Process for preparing virus disease live vaccines

Process for preparing a virus disease live vaccine by employing culture of a quail embryonated egg or tissue culture of a quail embryo fibroblast for cultivation of the virus. The vaccine obtained according to the present invention is effective for immunization of animals and humans against the virus disease. The vaccine is avirulent, scarcely subject to induction of unfavorable factors accompanying the present tissue used for isolation of the virus therefrom, and economically productible.

This invention relates to the preparation of a vaccine for animals and 
humans, and more particularly to a process for the preparation of a live 
vaccine for immunization of animals and humans against virus diseases by 
employing tissue culture of a quail embryo fibroblast or a quail 
embryonated egg. 
Beginning from the investigation by Harrison, the technique of tissue 
culture has been developed by A. Carrel, A. Fisher, G. Levi, etc. in the 
1910 to 1920's. Since Enders et al succeeded in the propagation of polic 
virus by the technique of tissue culture, this technique has been utilized 
for the propagation of various viruses. And nowadays, the tissue culture 
method is widely and advantageously used for the preparation of virus 
vaccines. 
With the tissue culture conventionally employed, there is such serious 
problem that, according to the kind of tissue supplying animal used, the 
unfavorable factors accompanying the parent cells often causes the culture 
cells to be stained therewith, leading to the induction of such favorable 
factors to the produced vaccines. In general, it is very difficult to 
previously detect non-manifestative infection of the tissue supplying 
animal with such unfavorable factors. For example, various simian viruses 
are often detected from the apparently healthy monkeys, leading to 
difficulty in the preparation of polio live vaccine. In addition, it was 
also reported that Newcastle virus was detected in the tissue cultures of 
the apparently normal chick embryo fibroblasts. It was also reported that 
the use of chick kidney as a medium for culturing Marek's disease virus is 
inevitably accompanied by danger of unfavorable induction of leukemia 
virus of wild chicken to the produced Marek's disease vaccine. 
For the reason as stated above, careful attention should be given to danger 
of induction of the accompanying unfavorable factors, in case of employing 
the tissue culture method in the preparation of vaccine. Moreover, the 
tissue culture methods conventionally employed have various disadvantages 
that the availability of materials is restricted in regard to quantity, 
and that they require complicated operation and assay. 
As a result of our extensive and intensive investigation to overcome such 
drawbacks as unavoidable in the conventional tissue culture, it has been 
found that the culture of a quail embryonated egg or the tissue culture of 
a quail embryo fibroblast is extremely suitable for cultivation of viruses 
for producing virus disease vaccines. 
It is one object of the present invention to provide a process for the 
preparation of a virus disease live vaccine which is very effective for 
immunization of animals and humans against the virus disease. 
It is another object of the present invention to provide a process for the 
preparation of a virus disease live vaccine which is extremely avirulent, 
ensuring safety of animals and humans inoculated therewith. 
It is a further object of the present invention to provide a process for 
the preparation of a virus disease live vaccine in which unfavorable 
factors are scarcely induced. 
It is a still further object of the present invention to provide a process 
of the character described, which is simple in operation and economical, 
thus enabling the production of virus disease vaccine to be economical. 
Essentially, according to the present invention, there is provided a 
process for the preparation of a virus disease vaccine which comprises 
employing a quail embryonated egg or tissue culture of a quail embryo 
fibroblast for cultivation the virus. 
By employing, as a culture medium for viruses, culture of a quail 
embryonated egg or tissue culture of a quail embryo fibroblast, not only 
the high quality virus live vaccine can be obtained but also the problems 
of poor availability of materials and complicacy of operation are 
advantageously eliminated. 
The advantages which the use of a quail embryonated egg (QEE) or a tissue 
culture of quail embryo fibroblast (QEF) has are as follows. 
1. Considerable kinds of viruses have an affinity to QEE and a tissue 
culture of QEF can grow and propagate to an extent sufficient to produce 
vaccine. 
2. QEE and a tissue culture of QEF are scarcely accompanied by danger of 
induction thereinto chicken leukemia virus, as opposed to a chick 
embryonated egg. 
3. QEE and a tissue culture of QEF are readily available at low cost. 
4. From the view point of tissue culture, the number of cells harvested 
from one embryo of a quail embryonated egg is approximately the same with 
that of a chick embryonated egg. Moreover, since a quail is smaller than a 
chicken, large number of quails can be bred in a given area, as compared 
with chickens. Breeding control with respect to SPF is readily conducted. 
5. Particulary for the preparation of chicken disease vaccine, superior 
vaccines having high safety can be produced by employing quails which 
rarely have therein microorganisms etiological to chickens. 
The isolation of disease viruses from the infected materials, the 
adaptation, growth and propagation the isolated viruses may be conducted 
in accordance with the ordinary methods well known to those skilled in the 
art. 
Examples of virus diseases include rubella, measles, mumps, influenza A, 
influenza B, distemper, vaccinia, Newcastle disease, Japanese 
encephalitis, dengue fever, rabies, Marek's disease and the like. 
As described, according to the present invention, superior quality viral 
diseases live vaccines can be prepared simply and economically. Moreover, 
it is particularly advantageous that live vaccines according to the 
present invention has scarcely danger of induction therein of unfavorable 
factors from the parent tissue.

The present invention is illustrated, by way of example only, with 
reference to the following examples. 
REFERENCE EXAMPLE 1 
The chickens and quails bred according to the ordinary method are examined 
with respect to infection with chicken leukemia virus (hereinafter 
referred to as "RIF") to which careful attention should be given 
especially when live vaccines are intended. The examination was conducted 
in accordance with the method by Rubin (Proc. Nat. Acad. Sci. 46 1105, 
1960) and the method by Sarma et al (Virology, 23 313, 1964). The results 
are shown in Table 1. 
Table 1 
______________________________________ 
Chicken Quail 
Number Number 
of of 
Number chickens Number quails 
of showing of showing 
test positive test positive 
chickens 
reaction quails reaction 
______________________________________ 
Anti-RIF of 
53 52 135 0 
parent bird 
Detection of 
83 3 163 0 
RIF from 
embryo 
Detection of 
67 2 85 0 
RIF from 
parent bird 
______________________________________ 
As clearly understood from Table 1, most of the adult chickens were proved 
to have anti-RIF, which showed the fact that most of chickens were 
infected with RIF. By contrast, all the test adult quails were proved to 
have not anti-RIF. The examination of bloods and visceral organs of embryo 
and adult birds with respect to detection of RIF, no RIF was detected from 
all the test adult quails. From the results, it can be fairly concluded 
that the probability of natural infection of quails with RIF is extremely 
low. 
REFERENCE EXAMPLE 2 
A chick embryonated egg and a quail embryonated egg were respectively 
treated in accordance with the ordinary method, to disperse the cells 
thereof. Then, the number of cells per one embryo was measured and shown 
in Table 2. 
Table 2 
______________________________________ 
Kind of embryo Number of cells 
______________________________________ 
Chicken 8.0 .times. 10.sup.7 -8.9 .times. 10.sup.7 
Quail 7.8 .times. 10.sup.7 -8.8 .times. 10.sup.7 
______________________________________ 
As apparent from Table 2, with regard to the numbers of the cells obtained 
through tissue culture of embryo, chicken and quail are approximately the 
same with each other. 
REFERENCE EXAMPLE 3 
Isolation of virus of Marek's disease (hereinafter referred to simply as 
"MD") 
As a material for isolating therefrom wild MD virus, there was employed 
whole blood or tumor tissues of chicken infected with MD. According to the 
ordinary procedure, the tumor tissue was treated with trypsin and the 
cells thereof were dispersed. Then, they were suspended in Eagle's culture 
solution. 
On the other hand, 7 to 15-day-old quail embryo was treated according to 
the ordinary procedure, to disperse the cells thereof. The thus obtained 
fibroblasts were plated at 2.4 .times. 10.sup.6 cells per 6cm-diametered 
dish. On the next day, the fibroblasts on dishes each were inoculated with 
1 ml. of the above-mentioned whole blood or tumor tissue suspension, 
whereupn they were cultivated in an incubator (containing carbon dioxide 
gas in an amount of 5%) at 37.degree. C. for 4 - 7 days. The results are 
summarized in Table 1 in which "+" and "-" show detection and 
non-detection of the MD virus isolated in and adapted to each fibroblast 
culture, respectively. 
For comparative purpose, the results respectively obtained by using chick 
kidney, duck embryo and turkey embryo as culture medium are also shown. 
Table 3 
______________________________________ 
Infected 
chicken 
(No. of 
Mater- 
test ial Dec .sup.1) 
QEC .sup.2) 
CK .sup.3) 
TEC .sup.4) 
sample) 
used CPE .sup.4) 
IF .sup.5) 
CPE IF CPE IF CPE IF 
______________________________________ 
13 Lever + + - - + + 
Spleen + + - - + + 
Kidney + + + + + + 
Blood + + + + + + + + 
22 Lever + + - - + + 
Spleen + + - - + + 
Kidney + + - - + + 
Blood + + + + + + + + 
23 Lever + + - - + + 
Spleen + + - - + + 
Kidney + + - - + + 
Blood - - - - - - + + 
76 Lever + + - - + + 
Spleen + + - - + + 
Kidney + + - - + + 
Blood + + - - + + + + 
105 Lever + + + + + + 
Spleen + + - - + + 
Kidney + + - - + + 
Blood + + + + + + + + 
______________________________________ 
Note: 
.sup.1) Duck embryo fibroblast culture 
.sup.2) Quail embryo fibroblast culture 
.sup.3) Chick kidney culture 
.sup.4) Turkey embryo fibroblast culture 
.sup.5) Cytopathic effect 
.sup.6) Immuno-fluorescence technique 
As apparent from Table 1, MD virus was detected in DEC, QEC and TEC as well 
as CK. It should be noted that MD virus could be isolated through QEC, and 
TEC also. From the other view point, it was noted that MD virus was 
separated from whole blood of affected chicken in a high proportion of 
4/5, and from liver, kidney and spleen each in a proportion of 5/5. 
The thus isolated viruses each have an affinity to cells of duck, quail and 
chick embryoes. It was also observed that these viruses can be further 
grown upon successive cultivation. 
As noted in Table 1, whether or not the embryo fibroblast used was infected 
with MD virus was determined by means of a cytopathic effect and 
immunofluorescence technique. The obtained result was further examined by 
means of an electron microscope. 
EXAMPLE 1 
The quail, duck and chick embryo fibroblasts prepared in the same manner as 
described in Reference Example 3, each were suspended in Eagle's culture 
solution in a concentration of about 5 .times. 10.sup.5 cells per ml., and 
then poured, by 100ml., in Roux's culture bottles. They were allowed to 
stand in an incubator at 37.degree. C. for 1 day. Whereupon the fibroblast 
cultures as prepared above, each were inoculated with BIKEN C strain of MD 
virus. Volume of inoculum per bottle was 1 ml. One day after the 
inoculation, the culture solution was removed, and then the cells attached 
to the bottle wall was washed with Hanks' solution, whereupon Eagle's 
culture solution was poured thereinto. Cultivation was further continued 
at 37.degree. C. for 4 to 7 days until alteration of the cells progressed 
to an extent of 40 to 45% based on the total cells. Then, cultivating 
operation was terminated. After termination of cultivation, the cells were 
taken to obtain infected embryo fibroblast cultures, which were suspended 
in Eagle's culture solution in a concentration of 5 .times. 10.sup.6 cells 
per ml. to obtain an infected culture suspension, and then stored upon 
addition of a suitable stabilizer, for example glycerin. 
Examination was done on each of the thus obtained infected embryo 
fibroblast cultures with respect to various properties, in accordance with 
the draft U.S. Standards for Approval of Vaccine to ensure the 
adaptability as vaccine. Then, they were formulated to commercial MD live 
vaccine. 
EXAMPLE 2 
MD virus isolated through DEC, for example BIKEN Q strain of MD virus was 
subjected to 6-passage, 36-passage and 46-passage successive cultivation 
using only QEC. On the other hand, BIKEN C strain of MD virus was 
subjected to 12-passage successive cultivation using DEC, and 24-passage, 
27-passage, 46 passage or 72-passage successive cultivation using QEC. 
The thus obtained two cultures each were treated in the same manner as 
described in Example 1 to obtain an infected culture suspension. The 
obtained infected culture suspensions were stored on addition of a 
suitable stabliizer, for example glycerin, whereupon its ability of 
immunization of chicken against MD was examined. 
The vaccines obtained according to the abovementioned experiments had a 
sufficient activity of immunization on storage at 4.degree. C. for 1 week, 
and even on storage at very low temperature, for example - 70.degree. C. 
for 2 months. 
Table 2 shows the effects of the vaccines obtained. 
Table 4 
__________________________________________________________________________ 
Age of 
Inoculation Age of 
chick in- 
of vaccine test chick 
oculated Age of 
when con- 
with viru- chick in- 
tacted 
Number 
lent strain oculated 
with of- 
of Rate of 
Number of 
of MD Successive 
with the 
fected 
Mortality 
Mortality 
Experiment 
chick virus cultiva- 
vaccine 
chick due to 
due to 
group used (day-old) 
tion 2) (day-old) 
(day-old) 3) 
MD 4) 
MD 
__________________________________________________________________________ 
A.sub.1 (Control) 
20 1 -- -- -- 14 14/20 
(70%) 
BIKEN C 
A.sub.2 
20 -- strain 1 -- 0 0/20 
DEC 12 
.fwdarw.QEC 46 
BIKEN C 
A.sub.3 
19 -- strain 1 48 0 0/19 
DEC 12 
.fwdarw.QEC 72 
B.sub.1 (Control) 
38 1 -- -- -- 15 15/38 
(39%) 
BIKEN Q 
B.sub.2 
20 -- strain 1 -- 6 7/20 
(35%) 
QEC 6 
BIKEN Q 
B.sub.3 
50 -- strain 1 -- 2 2/50 
(4%) 
QEC 36 
BIKEN Q 
B.sub.4 
50 -- strain 1 52 0 0/50 
QEC 46 
BIKEN C 
B.sub.5 
50 -- strain DEC 12 
18 -- 0 0/50 
.fwdarw.QEC 24 
BIKEN C 
strain 
B.sub.6 
48 -- DEC 12 1 49 1 1/48 
(2%) 
.fwdarw.QEC 27 
__________________________________________________________________________ 
(Note)- 
A: White leghorn 
B: De Carb 161 
The inoculations (0.2 ml. each) were effected to the abdominal cavity. Th 
period of time of observation was limited to 26-week-old. 
1) The vilurent strain of MD virus is a strain successively cultivated 
following of inoculation of chick abdominal cavity with blood of chick 
infected with MD. 
2) The "BIKEN C strain" and "BIKEN E strain" are the names of strains of 
MD viruses. Their infectivities are each 10.sup.4-5/ml. expressed in term 
of plaqueforming unit (PFU). The number attached after the abbreviations 
DEC and QEC shows the number of succession of cultivation. 
3) Test chicks were bred together with several chicks recognized to be 
affected with MD in the same poultry house. 
4) The affected and dead chicks were all subjected to autopsy and 
examined, and they are all proved to be of MD in view of pathology. 
As apparent from Table 2, the vaccine obtained on high passage (such as 
43-passage) successive cultivation using DEC showed a superior ability of 
immunization against MD. Whilst, the vaccine obtained by few passages 
successive cultivation using DEC followed by about 20 or more-passage 
successive cultivation, also showed a superior activity of immunization 
against MD as similar to that obtained on high passage successive 
cultivation using QEC alone. Thus, it has been statistically proved that 
MD virus is substantially made avirulent by successive cultivation using 
DEC and/or QEC and the resulting cultures are useful as MD vaccine. 
EXAMPLE 3 
A 7 to 12-day-old quail embryo was decapitated, amputated and viscerated, 
and then minced. 0.2% aqueous solution of trypsin was added thereto, 
followed by treatment according to the ordinary procedure to disperse the 
cells thereof. The obtained dispersion was subjected to a low speed 
centrifugalization to remove the aqueous phase, and then suspended in 
Earle's solution (containing 5% calf serum, 0.5% hydrolyzed lactalbumin 
and 50 mcg./ml. of erythromycin) in a concentration of 1.0 - 1.5 .times. 
10.sup.6 cells per ml. 
The thus obtained suspension was poured, by 100 ml., into Roux's bottles 
each having a capacity of 1000 ml. They were allowed to stand in an 
incubator at 37.degree. C. for 2 days. Whereupon the abovementioned 
culture solution was removed, and the cells attached to the bottle wall 
were washed with Hanks' solution to remove the calf serum content. Then, 
the fibroblast culture in Roux's bottle was inoculated with 1 ml. of 
measles virus-infected suspension [TANABE strain of measles virus (name of 
the strain measles attenuated by successive cultivation using 
chorio-allantoic membrane of chick embryonated egg)]. Whereupon, 100 ml. 
of Eagle's culture solution were added thereto, and then cultivation was 
further conducted at 36.degree. C. for 4 to 6 days. Upon termination of 
cultivation, the virus suspension was taken, and purified through 
centrifugalization or filtration to completely remove the culture cells. 
The infectivity of this virus suspension (measured by using FL cells) was 
10.sup.4.0 - 10.sup.5.0 TCID.sub.50/ml. (TCID.sub.50 m infectivity of 50% 
cultivated tissue culture) and was sufficient to prepare dry measles 
vaccine. 
On the other hand, the above-mentioned virus was subjected to 10-passage 
successive cultivation. At each passage of the successive cultivation, 
there was a virus suspension having an approximately equivalent and 
constant infectivity. 
The adaptability of the thus obtained virus suspensions was examined in the 
same manner as described in Example 1. Then, the suspension was formulated 
to commercial measles live vaccines. 
EXAMPLE 4 
Quail embryo was treated in the same manner as described in Example 3, to 
obtain a tissue culture, and then inoculated with the aforementioned 
TANABE strain of measles virus followed by successive cultivation. To the 
virus suspension obtained at the 10th passage of successive cultivation 
were added stabilizers of 3% of arginine, 5% of succharose and 10% of 
hydrolyzed gelatin. The resulting suspension was poured, by a given 
volume, to small reservoirs and then freeze-dried. Upon long storage of 
the obtained formulations, the change in infectivity of each formulation 
was examined. The infectivity was 10.sup.4.3 TCID.sub.50/ml. immediately 
after drying, and was changed extremely slightly even after stored at 
4.degree. C. for 12 months to 10.sup.4.2 TCID.sub.50/ml. Accordingly, the 
maintenance of infectivity can be said to be very excellent. This fact 
suggests that the present vaccine formulations are very stable with 
respect to its immunizing activity and can be stored for a long time. 
REFERENCE EXAMPLE 4 
TANABE strain of measles virus (which had been highly attenuated by 
successive cultivation using chorio-allantoic membrane of chick 
embryonated egg) was subjected to 1-passage successive cultivation and 
10-passage successive cultivation to obtain two kinds of virus 
suspensions, from which two kinds of vaccines were formulated. The 
comparison was made on these two kinds of vaccines and a vaccine 
(CAM-vaccine) prepared from viruses harvested from the chorio-allantoic 
membrane of chick embryonated egg which had been inoculated with the 
above-mentioned TANABE strain of measles virus. 
The thus obtained CAM-vaccine was examined with regard to its adaptability 
as vaccine in the same manner as described in Example 1. The qualified 
vaccine was inoculated to a little child (8-month-to 3-year-old) having 
therein no measles antibody. The results are summarized in Table 5. 
Table 5 
______________________________________ 
Number 
Number of child- 
of ren show- 
Infec- child- ing 
tivity/ Number ren rise in Number 
Inocu- of showing 
body of 
Deri- lation inocu- increase 
tempera- 
child- 
ration amount lated in neut- 
ture to ren 
of (log.sub.10 
child- ral anti- 
more than 
showing 
vaccine 
TCID.sub.50) 
ren body 39.degree. C. 
eruption 
______________________________________ 
Tissue 
culture 
of quail 
embryo 
fibro- 
blast 
1-pas- 
sage 
culti- 
vation 3.7 5 5 0 0 
Tissue 
culture 
of quail 
embryo 
fibro- 
blast 
10-pas- 
sage 
succes- 
sive 
cultiva- 
tion 3.5 7 7 1 0 
Con-* 
trol 
(CAM- 
vaccine) 
3.4 12 12 2 1 
______________________________________ 
Note: 
*Report at the general meeting of Society for Study of Measles vaccines i 
1969, entitled "Results of inoculation with highly avirulent measles 
vaccines"Report by shigeharu Ueda at the 16th general meeting of Society 
for Research of Virus in 1969, entitled "Study on measles vaccines 
As clearly understood from Table 5, the two kinds of vaccines prepared 
employing a tissue culture quail embryo fibroblast have not a significant 
difference in clinical reaction from the CAM-vaccine, which is an original 
strain of the above-mentioned two kinds of vaccines, derived from 
chorio-allantoic membrane of quail embryonated egg, and showed a 
sufficient immunizing activity. 
EXAMPLE 5 
Quail embryonated eggs were inoculated with measles virus and influenza 
virus, respectively. The obtained vaccines were compared with the vaccines 
prepared by inoculation of chick embryonated egg with measles virus. The 
results are shown in Table 6. 
Table 6 
______________________________________ 
Name of virus 
Measles .sup.1) Influenza .sup.2) 
______________________________________ 
Cultivation 
time 2 3 4 5 1 2 
Infec- 
Quail 4.2 5.0 5.3 5.6 6.0 8.5 
tivity 
Chick 4.0 4.7 5.5 5.5 6.5 8.5 
______________________________________ 
Notes: 
.sup.1) log.sub.10 TCID.sub.50/ml. 
.sup.2) log.sub.10 EID.sub.50/ml. 
The above table was obtained on the basis of the experiments as follows. 
With regard to the preparation of measles vaccine, 0.1 ml. of amniotic sac 
of a 7-day-old embryonated egg was inoculated with the suspension of the 
aforementioned TANABE strain, and then cultivated at 35.degree. C. for 5 
days. With regard to the preparation of influenza virus, A.sub.2 strain of 
influenza virus was inoculated to chorio-allantoic sac of a 8-day-old 
embryonated egg, and then cultivated at 37.degree. C. for 5 days. At given 
intervals, the viruses were taken, formulated to virus suspens, and 
examined with regard to infectivity. The yield of virus obtained by 
employing a quail embryonated egg was as good as that obtained by 
employing a chick embryonated egg. 
EXAMPLE 6 
In the similar manner to that described in Examples 3 to 5, quail 
embryonated eggs and tissue cultures of quail embryo fibroblasts were 
inoculated with various kinds of viruses. The growth and propagation of 
virus were measured by known assays. The results are shown in Table 7. 
As understood from Table 7, a quail embryonated egg and a tissue culture of 
quail embryo fibroblast are a superior culture medium for the adaptation 
thereto and propagation therein of various viruses. In addition, as 
described, a quail embryonated egg and a tissue culture of quail embryo 
fibroblast are scarcely subject to induction thereto of unfavorable 
factors, particularly chicken leukemia virus, and useful for produce high 
quality virus disease live vaccines. 
Table 7 
__________________________________________________________________________ 
Tissue 
Tissue 
culture 
culture 
of of Tissue 
Quail 
quail 
chick 
culture 
embryo- 
embryo 
embryo 
of 
nated 
fibro- 
fibro- 
monkey 
Virus 
egg blast 
blast 
kidney 
Assay 
__________________________________________________________________________ 
Rubella 
3.5 3.5 4.0 4.5 Tissue culture of 
African green 
monkey kidney 
(log.sub.10 I.sub.n D.sub.50/ml.) 
Tissue culture of 
Mumpus 
8.0 8.2 8.0 7.8 chick embryo fibroblast 
(log.sub.10 TCID.sub.50/ml.) 
Influenza 
8.5 7.2 7.5 7.5 Chick embryonated egg 
A.sub.2 (log.sub.10 EID.sub.50/ml.) 
Influenza 
8.0 7.5 7.2 6.0 " 
Tissue culture of chick 
Distemp- 
4.3 4.0 3.8 5.0 embryo fibroblast 
(log.sub.10 PFU/ml.) 
Vaccinia 
8.0 7.0 6.5 -- Chick embryonated egg 
(log.sub.10 POFU/ml.) 
New 8.0 8.0 8.2 -- Chick embryonated egg 
castle (log.sub.10 InD.sub.50/ml.) 
Japanese Tissue culture of chick 
encepha- 
7.8 8.5 7.5 8.5 embryo fibroblast 
litis (log.sub.10 PFU/ml.) 
Dengue 
4.5 5.0 4.5 5.0 Mouse 
fever (log.sub.10 LD.sub.50/ml.) 
Rabies 
7.3 4.0 3.8 -- Mouse 
(log.sub.10 LD.sub.50/ml.) 
Marek's 
3.5 4.8 4.1 -- Tissue culture of 
disease duck embryo fibro- 
blast (log.sub.10 PFU/ml.) 
__________________________________________________________________________