.

This transfer level is used as a vaccine charge. For vaccine administration, the contents of one vial are prepared by adding 0.5 ml of water or saline or a $ 5 sucrose solution and the administration is done as drops in the nostrils.

Clinical trials were conducted on 24 volunteers. Each volunteer received 2 nasal administrations of the vaccine R 75 strain prepared for use at 11 to 14 day intervals (ATCC VR789). With each administration, each person was given a viral concentration of 10 µl EID of the prepared vaccine in saline solution (5 drops in each nostril).

The serological results are summarized in the following Table V.

Table V

18 141926

Person Serum Titer (HI) before Serum Titer (HI) after _ vaccination_and vaccination 1 256 256 2 16 52 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 3 52 37 128 128 40 8 16 43 64 ^ 256 47 4 16 49 16 52 59 64 64 63 4 64 65 4 64 66 <4 16