A hepatitis A, B-combined adjuvanted vaccine is disclosed in this application, said vaccine comprising an inactivated hepatitis A virus antigen, a hepatitis B virus surface antigen and an adjuvant. The present vaccine is obtained by causing the antigens to be adsorbed to the adjuvant. According to the present invention, the infection associated with hepatitis A virus and with hepatitis B virus can be prevented without causing any interference due to the mixing of these antigens and any severe side-effects, and the anti-hepatitis A virus antibody titer is greatly enhanced by the mixing.

FIELD OF THE INVENTION 
The present invention relates to a hepatitis A,B-combined adjuvanted 
vaccine and, particularly, to a hepatitis A,B-combined adjuvanted vaccine 
comprising an inactivated hepatitis A virus (hereunder referred to as 
"HAV") antigen and a purified hepatitis B surface antigen (hereunder 
referred to as "HBs antigen") or an inactivated purified HBs antigen, 
which are adsorbed on aluminum gel, the inactivated HAV antigen being 
obtained by proliferating HAV, which has been isolated from the stool of a 
patient suffering from hepatitis A and which has been adapted to grow in 
Green monkey kidney cells, on a large scale according to cell culture 
technique, and then isolating and purifying it from the infected cells; 
the purified HBs antigen being produced by a recombinant (yeast), to which 
producibility of HBs antigen is imparted in accordance with a genetic 
recombination technique; and the inactivated purified HBs antigen being 
derived from the plasma of hepatitis B virus carriers (hereunder referred 
to as "carrier"). 
BACKGROUND OF THE INVENTION 
Hepatitis A is a disease which sporadically breaks out through oral 
infection with HAV. However, recent reports on its large-scale epidemic 
have become rare in advanced countries, because in those countries 
hygienic environment has been improved as a whole. Nevertheless, there is 
a report stating that 1 to 1.5% of patients suffering from acute hepatitis 
A become fluminant and, therefore, hepatitis A is believed to be a disease 
worth notice, epidemiologically and clinically. 
Recently, the number of people having anti-HAV antibody has been reduced 
year by year as the number of the reports on the epidemic has been 
reduced. As a result, most of people not more than 35-year-old are 
negative in anti-HAV antibody in the advanced countries. However, there 
become conspicuous, cases where such antibody-negative young people take 
passage to regions highly infected with indigenous hepatitis A and get 
infected. Taking into consideration the recent tendency that many 
enterprises branch out into the developing countries and that chances of 
traveling abroad have been increased, a preventive vaccine has been 
required to be immediately developed. However, any of such vaccines have 
not yet been put into practical use. 
On the other hand, hepatitis B is a disease caused by the infection with 
hepatitis B virus (hereunder referred to as "HBV") through blood or body 
fluid. Its prognosis is not good and this disease frequently shifts to 
chronic hepatitis, cirrhosis and even hepatocellular carcinoma. Until now, 
an effective means for treating hepatitis of this type has not yet been 
developed. Under such circumstances, a hepatitis B vaccine derived from 
plasma of the carriers has first been developed as a preventive means. 
Moreover, to overcome the difficulty in securing starting material, which 
is caused by the lack of the carrier plasma, there has recently been 
developed a technique comprising inserting a structural gene of HBs 
antigen, into yeast or animal cells as host cells in accordance with a 
genetic recombination technique to cause the expression, producing a large 
amount of only HBs antigen as a source material for vaccines for 
preventing the hepatitis, and purifying it to obtain highly purified 
antigen. 
It is believed that the number of hepatitis B carriers is about two hundred 
million in the world and that in the HAV indigenous regions such as 
Southeast Asia and Africa, the number of carriers almost reaches 10 to 15% 
of their population. This clearly shows highly latent possibility of HBV 
infection in the HAV indigenous regions. Therefore, in such regions, a 
means for preventing infection associated with hepatitis A virus and with 
hepatitis B virus has been eagerly requested to be developed. 
Recently, there have been actively conducted many attempts for developing 
vaccines capable of preventing a plurality of objective diseases through 
only one inoculation, i.e., polyvalent vaccines (combined vaccines) for 
the purposes of decreasing the number of inoculations, hence decreasing 
accidents possibly happening during its inoculation and reducing cost in 
preparing vaccines, when the vaccines are produced as a means for 
preventing various infectious diseases. However, such mixing sometimes 
reduces the immunogenicities of the vaccines (interference action). Now, 
this becomes a major obstacle in developing a polyvalent vaccine. 
SUMMARY OF THE INVENTION 
Accordingly, the object of the present invention is to provide a hepatitis 
A,B-combined adjuvanted vaccine which resolves the problems which are 
encountered when the infection with both hepatitises A and B is to be 
prevented, and which is safe and economic. 
The above object can be effectively achieved by a hepatitis A,B-combined 
adjuvanted vaccine comprised of an inactivated hepatitis A virus antigen, 
an HBs antigen and an adjuvant.

DETAILED EXPLANATION OF THE INVENTION 
As a HAV usable in the present vaccine, HAV obtained by tissue culture is 
employed. More specifically, a large amount of HAV can be obtained by the 
tissue culture utilizing an HAV high-producibility cell line, i.e., GL-37 
cell, which is established by cloning African Green monkey kidney cells in 
accordance with a colony culture technique, and also utilizing HAV KRM 003 
strain which is isolated from the stool of HAV infected-patients and is 
highly susceptive to the GL-37 cell. HAV thus obtained is purified by a 
proper combination of various methods for isolating and purifying 
biologically active substances, such as fractionation with polyethylene 
glycol, ultracentrifugation, treatment with organic solvents, enzyme 
treatment and gel filtration, to produce a purified antigen, which is then 
inactivated with formalin and used in preparing the combined vaccine of 
the present invention. 
On the other hand, the HBs antigen usable in the present invention includes 
those produced by a recombinant which is transformed in accordance with a 
genetic recombination technique to get an HBs antigen-producibility, or 
inactivated and purified HBs antigens derived from plasma of HBV carriers. 
The former antigen is produced as follows. First, a shuttle vector pAM82 
is prepared, which contains the replication origins of 2.mu. ori plasmid, 
pBR322 plasmid and yeast chromosome, the leu gene of yeast, the 
ampicillin-resistant gene of Escherichia coli, and the repressive acid 
phosphatase promoter region of yeast. Second, the HBs gene of HBV DNA, 
which is isolated from HBs antigen-positive and hepatitis B e 
antigen-negative plasma of blood donors and then cloned, is combined with 
the repressive acid phosphatase promotor region of this vector, to produce 
a shuttle vector pAM203. Third, this vector pAM203 is inserted into a 
yeast cell, to obtain a transformed yeast cell. Then, the cell is cultured 
to cause the cell to produce the antigen. The HBs antigen produced in the 
yeast cell is purified according to any combination of the following 
methods, such as breakage of cell body, extraction from broken substances, 
salting out, gel filtration, ion-exchange chromatography, sucrose and 
cesium chloride centrifugation or the like. In this connection, the 
details are described in Japanese Patent Un-examined Publication 
(hereunder referred to as "J.P. KOKAI") Nos. 59-31799 and 60-193925. 
The plasma-derived HBs antigen is prepared from HBs antigen-positive 
carrier plasma, as a highly purified antigen, by density-gradient 
centrifugation using sucrose and cesium chloride, or any combination of 
various ion-exchange chromatography techniques. The preparation is 
specifically described in Japanese Patent Publication for Opposition 
Purpose (hereunder referred to as "J.P. KOKOKU") No. 61-045610 filed by 
the inventors of this invention. 
An adjuvant usable in the present invention is not critical so far as it 
can enhance immune activity to a desired extent and does not give any side 
effects. Suitably used in the present invention is aluminum gel adjuvant, 
in particular aluminum hydroxide gel and aluminum phosphate gel adjuvants. 
One preferred embodiment of the combined vaccine of this invention is 
obtained by adsorbing to aluminum gel, the HAV inactivated antigen and HBs 
antigen prepared according to the foregoing methods. The concentration of 
the aluminum gel ranges from 100 to 1,000 .mu.g/ml and preferably 400 
.mu.g/ml. The final concentrations of HAV inactivated antigen and HBs 
antigen are not less than 50 ng/ml and not less than 2.5 .mu.g/ml 
respectively, and the mixing ratio is 1:20 to 1:200. The mixing may be 
performed in any manner, but preferred are the following 5 methods: 
1) The HAV antigen and the HBs antigen are mixed at a desired concentration 
and the mixed solution is brought into contact with an adjuvant to adsorb 
the antigens thereon. 
2) The HAV antigen is first brought into contact with an adjuvant to adsorb 
the HAV antigen thereon and then the HBs antigen is brought into contact 
with the adjuvant carrying the HAV antigen to adsorb the HBs antigen 
thereon. 
3) The HBs antigen is first brought into contact with an adjuvant to adsorb 
the HBs antigen thereon and then the HAV antigen is brought into contact 
with the adjuvant carrying the HBs antigen to adsorb the HAV antigen 
thereon. 
4) The HAV antigen and the HBs antigen are separately adsorbed on different 
adjuvants and then these adjuvants are mixed with each other. 
5) An aluminum gel adjuvant is prepared in a solution containing the HAV 
antigen and then the HBs antigen is brought into contact with the adjuvant 
to adsorb the HBs antigen thereon. 
The combined vaccine thus prepared is a useful pharmaceutical preparation 
which prevents both the infection with hepatitis A and B without any 
reduction in antigenic potency and any deterioration of its properties. 
Moreover, this preparation never causes interference between virus antigens 
due to their mixing, which interference is frequently observed, in 
particular in case of combined vaccines for animals (such as vaccines for 
Newcastle disease, infectious bronchitis disease, and for akabane disease, 
ibaraki disease). Thus the preparation has no immune response-inhibitory 
effect. Moreover, the combined vaccine preparation provides higher 
hepatitis A immunogenicity-enhancing effect than that observed when HAV 
antigen is used alone. 
In addition, according to the present invention, the amount of HAV antigen 
per unit dose can be reduced to a level lower than that required for 
vaccines in which no adjuvant is added, by utilizing an aluminum gel as an 
adjuvant. Moreover, the price of the preparation can be lowered to a level 
less than those comprising individual antigens, because the preparation of 
the present invention is polyvalent. 
Efficacy and Safety of Combined Vaccine 
Dr. Moritsugu et al. in National Institute of Health reported on the 
efficacy of a liquid type hepatitis A vaccine for marmosets, in the 33th 
Meeting of the Society of Japanese Virologists (1986) and in Report on 
Research and Development on Hepatitis A Vaccine (1985). These reports 
state that when the acquired antibody titer of the marmoset which is 
inoculated with an inactivated vaccine is not less than 1,000 mIU, the 
infection with a virulent virus strain of 10.sup.3 MID.sub.50 can be 
inhibited irrespective of whether the virus enters the body of living 
organism through the vein or the mouth. 
The inventors of this invention compared the antibody titers induced by 
immunization of the inactivated and purified antigens prepared by the 
inventors, with that of the inactivated HAV antigen (Reference) obtained 
from Dr. Moritsugu according to parallel line assay using a mouse. As a 
result, both linearity and parallelism with respect to Reference are 
confirmed and the relative potency is almost equal to that of Reference. 
Moreover, as will be described in the following Examples, it is 
demonstrated, by experiments using guinea pigs and mice, that when an 
aluminum gel was used as an adjuvant, the antibody-producibility equal to 
1,000 mlU or more can be induced by immunizing these animals with HAV 
antigen in an amount not less than 50 ng/dose. Regarding the safety of the 
purified HAV antigen, a test for freedom from abnormal toxicity was 
conducted according to "Minimum Requirement of Biological Products" edited 
by Ministry of Health and Welfare of Japan and an acute toxicity test was 
conducted according to "Japan GLP Guide Line." Any abnormality was not 
observed on these animals at all. 
On the other hand, the efficacy and safety of yeast-derived HBs antigens 
obtained according to a genetic recombination technique and those of the 
carrier's plasma-derived HBs antigens were already reported by the 
inventors of this invention in "KISO TO RINSHO (Clinical Report)," 1987, 2 
1, p. 259. In this report, an yeast-derived hepatitis B vaccine containing 
20 .mu.g of HBs antigen and 400 .mu.g of aluminum gel per 1 ml of the 
vaccine was subcutaneously injected into about 2200 persons three times in 
an amount of 0.5 ml (corresponding to 10 .mu.g of HBs antigen) per 
injection for adult and 0.25 ml (corresponding to 5 .mu.g of HBs antigen) 
per injection for infant, and it was found that the seroconversion rate 
was 94.5% for adult and 98.3% for infant. There were observed some 
side-effects such as local pain and itching for 11.6% of the whole 
subjects and malaise for 5.1% of the total subjects. However, these 
results are the same as those observed on the plasma-derived hepatitis B 
vaccine which have been put on market and whose efficacy and safety have 
been confirmed. Moreover, a test for freedom from abnormal toxicity was 
conducted on the combined vaccine according to "Minimum Requirement of 
Biological Products" and no abnormality was observed. 
As seen from the above, it is concluded that the present combined vaccine 
can be sufficiently put into practical use in light of its efficacy and 
safety. 
The present invention will be explained in more detail with reference to 
the following Examples and Reference Examples, and the effects practically 
attained by the invention will also be discussed. 
REFERENCE EXAMPLE 1 
Cultivation and Purification of HAV 
GL-37 cells which had been derived from African Green monkey kidney cells 
and which had been established by and distributed from Dr. Moritsugu in 
Japan National Institute of Health, was repeatedly passed in a roller 
bottle (cultivation area=approx. 700 cm.sup.2). At 19 to 23 serial 
passages, these cells were inoculated with HAV KRM003 strain derived from 
human stool, which strain had also been established by Dr. MORITSUGU so 
that the virus infectious dose per cell (M.O.I.) was equal to 0.1 to 1.0 
and then the cells were cultivated in Eagle's minimum essential medium 
(E-MEM) containing 2% fetal bovine serum (hereunder referred to as "FBS") 
for 2 to 3 weeks. After the completion of the cultivation, the cells were 
washed with phosphate buffered physiological saline (hereunder referred to 
as "PBS"), followed by adding 10 to 15 m l per roller bottle of a lytic 
buffer which contained 10 mM of tris-HCl buffer of pH 7.4 (containing 1% 
NP 40 (available from NAKARAI CHEMICAL CO., LTD.), 0.4% sodium 
deoxycholate and 50 mM of EDTA); and then cultivating the cells at 
37.degree. C. for one hour in a cell roller. After harvesting them, the 
cell debris was removed by centrifugation at 8,000 to 10,000 rpm for 30 
minutes. A five times concentrated polyethylene glycol 6,000 (available 
from WAKO JUNYAKU CO., LTD.) solution containing sodium chloride was added 
to the resultant supernatant in an amount of one volume per 4 volumes of 
the latter, and then the solution was stirred at 4.degree. C. for 2 to 3 
hours and was allowed to stand over night. Then, the solution was 
centrifuged at 8,000 to 10,000 rpm for 30 minutes and the resultant 
pellets were suspended in a lytic buffer. The suspension was further 
centrifuged at 20,000 rpm over night to pelletize the virus. The resultant 
virus pellets were resuspended in PBS and an equivalent volume of 
chloroform was added to the suspension to extract the virus at room 
temperature for 30 minutes. After collecting the aqueous phase (the virus 
phase), the residual chloroform was removed under vacuum and then the 
phase was treated with an enzyme. In the enzyme treatment, DNase I 
(available from TAKARA SHUZO CO., LTD.) and RNase A (available from Sigma 
Co., Ltd.), whose final concentrations were 20 to 40 .mu.g/ml 
respectively, and 50 .mu.g/ml of Proteinase K (available from Merck Co., 
Ltd.) were added to the aqueous phase for decomposing the protein 
components and nucleic acids derived from the host cells. This enzyme 
treatment was continued for 4 to 6 hours at 37.degree. C. To this solution 
treated with the enzymes, there were added an equivalent volume of 2.5M 
potassium phosphate buffer (pH 7.5) and 0.8 volume of a mixed solution of 
ethoxyethanol and butoxyethanol (2:1 v/v), to mix the solution several 
times. By this organic solvent treatment, the virus was concentrated in 
the middle phase to form a band. The virus phase was collected, suspended 
in 10 mM PBS (pH 7.4) containing 0.1% Tween 80 (available from WAKO 
JUNYAKU CO., LTD.) and 2 mM of EDTA, and then again treated with the 
organic solvent. The virus suspension finally obtained was centrifuged at 
10,000 rpm for 15 minutes and the resultant supernatant was passed 
through a gel filtration column packed with Sephacryl S 400 HR (available 
from Pharmacia Co., Ltd.) using PBS containing 0.002% Tween 80 as an 
eluent buffer. Antigen-positive fractions were collected, sterilized by 
filtration to obtain a purified virus solution, and then the solution was 
inactivated by treating it with formalin diluted by 2,000 to 4,000 time as 
a final concentration at 37.degree. C. for 12 days to obtain an 
inactivated purified antigen solution. 
REFERENCE EXAMPLE 2 
Preparation of Hepatitis A,B-Combined Adjuvanted Vaccine 
An aluminum gel as an adjuvant was prepared according to a method 
comprising addition of a 1N sodium hydroxide solution to a 10% aluminum 
chloride solution little by little to elevate the pH to about 7. The 
resulting gel was washed at least 5 times with PBS (pH 7.4) to remove free 
aluminum ions, and then suspended in the same buffer so as to adjust the 
concentration to 400 .mu.g/ml. The aluminum gel suspension was mixed with 
the inactivated HAV antigen and the HBs antigen so as to adjust the final 
concentrations thereof to 50 to 100 ng/ml and 2.5 to 10 .mu.g/ml, 
respectively. The mixed solution was stirred with a rotator at 4.degree. 
C. over night to adsorb these antigens to the aluminum gel. To confirm 
whether or not the HAV and HBs antigens were completely adsorbed to the 
gel, the supernatant obtained after adsorption was subjected to a 
quantitative analysis, more specifically an ELISA technique for the HAV 
antigen and an RIA technique for the HBs antigen, but the supernatant did 
not show any activity of both the HAV and HBs antigens. Therefore, these 
antigens were considered to be completely adsorbed to the gel. 
REFERENCE EXAMPLE 3 
Determination of Antigen Titer and Antibody Titer 
The HAV antigen titer was determined by an ELISA technique. More 
specifically, after coating a 96 well-microplate with anti-HAV rabbit 
serum as a first antibody and blocking it with bovine serum albumin 
(hereunder referred to as "BSA"), a specimen was reacted with the first 
antibody at 4.degree. C. over night. Then, the reaction product was 
reacted with a second antibody, which was an anti-HAV rabbit antibody 
conjugated with horseradish peroxidase, at 37.degree. C. for 2 hours and a 
solution of a substrate (o-phenylene-diamine) was added to let the 
specimen color-develop. After stopping the reaction, the absorbance at 492 
nm was measured and the antigen titer was evaluated from the calibration 
curve of a standard material. 
The anti-HAV antibody titer was determined according to a competitive 
inhibitory ELISA technique. More specifically, a well which had been 
coated with anti-HAV rabbit serum and blocked with BSA was reacted with 
HAV antigen at 4.degree. C. over night (as a control, a diluent was used 
in place of the antigen), an antibody as a standard sample or a specimen 
was added thereto to cause the reaction at room temperature for 30 
minutes. Then, a peroxidase-labeled anti-HAV rabbit antibody was added to 
cause the reaction at 37.degree. C. for 2 hours and the solution of 
substrate was added to cause the specimen to color-develop. After stopping 
the reaction, the absorbance at 492 nm was measured and the antigen titer 
was calculated as a titer at which the inhibition rate was 50% based on 
the calibration curve of a standard material. The antibody used as the 
standard material was prepared so that it showed a relative titer of 2 
IU/ml when the anti-HAV antibody titer of the anti-HAV Reference globulin 
No. 1 from Bureau of Biologics of U.S. Food and Drug Administration 
(F.D.A.) was set 100 IU/ml. 
The titer of the HBs antigen was determined utilizing an AUSRIA II kit 
(available from Abbott Co., Ltd.) and based on the calibration curve of 
the standard material. 
The titer of the anti-HBs antibody was determined by using an AUSAB kit 
(available from Abbott Co., Ltd.), preparing a standard sample on the 
basis of the WHO International Reference (IR-HBIG Lot. 26-1-77 50 IU/ml) 
and calculating it from the calibration curve. 
EXAMPLE 1 
To examine the response of the hepatitis A,B-combined adjuvanted vaccine 
prepared according to the same manner as in Reference Example. 4-week-old 
SPF guinea pigs (each group comprising 10 animals) were subcutaneously 
immunized with the vaccine at the back in an amount per dose shown in 
Table I. As a comparative test, each of hepatitis A and B vaccines was 
also administered. 
6 weeks after the immunization, the animals were bled. The anti-HAV 
antibody was detected by an ELISA technique and its titer (mIU/ml) was 
obtained as a titer at which the competitive inhibition was 50%. In 
addition, the titer of the HBs antibody (mIU/ml) was determined using an 
AUSAB kit. Each value was expressed as a geometric means. 
TABLE I 
______________________________________ 
Inoculated Amount and Antibody Response of A, B and A,B- 
Combined Vaccines 
Antibody Titer 
HAV-Ag HBs-Ag Al gel 
After 6 weeks 
Vaccine (ng) (.mu.g) (.mu.g) 
(mIU/ml) 
______________________________________ 
A 100 -- 200 220 
B -- 10 200 1870 
A,B-Combined 
100 10 200 1680 (A); 2639 (B) 
______________________________________ 
As seen from the results listed in Table I, the antibody titer of the 
A,B-combined vaccine was 8 times that of the hepatitis A vaccine alone in 
terms of the response of the anti-HAV antibody 6 weeks after the 
immunization, and 1.4 times that of the hepatitis B vaccine alone in terms 
of the response of the anti-HBs antibody 6 weeks after the immunization. 
No interference of the antibody responses due to the mixing of these 
antigens was not observed. In particular, the immunogenicity of the 
anti-HAV antibody was much increased due to the mixing. 
EXAMPLE 2 
In this example, the antibody response of the hepatitis A vaccine was 
investigated, when the amount of the HAV antigen was changed to 200 and 50 
ng/dose while keeping unchanged the mixing ratio of the HAV antigen to the 
HBs antigen (Test 1) and when the amount of the HBs antigen was changed to 
2.5, 5 and 10 .mu.g/dose while keeping constant the amounts of the HAV 
antigen and the aluminum gel (HAV antigen: 100 ng, aluminum gel: 200 
.mu.g) (Test 2). The amount of each vaccine inoculated and the results 
obtained by the immunization tests are summarized in the following Tables 
II and III, respectively. 
TABLE II 
______________________________________ 
Amount of Each Vaccine Inoculated 
Aluminum Gel 
Vaccine HAV-Ag (ng) HBs-Ag (.mu.g) 
(.mu.g) 
______________________________________ 
Test 1: A alone 
200 or 50 -- 400 (100) 
B alone -- 20 or 5 400 (100) 
A,B-combined 
200 or 50 20 or 5 400 (100) 
Test 2: A alone 
100 -- 200 
B alone -- 10, 5 or 2.5 
200 
A,B-combined 
100 10, 5 or 2.5 
200 
______________________________________ 
TABLE III 
______________________________________ 
Antibody Response and 100 mIU/ml Appearance-rate of Each 
Vaccine 
Vaccine Antibody Titer (mIU/ml) 
Appearance-rate 
______________________________________ 
(Test 1) 
(i) (ii) (i) (ii) 
______________________________________ 
A alone 598 360 2/5 0/5 
B alone 5241 363 -- -- 
Combined A 
1905 990 3/4 2/4 
B 4045 695 -- -- 
______________________________________ 
(i): HAVAg 200 ng + HBsAg 20 .mu.g + aluminum gel 400 .mu.g/dose 
(ii): HAVAg 50 ng + HBsAg 5 .mu.g + aluminum gel 100 .mu.g/dose 
(Test 2) 
Anti-HAV Anti-HBs 
______________________________________ 
A alone 318 -- 2/10 
Combined a 
743 1371 3/10 
b 2270 1086 5/8 
c 1190 809 4/8 
______________________________________ 
a: HAVAg 100 ng + HBsAg 10 .mu.g + aluminum gel 200 .mu.g/dose 
b: HAVAg 100 ng + HBsAg 5 .mu.g + aluminum gel 200 .mu.g/dose 
c: HAVAg 100 ng + HBsAg 2.5 .mu.g + aluminum gel 200 .mu.g/dose 
As seen from the results obtained by Test 1, the antibody titer of the 
hepatitis A vaccine was increased by mixing these two vaccines even when 
the amount of the HAV antigen was set 200 or 50 ng while keeping constant 
the mixing ratio of the HAV antigen to the HBs antigen (1:100). In 
addition, it was also evidenced that 1000 mIU/ml appearance-rate became 
also high due to the mixing of these vaccines. On the other hand, it was 
ensured that the titer of the HBs antibody was not adversely affected by 
the mixing. In Test 2, the amount of the HBs antigen was set 10, 5 and 2.5 
.mu.g while keeping unchanged the amounts of the HAV antigen and the 
aluminum gel, but in each of these groups, the anti-HAV antibody titer was 
higher than that observed when only the hepatitis A vaccine was 
inoculated. Moreover, there was no significant difference in 1000 mIU/ml 
appearance-rate when the amount of the HBs antigen was 10 .mu.g, while the 
difference was significantly large for the groups wherein the HBs antigen 
was inoculated in an amount of 2.5 or 5 .mu.g. 
EXAMPLE 3 
For the purpose of examining the influence of the aluminum gel and also the 
antibody responses after a first immunization with the hepatitis 
A,B-combined vaccine and after a booster, 5-week-old SPF guinea pigs (each 
group comprising 5 animals) were subcutaneously immunized with the 
hepatitis A,B-combined vaccine at the back in an amount per dose listed in 
Table IV and further immunized with the same amount of the vaccine 6 weeks 
after the first immunization. As a comparative test, the immunization 
tests with individual liquid type vaccines, an aluminum gel adjuvant 
vaccine and a liquid type combined vaccine were also carried out. The 
results obtained are summarized in Table V. The combined vaccine and the 
combined aluminum gel adjuvant vaccine did not indicate any significant 
difference from those of the individual vaccines in antibody titer both 6 
weeks and 10 weeks after the immunization, and no interference was 
observed on the antibody response due to the mixing. 
TABLE IV 
______________________________________ 
Amounts of Various Antigens Inoculated 
Al Gel 
Vaccine HAV-Ag (ng) HBs-Ag (.mu.g) 
(.mu.g) 
______________________________________ 
A Liquid 50 -- -- 
B Liquid -- 5 -- 
Combined A,B Liquid 
50 5 -- 
A-Al Gel 50 -- 100 
B-Al Gel -- 5 100 
Combined A,B-Al Gel 
50 5 100 
______________________________________ 
TABLE V 
______________________________________ 
Antibody Response against Various Antigens 
After 6 After 10 
weeks (mIU/ml) 
weeks (mIU/ml) 
Anti-HAV Anti-HBs Anti-HAV 
Anti-HBs 
Vaccine Antibody Antibody Antibody 
Antibody 
______________________________________ 
A Liquid 170 -- 320 -- 
B Liquid -- 2570 -- 190546 
Combined A,B 
160 1698 460 112202 
Liquid 
A-Al Gel 560 -- 2400 -- 
B-Al Gel -- 14454 -- 446684 
Combined A,B-Al 
620 4571 2400 245471 
Gel 
______________________________________ 
EXAMPLE 4 
The effects of the hepatitis A,B-combined vaccine of this invention were 
investigated on different kinds of animals. 4-week-old ddy mice (each 
group comprising 9 animals) were subcutaneously inoculated with the 
hepatitis A vaccine alone or the hepatitis A,B-combined vaccine, and then 
the animals were bled 6 weeks after the inoculation to determine the 
amount of the anti-HAV antibody and the 1000 mIU/ml appearance-rate. The 
results obtained are listed in Table VI below. There was not observed any 
interference even when the kind of the animal was changed from a guinea 
pig to a mouse and, as seen from the average antibody titer, and the 
immunogenicity of the combined vaccine was significantly increased 
compared with that observed when the hepatitis A vaccine was inoculated 
alone. 
TABLE VI 
______________________________________ 
Immunization Test for Mice (Anti-HAV Antibody Titer) 
Vaccine Antibody Titer (mIU/ml) 
Appearance-rate 
______________________________________ 
A alone 263 2/9 
Combined A,B 
889 4/9 
______________________________________ 
A alone: HAV 100 ng + Al Gel 200 .mu.g/dose 
Combined A,B: HAV 100 ng + Al Gel 200 .mu.g + HBs 10 .mu.g/dose 
EXAMPLE 5 
Method for Preparing Combined A,B Vaccine 
In this example, the method for preparing the combined A,B vaccine was 
studied with respect to the correlation between the adsorptivity of both 
the antigens to the aluminum gel and a kind of mixing processes and also 
the immonogenicity for mice. The results obtained are summarized in Table 
VII below. 
Mixing was performed according to several ways: the process (A Alum+B) 
comprises first adsorbing the HAV antigen and then the HBs antigen to the 
aluminum gel; the process (B Alum+A) comprises adsorbing these antigens in 
the reverse order; the process (A Alum+B Alum) comprises separately 
adsorbing these antigens to the different aluminum gel and then mixing 
them; the process (A in B out) comprises adding a desired amount of 
aluminum chloride to 0.15M acetate buffer containing HAV antigen (pH 5.2) 
and then adjusting the pH with a 1N sodium hydroxide solution to obtain 
aluminum gel in which the HAV antigen was included, and thereafter 
adsorbing the HBs antigen to the gel; and the process (B in A out) 
comprises exchanging the order of using the antigens. The amount of the 
antigens adsorbed on the aluminum gel was determined by mixing the vaccine 
with a 20% phosphate-citrate solution in a ratio of 1:1 (for the HAV 
antigen) or mixing the vaccine with 10% phosphate-citrate solution in a 
ratio of 9:1 (for the HBs antigen) to dissolve the aluminum gel and then 
determining the amounts of the antigens according to an ELISA technique 
for the HAV antigen and an RIA technique for the HBs antigen. As a result, 
it was found that almost all of the HBs antigens as charged were adsorbed 
to the gel under such preparation conditions. Moreover, the amount of the 
HAV antigen dissolved out from the gel was as low as about 60%. However, 
since it was not detected in the supernatant except for some of the mixing 
process, it is assumed that the solutions to dissolve the aluminum gel 
inhibited the determination of the HAV antigen by ELISA. 
In the immunological tests, the vaccines were intraperitoneally inoculated 
into SPF female mice of 4-week-old (ddy) (each group comprising 5 animals) 
and bled 6 weeks after the inoculation. The A,B-combined vaccine 
inoculated contained 50 ng of the HAV antigen, 5 .mu.g of the HBs antigen 
and 100 .mu.g of the aluminum gel per dose. The results are listed in 
Table VIII. In the mixing method composed of a combination of "in" and 
"out", the amount of the antigen which was not absorbed on the gel was 
great when the antigen was adsorbed to the gel according to the "out" 
method, in particular when the HAV antigen was absorbed to the gel 
according to the "out" method. The antibody titer of the HAV vaccine 
obtained by the process (B in A out) was almost the same as that of the 
liquid type vaccine simply composed of the HAV antigen, which supported 
the results in Table VIII. The mixing processes except for the process (B 
in A out) provided no statistically significant difference in the amount 
of the anti-HAV antibody from that induced by the vaccine composed of only 
the antigen. However, referring to calculated means of the titer, the 
combined vaccine obtained by the A in B out method showed high immune 
response compared with the vaccines obtained by the other methods. 
TABLE VII 
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Adsorptibity of Antigens 
HAV-Ag (%) HBs-Ag (%) 
Method of Mixing 
Supernatant 
Gel Supernatant 
Gel 
______________________________________ 
A Alum 0 64.6 -- -- 
A Alum + B 0 67.4 0.2 99 
B Alum + A 0 63.2 01 100 
A Alum + B Alum 
0 64.2 0.1 108 
A in B out 8.8 53.0 3.6 82 
B in A out 53.6 48.2 0.7 107 
______________________________________ 
TABLE VIII 
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Immune Test of Combined Vaccine 
Anti-HAV Antibody 
Anti-HBs Antibody 
Method of Mixing 
(mIU/ml) (mIU/ml) 
______________________________________ 
(A alone, Liquid) 
870 -- 
A Alum 3090 -- 
A Alum + B 2340 523 
B Alum + A 2400 372 
A Alum + B Alum 
2240 389 
A in B out 4070 905 
B in A out 660 219 
______________________________________ 
The foregoing results, show that the hepatitis A,B-combined adjuvanted 
vaccine of the present invention does not induce any interference on the 
immune response and the immunogenicity of both HAV antigen and HBs 
antigen, in particular, that of the former tends to increase, so long as a 
unit dose of inoculation comprises not less than 50 ng of the HAV antigen 
and not less than 2.5 .mu.g of the HBs antigen and so long as the combined 
vaccine is prepared according to the foregoing mixing processes except for 
the process (B in A out). These facts suggest that the preparation of this 
invention can be an effective polyvalent vaccine for protecting from the 
infection with both hepatitis virus of A and B types.