Increased Resistance to Tuberculosis in Mice Sensitizedwith Non-Specific Antigen
Mikio KOMATSU, Fuminori KAWATA and Shusuke TSUJI
The Second and Fifth Division of the Tuberculosis Research Institute, Kyoto University(Director : Prof. S. TSUJI)

Although there have been lots of investigations with reference to themechanism of tuberculous immunity since the finding of the socalled Koch'sphenomenon in 1891, the true mechanism has not yet been clarified. Some
degree of acquired resistance is noted in the host which has been previously
infected with living bacilli or injected with killed bacilli. It has not been clearly
determined that this acquired resistance is directly connected with an actual
immunological process in view of the antigen-antibody reaction.
Althoughantibodies are proved in the serum of immune animals or humans in the formof precipitation or agglutination reactions, it has been almost definite that thesea~ibodies

have no direct connection with the defense mechanism of the host.
Therefore, many authors have thought that immunity to tuberculosis occurs in
the form of cellular defensive power. At this point, immunity is mixed upwith allergy in tuberculosis. As a matter of fact, however, our serial investigations concerning the role of humoral factors in native and acquired resistanceto tuberculosis have shown that there is certainly a humoral defensive power
stimulated by immunizing procedures and that by further analysis this stimulated
power to inhibit the growth of tubercle bacilli in vivo £I nd in vitro is closely
connected with the alteration of the A : G ratio in serum.
Recently Takeoka,ll one of our associates, has demonstrated that the serumof animals treated by injection of bovine serum or typhoid vaccine --immunologically non-specific antigens-- has a more powerful inhibiting effect on thegrowth of virulent tubercle bacilli in vitro than the control serum of non-treatedanimals, when tested by the ring method.

This investigation suggests that thedefensive power of body fluids of animals against tubercle bacilli may be
stimulated by a non-specific mechanism as well as by specific immunity. It may
be of great interest to examine whether or not animals treated by these nonspecific procedures can be more resistant to tuberculous infection than non-treatedcontrol animals.
Recently, Dr. Le6n2 ) in Mexico produced a new Synthetic tuberculous antigen
and kindly sent it to our laboratory, requesting our examination of its immuno-2Mikio KOMATSU. Fuminori KA WATA and Shusuke TSUJI

genicity. This antigen is composed of aluminum phosphate adsorbing polysaccaride extracted from BCG bacilli in combination with r-globulin obtained fromeither rabbits's serum immunized with BCG or the serum from tuberculous
patients. According to Leon. mice injected with this antigen into the tail vein
3 or 4 times at two week intervals could elongate their average survival timeagainst tuberculous infection to 200 days, while non-treated control mice diedin 124 days. He concluded that this demonstrated the apparent immunogenicityof this antigen.
The protecting activity of this specific antigen against tuberculous infection
was compared with that of the non-specific treatments described above.
MATERIALS AND METHODS

8M mice (about 1.5 months after birth) are divided into four groups, asfollows.
I
19 mice immunized with Leon's antigen
II 18 mice sensitized with typhoid vaccine
III: 19 mice sensitized with bovine serum

IV: 12 mice, non-treated controls
5 mice each from groups II and III were used to determine the antibody
titer in the serum.
3 of group III died during sensitization because of toxicity of bovine serum<?r anaphylaxis.
The outline of the treatment in each group before the challenge with tubercle
bacilli is shown in Fig. 1.
challenge

Leon's Antigen

O.3C.c.

0.3
2w

2",

2w

O.lc.c. 0.2

Typhoid Vaccine

4dBovine Serum•

0.3

O.Se.c. 0.54d0.24<10.54d0.24d0.54dTlidT0.54<12d9d2dControlFig. 1.

The outline of the treatment in each group before thechallenge with tubercle bacilli
T : The day on which the antibody titer was determinedIncreased Resistance to Tuberculosis in Mice Sensitized with Non-Specific Antig2n3In group I, 0.3 ml of Leons antigen was injected into the tail vein 2 times
at two week intervals, and 2 weeks after the last injection O.lml of a suspensionof tubercle bacilli was injected into the tail vein.
In group II, 0.2 ml of typhoid vaccine was injected into the tail vein on the1st, 5th, 9th and 13th days, and the challenging bacilli were injected intravenouslyon the 21st day.
In group III, 0.5 ml of bovine serum was injected intraperitoneally 5 times
at 4 days intervals, and 11 days after the last injection tubercle bacilli wereinoculated into the tail vein.
Group IV was an untreated control group.
The time of the start of these pretreatments were controled so that thechallenge of tubercle bacilli could be done in the same day in all groups.
Tubercle bacilli of the H37Rv strain were used. The bacillary membrane
culture::l on glycerine-bouillon for 2 weeks was made into a suspension of 1 mg/ml
concentration with physiological saline solution, and 0.1 ml of this suspension
was injected into the tail vein.
Determination of the antibody titer was carried out using the serum collected
and pooled from 5 mice by cutting the neck artery on the 6th day after the lastsensitization in group II and on the 9th day in group III.
The Widal reaction was performed in group II, and the precipitation reaction
using 100 fold diluted bovine serum in group III.
According to the description by Leon, Leon's vaccine was prepared as follows.
"A polysaccharide is extracted from M. tuberculosis var. bovis strain BCG, bysuspending 1 mg. of semidried living cells per 100 ml of steril distilled water;
the suspension is kept at 4--6°C during two weeks, centrifuged several times
to get rid of the bacteria and three volumes of ethanol plus 3% sodium acetate
are added to the supernatant.

A precipitate is obtained that is redissolv~d in

distilled water and precipitated in the same way three times when a purepolysaccharide is obtained. This polysaccharide has the properties of a specificincomplete antigen or hapten. It fixes the complement in the presence of rabbit
anti-BeG serum or sera from human cases of tuberculosis; but it is unable tostimulate anti-body formation when injected parenterally to laboratory animals.
This polysaccharide is immunologically combined in optimal proportions withimmune gamma globulin from anti-BGG rabbit serum, and the complex polysaccharide-gamma-globulin so obtained is addsorbed to particles of aluminium
phosphate by mixing, shaking and incubating at 3TC for 3 hours, equal partsof a dilution in steril distilled water of the complex polysaccharide-gammaglobulin, at a concentration of 1 : 20,000 of the polysaccharide, and 2 : 1000 ofaluminium phosphate. The complex so obtained now has the properties of a
specific complete antigen."4

Mikio KOMATSU, Fuminori KAWATA and Shusuk2 TSUJI

RESULTS(1)

Antibody Titer

In group II, a positive reaction was noted at 1 : 800 against typhoid, at 1 : 800-against para-typhoid A, and at 1 : 400 against para-typhoid B.

No other increasein antibody titer was noted. In addition, it was noted that in the non-treated·control mice, the Widal reaction was completely negative even at 1 : 25 concentration. Therefore, it may be reasonable to think that in mice the Widal titer
can not go very high because of the innate characteristics of this a.nimal species,
and that a titer as high as 800 may have sufficient significance in the sensitization with typhoid vaccine.
In group III, there was a 2 6 precipitin titer.

Antibody-titers in both groups

were thought to indicate that sensitizations had given to the animals a fairly
strong influence.
(2)

Survival
100r----\1""..,.-....,..----------.,
%i
L..,

90

\i\\the day when 50% of the deaths~'-'\70

occurred,ii' -~'--'-'-'-'-'-'-,
I li -'~~~ ~,\ L..._. ~ .._.... ,L

150

50J.

--------------\--:\i...,.--- •.•. - •• --.

i'l'yphoill Vaccine

10-.-.-... - ... -~1

groupII(Typhoid

pointed out, but a relative slight\Control

34.5 days,

rate, no distinct difference between group I and group IV can be~----

L_'_" lBovine 'Serumfollowing(Bovine serum) over 60 days.
From the pattern of survival

I\Leon's Antigenthevaccine) 46.5 days and group III\302li_\... __ .,tookorder: group IV (control) shortest(27 days), group I (Leon antigen)\1.. __ ,~40Fig. 2 shows the survival rate
in percentages in the ordinate
and the survival time in days in
the abscissa. It is evident thati80Survival Times

but well-defined prolongation oflife is seen in group II, and the10203U40GO6070 dayFig. 2. Survival rate and time in -micetreated with various antigens

most evident prolongation is ingroup III.

DISCUSSION

In order to determine the degree of immunogenic property using the-survival time of mice as indicator, attention must be paid to the technique and-conditions of the experiment.

It would be desirable to make the experimetal

Increas?d Resistance to Tuberculosis in M!ce S?nsitiz?2 with Non-Sp2dfic Antigen

5results accurate and significant to have a majority of the mice in a group diewithout large differences in survival time. For this purpose the selection of
the strain of animals and bacilli, the mode of inoculation of bacilli, and thecondition of the breeding of animals are important. In examining the effect ofa chemotherapeutic agent it has been generally thought to be con7enient forthe control animals to die within a relatively short period after being challenged
by bacilli, and the experimental results become inaccurate if average survival
period is very long. In Leon's experiments the average survival time of thecontrol group was 124 days and that of the immunized group 200 days. Theseexperimental conditions may not be adequate, for the reason described above,for determining the immunogenicity. We considered this point and planned tokeep the average survival time of control animals to under 50 days. As theresults a slight prolonging effect of Leon's antigen was recognized in the lengthof time it took for 50% to die. However, there was some lack of uniformity ofresults also in this experiment showing that more animals in the control groupremained alive in the late period of the experiment than in the immunizedgroup, and from the stand point of average number of days of survival there
was almost no difference between the two groups.
The following points may be considered as the cause of the lack of uniformity in this experiment.

The H37Rv strain used in this experiment was lessvirulent than the Kurono strain which is usually used for testing chemotherapeutic agents, and, in addition, ~,M mice were less susceptible than DD mice~so the average survival period was somewhat prolonged.
Anyway, we could not confirm the ability of Le5n's antigen to act as a
powerful antituberculous vaccine, contrary to Leon's opinion. However, it may
be that the vaccine changed in some of its propertie3 during transportationfrom Mexico to Japan.
It is of great interest that non-specific sensitization using bovine serum
or typhoid vaccine has given animals powerful and significant resistance to·tuberculosis.
Non-specific resistance brought about by infe~tion or vaccination with one
species of bacteria against other kinds of pathogen3 has become one of theinteresting problems in experimental biology.
Nukada 3 ) and his associates.
reported on many of their experiments in which the injection of bacillary
autolysates of one strain of bacteria (heterasate) is capable of protecting animals against infection by other strains of pathogens, including tubercle bacilli.
Dubos and ~,chaedler4) found that pretreatment with endotoxins of various origins.
including Salmonella typhosa influenced infections by various other strains ofbacteria including tubercle bacilli and acted as a protection if the infection was
not induced too early after the pretreatment. They,,)(j) also observed that the6

Mikio KOMATSU, Fuminor! KA WATA and ShusukJ. TSUJI

cellular components of mycobacteria acted either to protect against or to enhance infections of other sorts (staphylococuss aureus) according to the mode oftreatment. Elberg, Schneider and Fong 7 ) found that there was a sort of crossimmunity between Brucella melitensis and Mycobacterium tuberculosis. Fenner~llreported a similar observation between different strains of mycobacteria. In
short, it may become more important t~) search for the non-specific mechamismof the protective power of animals in natural resistance or immunity.
In our laboratory, 9) investigation of the role of the protein fractions of theserum revealed that non-specific change --reduction in the growth-promoting
power of the albumin fraction and a change in the A : G ratio (albumin decreases and globulin increases) - - might be more significant than a specificchange in the globulin fraction in the immunity to tuberculosis.
Recently,
Takeoka demonstrated that sensitization of rabbits with bovine serum or typhoidvaccine brought about in the protein fraction of the serum the same change ofeffect on growth of tubercle bacilli in vitro as was found in the serum of immune animals.
In this investigation, it has been clearly demonstrated that mice were also
influenced by these non-specific sensitizations so as to increase their resistanceto tuberculosis in vivo.
It appears from these results that the non-specific mechanism plays a more

important role than specific immunological change in tuberculous immunity, atleast in humoral immunity.
Further experiments using other specific vaccines (BCG or killed vaccine)
may be necessary in order to reach a conclusion.
SUMMARY

Mice (SM strain) were sensitized non-specifically with either bovine serum
or typhoid vaccine and the immunogenicity of these treatments to tuberculosis
was compared with that of a specfic antituberculous vaccine prepared by Dr.
Leon in Mexico, using the survival time of amimals as an indicator.
(1) Contrary to Le:Sn's opinion, the presence of a significant immunogenic
property could not be confirmed in Leon's vaccine.
(2) An apparent increase in resistance is observed in animals treated withbovine serum and to lesser extent with typhoid vaccine.
e3) A non-specific mechanism can be thought to play a very importantrole in addition to the specific immunological mechanisms in antituberculousimmunity.
Acknowledgment: We express thanks to Dr. Le:Sn for his kindness of sending us his vaccine.

Increased Resistance to Tuberculosis in Mice Sensitized with Non-Specific Antigen

7

REFERENCES
1)

Takeoka, A. : Japan. J. Tuberc., 6, 127, 1958.

2)

Leon, A. P. : Personal communication, 1958.

3)

Nukada, S., Kimura, T., Yasaki, Y. and Utsunomiya, S. : The Tokyo Journal of
Medical Sciences, 61, 183, 1953.

4)

Dubas, R. J. and Schaedler, R. W. : J. Exper. Med., 104, 53, 1956.

5)

Dubos, R. J. and Schaedler, R. W. : J. Exper. Med., 1e6, 703, 1957.

6)

Schaedler, R. W. and Dubas, R. J. : J. Exper. Med., 106, 719, 1957.

7)

Elberg, S. S., Schneider, P. and Fang, J. : J. Exper. Med., 106, 545, 1957.

8)

Fenner, F. : Am. Rev. Tuberc., 76, 76, 1957.

9)

Oshima, S., Fujita, Y., Takeoka, A., Nakajima, M. and Tsuji, S. : Am. Rev. Tuherc.,
78, 884, 1958.