DE2337134A1 - METHOD OF MANUFACTURING A FLU VACCINE - Google Patents

Description

Dipl.-Ing. H.Weickmann, Dipl.-Phys. Dr. K. Fincke

. Dipl.-Ing. F. A. Weickmann, Dipl.-Chem. B. Huber

73T7T34

S MUNICH «6, DEN

PO Box 860 820

MÖHLSTRASSE 22, CALL NUMBER 98 39 21/22

AGENCE NATIONALE DS VALORIZATION DE LA RECHERCHE

(ANVAR)

922oo Neuilly-Sur-Seine / France 13, rue Madeleine Michelis

Process for making a flu vaccine

The invention relates to new vaccines or vaccines for control of viruses, in particular to control epidemic flu or influenza diseases. The invention relates to furthermore a process for the production of such vaccines.

Influenza or influenza is an acute infectious disease of the respiratory tract of humans, which is usually epidemic Form occurs and is caused by a flu virus. The illness known as the flu covers many centuries of epidemic diseases that occur explosively in the population and spread rapidly and to a large extent spread large areas.

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Various pandemic or global flu infections have already occurred. For example, between 1800 and 1875, 1889 to 92 and in the years 1918-19, serious pandemic or general flu illnesses occurred. Influenza illnesses occur in waves, beginning in certain parts of the world and then spreading to others. One of the most peculiar features of influenza epidemiology is its periodicity. In North America and Europe, for example, all ZV / ei occur or three years, usually between December and ^Β Λ ε-ary flu outbreaks. A division into three distinct immunological types of influenza viruses, namely Λ, Β and C has been made. The severe outbreaks are almost always caused by the flu virus A. The severity of the general flu epidemics has led to the establishment of the World Influenza Center in London and the American Tribal Investigation Center in New York. These institutes monitor and classify the viruses and carry out appropriate research. In 1933 the influenza virus was isolated and named the original or classic A strain (WS) or PR8 strain. In the years 1946 to 4-7 a new mutant or an antigenic variant became predominant, which contained the name Α variety (A prime) · The first recognized outbreak of the Α variety occurred in Japan and Korea in 1946. The strains of Α cultivars, possibly with a few exceptions, have supplanted the original strains as causative agents of influenza epidemics in different parts of the world.

From 1946-47 to 1957, several different strains of örlppvirus of the Λ variety appeared in different parts of the world, with diseases breaking out that progressed from one part to another. From 1957 to 1958, a new variant of type A, A ₂ , caused widespread infection in Asia. This variant was soon introduced to Europe. This epidemic has been known as Asian flu. The A-

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BATH ORIGINAL

zelnon of these iutanton lasted about Io bio 1 · ί years. Another type of flu virus emerged in 1968, the Horgkcng-type A _HK virus, which reached Europe in 1969 after spreading through the UOA. There, more than 40 million people of various degrees of severity were infected by him, with more than 35,000 diseases resulting in death. The flu is therefore one of the main causes of very significant economic losses as people have to stay away from their normal productive work.

The Hong Kong virus is the predominant subtype of the flu virus, the number one prevails in the world. It is believed, that this was the infectiously active type until at least about 1978 until 1980 will be.

The invention now provides a means against this type of influenza pathogen.

In the description of the present application, the nomenclature is for the various viruses those used by the Virus Subcommittee of the International Nomenclature Committee Is accepted.

In Table I, "Influenza Viruses" below, a partial List of the different types or sub-types of viruses with made of the accompanying designation to which reference is made in the description of the invention.

Table I influenza viruses

A / Hong Kong / _{1/68 (H 5} N ₃ ) A / Hong Kong Io7 / 71 (H ₃ N ₂ )

A / NT / 60 / I90S (H ₃ N ₂ ) A / £ ngland / 42/72 (H ₃ N ₂ )

A / England / S7S / 69 (H ₅ N ₃ ) A / Pinland / 2/72 (H ₃ N ₂ )

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BATH ORIGINAL

One of the most confusing and frustrating merlanals in the world Influenza virus is its continuous evolution or its so-called "plasticity". It shifts from year to year the virus strain at hand, gradually evolving so that chemical antibodies or in the body through introduction antibodies produced by antigens due to disease or by vaccination with the original strain are increasing become ineffective. The vaccine manufacturers are on a continuous basis A race has begun to compensate for this virus shift, for which new strains are being collected, as in different ones Occur in parts of the world and vaccines are made from them.

The protection conferred by one natural immunity or by vaccination becomes constant by another viral Variant outdated. In fact, the structure of the flu virus appears to vary slightly from year to year, so that the patient who has been vaccinated with the virus of the previous year or has suffered a corresponding illness, against the new variant finds itself defenseless or with disabled defenses. This explains why a patient is already has had the flu, although he may get it repeatedly due to occurrences of different sub-strains is vaccinated. This also explains the fact, so far confusing, that a vaccine that is effective against a certain sub-strain is considered, in reality after inoculation in the patient is no longer effective to the To give the desired immunity, as the patient may already be infected with a sub-strain that was administered to it Vaccine is already one step ahead.

The changeability of the flu strains is evident from the fact that the mutants A _Q , A ₁ , A ₂ and A _{HR have been} known since 1933 and that a number of variants or sub-

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types exist that are sufficiently different from the initial stem, that the natural defenses or immunization of the patient is overcome. As stated above there is currently only one subtype or variant that has established itself in the world, namely the Hong Kong sub Typ. Serological examinations of the population have shown that an infection by a new variant is effective immunized against the previous variants of the same subtype, however, these preceding variants neither against the presently predominant strains nor against the following ones Give strains effective immunity. This strain is therefore one step ahead of the vaccine. Indeed it seems to be such that immunity plays the role of a selection agent that promotes the survival of new subspecies mutants, which then become free and spread to epidemic proportions. These mutants are opposite to that at this point used vaccine more resistant. This is a property which the forerunners do not have. In fact, each new generation of sub-tribes replaces its previous generation and she will not be attacked by the vaccine, at least in part, until she is in turn attacked by a new generation of sub-tribes is replaced.

The gradual and continuous development of a strain to a certain stage, which is very frustrating, is said to be due to the development of the structure, which is responsible for the antigen-determining factors that are specific for the hemagglutinin of the virus that is responsible for the ability of the virus, red blood cells, for \ i coagulate responsible. This is a distinctive feature of the flu viruses and also of the Hong Kong type, which is specifically considered herein. A vaccine that gives the desired immunity has so far eluded the researchers.

A suggested explanation of the gradual described above

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and evolution is based on the theory that it can be assumed that the flu virus has an antigenic area on a viral coating protein which is identified as a small number of amino acid side buttons can be thought of which wcgapringen from the surface.

If the virus strain changes from year to year, then it doesn’t one assumes that the amino acid arrangement extends down to a structure varies, polygamy no longer results in a further possibility of displacement, so that the next change is then in a fundamental one Mutation exists, which has a completely different :: Subtype of tribes results.

It has been found, however, that within the same subtype, the antibodies raised by the antigen fraction of an original strain of the Hong Kong subtype from 1968 are less able to combine or reconcile with the variant which this followed, which, if one follows the above explanation, can be traced back to the change in the amino acids of the antigen. The greater the changes in the amino acid structure, the lower the ability of the antibodies that were active against the antigen fraction of the original strain to combine with the corresponding variants or to reconcile them, so that their ineffectiveness against the subsequent ones Variants, ie, in the chosen. "Example, against the flu viruses that appeared in the world in 1970, 1972, expected from danon vr ^!: -D that they will emerge from 197 ^ etc. to .980, is correspondingly larger.

However, it has been found, conversely, that antibodies are raised in a living being's body as a result of exposure newer tribes are being developed against the past Strains of the same subtype can be effective.

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BAU ORiQfNAL

For the purposes of classification, the strains and the variants of the same subtype are distinguished from one another herein by different degrees of "rank level", with the newer strains being "higher in rank" than the preceding strains of the subtype in question. Conversely, a given strain that preceded the newer strains in the development of the subtype is "inferior" to the latter. The antibodies induced by a given strain are not only effective against the strain itself, but against all strains that are "lower in rank" with respect to the given strain. For example, a recently isolated Hong Kong strain, namely variant A ₁₁₁ , England 42/72, causes the formation of antibodies in humans that

are effective not only against this tribe, but also against all other tribes of the primitive Hong Kong subtype that contain this have preceded or are of lower rank than this. However, it is expected that / 3 those from the tribe A "". England 42/72 raised antibodies against the Hong Kong subtype strains which will appear in nature and which will thus are higher in rank than the tribe A ^ England 42/72, only to a limited extent or not effective at all.

Several attempts have been made to artificially mutate influenza viruses belonging to the above-mentioned A _Q or PR8 strain. For example, Paul Gerber et al

in "Hie Journal of Experimental Medicine 1956 * lc-3, page up to 424 "describes the isolation of a number of J5 variants, each of which is derived in sequence from the above, (being the first variant from the original PR8 strain derived). These variants were obtained by serial runs in the lungs of mice against the previous one Strain had been immunized. Although they have a progressively decreased ability to react with the antisera of the previous variants and the original PR8 virus, they retained their ability to resist the previous variants and the strain PR8 virus antibodies

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to evoke.

However, the techniques used were not sensitive enough to to obtain and characterize the variants under conditions that can be considered reproducible and to exclude the presence of low proportions of other strains along with the described variants. This technique was also fraught with considerable difficulties, particularly in relation to the upright shark management and development of the variants. In all cases it was necessary to document the homologous passage of the virus in the animals by hemagglutination inhibition tests - low antibody titers to begin with. For some variants it was necessary to alternating the virus in immunized and unimmunized animals to get the strain maintain. Although the authors thus received variants that correspond to the character of the previous strains showed, but there is no evidence that these variants were also higher in rank than the tribes, which developed in nature as a result of a natural shift in Pns. There are no signs of it either suggested that this method was suitable to generate mutants that the natural development of PR8 and naturally also anticipate tribes belonging to another subtype.

A closer approximation of the problem of making more effective vaccines can be found in an article by S. Fazekas de st. Groth (Bull.WId.Hlth. Org. 1969, kV, 651, 657). In this article, 3. Fazekas de St. Groth establishes the basics of the virus classification described above. The author also reports that when a lower-ranking virus Pns is cultivated in the presence of 2.5 to 1.5 times the amount of the homologous antibody, the 50% neutralization doses

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clones were isolated which appeared to be mutants, all of which were superior to the parent antigen. The author has therefore suggested that there should be an epidemic Strain should take, should select its higher-ranking mutants and a mixture of these future antigens as a vaccine should use. If one had followed this suggestion, one would have arrived at, at least in theory, a vaccine that would have been better than a vaccine made from the epidemic strain itself.

The practical problem, however, is that the former vaccine is only one step ahead of the latter vaccine which would not be sufficient to account for the amount of time it would take to obtain the mixture of mutants is required. This period could not be much less than what it would take for a natural one Shift mutation of the epidemic strain occurs, so that much of the progress that may have been made is lost would.

The prior art is described, for example, in the following references: Hoyle, The Influenza Virus, Virology Monographs, 4, Publisher Springer New York., 1968 (Library of Congress Catalog Card Number 68-54o69), hereinafter referred to as Hoyle, Francis and Maassab, Influenza Viruses, in Viral and Rickettsial Infections of Man, Editors Horsfall and Tairnn, 4th Edition, J.B. Lippincott Co. An overview of the antigenic changes caused by passage in partially immune animals or in the presence of an immune serum is given in Hoyle on pages 179-180.

The main object of the present invention is, intentionally or systematically, prior to their natural occurrence in nature a number of individualized mutants of the predominant

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the Hong Kong type, the mutants being a targeted one Type of antigenic components are supposed to have. Farther dominant strains of virus are to be formed, which are able to generate antibodies that have an In.:r.unity compared to variants can give, of which anorrjnon that they will occur in nature in the foreseeable future. Furthermore, according to the invention, a dominant stable Mutants are trained. According to the invention, a future antigen is also to be made available and a vaccine which contains such an antigen and which, when injected into animals, generates antibodies which are responsible for this are effective to oppose other sub-tribes throughout the Hong Kong era, namely the past prevailing and future To give protection to tribes. Finally, according to of the invention, a non-virulent strain can be made available that can be used in all stages of a manufacturing process of such Vaccines are safe to use.

The invention makes vaccines available, that are effective against the prevailing and future strains of flu. The vaccines of the invention have antigens differently than the outdated antigens of conventional vaccines that are capable of providing immunity to the prevailing ones Strains previous and notably also against the mutants of the current subtype expected that they will continue their development from around 1978 to 1980, their development is several steps ahead of that of the viruses currently predominant in nature. This represents a dramatic breakthrough in immunology and medicine.

The invention thus provides an antigenic component and vaccines containing this component which are against all and any type of strain of flu in a given one

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BATH ORIGINAL

-Ix-

Grant subtype-ftra an immunity. Through the invention New strains are also made available that are within the same subtype versus the currently prevalent and the future flu viruses rank higher within this subtype era. The effectiveness of the vaccines according to the invention, which are made from such strains will be made by shifting or changing strains like those above were no longer adversely affected.

The invention therefore relates to a process for the production and isolation of higher-ranking strains from a strain of an influenza virus which is capable of a mutation leading to the formation of higher-ranking strains, which method is characterized in that operating cycles are provided, each of the cycles comprising: the preparation of a homologous serum of the virus strain, the determination of the relative dosages of this homologous serum which are adapted to neutralize in vitro the normal development of a corresponding culture of the same virus, the preparation of η individual individual cultures of the above virus in the presence of the homologous serum, the concentration of the virus used in each of the individual cultures being lower than the mutation frequency of the strain in question, the number η of cultures being sufficiently large that on the one hand the product of the above-mentioned initial concentration of the virus and the number η at least ns is the mutation frequency of the cultured virus and that, on the other hand, the probability of the formation of two mutants in the same culture is practically negligible, the dose of the homologous serum used in each culture being greater than that required to achieve the above to produce said neutralization and is selected such that, however, the development of 1 to £ of the above η cultures is obtained, where £ is an integer which is a function of the above mutation frequency and is sufficiently low with respect to η that it is likely that the grown crops as

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Mutants can be considered, and finally the characterization of the individual cultures 1 to £, in particular by comparing the measured titers of the test sera in hemagglutination inhibition tests with regard to the above-mentioned cultures 1 to ρ and the strains of the cultivated strains, and the extraction is of higher rank Mutants of those of the above-mentioned cultures 1 to p_, which have a reduced sensitivity with regard to the inhibitory effect of the reference sera, provided that the differences between these titers and those of the strains are greater than preselected threshold values, the cycles with those obtained in this way being of higher rank Mutants, in particular with the least sensitive mutants of cultures 1 to p_ isolated in the previous cycle, are repeated and these cycles are repeated essentially until essentially in the presence of doses of the homologous serum as defined above There is no further development of significantly different strains from the strain used in the cycle in question.

It is important to have a total in each of the above cycles of the virus sufficient to cause the appearance of a sufficiently large number of mutants. Furthermore, it is essential to work under such conditions that the probability that 2 mutants occur is practically zero. When attempting a culture that contains a mixture of mutants in the above described Conditions to subject a new culture cycle would give erroneous and non-reproducible results without that the person carrying it out would be able to observe them quickly and effectively. It is therefore essential to adjust the amount of to fractionate the virus applied sufficiently, or in other words, a sufficiently high number of cultures at the same time to produce that the possible individual mutants only in

separate cultures occur.

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233713A

In the case of the flu virus, the likelihood of mutation is

in the area of Io when under the experimental conditions used is related to the infectious units of the virus. In the event that the individual individual cultures at the beginning contain no more than about Io 'infectious units of the virus, the probability that 2 mutants will occur in the same culture is practically negligible.

In practice it has been shown that it is advantageous from two to 3 ° o cultures of about Io 'infectious units of the Virus to carry out the production in the presence of the homologous serum.

It must also be emphasized that the previous preparation of the homologous serum of the strain cultivated in each culture cycle and determining the relative doses of this homologous serum which will neutralize the normal development of a culture of the the same virus are adapted in vitro, represent essential steps of the method according to the invention.

The homologous antiviral immune sera are produced by immunizing laboratory animals, for example rabbits. This is done, for example, by inoculation by repeated injections of high doses of the virus by the intravenous route at monthly intervals, and by taking the sera about 1 week after the last injection, the sera being selected according to their titer. If desired, antibody fractions Ig.G can be extracted from the Gesamtseren, c _e as by precipitation with methanol and isolation by immuno-absorption "Vonspraktischen point of view it is preferred, however, to work with total serum, as these extraction techniques are very time consuming .

In order to obtain mutants under the conditions given above, it is necessary, at an earlier stage, to

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define the limits of the serum to be used. This stage includes the preparatory titration of the Serurr.rj and iiodunn die Determination of its neutralizing power. For this purpose 'successive Dilutions of the serurr. In the opposite of variable doses of the above virus in the forr ;. one brought pure and diluted infected allantoin liquid. After a preselected contact time, the remaining 'Virus titrated. In this way it is possible to adjust the dose dea To determine serum capable of neutralizing a given dose of virus.

It then becomes possible to determine the "selection zone", i.e. the interval of serum doses that must be used to to inhibit the development of cultures that do not contain mutants. Naturally, the doses of the serum used are allowed do not exceed those that would neutralize the mutant itself. In practice it has been shown that can be used with advantage on doses of the homologous serum, which are about 1.8 to about 2.5 times the amount from that which is needed to neutralize the initial strains. Only the develop mutants possibly present to the extent that they have acquired a sufficient degree of precedence with respect to the initial strain that they are opposed to the inhibitory effect of the homologous serum of the cultivated strain is less sensitive are.

Except for the fact that * it is convenient to eat in every cycle a large number of virus cultures (several hundred) with an initial infectious dose in the area of etv / a Io for To prepare each of the cultures, it is preferred to work with several serum concentrations within the range previously determined Selection zone are included, these concentrations jev / er'.ls by diluting the initially titrated serum.

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2 3 3 713 Λ

Go to beispielnv; oise ¹ I different dilutions of the serum and groups that sufficient numbers of cultures included in hergestellt- Gegenv.Virt the various respective oerumverdünnungen to mindester :: a Kutante per group of cultures to it.

The mutants have to appear just fine in the cultures thus produced. Obtaining a number of mutant strains in any group of cultures that is zero or that is contrary to significant is due to an incorrect selection of the doses of the Attributed to immune serum. In such a case it is necessary establish new cultures in the presence of adjusted doses of the serum.

It should be noted that repeated application of this cycle to tribes presently ruling in nature will result in the production of has made possible new mutant strains, which with respect to the original strains and also in particular with respect to the strains themselves of the newest strains up to February 1975, those for the last Influenza epidemic are held responsible, have a distinctly higher-ranking character. It turns out that it doesn't is possible to go beyond a maximum degree of rank within a given subtype. Sin tribe that apparently has reached the level of its evolution, should therefore be regarded as practically stable.

With regard to the characterization of the mutants in a selection test any strain assumed to be a mutant must match the strain from which it emerged in cross-hemagglutination inhibition tests with respect to reference immune sera be compared. Such a strain is not considered a mutant unless it is determined to be real has the character of a higher rank or a dominance in relation to the parent family.

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BATH ORIGINAL

ZJ3713A

To determine whether - and to what extent - a particular one Virus strain versus another strain or strains is dominant, an index, namely the hierarchical index, is used according to the invention. The more positive the hierarchical Index of the respective tribe, the higher the rank of the tribe compared to another tribe of the same subtype.

The hierarchical index is derived from certain expressions the actual hemagglutination titers, which are defined below. A homologous titer is the titer of a serum with respect to the homologous antigen. A heterologous titer is the titer of an oerurn for an antigen other than the homologous antigen. The normalized titer of an antiserum against a given antigen is the logarithmic value of the measured one Titer of this serum against the given antigen minus the logarithmic value of the homologous titer. The normalized Accordingly, the titer is the logarithmic value of the ratio of the heterologous titer to the homologous titer. When the antigen is homologous to the serum, then the normalized titer is zero.

The hierarchical index of 2 related stems is the algebraic difference between on the one hand the normalized titer of the antiserum that is homologous to one of the strains with respect to the other strain and, on the other hand, of the normalized titer of the serum homologous to the other strain relative to the first.

From the above it can be seen that the hierarchical index is a measure of the extent to which in comparison a subtype virus strain was developed for another subtype virus strain with regard to the respective immunological properties has or is advanced. It has been determined experimentally

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if the sera used consisted of total rabbit serum by estimating the standard deviations that if the hierarchical index of 2 tribes compared with each other are above 0.4o units, then there is a significant immunological difference between these two strains Properties, especially the antigenic properties, develops. The value of o, 4o is therefore considered to be a measure of a significant Considered difference. It is chosen herein as the determinant value for practical selection purposes.

At this point it should be noted that the term used herein hierarchical index is not an inventive feature of this invention, but merely a suitable measure of the dominance or the rank. Other suitable measures of these properties can also be used. It should also be noted that if the difference in the index is less than 0.4 units, this is not intended to mean that there are no differences in the antigenic properties would be present that would not be brought out by further experimentation. However, it was within limits for reproducibility for practical purposes the value of o, 4 selected.

In the following Table II typical hierarchical indexes are given, which the relationship of a typical strain according to the present invention compared to other strains. This table shows the antibody titers, the normalized titers and the hierarchical indices as well as the mean values thereof.

From the right column it can be seen that the matrix strain shows hierarchical indices against the other tribes that are greater than the threshold value given above.

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2,437

Anti
NT 60/68 Table II -0.06 0.08 Serums Anti
42/72 At '^ i
30 c antibody 0.00 0.00 Anti
HK 107/71 Tit he 3.94 Anti
ENG / 69 -0.63 -0.30 3.41 4.25 Antigens 3.8o -0.09 -o, 39 3.65 3.65 4.25 1968 NT / 60 3.62 3.62 -0.63 -o, 95 3.65 3.1ο 3.5o I969 ENG / 878 3.41
2.99 3.68 Anti anti
NT 60/68 ENG / 69 -o, 39 3.98 2 ^ 87 4.22
3.92 I97I HK / I07 3, o5 3.35
2.87 Anti
42/72 Anti
3 ° c 1972 42/72
- 30 c 3.59 0.00 Serums
Anti
HK 107/71 Normalized
Titer Anti anti
NT 60/68 ENG / 69 -0.08 0.06 o, 32 Antigens «O, ol -o, 33 o, 3o o, 32 1968 NT-60 0.00 -o, 59 -o, 33 -o, 25 -o, 42 1969 ENG / 878 -o, l4 -1.27 0.00 0.00 0.30 I97I HK / I07 -o, 32 -o, 46 -0.63 -0.48 0.00 1972 42/72 -o, 53 -1.11 Anti
42/72 Anti
30 c - 3o c -o, 95 Serums
Anti
HK 107/71 Hierarchical
matrix o, 59 1.27 Antigens ο, οΐ o, 39 o, 95 1968 NT / 60 o, 3o o, 38 0.69 1969 ENG / 878 ο, οο 0.00 o, 78 1971 HK / I07 -0.38 -o, 78 0.00 1972 42/72 -0.69 + o, l4 + o, 92 - 3o c - o, 19 Mean values

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ORfGlNAL INSPECTED

Ε :? should be noted that the first cycles of culture, carried out on a given initial strain could result in a series or generation of mutants, which, although they can all be higher in rank than the original tribe, can form several families whose members Ilemagglutins can have that to some extent vary from one family to another. It was found, however; which cannot be foreseen due to the state of the art was that the hair agglutins of the new families that were called Result of further and repeated operating cycles with the Mutants of the above-mentioned first families were formed, rapidly approached each other. In such a case it is it is useful for the correct determination of the orientations of the further mutations in cross-hemagglutination inhibition tests compare the mutants of the first successive families with those of the first mutants of the original Tribe that has occurred or occurs in nature to select those of the families or mutants, which appear to be closest to the first natural mutants mentioned above and these cycles with the selected To carry on mutants. These comparisons are continued until all mutants obtained are only one family that is, the hanging agglutinine of all the links of this family no longer differ significantly from one another. These cycles are then continued until none further development is obtained from families which differ significantly from those to which the cultured Tribe heard.

In a preferred embodiment of the method according to the invention, the strain A / Australia IiT 60 / 6P / ft, N _{2 is} used as the original strain, which is referred to below as NT 60 (A ¹ I ¹ CC ITr. VR 753). v; ird. This Starr.ir. is freely available from the National Flu Center of Frank.eich, which is located in the Fasteur-Inntitut. This 3ta.v.::i turned the. · Infected throat into a; -;

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BATH ORIGINAL

Patients isolated and cultured by culturing a taking place in the seed coat inoculation onto chicken embryo in the egg ,, an incubation at 37 ⁰ C, a decrease in the allantoic fluid and a repeated passage through chick embryos, or other conventional method described in the book by Hoyle method whereby highly infectious solutions were obtained. The latter were then subjected to the process according to the invention under the conditions described.

In the first cycles of the procedure, a first generation of 150 mutants was obtained in the presence of homologous sera. These mutants were identified by their antigenic properties, which differed slightly, and were divided into several Families classified. From these families those were chosen whose generic spectra came closer to those of strains which occurred in nature in 1969 and from there were isolated. In Table III, the respective effectiveness of the antisera from representative strains is exemplified shown by two different families A and B versus the natural original strains HK 68 (NT60) and England I969. The immune antisera of family A - although these are more effective than the HD 68 antiserum - but are slightly less effective against England 1969 strain than the antisera of the family B. The latter strain was therefore chosen for the development of future generations.

Table III

Rabbit immune serum antigen

60 Anti HK 68 -M- Anti A Anti B NT 69 5, 5 7.6 7.6 CLOSELY 4,
U 1
00807 6.9
-e-2-1 7.3

Titers are reported as log 2

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In a similar manner, a second generation of mutants was cultivated in a multiple cycle. The mutants of this generation were already closer to each other, several of which did not appear to be significantly different from the strain HK Io7, which was isolated in nature in 1971. Further cycles of culture yielded new mutants, none of which were significantly different from one another. The orientation of the mutation was thus established. Further cycles resulted in further higher-ranking mutants, among which, for example, the following strains were 29 C / M ^ and J> ¹ \ C / M ^ in succession. The final stage resulted in a family or group of strains, of which the most senior or dominant strain was 3 ° C, also referred to as 30 - C or J> o C / X5 (ATCC No. VR 760). This has been found to be stable under the conditions given below.

Obtaining mutants with a hemagglutinin that does not was significantly different from that of strain England 42/72 is, a year ahead of time confirms the value of the chosen orientations. The tribe Jo c therefore has as outlined below will have antigenic properties not significantly different from those of the latest strain which can be expected in the latter part of the Hong Kong era.

Thus, if necessary, encompasses the method of the invention according to a further feature of the present invention in the first of the above-mentioned cycles and up to the point in time where the different mutants formed and obtained in a culture cycle essentially in terms of their antigenic properties be similar, comparing each of these mutants with those which are derived from the original strain and which are in the Nature occur, whereby the above-mentioned cycles are repeated with those mutants which are closest to the bex above comparison natural strains used.

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Thus the invention provides a method of man-made production of strains available, that have antigens that several generations with regard to the last strains that have recently appeared and been isolated in nature are ahead.

Such strains are cultivated according to the known methods. The following recognized practices are useful in connection with the invention, reference being made to the appropriate page number of the Hoyle textbook.

(1) Cultivation in the allantoin bag, page 29

(2) Cultivation on the outer surface of the chorioallantoinic Diaphragm, page 28

(5) Hemagglutination Reaction, page 81

(4) Inhibition of the hemagglutination reaction, page 95

(5) Complete fixation test, page lol

The invention also relates to the strains produced by the Processes according to the invention are formed, and all of which are superior to the tribes predominating in nature. The strains of the invention are related to the Hong Kong subtype, what is the result of their ability to raise antibodies against all natural strains of the Hong Kong subtype, which have been isolated and which may occur in nature are effective. The strains of the invention are expressed in a consistent hierarchical order in terms of rank with respect to the natural tribes and among each other characterized. In particular, they all rank higher than the comparative strains in Table IV. These were due to their antigenic diversity selected, as they are by the cross-inhibition reactions of the strains of the invention and the Reference strains and their respective sera are shown. The homologous sera of the reference strains are given below

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referred to as reference sera.

Table TV reference sub-stems

Tribe name abbreviation

(1) A / AUSTOALIA / NT6o / 68 / H, N ₀ ΝΤβο

(2) A / AICHI. / 2/68 / H ₃ N ₂ AICH

O) A / ENGLAND / 878/69 / H ₃ N ₂ ENG

(4) A / HONG K0NG / lo7 / 7l / H ₃ N ₂ HK Io7

(5) A / PARIS / 22 / 7l / H ₃ N ₂ DJEM

(6) A / PARIS / 21/71 / H ₃ N ₂ ALV

(7) A / AUBERVILLIERS / l / 72 / H ₃ N ₂ LASN

(8) A / ENGLAND / 42/72 _{/ H 3} N _{2 42/72}

The terminology used corresponds to the terminology of the National Influenza Center at the Pasteur Institute.

The following reference strains are of particular significance: NT60, ENG 69, HK Io7 and 42/72.

One especially for a first group of strains according to the invention representative strain is strain J54c / Mj5. The following Table V shows the inhibition titers of hemagglutination which measured relative to strain 34C / MJ5 in some of the reference sera have been. The other strains of the same family show hemagglutination titers sufficiently close to those of this one selected representative tribe so that they can be considered to have the same degree of rank.

409807/1021

- 24 -

Table V

Serums

mean titer of the antibody titer of the serum with respect to the homologous strain

NT 60 AICH ENG 69 HK I07 DJEM ALV
LASN.

3.29 3.44

3.14 2.92 3.83

3.79 3.74 3.96 3.81

3.99 3.87 3.81 3.70 3.9o

The titers are given as log _lo .

The strain 29C / M3 is for another family of the invention Mutants representative. Table VI gives a number of measured titers of some of the reference sera.

Table VI

Serums

mean titer of the antibody titer of the serum with respect to the homologous strain

NT 60

ENG 69 HK Io7

3.65 3.68 3.68 3.38 3.90 3.80 3.5o 3.96 3.81 3.99

3.87 3.81 3.7o 3.90

The titers are in logarithmic units

) specified.

The 3oC / W3 strain is for a third family of the invention

409807/102 - 25 -

Mutants representative. In Table VII, measured titers of some of the reference sera against 3oC / M3 (or 3 ° C) are given.

Table VII

Sera mean titer be the titer of the serum

Antibodies in addition to the homologous

Tribal

NT 60 5.08 3.96

AICH. . 3.17 3.81

ENG 69 3.05 3.99

HK I07 2.36. 3.87

AJEM 3.14 3.8l

ALV · .2.99 3.7o

LASN 2.84 3.9o

The titers are given in logarithmic units (log ^).

Table VIII shows the hierarchical matrix of the hierarchical Indices of the strains according to the invention and the reference strains in comparison with one another, the values given being based on the Partly derive from the actual titers given in Tables V, VI and VII. Such a representation enables it is to read directly the degree of rank of a given tribe. The degree of rank of any tribe can be found in the last row of the hierarchical matrix.

The most positive value of the hierarchical index shows the minimum response to the homologous sera of the other strains at. The most positive value therefore indicates the highest level of rank. In the first column from the right corresponds to the highest Worth the most effective vaccines. From this it can be seen that the 30C / M3 strain is the highest ranking strain and the best vaccine results.

409807/1021 - 26 -

The variant J5oc is a representative example of a family or class of strains which is stabilized to the extent that attempts have been made to cultivate this strain in accordance with the invention Subject to procedures do not result in mutants whose immunological properties are considered to be of Joe can be considered significantly different. the hierarchical indices of the tribes in positive cultures that, if any, are preserved in such cultures can, are in all cases below 0.4o units. The hemagglutinin values need to be practically not different be considered by those of 3> oc.

Thus, in other words, the tribes of the family or the Class that are considered equivalents than those Strains are defined, their hierarchical indices in cross titrations in the inhibition of hemagglutination tests in relation to 3oc are below 0.4o units (if their corresponding homologous sera from rabbit sera).

The equivalence of the other phyla with the ^ oc class of phyla according to the invention is justified by the fact that this class of strains induces the formation of antibodies, those with regard to the strains HK / 68 and the pandemic strains of I97I / 1972 (England / 72) and also against the strain itself Inhibit hemagglutination.

As can be seen from Tables II and VIII, the hierarchical index of the strain is; 5oc (or of strains of these Class) compared to the strain ΝΤβο 1.27 (Table II) or 1.16 (Table VIII). These tables refer to different series of measurements. Taking into account the deviations that occur in different chewing processes can be observed, then the hierarchical indices can be compared to ΝΤβο as approximate denote l, 2o + o, 2.

409807/1021

- 27 -

233713A

As can be seen from Table II, the strain Joe is still considerably ahead of the recently isolated natural strains such as A / ENGLAND / ^, 72 / H, N _2.

The tribes of the Joe family rank higher than any of the tribes or dominant, currently occurring in nature and believed to be until at least 1978 to I9B0 occur in nature and which may be considered representative Examples of the last stage of the Hong Kong subtype can be viewed. Thus there is the possibility that the hemagglutinin part of the last tribes, which develops in this era, approaches that of the ^ oc-Stamroes, or is closer to each other even essentially does not differ from it. Thus, a preferred embodiment of the invention with respect to the Hemagglutinin antigen of this sub-type as synonymous with the artificial generation of the final stage of the development of the Hong Kong sub-type be considered.

Class 3oc strains thus include future hemagglutinins, which can be regarded as essentially stable.

Other characteristic properties of the Joe class strains reside in that they are essentially non-virulent and thus have very reduced pathogenicity. V / pus Properties reside in that all of its antigens other than the hemagglutinatinin antigen are essentially the are the same as the corresponding antigens of the Νϊβο strain and that they are resistant to non-specific inhibitors.

A09807 / 1021

Table VIII

Hierarchical matrix of cross-reactions. Tested antigens

- 28 -

. ■■! Cren NT 60 AICH ENG 69 HK Io7 34C / M, 29C / M, DJEM ALV LASN 30C / M, mean value

MT 60 -0.0 0.63 -o, 29 0.09 0.95 o, 38 I, o5 1.14 0.72 . 1.32 0.66 AICH -0.63 0.0 -o, 54 -o, 12 o, 54 0.06 o, 42 0.60 0.45 I, o5 o, 2o ENG 69 o, 29 -o, 54 0.0 I, o8 o, 77 o, 39 0.88 0.95 0.62 1.41 o, 77 HK 1ο7 -0.09 o, 12 -1.08 -0.0 o, 51 o, 24 I, o5 1.26 I, o2 1.35 O, 49r 34C / M ^ -o, 95 -o, 54 -o, 77 -o, 51 0.0 o, 15 o, 15 o, 28-o, o2 1.34 -o, lo 29c / M ₃ -o, 38 -0.06 -o, 39 -o, 24 -o, 15 0.0 o, 42 0.38-0.18 I, o8 o, o5 DJEM -I, o5 -o, 42 -0.88 -I, o5 -o, 15 -o, 42 0.0 o, 21 0.03 0.87 -6.32 ALV -1.14 -0.60 -o, 95 -1.26 -o, 28 -0.38 -o, 21 0.0 -o, 49 0.80 -o, 5o LASN -o, 72 -o, 45 -o, 62 -I, o2 o, o2 o, l8 -0.03 o, 49 0.0 1.22 -o, lo 5? 30 CM, -1.32 -I, o5 .-1.41 -1.35 -1.34 -I, o8 -0.87 -o, 8o-l, 22 0.0. -1.16 ' middle value -0.66 -o, 2o -o, 77 -o, 49 -o, lo -0.05 o, 32 0.50 o, lo 1.16

The values are given in logarithmic units (log)

409807/1021

- 29 -

ro CO Ca)

CJ

Strains contemplated according to the invention are also those that in a combination or recombination of the strains according to the invention with virus strains of different Types result. Thus, the hemagglutinating antigen - its significance for the production of vaccines What is essential is - the strains according to the invention with known strains like Pns, WS, MEL, FMI, SI, Jap / 3o5 / 57, SWINE and others or Mutants or derivatives thereof are combined or recombined. The combination or recombination of the invention "Strains" with other viruses allows the production of strains which, in addition to the desired immunogenicity, also have preselected others Possess properties. The recombination can offer changes, e.g. a sufficient growth rate in the Cultivation cycles to allow for the massive manufacture of vaccines or alterations in the antigenic components of others than the hemagglutinin antigen - the strain etc. The recombination is advantageously carried out using the classi-r see methods carried out by Tumova and Pereira, Virology, 27, 253, 1965 or by Kilbourne et al., Perspect. Virol., £, 87, 1967.

The invention particularly relates to the recombinant strain ATCC No. VR 752 obtained by coculturing the 3 ° c and the classic PR8 strain is obtained in the egg. The recombination strain mentioned consists essentially of the PR8 strain, however, its hemagglutinatin part replaced by that of 30c has been replaced.

The recombination strain according to the invention thus has the immunological Properties of the jtoc-type family of the invention. It has the ability (like the PR8 strain) to grow under comparable conditions with higher yields than a trunk of the type

The invention also relates to vaccines made with the above future and dominant antigens are formed. These

409807/1021

- 3ο -

Vaccines are thus able to protect against the variants of the Hong Kong subtypes that will appear in nature in the future years by the end of the Hong Kong era, probably no earlier than 1978 »more likely 1980, immunity to occur to lend.

More details about the invention are given below Procedures listed for the respective operating cycles in connection with preferred embodiments of the invention:

A. Isolation and culture of the initial viral strains

The initial virus strain, particularly the NT60 strain or a selected strain that is superior to NT60, and which in a series of operating cycles according to the method of of the present invention, with the first cycle applied to NT60, is first cultivated as follows:

Embryonated chicken eggs which have previously been incubated for 11 days are mixed with 0.05 ml of a suspension of the allantoin route concerned virus inoculated, which io to lo- ^ infectious Contains units of 50 # / ml. After a 24 hour incubation at 35 ° C the allantoin liquids are taken up separately and titrated in hemagglutination inhibition tests. Those whose Titers higher than looo are collected, filled into ampoules, frozen and stored at -7o ° C. The infectious titer, which is determined on the chorio-allantoic membrane, must go beyond 10 "infectious units 50 / £ / ml. The cultivation the virus can also be carried in calf kidneys in a known manner will.

B »Production of homologous immune sera

The homologous antiviral immune sera are obtained by immunizing rabbits. You will be having a first injection

409807/1021

- 31 BAD ORIGINAL

by intravenous route with 5000 hemagglutination units des Virus inoculated and a month later reinforced with a second injection of 10000 units. The rabbits will bleed out and the sera are 4, 6, 8, io and 12 days added after the second injection. Each serum sample should be in the region of 20 ml. The serums that are the highest Haema ^ glutination inhibition titers have been obtained and collected.

C. Determination of the neutralization titer of the serum for the virus

o, 25 ml of a solution of the serum, diluted in a special nutrient medium, which is referred to below as "SM medium" (in the ratio of o, lo ml of serum per o, 9o ml of SM medium) are at O ⁰ C in the individual tubes of 4 rows with 8 tubes each. introduced. Each tube contains 0.5 ^ ml of the SM medium. The sera are diluted in each of these series of tubes so that increasing 3.16-fold dilutions are obtained. In this way, dilution ranges from lo ~ '-> to lo ~ - * of the original serum concentration are obtained.

That described in J. of Hygiene, Vol. 56, No. 1, page 151 SM medium has the following composition:

NaCl 8.0 g, KCl 0.6 g

CaCl ₂ 0.8 g MgCl ₂ 0.05 g glucose 0.3 g gelatin (acid-free) 2, ο g chloramphenicol o, lg phenol red (o, ol £ solution) 25 ml

409807/1021

Distilled and demineralized water to 100 ml

Before use, the pH is adjusted to 7. o with sodium hydroxide solution and the medium is sterilized for> o minutes in an autoclave at 115 ° C.

o, o5 ml of the initial highly infectious allantoinic solution (titer greater than lo "IDc ₀ ZmI) are poured into the tubes of the first row. o, o5 ml of dilute solutions of the virus, the concentrations of which are Io, Io ^ and Io ^J the beginning - Corresponding to the virus concentration are entered into the tubes in the second, third and fourth row, respectively.

After incubation for 15 minutes at 0 ° C, 0.05 ml of des Medium of the individual tubes then in contact with o, jk> ml of the SM medium in question are inoculated onto chorio-allantoic membranes attached to the shell pieces. These operations are carried out on bowls, each containing 8o cups, which can be locked and brought to 37 ° C.

The dishes are sealed and placed at 35 ° C for 72 hours Shaking incubated. The shell pieces are then removed from the individual preparations contained in the cups and in each beaker, 0.25 ml of a 5 # solution of red Given to blood cells from chickens.

Examination of the beakers allowed after being allowed to settle for 25 minutes of the cells at ambient temperature, the determination of the neutralization concentration of the serum in relation to that indicated above Dose of the virus.

D. Determination of the selection area

Using the same technique as above, a large number of cultures are prepared in order to determine the total amount of virus that the

409807/1021

Selection test is subjected to fractionation: Individual

Cultures less than Io, preferably no more than

Io ', containing infectious entities, are in the presence of a homologous immune serum prepared in concentrations determined in the previous step, i.e. from about 1.8 to about 2.5 times the neutralization concentration of the serum. These cultures are within the above limits carried out in the presence of at least 4 serum concentrations.

The selection zone is created by using, for example, at least 4 bowls each with 8o cups. This makes one Cultivation of a total of 4 χ 8o χ Io 'infectious units. The reaction is then fractionated into 52o separate systems, this fractionation on the one hand measuring the frequency of occurrence of the antigenic mutants sought and on the other hand, the separate insulation is made possible. There is a risk that two mutants will grow in the same cup practically zero. The mutation frequency that occurs in the

q search conditions are determined, includes about 1. to about 5.Io, it should be taken into account that each dish should have a small proportion of positive cultures. If someone has a greater number of positive cultures than 5 of these, then this can be discarded as it is unlikely that that the cultures that have grown are mutants.

The operations or modes of operation, the principle of which has just been described, can be carried out, for example, as follows, assuming, for example, that the neutralization titre of the serum is related to the amount of the cultured virus that was determined in the previous step (determination of the neutralization titer of the serum with regard to the virus) corresponds to a dilution of 800. Experience shows that the selection range normally lies between the dilutions 8oo = 548 and 8oo = 445.

409807/1021

4 bowls with 80 cups are made, each with a chorio-allantoic membrane attached to the corresponding Shell piece is attached.

Dilutions of serum in 4,5o ml of SM medium v / ground prepared in tubes so that dilutions of l /.? 4o, I / 370 l / 4LO and I / be 450. 0.50 ml of the allantoic solution of the highly infectious virus (which contains Io 'infectious units of the virus) is added to each of these tubes, which had previously been placed in an ice bath. The tubes are ^{incubated at 0} ° C. for 30 minutes.

The beakers of the 4 dishes (one dish per dilution) are inoculated with 0.05 ml of the virus solutions obtained in each case. The dishes are sealed and incubated with shaking at 35 ° C for 72 hours. The shell pieces are then removed and 0.25 ml of a 5% solution of red blood ropes from chickens are placed in each of the beakers. The dishes are then left to stand for 35 minutes.

If the dilutions chosen are to be appropriate, then at least some of the dishes should have 1 to 5 cultures develop (namely in those in which agglutination is observed). If there is no culture in a given bowl developed, then the corresponding dilution that was selected was too low. If there are more than 5 cultures have developed, then the corresponding dilution was too high. If necessary, the measures described are included Repeated a readjusted series of serum dilutions until dishes containing only 1 to 5 positive cultures are obtained contain.

The strains that have developed in the latter dishes are recovered by removing the liquid and the agglutinated red blood cells themselves are taken away.

409807/1021

- 35 -

The isolated menses of the virus are generally too low than that the following identification test could be performed. They are therefore first as described above under A, in one cultured in an embryonic egg or in a calf's kidney until the hemagglutination inhibition test gives a titer equal to is sufficient to carry out the subsequent characterization of the mutants.

E. Characterization of the mutants

The characterization of the mutants comprises the comparison of the individual strains isolated in the above manner which are identified as one Mutants are considered with at least 3 reference strains, in particular with the starting strain, i.e. with the strain from which the assumed mutant was obtained under the specified conditions has been. The characterization further includes a comparison with two heterologous strains which, however, belong to the same family or belong to a class like the parent strain above. In this comparison are hemagglutination inhibition tests with immune sera provided, which have been established opposite the parent strain and the neighboring strains.

The following table summarizes typical results that were obtained.

Test sera

Tribes homologous starting
tribe Heterologous
tribe Heterologous
tribe Isolated trunk
number 1 5.9 3.5 3, o Isolated trunk
No. 2 5, o + 3.6 3, o Isolated trunk
No. 3 5.3 + 2.5 + 2.9 Output level 6, ο 3.5 2.9 A098 ° 07/1021 3.7 2.9

(The titers are in logarithmic units based on 2 expressed; the tests were always carried out twice).

This table gives an example of J> strains that are derived from a selection test and that may be mutants. Comparison by cross-hemagglutination inhibition tests with immune sera prepared against the reference strains shows that the three isolated strains are not identical. If the statistical and experimental deviations are taken into account, strain no. 1 behaves like the starting strain, so that it cannot be one of the antigenic mutants sought. However, strains No. 2 and 3 are less sensitive to the homologous antibodies of the parent strain and could therefore have the desired quality. In addition, it can also be stated that these two mutants are not identical to one another, since No. J5 is less sensitive than No. 2 (and the parent strain) to heterologous antibodies corresponding to one of the reference strains.

The mutants whose characteristics are most distant from those of the parent strain and those opposite are less sensitive to the inhibiting effect of the immune serum and are therefore considered to be of higher rank than the original strains will be retained.

In the table above, those titers are indicated by plus signs which indicate the possibility that the corresponding strains have a pronounced character of rank with regard to the starting strain, whereby the difference between the respective titers and the titer of the test serum with respect to the starting strain ( or the parent strains) can be observed. When cen-described experimental conditions, can only be seriously believed that one isolated strain is a mutant when between the

Sow // -j £

- ΎΙ -

Titers of at least one of the reference sera with respect to the isolated strain and from one or more output strains one significant difference greater than 0.84 log units on the basis of 2 or if the titers of at least J5 reference sera with respect to the isolated strain and the parent strains significant differences greater than 0.42 logp units exist.

The tribes that seem to have the most senior traits are then selected and the homologous immunoscres of these strains are prepared under the conditions set out in Part B above (Production of the homologous immune sera) have been described. These imperfections permit a stricter identification and Characterization of the isolated strains in cross-serological studies Tests that then determine the hierarchical index of these strains with respect to the parent strains and with respect to of the strains that have passed in the operating cycles prior to the method according to the invention.

The trunk selected at the end is then in the presence of a previously certain - dose of the homologous serum which normally has to inhibit the development of this strain, but without that which, if any, higher-ranking mutants that might appear in the middle of the corresponding cultures is inhibited again subjected to fractional culture.

By using the procedure described, it is possible, starting from the stem ΝΤ6ο, to end up with the stem ^ oC / fl ^ or; get 5oc. The strains J54c / M3 and 29C / M3 only represent Are intermediate strains isolated in the course of this procedure.

F. Production of a recombinant strain of j5oc and PR3 (ATCC No. VR 752)

The two strains were in the egg under A (isolation and

409807/1021

- 33 -

2 3 3 71 3 A

Culture of the initial stems e) conditions like one: .. m cultivated.

The viruses obtained consisted of ΡΠ8, j5 ° C and the fast growing hybrid of 3 ° c and PR8.

Then, according to the procedure under B (production of homologous irr.rr.our) procedure described the PR8 homologous Inirnour. ·. manufactured. The neutralization titer of the serum for PR8 was essentially under conditions C (determination of the Neutralization titer of the serum with respect to the virus).

A sample of the culture obtained was subjected to a cloning operation in the egg at a limiting dilution with the anti-Pns rabbit immune serum, i.e. in the presence of the amount of serum that inhibits the development of Pns. The trunk won in the end passed essentially from the hybrid PR8-3oc, which has 3 ° c hair agglutinin. It contained small, if any, amounts of 3oc since the latter grows considerably more slowly than the hybrid.

The invention also encompasses vaccines and vaccine preparations made from the above strains, in particular from any strain containing the hemagglutinin of the 3oc class strains. The trunks can be made according to the conventional Methods treated to obtain vaccines and in the form of to control flu, especially human flu, administered in a suitable form of preparation.

The vaccines of the present invention are effective for humans against past, currently developing pathogenic strains of flu as well as against strains that end up in nature the Hong Kong era will appear to protect. Such vaccines are used in amounts that are effective in eliminating error by causing sufficient antibody production for this purpose.

409607/1021

- 39 -

ORIGINAL INSPECTED

Below are some non-limiting examples of Vaccine preparations given:

- Vaccines formed from suspensions of the aqueous or oily injectable sterile medium of purified and inactivated virus (by formaldehyde, ß-propiolactone or another inactivating agent is inactivated). Each dose of the injectable vaccine can be 15,000 to 60,000 uIIA units, e.g., 2o, ooo uHA (or 9oo ul) units contain. The strains can be pure or in combination or mixtures with other strains with or present without auxiliaries.

- Inactivated vaccines, formed from aqueous suspensions of the type mentioned above, but in spray form or in instillation form for nasal administration, or for oral administration.

- Vaccines that contain viral subunits that have been obtained by combining the purified virus with compounds such as ether, sodium deoxycholate, sodium dodecyl sulfate or another detergent or with a lipid solvent compatible for the intended purpose has been.

- Weakened live vaccines, in which the characteristic antigenic properties have been retained.

It is noteworthy that the recombinant techniques for living or weakened vaccines according to the invention which contain, for example, live viruses which at body temperature (such as up to about 37 to 40 ° C) to grow are incapable, but the immunological spectrum of the present Invention. The recombination can be done according to the classical

4 0 9 8 0 7/102 '

- 4ο -

Methods from Tumova and Pereira or Kilbourne et al as noted above can be obtained. Reference is also made to "The Recombination Phenomenon" on page 16l of the book by HoyIe .

The following test values demonstrate the effectiveness of the invention Vaccines.

Various series of mice were made with those in the left Column of Table X specified viruses. Groups of mice selected in each of the series were then called strains inoculated.

Table Io shows that the vaccine Hong Kong / 68 mice gives insufficient protection against the epidemic strain 1972, while strain 3oc is effective against the initial strain and protect the newest tribe. This shows that the 3oc strain is efficaciously superior to the other strains.

Table X

Mice vaccinated with virus following virus following evidence of inoculation were Hong Kong Paris / 72 3oc

Hong Kong / 68 loo 8 23

Paris / 72 117 loo 39

3oc 92 J55o loo

Protection given with the various vaccines over the test vaccine. The comparison with the homologous Protection is set as loo).

Similar results were obtained in ferrets. Ferrets vaccinated with A / Eng / 42/72 were against one recently isolated natural strain, i.e. A / Finland / 2/73 not

A09807 / 1021

- 41 -

- 4l -

233713A

effectively protected. The ferrets vaccinated with J5oc were against it protected against this newest strain as well as against the strain A / Eng / 42/72.

The following clinical tests demonstrate the effectiveness of a vaccine containing formaldehyde-inactivated J5oc, as evidenced by the serum conversion to Eng / 42/72 after vaccination with the 3 ° c vaccine.

95 adults whose blood (as measured in hemagglutination inhibition tests) had an initial antibody titre against Eng / 42/72 of less than Io were vaccinated with one dose of the vaccine (600 µl). Blood samples were taken three weeks after vaccination removed.

Table XI shows the number of people in whom the increases in antibody titers were greater than 2o resp. is. This table also includes the results obtained in 26 unvaccinated adults.

Table XI

Number of people

Increase in antibody titers by more than #

Increase in antibody titers by more than #

vaccinated
not vaccinated

95 26

71
1

65 1

Similar results were obtained in adults who received a spray injector with a dose of 600 µl of the vaccine had been vaccinated. Their pre-vaccination antibody titers to Eng / 42/72 were below Io. Table XII shows that in the majority of vaccinated people the increase in antibody

409807/1021

Titers 3 weeks after vaccination were greater than 2o, "1 and 4o pounds, respectively.

Table XII

Number of increases in anti-increase in anti-person body titcr by body titre by more than 2ofo more than 4ofS

vaccinated 36 35 51

Table XIII shows the results obtained with adults which already showed significant titers of antibodies against Eng / 42/72 before vaccination. The main part of the vaccinated Adults showed more than 4 times the pre-vaccination antibody titers measured 3 weeks after vaccination.

Table XIII

Antibody titer

Number of people more than 4 times the original titer

vaccinated 34 29

not vaccinated 19 0

Corresponding results obtained with adults taking 2 doses of the vaccine 3 weeks apart (600 ui + 600 ui) are shown in Table XIV.

Table XIV

Antibody titer

Number of persons more than the 4-facne of the original titer

vaccinated 36 29

not vaccinated A098fc? 7/1021 °

- 43 -

233713A

Children (4 to 11 years old) were vaccinated with 2 doses of vaccine 3 weeks apart (300 ui + 300 ui). Table XV indicates the number of children in whom the antibody titre against Eng / 42/72 3 weeks after the second vaccination with either the children who had already shown significant levels of antibodies before vaccination - four times greater than before Vaccination or - in children whose original titers were below Io - an increase in antibody titers of more than 4o # had taken place. Table XV also gives the geometric mean values of the titers of the antibodies before and after vaccination at.

Table XV

Number of subjects more than 4 times the geometric mean original titers values of the titers or an increase in the

Titers greater than before after

Io 6.8 82.3

All of the above tests prove the effectiveness of 3oc as effective Principle for the formation of vaccines.

The preceding description has essentially dealt with higher-ranking strains and vaccines based on the Strain NT6o. It is noteworthy, however, that the present invention also enables the production of higher-ranking viruses of the type of flu originating from any other original strain. It should also be noted that the invention is applicable to the production of senior and dominant strains that of strains of new subtypes which may be isolated in the future and which result from a fundamental mutation of the subtype viruses.

409807/1021

- 44 -

stir, which is currently usually distributed throughout the multitude are.

The invention is not limited to the human flu viruses described above. Rather, it is also on animal flu viruses, e.g. of the horse type, such as A / equi / 56, A / equi / 63, A / equi-2 / Miami 63 and others applicable.

As described herein, the invention is applicable to human and animal influenza viruses in general, and it is also on the method of developing strains of viruses using. an antigen that ranks higher than an antigen that is already present is applicable. Finally, the invention also includes such strains and the corresponding vaccines.

It was further described that according to the invention the antigens can be transferred to animals such as ferrets, mice and rabbits as well as chicken eggs and that confer immunity but immunity can also be given to other rodents such as hamsters or other pets such as dogs. Of greater economic importance is the application of the invention to animals that live in herds or groups such as Pigs, horses and cattle. Equine flu is known to be very infectious and spreads in an infected stable on most horses.

A known strain A / Equi / 2 / Miami / 6; 5 is selected to produce a vaccine to control equine flu. It will proceeded according to the procedure of the above examples to develop an antigen-bearing strain, which in the hierarchical index is higher in rank than the selected parent tree. When horses are inoculated with a vaccine containing this advanced strain they will develop Animals antibodies that confer immunity against this disease. ·

-409807/1021

A similar procedure is used to obtain a vaccine from A / Equi / l / Prague / 56 and A / Equi / l / Cambridge / 63, which is suitable for vaccinating horses with the vaccine. For the same purpose, a vaccine is suitable that a Contains mixture of the advanced higher-ranking tribe. More details on the use of the vaccine at of equine flu can be found in U.S. Patent 3,518

It has been shown that pigs are also immune to infection with different types of flu viruses, among which the so-called pig virus falls, are sensitive. Senior tribes that develop from it can be used to to give immunity to pigs. Although it is obvious that a vaccine for humans is greater than that It is important because the use of a vaccine according to the invention for veterinary medicine is also of great interest,

Finally, the invention is also applicable to preparations from the highest-ranking strains which differ from other pathogenic Inferring strains as influenza strains and which are also shift mutations are subject to, applicable.

- patent claims -

409807/1021

- 46 -

Claims (1)

Claims Ql). A method for the production and isolation of higher-ranking strains from a strain of an influenza virus which is capable of a mutation leading to the formation of higher-ranking strains, characterized in that operating cycles are provided, each of the cycles comprising: the preparation of a homologous serum of the virus strain, the determination of the relative doses of this homologous serum which are adapted to neutralize in vitro the normal development of a corresponding culture of the same virus, the preparation of η individual individual cultures of the above virus in the presence of the homologous serum, the concentration of the Virus used in each of the individual cultures is lower than the mutation frequency of the strain in question, the number η of cultures being sufficiently large so that on the one hand the product of the above-mentioned initial concentration of the virus and the number η at least the mutation frequency of the cultivated virus us is the same and that, on the other hand, the probability of the formation of two mutants in the same culture is practically negligible, the dose of the homologous serum used in each culture being greater than that required to produce the above-mentioned neutralization and so selected is that, however, the development of 1 to £ of the above η cultures is obtained, where £ is an integer which is a function of the above mutation frequency, and with respect to η is sufficiently low that it is likely that the grown cultures can be regarded as mutants and finally the characterization of the individual cultures 1 to £, in particular by comparing the measured titers of the test sera in hemagglutination approach tests with respect to the above cultures 1 to £ and the strains of the cultivated strain continued and the extraction as higher ranking mutants from those of the above 409807/1021 - 47 - - hi - Cultures 1 to £ relating to the inhibitory effect of the reference sera have decreased sensitivity provided that the differences between these titers and those of the parent strains · are larger than the preselected ones Thresholds, with the cycles ranked higher with the ones so obtained Mutants, especially the least sensitive mutants of those isolated in the previous cycle Cultures 1 to £ is repeated and these cycles are repeated essentially until in the presence of doses of homologous serum as defined above, essentially no further development of significantly different strains is made from the strain used in the cycle concerned. 2. The method according to claim 1, characterized in that one obtained in the first cycles Mutants grouped according to their hemagglutinins, if the latter differ to a certain extent from one family to another, that one has the different families, in cross-hemagglutination inhibition tests with natural mutants of the original strain that have occurred in nature or that are supposed to occur in nature compares that nan those of the families of the mutants involved in the above appear to be closest to natural mutants, selecting and re-cycling using the above comparison the new mutants formed in further cycles with natural mutants and the above-mentioned selection is carried out, until all mutants obtained at the end of the further cycles form only one family, the mutants Have Ilärnagglutinin which are not significantly different from each other. ; 5 · The method according to claim 1 or 2, characterized in that each individual culture 409807/1021 Beginning of each cycle no more than about Io 'infectious units of the virus. 4. The method according to claim 1 or 2, characterized in that in each cycle 2 to 3oo Cultures with about 10 'infectious units of the virus in the presence of the homologous serum. 5. The method according to any one of claims 1 to 4, characterized characterized in that the cultures are carried out in the presence of doses of the homologous serum in each cycle, which are about 1.8 to about 2.3 times that required to neutralize the initial strain are. 6. The method according to any one of claims 1 to 4, characterized in that there are several in each cycle Forms groups of cultures, the cultivations in the presence of different in comparison to the homologous serum removed various doses, with the doses in a range of pre-estimated doses corresponding to the the dose required to neutralize the initial strain, but insufficient to achieve the neutralizing mutants that may have formed, includes harvesting cultures for characterization purposes, which have only developed in those of the groups that have a number between the preselected minimum and maximum numbers of positive cultures, or in the case where none of the groups include a number of positive cultures between those mentioned has included minimum and maximum numbers, readjusts the range of pre-estimated doses of the homologous serum, and that one carries out the cultivations again until in the presence of the appropriate doses of dea homologous serum in at least * £ $ 307/1021 - 4 C - one of the groups a number of cultures between those desired Limits has grown. 7. The method of claim 6, characterized in that said first said pre-estimated range between about 1.8 to about 2.3 times the dose of the serum required for neutralization of the initial strain. 8. The method according to any one of claims 1 to 7, characterized in that the characterization in such a way that one compares the mutants isolated in each cycle with at least 3 reference strains, which include the parent strain from which the assumed mutant was obtained in the cycle in question. 9. The method according to any one of claims 1 to 8, characterized in that the serum is rabbit total serum used. 10. The method according to claim 9 *, characterized in that a strain isolated in the above cultures is then regarded as a mutant which is accordingly subjected to new culture cycles if a significant difference greater than 0.84 logarithmic units on the basis of 2 is , between the titers of at least one of the reference sera with respect to the isolated strain on the one hand and the one or more initial or strain strains on the other hand, or again significant differences above 0.42 log ₂ units between the titers of at least 3 Reference sera relating to the isolated strain and the parent or parent, originate on the other hand. 11. The method according to any one of claims 1 to lo, characterized marked that one as the initial strain 409807/1021 - 50 - the strain NT60 is used in the first operating cycle of the process. 12. The method according to any one of claims 1 to 11, characterized in that the obtained End stem with a further stem with selected properties, E.g. Pns cultivated together and that the recombination hybrid, e.g. by a cloning operation, in the egg with the limiting dilution with the homologous serum compared to the other Tribe wins. 13 · Serum, characterized in that it is the homologous serum of a strain produced according to claims 1 to 12. 14. Vaccine, denoted by it, rejects, that it contains the antigenic part of a strain produced according to claims 1 to 12 as the active ingredient principle. 15. Vaccine according to claim 14, characterized in that the active ingredient principle with a physiological acceptable medium is present. 16. Vaccine according to claim 15, characterized in that the physiologically acceptable medium is sterile and is injectable. 17. Vaccine according to claim l6, characterized in that it is in unit dosage form, which contains about 15,000 to 60,000 uHA. 18. Vaccine according to one of claims 14 to 17, characterized in that the strain is weakened.  / 409807/1021