CA1050886A

CA1050886A - Preparation of pyrogen free virus sub-unit vaccines

Info

Publication number: CA1050886A
Application number: CA211,654A
Authority: CA
Inventors: Ian G. S. Furminger; Margaret I. Brady
Original assignee: Duncan Flockhart Co Ltd
Current assignee: Duncan Flockhart Co Ltd
Priority date: 1973-10-18
Filing date: 1974-10-17
Publication date: 1979-03-20
Also published as: DK543974A; NL181553C; ATA835074A; JPS5082228A; SE7413113L; DK138581C; DE2449530C2; DK138581B; AU7444974A; NO142024B; GB1486557A; DE2449530A1; IL45870A0; CH611518A5; NO743758L; NO142024C; IL45870A; IE40187L; SE426439B; NL7413642A

Abstract

PREPARATION OF PYROGEN FREE VIRUS SUB-UNIT VACCINES
ABSTRACT

Virus sub-unit vaccines which are substantially free from pyrogens, virion nucleic acids and unwanted antigenic material are prepared by introducing inactivated whole virus into a continuous loading zonal ultracentrifuge provided with a density gradient containing a haemolytic surfactant and banding the split sub-units isopycnically. All ether-sensitive viruses can be used, especially influenza virus.

Description

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This invent.ion relates to the preparation of pyrogen free virus sub-unit vaccines, in particular inf luenza vacc:inesO
The influenza vAccines generally used at present are inactivated whole virus v~cclnes which contain, besides the influenæa virionsg some egg protein derived ~ -i from the allantoic ~luid in which the virus is harvested On injection, this type o~ vaccine can give rise to : hypersensitivity caused by the egg protein and pyrogenic ;~
; 10 ef~ects caused by the virions.
The influenza virus, and similar viruses of the : same type, i.eO orthomyxoviruses an~] paramyxoviruses, are ch~rac~erised ~y an ou~er membrane carrying "spikes" ~ .
of antigens having haemagglutinating and neuraminidase -;. ;
activity. This outer membrane is capable of being dis- :
rupted by solvents such as.ether or by surfactants to release as sub-units such antigens having haemagglutinating ~nd neuraminidase activity. The so-called t split virus' vaccines w~ich have hither~o been prepared in this way are found to have lower pyrogenicity than corresponding whole virus vaccines even tholl~h no attempt has been . made to separate the antigens having neuraminidase and .
haemagglutinating activity from pyrogens and other un-wanted material such as ether antigens and nucleic acids.
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We have found, however, that the antigens h~ving haemagglutinating and neuraminidase activit:y are not themselves pyrogenic or otherwise toxic and that consequently a still further reduction in pyrogenicity can be acllieved by obtaining the desired antigenssubstantially free from other unwanted material.
We have attempted to separate the components of a split virus preparation by zonal ultracentrifugation.

satchwise loading of an ultracentrifuge rotor was however, found to be irnpracticable for large scale production and it was found necessary to use a continuous loading zonal centrifuge wherein the relatively dilute suspension ~ the virus subunits is passed into the rotor and flows across the ~ . .
centripetal end of a concentration gradient. It was ~ound, however, ,. :
that thevery small particles of the desired antigens having haemagglutinating and neuraminidase activity were reluctant to transfer onto the gradient at flow rates consiQtent with lar~e scale production, ..
We have now found, however, that viruses of the - in~luenza and assoGiated types, wh;ch can readily be intro-duced into the gradient of a continuous loading zonal ultracentrifuge, can suc~essfully be split while passing - ` . ' . , ~ ~' `:
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through the gradient if a surfactant is present in ~he . - -~
gradient solution. This.facilitates the .large-scale preparation of sub-unit vaccines substantially Eree from egg : :
protein pyrogens and virion nucleic acids and containing substantially only the desired protective antigens having haemagglutinating and neuraminidase activity. The haemagglutinin found in the so-called 'spikes' is itself split by the .
surfactant to a monovalent form which no longer exhibits `~
haemagglutination but does possess the required antigenic . ~ ;
.. . . .
: lO properties. . `
In general, all the ether-sensitive viruses can be .
split by treatment with detergents and in addition to the : above ortho and para myxoviruses, the following viruses can . yield sub-unit vaccines, containing.protective antigenic components analogous to haemagglutinin and neuraminidase7 : namely~
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a~ Togaviruses ;. . b) Rhabdoviruses ;~
c) Leukoviruses .
.
: 20 d) Coronoviruses ., :~
~ e) . Arenoviruses ~ .
~ .
~ f) Herpesviruses . . :
~; .
. g) Poxviruses :
- According to one eature of the present invention, :~

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i' '.,.' . ' i` ' " ~. ` . ' . ' ' ' ' ' - 1~5~81~6 therefore, we provide a process for the preparation of ~irus sub-unit vaccine containing an-tigens having haema~glutinating and neuraminiclase activity ox analagous protective antigenic componen-ts derived from an ether-sensitive virus capable of releasing said antigens or components, and being substantially free from pyrogens, virion nucleic acids and unwanted antigenic material, inclucling the step of introducing a liquid medium containing the inactivated whole virus into a continuous loading zonal ultracen-trifuge provided with a density gradient solution containing a l~aemolytic surfactant, whereby the virions enter the density gradient and are split by the surfactant and the `
antigenic sub-units are banded isopycnically, the ~raction or fractions containing the protective antigen sub-units sub-sequently being recovered. ~ ;
While it is possible to load the whole virions directly from a relatively dilute culture medium such as allan- -toic fluid, it is generally preferred to effect some purification of the whole virus preparation before splitting on the ultracentrifuge.
For such purification, the whole, intact, inactivated virus may be isolated using conventional means. Thus, for `
example, influenza virus may be grown in 10 to 11 day old embryonated hens eggs. The harvested allantoic fluid may then be treated, e.g. with ~-propiolactone and/or formalin, to inactivate or "kill" the virus and then clarified by centrifugation, e.g. in a Westphalia (trade mark) continuous .' ' ~ '' '' '`~ ' ' ~
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flow centrifuge.
The virus may then be further purified. While this can be achieved using continuous sedlmentation centrifugation, treatment with fluorocarbons, gel filtration or adsorption onto red cells or minerals such as barium sulphate and subsequentelution, it is preferred for speed and ease of handling in bulk to use continuous zonal centrifugation for example using a KII apparatus (originated in the ~ ;~
Molecular Anatomy Program, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, U S.A. and described by Anderson et al in Anal.ytical Biochem 32 (1969) p. 400-494 a-ld 495-511, Electro_NucleOnic8~ Inc., Fairfield, N.J. U.S.A.) A KII rotor of 3.2 litres capacity with a sucrose density gradient is particularly effective. The method used may be . ~ . ~............................... .. ~ .
that of Reimer et al (Journal of Virology Vol. 1, No 5 pages 1207-1216). After purification the fractions containing .,. ~ ,, ,: .
virions, as determined by haemagglutination titre may then be pooled and diluted or dialysed to reduce the density to a level suitable for introduction onto a gradient in the , , second ultracentrifugation step.
For the step of splitting the virus, the virions are . , ~ loaded, preferably after purification as described above, onto the density gradient solution of a continuous loading zonal ultracentrifuge. As for the virus purification7 a ,. . . .
convenien~ medium for the gradient is a solution of a sugar, in particular sucrose, preferably in bufered saline at a suitable pH for the virus. In general a pH of a little ~bove .

- 6 - ;
. . .

~ ~ 5~ ~ ~ 6 7 is desirable and it is convenient to use 0.01 M phosphate as buffer at pH 7.2. A density grad-Lent of about 1.02 to about 1.24 g /ml is suitable. Other ~olutions which can be used to form a density gradient o~ this order include polyols such as glycerol and salts such as tartrates or ~ caesium chloride. The gradient may be prepared by half - filling the rotor with a solution at the lowest density of ;~ the desired gradient and adding an equal volume of a solution at the highest density. Spinning of the rotor at moderate speeds will then establish the gradient.
As stated above, the density gradient solution contains a surfactant in order to split the virus while it traverses the gradient. In general, the surfactant, ~-in aqueous solution, may be introduced into the gradient and lS the centrifuge rotor is spun for a time to ensure penetration of the surfactant into the gradient solution. Alternatively, the surfactant may be present in the gradient solution when it is loaded into the rotor. Many o~ the haemolytic surfactants which may be used in this invention form distinct bands in the gradient although banding is not an essential characteristic.
The posi~ion of the band on the gradient is not cri~ical since -the virions, beil~g relatively dense, will traverse the gr~dient until they reach this band; af~er splltting, sub-units may move to zones of appropriate density which may be on the low density side of the surfactant band.
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As stated, the surfactant should be a haemolytic :-: surfactant, By this we mean a s~rfactant which will give a positive result in the following test: ;~
Chick red blood cells (0.5% V/V in t).Ol M phosphate buffered saline pH 7.0; 0.25 ml) are added to a 1% W/V `~
aqueous solution of the surfactant (0.25 ml). For a . :;~
positive result,.complete haemolysis must occur within ~:
5 minutes at 22C. `~
Included within this category are the following types ~ :
of surfactant~
NONIONICS

Aryl Ether Adducts of Ethylene Oxide ` ~.
` General names: Alkyl Phenol Ethylene Oxide Condensates '~
or ~.
Alkyl Phenol polyoxyethylenes .-~ :
Polyethoxy Alkyl Phenols e.g. Nonidet P40 ~ Octyl Phenol Polyoxyethylene (Shell) or ` 20 Polyethoxy octyl phenol ` Tergitol(~ Nonyl Phenol 10.5 oxyethylene (Union NPX or Carbide~
10.5 Ethoxy nonyl phenol Triton N101 ~ Nonyl Phenol 9-10 oxyethylene (Rohm & Haas) 9-lO Ethoxy nonyl phenol .`` `
Renex 69B ~ Nonyl phenol 9-9.5 oxyethylene (Atlas) or .~ 9-9.5 Ethoxy nonyl phenol selloid EMP ~ Octyl phenol polyoxyethylene (Geigy) or Polyethoxy octyl phenol . .
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~ ~050~86 Adinol C0630 ~ Polyoxyethylene nonyl phenol (Fine DyeStuffs)~
Aliphatic Ether Adducts_of ~
General names Aliphatic Alcohol Polyoxyethylenes ~ ;
or Polyethoxy Aliphatic Alcohols e.g. Bri~96 Oleyl alcohol 10 oxyethylene (Atlas) or ` `~
10 Ethoxy oleyl alcohol - -Tergitol TMNiO Trimethylnonanol :LO ethylene oxide Tergito 15-S-7 Linear alcohol Cll to C15 7-eth~lene oxide Ester Adducts of Ethylene Oxide General names Fatty acid ester Polyoxyethylenes or Polyethoxy esters of fatty acids ;~
e.g~ Cithrol 4ML 400 Polyethylene Glycol monolaurate (Croda) or Lauric acid 400 Polyethylene glycol ester ~

Amine ~dduct~ ~ e Oxide ~ ;
:
, General names Fatty Amine Polyoxyethylenes or x Polyethoxy Fatty Amines `~ e.g. Ethomeen~ Stearyl Amine 15 Oxyethylene (Armour) ` ' -~ 15 Ethoxy Stearyl amine ;
Ethomeen~ Coco Fatty amines 15 oxyethylene(Armour) C/25 or - ~ 15 Ethoxy Coco fatty amines (Av. Mol, Wt. 860~ `

S/15 Soya fatty amines 5 oxyethylene(Armour) 5 Ethoxy soya fatty amines ;,.~ ~ ~
General names Alkanolamide Polyoxyethylenes or Polyethoxy Alkanolamides .: ~
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_ ~ ~ 50 e.g, Conox J 754 Exact composition not known ANI
e.g. Texapon~ Sodi~ Salt of sulphated lauryl(Dehydag) N25/S alcohol dioxyethylene or ; ~, Dioxyethylene sodlum lauryl sulphate Adinol T~ Sodium N-methyl N-oleyl taurine Solumin ~ Sodium Salt of Sulphonated fatty 45 S alcohol 45 oxyethylene :~
or 45 Ethoxy fatty alcohol sodium sulphonate Teepol 610 Sodium secondary alkyl sulphate (Shell) , ::
Sod ium deoxycholate Anionic bile salt Pentrone~A4 Amine salt of alkyl aryl sulphonic acid ~Glover) -~
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MISCELLANEOUS
e.g. Belloid M3 Polyoxyethylene 3 condensate (Geigy) ;-with an aliphatic alcohol ~-Galoryl MT5 (CFPI~
~rance~

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In general, the minimum effective quantity of ; surfactant should be used, to reduce problems of removal : later on. For the preferred surfactant, an aryl ether adduct of ethylene oxide, especially Triton N101, the .
overall concentration in the gradient is preferably at ::
least 0.5~ v/v, advantageously 1.0% v/v. The concentration ~ ~:
in the localised bana on the gradlent will, of course, be : hlgher than this.
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1 05~ 6 Some surfactant will normally be present throughout the gradient where desired sub-u~its of the virions band and this prevents or minimises re-aggregation of the sub-units. I
such aggregation occurs the separation from unwanted S antigenic material is poor. -The medium containing the purified virions is then ;
flowed through the zonal ultracentrifuge so that they enter ;~
the gradient and are split by the surfactant, eventually banding as separated sub-units. In general, ~he vlrions ;
should be loaded at a rate of about 2.8 rotor volumes per hour e.g. about 10 litres per hour for a 3.6 litre KII rotor.
(When puriying virions, without splitting, the loading rate may be higher e.g. 30 litres per hour ) In general the ;~
, .
- ~ rotation speed during loading o~ the gradient may be at -~
least 20,000 r p.m , conveniently 35,000 r.p.m. giving ;~
O,000 g in the KII centrifuge. After completion of ~; "loading", the ultracentrifuge rotor is spun for a further period, e g. 2 to 3 hours, to further separate and consolidate the bands and, after the rotor has been slowly decelerated and brought to a halt, the contents of the gradient are run ~ff `~; in fractions. Those fractions containing the desired sub-units may then be pooled.
; In the case of viruses liberating neuraminidase, ~ ~-. .
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or analogous material this may be detected by standard assay methods, e.g. the method of Warren (J.Biol~ Chem 234, 1959, p 1971).
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In the case of viruses llherating mono~valent haemag~lutlnin, or analogous material -this cannot be identified by as~.ay of haemagglutination as the haemagcJlutinin obtained is in the ~; monovalent form and does not exhibit such activity. It i5 . ~` ~ ' . .
necessaxy, therefore, to assay the antigenic ef~ect of the samples (e.g. by the haemagglutination inhibition titre ox the single radial di~fusion test) or measure a physical characteristic such as optical density, u.v. absorption etc. In some cases haemagglutinin will be found in the same fractions as ' 10 neuraminidase. ~ ~
'' ` ' ' ' ':. ~'' ~; The virus used is ether-sensitive and as such will . have the necessary strippable coat containing antigenic material. The preferred virus iB an influenza virus, but other suitable viruses include measles and mumps in humans and ~` 15 New~astle Disease virus (NDV) in poultry(which are all `: : :
myxoviruses)and bovine rhinotracheiti B virus(which iB a Herpes ; virus~
For use as a vaccine, the pooled antigenic sub-unit ~ .
~ fractions must be substantiaIly free from any surfactant -~ 20 and this can be achieved in the case of non-ionic suractants, such as Triton NlOl, by depression of the cloud point by addition of ionic material to the diluted aqueous medium so ~ .
that the surfactant separa~es as a new phase. However, it ;~

is ound advan~ageous to separate the sub-units by .: , :
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1~5~81~6 ~ ` ~
selective adsorption onto colloidal aluminium hydroxide (e.g. Alhydrogen~ ) leaving the surfactant in solution.
This process can desirably be effected as a second step after cloud-point depression, where applicable. The use of the aluminium hydroxide has the advantage that the `~
; antigens are obtained in an adjuvanted form which is usable ~ ~ -;~ as a vaccine as such. Alternatively, the antigens may after removal of surfactant, be adjuvanted with other convenient adjuvants such as oil emulsions e.g. emulsions -of water in an oily fatty acid glyceride such as peanut oil. Such emulsions may contain non-ionic emulsifiers such as sorbitan tri-oleate and a stabilis~r such as aluminium monostearate.
The potency of 'Alhydrogel'-adsorbed influenza i :, sub-unit vaccine was compared with aqueous whole influenza virion vaccine. The results are shown in the following .. : , , Table The potency was estimated by inoculating grou~s of 5 chickens intramuscularly in the leg with 0.5 ml of vaccine dilutions on day 0 and day 14 and bleeding on day 28. ;
;: .
Haemagglutination inhibition and anti-neuraminidase antibodies :. : . :
were estimated.
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TABLE

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VACCINE ~laemagglutination Anti Neur~
InhibLtLon Titre aminidase .- ~Influenza virus strain A/ Titre Hong Kong/1/68X) Geometric Mean Titre (GMT) G M T. :~

: 2 weeks 4 weeks 4 weeks ~ ~.

Whole virus aqueous single strength 291 .2027 75 " " " 1 in 4 dilution 22 . 582 27 :
" " " 1 in 10 " 11 291 0 .
1l 1l 17 1 in 100 1- 0 6 0 . : , Sub,unit adsorbed slngle strength 167 1536 137 .
" " " 1 in 4 dilution 24 1536 26 :~
. : " " " 1 in 10 " 1 1163 36 ` `~
, .. " '~ " 1 in 100 " 0 18 0 ~ : ~
:
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Single streng~h aqueous virus, based on haemagglutination titre, contained 400 i.u./ml. The single strength sub-unit ~ ;
vaccine contained an equivalent amount of antigen. These ~ res~lts show that the sub-unit adsorbed vaccins is similar -~
.:: or better in antigenicity than whole virus aqueous vaccine.
According to a further feature of the present invention5 therefore, we provide a process for the preparation :~
:.................... . . .
. of an adjuvanted virus sub-unit vaccine con~aining protective . ~ .
., , ~ ' ,,., ' ~ ' ' ' :
' ' ' :, ~ ~ 5~ ~ ~ 6 antigenic components and being su~stantailly free from pyrogensnucleic acids and unwanted antigen~ including the st~p of adsorbing onto colloidal aluminium hydroxide the required protective antigenic component(s) obtained by the foregoing procedure according to the invention, preferably after first removing most of any remaining suractant. Non-ionlc surfactants may, for example, be removed by clou~-point depression.
The colloidal aluminium hydroxide used conveniently contains about 2% as Al(OH)3, e.g. Alhydrogel. The final concentration of the 2% aluminium hydroxide gel in the vaccine should,in general, preferably be from 6.0% to 18~/o v/v~
preferably about 12 5% v/v The adsor~ed vaccine may, if desired, be washed free of any associated impurities, e.g.
; 15 residual surfactant, by centrifuging the aluminium hydroxide and resuspending in fresh phosphate buffered saline. The optimum pH for the pH for the.final vaccine is from 7 ~o 8, advantageously about 7.6.
The following Examples illustrate the invention further.
Influenza virus strain A/Hong Kong~1/68X was grown in 11 day old embryonated hens eggs. The allantoic fluid was ;
. , , .~.
harvested after 48 hours. inactivated with ~-propiolactone ~1 ml per 1000 ml allantoic fluid) overnigh~ at room ¦~

: ' ' . :: ' 86 : ~
`~ temperature and then clarified through a T.1,'estphalia ` (Trade Mark) continuous flow centrifuge. 'I`he clarified . , .
Lnactivated allantoic ~luid was flowed at 10 litres per hour over a gradient prepared from 60% w/w sucrose in 5 0,OlM phosphate buffered saline pH 7.2 (1.8 litres) and pure O.OlM phosphate buffered saline pH 7.2 (1.4 litres) in a KII rotor, The rotor speed was 35,000 r.p.m. (90,000g) and it was spun for 2 hours after all the allantoic fluid ~ ~
; has been introduced, The fractions containing the influenza ~;
virions as determined by their haemagglutinin titre, were pooled (13,500 i.u. per ml; 1500 ml total), The virions were present in the frartions containing 40% w/w sucrose, and were diluted 1 in 8 in O.OlM phosphate :~J ,~j~ Sal~qe i buffered~ L~ pH.7,2 to a sucrose concentration o 5%
lS wjw. The diluted virions were flowed over a sucrose gradient .. , ~ ;
containing 1% Triton N101 in the KII rotor at 10 litres per hour, The sucrose gradient was prepared by filling the stationary rotor with 1,8 1itres of 60% w~w sucrose in O.OlM
; phosphate buffered saline pH 7.2 also containing Triton N101 (1% v/v) and 1.4 litres of O.OlM phosphate buffered saline ~ `
~$
; pH 7,2 on top and then accelerating the rotor to 90,000g (35,000 r.p.m.). The rotor was spun at 35,000 r.p.m. ~or 2 hours after alI the influenza virus~had been introduced.
The fractions containing the Iarge neurami~idase and Triton '.~ . ~:.
~ . .
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~ ~ 50 ~
peak were pooled; haemagglutinin was also present (50% of original level by the haemagglutinin titre). To 3 parts of ;~
this pool was added 1 part of 1.6M phosphate buffer pH 7.6. ~.
`~ The mixture becerne cloudy and the phases were separated by spinning at 2000 g for 20 mins. The lower phase was collected and contained the neuraminidase andhemagglutinin with only ~:
about 0.05% v/v Trito ~ N101 (yield 40%). To an aliquot ~lOml) of this an equal volume of 'Alhydrogel' was added and the mixture allowed to adsorb overnight at 4C.. Dil.utions were made and the potency of the dilutions estimated in chickens. After the dilution to give a vaccine of.adequate potency had been determined, all the vaccine was diluted :
to this strength but the final 'Alhydrogel' concentration ;~
was kept at 12.5%. Before the vaccine was adjuvanted the ~15 potency was estimated by the single radial diffusion method of Schild. (J. Gen. Virology. 16 (1972) 231-236).
! ' ~` Example 2 ` The process of Example 1 was repeated except that ~ :
Ethomeen 18/25 replaced Triton N101 inthe gradient. ~ ::
Example 3 ~, . The process of Example 1 was repeated but the Triton N101 was not separated by cloud-point depression. Instead., to the fractions from the KII centrifuge containing the -haemagglutinin9 neuraminidase and Triton N101 was added . . .

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105~386 ;:
- 12 5% v/v 'Alhydrogel'. Thie mixture was left at 4C overnight and the 'Alhydrogel' spun off. The supernatant contained the Triton N101. The pellet wais suspended in lts original volume of O.OlM phosphate buffered saline pH 7.2, spun S and the pellet resuspended in its original volume of O.OlM ;~
; . ."
phosphate buffered saline. The potency of the vaocine was determined, as in Example 1 and the vaccine diluted accordingly keeping the final 'Alhydrogel' concentration I2.5 v/v.
Exam~le 4 :
The process of Example 1 was repeated with influenza ~;
-.; :
virus strain B/Hong Kong/8/73 to give a potent vaccine.
, The process of Example 1 was repeated with influenæa -virus strain BlVictoria/98926/70 to give a potent vaccine.
Example 6 :: :
The process of Example 1 was repeated with influenza virus strain A/Englsnd/42/72 to give a potent vaccine.
Example 7 ; Newcastle Disease virus was grown in 10 day old embryonated hens eggs. The allantoic fluid was harvested ;~
~,.:;:: . , ~ after 96 hours, inactivated with ~-propiolactone (1 ml per ~ ~
~ . . . .
000 ml ~luid) for 2 hours at 37C. The solution was cen~rifuged at lO,OOOg at a flow rate of 20 litres~per hour to clarify and remove urates from the solution.
.
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The clarified allantoic fluid was flowed over a sucrose gradient prepared as ln Example 1 in the KII rotor at ~0 litres per hour. The rotor was cenl:rifuged at 35,000 r,p.m. or 2 hours after all the allantoic ~luid had been introduced, The fractions containing the subunits, as determined by neuraminidase content, were pooled. The rest of the method was as for Example l.
Example 8 `~ -~.
The fractions containing influenza neuraminidase and haemagglutinin from Example l after ~he Triton N101 had been removed by cloud-point depression were pooled and diluted with O.OlM phosphate buffered saline pH 7.2 to give a fluid containing 1600 i.u, of haemagglutinin per ml. It was then -homogenised in the.following proportions. ~ ;~
Virus subunits fluid 39.5 ml Vegetable oil 53 ml Sorbitan trioleate S ml Aluminium monostearate 2.5 ml (The aluminium monostearate was heated in the vegetable oil to disperse and the sorbitan triolea~e added before mixing with the aqueous phase).
,:
The antigenicity of this vaccine was confirmed in chîckens.
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Claims

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

1. A process for the preparation of virus sub-unit vaccine containing antigens having haemagglutinating and neuraminidase activity or analogous protective antigenic components derived from an ether-sensitive virus capable of releasing said antigens or components, and being substantially free from pyrogens, virion nucleic acids and unwanted antigenic material, including the step of introducing a liquid medium containing the inactivated whole virus into a continuous loading zonal ultracentrifuge provided with a density gradient solution containing a haemolytic surfactant, whereby the virions enter the density gradient and are split by the sur-factant and the antigenic sub-units are banded isopycnically, the fraction or fractions containing the protective antigen sub-units subsequently being recovered.

2. A process as claimed in claim 1 in which a continuous loading zonal ultracentrifuge of the KII type is used.

3. A process as claimed in claim 1 in which the density gradient solution is a solution of sucrose.

4. A process as claimed in claim 1, 2 or 3 in which the density range of the density gradient is from 1.02 to 1.24 g/ml.

5. A process as claimed in claim 1 in which the surfactant is an aryl ether adduct of ethylene oxide.

6. A process as claimed in claim 5 in which the surfactant is nonyl phenol 9-10 oxyethylene.

7. A process as claimed in claim 6 in which the overall concentration of the surfactant in the gradient is at least 0.5% v/v.

8. A process as claimed in claim 7 in which the concentration of the surfactant is about 1%.

9. A process as claimed in claim 1 in which the virus is an influenza virus.

10. A process as claimed in claim 1, 2 or 3 in which the virus is a Herpes, mumps, measles or Newcastle Disease virus.

11. A process as claimed in claim 1 in which surfactant is removed from the suspended protective antigenic sub-units by separation as a further phase.

12. A process as claimed in claim 1 in which a non-ionic surfactant is removed from the suspended protective antigenic sub-units by separation as a further phase by adjusting the ionic strength of the suspension to the cloud point of the surfactant.

13. A process as claimed in claim 1 in which the virus sub-units are subsequently formulated with a vaccine adjuvant.

14. A process as claimed in claim 13 in which the protective antigenic sub-units are both purified and adjuvanted by selective adsorption onto colloidal aluminum hydroxide.

15. A process as claimed in claim 11 or claim 12 in which the colloidal aluminum hydroxide contains about 2% as Al(OH)3 and the concentration of the colloidal aluminum hydroxide in the vaccine is from 6.0% to 18% v/v.

16. A process as claimed in claim 1 in which the adjuvant is an emulsion of water in an oily fatty acid glyceride.

17. A process as claimed in claim 16 in which the adjuvant is an emulsion of water in peanut oil containing sorbitan tri-oleate as emulsifier and aluminum monostearate as stabiliser.

18. A virus sub-unit vaccine containing antigens having haemagglutinating and neuraminidase activity or analagous protective antigenic components derived from an ether-sensitive virus capable of releasing said antigens or components, and being substantially free from pyrogens, virion nucleic acids and unwanted antigenic material, whenever prepared by the process according to claim 1, 2 or 3 or an obvious chemical equivalent.

19. An influenza virus sub-unit vaccine containing protective antigenic sub-units of an ether-sensitive influenza virus and having haemagglutinating and neuraminidase activity and being substantially free from pyrogens, virion nucleic acids and unwanted antigenic material, whenever prepared by the process according to claim 9 or an obvious chemical equivalent.

20. A process as claimed in claim 1 wherein the virus is an influenza virus liberating neuraminidase and haemagglutinin.

21. An influenza virus sub-unit vaccine containing a mixture of protective antigenic sub-units which have haemagglutining and neuraminidase activity and which is substantially free of other undesirable components of the virus particle, whenever prepared by a process according to claim 20 or an obvious chemical equivalent.