CA1271708A - Process for the preparation of a rabies vaccine and the vaccine obtained by this process - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The invention relates to a process for the preparation of a rabies vaccine by multiplication of rabies viruses in animal nerve tissue or poultry embryos, harvesting of the viruses from the nerve tissue or from the heads of the embryos, enriching the viral preparation, inactivating thereof, and preparing a vaccine. In one aspect, the process comprises homogenizing the nerve tissue or embryo heads and harvesting of the viruses therefrom by avoiding the use of a mixer, and thus preventing damage to and fragmentation of the viruses. The nerve tissue, the embryo heads or their contents are comminuted in a manner which preserves cell and viral integrity. The preparation is then treated by separating the complete live viruses which are capable of multiplication from the resulting cell suspension, delipidating by extraction with a water-immiscible organic solvent and then further selectively concentrating the viruses thereof.
The invention also relates to a myelin-free rabies vaccine which has been obtained by the process described above from animal were tissue or poultry embryo head tissue containing rabies viruses.

Description

1~'71~

BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to a new, economic process for obtaining a rabies vaccine comprising obtaining whole live viruses and rendering the viruses thereof incapable of replicating by chemical treatment. This invention also relates to a vaccine obtained by this process, which is by reason of ~ts high purity, distinguished by a high specific activity and the absence of undesirable secondary reactions when inoculated to human sl~bjects.

Descri~tion of the Back~round Most rabies vaccines have up until the present time been obtained by multiplication of the rabies virus in living animals such as mice, rats, rabbits, sheep, etc. However, lS the thus obtained virus-containing preparations contain considerable amounts of myelin and elicit detrimental side effects.
In recent times, rabies vaccines have also been obtained from viruses multiplied in poultry embryos~ This method has the advantage, in principle, that the thus obtained virus-containing tissue contains hardly any in~urious myelin.
After multlplication o~ the viruses ln poultry embryos, these embryos are completely homogenized ln toto ln a mlxer or blender. In this manner, however, it is only possible to incompletely separate from this pasty homogenate the virus ~ 7~

constituents from heterologous protein which may initiate undesired secondary reactions upon inoculation. This is also the case with vaccines obtained from brains of living animals which have been infected with rabies. On repeated inoculation - indispensable in the case of huntsmen, forestry workers, veterinarians, etc. - these secondary reactions may increase considerably and result in violent allergic defense reactions against the heterologous proteins.
The quality of embryo vaccines has been somewhat improved by using only the heads of the embryos to obtain the vaccines. Since, in comparison, embryo heads carry an essentially higher concentration of the viruses, the vaccines prepared only from embryo heads have a correspondingly lower content of byproducts and cause fewer side effects (German Patent 3,009,064; U.S. Patent 4,255,520).
However, in the course of preparing vaccines from nerve tissue of animals (from embryos or from embryo heads), viruses are often damaged or fragmented when the virus-containing tissues are homogenized with a mixer or blender.
This considerably reduces the activity of the vaccine prepared from homogenates of this type and makes its purifi-cation more difficult since large amounts of proteins and li~uids are released from the fragmented cells.
A slightly better vaccine has only been obtained by multiplication of the rabies viruses by in vitro culturing human diploid cells (HDC)(H. KoProwski/ "Vaccine for man prepared in human diploid cells", Laboratory Techniques in Rables by M . M . Kaplan and H. Koprowski, WHO Monograph Series No. 23, Chapter 28, pp. 256-60 (1973); T.J. Wiktor, Develop. Biol. Standard, Vol. 37, pp. 256-66, S. Karger, Basel 1978, "Production and control o~ rabies vaccines made on diploid cells"; T.J. Wiktor et al. "Development and clinical trial of rabies vaccine of tissue culture origin", Develop. Biol. Standard, Vol. 40, pp. 3-9 (1978)). The thus obtained vaccines contain human protelns as contaminants.
Such proteins, however, although producing fewer secondary reactions than do heterologo~s proteins, still produce some.
A considerable disadvantage of this method is the relatively low multiplication rate of the rabies viruses in diploid fibroblast cells. This re~uires the use of a 10- to 25-fold greater concentration of the vaccine. Hence, this method is not efficient enough to meet world-wide demand for rabies vaccine in an economically feasible manner.
The preparation of a rabies vaccine in duck embryo cell cultures is described in U.S. Patent 3,674,862. In this process, however, the multiplication rate in cell cultures is limited.(U.S. Patent 3,973,000 describes a method for the enrichment of rabies viruses by density gradient centrifuga-tion M. Rolle and A. MaYr: Mikrobiologie, Infektions- und Seuchenlehre, Stuttgart (Microbiology, infection and epidemiology):489-493 Stuttgart (1978) describe the traditional preparation of duck embryo rabies vaccine).

1~ 71 ~3~

Thus, there is a pre~ing need for a new and highly active rabies vacclne which contains mechanically intact vlruses with fully retalned anti~enic activlty, whlch is 6traightforward to prepare and thu~ not too costly, and free of ~lde effect6. Such vacclne would be an effective and well-tolerated vaccine which has long been sought for world-wlde control of the fearsome and fatal rabies d~sease.

SUMMARY OF~THE INVENTION
The present invention provides a proce6s for obtain~ng inactivated rabies viru.~es which are substantially myelin-free, comprising:
(1) intracerebrally inoculating an experimental animal with whole live rabies viruses;

(2) allowing for ~aid viruses to multiply;

(3) comminuting nerve tissue from the animal's brain to obtain a cell suspen~ion, 6aid comminution belng conducted in the absence of a mixer to pre~erve the integrity of the viru~es;

(4) separating live whole viruses from the cell 6uspension;

(5) dellpidatlny the live whole vlrufie6) and

(6) 6electlvely concentrfltlng the viru~eGJ whereln step6 (1) through (4) are conducted at lea6t once and up to 3 tlmes.

'7~7~8 ~n ~ddltlon, thi~ lnv~ntlon alro provldes a proc~s for obt~lnlng inactiv~ted rabies viruses which are sub~tantlally myelln-free comprlslng:
(1) lnoc~latlng a poulery ambryo ~g~ wlth whol~ llv~
S rabie~ vlruses;
(2) allowlng for ~aid vlruse~ to multlply~
~ 3) commlnutlng the embryo from ~he poulery egg to obtaln ~ cell 6u~pensiont cald commlnutlon bæing conducted ln tho ~bsence of a mlxer to ~r~s~rve the lntegrity of the v-ruse~
~ 4) separ~ting llve whole viruses from the cell su~penslon;
(5) del~pidatlng the llve whole vlruses; and (6) selectively concentratlng the attenuated vlruse6;
whereln steps ~1) throu~h 14) ~re performed at lea~t once and up to 3 times.

Thls lnvent1on al~o provl~s a r~blc~ vacclne comprlslng inactivated r~bies viru~es which are ~ub~tantially myelin-free, cald vlruses bslng prosent ln ~n ~mount eff~ctlvo to ellclt ~n lmmunl~lng r~pon~e when admlnl~tere~
to a ~ub~ect. The pre-ant vacclne m~y b~ obta~na~ by th~
her~lnabove proce~se~.

~71~

DESCRIPTION OF THE INVENTION
The present invention relates to an esonomic process for preparing a rabies vaccine which is of the highest quality when compared to vaccines obtained fxom viruses multiplied in diploid human cell cultures.
In one aspect of this invent~on the process comprises (1) isolating the rabies viruses which have multiplied in animal nerve tissue or poultry embryos avoiding mechanical damage to or fragmentation of the viruses thereof;
(2) removing lipids from the resulting viruses by extraction with a water-immiscible organic solvent such as volatile paraffin hydrocarbons or halogenated hydrocarbons such as fluorinated hydrocarbons;
(3) enriching the delipidated viruses by density gradient centrifugation;
(4) precipitating the viruses by addition of a polyethylene glycol (e.g., PEG 6000) and concentrating by centrifugation and purifying the live whole viruses.

Step (1) entails extracting the rabies virus by cautious comminution (preserve cell and viral integrity) of the nerve tissue or poultry embryo heads (e.g., duck, chicken or quall), and washing the tissue fragments with ~ buffer, e.g., phosphate-containing buffer. This step is supexior than homogenizing in a mixer or blender since foreign proteins and lipids are solubilized to a lesser extent, the occurrence of 1~ 7~ 8 oxidation products of antigens, proteins and lipids is avoided, and the content of intracellular, incomplete and non-immunizing rabies an~igen is diminished. The vlrus-containing suspension obtained by washing the tissue frag~
ments with an agueous buffer solution is then removed by differential centrifugation.
At least 95% of the residual protein is discarded by operations 3 and/or 4.
The viruses are then finally inactivated in a known manner, for example by addition of ~-propionolactone or tri-(n-butyl)phosphate.

DESCRIPTION OF THE PREFERRE~D_EMBODIMENTS
The individual process steps of the inventive process are performed in such a manner that a surprisingly good lS overall result is achieved. Harvesting viruses only form the heads of the embryos entails producing a high basic concentration of the viruses. The mild treatment of the virus-containing tissue material gives a fine paste, especially upon avoiding the homogenization thereof with a mixer or blender, provides a viral suspension which exhibits substantlally no mechanically d~a~ed or fragmented viruses with incomplete antigen content, and which, moreover, contains far fewer foreign materials such as cell debris, proteins and lipids. The remaining lipids can be removed from this viral suspension by extraction, and the proteins "1~717<~

c~n be f ~r more complet21y r~moved by eelectlv~ concentratlon and/or prec~pltatlon of the vlru~ th~n from a p~ty homo~enate. ~he vaccln~ obtalned by the proce~- comprlslng the s~u~nce of ~eps d~crlbod 18 lmprov~d by around 90-fold S compsred wlth the conventlon~l duck embryo rabfeL vacclno.
When the preparaelon of r~ble~ vacclne 1~ obtalned by pa~sAg~n~ on nerve tl-~u~6 of anlmal~ cuch as mlce, rat6, rabblt6 and cheep, the vlruce6 are multlplled ln the llvln~
an~mal by lntracerebr~l lnocul~tlon of rablec viru~e~
of standardl~ed ~eed straln6. It mu6t, however, be noted that the multlpllcatlon of the vlru~ec ln llvln~ anlmal6 ha~
the dlcadvantage, comp~red wlth the multlpllcatlon of the vlruse6 ln poultry embryo6, that the nerve tl66ue~ of ll~ln~
anlmal~ conta~n myelln. Thl~ proteln ls known to glve rlse to secondary reactlons when the vaccine 16 u~ed, lncludlng enceph~lltlo.
A~ter slaughterlng the animal6, whlch were prevloucly lnocul~ted wlth llve whole rablec vlru6es, thelr bralns sre r~moved, commlnuted ln a manner whlch precerve6 the wholene~
20 of the cells and the vlrucec, and a vacclne 1c propar~d from the r~ultln~ oell su-pcn~on by the proc~c do-crlbed her~ln~bovc. Owlnu to thc voldancc of coll fra~ment-tlon dur~n~ tho commlnutlon of the vlru~-contalnln~ nerve tl-~ue~, lc~ myelln 16 rele~ed than ~urlng commlnutlon wlth a 25 mlxer. Durlng the extrac~lon of the llpld6 wlth ~n or~nlc olv~nt ln a later ~tep of the proce~c a further p-rt of the ~ 2 7~

8tlll pre~ent myelin 1~ removed ln Duch a manner that the vacclno whlch 18 finally o~alnod cause~ only mlnlmal, lf ~ny, ~condary reactions, and ~ho-e whlch ~re caused are ~tlll hlghly tol~rable.
The ~teps of the proces~ must b~ conduct~d nvoldlng ~he use of a mlx~r or blender ~or the homo~en~atlon of nerve tl88u08 or of embryo6 or embryo head~ on harve~tlng of the vlruse~. Thls preven~s damage to and fragmentatlon of the vlrus2s by commlnution of the n~rve tl~sue~ or of the embryos or embryo heads and thetr co~tents ln a manner which preserves the wholene~s of the cell8 and the viruse~, ~eparattng the complete llve vlru~os whlch are c~pable of multlpllca~ion from the re~ultlng cell 6uspenslon, and purlfylng the resultlny vlral suspenslon, delipidatlng by extractlon wlth a water-~mmlw lble organlc solvent and then 6electlvely concentratlng the vlral prepsration.
The commlnution of the nerve tls~ue, the embryos or embryo heads or thelr contentE ~ i8 c~rrled out wlth the ald of a meat mlncer on ~ cour6e ~ettin~, by cuttlng up or by openlng of the heads and comm~nutln~ the removed brain tlssue ln a manner wh~ch pre6erve6 the lntogrlty of the cell8, and th~refore the vlruse~.
Washing or extractlng the vlru~eo from the commlnut~d tl~8ue 18 carrled out wlth a buffer solutlon, pref~rably wlth an aqueou~ phosphnte buffer of about pH 7-8, a~ 18 ~nown ln the art. The removal of the llpld6 18 carrled out by 127~7~)8 extractlon wlth a water-immisiclble solveht, xuch a~ liquid, volatile, optionally halogenated hydrocarbons. Sultable ~olvents are petroleum ethers such as heptanes, fluorlnated and chlorinated ethanes and homologs thereof. However other solvents can al80 be used. Th~ further concentration of the delipldated vlr~l suspension can be carried out by density gradient centrlfugation and/or precipitation with a polyethylene glycol, preferably w~th PEG 6000, as is known in the art. Suitable t~pes of embryo poultry eggs for the multiplication of rabies viruses are in particular those from ducks, chickens and quails. In general, incubated duck eggs are preferred as the tissue for the multiplication of the vlruses. The myelin-free rabies vaccine provided herein may be obtalned from poultry embryo head tissue which lS contains rabies viruses by the process which is described above, which process fully preserves viral integrity.
The processes according to the invention results in a rabies vaccine which, compared with the vaccines obtained by processes hitherto known, exhibits a far better ratio of antigen contant to proteln content, contain subst~nti~lly no foreign lipid~, and ~pproach in ~uallty an ldesl HDC vaccine.

717~)8 DETAILED DESCRIPTION OF THE PROCESS
Now the process will be described in relation to each separate step.

Ste~ 1:
A rabies virus strain which is suitable for the preparation of the vaccine is adapted to the intended viral host by appropriate passages on the embryonal cells of poultry eggs or in mice, rats, rabbits or sheep, among others.
Attenuated rabies viruses are, for example, inoculated into the yolk sac of fertilized poultry eggs which have undergone initial incubation and in which an embryo has started to develop. After about two weeks, the embryos are removed and their heads are harvested. The embryo heads are comminuted in a manner which preserves cell and viral integrity in a meat mincer.
Alternatively, the head of the embryo is cut open and the brain tissue is removed and comminuted. The multiplication of the viruses may also be undertaken in living animals. In such case, animals whlch are only a few days old (mice, rats, rabbits, lambs, etc.) are usually inoculated intracerebrally with the same 6pecles-specific attenuated seed virus.
After about 10-30 days the animals are sacrificed, and the brains are removed by operation and comminuted in a ~ 71 7~

manner which pre~erves cell and viral integrity. The extraction of the rabie~ viru~ from the comminuted tlssue is c~rried out by wa~hlng the tl6~ue fragment~ wlth a phosphate-contalnln~ buffer. A suitable pho6phate buffer S 1E one comprislng, e.g., 0.75~ by weight of disodium hydrogen pho~phate (Na2HP04), 0.145~ by weight of potassium dihydrogen phosphate (KH2PO4) and 0.48~ by welght of ~odium chlorlde in distilled water, pH 7.~. However, other buffer solutions known ln the art may al80 be used. It i8 egually possible to use ~or the extr ction, ~tabilizers and salt ~olutions which ase cu~tomar~ly u~ed for the preparation of viral vaccine ~u~pensions, or even deionized water as long as the pH is in the range between 7 ~nd 8.
The cuspension containing the viral antigens is 6eparated from the ti~ue by differential centrlfugation at about 10,000-15,000 x g (g bein~ the acceleration of gravity). The remaining ti6 ue 6ediment can be used for further extraction~, by which means a yleld of about 30~ of vlral antigen i5 po66ible. The two or more virus-containing extracts are comb~ned and then f~ltered.

SteD 2:
The forel~n lipld~ stlll remalnln~ ln the vlr~l ~uspenslon are removed by extractlon wlth a water-lmmi6clble organic 601vent, 6uch as, e.g., with an hydrocarbon, optionally halogenated and preferably fluorinated.

1~ 7 17 ~

Subse~uently, the antigen extract (the viral suspension) is enriched by density gradient centrifugation in a manner known per se at 15,000-90,000 x g using a buffer and sugar solutions of various concentrations, by increasing the sugar 5 concentration in the buffer in a manner known in the art.
Alternatively, the viral suspension can be concentrated by precipitation with polye~hylene glycol.

Ste~ 3:
The density ~radient centrifugation is carried out in a manner known per se at 15,000 to 19,000 x g using sugar solutions of various concentrations and buffer solutions, by increasing the ~ugar concentration in a buffer solution. For this purpose, the prepurified suspension is pumped ~t 15,000 to 90,000 x g at a flow rate of, e.g., 4 litres/h over a step gradient of an increasing concentration of sugar (usually, sucrose from 15 to 55 %) which has previously been introduced. The fractions collected from the various densities are then subjected to tests for density, the contents of lipids, nucleoproteins and glycoproteins, and 6terility.
The antlgen-containing fractions are pooled, tested once more, and then processed further to obtain the vaccine.
Physiological saline solutions of any type, e.g., the phosphate buffer mentioned above, can be used for dilution in a manner known per se (Duck embryo rabies vaccine:

~27:~7~3~3 J.M._ Hoskins, Laboratory Techni~ues ln R~bles by M.M. Kaplan et al., WHO Geneva 1973, Chapter 27, page~ 243-55; Density gradient centr~fugation: J. Hilfenhaus et al., J. Biol. Standard. 4:263-271 (1973); M. Ma1er et al., 5 Develop. Biol. Stand~rd. 37:267-271 ~1977); and P. Atanasiu et al., Develop. Biol. Standard. 40:35-44.

Ste~ 4:
In addition or alternative to the enrichment of the lO virus concentration by density gradient centrifuqation, the prepurified, and usually enriched, viral suspension can be further concentrated and purified by precipitation with a polyethylene glycol, preferably free of heterologous protein. For this purpose, the pH of the viral suspension 15 can be adjusted to about 8. After addition of a polyethylene glycol (e.g., PEG 6000) to a final concentration of 6% by weight, the suspen~ion is stirred for at least one hour and the virus is precipitated by ~ubsequent centrifugation at 10,000--15,000 x g. The viral sediment is then re~uspended 20 in a stabilizer composed of a ~olutlon containing lactose and physiological gelatin ~E.M. Mikhailov6ky,e~t al., Ann. Inst. Pasteur 121:563-568 (19'71)S J~me6 McSlarrv et al., Virology 40:745-746 (1970).

1~ 7 Step 5:
The lntact llve vlruse6 capable of mult~pllcatlon whlch are present in the re~ultln~ vlral concentrate are now lnact~vated. ~eta-pxopionolactone (~PL) lfi u~ually u~ed 5 for the lnactivation (G.A. LoGrl po, Annals New York ~a~. of Sci. 83:578-94 (1960). However other substances are ~lso ~ uitable for this purpose such as tri(n-butyl) pho~ph~te (H. T~nt et al., Sympos1a ~erles ln "A new tis~ue culture lO r~bles VACCine, lnactivated and di~ggregated w~th tri-(n-butyl) phosphate" Immunoblol. standard. (Kar~er, Basel) 21:132-144; T.J. Wiktor et al., Develop. Biol.
Standard. 40:3-9 (1978).
The vaccine concentrate obtained by the new process dlffer~ from commercially available rabies vacclne6 in lt~
hlgh content of antigen value units per mg of nitrogen (meas-ured using the standard NIH test in mice and the antibody bind-ing test in the RFFIT). Preferavly, the vaccine contains more than antiqen value unl~s per m~ of nitrogen, and stlll more preferably more th~n 15 unit6 per mg of nltrogen, butalw~y~ more than 8 unlt6 per mg of nitrogen. A~ a rul~, the ~ame can ~e obtained u~lng unb~rn embryo~ wh~ch ~o not a6 yet feel paln and ln which the braln tlssue, whlch 1B ~ust ln the process of development, appear~ to be stlll fr~e of 25 myelln (M. Abdussalen et ~1-, "The problem of antl-rable6 vacclnation", ~nternatlonhl conference on the appllcatlon of ~ 7~

vaccine against assay viral rickettsial and bacterial diseases of man, Pan. Am. Health Org. (PAHO), Sc. pub. No.
226:54-59 (1970); and P. Fenie, "The status of existing rabies vaccines", ibid. pages 60-65).

SteD 6:
~ he vaccine resulting after the inactivation can be dispensed into vials and can then be freeze-dried. It may be reconstituted for use by dissolution or suspension using distilled water.
As is well understood by those skilled in the art of viral purification, additional steps may be included to further purify the rabies virus.
It is possible by the process which has been described herein to prepare unlimited, or at least adequate, amounts of a valuable and innocuous rabies vaccine in an economic and relatively straightforward manner. The preparation of such quality rabies vaccine by multiplication of the viruses in human diploid cell cultures (HDC) is highly impossible as a conseguence of the low efficiency o~ the substrate.
~t i8 noteworthy that by an order of February 1979, the CDC has restricted the use of human diplold cell rabies vaccine to people having developed ll~e-threatenlng side effect6 after administration of the duck embryo vaccine or who were incapable of acquiring an appropriate titer of antibodies. The reason given for this is inade~uate 1~ 7~ 7~

productivity of the human diploid cell cultures (See also, Morbiditv and Mortalitv Weekly~llæe2E~ (MMWR) 27:333, 413 (1978)).
The rabie~ vaccine prepared by the process according to the invention is at least equivalent to an HDC vaccine in which the viruses have been multiplied in human diploid cell cultures (See, Example 1 hereinbelow). No side effects have been observed upon administration of this vaccine up to the present time, thereby making available for medical use a rabies va~cine of excellent value and effectivepess and which has negligible side effects.
When the antigen of this invention is used to induce immune response in a human or animal, it is administered in an amount sufficient to elicit an immunizing response. The amount of antigen may be adjusted by a clinician doing the administration, as commonly occurs in the administration of vaccines and other viral agents which induce immunizing responses. Suitable vaccine unit amounts are between about 2.5 units and lO units, preferably between about 4 units and 6 units. Although a single admini~tration induces an immune response, multiple administrations may be carried out if desired or if 60 re~uired in accordance with schedules known per se. The route of administration can be any of the routes generally used for rabies vacclnes, 6uch as by injection subcutaneously, intramusculary and the like.

3 27~

Havlng now generally described this invention, the same will be better understood by reference to certain ~pecific examples, which are included herein for purposes of illustration only and are not intended to be limiting of the lnvention or any embodiment thereof, unless so 6pecified.

EXAMPLES

Example 1 - PREPARATION OF A PURIFIE~ DUCK EMBRYO RABIES VACCINE

I. Preparation of the virus suspension (a) The "Wistar rabies, PM (Pitman-Moore) 8HDCS" virus strain from the Wistar Institute, Philadelphia, or another rabies virus strain suitable for the preparation of a vaccine was adapted to the embryo cells before actual use by intracerebral passage in mice and repeated passage by inoculation in duck eggs which have undergone initial incubation. The viruses used for the preparation of the vaccine are those from a passage with a particularly high titer and which h~ve already proved to be suitable in the preparation of rabies vaccine in accordance with the method of J.M. Hos~ins, "Laboratory Techni~ue~", in Rables by Kaplan et al., W~O, 27:243-55 Duck Embryo V~ccine (1973).
Fertilized duck eggs from healthy stocks were incubated at a temperature of 36C + 1C and a humidity of 65-70%. After six dsys they are candled with W light and ~.2~7~7(~8 unsultable eggs are rejected. On day 7 of incubation, the rables virus was inoculated directly into the yolk sac of the eggs in which an embryo was developing. The incubation was continued and 10-14 days later the eggs were again candled 5 with W light. The eggs in which the embryos continued to develop well were opened under sterile conditions, and the embryos were removed and decapitated. The heads were stored individually under sterile conditions in the vapor phase over liguid nitrogen until the ~terility tests were complete. Groups of 40-60 of the sterile heads were combined into a pool with the addition of a defined amount of a stabilizer. The sterility of each pool was again tested. In addition to the stabilizer, it was also possible to use a NaCl/phosphate buffer comprising 0.75% disodium hydrogen phosphate, 0.145% potassium dihydrogen phosphate and 0.48~
sodium chloride in distilled water, or other saline solutions as are customary for the purpose of diluting vaccines, even desalinated water, as long as the pH was in the range between

7 and 8.
The rabies virus extract was obtained by comminution of the above mentioned sterile embryo heads using a meat mincer. The tlssue fragments were washed twice with a phosphate-containlng buffer. After centrifugation at 10,000-15,000 x g and at a temperature of 2-8C the infectious virus was collected in the supernatant fraction.
Remaining brain particles or other lipid-containing tissues i~7~08 were removed by 6u~sequent ~iltr~tlon throu~h a ~auze fllter ~ystem. The remalning re~idue6 of head ti6~ue can be extracted once more and flltered by u~e of the same proce~s, by whlch means hlqher ~nti~en yield of about 30~ ls 5 achleved. The 6edlment wa~ a~aln 6u~pended in a phosphate-containlng bu~er and ~tirred for at least one hour at low temperature (1-4C) before the centrlfugatlon and filtration.
(b) A subsequent, virtually complete dellpidation 10 was carried out by mlxing the re6ultln~ vir~l ~uspens~on with an inert liquid hydrocarbon solvent with a rela~lvely low den~lty, such a~, for example, n-heptane.
Homogenization wa~ carrled out ln every ca~e under a ~la~
bell contalning nitrogen ~a~. The viral 6uspen~ion was pumped through a mixer system, e.~., Vlrtl~ mlxer, at a constant flow rate of, e.g., 500 ml/min. At the ~ame tlme, the n-heptane was pumped lnto the mixer ~ystem ~t a ra~e of 50 ml/mln. The llpld-contalnln~ phase was removed by centrifu~atlon at 10,000-15,000 x ~. Traces of the dl6solved hydrocarbon solvent were then removed from the dellpldated vlrus extract by allowing an lnert ~as cuch ~6 , e.g., nltro~en, to bubble through the ~queouc pha~e and malntalnln~
the acqueous pha6e under vacuum ~t 4-C for ~ p~rlod of about 15 hours.
(c) An alternatlve proces~ for the hydroc~rbon dellpldation i~ as follow~. Ster4le embryo head~ may be ~ 7 17 0~

comminuted, ex~racted and flltered a~ d~ cribed under (la).
The removal of the forel~n lipid~ may then be carrled out by u~ing the fluorln~ed hydrocarbon ~olvent 1,1,2-trichlorotrlfluoroethane. ~he indlvldual workin~
~tep~ re~in ~he ~me.

II. Concen~ration and further purlfication of the viru~
suspenslon (a) The prepurified rable~ vlr~l uspen~lon prepared by the proce~ de~cribed above had a v~ral tlter between 107 ~nd 1o8 MLD50/ml. ~his m~ter~al wa~ further purifled and concentrated by centrifugatin~ once or twice on a linear sucrose gradlent (15-55~) at 75,000-90,000 x g. A concentration factor of lOOol wa~ attained ln thls manner. The glycoprotein and nucleoproteln content (before and after solubllizatlon of the viru~ membrane with Triton X 100, that is to ~ay election of the ~nt4ct virion~), the VlrU8 titer, the dens$ty and the 6~erlllty of the gr~dlent fraction6 were tested. Sterlle ~raction~ wlth a ratio of rable6 glycoproteln to nucleoproteln which correspond6 to th~t of the purlfied whole vlrlon solution, ~nd with a very hlgh lnfectlous tlter (for ex~nple 109-101 MLD50/ml) were comblned and reserved for further proce~slng.
(b) A further purlflcatlon and concentra~ion of the viral 6uspen~1On can be ~chleved by poly~thylene glycol (PEG) proclpltatlon. For thi6 purpo~e, PE~ 6000 lSie~frled A.G., ~27~708 Zoflngen, switzerland) was dls~olvod ln a 30~ ~tren~th pho~hate-contalnln~ buffer ~olutlon (pH 8.0). Tht6 ~tock PEG
~olutlon was ~terlll~d ln an autoclave ~nd ~or~d ~t 4DC.
The vlral ~uspen~lon wh~ch WaB ~dJu~t~d to a pH of 8.0 S wlth ~ 10~ NaO~ ~olutlon wa~ then pr~clplt~ted wlth tho g~ock P~G solutlon ~t a flnal dllutlon of 6~. The mixture wa~
~tlrred ~t a temperature of 4-C for at lea~t one ~our. The rables vlru~ can then be ~edlmented by centrlfu~atlon at 6peed of lO,OQO-15,000 x g over a perlod of 30 mln. The removed vlru6 wa~ agaln 6u6pended wlth a ~tablllzer to the flnal volume and wa6 re6erved for further proce~ing.

III. Formulatlon of the viral concentrate~
Pretested vlr~l concentrate~ were comblned and dlluted with a 6ulta~1e stabllizer, for example ~odlum phosphate bu~er (pH 7.4), wlth a phy6iologlcal sodium chlorlde ~olutlon, or w:Lth ænother st~blll~er whlch hn~ already been descrlbed (6ee, Hoskln~, l.c.) to ~ concentratlon of ~bsut MLD50/ml. Sterllity and vlru~ tlter were te~ted agaln.

IV. Inactlvatlon of thQ viru~e~
For the lnactlvatlon wlth beta-proplonolActone, the flnal vol~me of the vlr~l Eu~pen~lon war m~lntnln~ ~t A
temperature of 1-4-C wlth contlnuou~ ~tlrrln~. Fr~hly prepared, lae-cold a~ueou~ bet~-proplonol~ctone ~olut1on wa~

- ~3 -~ 2 ~ 7 added in an amount ~uch ~hat a concentration of 1:4,000 wa~
attained. After the suspen~lon wa~ ~tirred ~t a temperature of 4C for 5 min, it wa~ tran6ferred into a s~co~d ve~sel and ~t~rred for 8 further 40 hour~ the pH and tempera~ure were S contlnously monitored. A decrea6e in the p~ wn~ taken a~ a mea~ure of BPL hydroly61s. As recorded, the pH fell from about 8.0 to about 7.4. At the end of the lna~tivation, thlomersal ~o-(ethylmercurythio)-benzoic ac~d) was added until the concentration of thi~ antiseptic 6ub6tance was 1:10,000.

V. Freeze-dryina The inactivated viral suspension obtained in accordance with sectlon IV was dispensed in sinsle doses of 1 ml into 3 ml vials, ~reeze-drying stoppers were placed 1006ely on top, and the vaccine was freeze-dried in vacuo. When the drying process wa~ completed the ~toppers were pu6hed ln tlght and the vial6 were clo~ed wlth metal caps to assure the tightness of the vials. The vlals were then stored at a temperature of -20C.

VI. Reconstitution to ~lve the vaccine readY for uoe, and use of thi~ vacclne Prior to its use, 1 ml of 6terile di~tllled water WaE
ln~ected through the rubber ~topper into each vial. The vlal Wa6 then shaken cautlously, wlthout formln~ a foam, until the vaccine was completely dis olved. The entire content of the vial was then injected subcutaneously into the upper arm of the subject.

VII. ~uality control of the final product - tests The quslity control procedures comprised: the determination of the antigenicity, sterility, inactivity, innocuousness ~nd contents of nitrogen, cholesterol, NaCl, BPL residues and thiomersal.

Antigenicity:
Antigens were tested in accordance with standard instructions of the National Institute of Health, USA. Their ability to bind antibodies in the RFFIT test was also measured (R.J. Arko et al., Laboratory Technigues in Rabies, 3rd edition, WHO Monograph Series 23:265-267 (1973); and J.S. Smith et al., Lab. techn. in Rab., 3rd edition, WHO Monogra~h Series 23:354 to 357 (1973~.

`` ~27~708 Sterilit~:
All the final products for use were proven to be sterile.

Inactivitv:
This was tested in every case on three young rabbits and ten mice, which, after intracerebral inoculation of the reconst~tuted vaccine, were observed for 14 days. The animals showed no signs of disease in any case.

Innocuousness:
Three guinea pigs received 5 ml intraperitoneal doses of the reconstituted vaccine solution, and 3 mice received 0.5 ml i.v. doses.
In no case did the animals show reactions differing from normal.
The stability of the vaccine obtalned in accordance wlth the above description in the freeze-dried form was also tested. Efficacy (AGV-U/ml as a percentage of the initial figure (0)) was preserved after storage at the stated temperature for ~ months.
(a) Stability of the vaccine obtained in accordance with Example 1 (concentration in accordance with 2a) in the freeze-dried ~orm. The ~;~7~7~

activity (AGV-U/ml) as a percentage of the inltial figure (0 figure) is shown in Table 1, hereinbelow.

Table 1: Activity of the Vaccine (Example 1-2a) Batch 0 figure ~ 37C ~ 37C
numb~r AGV-U/ml 1 month 2 months 83 Ly III T16 6.7 100% 110%
83 Ly III T18 7.3 93% 92%
.

(b) Stability of the rabies vaccine obt~ined in accordance with Example 1 (concentration in accordance with 2b) in the freeze-dried form. The activity ~AGV-UJml) as a percentage of the initial figure (0 figure) is shown in Table 2 hereinbelow.

Table 2: Activity of the Vaccine (Example 1-2b) Batch 0 figure + 37C + 37C
number AGV-U/ml 1 month 2 months 83 Ly III T15 5.0 148% 98%
83 Ly III T19 8.2 107% 104~

83 Ly III T20 9.7 144% 76%
83 Ly III T2115.3 ~124% 92~
83 Ly III T22 8.5 165% 105%
83 Ly III T2313.4 105% 112 83 Ly III T23 9.5 147% 126 ~._ ..

~ 7 17~

(c) The activity of the rabies vaccine obtained in accordance with Example 1 in a dog after s.c. inoculation is shown in Table 3 hereinbelow..

Table 3: Activity of Vaccine (Example 1, dog) Number of inoculated Vaccine Vaccine animals with more thanaccording toaccording to (sa), 7 dogs (2b), 8 dogs 0.5 IU 100% 100%
1 86% 88~
2 43~ 75%

-IU = international units of antibody content (d) Comparative activity in human~ of the rabies vaccine obtained in accordance with Example 1 and the HCD vaccine (Behring).

The activity of the new vaccine was compared with that of the HCD vaccine (Behring). Table 4 hereinbelow ~hows the percentage of sub~ects which immunolo~lcally reacted by forming antibodies after administration of one of these vaccines, in general 0.' IU being regarded as conferring protection (inoculation on days 0, 3, 7, 14 and 28).

~27~708 Table 4: Comparative Activity of InYentive vaccine and HCD Vaccine _ Antibody titer Vaccine Vaccine HDC vaccine (RFFIT) (Ex. 1 (2b)) (Ex. (2a))( ehrinq-Werke) (day 14) 3 consecu- 2 consecu-AGV: 6.3 IU
tive batches tive batches 20 subjects 54 subjects lS subjects O.S IU 100% 100% 100%
2 98~ 100% 100%
80% 80% 80%
45% 60% 50%
28% 47% 30%
.
IU = $nternational units of antibody content Results The rabies vaccine prepared by the process according to the invention proved in the clinical trial to be of at lea~t egual quality to an HDC vaccine, wherein the viruses had been multiplied in human diploid cell cultures (HDC).

Example 2 - Preparation Of A Purified Duck Embryo Vaccine.
Rabie~ viruses were multiplied in duck eggs whlch have undergone initial incubation as described in Example 1. The viruses were separated from the embryo heads by cutting the heads open, removing the brain tissue and comminutlng in a ~ 7~ 70~

manner preserving cell and viral integrity, and were harvested by resuspension in a phosphate buffer ~lution.
The viral suspension was further processed to give a vaccine as in Example 1~

Exam~le 3 - Purified Duck Embryo Vaccine.
A highly concentrated viral suspension was prepared in accordance with Example 1 and was inactivated by treatment with tri-(n-butyl) phosphate. After inactivation, ~he concentrate was freeze-dried.

Example 4 - Preparation Of Purified Chicken Embryo Vaccine.
Chicken eggs are incubated at 36C + 1C under a humidity of 60-75% for 7 days. On day 7 of the incubation, the inoculum virus was directly inoculated into the yolk sac of the embryos undergoing development in the eggs. The incubation continued. Seven days later, the eggs were opened and the embryos were removed and dismembered. ~he heads, the spinal cord and the trunks were processed separately. They were comminuted in a manner such that they provided a 10%
strength tissue su6pen~ion (that i6 to 6ay a suspenslon o 10% by welght of embryo tlssue). The viral concentratlon was tltr~ted using Antibody binding test in the RFFIT (rapid Fluorescent focus Inhibition Test). Three series of tests were carried out; the results of which are shown in the table hereinbelow.

~7~08 Table 5: Preparatlon of Chlcken Embryo Vaccine Vlral concentration (ID 50/ml) Head 7.2s / 7.4 & 7.8 Sp~nal cord 6.55 & 7.8 Trunk 6~0 / 6.6 & 6.8 -- , ID = 50/ml = vlru~ titer x log 10 for the minimum con~entration for lnfectlon of 50% of the tis~ue culture~.

It was found that the ti~sue of the central nervous ~y~tem (CNS) contained about 10 times a6 much viruses a~ the trunk without the CNS.
on the ~asls of the6e re~ults, the preparation of the chicken embryo vaccine by the method described in Ex~mple 1 wa~ carrled out only wlth ~he embryo head6. In the pre~ent ca~e, the rabies viruse6 whlch had been adapted to chicken cell~ wa6 multiplied in partially incubated chicken egg~, e.g., by the method of H. Koprowskv (Laboratory technique ln Rable~ by M.M. ~aplan et al., WHO Geneva, Chapter 26, pa~eo 235-242).
The eggs were lnocul~ted on d~y 7 of lncubatlon and lnGubation was contlnue~ the next day. Seven to nine days af~er inoculation of the virus into the yolk 6ac, the heads 127170~3 of the chicken embryo~ were removed and proce~sed in accordance with the method de~cribed in Example 1. This entailed the final concentration of the viruses being carried out by precipita~ion with polyethylene glycol. The vaccine prepared in this manner was sub~ected to the quality con~rol test~ described under I through VII. This vaccine also proved to be fully active in humans.

Exam~le S - Preparation Of Quail Embryo Vaccine.
In a manner analogous to that described i~ Example 1, attentuated rabies viruses were multipl$ed in quail eggs whlch had undergone initial incubation, and were harvested and processed to obtain the vaccine. The resulting vaccine proved to be fully active in animal experiments.

Example 6 - Preparation Of A Rabies Vaccine From Viruses Multiplied In Mice.
Five-day old mice were inoculated intracerebrally with attenuated seed rabies viruses. After ten days, the mice which were still alive were sacrified. The brains of the anlmals were removed and comminuted in a manner which 20 pre~erved cell and viral lntegrlty. The cell suspension was proces6ed to glve a vacclne by the process of Example 1.

~7~7~

Exam~le ? - Preparation Of A Rable Vacclne From Viruses Mult~plied In Rats.
3-4 day old rats were inoculated intracerebrally with attenuated ~eed rabie~ vlruses. After 12 day~, the rats which were ~till ~urvlvin~ were ~acrified. The brains of the animal6 were processed to glve the vaccine in analogy to Example 6.

ExamPle 8 - Rables Vaccine From Viruses Multiplied In Rabbits.
lG Six day old rabbi~s were inocula~ed intracerebrally with attentuated ~eed viruses. After 15 days the rabbits were sacrified. The brains of the animals were processed to give a vaccine in analogy to Example 6.

Example 9 - Vaccine From Viruses Multiplied In Lambs.
lS 8 10 day old sheep were inoculated intr~cerebrally with attenuated seed rabies viruses. After 30 days, the lamb~
were sacrified. Their brains were removed and processed to give a vaccine in anAlogy to Ex~mple 6.

1 ~ 7~ 7~

Having now fully described the invention, it w~ll be apparent to one of ordinary ~kill in the art that many changes and modifications can be made thereto without departin~ from the spirit of the scope of the invention as 6et forth herein.

Claims (38)

WE CLAIM:

1. A process for obtaining inactivated rabies viruses, comprising:
(1) intracerebrally inoculating an experimental animal with whole live rabies viruses;
(2) allowing for said viruses to multiply;
(3) comminuting nerve tissue from the animal's brain to obtain a cell suspension, said comminution being conducted in the absence of a mixer to preserve the integrity of the viruses;
(4) separating live whole viruses from the cell suspension;
(5) delipidating the live whole viruses; and (6) selectively concentrating the viruses; wherein steps (1) through (4) are conducted at least once and up to 3 times; said viruses being substantially myelin-free.

2. The process of claim 1 wherein the viruses are selectively concentrated by density gradient centrifugation or precipitation with a polyethylene glycol.

3. The process of claim 1 further comprising inactivating the viruses.

4. The process of claim 1, wherein the viruses are separated from the cell suspension by washing and suspending thereof in a physiological buffer solution having a pH about 7 to 8.

5. The process of claim 1 wherein the viruses are dilapidated by adding a water-immiscible liquid organic solvent.

6. The process of claim 1, wherein the solvent is a hydrocarbon selected from the group consisting of water-immisible liquid hydrocarbons or halogenated hydrocarbons.

7. The process of claim 6 wherein the delipidating hydrocarbon is an halogenated hydrocarbon.

8. The process of claim 6 wherein the hydrocarbon is selected from the group consisting of a petroleum ether, fluorinated or chlorinated ethane and homologues thereof.

9. The process of claim 8 wherein the petroleum ether is heptanes.

10. The process of claim 1 wherein the animal is selected from the group consisting of mice, rats, rabbits and sheep.

11. The process of claim 1 further comprising precipi-tation and concentrating the viruses.

12. The process of claim 3 wherein the viruses are inactivated by adding a virus-inactivating amount of .beta.-propionolactone or tri-(n-butyl)phosphate.

13. The process of claim 1 further comprising placing said viruses in a sterile vial and freeze-drying thereof.

14. A rabies vaccine comprising inactivated rabies viruses which are substantially myelin-free in an amount effective to elicit an immunologizing response when administered to a subject.

15. The rabies vaccine of claim 14 having at least 10 antigen value units per mg of nitrogen.

16. The rabies vaccine of claim 14 in dosage unit form.

17. A rabies vaccine comprising attenuated rabies viruses obtained by the process of claim 1, said vaccine being substantially myelin-free and said viruses being present in an amount effective to elicit an immunizing response when administered to a subject.

18. The rabies vaccine of claim 17 having at least 10 antigen value units per mg of nitrogen.

19. The rabies vaccine of claim 14 in dosage unit form.

20. A process for obtaining attenuated rabies viruses comprising inoculating a poultry embryo egg with whole live rabies viruses;
allowing for said viruses to multiply;
comminuting the embryo from the poultry egg to obtain a cell suspension; said comminution being conducted in the absence of a mixer to preserve the integrity of the viruses;
separating live whole viruses from the cell suspension;
delipidating the live whole viruses; and selectively concentrating the viruses; wherein steps (1) through (4) are performed at least once and up to 3 times; said viruses being substantially myelin-free.

21. The process of claim 20 further comprising inocu-lating the viruses.

22. The process of claim 20 further comprising conduct-ing the following steps at least once prior to inoculating the poultry embryo intracerebrally inoculating an experimental animal with live whole rabies viruses;
allowing for the viruses to multiply; and separating live whole viruses from brain tissue.

23. The process of claim 20, wherein the attenuated viruses are selectively concentrated by density gradient centrifugation or precipitation with a polyethylene glycol.

24. The process of claim 20, wherein the viruses are separated from the cell suspension by washing and suspending thereof in a physiological buffer solution pH about 7 to 8.

25. The process of claim 20 wherein the viruses are delipidated by adding a liquid water-immiscible organic solvent.

26. The process of claim 25, wherein the solvent is a hydrocarbon selected from the group consisting of liquid, volative hydrocarbons or halogenated hydrocarbons.

27. The process of claim 26 wherein the delipidating hydrocarbon is an halogenated hydrocarbon.

28. The process of claim 26 wherein the hydrocarbon is selected from the group consisting of a petroleum ether, fluorinated or chlorinated ethane and homologues thereof.

29. The process of claim 28 wherein the petroleum ether is heptane.

30. The process of claim 22 wherein the experimental animal is selected from the group consisting of mice, rats, rabbits and sheep.

31. The process of claim 21 wherein the viruses are inactivated by adding a virus-inactivating amount of .beta.-propionolactone or tri-(n-butyl)phosphate.

32. The process of claim 20 further comprising pre-cipitating and concentrating the viruses.

33. The process of claim 20 wherein the embryonic poultry eggs are selected from the group consisting of embryonic duck, chicken and quail eggs.

34. The process of claim 20 further comprising placing said viruses in in sterile vial and freeze-drying thereof.

35. The process of claim 20 wherein the cell suspension containing the viruses is obtained by comminuting the beads of the embryos.

36. A rabies vaccine comprising inactivated rabies viruses obtained by the process of claim 20 said vaccine being substantially myelin-free and said viruses being present in an amount effective to elicit an immunizing response when administered to a subject.

37. The rabies vaccine of claim 36 containing at least antigen value units per mg of nitrogen.

38. The rabies vaccine of claim 36 in dosage unit form.