Note: Descriptions are shown in the official language in which they were submitted.
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.
.
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
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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
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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~
.1 .
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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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,
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~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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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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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
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~ . . 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
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to 22 carbon atoms,
or R; signifies methyl and R2 signifies
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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
/ \
C6HSCH2 R4
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,
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: 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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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
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employed include benzalkonium chlorides and bromides, fcr
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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
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.residual intact sub-viral particles using conventional
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~0499L~
~ 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.
: .
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
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;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-
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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
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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-
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- EXAMPLE 2:
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: -
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~ ~ ; 20 An aqueous solution ~0.5~) of cetylpyridinium
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~; bromide is added to influenza virus of the type
A/Pasteur/30 C ('iMutagrip", Institut Pasteur) ~hich has
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~ been lnactivated with formol, up to a final concentration
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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.
~ . .
EXAMPLE 7-
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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EXAMPLE 9:
.
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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