Note: Descriptions are shown in the official language in which they were submitted.
SPEClFICA'l`:lON:
I`his invention relates to a vaccine agai.nst
Varicella and -to methods :for the preparation of` such a
vacci.ne.
In par-tlcular, the inven-tion re].ates -to a safe
.L:i.ve nttcnllated v~lricella virus vaccine and a method of
producing the vaccine by seria.l propagation of varicella
v:irus in ce:Ll cul-ture systems.
Varicella, or chicken pox, is a high communicable
disease primarily of childhood. The illness is
characterized typically by fever and development of a mac-
ular rash which rapidly evolves through stages of papule,
and vesicle formation. Recovery is usually without inci-
den-t, but the disease even in its mild form is unsightly and
can resul-t in considerable scarring from ruptured vesicles.
In some cases, the illness may be quite severe in that
primary varicella pneumonia may occur in children or adults
Central nervous system complications such as acute cere-
bellar ataxia with tremors and muscular hypotonia may
precede, accompany, or follow varicella infection. More
rarely, there is generalized involvement of` the central
nervous system with hemorrhage, perivascular round cell
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infiltration, and demyelinization. Neuritis and myelitis
2 may also occur. The skin involvement may also be trouble-
3 some with appearance of hemorrhagic, bulbous or gangrenous
4 lesions.
Thu~ prevention of the disease by vaccination is
6 justified to preclude such events, and it i8 an object of
7 this invention to provide a safe live attenuated varicella
d virus vaccine for this purpose and a process for its prep-
9 aration.
Several investigators, in the past, have attempted
11 vaccination of susceptible human volunteers with variable
12 results with respect to "takes", and much disagreement con-
13 cerning the protective efficacy. However, there was a lack
14 of signiicant untoward effects in spite of the fact that
these a-ttempted vaccinations were of susceptible volunteers
16 with fully virulent virus in vesicular fluid from varicella~
17 zoster cases.
18 The prior art has reported the propagation of
19 varicella in various cell culture systems such as human
amnion, human embryo fibroblasts, HeLa cells and human
21 thyroid, but prior to this invention it was generally
22 believed that the only source of viable, cell-free varicella
23 virus was vesicular fluid. Caunt et al., Journal of
24 Hygiene, 62, 413-424 ~1964~ reported the isolation of cell-
free vixus from human thyroid tissue and Brunell, Viroloyy,
26 31, 732-4 ~1967) described the isolation of cell-~ree
27 virus rom human embryo fibroblasts. Vesicular fluid is
28 obviously an unsuitable source of live virus for production
29 of a vaccine because of the limited supply and virulence.
30 Human thyroid tissue is a primary human cell-line, and ~ -~
31 hence unacceptable as a tissue culture system for the `
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1 propayation and attenuation of viruse5 for the production
Z of live virus vaccines.
3 Surprisinyly, it now has been discovered that
4 call-free passage can be achieved in suitable tissue cul-
ture pxepara-tions, extra~cellular living varicella virus
6 useful as an antigen can be obtained in quantity, and a
7 live attenuated varicella vaccine which will evoke in man
8 an antibody response against a virulent varicella virus
9 without causing the severe clinical manifestations of the
~isease can be prepared therefrom.
11 The process of this invention comprises serial
12 passage of virulent varicella virus in a tissue culture
13 system. The tissue culture system can be any one suitable
14 for vaccine production and known to support growth of the
virus, such as human diploid cell strains as exemplified
16 by WI-38 and MRC-5, animal diploid cell strains (fetal
17 rhesus) and primary cells of simian origin. The preferred
18 systems are of monkey testicular tissue and diploid human
19 fetal lung tissue such as WI-38 prepared by known methods.
~ The culture is incubated at 30-38C.~ preferably
21 at about 32C. to 36C. for from 3 to about 10 days. The
2~ actual time of incubation varies and is determined by the
23 extent of viral propayation as indicated by microscopic
24 observation (e.g. CPE). The inoculum can be an infected
cell suspension or virus released by conventional methods
26 (e.g. sonication~
27 when the appropriate degree of infectivity is
28 observed, usually after from about 10 to about 40 serial
29 passages of the virus, the maintenance fluids are aseptic-
ally removed. It should be noted that the number of serial
31 passages required may depend upon the particular isolate '!. '
3~ or source of virus utili~ed in the preparation of the ~ -
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1 vaccine. The cell slleets are then rinsed several ~imes
2 witi~ a serum-free physiological solution such as phosphate
3 ~uffer, saline ~PBS) or Hanks balanced salt solutioll. A
4 suitable s~abiLizer sucil as a composition comprised of
sucrose, albumin, ylutamine and pilospha-te or the like is
6 added in a volume of from about 5 to 30% of the original
7 fluid volu~e.
Tbe infeeted cells are removed from the vessel surface
g into che stabilizer medium by appropriate means (e.g.
freeze-thawing or eell seraping). Tile stabilizer-cell
11 suspensions are then pooled and the infeeted eells are
12 further disrupted by appropriate means sueh as sonication.
13 The resultant disrupted suspension is then clarified by
14 filtration to remove the cell debris to insure a cell-
free virus preparation. The resultant virus preparation
16 is then subdivided into a suitable container for dis~
17 tribution as a vaccine. The dose can be from about 0.1
18 to about 2.0 ml. containing from 100 to about 1000 infected
19 units in flame-sealed ampules, or as a freeze-dried product
in stoppered vials ready for reconstitution in sterile
21 wa-cer. Administration can be by scarification (mul-tiple
22 puncture) intradermal, or subcutaneous routes.
~r3 The seed virus for the above process is isolaced
24 in vesicle fluid from a elinical case of chicken pox and
stored in veal infusion broth at -70C.
26 EXAMPLE
27 Live attenuated, eell free varicella vaecine prepared by
2~ 20 serial passayes in WI-38 _
29 Nineteen ~19) serial passages of varieella virus
30 in Wl-38 tissue eulture was conducted as follows:
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1 Initial isolation of varicella virus was made from
2 a vesicle ~luid specimen ob~ained from a child with clinical
3 chicken pox. The vesicle flui~ specimen was collected in
veal infusion broth and .stored at -70C.; at the time of
isolation the specimen was thawed, diluted with an equal
6 volume of veal inEusion broth containinglO0 mcg of neomycin
7 and 100 mcg of polymixin per ml and incubated for 30
8 minutes at 25C. The specimen was inoculated into estab-
g lished WI-38 cell cultures of human fetal diploid origin
and incubated at 32C., thus constituting passage one of
11 a set of serial sub-cultures.
12 Progressive cytopathology -typical of uaricella
13 was observed and when sufficiently advanced (13 days post
.~ 14 inoculation) the overlying fluids were decanted and the
15 infected cells harvested by trypsinization. Following ~:
16 removal of trypsin by centrifugation, the cells were
17 resuspended, counted and re-inoculated to a fresh set of
: 1~ WI-3~ cell cultures, constituting the second serial passage
: 19 at 32C. Subsequent passages of infected cell suspensions
20 continued in the same fashion. Passage of virus could also ~.
21 be initiated from infected cell suspensions stored at -70C. .
22 with cryogenic protective additives such as glycerol, ~;
23 sorbitol or DMSO, known to preserve cell viability. ~.
24 Using an infected cell suspension of the l9th
... 25 passage as seed virus, a live attenuated cell free vari-
26 cella vaccine was preparPd as follows~
~ 27 WI-38 cell cultures were prepared in glass rol~
28 ler bottles using ~BME, a commercial preparation of Eagle's
29 medium in Earle's basal salt solution containing 10%
. 30 unheated fetal calf serum as growth medium. Two days
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1 post-planting, the grow-tn medium was discardad and bottle
2 culture~ were inocula-ted with a~proximately 2 x 106 infec-ted
3 cells per bottle and refed wi-th 100 ml. of ~ E containing
4 2~ inac-tivated agamma calf serum.
Three days post inoculation the system was refed
6 with 100 ml. of EMBE containing 2% inactivated agamma calf
7 serum. Incubation was at 32C. in a rolling apparatus.
8 Seven days post-seeding, the bottle cultures were rinsed
g two times witn phosphate buffer saline, 100 ml. per rinse.
Six-teen ml. of SPGA stabilizer was added to each bottle.
11 The SPGA stabilizer is comprised of the following:
12 Sucrose . . . . . . . . . . . . . . . . 74.621 grams
13 Mono-potassium phosphate . . . . . . . 0.45 grams
14 Di-potassium phosphate . . . . . . . . 1.35 grams
Mono-sodium L-glutamate . . . . . . . . 0.956 grams
16 Human albumin ~25% solution of
17 albumisol) . . . ~ . . . . . . . . . . 40 ml.
18 Sterile distilled water . . . . . . . . q.s. to one liter
19 The stabilizer is prepared by dissolving the
ingredients in 800 ml. of sterile distilled water and
21 bring up the volume to one liter with additional distilled
22 water. The pH of the composition should be between 6.9-7.1.
23 Neomycin at a concentration of 50 mcg/ml was incorporated
~4 in the growth, rinse, maintenance and stabilizer media.
Infectivity titrations of each harvest were perormed in
26 WI-38 cell cultures.
27 After addition of the stabilizer, contents of
28 individual bottles were shell frozen in a CO2-alcohol bath
29 (ca. -70C.~. The frozen contents of the bottles were
rapidly thawed with vigorous shaking, partially disrupting
31 the cell sheet and releasing the cells into the fluid
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1 stabilizer. The bo-ttle contents wexe pooled, sampled for
2 sterility and infectivity (determined afker batch sonication
3 of a small sample) and stored at -70Co in a mecilanically-
4 operated Ereezer. Three weeks later, when preliminary
sterility and infectivi-ty tests were satisfactory, the
6 pooled stabili~er-cell suspensions were rapidly thawed and
7 transferred to a flow sonication apparatus.
8 Alternatively, storage of the pooled stabilizer-
g cell suspension at -70C. could be omitted and the process
continued with flow sonication and filling completed in a
1 single continuous operation.
12 The flow sonication apparatus had the following
13 components:
14 1) Sonifier cell disruptor and attachments ~wattmeter,
1/2" tapped ~isruptor horn and stainless steel con-
~- 16 tinuous flow attachment);
17 2) Special water-jacketed four liter pyrex glass vessel; .~ ~ :
18 3) Heat exchanger of stainless steel tubing, spirally
19 wound and jacketed, ~ .
4~ Refrigerated bath and circulator;
: 21 5) Flowmeter; and -
22 6~ Miaro-pump, variable speed. : .
23 The conditions of operation were:
24 1~ Power input: 120 watts as shown on the ultrasonic
wattmeter attachment
.. 26 2) Flow rate: 150 ml/min. tmeasured on flowmeter and ~-~
` 27 controlled externally by the micro-pump);
28 3) Cycles of sonication: four, e.g. total volume dis-
29 placed through the sonicator four times; and .
30 4) Temperature of operat.ion: 0-4C. ~.
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1 The continuous flow attachment, four~ ter holding
2 vessel, heat exchanger, 10wmeter and the micro-pump
3 intake were assembled as a unit before use and sterili~ed.
4 External connections between the rerigerated circula-ting
bakh and -the jacketed portions of the apparatus allowed
6 the entire apparatus to be chilled to 0-4C. and maintained
7 at that temperature throughout the sonication process.
d Following sonication, which resulted in
9 essentially 100~ cell disruption, the pre-clarified bulk
vaccine was sampled for control and safety testing. The
11 remaining vaccine was clarified by filtration through a
12 1" by 8" medium (15 micron) porosity sintered glass filter
13 candle and collected in a pre-chilled container. Prior to
14 use, the candle-filter was primed with one liter o~
stabilizer. Post-clarification samples were removed for
16 animal safety testing and a 50 ml. sample was centrifuged `;
17 and the sediment resuspended in 0.5 ml. of stabilizer
18 (hundred-fold concentrate) and transferred to slides for
19 microscopic examination for presence of intact cells.
None were observed.
21 The final cell-free live varicella vaccine
22 was dispensed in .7 ml. - 1.2 ml. amounts and frozen at
23 -70C. or frozen at -70C. in rubber stoppered vials for
24 subsequent preservation by lyophilization, employing
techniques well known to the art as set forth in Calnek
26 et al., Applied Microbiolo~y, Nov. 1970, Vol. 20, No. 5,
27 pgs. 723-726. Infectivity was demonstrated in wet-frozen
28 (-70C.) or lyophili2ed vaccine preparations for at least
29 six months following preparation.
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A convenient dosage form may be prepared by re-
constituting the lyophilized material to the original fill
(.7 - 1.2 ml.) by the addition of sterile distilled water and
employing from about 0.1 ml. to 1.0 ml. of said reconstituted
material for parenteral admlnistration as a varicella vaccine.
The wet-frozen Form of the vaccine which contains .7 to 1.2 ml.
of vaccine may be thawed prior to use with a dosage of from
about 0.1 ml. to 1.0 ml. of the vaccine being administered
parenterally.
EXAMPLE 2
Live attenuated, cell Free varicella vaccine prepared by 15
serial passages in WI 38
A live attenuated, cell free varicella vaccine is
` prepared by employing the procedure substantially as described
in Example 1, except that a total of 15 passages in WI-38
tissue culture is performed.
EXAMPLE 3
Live attenuated, cell free varicella vaccine prepared by 15 - -
serial passages in WI-38
` 20 A live attenuated, cell free varicella vaccine is `~
prepared by employing the procedure substantially as described
in Example 1 except that following six serial passage levels
at 32C., a new series of passages at 36C. was initiated at
` the seventh passage level and continued at 36C . through the
fourteenth passage level for preparation of a vaccine at the ~ ~ -
; fifteenth passage.
EXAMPLE
Live attenuated, cell free varicella vaccine prepared by 20
serial passages in WI-38 _
A live attenuated, cell free varicella vaccine is
prepared by employing the procedure substantially as described
in Example 1 except that following six serial passage levels
at 32C., a new series of passages at 36C. was initiated at
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the seventh passage level and continued at 36C. through the
nineteenth passage level for preparation of a vaccine at the
twentieth passage.
EXAMPLE S
Live attenuated, cell free varicella vaccine prepared by 30
serial passages ln WI-38
A live attenuated, cell free varicella vaccine is
prepared by employing the procedure substantially as described
in Example 1 except that following six serial passage levels
at 32C , a new series of passages at 36C. was initiated at
the seventh passage level and continued at 36C. through the
: twenty-ninth passage level for preparation of a vaccine at the
thirtieth passage.
:. EXAMPLE 6
Live attenuated, cell free varicella vaccine prepared by 40
serial passages in WI-38 _ :
A live attenuated, cell free varicella vaccine is
prepared by employing the procedure substantially as described
in Example 1 except that following six serial passage levels
at 32C., a new series of passages at 36C. was initiated at
- the seventh passage level and continued at 36C. through the:; thirty-ninth passage level for preparation of a vaccine at the
fortieth passage.
EXAMPLE 7
- Live attenuated, cell free varicella vaccine prepared by 20 ~ ~
:` serial passages in monkey testicular cell tissue culture ~ :-
- A live attenuated, cell free varicella vaccine is
-, prepared by employing the procedure substantially as described
in Example 1 except that a known monkey testicular cell tissue
30 culture is used in place of the WI-38 culture system, with a `~
total of 20 serial passages.
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EXAMPLE 8
Live attenuated, cell free varicella vaccine prepared by 20
seria~assa~es in monkey testicular cell tissue culture
A live attenuated, cell free varicella vaccine is
prepared by employing the procedure substantially as described
in Example 3 except that a known monkey testicular cell tissue
culture is used in place of the WI-38 culture system, with a
total of 20 serial passages.
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