CA1049406A - Isolation of hemagglutinin and neuraminidase from influenza virus - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The invention provides a novel method of isolating the immunogeric components from influenza virus and to vaccines comprising such components. In particular, it provides a method of isolating the hemagglutinin and neuraminidase components from influenza virus, comprising treating influenza virus in an aqueous medium with a cationic detergent to selectively solubilise such components, and separating the resulting solubilised such components from residual sub-viral particles.

Description

Case 900-9102 ~4g~06 IMPROVEM~NTS IN OR RELATING TO ORGANIC COMPOUNDS

The present invention relates to influenza vaccines, in particular influen~a sub-unit vaccines, and their production by selective solubilisation and iso- -lation of the immunogenic components of influenza vixus.

Flgure 1 is a schematic representation of the influenza virus particle. The genetic material, riko-nucleic acid (RNA), associated with the group specific nucleoprotein is surrounded by a double membrane con-sisting of an inner layer of protein and an outer layer ; 10 of host-derived lipid material. Two glycoproteins, hemagglutinin and neuraminidase, appear as projections ~r spikes on the surface of the viral envelope.

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It is now well-esLablished that the two glyco-proteins, hemagglutinin and neuraminidase, are the major immunogenic components of the influenza virus, all other components, including other virus proteins, nucleic acid and lipids, being non-essential for the induction of .. ~
lmmunity. However, the presence of such non-essential materials in an influenza vaccine may lead to undesired side effects and, in any event, limits the dosage of the Yaccine which can~be administered and, consequently, the ~ . .
,! : level of immunity which can be achieved.

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- 2 - 900-9102 The ideal lnfluenza vaccine should, thereforet contain the two essential immunogens, hemagglutinin and neuraminidase, in the absence or substantial absence of non-essential components of the viral particle. Previ- J
ous attempts to separate the influenza immunogens have involved as an initial step, substantially complete - disruption or solubillsation of the virus particle, - for example with anionic detergents, such as sodium desoxycholate or sodium dodecyl sulphate, such that all or the major portion of the viral components are liber-ated and go into solution with the immunogens. A
subsequent purification or partial purification of the desired immunogens is necessary, and is very elaborate and laborious and the yields are usually low.

The present invention provides a method for `J . isolating the hemagglutinin and neuraminidase immunogens, involving selectively solubilising these components while leaving residual subviral particles consisting o~
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the intact lipid/protein membrane enclosing all other l 20 non-essential viral components. The difference in size Z or density of the solubilised immunogens and the residual .
sub-vir 1 particles permits ready separation of the lmmunog ns by conventional separating methods utilising ~3 ~ such differences in physical properties.

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- 3 - 900-9102 .
It has thus been found that such selective - solubilisation of the hemagglutinin and neuraminidase components can be achieved by treatment of the influenza virus ~ith a cationic detergent.
The present invention accordingly provides a method of isolating the hemagglutinin and neura~inidase components from influenza virus, comprising treating influenza virus in an aqueous medium with a cationic detergent to selectively solubilise such components~
and separating the resulting solubilised such compo~nents from r~sidual sub-viral particles.

The method of invention may suitably be applled to influenza Type A, Al, A2 or B viruses or mixtures thereof. The particular strain employed will, of course lS depend on the immunity desired from the immunogens to be isolated but the following may be mentioned as examples:-i strain A2/ALchi/68, MRC-2 ~recombination of Type A2/
England/42/72), MRC-ll (recombination of Type A2/Port Chalmers~733, A/Pasteur/30C ("Mutagrip", Institut Pas~eur) and B/~ass/67.
The influenza virus to be treated is suitably multiplLed in conventional manner, for example by inoc-ulation in ll day vld embryonated ch~cken egys, ar.d , 1 in~ubation for a suitable period at a suitable temperature, .
~5 for example for ~-days~at 37C. The harvested allantoic . ~, -',:~ : .

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fluids are then suitably pooled and the virus suitably concentrated and purified by ultracentrifugation followed by xesuspension of the virus in, for exa~ple, phosphate buffered physiological saline, or by centri~uging in a continuous ~flow zonal centrifuge using, for exzmple, a sucrose gradient in phosphate buffered physiological saline, followed by lowering of the sucrose content to, for example, less than 5~O, suitably by dialysis asainst physio-logical saline, or by Sephadex~ chromatography or diluting.
The concentration of the starting virus is not critical and can be adjusted dèpending on the desired yield of immunQgens .

The pH of the virus concentrate is suitzbly from 6.5 to 8.5, using buffers, such as phosphate buf~er, where required, prior to the addition of the cationic deterqent, and the concentrate may also be inactivated, e.g. by the .
addition of formaldehyde. The cationic detergent is then suitably added to the virus concentrate in the ~orm o~ an aqueous solution. The appro~riate quantity of cationic detergent to be addèd will depend, for exa~.ple on the particular detergent employed. Ho~ever, in general, the cationic detergent is suitzbly added in ..

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, . ~ . , ~L049~LO~; goo-9l02 such a ~uantity that the weight ratio of detergent to protein in the resulting mixture is from 1:2 to 1:10, particularly from 1:3 to 1:5. After addition, the mixture ls suitably allowed to stand, for example for a period of 30 minutes to 16 hours at a temperature of, for ; . example 4C to 37C, the higher temperatures requiringthe shorter standing times. Preferably,the mixture is allowed to stand for 30 to 60 minutes at room temperature, or overnight at 4C.
The cationic detergent employed ~.ay be any cationi.c detergent sufficiently active to solubilise the hemagglutlnin and neuraminidase components, but lnsuffi.ciently actlve, under the conditions employed, to disrupt the whole virus particle.

Such cationic detergents may be selected from the well-known class of formula I~
R R
1 6~/ 3 X
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., R2 4 3 in whlch R4 signifies alkyl or aryl, ;

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~ ~ ' - - 6 - ~ ~49~ 900-91~2 Rl, R2 and R3 are the same or different - and each signifies alkyl or aryl, or Rl and R2, together with the nitrogen atom to ~Ihich they are attached form a S- or 6-membered saturated heterocyclic ring, and R3 signifies alkyl or aryl, - or Rl, R2 and R3, together with the nitrogen atom to which they are attached, signiy a 5- or 6-membered heterocyclic ring, ; lO unsaturated at the nitro~en ato~., : and X signifies an ~nion.
Representative compounds of formula I include ' those o~ formula Ia, .

R \ ~ / CH3 Ia ., . . ~ ~
~ . . R3~ R4 ~ ~

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ln which X is as defined abovel and R4 signifies alkyl of 8 to 22 carbon atoms, and el~her Ri and R2 are the same or different and : each signifies methyl or alkyl of 8 ,~ .
to 22 carbon atoms, or R; signifies methyl and R2 signifies ; . benzy1g ~ :

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~G149406 goo glO2 in particular compounds of formula Iaa, 3 \ f 3 / N \ X ~ Iaa c~3 R4 ir which R~ and X are as defi.ned above~

~ or of formula Iab, :~ CH3 ~3 \.N X Iab / \

ln which R4 and X are as defined above. .

~ . Further representative compounds of formula I
are those of formula Ib, R ~ X ~ }b in which X is as defined above, .. . . .
: R4 signifies alkyl of 12 to 18 ;carbon . atoms, and R5 signifies hydrogen or methyl, ~;3 ~ preferably~hydrogen.
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Preferred alkyl radlcals of 8 to 22 carbon atoms:contain 12 ~o }8 oarbon atoms. Preferred alkyl radlcals o~ 12 to 18 carbon atoms include lauryl, !~ 15 ~: myristyl, cetyl and stearyl.

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- 8 - g00-gl02 In the above formulae, X preferably signifies - such anions as chloride, bromide, sulphate, or acetate, particularly chLoride or bromide The preferred compounds of for~.ula Iaa include myristyltrimethylammonium and cetyltrimethylammonium salts, in particular chloride or bromide, more partic-ularly bromides. Preferred compounds of formula Iab include stearyl~imethylbenzyla~.onium salts, in partic-ular chloriae. or bromide, more particularly bromide.
lD The preferred compounds of formula Ib include cetyl-pyridinium salts, in particular chlorlde or bromide, more pa.rticularly bromide.
Other cationic detergents which may suitably be . - .
employed include benzalkonium chlorides and bromides, fcr ,, .
example benzethonium chlorlde or methylbenzethonium chlor-~i ide, as well as such agents as decamethonium chloride.

; The preferred cationic detergent for use in the process of the invention is cetyltrimethyla~onium ~ -bromide.
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! 20 Upon completion of the process, the hemagglutinin :. , .
i ~ ~ and neuraminidase components may be separated from .residual intact sub-viral particles using conventional ., ~ :
~ metho~s~or the separation of materials having different . ~1 .
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~ 900-9102 sizes or density, for example by gradient centrifuying, uslng sucrose or sodium glutamate media, followed by fractionation of the gradients, by sedimentation, by molecular sieve chromatography or by pelleting in an ultracentrifuge.
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The mixture of immunogens produced in accord-ance with the process of the invention are suitable for use in influenza vaccines. ~or this purpose, the hemagglutinin and neuraminidase components isolated as described above are suitably resuspended in a conven- ;
tional diluent, ~or example a physiological isotonic solution, e.g. a 0.9% sodium chloride solution, optlon-ally buffered, e.g. with phosphate buffer. Sucrose remaining from the purification of the initial virus or from the -15 separation of the so~ubilised components, should suitably be reduced to less than 5%~by weight in the vaccine, for example by dialysis. Likewise, the content o~ cationic detergent remaining should largely be removed, for exa~ple reduced to less than 0.01% in the vaccine~ for ., . - .
;l 20 example by dialysis or gel chromatography.

j~ Ig desired, preserving agents or inactivating agents, such as ~ormaldehyde, may be added to the vac-1 . . :

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- 10 ~ 494~6 goo-gl02 cines, in conventional amounts, for example at a ratio by weight at 1 part to 10,000 parts.

Immunogenicity of the vaccines of the invention may also suitably be improved by inclusion of corventi-onal immunological adjuvants, such as aluminium hydroxide or aluminium phosphate,in conventional amiounts, for example, - by inclusion of o.2% of aluminium hydroxide.

- As indicated, the vaccines produced in accord-ance with the invention are useful as vaccines agalnst ln1uenza viruses, for-example those mentioned above, as shown, for examp~le, by comparlson with whole virus ' vaccines, having the same immunogenic content, in the ,~
mouse protection test. Separate groups of 30 mice are administered, i.p., 0.25 ml of whole virus vaccine and :i - . .
lS sub-unit vaccine of the inventionj each having a hemagglu-tLnin content of about 2 . Separate groups are infected, 3,4 and 8 weeks after immunisation, with a virulent vir~us i by spray application. On the ninth day after infection the , ~ protection against mortality and against lung lesions is ;~ ~ 20 evaluated in each group~ The test is repeated using differ-ent antigenic contents in the vaccines. The results indicate that the sub-unit vaccines of the invention produce a more ~3~ prolonged immunity against the infecting virus but other .
~J~ wise have parallel effects to the whole virus vaccine.
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For such usage the dosage to be administered will, of course, vary. Ho~Jever, in general, satisfactory i :: .
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~ 94~6 900-91~2 - results are obtained when administered at a single dose of from about 9 to 43 international units per kg of animal body weight. For the larger mammals, a single dose of from 600 to 3000 internaitonal units is indicated.

The dosage is suitably administered sub-cuta-neously or intramuscularly.
The folLowing Examples i:Llustrate the invention.

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EXA~LE 1 Influenza virus of the antigen type X-31 (reco~ ation of the strain A2/Aichi/68) is multiplied in embryonated chicken e~gs by incubation at 37~C for two days. The eggs are then chilled at 4C overnight and the harvested infected allantoic fluid pooled.
The virus is subsequently concentrated and ~urified from the infected allantoic liquid by centrifuging in a _ontinuous flow zonal centrifuge (model RK, Electro-Nucleonics) uslng ~ sucrose gradient in phosphate buffered saline.
The virus concentrate obtained after reduction of the sucrose content to less than 5% by dialysis against ', phosphate buffered saline in the cold, has a hemag-glutination titre of 1: 217 and a protein content of ,~ 15 O.7 mg/cc. The im~unogens are split of by adding to , the virus suspension 1/50 of its volume of an aqueous detergent solution (cetyltrimethylammonium bromide, 1~
~' solution). After 30 to 60 minutes ~room temperature) the reactlon mixture is worked up by zonal ~adient centrifuginc using a preformed linear sucxo~e gradient and subsequ-,~ ent fractionation o~ the gradients with a peristal~ic pump. Hemagglutinin and neuraminidase are solubilized .
quantitatively and are present in the upper part of the gradient, well separated fxom the virus residual part-J
~t, ~ ~, 25 1~1e which forms a sediment much more rapidly~
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Multiplication, concentration and cleavage of the virus are e~fected as described in Example 1.
Working up is effected by equilibrium centrifuging in a preformed sucrose gradient. After adjusting equilib-rlum, the gradient is fractionated and tested: hemag-glutinin and neuraminidase are present in the lighter part of the gradient, well separated from the more dense ~irus residual partic~e.
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EXAMPLE 3:
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~', The process is effected as described in Example 1 or 2, except that influenza strain M~C-2 ~recombination of type A2/Enyland/42/72) or MRC-ll ke~
combination of type A2/Port Chalmers/73) is used.

;l 15 EXAMPLE 4: ~

The process is efected as described in Example 1 or 3, except that the reaction mixture is worked up by molecular sieve chromatography.

EXAMPLE 5: -"'i ' -- ~ , .

~ ~ ; 20 An aqueous solution ~0.5~) of cetylpyridinium , , . . :
~; bromide is added to influenza virus of the type A/Pasteur/30 C ('iMutagrip", Institut Pasteur) ~hich has . ~j: . , ~ been lnactivated with formol, up to a final concentration . 3 ~',.i ~
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- of 0.02 to 0.1~. Working up is effected in a manner analogous to that described in Example 1 t 2 or 4.

EX~PLE 6:
. . _ The process is effected as described in Example 1, 2, 4 or 5, except that the influenza strain B/~ass/67 is used.
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The process is effected as described in .
Example 1, 3, 5 or 6, except that the cleavage mixture is worked up by pelleting in an ultra-centrifuge. :.
This may, for example, be effected in a ~eckmann L-2-65 B
centxifu~e kotor 60 Ti, 35 000 r.p.m., 90 minutes).
The solubilzed i~.munogens are present ir. the supernatant..
fraction.
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j 15 EXA~PLE 8:
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The procedure o any of ~Y.a~les 1 to 7 is repeated but employing, in place of the cetyltrimethyl-ammonium bromide solution, a 1% solution of myristyl-trimethylammonium bromide, benzethonium chloride, methyl-benzethonium chloride, decar.ethonium chloride or stearyl-dimethylbenzylammonium bromide. Similar results are : obtained.
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- 15 - ~0~94~6 900-9102 EXAMPLE 9:
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An influenza vaccine of the invention may be formulated as follows:-Immunogenic mixture :- 700 international units ~hiomerosal :- 1 part in 10,000 parts Phosphate buffer in 0.9% physiological :- to 0.5 ml.
saline The immunogenic mixture may be produced in accordance with any one of the preceding Examples, for example that .. ..
produced in Examplè 3 from the influenza strain MRC-ll (recombination of type A2/Port Chalmers /73).

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Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of isolating the hemagglutinin and neuraminidase components from influenza virus, comprising treating influenza virus in an aqueous medium with a cationic detergent to selectively solubilise such compon-ents, and separating the resulting solubilised such components from residual sub-viral particles.

2. A method according to Claim 1, in which the influenza virus is an influenza Type A, A1, A2 or B virus, or a mixture of any two or more thereof.

3. A method according to Claim 2, in which the influ-enza virus is strain A2/Aichi/68, MRC-2 (recombination of Type A2/England/42/72), MRC-11 (recombination of Type A2/Port Chalmers/
73), A/Pasteur/30C (Mutagrip) or B/Mass/67.

4. A method according to Claim 2, in which the cationic detergent is added in such a quantity that the weight ratio of detergent to protein in the resulting mixture is from 1:2 to 1:10.

5. A method according to Claim 4, in which the cationic detergent has the formula Iaa, lab or lb, Iaa Iab in which R4 is alkyl of 8 to 22 carbon atoms, and X is an anion, Ib in which R? is alkyl of 12 to 18 carbon atoms, R5 is hydrogen or methyl, and X is as defined as above, or is a benzalkonium chloride or bromide, or decamethonium chloride.

6. A method according to Claim 5, in which the cationic detergent is selected from myristyltrimethylammonium chloride or bromide, cetyltrimethylammonium chloride or bromide,stearyl-dimethylbenzylammonium chloride or bromide, cetylpyridinium chloride or bromide, benzethonium chloride, methylbenzethonium chloride, and decamethonium chloride.

7. A method according to Claim 6, in which the cationic detergent is cetyltrimethylammonium bromide.

8. A method according to anyone of Claims 5 to 7, in which the cationic detergent is added, in the form of an aqueous solution to a virus concentrate having a pH of 6.5 to 8.5 and, after addition, the mixture is allowed to stand for 30 minutes to 16 hours at 4° to 37°C.