CA1045546A - Highly attenuated cytomegalovirus vaccine and production thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A cytomegalovirus (CMV) vaccine, capable of inducing immunity in humans against cytomegalic inclusion disease (CID), without spread to contacts and with minimal excretion of the virus, is prepared by serially passaging virulent CMV in human diploid lung fibroblasts.

Description

~ ~ 5 ~ ~ 6 Cytomegalovirus (CMV) infection n utero is an important cause of central nervous system damage in newborns. Although the virus is widely distributed in the population, about 40~ of women enter pregnancy without antibodies and thus are susceptible to infection. About 1~ of these women undergo primary infection in utero. Classical cytomegalic inclusion disease is ra~e;
howcver, a proportion of the infected infantsJ including those who were symptom free, are sub~equently found to be mentally retarded (Lancet Jan. 5, 1974, pp. 1-5).
lo Prel~minary estimates based on surveys of approx~ately 4,000 newborns from several ~eographical areas indicate that the virus causes significant damage of the central nervous system leading to men~al deflciency in at least 10%, and perhap~ as high as 25~, of infected infants. Assuming that about 1~ of newborn infants per year excre~e CMV and that about one fourth of those develop mental deficiency, in the United State~ this means approximately 10,000 brain-damaged children born per year.
T~i8 iS a fonmidable number, particularly in view of the ability of these children to survive ~J. of Infect, Dis. ~, No. 5, 555 (May 1971), ) ;
In view of the seriousness of the problem~ research . . . .
directed toward the developmen~ of an efective CMV vaccine has been stimulated in recent years. The problem for vaccination is to provide cell-mediated and humoral immunity without allowing spread o~ virus throughout the body of the vacinee and without causing ill effects. The vaecine shc~uld be u~eful ill protectin~5 women agains~ infection during pregn~ncy. Vaccination of adolescent girls should reduce ~he incidence o~ pr:Lmary CMV infecltion In

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pregnancy and eliminate fetal brain damage due ~o ~his cause.
DESCRIPTIC)N OF ~HE INVENTION
The present invention provides a vaccine which is capable of inducing immunity against CMV ~nd thus i5 useful in protecting women against infection during pregnancy, whereby dama~e to the central nervous system, including mental retardation of newborn~
may be greatly reduced~ The vaccine of this invention advantageously does not evidence spread of the virus to contacts.
Furthermore, the vaccine~ by reason of its mode of preparation, lo does not possess ~h~ capabili~y of transmitting latent animal virus to vaccinees. The invention also consists in a novel process ~or preparing the vaccine.
The vaccine o~ the present invent~on pos~ess the above-described advantages by virtue of lts mode of preparation. An essential feature of ~he proces~ for the preparation of the vaccine is the serial passaging of CMV in human diploid lung fibroblasts, particuLarly WI-3~ and ~RC-5 fibroblasts, preferably the ~ormer.
The WI-~8 fibro~lasts were originally derived from a single h~man lung; they are pedigreed in the sense that they have been extensively characterized biologically, biochemically~ viro-logicallyJ and geneticallyO The ~RC-5 fib~oblasts were similarly derived from a single human lung, but of a different individual, and have been pedigreed in like manner. These two cel:L llnes are standardlzedg in contrast to conventionally u~ed primary animal cell~. WI-~8 ha~ been descri~ed in Exper, Cell Res, 25~ 585 (1961) and has been deposited with the kmerican Type Culture : -Collect~oll ~nd assigned the designation ATCC CCL-75, MRC~5 has ,: , ~ ~.

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been described in Nature ~1 168 (July 11, 1970). WI-~8 has been made available to laboratories and may be obtained from ~he collection. The use of these cell l~nes for the propagation of the virus minimizes the likelihood of latent animal viruses being ~:
transmitted to a vaccinee by means of the vaccine.
The To~ne strain of CMV, a preferred strain for use in preparation of the vaccine because of its broad antigenic spectrum, was isolated from the urine of a two month old male infant with cytomegalic inclusion disease (symptoms - central lo nervous system damage and hepatosplenomegaly)0 This strain of CMV was Isolated by Stanley A. Plotkin, M.D. of the Wistar Institute of Anatomy and Biology, Philadelphia, P~nnsylvania, and is described in J. Virol. 11 No. 6, 991 (June 1973). However, other strains of CMV may be used.
Propagation of the human diploid lung flbroblasts may be carried out by any of the standard methods described in the literature. Speciic examples of such propagation techniques are disclosed in Exper. Cell Res. 25, 585 (1961), and Virology 16, 147 (1962), The tissue culture system uæually comprises Eagl~s basal medium (BME) or Eagle's minimal essential medium (MEM) in Eagle's balanced salt solution supplemented with pre-screened calf ser~n con~aining a sterilizing amount o an an~
biotic such as penicillin, streptomycln, chlor~etracyline~ or other an~ibiotic, or mixtures thereof, the system being buf~ered at a pH of about 6.8 ~ 805 with a conventional biological buffering agent such as an alkali metal bicarbona~e, carbonate or hydrogen phosphate.

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The CMV is cultivated for vaccine use by inoculating human diploid lung fibroblasts with CMV, Urine containing CMV is a particularly useful starting material for inoculating the fibro- ;
blasts. The trypsinized infected cells are harvested and serially passaged in human diploid lung ~ibroblasts. Each incubation proceeds for a period of 7 day~ and ~s conducted at a temperature of about 37C, The number o~ passages is ~uch as to produce first a strain of CMV which release~ large amounts of cell-free virus, followed by attenuation of the cell-free virus. At lo least about 50 and preferably about 125 to 150 total pa~sages are used. The attenuated virus is then harvested and subjected to standard sterility tests for the presence of ba~teria~ fungi, mycoplasma, and other contaminating agents.
The term l'attenuated virus" as employed herein refers to a virus of which the virulence has been al~ered by the method of culture so that it does not produce severe symptoms when inoculated into humans although retai~lng its antigenicity, that is, its ability to ætlmulate production of antibodies.
The attenuated virus is u~ilized as a vaccine by filtering the harvested material in orde~ to remove cells or bacteria, and the filtrate ls either used as is~ frozen for later use, or lyophilized and subsequently reconstituted with a solvent such as water. Preferably a stabilizer such as albumen is used where the filtrate i5 lyophilized. The vaccine may be administered sub- ; -cutaneously. The dosage whi,h may be used is a minim~ of loo TCIDSo of attenuated ~MVg and may ~e as ~igh as lo~ooo TCID50.
l~e attenuated virus may be produced in lar~er quan~i~ie~
by inocula~ing human diploid lung fibrob~asts ~lth the virus~ and .
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~ ss~growing the attenuated virus therein. The attenuated virus may be harvested at the time of maximum titer, e.g. about one week.
The following description of the preparation and testing of the CMV vaccine is intended to illustrate the invention, but is not to be construed as limiting its scope.
PROCEDURE FOR OBTAINING VIRUS
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The CMV used is obtained from the urine of a two month old male human infant with cytomegalic inclusion disease. This strain i8 designated Towne and has been de~cribed h~reinabove.
lo The urine is inoculated onto WI-38 human diploid lung fibroblasts.
The nutrient medium used or tissue culture of the virus is Eagle's minimal essential me~ium (MEM) with 2~ of pre-screened calf serum added. The concentration o antibiotics in each ml, of medium is loo ug of gentamicln, and 2 ug of amphotericin-B. Other antibiotics may be used in place of those specifically enumerated above.
PROCEDURE FOR PREPA]RING ATTENUATED ~MV
The har~est comprising supernatant ~luid, and virus in-fected cells removed from the surface of the vessels using 0.25%
trypsin in a physlological sallne solution is inoculated on ~ta~ionary WI-~8 human diploid lung ~ibroblasts to initiate in- -~ection of these ce71s and then subsequently pasæaged. The nutrient medium used to initiate cell infection and in all sub- ;
sequent paæ~ages is ~he same as tha~ employed for tissue culture of the viru~, described above. me temperature used in each passage i8 ~7C. HoweverJ temperatu~e~ ~omewha~ below thls temperature, e.g~ temperatures in the range of abou~ 30" ~o 37C.
can also be usedO The dura~ion of each passage is seven days~
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but passages of a somewhat shorter or longer period~ e.g. 5 or lo days~ may be usedO
In the initial passages, which are albou~ 10 in number, supernatant fluid contalning virus infectecl cells harvested from each prevlous passage is passaged on fresh WI-38 hu~an diploid lung fibroblasts. Approximately 25% of cell-containing fluid from a preceeding passage is used in each passage. The number of passages selected is such as to obtain a s~rain which produces a substantial amount of cell-free virus, ~MV ordinarily being cell lo associated. Thus, a somewhat smaller or greater number of pas~ages may be used.
The superna~ant fluid from the ~enth passage is obtained by decanting and i8 centrifuged at 1~200 rpm to obtain a fluid containing cell-~ree virus. The cell-free fluid contairling cell-free virus is then serially passaged 28 times on fresh WI-38 -human diploid lung fibroblasts~ 1 ml. of 1uid being used to inoculate vessels each having a surface area of 75 cm . Although 28 passages are used, the number of passages need merely be sufficient to provide the desired ~mount of cell-free CMV, mus~ :
a greater or le~ser number of passages of the cell-free fluid could be used, The 6upernatant fluidJ which may also contain virus re-leased from cells by sonication, is harvested and then serially passaged on ~resh WI-~8 a sufficient number of times, e~g. about 115 times, to attenuate the virus. The total number of passages used in the overall process`hereinabove described will ordinarily be between abou~ 50 and 150, preferably about 125 to ]L50.

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Cloning of the attenuated virus by terminal dilution is performed at 50, 60 and 70 pa~sages.
The virus for vaccine pools is harvested from the super-natant fluids of infected WI 38 cultures at the time of maximum virus titer, generally about one week. By this procedure a larger amount of attenuated CMV is obtained from that initially prepared.
CHARACTERISTICS OF THE CMV VACCINE
The CMV vaccine grows on human fibroblasts such as WI-38 lo cells. Its cytopa~hic effect is characteristic of human CMV
described in the literature, It can be readily propagated in cell-free form by fil~ering the supernatant o~ infected culture (~ micron filter) and inocul~ting on fresh WI-~8 ceLLs. CPE
occurs withln 4-lo days depending on the multiplicity of inection, Inclusion bodies form as deseribed in the literature and can be stained with Haematoxylin-Eosin or Giemsa. The attenuatled virus forms plaques with a nutrient agarose overlay.
~ he vaccine should be stored at -70C. in a solution of 70~ sorbitol in distilled water (one Yolume of virus to one 20 volume of sorbitol solution).
The cell~free attenua~ed VirUB can be seroneutralized with `~
commercially available human antiserum (Flow LabsO, Be~he~da, Maryland), CF titre 1:16 (diluted l:lo). oOl ml. diluted ~erum neutralLzes 250 TClD~oO Hyperimmune guineapig serum against AD
169 strai~ may also be used.
The following ~able sJ~mmarlzes the serum neutralization (SN) titres alone and in the presence of complement, SN was done in microplaques (Linbro): 0 025 ml. of suspension of 60 .: , . ~ . . , - :

TCID50 Towne 125 plus 00025 ml~ dilu~ed ser~m. After one hour 0.250 ml. containing 104 WI-~8 cells in EagLe's modified basal medium with lo~ unheated calf serum is added. Mieroplaques are read after 5-7 days' incubation at 37C. Eight wells per dilution were used.

Neutralization Serum S~ple _ Alone with C

Rabbit No. 1 8 8 No. 2 8 ~4 No. ~ 8 ~2 No. 4 8 64 ~ . .
lo Guinea Pig 20- loo loo Human Antiserum 40 40 . . . .~ ., . , _ . . ... . .. .. , ,.. ~ . . . .. . .
PROCEDURE FOR I~STING V~CCINE
Each pool used for inoculation was subjected to tests for the presence of bacteria, ~ungi, and my~oplasma by inoculation onto appropriate artificial media. Tests for safety in animals included injection of aliquots of the pool into adult mice (intraperitoneally and intracerebrally)3 suckling mlce (intra~ :
peritoneally and intracerebrally), guinea pigs (intraperi-toneally)~ and rabbit~ ~intradeImally). Examination of the -animals at di~ferent period~ of time (all several weeks) after inoculation~ revealed that all of ~he anim~ls remained well.
Twenty cercopithecus monkeys were inoculated with ~he vaccine~ o.5 ml. intraspinally~ intracerebrally~ respectively, and 1 ml. intramuscularly~ ~ney were all ~eronegative to loo ~ -T~ID50 CMV neutralization, bo~h in the presence and ab~ence o~ ~ -fresh guinea pig complement. The monkeys were tested again three wee~s la~er. No seroconverslon was demonstrate~l by ~he ~ame monkeys upon clinical or pathological examirla~ion3 during '' ~;

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Fur~her tests for identity and for the absence of con-taminating agents were performed in tissue culture. Primary African green monkey kidney, human embryo kidney, prim~ry rabbit kidney, and WI-38 cells all were inoculated with aliquots of virus .: . .
either directly or after neutralization for 1 hour at 37C. with rabbit CMV s~rum. There was no evidence of any agent other than CMV vaccine in the pool. Titration of pools for quantity of CMV
virus was perfonmed by end point dilution assay on WI 38 cell~.
lo RESULTS 0~ TESTING VACCINE IN HUMANS
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The vaccine was teæted on adults, pretested for antibody to CMV and only seronegative~ were lncluded in the testsO Ten seronegative adults were given 103- TCID50 intrana~ally. There were no ~eroconversions, demonstrating that the viru~ was no -longer infectious by the usual route. Nine other adulks were given the same dose subcutaneously, and there was seroconversion in all cases. No symp~oms were observed, nor was virus recovered from urine, throat or blood, In obtaining the aforeæaid re~ul~s, the viruses were inoculated in a volume of 1 ml. subcutaneously, Throat ~wabs were made with cotton-tipped applicator sticks moved over the posterior pharynx. Both types of swabs were placed ~mmediately in screwcap tubes containlng Hanks' medium with o.l percent gelatin, looo ~g o streptomycin and 400 ug of mycostatin per ml.
The 3pec~mens either were stored at 4C. until inoculated into tis~ue cultures on the same day3 or at -20C, until testled within two weeks. Urine was inoculated direc~ly on WI-38 celLs. Test~
f~ vlremia were per~ormed by collect:ing heparinlzed blood and _ 10~

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allowing it to settle by gravity at 4C. until leucocyte-rich plasma was obtained. After sonication for l~o minutes, the plasma was inoculated into tissue cultureO Antibody studies were done on serum from clotted blood specimens using complement-fixation and fluorescent antibody tests.
In a subsequent test nine new seronegative adul~s were given the same dose ( lo TCID50) subcutaneously. No systemic symptoms were observed and all nine developed antibodies to ~MV.

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Claims (7)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing an attenuated cytomegalovirus vaccine comprising serially passaging in WI-38 human diploid lung fibro-blasts a strain of virulent cytomegalovirus having a broad antigenic spectrum a sufficient number of times to obtain a strain of cytomegalovirus which produces a substantial amount of cell-free cytomegalovirus, and serially passaging said cell-free cytomegalovirus in WI-38 human diploid lung fibro-blasts a sufficient number of times so that the virus, when administered to humans, induces immunity without producing severe symptoms or more than minimal virus excretion and spread to contacts, the total number of passages employed in preparing said vaccine being from about 50 to about 150.

2. A process for preparing an attenuated cytomegalovirus vaccine comprising growing said attenuated cytomegalovirus of claim 1 in WI-38 human diploid lung fibroblasts for a sufficient length of time to produce a larger amount of said attenuated virus and harvesting the resulting attenuated virus.

3. A process according to claim 1 in which the passages are carried out at a temperature of 30° to 37°C for 5 to 10 days.

4. A process according to claim 3 in which the total number of passages is about 125 to 150.

5. A process according to claim 4 in which trypsinized cells infected with virulent cytomegalovirus are passaged 10 times on fresh human diploid lung fibroblast cells, followed by subsequent viral passaging of cell-free virus on fresh human diploid lung fibroblast cells.

6. A cytomegalovirus vaccine comprising an attenuated cyto-megalovirus prepared by growing said attenuated cytomegalovirus of claim 1 in WI-38 human diploid lung fibroblasts for a sufficient length of time to produce a larger amount of said attenuated virus and harvesting the resulting attenuated virus.

7. Cytomegalovirus vaccine comprising a carrier and an effec-tive amount of attenuated cytomegalovirus prepared by serially passaging in WI-38 human diploid lung fibroblasts a strain of virulent cytomegalovirus having a broad antigenic spectrum a sufficient number of times to obtain a strain of cytomegalovirus which produces a substantial amount of cell-free cytomegalovirus, and serially passaging said cell-free cytomegalovirus in WI-38 human diploid lung fibroblasts a sufficient number of times so that the virus, when administered to humans, induces immunity without produc-ing severe symptoms or more than minimal virus excretion and spread to contacts, the total number of passages employed in preparing said vaccine being from about 50 to about 150.