CA1054054A - Attenuated cell-free varicella virus vaccine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
Preparation of safe live attenuated varicella virus vaccine by serial propagation of varicella virus in tissue call culture systems.

Description

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 : : :

' ~

" ~:
..
' '~ ~; ' ' `

r~ 15595IA

~C15~5~L

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 `

~LOS~3S~L
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 ~ -S~S4 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.

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:

_4 _ .`~ . , . , ' ~ ~ 15595~

~S~S~

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 ;'

-5-- . .

)5~l~S~

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 ~ , ~..~

~ QS~54 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. ~.

.'' . :
. , ~ .

:: :
.

~OS9OS~L

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.

:'~

`

~5~
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.

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.

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 ;,'' ~, g : . . . ~ , , . . ., ': . ` : : . , - ~ , . - .
. . . .
. . . , ,. , -: , . . . " , . : :-1 55g51A
:

l,~S~

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.

- 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.
:

~'~. 1 0 ..

. . , . : ' ; . ., , ~.,, ! ' ~05~

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.

.:
:

, . . ~ ,~

'

Claims (20)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A process for the preparation of a live attenu-ated, cell-free varicella virus useful as an antigen in a vaccine, which will evoke in man an antibody response against a virulent varicella virus without causing the severe clinical manifestations of the disease which comprises serially passaging the virulent virus at 30-38°C. in a tissue culture preparation selected from the group consisting of human and animal diploid cell strains and primary cells of simian origin a sufficient number of times to attenuate the virus followed by release of the attenuated virus from the infected cell by sonification in the presence of a stabilizer.

2. The process of Claim 1 wherein the temperature is 32°C.

3. The process of Claim 1 wherein the temperature is 36°C.

4. The process of Claim 1 wherein the tissue culture preparation is of human diploid cell strains.

5. The process of Claim 4 wherein the human diploid cell strain is human fetal lung tissue.

6. The process of Claim 5 wherein the human fetal lung tissue is WI-38.

7. The process of Claim 1 wherein the tissue culture preparation is of primary cells of simian origin.

8. The process of Claim 7 wherein the primary cells of simian origin are of monkey testicular tissue.

9. The process of Claim 1 wherein the tissue culture preparation is of animal diploid cell strain.

10. The process of Claim 9 wherein the animal diploid cell strain is of fetal rhesus tissue.

11. The process of Claim 6 wherein the stabilizer is SPGA.

12. A live, attenuated cell-free varicella vaccine comprising as its essential ingredient an immunologically effective amount of a live attenuated cell-free varicella virus prepared by the process of Claim 1, 2 or 3.

13. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 4 or by an obvious chemical equivalent.

14. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 5 or by an obvious chemical equivalent.

15. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 6 or by an obvious chemical equivalent.

16. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 7 or by an obvious chemical equivalent.

17. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 8 or by an obvious chemical equivalent.

18. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 9 or by an obvious chemical equivalent.

19. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 10 or by an obvious chemical equivalent.

20. The live attenuated cell-free varicella vaccine, when prepared by the process defined in Claim 11 or by an obvious chemical equivalent.