CS202545B2 - Method of preparing living vaccine from influenza virus for nasal application - 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

. The present invention relates to a method for the preparation of a live influenza virus vaccine for pronasal administration comprising, as an active ingredient, a attenuated, steady-state strain of influenza B virus or an attenuated, steady-state strain of influenza A virus administered nasally and method. preparing said vaccine from a attenuated, steady influenza virus. »·

Protection against respiratory viral infections has been shown to be related to the local immunity of the respiratory mucosa. This aspect has been elaborated recently in the form of a review by Rossen sp. In recent years, several attempts have been made to induce influenza immunity by nasal administration of inactivated vaccines (see, for example: Waldman et al., Nature, 1968). 218, 594; JPMA 1969, 207; 520; WHO Bull. 1969, 41, 543). The results were inconsistent, probably due to insufficient stimulation of the immune system by the activated antigens (TyreH, et al., J. Hyg. 19, 70-68, 359).

Live influenza virus vaccines. B are known, but provide a variable degree of protection. Numerous attempts to reduce the pathogenic properties of the virus have been made, for example, by serial passages on eggs (see, for example, PP SmoooOntsev, Proc. Symposium on acu.te respirators Diseases, Zagreb 1969, 391-404) or using low temperature mmu-ant (P. · M. Davenpprt et al., Proc., Symposium on Live Influenza Vaacines, Zagreb 1971, 105-113). .

Recombination of influenza B virus strains has also been described in the literature (see, for example, E. D. Kilbourne: Progr. Med. Virol. 2, 79-126, 11963). ‘

For example, G. · C. Schildem sp. : Brit. Copper. J. 4, 127-31, 197.3, the antigenic variation of its surface antigens is one of the most important chaaaktteistics of influenza virus and viralities, which presents numerous problems in attempts to control influenza by vaccination.

The present invention provides attenuated, stable strains of influenza B virus valuable for vaccine use or a method for their preparation, characterized in that the virulent strain of influenza virus V is attenuated to such an extent that it obtains a attenuated and vaccine strain of influenza B virus, said attenuated virus. and the vaccine strain is recombined with the influenza virus isolate type V, and the recombinant having at least one feature of the attenuated parent strain not shared with the second parent strain and the antigenic composition of the second parent strain is isolated by selective pressure. By selective pressure is meant here any selective activity carried out by the virocid and impinging on the survival of viruses having one or more particular hereditary facts. viruses, while viruses that do not have these factors are eliminated.

The attenuated and vaccine influenza strain of type V is particularly a strain also referred to herein as Brigit (deposited in the American Type Culture Collection, 12301 Parklawn Driwe, Rockville Md 20852 under deposit number ATCC VR786) and was obtained by passage of influenza strain B / Russia / 69 / ATCC VRT90 in the allantoic cavity of chicken embryos in the presence of porcine serum up to the isolation of the attenuated influenza virus type V virus with the following characteristics:

- is completely resistant to serum inhibitors,

shows a positive sign of allantoic on shell (AOS), (titration on the allantoic membrane on the shell is described by S. · Fazekas, St. Groth and D. O. White: J. Hyg., 56, 151-62, 1958).

- it is antigenic in the ferret tests without side effects,

- exhibits a positive hemagglutinin thermosensitivity at 60 ° C,

- exhibits unstable haemagglutination of sheep erythrocytes at 30 ° C.

In said method of preparation, the recombination step is carried out in any known influenza B virus growth substrate, i.e. chicken embryo material or on bovine kidney tissue culture, particularly in the allantoic cavity of chicken embryos. Recombination can be carried out with any desired Type B virus isolate, particularly with the novel B / Hong Kong / 5/72 (ATCC VR79I) or B / Hong Kong / 8/7/73 (ATCC VR792) isolate ./

The recombinant strains thus obtained can be cloned or subjected to several dilution passages without altering the spirit of the invention.

Recombinants obtained according to the invention from Brigit strain (ATCC 7R786) with either isolate B / HK / 5/72 (ATCC VR79D) or isolate Β / ΗΚ / Θ / 73 (ATCC VR792) were identified as influenza virus strain type R 22 (ATCC VR788), strain R5 (ATCC VR787) and strain R75 (ATCC VR789).

The virus recombinants obtained by the preparation method of the invention are non-pathogenic, immunogenic, valuable for the preparation of a live influenza virus type V virus using any known process for preparing and / or stabilizing a live influenza virus vaccine. Accordingly, the invention also relates to an attenuated influenza virus type vaccine comprising at least one said recombinant influenza virus type V and a method of preparing said vaccine from said recombinant.

Accordingly, the invention relates to a method of preparing a vaccine from an attenuated influenza virus; characterized in that the influenza virus type B strain obtained by the above process is allowed to grow in the allantoic cavity of the chick embryos for a sufficient period of time allowing the growth of a large amount of virus and obtaining the resulting viral material.

The attenuated influenza virus vaccines thus obtained are administered topically in the nasopharynx in an effective dosage unit (ie at least 10 ΙΏ of virus), said administration repeated as necessary.

For vaccine use, the virus is preferably maintained in the form of lyophilisate and the vaccine is reconstituted if necessary by the addition of water or other pharmaceutical solvent or mixture known for the preparation of nasal preparations such as drops or spray. The vaccine form may, of course, contain a stabilizer, such as peptone, sugar and other known substances used. '’

In accordance with another aspect of the invention, the influenza B virus strain obtained as described above may be combined with a nasally administered influenza A virus vaccine.

The invention therefore relates to a method of preparing a live influenza virus vaccine for nasal administration, the active ingredient of which comprises at least an effective amount of a recombinant attenuated strain of influenza B virus, characterized in that it is recombined in a known influenza B virus growth substrate, i.e. chicken. germs or in tissue culture from bovine kidney, preferably in the allantoic cavity of chicken embryos / attenuated and vaccine strain of influenza B virus, i.e. Brigit influenza virus type В (ATCC VR786) with influenza B virus isolate, ie with isolate B / HK / 5/72 (ATCC VR791) or with B / HK / 8/73 (ATCC VRT92) and a recombinant, i.e. a strain of influenza virus type V R 22 (ATCC VR788) or strain R 75 (ATCC VR789) or strain R 5 (ATCC VR787)> having at least one feature of an attenuated parent strain that does not share with the second parent strain and the antigenic composition of the second parent strain is isolated by selective pressure, said recombinant is allowed to grow in the allantoic cavity of the chicken embryos and, if necessary, a stabilizer is added and the mixture is lyophilized.

The following examples illustrate the invention without limiting it.

He did

A sample of the influenza strain, B / Russia / 69 (ATCC VR790), was subjected to three final dilution passages for purification on eggs free of specific pathogens. The last passage sample is used as an inoculum for the preparation of a test batch that has been found to be free of pathogenic avian agents and susceptible to serum inhibitors and showing residual pathogenic 1 1-3-3-7 tu for humans. Different concentrations (i.e., 10, 10, 10 'and 10') of the inoculum viral material in saline are mixed with different concentrations of sterile pig serum (undiluted, 25% and 5%) exposed to the boiling water bath temperature for 15 minutes. , homogenized and centrifuged at 2000 rpm. for 30 minutes. The filtrate is used for the next step of incubating the virus, serum mixture for 1 hour at 37 ° C before inoculating 0.2 ml of this mixture into the allantoic cavity of eggs with chicken embryos previously incubated for 8-11 days at 37 ° C and filtered through a candle (only surviving eggs were inoculated). The eggs are then incubated further for a period between 24 and 96 hours.

After completion of the incubation, candle filtration is performed and the surviving eggs are cooled to _x 4 ° C. Allantoic fluid from all surviving egg series is isolated separately and tested by the hemagglutination method for the presence of influenza virus. The isolated virus produced the largest viral inoculum dilution in the presence of highest serum concentration exhibiting hemagglutinační activity (i.e. virus dilution 10 ^_i> serum concentration 25%) was used for the second passage, under the same working conditions.

A total of 5 passages are performed as described above.

The virus, isolated after 5 passages, produced by the inoculum of the highest virus dilution (i.e. 10 ° C) in the presence of undiluted serum, shows haemagglutination activity and is resistant to serum inhibitors.

Three additional passages at final dilution are performed in the absence of serum to clone a resistant mutant and stabilize its resistant character. The virus obtained from each of these three passages is resistant to normal serum inhibitors. The last passage level virus named Brigit ”and deposited in the ATCC under deposit number VR786 is used as an inoculum to prepare a batch of viral inoculum. The resulting allantoic fluids are collected and pooled, tested for sterility and safety, and mixed with the peptone until a final concentration of 5% peptone is reached. The volume of 0.5 ml virus suspension is filled into 3 ml vials, lyophilized to yield a batch of Brigit strain (ATCC VR786).

In vitro and in vivo characteristics of the modified virus (Brigit strain):

1) Inhibitor resistance 's *' ·

To assess the resistance to inhibitors in normal, warmed animal serum (previously heated to 75 ° C for 1 hour), serial two-fold dilutions of the warmed serum were mixed with 4 htg / ml units. of the parent strain and. modovrnéht virus. After in, cubing for 1 hour at normal temperature, chicken erythrocytes were added and the results evaluated. The results show complete resistance in the inoculation lot as well as in the inoculation lot experimental batches prepared. In the present specification, eeaeo is said to be positive. .

2) Antigenicity and side effects.

The group of two ferrets was inoculum nasally with 10? -10® EID? Q Brrgit strain (ATCC VRR86 ·).

Animal temperature controlled daily for 8 days p.i. No significant was observed. temperature increase (maximum temperature 39.6 ° C). A second group of two ferrets was inoculated nasally with 10? -10 ® EID ® q of the parental strain B / Ruusia / 69 (TCTCC VR790). Two days after inoculation, the animals are allowed to attain a temperature of 40.2 ° C. Tests on atmospheric inhibition showed that the decoded strain induced an antigenic response in ferrets: 14 days after nasal inoculation, serum antibody titers of vaccinated animals were very high (titer: 1/256) versus the control, unvaccinated groups (tit ^). ?: <11/8).

3) E ^ / UOS ratio]] ^ (AOS character) '. . .

The same inoculum is titrated on chicken embryos and in the allantoic membrane system on the shell. (Strace on rllanetic membrane · on · shell is described by S. Faztkasto, St. Grotil and DO · Wh tem: J. Hyg. 5) 151-62, 195B.,) · EID ^ q / C ratio AOSJID-q for Brigit strain ^(AT CC ' ^VR 786 ⁾ was found to be 1 ° ² . ^{In Pla dl} marries ^{E p} ec ^CF esterification ratio is ^410,214 uváděnjato positive. ,

4) Hemoglucination eatroozettittvitr ·

The healing assay was performed in an infected allantoic cavity, incubated for 1 hour at 60 ° C. After this intervention the ΒτΊ.β ± ^ strain (ATCC VR786) completely lost the intrinsic property. This th mass is referred to herein as positive.

5) Haemogluluenea test with sheep eryeaascyey at 30 ° C

The heeoglucination test was performed with sheep eryearocyte at 4 and 30 ° C.

specimens at 4 ° C were normal, while at 30 ° C rapid excretion occurred so that no clear borderline was observed. By observation 20 hours after the start of the assay, it was found that htmariuUinact had completely disappeared. This character is unstable here.

Example2

A sample of influenza B virus strain B / HC / 5/72 (ATCC VR791); whose aemargiumUinin differs from the Brigit strain (ATCC VRR78), is inoculated in a rllattic cavity of 10-11 days old chicken embryos (um inoculum: 0.2 ml / egg), subjected to three passages to obtain subtractive mn The virus branch. At the end of the ^P and ^to E virus sacrificing ^ s ^{p 1} sus ^Enns, its titre ^Iz · oíioovíí ¹ W®

EID-q / diI. An appropriate viral suspension (0.2 m) of the undiluted viral suspension is inoculated in the SPF of the fetal cavity (free of specific paehthienes) of the chicken embryos. After incubation for 2 hours at 33 ° C was inoculated into eggs stejrý ^ Brig strain sus ^ n ^ ^(ATCC VR78 ⁶⁾ There are three different ^ · ^L0? ' ⁴ EID ⁴ / oI. ^In the ejce ^p and ^p inkubuuí ^{16 h} Odin suits. 33 ° ^C and the obtained allantoic ^Í ^ Kuhn.

An equivalent amount (0.25 ml) of anti-BrigiVtaieoe serotype rabbit serum previously heated to 56 ° C for 1 hour is mixed with an equal volume of normal serum. An appropriate amount (0.25 ml) of this serum mixture is added to the obtained allanolic fluid. The mixture was maintained at 37 ° C for 1 hour. Eggs with chicken embryos are inoculated with 0.2 mL of incubation mixer (2 eggs per mix) and incubated for 48 hours at 33 ° C. Allitritic fluids are obtained from each inoculated egg, passaged in chicken embryos . (0.2 ml per egg) and incubate for 72 hours at 33 ° C, leaving 2 eggs for each sample. After 72 hours of incubation, they are recovered from the ... fluid and examined for:

- hey ^ m ^^ u ^ na! sheep erythrocytes,

- anti-Brigit serum and anti-B / HV5 / 72 serotype,

resistance to non - specific serum inhibitors,.

- growth in eggs and on AOS (Ι Ιε ^ ^ ί ά na na na na na na na).

One of the virus suspensions thus obtained, which has, as shown in the successors of Table I, the serotype of one parent (B / HM5 / 72) and the infectious markers in the AOS system and the serum ichibitor mark. (ATCC VR788) and subjected to 2 passages on chicken embryos to obtain substantial multiplication of the recombinant virus R 22. The obtained allactic fluid is supplemented with peptone up to a final concentration of 5% and lyophilized. '

T a b u 1 k · a ’1‘

* Tribe Serotype Inhibitor resistecce EID50 / (AOS) ID ₅₀ 0 . Hemagglutination thermosensitivity Hemmagiu uinac e  of sheep erythrocytes at 30 ° C Brigit • B / НМ 5/72 Brigit + - + + ·, + · unstable B / HK / 5/72 - + ’ • ₍ - - -. stable. гeCob ^ 1 ^ na ^ t - + + - stable ' R 22 (ATCC VR788) Example 1 a d · 3 Slejcé volumes suspension Brigit tribes (ATCC VR786) influenza type B and • B / Hong Kong / 8/73 strains (ATCC VR792) (the titer of each is · 10 ',? ЕИ ₅ 0 / ь 1) They were mixed and pre-injected for 72 hours

hours at 4 ° C. The mixture is then used to inoculate in the allaitoic cavity of the SPF of chicken germs (0.2 ml per egg). After an incubation time of 16 hours at 33 ° C, the allaito fluids are isolated and sonicated for 30 seconds.

Isolated liquids · undiluted and diluted to 10 and 10 are left! to react for 1 hour at 37 ° C · with differently diluted rabbit serum of the arti-Brigit strain, which was previously heated to 56 ° C for 30 minutes. RezMlní! the mixture was seeded on an alleilized system and incubated for 72 hours at 37 ° C. The samples of the last positive virus dilution (10 ² ) are numbered 1 to 7.

The titers and the neutralization of aiti-Brigit serum in the AOS system for the 7 samples are compared to the two parent strains.

Samples that are not neutralized by acti-Brigit serum and which report;)! The (AOS) IDg _O titer by at least equivalence of the Brigit strain (ATCC VR7 / B6) is isolated at the highest positive dilution.

The individual samples obtained are diluted to 10 µl and 1 x on SPF eggs.

One of these viral suspensions, exhibiting serotype and hembagiutinic therapy of strain B / Hong Kong / 8/73 and EHD5q / (A0S) ID50 ratio of the Brigit strain, is designated R 5 (ATCC VR777). .

The following Table II summarizes the characteristics of the R 5 recombination and compares 8 weeks for Brd-git (ATCC VJR786) and B / HK8 / 73 parents. (ATCC 'VR792). . . _t

T a b at 1 k a I I.. · . - Strain Ér Serotype EID _5a / (AOO) lD _5a ' HeInbgglutinační Brigit 8/73 tuetbossrsiíd.vi'ta Brigit  + -. +. +. B / HK8 / 73. -. + - R 5 ATCC VR787 - + + ·  -

The R 5 recombination is sustained by two limers. thin passages (in SPF chicken embryos) to eliminate any potentially harmful. avian agents and other passages to obtain a substantial amount of recombinant R 5 virus. The obtained allentic fluid is supplemented with peptide to a final concentration of 5% and lyophilized.

Example ‘

Starting from the lyophilisate obtained in Example 1 (ie Brigit strain, ATCC VR786) as an inoculum. for large scale vaccine preparation, another passage is performed in an allantoic fluid for another series of chicken germs which are incubated for 3 days at 35 ° C.

The allantoic fluids are obtained, pooled, tested for sterility and safety, mixed with peptone as a stabilizing agent to achieve a final peptone concentration of 5% and distributed in 3 ml glass vials to obtain the desired number of virus units (i.e. at least

10 'EID (q). The product is then lyophilized and the vials are sealed. ·

This passage level is used as a batch of vaccine. The contents of one vial are reconstituted by adding 0.5 ml of water, saline or 5% sugar solution and administered as drops into the nasal cavities.

Alternatively, preparations containing allantoic fluids and peptone are dispensed into larger glass vials to obtain a virus dosage unit corresponding to the bmltildvaccine vaccine preparations.

Vaccination with attenuated influenza virus type V (Brigit strain).

Clinical trials were performed on 46 volunteers with a pre-vaccination HI titer (hemagglutination inhibition) against influenza B virus of less than or equal to 32. Each volunteer sewn! two intranasal administrations within 8 or 14 days. During each application, they were given a Brigit strain with a concentration of 10 ' EID &apos; (5 drops into each nasal opening of a freshly reconstituted vaccine in 5% aqueous sugar solution). Serum changes were observed in 40 cases (ie 87% of cases); only mild clinical reactions (runny nose or nasal congestion for 1-2 days) were observed. Influenza virus vaccine transmission was not observed in experiments in which co-groups were available. Attempts to isolate the virus from cervical or nasal swabs were unsuccessful, after two blind passages on chicken embryos the samples remained negative. .

Example 1 - d * 5 * '

Starting from the Lyorlizate R 22 strain (ATCC VR778) obtained in Example 2 as a batch inoculum for large-scale vaccine preparation, the next passage is performed in allantoic fluid for another series of chicken embryos which are incubated for three days at 35 ° C.

Allantoic fluids are isolated, decanted, tested for sterility and safety, mixed

With peptone stabilizer to reach a final concentration of 5% peptone and distribute 7 into 3 ml glass vials to obtain the desired amount of virus (i.e. at least 10 ' EID &lt; 6 &gt;). The product is then lyophilized and the vials are sealed.

This passage level is used as a batch of vaccine. The contents of one vial are reconstituted by adding 0.5 ml of water, saline or 5% sugar solution and administered as drops into the nasal cavities.

Alternatively, preparations containing allantoic fluids with peptone are distributed into larger glass vials to obtain dosages of virus units corresponding to multi-dose vaccine preparations.

Vaccination with attenuated R 22 influenza vaccine strain type V

Material and methods

Six volunteers with an antibody HI titre of less than or equal to 32 were selected for the experiment. Two subjects were used as control, 6 cases received two to 14 days of freshly reconstituted vaccine, R 22 strain, at 1 to 14 day intervals. At each administration, all subjects received 5 drops of vaccine (10 µ * EID 4 q).

Blood samples for antibody determination were taken on the day of vaccination and 10 and 14 days after the first inoculation and 14 days after the second administration. Nasal swabs for local antibody determination were collected 14 days after the second inoculation.

Clinical symptoms ₄

None were recorded.

Virus isolation

Nasal swabs were collected on days 3 and 6 in three cases. After two blind passages in chicken embryos, all samples remained negative.

Serological results and local antibodies

In 5 of the 6 volunteers, neither serum nor. nasal reactions or combinations thereof. The results are summarized in Table III below. T a b u 1 III Subject Serum titre (HI) Serum titer (HI) Local antibodies Local antibodies before vaccination after the 2nd vaccination before vaccination (SN) after 2nd vaccination (SN) 1 32 32 ' 2 24 2 8 8 2 2-4 3 32 64 2 12 4 8 32 of 2 2 5 8 8 ND ND 6 8 16 ND ND

ND = not determined

202544 8 ·.

Example 6 * '.

Starting from - the lyophilisate obtained in Example 3 - (7 5 strain, ATCC VR787), as a large-scale inoculum for the preparation of a vaccine, the next passage is carried out in allantoic fluid of another series of chicken germs which - are incubated for 3 days at 35 Noc: 2 ° C. .

The allantoic liquids are obtained, combined, tested for sterility and safety, mixed with the peptone as a stabilizer to achieve a final concentration of 5% peptone and distributed. into 3 ml glass vials to obtain the desired number of virus units (i.e., at least 10 EID ₁₀ ). The mixture is then lyophilized, the vials sealed or tightly closed.

This passage level is used as a vaccine batch. For adninnasraci. For the vaccine, reconstitute the contents of one vial by adding 0.5 ml of physiological water or 5% sugar. Alternatively, preparations containing allantoic fluids and peptone are distributed to the nasal cavities. larger glass vials, in order to obtain a dosage of virus units corresponding to the multidose vaccine preparations. Example 7

To a single dose of vaccine (Brigit strain) ATCC VR776), an influenza B virus prepared according to Example 4, is added a dose of live, attenuated influenza A virus (Alice strain, ATCC VR776) in 0.5 ml of 5% - of a sugar aqueous solution. The thus - obtained bivale ^ ^<rs vaccine is given - such as drops. of nasal cavities - and administration is repeated after 14 days.

Clinical trials were performed on 22 volunteers having a pre-vaccination HI titer (hemagglutination-inhibition) against influenza virus A or B, less than or equal to 64. Each volunteer received two nasal administrations (10? EID ^ q Brigit strain and EID ^ q) Alice strain) with 8 and 4 day interval. .

Changes - in serum against influenza A virus observed in 80% of cases, against influenza B virus in 73% - cases.

Only mild clinical reactions observed. No transmission of influenza virus vaccine. Example.

K - -dávce one (10? 'EIDc ^ _n) lyofiizoovené vaccine, influenza virus type B (7 22 strain, ATCC

5

VR778), prepared according to Example -5, was added a single portion (10 × 5 eid _{q q} ) of live attenuated influenza A virus (Alice strain, ATCC VR776) in 0.5 ml of 5% sugar solution.

The bivalent vaccine thus obtained is administered as drops into the nasal cavities and repeated 14 days later. A change in serum -for -compensation (type B and type A) is observed 16 days after the second administration.

Example 9 -,

A sample of influenza virus type B strain B / HK5 / 72 (ATCC VR791) with a different hemagglutinin compared to the Brrgit strain is inoculated in the allantoic cavity of 10 to 11 day old eggs with chicken embryos (0.2 ml inoculum per egg) and subjected to the three passages to - obtain substantial ^Star íiožžsví virus. The third ^or FONCA - ^{ASA P} e from s ^{to d} and viral sus ^p Enns ^tit it ^iz r is ₀ EID5 ® ¹⁰ / m .. portion (0.2 ml) undiluted suspension SE- 'inoculated allentoické - cavity SPF eggs and chicken ^{c IMI} s ^ro affordable dk ^y s ^{p p} ecifických ^{athžienů); p} o intobaci for 2 hours, ^and c ^h at ³³ ° C was inoculated in the same part of the eggs (0.2 mL) suspension of strain Brigit (obtained - in Example 1) with ^Ti-1 Treml KDC ⁰ ^ II.

. . '. 9 /. 202545 ·

The eggs are then incubated for 16 hours at -33 ° C to obtain allantoic fluids.

a portion (0.25 ml) of rabbit. sera>, of the an.ti-Brigit strain, heated previously · 1 hour .. · to 56 ° C, are mixed with an equal volume of normal. sera. Portions (0.25 ml) of this serum are added to the allantoic fluid obtained. . The mixture is then held at 37 ° C for 1 hour. SPF eggs with chicken embryos are inoculated with 0.2 ml of this mixture and incubated for 48 hours at 33 ° C. The allantoic fluids of each incubated are collected, passaged in SPF eggs with chicken embryos (0.2 ml · per egg) and incubated for 72 hours. at 33 ° C. Two eggs per sample are used.

After 72 hours of incubation, positive allantoic fluids are isolated and tested for:

- anti-Brigit serotype and anti-B / HK / 5/72 serotype, '.

- growth in eggs and AOS (allantoic memiban in shell).

One of the virus suspensions thus obtained, showing serotype B / H // 72 of the parental strain a. infectivity of the Brrgit parent strain in the AOS system is called R 75. (ATCC VR789).

Rekomminant 7 75 is subjected to two passages in the SPF of chicken embryos to obtain a substantial amount of recombination 7 75.

The descendant of Table IV summarizes the recombinant description and compares it with Brigit and B / HK / 5/72 parents.

Table IV Strain Serotype EID5 ₀ / AOS / eemggluUininsvá // mmggiuUsing sheep Brigit B, K / 5/72  ID50 thermosensitivita erythrocytes at 30 ° C Brigit +. - · + + ’ unstable В / ШК5 / 72  - + , - '-. stable recombinant - + · ' '- - · stable * 7 75

Př.í.klaao. .. '. The allantoic liquids obtained in Example 9 are tested for sterility and safety, mixed with peptone as a stailase to achieve a final conenenration of 5% peptones, and dispensed into 3 ml glass vials to prepare a dose of virus (i.e. at least 10 µl). q). The mixture is then lysophilized, the vials sealed or sealed.

This passage level is used as a batch of vaccine. Prior to administration of the vaccine, the contents of one vial are reconstituted by the addition of 0.5 ml of water or saline or 5% sugar solution and administered as drops into the nasal cavities.

Clinical trials were performed on 24 volunteers. Each received two nasal doses of a freshly reconstructed 775 strain (ATCC VR779) at 11-14 days intervals. At each administration, volunteers completed a viral concen- tration of 10 &lt; ELD &gt; ELISA saline (5 drops into each nasal cavity). . .

Serological results are collected in the following table.

202545 · 10 ' Table IN Case ' Serum titre (HI) před vakcinačS Serum titer (HI) after the second vaccination , 1, 256 256 '. · 2 16 32 . ', · 3 16 32. 5. '' · <4 32 9 16 32. 13 <4 32 23 8 16 25 4 4 64 27 Mar: 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 16. 66 <4 16

A change in serum (4-fold increase in HI titer) was observed in 75% of cases with pre-vaccination titre 416.

Peak 1 and dl, 1 '·

A suspension of 75 influenza B strain (ATCC VR789) and influenza A Alice strain (ATCC VR777) is mixed with the peptone as a stabilizer to a final concentration of 5% peptone and the mixture _in 7 is dispensed into 3 ml glass vials. such that the content of each virus is at least 10 ' Mixture. The vials were lyophilized and the vials sealed or sealed. Immediately before administration of the vaccine, the contents of the vial are reconstituted by the addition of 0.5 ml of water, saline or 5½ of sugar solution and administered as drops into the nasal cavities.

Claims (9)

SUBJECT OF THE INVENTION A method for preparing a live vaccine from influenza virus for nasal administration, wherein the active ingredient comprises at least an effective amount of a recombinant, attenuated strain of influenza B virus, characterized in that the attenuated and vaccine strain of influenza virus ts in a growth substrate is recombined an influenza B virus, which is preferably a chicken germ material or a kidney tissue culture of a bovine fetus, and in particular an allantoic cavity of the chicken germ, with a influenza virus isolate B and selective pressure isolating a recombinant having at least one characteristic of attenuated parental strain, which does not share with the second parent strain and the antigenic composition of the second parent strain and the recombimant is allowed to grow in the allantoic cavity of the chick embryo and, if necessary, a stabilizer is added and the mixture is lyophilized. . ' no. 202545 2. The method according to item 1 _t wherein the attenuated vaccine and influenza type A virus strain is a strain В Brigit В influenza type A virus (ATCC VR7S6). 3 ·. 2. A method according to claim 2, wherein the recombination is carried out in the allantoic cavity of the chickens. '> · _ 4. The preparation method of items 1 and 3, wherein the isolate is a B / HK / 8/73 isolate (ATCCVR792) and the recombinant is an R5 influenza virus type V strain (ATCC VR787). 5. The preparation method of items 1 and 3 _f, wherein the isolate is a B / HK / 5/72 isolate (ATCC VR791) and the recombinant is an R 22 influenza virus strain of type В (ATCC VR788). 6. The preparation method of items 1 and 3, wherein the isolate is a B / HK / 5/72 isolate (ATCC VR791) and the recombinant is an R 75 influenza virus type V strain (ATCC VR789). 7. A process according to claim 1 wherein the stabilizer is peptone or sugar. 8. The method of claim 1, wherein the active ingredient also comprises an effective amount of an attenuated influenza A virus vaccine that is added after obtaining a influenza B virus recombinant. 9. The method of claim 8, wherein the attenuated influenza A virus is an influenza A Alice strain (ATCC VR776).