IL23187A - Production of vaccines - Google Patents

Description

•ninii jna τιιηι«»η ·*π ^ i V PATENTS AND DESIGNS ORDINANCE SPECIFICATIO Improvements in the production of vaccines o»a *3 n TIS» *-» o»>i 3w I (we) THE WELLCOME FOUKDATIOK LIMITED, a British company, of 183-193, Bus on Hoad, London, H.W.I, England do hereby declare the nature of this invention and in what manner the same is to be performed, to be particularly described and ascertained in and by the following statement : - 333.87/2 fills invention relates to the inaetivation of mixoviruses, and in particular to the productio of vaccines therefrom* 'Jfhe myxovirus group includes the viruses of the influenza group influenzae-A. M. influenzae**!** M. influenzae-O and H« multiforfflae {Se eastle disease virusj/* the parainfluenza group pa ainfluenzae-1 (formerly M. influenzae-!? of Sendai virus), M. parainfluenzae-2 (acute laryngotracheobronchitis virus), M. paratofluenzae-3 (hemadsorption virus) and 1»parainfluenzae-4 (M.25 j¾, pestigalll (fowl plague virus), ig. oarotidia (mumps virus), measles virus, distemper virus, rinderpest irus respiratory syncytical virus and allegedly rubella virus (German measles)* These viruses are pathogenic, and cause overt or in-apparent infections, which are frequently associated with an inflammation in the respiratory tract* She diseases caused by myxoviruses are associated with an immune response, thus susceptible animals or persons can be protected, against the disease by vaccination. To produce a vaccine the virus must be deprived of its infective properties without losing its antigenic activity, i.e. the ability to stimulate the production of antibodies in the tissues of susceptible animals or persons. This may be effected by attenuation, which onl alters the virus so as to lose its pathogenicity, or by inaetivation, which also deprives the virus of its ability to multiply. When this inaetivation is carried out with formaldehyde, considerable losses in antigenicity can be observed and also neutralising agents have to be added subsequently to the system* 23187/2 It has been shown that several members of the myxovirus group can be inactivated in aqueous suspension by contacting the suspension with diethyl ether in the presence of a pol oxy thylene sorbitan mono-oleate This method, however, suffers from the disadvantage that special and expensive equipment is needed to reduce the risk of fire owing to the inflammability of diethyl ether, and that about 7$ diethyl ether remains dissolved in the aqueous medium* It has now been discovered that memb ers of the myxovirus group can advantageously be inactivated without significant loss of antigenic activity by contacting the aqueous suspension of the virus with certain non-inflammable organic solvents , which are immiscible or partially miscible with water, in the presence of a non-ionizing hydrophilic surface active agent , such as a polyoxyethylene sorbitan mono-oleate.

Organic solvents which have been found useful in carrying out the method according to the invention are fully or partially chlorinated, or chlorinated and fluorinated lower hydrocarbons other than chloroform.

They include tetrachloro-methane , trichloro-ethylene , tetrachloro-ethylene , 1 ,1 ,2-trichloro-1 ,2,2-trifluoro-ethane , 1 ,1 ,1 ,3,3,3-hexachloro-propane and hexachloro-but -1 ,3-diene. TTOien inactivating measles virus by the method of the present invention , best results , i. e . highest antigenicities , have been obtained with tetrachloro-ethylene . Not infrequently higher antigenicities have been observed after inactivation than before .

To provide the non-ionizing hydrophylic surface active agent , a polyoxyethylene sorbitan mono-oleate, such as marketed by Messrs . Atlas Manufacturing Co. under the trade name Tween 80, may conveniently be used.

Other agents falling into this class , such as polyoxyalkylene ethers of partial lauric , palmitic or stearic acid esters (Tween 20, 0 and 6θ) , as well as other oleic acid esters have also been found satisfactory for the purpose. To be effective , the concentration of the agent should be at least about 0.05%, Below this , increasing losses in antigenicity may be observed. Within reasonable limits relatively high concentrations of out the agent may be used with/ any harmful effect , but above 0.2% no further improvement can usually be observed. The pH of the aqueous suspension is adjusted to a value between pH 6.0 and 8.0, preferably between pH 6.5 and 7.5.

The volume of the solvent , relative to the volume of the aqueous suspension containing the virus , is unimportant so far as two distinct phases are formed and the proportion of volumes is not excessive so as to make contacting inconvenient or inefficient. It may conveniently be between Q and >Qlf of the volume of the aqueous suspension. To secure a satisfactory contact with the virus, the two phases are thoroughly mixed, preferably for at least one hour.

For the purposes of the present invention any strain of the selected virus, which is substantially free of contaminating viruses, may be used. In the case of measles virus the Edmonston strain has been found satisfactory, and has been preferred because it is commonly used in the production of measles vaccines. This strain may for instance conveniently be grown on a chick embryo tissue culture or a monkey kidney tissue culture, or on a culture of a stable diploid cell line as the Wist^r strain. The tissue culture fluid (TCF) is usually harvested and clarified by slow centrifugation or filtration. The fluid is then treated and the virus therein inactivated by contacting it with a solvent according to the present invention. Subsequently the solvent is removed for instance by centrifugation and decantation. The suspension containing the inactivated virus particles constitutes a primary vaccine in which fragments of the virus particles carry the 'antigenic activity.

Viruses belonging to the influenza group (A, B, C and M. multi ormae may also be grown on a chick embryo tissue culture or a monkey kidney tissue culture, or they may conveniently be grown in embryonated hens' eggs In the latter case the harvested allantoic fluid is treated with a solvent, according to the invention. After the removal of the solvent, the aqueous suspension constitutes the primary vaccine.

The primary vaccine itself may be used for inoculation, or it may be further improved by purification or concentration. Stabilizers, such as polyvinyl pyrolidine or dextran may also be added to the vaccine.

It has also been found that a primary vaccine containing myxoviruses inactivated by the present method can be purified, and if desired, presented in a concentrated form by adsorbing the fragments of virus on particles/to an aluminium salt, which is not more than slightly soluble in a neutral medium. Examples of such a salt are aluminium phosphate, a pharmaceutically acceptable alum, aluminium hydroxide or oxide. The aluminium salt, with the antigenic substance adsorbed on it, can be separated and resuspended in a new medium, which may have a smaller volume than the volume of the primary vaccine.

The adsorption may be carried out at a pH between pH 6 and pH 8 preferably between pH 6 and 7, For example, a primary measles vaccine may conveniently be adjusted to the required pH, and a suspension of the rJ.uminium salt may then be added in an aqueous solution adjusted to a pH close to the required pH value. The mixture may then be shaken for about an hour, and the adsorbent sedimented, separated, and resuspended in a new medium, usually consisting of a saline solution buffered to a pH between 6.0 and 8.0.

In the case of potassium alum as an adsorbent, a soluble aluminium salt may be added in solution to a primary vaccine and then the alum is precipitated from the solution by the addition of an appropriate potassium salt and adjustment of the pH to the required value.

The concentration of the adsorbent may be chosen according to the concentration of the antigen, and may conveniently be around 1.0 mg./ml. at the e.dsorption stage and around 4.0 mg./ml. in the concentrated vaccine.

Protamine sulphate may also be added to the primary vaccine before the addition of the adsorbent; protamine is also partially or fully adsorbed on the aluminium salt, and is not eluted therefrom when resuspended in new medium at a pH around pH 6.0.

A purified or concentrated vaccine produced according to the invention may be mixed with other vaccines, or the aluminium salt may also be used to adsorb other materials having different antigenicity, provided that the constituents are mutually compatible. 23187/2 The antigenic activity of a tissue culture fluid or a vaccine may measles virus an assay of these sera may be carried out by methods well known in immunology such as by neutralising the infectivity of a standard pool of measles virus (Neut. test), or by the haemagglutination inhibition test (HAI. test), in which a diluted series of the serum is mixed with a standard pool of haemagglutinating measles virus, and the highest dilution causing complete inhibition of haemagglutination is recorded.

In the case of measles virus grown on monkey kidney tissue cultures, it has also been possible to test the antigenicity of these suspensions in vitro by the haemagglutination activity test (HA. test), as this type of virus has such an activity. It has been proved and is accepted in immunology that the haemagglutinating activity, and thus the HA. test is positively correlated with the antigenicity of this family of viruses, and can therefore be used as 'a measure and in vitro test of antigenicity..

Measles virus grown on chick embryo tissue culture has no haem - agglutinating activity, and no suitable method has hitherto been 'available for the efficient in vitro testing of these vaccines. ■ ' The antigenic activity of tissue culture fluids and vaccines containing measles virus having no haemagglutinating activity can now be tested in vitro by the Antibody Combining Test (AbC test). In this test a diluted series of samples of the culture fluid or vaccine is mixed with a standard dilution of antiserum, havin /determined antibody content.

After incubation, standard haemagglutinating/ antigen and a suspension of Erythrocebus patas red blood cells are added, and the samples are again incubated. Where the test antigen is present in a sufficient amount, the haemagglutination inhibiting action of the serum is blocked and the red cells are agglutinated.' The test results obtained by this method 23187/2 ' ! . ' have been found to be positively end adequately corelated with the direct tests of antigenic activity, using the serum of inoculated animals.

The infectivity of tissue culture fluids and that of the vaccine is determined by the highest dilution that produce infection in 50 of the standard tissue cultures (TCD 50.). It has been found that-ihe infectivity of the virus after inactivation by the treatment according "to to the present invention has been reduced/below detectable levels.

According to the present invention in oi¾3 aspect therefore, there is provided a method for the inactivation of myxoviruses which method . ■ comprises"contacting the viruses in an aqueous suspension at a pH between 6 and 8 in the presence of at least about 0>05 w/v of a non-ionizing hydrophilic surface active agent with a fully or partially chlorinated, or chlorinated and fluorinated lower hydrocarbo other , than chloroform, which is liquid at room temperature. In another aspect there is provided a primary vaccine which contains. ntigens derived from ^ myxoviruses inactivated by the method of the present invention.

In a further aspect, there is provided a method for producing a purified or concentrated vaccine, which method comprises adsorbing fragments of myxovirus particles, obtained by inactivating the viruses as. hereinbe ore described, from an aqueous medium at a pH between 6 and 8 on to^ n aluminium salt, which is pharmaceutically acceptable and is not more than slightly soluble at this pH, separating the salt and resuspending it in a new medium. In another aspect there is provided a purified vaccine, comprising an aluminium salt, having fragmented-' myxovirus ■ 'particles adsorbed on it, suspended in an aqueous medium.

■ The following examples illustrate the invention.

Exam le 1 ■ fV> ; M Edmonston strain of measles virus was grown and propagated in a chick embryo tissue culture maintained on a serum free medium (S.M. 199) The harvested tissue culture fluid (TCF) was clarified by centrifugation at a low speed.

A 6.2 w/v solution of T een 80 (Atlas Manufacturing Co. ) (9 ml. ) was added to the clarified tissue culture fluid (441 ml. ) j ami "the fluid was then thoroughly mixed with tetrachloro-ethylene (50 ml. ) at room temperature for one hour.

The solvent was subsequently removed by centrifugation. The aqueous phase collected contained less than 0.01$ tetrachloro-ethylene and constituted a primary vaccine.

The vaccine was assayed for inf ectivity, and also for antigenic activity by injecting it twice (10 days apart ) into guinea pigs, bleeding, the animals for antibody on the 7 h day after the second injection, and testing the antibody content of the sera by the Neutr. test and ΗΔΙ. test methods. The vaccine was also tested by the Antibody Combining Test in vitro. Results were as follows.

Inf ectivity Geometric Mean Titre of G-uine In vitro Sera AbC HAI test Neutr. test test TCP 1 0 , .7 28 68 16 Primary vaccine 0 2 83 16 Measles virus tissue culture fluid (1 176 ml. ) prepared as in Example 1 , was clarified by filtration. To this clarified fluid a 5% solution of Tween 80 (24 ml. ) and tetrachloro-ethylene (106 ml. ) was added. The fluid was then thoroughly mixed with the solvent for one hour, and was subsequently separated from it by centrifugation.

The primary vaccine was assayed for infectivity and antigenic activity as in Example 1 . Results were as follows.

Inf ectivity G- .M .T. of Guinea pig sera HAI test , Neutr. test TCP 10 ,4.5 128 168 Primary vaccine 0 128 98.5 An Edmonston strain of measles virus was grown and propagated in an Erythrocebus patas monkey kidney tissue culture maintained on a serum free medium (S.M. 1 99 ). The harvested tissue culture fluid was clarified by slow centrif ugation , and Tween 80 ms added to a final concentration of 1 .25 mg./ml. The fluid was then shaken with an equal volume of tetra-chloromethane for hours at room temperature. After shaking, the solvent was removed by centrif ugation , and the aqueous layer was tested for infectivity and for antigenic activity by the HAI test and Neutr. test as in Example 1 , and also by the HA. test . Results were as follows.

Infectivity G- .M.T . of Guinea pig sera in vitro HAI test Neutr. test HA. test TCP 1 0 ·7 51 325 12 Primary vaccine 0 28 365 16 Example 4 To a tissue culture fluid prepared as in Example , a solution of Tween 80 was added to a concentration of 1 .25 mg./ml. The fluid was then shaken with an equal volume of tetrachloro-ethylene. The solvent was then removed and the aqueous layer tested. Results were as follows.

Infectivity HA. test TCP 10 A-.7 4 Primary vaccine 0 4 Samples of a tissue culture fluid, prepared as in Example 3> were shaken with 10 v/v trichloro-ethylene, 1 ,1 ,1 ,3,3,3-hexe>chloropropane, hexaohloro-buta-1 ,3-diene and ,1 ,2-trichloro- ,2,2-trifluoroethane, respectively.

The solvents were removed by centrifugation and the samples were assayed in vitro for antigenic activity by the HA. test. In all cases at least 50$ of the HA. activity was retained and no infectivity was shown after inactivation.

Example 6 To a tissue culture fluid (40 ml.) prepared as in Example 3, a 6.2 $ solution of Tween 80 (9 ml.) a 5$ solution of polyvinyl pyrrolidine (40 ml.) and tetrachloro-ethylene (50 ml.) were added.

The mixture was homogenised for 4 hours, and the solvent was subsequently separated. The vaccine was assayed for infectivity and antigenic activity by the HAI. test. Results were as follows.

Infec ivity HAI. test 4.0 TCP 10' 40 Primary vaccine 0 64 A Herts, strain of Newcastle disease virus was cultivated in embryonated hen's eggs. To a sample of the allantoic fluid (9.8 ml.) a $ solution of Tween 80 (0.2 ml.) and tetrachloroethylene (1 ml.) or, in another test, ,1 -trichloro-1 ,2,2-trifluoro-ethane (l ml.) was added.

The samples were shaken for 4 hours and were then centrifuged. The aqueous l HA. test Original allantoic fluid 2048 Tetrachloro-ethylene treated vaccine 096 1 ,1 ,2-trichloro-1 ,2,2-trifluoro- 1 024 ethane treated vaccine Example 8 Three other representatives of the myxovirus group, namely, The aqueous layers after extraction were assayed for fowl red blood cells haemagglutinating activity.

HA. test Sendai Original Allantoic Fluid 1 024 tetrachloro-ethylene treated 2048 suspension trichloro-trifluoro-ethane treated 51 2 5 2 4096 suspension Example 9 A primary vaccine (90 ml. ) , prepared from measles virus grown in a chick embryo tissue culture as described in Example 1 , was acidified with 0.1 hydrochloric acid to pH 6.0, and aluminium phosphate (1 0 mg. ) suspended in water (1 0 ml. ) was added. The mixture was stirred for an hour at room temperature and then allowed to settle overnight at 4°C .

The clear supernatant fluid was decanted, and the aluminium salt further sedimented by centrif ugation. After the remaining supernatant fluid was removed, the aluminium salt was resuspended in normal solution (1 00 ml. ) buffered to pH 6.0 by a 0.017 M S rensen ' s phosphate buffer.

This constituted a purified vaccine , adsorbed on aluminium phosphate.

The vaccine was tested for antigenic activity and the results were as follows.

Primary vaccine 21 5 83 Purified vaccine 1 94 92 To the acidified primary vaccine of Example 9 (85.5 ml. ) a 0.2^ w/v protamine solution (4.5 ml. ) and subsequently aluminium phosphate (1 0 mg. ) suspended in water (1 0 ml. ) were added. The mixture was stirred, sedimented and resuspended as in Example 1 .

The product constituted a purified vaccine, adsorb ed on aluminium phosphate with protamine . The final concentration were approx. 1 mg. AlPO^/ml. and 00 μ . protamine sulphate/ml. The vaccine was tested for antigenic activity and the results were as follows .

HAI. test Neutr. test Primary vaccine 2 83 Purified vaccine 223 28 with protamine Example 11 A primary vaccine (400 ml. ) , prepared from measles virus grown in a chick embryo tissue culture as described in Example 2, was adjusted to pH 6.0 with N hydrochloric acid (18 ml. ) . Aluminium phosphate (1 .60 g. ) suspended in water (220 ml. ) was added, and after stirring the mixture was allowed to settle. After sedimentation and separation the aluminium salt was resuspended in normal saline solution (400 ml. ) buffered to pH 6.0.

The product constituted a purified vaccine adsorbed on aluminium phosphate, containing 4 mg. The vaccine was tested and the results were as follows .

HAI. test Keutr. test Primary vaccine 1 28 98.5 Purified vaccine ■ 180 332 The purified vaccine of the . aluminium phosphate protamine type , prepared in Example 1 0 (320 ml. ) was sedimented by centrifugation, and the separated aluminium salt was resuspended in normal buffered saline solution (80 ml. ) to yield a fourfold concentrated and purified vaccine. The final-concentration were approx. mg. AlPO^/ml . and 400 μ¾. protamine sulphate/ml. The vaccine was tested and results were as follows.

Neutr. test Purified vaccine 223 128 Purified & cono . 5 2 51 2 vaccine Example 13 A primary vaccine (90 ml. ) prepared in Example 2, was acidified with N hydrochloric acid, to pH 6.5 , end aluminium phosphate (9.8 mg. ) suspended in water (1 0 ml. ) was added. The mixture was stirred for 2 hours at 4°C , end then left to settle overnight at 4°C.

The clear supernatant fluid was decanted, and the aluminium salt further sedimented by centrifugation. Then each half of the deposit was re-suspended in a) a normal saline solution (50 ml. ) buffered to pH 7.4 and b ) the same buffer (1 2.5 ml. ) to yield a fourfold concentrated vaccine.

Pinal concentrations were a) 0.98 mg. AlPO^/ml. and b ) 3.8 mg.

The vaccines were tested for antigenic activity and the results were as follows .

HAI . test Primary vaccine 31 .75 Purified vaccine (a) 90.2 Purif . & cone. 640.0 vaccine (b ) Example 14 A primary vaccine (45 Ε1· ) > prepared from measles virus grown in s Erythrocebus patas monkey kidney tissue culture as described in Example 6 was acidified to pH 6.5 with hydrochloric acid. Aluminium phosphate ( 0 mg. ) suspended in water (5 ml. ) was added, and the mixture was shaken at 4°C for 1 hour. The aluminium salt was then sedimented by centrifugation, and was resuspended in serum free (S .M. 199) medium 0 ml, ).

This constituted a purified vaccine. The vaccine was tested and results were as follows .

HAI, test Primary vaccine 64 Purified 80 vaccine An Edmonston strain of measles virus was grown and propaged in chick embryo tissue cultures maintained on a serum-free medium (SM 199) .

To the harvested infected tissue culture fluid (20, 0 l) was added a 5% solution of Tween 80 (400 ml. ) in normal saline and tetrachloro-ethylene (2.0 1 ) .

The mixture was homogenised for two hours. Samples were removed at 20, 0 and 60 mins. for virus assay. The samples were centrifuged to remove tetrachloro-ethylene and 0.5 ml. undiluted fluid was added to each of 20 culture tubes of Patas monkey kidney. These tubes were observed for 1 days. Wo cytopathic effect was observed in any culture.

After the homogenisation, the bulk vaccine was clarified and freed of tetraoiiloro-ethylene by continuous-flow oentrifugation. 25Ο ml. samples of the primary vaccine were tested in chick-embryo tissue cultures and Patas monkey kidney tissue culture.for the presence of live virus. No cytopathic effect was observed in any culture after 14 days incubation.

The primary vaccine (12 l) was acidified to pH 6.0 by the addition of 0.1 N hydrochloric acid in normal saline (l80 ml.). To the acidified vaccine an aluminium phosphate stock solution (I46O ml.) containing 8.13 ng. AlPO^ml. was added. The mixture was stirred for 1 hour and.allowed to settle overnight at 4°C. The supernatant fluid was then removed and the aluminium phosphate suspension made up to 3.O L. by the addition of normal saline buffered at pH 6.0 by Hi/so S rensen1 s phosphate buffer. The final concentration of aluninium phosphate in the concentrated vaccine was estimated at 3.84 mg./ml.

G-uinea-pig Potency Tests were carried out on samples taken throughout the process, and the results were as follows: - G-uinea-Pig Test Results S mple Diln. HAI N.T.

Original tetrachloro-ethylene 19 † V4 3.4 Final Vaccine (4 times 1/1 32 concentrated) V4 6.7 Supernatant from Adsorption 4 Repeat Test Final Vaccine (four times Vi 84.4 304 Example 16 The PR-8 strain of type A influenza virus was grown and propagated in embryonated chicken eggs. The virus-containing allantoic fluid was harvested and the virus removed by centrifu-gation. The sedimented virus was resuspended in isotonic saline buffered to pH 7- 2 and clarified by low speed centrifugation.

The clarified virus suspension 400 ml. was brought to 0.1 (w/v) of Tween 80 and 00 ml. of tetrachloroethylene (400 ml.) was added. The resultant mixture 'was agitr.ted in an ice bath for -five hours.

The solvent was subsequently removed by decantation and centrifugation. The aqueous phase which constituted the vaccine, contained less than O.Olo tetrachloro-ethylene.

The vaccine and a sample of untreated virus were assayed for their chicken cell agglutinating titers. The results were as follows: - Untreated virus - 1308 CCA units/ml. 1%E treated virus - 2781 CCA units/ml.

The Ann Arbor strain of type A-l influenza virus was grown and propagated in embryonated chicken eggs. The virus-containing allantoic fluid was harvested and the virus removed by centrifugation. The sedimented virus was resuspended in isotonic saline buffered to pH 7· 2 and clarified by low speed centrifugation.

The clarified virus suspension (450 ml.) was brought to 0,1% (w/v) of Tween 80 and tetrachloro-ethylene (450 ml.) was added. The resultant mixture was agitated in an ice bath for five hours.

The solvent was subsequently removed by decantation and centrifugation. The aqueous phase, which constituted the vaccine, contained less than 0.01 tetrachloro-ethylene.

The vaccine and a sample of untreated virus were assayed for their chicken cell agglutinating titers. The results were as follows: Intact virus - 1717 CCA units/ml. fCE treated virus - 2605 CCA units/ml.

Example 18.

The Japan 170 strain o:- type A-2 influenza virus was grown and propagated in embryonated chicken eggs. The virus-containing allantoic fluid was harvested and the virus removed by centrifugation The sedirnented virus was resuspended in isotonic saline buffered to pH 7.2 and clarified by low speed centrifugation.

The clarified virus suspension (825 nil.) was brought to 0.1 (w/v) of Tween 80 and tetrachloro-ethylene (825 ml.) was added. The resultant mixture was agitated in an ice bath for five hours.

The solvent was subsequently removed by decantation and centrif gation. The aqueous phase, which constituted the vaccine, contained less than 0,01$ tetrachloro-ethylene.

The vaccine and a sample of untreated virus were assayed for their chicken cell agglutinating titers. The results were as follows: - Intact virus - 1331 CCA units/ml.

"FCE treated virus - 2171 CCA units/nl.

Example 19 The Maryland strain of type B influenza virus was grown and propagated in embryonating chicken eggs. The virus-containing allantoic fluid was harvested and the virus removed by centrifugation The sedirnented virus was resuspended in isotonic saline buffered to pH 7.2 and clarified by low speed centrifugation. fhe d&rlfled viae suspension (300 ml.) rought to 0.1$ (w/v) of Sween 80 and tetracnloro-ethylene (300 ml.) as added* fhe resultant mixture s agitated in an ice bath or five hours* She solvent was subsequently removed by deoantation and centrifugatlon. $he aqueous phase which constituted the vaool , contained less than 0.01$ tetraohloro-e hylene· he vaccine and a sample of untreated virus were assayed for their chicken cell agglutinating titers. She results were as followsss Untreated virus - 1794 OCA units/ml.

TOE treated virus - 1767 COA unite/ml.

Exampl ¾0 A "JUDITH" (laearthy) strain of rubella virus was propagated in an BE 13 rabbit tissue culture maintained in an augmented Sagle»s medium, fo the harvested tissue culture fluid (17.3 cil* allquots) was added a 5 fween 80 solution (0.35 ml. to each), and the fluid s shaken for 15 minutes. Setrachloroethylene (1.75 ml. to each) was then added and the mixture was shaken.

One aliquot mixture was then eentrifuged for 15 minutes after shaking for 109 20 and 60 minutes and the supernatant liquors were tested for residual infectivlty by inoculation into Bryfehrocebtts patae monkey kidney tissue culture. After 7 days the cultures were challenged with a measured dose of ΙΪ.6. "ΒΟΒΟ* virus and incubated for a further 6 days. $ e Jesuits were estimated by using the interference phenomenon, and were as followst freatsent flme lnfectlvity/0«5 ml* (minutes) Untreated suspension - 104.1 ith 2ween 80 only 60 103.3 Tween 80 + CB 10 101,7 I» 20 ioX n 60 0 (10°) She original tissue culture fluid and the 60 min* extract were tested for antigenicity in the usual Banner in rabfcits ed guinea-pigs* Mo significant loss in antigenic potency was demonstrated.

Claims (4)

1. HAVIHG NOW particularly described and ascertained the , nature of our said invention and in what manner the same is to be performed, we declare that what we claim is: » 2(-· 1. A method for the inactivation of myxoviruses, which comprises contacting the viruses in an aqueous ^suspension at a pH between 6 and 8 in the presence of at least about 0.05¾ w/v of ΆΚ ionizing hydrophilic surface active agent with a fully or partially chlorinated, or chlorinated and fluorinated lower hydrocarbon other than chloroform, which is liquid at room temperature.

2. A method according to claim 1, in which the non-ionizing surface active agent is a polyoxyethylene sorbitan mono-oleate.

3. A method according to claim 2, in which the concentration of the polyoxyethylene sorbitan' mono-oleate is between 0,05 and. Z. A method according to any of the preceding claims, in which the pH of the suspension is between pH 6.5 and 7·5· 5. A method according to any of the' preceding claims, in which the fully chlorinated lower hydrocarbon is tetrachloro-ethylene. 6. A method according to any of the claims 1 to k-, in which the fully chlorinated lower hydrocarbon is tetrachloro-methane. 7. A method according to any of the claims 1 to 4, in which the fully chlorinated lower hydrocarbon is hexachloro-buta-l,3-diene. Λ v 8. A method according to any of the claims 1 to > n which the partially chlorinated lower hydrocarbon is trichloro-ethylene. A 254 9½ A method according to any of the claims 1 to » in which the partially chlorinated lower hydrocai'bon is ljl,l, 3> 3> 3j-hexachloro-propane, 10. A method according to any of the claims 1 to » in which the chlorinated nd fluorinated lower hydrocarbon is 1, 1, 2-trichloro-. 1, 2, 2-trifluoro-ethane. 11. Δ method for the inactivation of myxoviruses substantially as described with reference to any of the Examples 1 to 19. 12. A method for the production of a primary vaccine, which comprises inactivating myxoviruses according to a method claimed in any of claims 1 to 11, separating the organic phase and presenting the aqueous phase as the primary vaccine. 13. method according to claim 12, in which a stabilizer, such as polyvinyl pyrrolidine, is added to the vaccine. 14· A method for the production of a purified or concentrated vaccine, which comprises adsorbing fragments of myxovirus particles, as obtained by inactivating the viruses according to a method/claimed in any of claims 1 to 11, from an aqueous medium at a pH between 6 and 8 on to an aluminium salt, which is pharmaceutically acceptable and is not more than slightly soluble at this pH, separating the salt and resuspending it in a new medium. 15. A method according to claim 14, in which the aluminium salt contains aluminium phosphate. 16. A method according to claim 15, in which the aluminium salt is resuspended in s S/5renson phosphate buffer having a pH & pH 6, 0. 17· A method according to any of claims 14 to 16 in which the medium containing fragments of wyxovirus particles contains also protamine. 18. A method for the production of a purified or concentrated vaccine according to Claim 14 substantially as described with reference to any of the Examples 9 to 15· 19i A method according to any of the prceding claims in which the myxovirus is measles virus. 20· A method according to claim 19» in which the measles virus has been grown and propagated in a chick embryo tissue culture* 21, A method according to claim 19» in which the measles virus has been grown and propagated in a i&rythrocebus patas monkey kidney tissue culture. 224 A method according to any of claims 19 to 21, in which measles virus of the Edmonston strain is used* 2 « A method according to any of claims 1 to 18· i& which myxoviruses of the influenza group are used* 24» A method according to claim ?» in ¾rhlch M* Influenzae-A viruses are used* 25. A method according to claim 23, in which M. Influenzae-B viruses are used. 26· A method according to claim 23 in which M. multiformae (Newcastle disease) viruses are used* 27· A method according to any of claims 1 to 18, in which niyxoviruses of the parainfluenza group are used. 28. method according to claim 27, in which M. in luenzae-D (Se¾dai) viruses are used. 29. Inactivated myxoviruses, whenever produced by a method including the step claimed in any of claims 1 to 11. 30. Inactivated measles viruses, whenever produced by a method including the step claimed in any of claims 1 to 11, and any of claims l^to 22. 31. Inactivated viruses of the influenza group, whenever produced by method including the step claimed in claim 23 and any of claims 1 to 11. 32. Inactivated M. influenzae-A viruses whenever produced by a method including the step claimed in claim 24, and any of claims 1 to 11. 33 · Inactivated influenzae-B viruses, whenever produced by a method including the step claimed in claim 25» ?-nd any of claims 1 to 11. 34· Inactivated guJLtiformae (Newcastle disease) viruses, whenever produced by a method including the step claimed in claim 26, and any of claims 1 to 11. 35· Inactivated viruses of the parainfluenza group, whenever produced by a method including the step claimed in claim 27, and any of claims 1 to 11. 36» Inactivated Μ. par influenza-I (Sej¾dai) viruses, v7henever produced by a nethod including the step claimed in claim 28, and any of claims 1 to 11, 37· -A primary vaccine, containing antigens derived from nyxoviruses inactivated by a method including the step claimed in any of claims 1 to 11. 38. A primary vaccine, containing antigens derived from measles viruses inactivated by a method including the step claimed in any of claims 1 to 11, and any of claims 19 to 22. 39. A purified vaccine, 7/henever produced by a method including the step clained in any of claims 14 to 18. 40. A purified measles vaccine, whenever produced by a method including the step clained in any of claims 14 to 18, and any of claims 19 to 22. 1. A purified influenza vaccine, vhenever produced by a method including the step claimed in 14 to 18, and any of claims 23 to 2^>. 42. A purified vaccine, comprising an aqueous suspension of an aluminium salt bearing antigenic fragments of myxoviruses adsorbed on to it. 43 · A purified vaccine according to claim 42, in which the fragments are those of measles viruses. 4

4. A purified vaccine according to claim 42, in τ/hich the fragments are derived from viruses of the influenza group. 45» A purified vaccine according to any of claims 42 to 44» in which the aluminium ealt is aluminium phosphate* 46· method according to any of claims 1 to 18, in ¾hieh the mixovirus is the rubella virus* 47» Inactivated rubella virus, whenever produced by a method including the step claimed in claim 4$t and any of claims 1 to 11· Sated this 23th larch 65· For the Applicants DR. KBimow oom & GO.