WO1981002107A1

WO1981002107A1 - Highly attenuated varicella virus vaccine and production thereof

Info

Publication number: WO1981002107A1
Application number: PCT/US1981/000075
Authority: WO
Inventors: S Plotkin
Original assignee: Wistar Inst
Priority date: 1980-01-25
Filing date: 1981-01-21
Publication date: 1981-08-06
Also published as: EP0043847A4; GB2077590A; JPS57500149A; EP0043847A1; IT8147637A0

Abstract

A live attenuated varicella virus vaccine, capable of inducing immunity in humans against varicella without spread to contacts, is prepared by serially passaging virulent varicella virus in human diploid lung fibroblasts.

Description

Highly Attenuated Varicella Virus Vaccine and Production Thereof

Background of the Invention Varicella, commonly referred to as chickenpox, is an acute, highly communicable disease, principally of young children, caused by the virus Herpes varicellae. This disease is marked by slight fever and an eruption of macular vesicles, which appear in crops, and are super- ficial and rarely umbilicated. They rarely become pustular, but dry up, and are only occasionally followed by scars. The duration of the disease is about a week.

Although varicella is a relatively mild illness and rarely causes complications in normal children, when it occurs in a ward of a children's hospital, it may be serious and sometimes lethal in patients with leukemia or other malignancies, as well as in patients receiving immunosuppressive treatments and those with congenital immunodeficiencies. Because of such problem, numerous attempts have been made to prevent or modify the disease by passive immunization with convalescent serum, gamma- globulin, or zoster immune globulin. Adenenine arabinoside has also been used for treatment of disseminated zoster and varicella in compromised patients. However, effective- ness of these treatments varies, and in some instances clinical use of these agents may cause problems.

Until quite recently there has been little progress in developing a varicella vaccine. In about 1974 researchers in Japan attenuated a strain (Oka strain) of varicella virus by cultivating it serially in human embryonic lung cells and then in guineapig embryo (G.P.E.) cells. It is reported that subcutaneous injection of the virus stimulated the production of complement-fixing (C.F.) antibody without clinical reactions in normal susceptible children. The virus propagated in human diploid lung

OMPI fibroblasts ( I-38) , after passage in G.P.E. cells, reportedly was also effective in inducing immunological response without clinical reactions in normal susceptible children (see Takahashi et al. "Live Vaccine Used to Prevent the Spread of Varicella in Children in Hospital" Lancet (Nov. 30, 1974) 1238-90).

Description of the Invention

The present invention provides a vaccine which i capable of inducing immunity against varicella, and thus i useful in protecting humans, especially children, against this source of human misery. Of particular importance is the immunity the vaccine provides in hospital patients , such as those with leukemia or other malignancies, as well as in those patients receiving immunosuppressive treatmen and those with congenital immunodeficiencies, in which patients the disease may be serious, and, in some instance lethal. The vaccine of this invention advantageously does not evidence spread of the virus to contacts and does, not produce clinical reactions in normal susceptible children. Furthermore, the vaccine, by reason of its mode of preparation, does not possess the capability of transmitting latent animal virus to vaccinees. The invention also consists in a novel process for preparing the vaccine. The vaccine of the present invention possesses the several above-described advantages in large measure by virtue of its mode of preparation. An essential featur of the process for preparation of the vaccine is the seria passaging of the varicella virus in human diploid lung fibroblasts, particularly WI-38 and MRC-5 fibroblasts .

The WI-38 fibroblasts were originally derived from a single human lung; they are pedigreed in the sense that they have been extensively characterized biologically biochemically, virologically, and genetically. The MRC-5 fibroblasts were similarly derived from a single human lung, but of a different individual: and have been pedigreed in like manner. These two cell lines are standardized, in contrast to conventionally used primary animal cells. WI-38 has been described in Exper. Cell Res. 25, 585 (1961) and has been deposited with the American Type Culture Collection and assigned designation ATCC CCL- 75. MRC-5 has been described in Nature 227., 168 (July 11, 1970) . WI-38 has been made available to laboratories and may be obtained from the collection. The use of these cell lines for the propagation of the virus minimizes the likelihood of latent animal viruses being transmitted to a vaccinee by means of the vaccine.

The Webster strain of varicella virus, a preferred strain for use in preparation of the vaccine, was isolated in 1970 from a vesicle of a three year old female child at the onset of varicella (symptoms: fever and eruption of macular vesicles, in crops). This strain was isolated by Stanley A. Plotkin, M. D., and has been deposited with the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania, to afford permanence of the deposit and ready accessibility thereto by the public should a patent be granted on this application. However, other strains of varicella virus may be used.

Propagation of the human diploid lung fibroblasts may be carried out by any of the standard methods described in the literature. Specific examples of such propagation techniques are disclosed in Exper. Cell Res. 25_, 585 (1961), and Virology 1(5, 147 (1962) . The tissue culture system usually comprises Eagle's basal medium (BME) or Eagle's minimal essential medium (MEM) in Eagle's balanced salt solution supplemented with prescreened calf serum containing a sterilizing amount of an antibiotic such as penicillin, streptomycin, chlortetracycline, or other antibiotic, or mixtures thereof, the system being buffered at a pH of about 6.8-8.5 with a conventional biological buffering agent such as an alkali metal bicarbonate, carbonate, or hydrogen phosphate.

In preparing the vaccine, the virus is serially passaged in human diploid lung fibroblasts. In each passage the trypsinized infected whole cells from the

OMPI previous passage are harvested and used to inoculate an uninfected culture. Each incubation proceeds for a perio of about 7 days and is conducted at a temperature of abou 35° C. After about 25 passages, the trypsinized culture of infected cells is subjected to sonication, and after centrifugation the cell-free supernatant containing infectious varicella virus is used to inoculate the next uninfected culture of human diploid lung .fibroblasts . Thi latter procedure is repeated for about 35 passages, each lasting about 4 to 7 days and conducted at a temperature o about 35° C. The total number of passages should be on t order of about 50, and preferably at least about 70, of which at least about 35 or more involve inoculation of eac uninfected culture with cell-free virus . The attenuated virus is then harvested and subjected to standard sterilit tests for the presence of bacteria, fungi, ycoplasma, an other contaminant agents .

The term "attenuated virus" as employed herein refers to a virus of which the virulence has been altered by the method of culture so that it does not produce clinical symptoms when inoculated into humans although retaining its antigenicity, i.e. its ability to stimulate antibodies .

The attenuated virus utilized as a vaccine is obtained by filtering the harvested material to remove cells or bacteria, and the filtrate is either used as is, frozen for later use, or lyophilized and subsequently reconstituted with a solvent such as water. Preferably a stabilizer such as sorbitol is used where the filtrate is lyophilized. The vaccine may be administered subcutaneous the minimum dosage of attenuated varicella virus being about 500 TCIDC_Q. However, the dosage may be as high as 1000 TCID₅₀.

The following description of the preparation and testing of the varicella vaccine is intended to illustrate the invention, but should not be construed as limiting its scope.

OMP Procedure for Obtaining Virus

The varicella virus used is obtained from a normal three year old female child at the onset of clinical varicella. This strain is designated Webster and has been identified above. The vesicle fluid from the skin of the child is inoculated onto WI-38 human diploid fibroblasts. The nutrient medium used for tissue culture of the virus • is Eagle's minumum essential medium (MEM) with 2% of pre-screened calf serum added. The concentration of antibiotics in each ml. of medium is 60 eg. of penicillin and 10 eg. of gentamycin. Other antibiotics may be used in place of those specifically named.

Precedure for Preparing the Vaccine

The harvest comprising supernatant fluid and virus infected cells after being trypsinized with 0,25% trypsin in a physiological saline solution is inoculated on stationary WI-38 human diploid lung fibroblasts. The nutrient medium used is the same as that employed for , tissue culture of the virus described above. The duration of each passage is 7 days, but passages on- the order of 4 to 7 days may be used. The temperature used in each passage is 35° C. However, temperatures in the range of 30° C to 37° C may be used.

In the initial passages, which preferably are 25 in number, supernatant fluid containing virus infected cells is harvested from each preceding passage, after trypsinization is passaged on fresh uninfected culture comprising WI-38 human diploid lung cells . Approximately 507o of the cell-containing fluid from a preceding passage is used in each succeeding passage. The number of initial passages, also referred to as the first series of passages, is such as to obtain a laboratory adapted virus. Thus, a somewhat smaller or greater number of passages than 25, e.g. 20 to 30 passages, may be used. The supernatant fluid containing virus infected cells from the 25th passage is subjected to ultrasonic cell disruption, commonly referred to as sonication. This may be accomplished by placing the supernatant fluid in a tub into which is inserted the tip of a sonicator, such as th made and sold by Branson Ultrasonics Corporation. Sonication generally takes place in about 30 seconds when such a sonicator is on setting No. 6. The resultant sonicate is centrifuged at 2000 rpm for about 20 minutes t separate cellular debris, and the cell-free supernatant which contains the varicella virus is recovered. This supernatant, after trypsinization, is then used to inocul fresh uninfected culture. This passaging procedure, referred to herein as the second series, is repeated for at least about 35 passages. If desired a somewhat lesser or greater number of passages employing the sonication st may be from about 30 to 50. In these passages, the temperature may vary from 30° to 37° C; preferably a temperature of 35° C being used. The duration of each passage will be on the order of 4 to 7 days, a passage duration of 7 days being preferred.

The cell-free supernatant containing attenuated varicella virus from the final passage, preferably at the time of maximum titer, and, as previously noted, after separation of cell debris, is used as such as a vaccine, is frozen and kept at a temperature of -70° C until time of use, or lyophilized for subsequent reconstitution with a solvent, e.g. water.

Characteristics of the Varicella Vaccine

The varicella vaccine grows on human fibroblasts such as WI-38 and MRC-5 fibroblasts. Its cytopathic effec (CPE) is characterized by the lysis of cells into formatio of elliptical plaques of 1-2 mm. in diameter. The nuclei sometimes contain inclusions.

The vaccine should be stored at -70° C in a solution of sorbitol (one volume of virus to one volume of stabilizer) .

SUBSTITUTE - SHFΕ s-T i Procedure for Testing Vaccine and Results of Testing on Humans

The vaccine is tested according to the procedures described by the Bureau of Biologies for testing live virus vaccine in tissue culture and in animals .

Children of school age were selected whose serum was free of antibodies to varicella. The vaccine (Webster strain) produced as a result -of a total of 70 passages (25 first series, 45 second series as described above) was administered subcutaneously (1000 plaque-forming units) , and each patient was observed daily for one month. Serum specimens were taken 42 days after inoculation and subjected to varicella-zoster antibody to membrane antigen (VZ-FAMA) testing. The test procedure used is as follows:

Propagation of Normal Cells

MRC-5's (human embryonic lung fibroblasts) are propagated in Falcon T-75's with growth medium consisting of minimum Eagle's Medium, supplemented with 7.5% fetal calf serum, 1.25% glutamine and penicillin-gentamycin- amphotericin-B, Monolayers are routinely split from 1<2 to 1:5 once a week. Confluent monolayers are refed with maintenance medium as above with 2% fetal calf serum. The cells are incubated at 37° C.

Preparation of Infected Cells Ellen strain of varicella-zoster virus, which has been deposited with the American Type Culture Collection and designated ATCC-VR 586, is passed undiluted when CPE reaches 90% of the cells into a T-75 with a confluent (4-5 day old) monolayer, containing maintenance medium. The culture is incubated at 37° C for 3-5 days, until

80-90% of the cells are affected. The infected culture is harvested by washing for 10 seconds with 5 mis, of 0,25% trypsin - 0.1% versene at 37° C and then incubating the cells at 37° C until the cells detach (<5 minutes), The infected cells are collected in 3.0 ml, of growth medium, of which 0,3 ml. is used to infect a new monolayer and the rest for FAMA as described below.

The cells may also be stored. frozen in a suspension of growth medium, with 10% DMSO at -70° C and quick-thawed immediately before use. Frozen cells can be stored for FAMA testing for up to 2 months. They are more convenient to use than fresh cells, but preparations made with fresh cells are easier to read.

The Test

FAMA testing is done in microtiter U-plates (Linbro, polystyrene) - A microtiter dropper is used to add 0.025 ml. PBS to each well. Serum is transferred by microtiter loop (0.025 ml.) which is then used to make two-fold dilutions of the serum from 1:2 to 1:64, or further, as necessary. A positive and negative control serum are included per plate.

0.025 ml, of the infected cell suspension is added to each well by dropper. The plate is covered by a plate sealer and mixed for 10 seconds on a platform shaker It is then incubated at 37° C for 1 hour. The cell suspensions are centrifuged at 1200 rpm for 10 minutes

(Sorvall) and the supernatant is drawn off. The cells are resuspended in 0.025 ml, PBS/well, again centrifuged and the supernatant discarded. 0.025 ml. of the working dilution (usually 1:30 - 1:40 in PBS) of fluorescein- labelled goat antihuman IgG (heavy and light chains) is sealed, mixed and incubated at 37° C, for 1 hour. The cel are washed as above and resuspended in cold (+4° C) PBS with 10% glycerol. The plate is either read immediately or stored overnight at +4° C. The cells are prepared for microscopic examinati by placing one drop of the cell suspension on a glass slid and covering it with a cover slip. The slides are examine with a Leitz fluorescent microscope. Positive cells appe rounded with a bright apple-green halo of fluorescence on the perimeter. A field is considered positive if the cell fluoresce with an intensity of at least +2 on a scale of 0 to +4. The fluorescence should diminish with dilutions of serum and a distinct end-point dilution should be observed.

Results

The results obtained are set forth in Table I, below:

Table I

VZ -FAMA titer

Patient No. Clinical

Pre--Inoculation Post-Inoculation Reactions

1 <2 32 None

6 <2 32

10 <2, 16* 16

12 <2 N.D.

14 <2 <2

21 <2 _8

24 <2 <2

25 <2 16

26 <2 64(8)**

* Two laboratories disagreed on titer ** CF titers

Claims

1. In a process for preparing an attenuated varicella vir vaccine by serially passaging in human diploid lung fibroblasts a strain of varicella virus, the improveme which comprises carrying out said serial passaging wit a strain of varicella virus characterized by plaque neutralization, as evidenced by positive fluorescence when subjected to fluorescent antibody to membrane antigen testing, a sufficient number of times so that the virus, when administered to humans, induces immuni from varicella without producing severe symptoms .

2. The process of claim 1 in which said human diploid lun fibroblasts are selected from the group consisting of those designated WI-38 and MRC-5.

3. The process of claim 1 in which said human diploid lun fibroblasts are those designated WI-38.

4. The process of claim 1 in which the total number of passages Is at least about 50.

5. The process of claim 1 in. which said passages are carried out at a temperature of from about 30° to abou 37° C for a period of from about 4 to about 7 days.

6. In a varicella vaccine, the Improvement comprising attenuated varicella virus prepared by the process of claim 1.

7. The process of claim 1 in which there are first and second passage series, in which in each of the passages of said first series trypsinized supernatant fluid containing virus infected cells from the previous passage is used to inoculate the uninfected fibroblast culture of each succeeding passage, and in which in each of the passages of said second series trypsinized cell-free supernatant fluid containing said virus is used to inoculate the uninfected fibroblast culture in each succeeding passage.

OMPI

8. The process of claim 7 in which said human diploid lung fibroblasts are selected from the group consisting of those designated WI-38 and MRC-5.

9. The process of claim 7 in which the total number of passages in said second series is from about 30 to 50.

10. The process according to claim 9 in which said passages are carried out at a temperature of from about 30° to about 37° C for a period of from about 4 to about 7 days^'.

11. The process according to claim 7 in which said human diploid lung fibroblasts are those designated WI-38, the total number of passages in said first series is about 25, the total number of passages in said second series is about 45, and said passages are carried out at a temperature of about 35° C for a period of about 7 days.

12. In a varicella virus vaccine, the improvement comprising attenuated varicella virus prepared by the process of claim 11. , .

13. In a process for immunizing humans against disease caused by varicella virus, the improvement comprising subcutaneously administering to humans at least an immunizing effective amount of attenuated varicella virus vaccine prepared by the process of claim 1.