감마선(Co60)조사 결핵균(H37Rv)의 면역학적 연구

Abstract

[한글]

Immunological Studies on Vaccine Prepared from Gamma-irradiated (Cp**60)

Mycobacterium tuberculosis(H^^37 Rv)

Sei Yung Chun, M.D.

Department of Microbiology College of Medicine, Yonsei University Seoul, Korea

(Directed by Joon Lew, M.D., Ph.D.)

The use of reduced toxin or of killed bacteria as antigens for the prevention of

many infectious diseases is widely accepted today. It is also well known that

antigenicity of bacteria may be reduced or destroyed by heating, chemical changes,

or by the process of manufacture of the antigen so that the immunologic response to

the prepared antigen is much less than the response to the use of living attenuated

organisms or to an active infection.

The most desirable or effective antibody is one which will produce an immunity

which is equal to that produced by living bacteria, but which will not produce any

pathologic changes.

In an attempt to find a more ideal antigen against tuberculosis, the author used

Gamma-irradiated(Co**60) Vaccine of Mycobacterium tuberculosis (H^^37 ,Rv)

(GIV-TB).

This form of the Mycobacterium has lost its ability to multiply and also uses

only a minimum of oxygen. The immunologic response to the irradiated vaccine was

compared with that following the use of BCG in human hosts.

The Gamma-irradiated Vaccine of Mycobacterium tuberculosis used in this

experiment was supplied by Dr. C. M. Carpenter the initiator of the vaccine, at the

Department of Infectious Diseases, University of California, Los Angels,

California.

The vaccine was used to human hosts after the thorough laboratory tests in vitro

and in vivo.

Under aseptic conditions two antigens were prepared, the first in physiologic

saline and the second in two per cent aluminum hydroxide aqueous solution. Both

contained lyophilized GIV-TB which had been aseptically ground in a Agate motar and

pestle. Suspensions, one mg per ml, of the two vaccines were kept under

refrigeration and used within two days after preparation. The control antigens were

liquid suspensions of BCG produced by the National Institute of Health in Korea,

and a lyophilized BCG from Japanese BCG research institute. Liquid suspensions of

the two control vaccines were also kept under refrigeration and used with고 five

days after preparation. Tuberculin (1 : 2,000) which was used in this experiment

was produced by the National Institute of Health in Korea, and the lepromin used in

this study was produced by this Department following the international

standardization.

163 leprosy born children (non infected) and 128 patients with the lepromatous

type of Hansen's disease were selected, and divided into two groups. These groups

had negative reaction in tuberculin and lepromin tests. The first group received

the GIV-TB and second group received the BCG. After six weeks, these groups were

also tested for their tuberculin and lepromin reactions.

128 patients with Hansen's disease and 50 leprosy born children were divided

according to age and sex into three nearly equal groups. The first group received

the suspension of GIV-TB in physiological saline, the second the suspension in 2%

aluminum hydroxide, and the third, the control group, received the saline

suspension of BCG from the National Institute of Health in Korea.

As to the method of inoculation the GIV-TB was given on the surface of the left

shoulder through 50 needle multiple skin punctures and the BCG was injected

intradermally in a 0.1 ml dose. Six weeks after the above GIV-TB and BCG

inoculations, the tuberculin skin reactions were tested.

Another 113 leprosy born children were divided according to age and sex into two

approximately equal groups. The first group received a saline suspension of GIV-TB

and the second group received a saline suspension of lyophilized BCG obtained from

Japan. Inoculation was given intradermally, after six weeks this group was also

tests for their tuberculin and lepromin reactions.

After the inoculation of GIV-TB or BCG the following changes in the tuberculin

and lepromin skin reactions (to positive conversion) were noted.

Out of 128 leprosy patients and 50 leprosy born children in So Rok island,

positive conversion rates of tuberculin reactions were found in 70% of the group

who received GIV-TB in physiological saline, in 60% of the group who received

aluminum hydroxide suspension, and in 48% of control group who received liquid

suspension of BCG. Females showed a lower number of positive changes than males. No

reason has been known for this sex difference. No one under four years of age

showed a positive conversion. The rate of positive change was increased

progressively with age.

Thus, in summary, the group which received the GIV-TB showed higher positive

conversion rate than the group received liquid suspension of BCG.

Positive conversion rate of lepromin reaction was lower than 40% in each groups.

It also showed that the leprosy patient group was slightly lower in the positive

conversion rate than leprosy born children group.

In another 116 leprosy born children, positive conversion rate of tuberculin was

about 61% in GIV-TB received group, and about 56% in Japanese lyophilized BCG

received group.

The positive conversion rate of lepromin reaction was about 85% in each group.

In 1939 Fernandez first used BCG for a preventive inoculation against Hansen's

disease. This was on the basis of his finding that previously negative reaction to

tuberculin and lepromin test, became skin positive to both following BCG

inoculation.

Many workers have proved that BCG might be used to increase the resistance to

Hansen's disease. However, marked difference was noted in the findings of many

investigators who have studied that positive conversion rate in lepromin test after

BCG inoculation in patients with Hansen's disease, in leprosy born children and in

normal children. These differences in positive conversion by various investigators

might have been due to the difference in host parasite relationship. Marked

difference was also noted in the findings of positive conversion rate in tuberculin

reaction after the BCG inoculation. This indicates that the variations of BCG

strain used, the preparation methods of the vaccine, and methods of the vaccine

storage are greately influenced by the workers.

In 1950, barber and his coworker found that the effect of BCG vaccine was very

unstable, but it became reliably constant when the vaccine made of human strain of

Mycobacterium tuberculosis which was irradiated with ultra-violet rays and could

secure positive conversion rate of the lepromin test.

Though much progress has been made in the method of preparation of BCG vaccine,

it is generally accepted that the liquid suspension of BCG is unstable and its

efficacy decreases as the duration of storage prolongs. The lyophilized BCG is

considerably more stable than the liquid suspension of BCG. however, this is also

rather far from out satisfaction.

In view of the above facts, it is highly desirable to have the vaccine which is

simple to prepare, high degree of stability in storage and high potency in

immunity.

The GIV-TB used in this experiment had been preserved more than one and half year

after manufacture prior to the injection. The result of the vaccine was fully

comparable to the results of many previous workers who employed lyophilized BCG.

This proves that the GIV-TB had high degree of stability and potency of immunity.

Even if the positive conversion rate of tuberculin test after GIV-TB inoculation

is of the same degree as the positive conversion rate after BCG inoculation, the

former should be interpreted as more significant as to its subsequent

hypersensitivity and resistance to human tuberculosis, because GIV-TB antigen is

prepared from pathogenic human strain itself, and BCG from alternated bovine

strain.

It is stressed that more studies will be made in the future on the prospect of

GIV-TB vaccine which is simple to prepare, and has higher stability and render more

effective resistance and hypersensitivity against human tuberculosis than the

presentday BCG vaccine.

[영문]

The use of reduced toxin or of killed bacteria as antigens for the prevention of many infectious diseases is widely accepted today. It is also well known that antigenicity of bacteria may be reduced or destroyed by heating, chemical changes,

or by the process of manufacture of the antigen so that the immunologic response to the prepared antigen is much less than the response to the use of living attenuated organisms or to an active infection.

The most desirable or effective antibody is one which will produce an immunity which is equal to that produced by living bacteria, but which will not produce any pathologic changes.

In an attempt to find a more ideal antigen against tuberculosis, the author used Gamma-irradiated(Co**60) Vaccine of Mycobacterium tuberculosis (H^^37 ,Rv) (GIV-TB).

This form of the Mycobacterium has lost its ability to multiply and also uses only a minimum of oxygen. The immunologic response to the irradiated vaccine was compared with that following the use of BCG in human hosts.

The Gamma-irradiated Vaccine of Mycobacterium tuberculosis used in this experiment was supplied by Dr. C. M. Carpenter the initiator of the vaccine, at the Department of Infectious Diseases, University of California, Los Angels, California.

The vaccine was used to human hosts after the thorough laboratory tests in vitro and in vivo.

Under aseptic conditions two antigens were prepared, the first in physiologic saline and the second in two per cent aluminum hydroxide aqueous solution. Both contained lyophilized GIV-TB which had been aseptically ground in a Agate motar and pestle. Suspensions, one mg per ml, of the two vaccines were kept under

refrigeration and used within two days after preparation. The control antigens were liquid suspensions of BCG produced by the National Institute of Health in Korea, and a lyophilized BCG from Japanese BCG research institute. Liquid suspensions of

the two control vaccines were also kept under refrigeration and used with고 five days after preparation. Tuberculin (1 : 2,000) which was used in this experiment was produced by the National Institute of Health in Korea, and the lepromin used in

this study was produced by this Department following the international standardization.

163 leprosy born children (non infected) and 128 patients with the lepromatous type of Hansen's disease were selected, and divided into two groups. These groups had negative reaction in tuberculin and lepromin tests. The first group received

the GIV-TB and second group received the BCG. After six weeks, these groups were also tested for their tuberculin and lepromin reactions.

128 patients with Hansen's disease and 50 leprosy born children were divided according to age and sex into three nearly equal groups. The first group received the suspension of GIV-TB in physiological saline, the second the suspension in 2%

aluminum hydroxide, and the third, the control group, received the saline suspension of BCG from the National Institute of Health in Korea.

As to the method of inoculation the GIV-TB was given on the surface of the left shoulder through 50 needle multiple skin punctures and the BCG was injected intradermally in a 0.1 ml dose. Six weeks after the above GIV-TB and BCG inoculations, the tuberculin skin reactions were tested.

Another 113 leprosy born children were divided according to age and sex into two approximately equal groups. The first group received a saline suspension of GIV-TB and the second group received a saline suspension of lyophilized BCG obtained from

Japan. Inoculation was given intradermally, after six weeks this group was also tests for their tuberculin and lepromin reactions.

After the inoculation of GIV-TB or BCG the following changes in the tuberculin and lepromin skin reactions (to positive conversion) were noted.

Out of 128 leprosy patients and 50 leprosy born children in So Rok island, positive conversion rates of tuberculin reactions were found in 70% of the group who received GIV-TB in physiological saline, in 60% of the group who received

aluminum hydroxide suspension, and in 48% of control group who received liquid suspension of BCG. Females showed a lower number of positive changes than males. No reason has been known for this sex difference. No one under four years of age showed a positive conversion. The rate of positive change was increased

progressively with age.

Thus, in summary, the group which received the GIV-TB showed higher positive conversion rate than the group received liquid suspension of BCG.

Positive conversion rate of lepromin reaction was lower than 40% in each groups.

It also showed that the leprosy patient group was slightly lower in the positive conversion rate than leprosy born children group.

In another 116 leprosy born children, positive conversion rate of tuberculin was about 61% in GIV-TB received group, and about 56% in Japanese lyophilized BCG received group.

The positive conversion rate of lepromin reaction was about 85% in each group.

In 1939 Fernandez first used BCG for a preventive inoculation against Hansen's disease. This was on the basis of his finding that previously negative reaction to tuberculin and lepromin test, became skin positive to both following BCG inoculation.

Many workers have proved that BCG might be used to increase the resistance to Hansen's disease. However, marked difference was noted in the findings of many investigators who have studied that positive conversion rate in lepromin test after BCG inoculation in patients with Hansen's disease, in leprosy born children and in normal children. These differences in positive conversion by various investigators might have been due to the difference in host parasite relationship. Marked difference was also noted in the findings of positive conversion rate in tuberculin

reaction after the BCG inoculation. This indicates that the variations of BCG strain used, the preparation methods of the vaccine, and methods of the vaccine storage are greately influenced by the workers.

In 1950, barber and his coworker found that the effect of BCG vaccine was very unstable, but it became reliably constant when the vaccine made of human strain of Mycobacterium tuberculosis which was irradiated with ultra-violet rays and could

secure positive conversion rate of the lepromin test.

Though much progress has been made in the method of preparation of BCG vaccine, it is generally accepted that the liquid suspension of BCG is unstable and its efficacy decreases as the duration of storage prolongs. The lyophilized BCG is considerably more stable than the liquid suspension of BCG. however, this is also rather far from out satisfaction.

In view of the above facts, it is highly desirable to have the vaccine which is simple to prepare, high degree of stability in storage and high potency in immunity.

The GIV-TB used in this experiment had been preserved more than one and half year after manufacture prior to the injection. The result of the vaccine was fully comparable to the results of many previous workers who employed lyophilized BCG.

This proves that the GIV-TB had high degree of stability and potency of immunity.

Even if the positive conversion rate of tuberculin test after GIV-TB inoculation is of the same degree as the positive conversion rate after BCG inoculation, the former should be interpreted as more significant as to its subsequent

hypersensitivity and resistance to human tuberculosis, because GIV-TB antigen is prepared from pathogenic human strain itself, and BCG from alternated bovine strain.

It is stressed that more studies will be made in the future on the prospect of GIV-TB vaccine which is simple to prepare, and has higher stability and render more effective resistance and hypersensitivity against human tuberculosis than the

presentday BCG vaccine.