Note: Descriptions are shown in the official language in which they were submitted.
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
-- 2 --
~ 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
- 10 -
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.
- 11 -
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
- 12 -
~ 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.
- 13 -
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:
- 14 -
~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).
-- 15 --
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
- 16 -
~ 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
- 19 -
~.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
- 20 -
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
- 21 -
~ 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
- 24 -
1708
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~.
- 25 -
`` ~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
- 26
~;~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
~._ ..
- 27 -
~ 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).
- 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
- 29 -
~ 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.
- 30 -
~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
- 31 -
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.
