CHEMO-IMMUNOLOGICAL STUDIES ON CONJUGATED
        CARBOHYDRATE-PROTEINS

V. THE IMMUNOLOGICAL SPECIFICITY OF AN ANTIGEN PREPARED BY
   COMBINING THE CAPSULAR POLYSACCHARIDE OF TYPE
      III PNEUMOCOC~US WITH FOREIGN PROTEIN

  BY OSWALD T. AVERY, M.D., AND WALTHER F. GOEBEL, PH.D.
   (Front the Hospital of Tke Rockefeller Institute for Medical Researck)

        (Received for publication, June 2, 1931)

The fundamental studies of Landsteiner and his coworkers (1) on
complex antigens have established the important principle that the
introduction into the protein molecule of a simple non-protein radical
confers a new immunological specificity on the antigenic compound.
Furthermore, this newly acquired specificity has been shown to depend
upon the nature of the new chemical grouping thus introduced.
Previous studies from this laboratory (2) on the chemo-immunologi-
cal properties of "synthetic antigens," prepared by combining a simple
carbohydrate radical with protein, have shown that the specificity
of the newly formed compounds is determined in each instance by the
chemical individuality of the reactive carbohydrate, irrespective of
the protein to which it is attached.  Simple derivatives of glucose
and galactose, which by themselves are non-antigenic will, when
coupled to a common protein, stimulate the formation of antibodies
that are specific for the particular sugar used. Antisera, produced
by immunization with the conjugated sugar-proteins, invariably re-
flect the controlling influence of the garbohydrate on the specificity
of the whole antigen.  So sensitive is this chemo-specific effect, that
mere differences in the spatial arrangement of the groups on a single
carbon atom in two glucosides otherwise identical, were found suf?ici-
ent to change completely the antigenic specificity of the respective
compounds.
In order to test the possibility of synthesizing a specific antigen by
                   , -     437


438       CONJUGATED CARBOHYDRATE-PROTEINS. V

combining a bacterial carbohydrate with a foreign protein, the specific
capsular polysaccharide of Type III Pneumococcus was purposely
chosen, since in its purified form it contains no nitrogen and may be
regarded as a definite chemical entity.  Further, if results were ob-
tained by the use of this particular bacterial polysaccharide, they
would be the more significant, since the isolated pure substance alone
has never been found to elicit antibodies in rabbits, and even the in-
tact bacteria1 cells from which it is derived commonly fail to incite
the formation of type-specific antibodies in these animals.

From a chemical <point of view, the difficulty lay in synthesizing
the appropriate derivative of the capsular polysaccharide.  It must
be one capable of coupling with protein on diazotization and one in
which the chemo-specific groups of the bacterial sugar are not masked
or destroyed in its preparation.  The details of the chemical methods
used in the synthesis of the aminobenzyl ether of the capsular poly-
saccharide of Type III Pneumococcus have been described in the pre-
ceding paper (3). The diazonium derivative of the polysaccharide
was coupled with globulin prepared from horse serum, and the newly
formed compound was tested for antigenicity by repeated intravenous
injection in rabbits.  The sera of the immunized animals were tested
for the presence of type-specific agglutinins and protective antibodies
against encapsulated, virulent strains of Type III Pneumococcus and
for precipitins against the type-specific capsular polysaccharide and
its aminobenzyl derivative.  The treated rabbits were subsequently
infected with a rabbit-virulent strain of Type III Pneumococcus to
determine whether, as a result of immunization, they had acquired
active immunity against Type III infection.

In evaluating the immunological findings presented in this paper,
the fact should be borne in mind that the antigen used in the experi-
ments has in common with the Type III Pneumococcus only the spe-
cific capsular polysaccharide, and that the protein with which it is con-
jugated is of widely remote biological origin.

                      EXPERIMENTAL

                 Mefhods
Immu~zization.-Rabbits were immunized by the intravenous injection of from
1 to 2 cc. of solutions of the conjugated carbohydrate-protein antigen daily for


OSWALD T. AVERY AND WALTHER F. GOEBEL     439

six doses, and the course of injections was repeated a second time after a rest pe-
riod of 7 days.  8 days after the last injection the rabbits were bled and the serum
tested for type-specific pneumococcus antibodies.

Antigen.-The antigen, prepared as described in the preceding paper, was pre-
served by the addition of 0.25 per cent tricresol.  Each preparation was standard-
ized on the basis of nitrogen content, SO that 1 CC. of the solution contained 5 mg.
of protein.  This method, however, does not indicate the amount of bound car-
bohydrate and hence is not an accurate measure of the effective antigenic com-
plex. The protein, to which the aminobenzyl ether derivative of Type III poly-
sac&ride was bound by the diazo reaction, was globulin prepared from horse
serum by precipitation with ammonium sulfate.

Technic of Immunity Reactions.-In the precipitin reactions, the immune serum
was diluted @ the proportion of two parts of serum to three of salt solution and
0.5 cc. of this dilution containing 0.2 cc. of original serum was added to 0.5 cc. of
varying dilutions of reacting substance.  The final concentration of precipitino-
gen in the reaction mixture is shown in the protocols. The protection tests were
done by the usual technic; mice were injected intraperitoneally with 0.2 cc. im-
mune serum together with varying dilutions of an actively growing culture of
Pneumococcus.  The dilutions were so made that the total volume injected was
1 cc. in all instances.

I. Type-Specijc Antipneumococcus Antibodies

1. Precipitk-The sera of rabbits immunized with the Type III
"synthetic antigen" were tested for the presence of precipitins for the
original capsular polysaccharide and for the aminobenzyl ether deriv-
ative used in preparing the antigen.  The introduction of the para-
aminobenzyl radical into the polysaccharide molecule did not destroy
the immunological specificity of the derivative, since the latter reacted
with Type III antipneumococcus horse serum in high dilutions,
showing a specific reactivity comparable to that of the original poly-
saccharide from which it was derived.  It was of interest, therefore, to
determine whether this specific carbohydrate derivative, when bound
with protein, would stimulate the formation of precipitins reactive
not only with the amino derivative but also with the polysaccharide
itself, when both substances were used in the free, soluble form.

The precipitin reactions of immune rabbit serum with the original
and modified form of the Type III capsular polysaccharide are com-
pared in Table I.  The results show that the serum of a rabbit im-
munized with the "Synthetic antigen" reacts with the native polysac-
charide and with its aminobenzyl ethet in equalIy high dilutions.


440      CONJUGATED CARBOHYDRATE-PROTEINS. V

Similarly, as previously pointed out, the serum of a horse immunized
with the intact bacterial cells containing the natural Type III antigen
precipitates the polysaccharide derivative as well as it does the poly-
saccharide itself. The evidence is, therefore, that. the specifically
reactive groups of the polysaccharide have not been chemically masked
in forming the new derivative, and that when this specifically reactive
carbohydrate is chemically bound with protein to form a new antigen,
it orients the immune response as specifically as does the polysaccha-
ride itself in the form in which it exists as a natural antigen in the en-
capsulated cells.

TABLE I

Precipitins for the Capsdar Polysaccharide oj Type III Pneumococcus ilz Serum
     oj Rabbits Immunized with Type III Synthetic Antigen

Dilution of carbohydrate

Purified preparation of Type III
    polysaccharide      Aminobenzyl ether of Type III
                    pc+saccbaride

1: 100,ooo           ++=t             +++
1:2oo,ooo           +++             +++
1:400,000            ++*             +++
1:8oo,ooo            +*              +
1:1,6oo,ooo            +                   f

+ + + = complete precipitation with compact disc.
++ - diffuse turbidity.
+ = slight turbidity.

2. Agglutinins.-The sera of rabbits, prepared by serial injections
of the chemically combined antigen, specifically agglutinate encapsu-
lated strains of Type III pneumococci.

The results of the agglutination reactions, as illustrated in Table
II, show that while the titre of agglutinins is not high, the serum is
type-specific in that it agglutinates only Type III pneumococci, and
fails to react with organisms of Type I and Type II.  The fact that
the irmnune serum does not agglutinate the non-encapsulated R forms
of Pneumococcus was to be expected, since the antigen contains none
of the somatic constituents commonly present in all pneumococci.
The lack of these cellular substances in the artificial antigen is reflected
in the serum by the absence of the species-specific antibodies, which


        OSWALD T. AVERY AND WALTHER F. GOEBEL      441

are ordinarily present when the whole cell is used as the immunizing
agent.

TABLE II

Specificity of Agglutination Reaction with Serum of Rabbit Immunized with Type
              III Synthetic Antigen

                                    Immune serum
      PllcUmOC~US
                         1:s        1:lO        1:zo

TypeI....................:. -
TypeII..................... -         -         -
TypeIII....................  +++-F    +++      +
"R" strain*                  -                  -
       . . . . . . . . . . . . . . . . . .

+ + + + = complete agglutination with disc formation.
- = no reaction.
* Derived from Pneumococcus Type II.

1:40





f

TABLE III

Protective Action in Mice of Sera of Rabbits Immunized with Type III Synthetic
                 A?l.tigert

PW3UlXXOCCUS
Type III        (a)

   cc.
0.01          - -
0.001         - -
O.oool       s s
O.ocQol       s s
O.oooool      s s
O.ooooool - -
O.ooooooOl - -

S = survival.

Immune rabbit sera (0.2 cc.,

(b)        Cc)

- -   D 22 D22
- -     s s
s s     s s
s s     s s
s s     s s
- -      - -
- -      - -

Cd)

D7 D24
s s
s s
`S s
s s
- -

- -

Virulence
contmls
No serum

-

-

   -

D 42  D 42
D 42  D 4.5
D 75  D 45

D = death of animal, the numeral indicates the number of hours elapsing before
death.
- = not done.

3. Protective Antibodies.--To ascertain whether passive immunity
against pneumococcus infection could be conferred on mice by the
injection of the serum of a rabbit immunized with the artificial Type


442      CONJUGATED CARBOHYDRATE-PROTEINS. V

III antigen, protection tests were carried out by the technic described.
A constant amount of immune serum (0.2 cc.) was injected intraperi-
toneally into mice together with varying quantities of a virulent cul-
ture of Pneumococcus.

The results given in Table III show that the immune rabbit sera
were effective in protecting mice against 1O-J cc. of a virulent culture

TABLE IV

Type Specifiity of Protective Antibodies in the Sera of Rabbits Immunized with
            Type III Synthetic Antigen

Immuue serum     Amount of culture

cc.            cc.

0.2          10-a
0.2           lo-'
0.2          10-b
0.2          10-s

Viiulence controls

10-6
lo-'
10-n

   -          -
D 28 D 28   D 28 D 28
D% D28   D28 D%
D28 D%   D 28 D 28


  D%       D 28
  D 72       D%
  St      D Wt

Type III

s s
s s
s s
s s

D%
D%
D Wtt

S = survival.

D = death of animal; the numeral indicates the number of hours elapsing
before death.
- = not done.
7 The number of colonies developing in blood agar seeded with this inoculum
= 0.

tt The number of colonies developing in blood agar seeded with this inoculum
= 3.

ttt The number of colonies developing in blood agar seeded with this inocuhun
= 2.

of Type III Pneumococcus, of which lo-* cc. was invariably fatal
when injected alone into untreated control animals. Although the
maximum degree of protection afforded by the serum was not high,
the special interest of these experiments lies in the fact that the pro-
tective antibodies were produced in response to a combined- antigen
containing a specifically reactive carbohydrate which by itself fails
to evoke any immune response in rabbits.

,


OSWALD T. AVERY AND WALTHER F. GOEBEL      443

The specificity of the protective antibodies in the sera of rabbits
immunized with the Type III synthetic antigen is shown in Table IV.

The protective action of the immune serum is strictly type-specific.
Mice receiving the immune serum survived infection with Type III
Pneumococcus, but succumbed promptly to minimum doses of Type
I or Type II culture.  The marked specificity of the protection af-
forded by the serum is further evidence that the antibodies concerned
in the reaction derive their specificity from the polysaccharide com-
ponent of the whole antigen.  The protein constituent of the antigen
appears to contribute nothing to the antibacterial properties of the
serum, except in so far as it renders the carbohydrate antigenic.

4. Active Immunity.-Of the series of rabbits injected at various
times with effective preparations of antigen, none have failed to
respond with the formation of type-specific antibodies. It was of
interest, therefore, to ascertain whether rabbits in the sera of which
Type III Pneumococcus antibodies were demonstrable had acquired
active immunity against infection with this organism.  In order to
determine this point, several of the treated animals were subsequently
infected with multiple lethal doses of a virulent culture of Type III
Pneumococcus.

11 days after the last injection of autigen, four immunized rabbits whose serum
contained type-specific antibodies were infected by the iutradennal injection of
0.2 cc. of an 18 hour blood broth culture of a rabbit-virulent strain of Type III
Pneumococcus. The virulence of this strain, maintained by repeated rabbit
passage, was such that 0.00001 cc. of the culture alone injected into the skiu proved
fatal witbin 72 hours.



Four rabbits, which had received repeated injections of the "synthetic
antigen," were reserved and later tested for active immunity. All
four of these rabbits survived infection.  In the two animals which
had the highest titre of type-specific antibodies in their serum, the
infection ran a practically afebrile course with only a slight inflam-
matory reaction at the point of inoculation; in the other two rabbits,
the temperature remained- relatively low and the skin lesion was not
severe.  At no time during the course of the infection were pneumo-
cocci present in cnltnres of the blood of the immunized rabbits,
whereas in the normal controls the bacteremia invariably increased
up to the time of death.


444       CONJUGATED CARBOHYDRATE-PROTEINS. V

The results demonstrate that an antigen prepared by combining
the Type III capsular polysaccharide with an animal protein is cap-
able of inducing in rabbits an active immunity against infection with
a virulent culture of Type III Pneumococcus.  The only constituent
of Pneumococcus in the artificial antigen is the capsular polysaccha-
ride.  Since this carbohydrate alone is non-antigenic in rabbits, the
antibacterial immunity induced by the combined antigen can be
ascribed only to the antigenicity acquired by the polysaccharide
through combination with the foreign protein. The results bring
evidence that an effective, active immunity can be developed in which
the only antibacterial antibodies formed are those directed against
the capsular component of the pneumococcus.

II. Antiprotein Antibodies

In the conjugated antigen, the protein used in combination with the
Type III capsular polysaccharide was globulin derived from normal
horse serum.  The total protein of the antigen was estimated by de-
termining the nitrogen content of the various preparations. This
method of standardization, while affording a measure of the total
globulin present, does not indicate the amount of protein that is bound
with the polysaccharide to form the effective type-specific antigen.
The sera of immune rabbits invariably contained not only type-
specific antibodies for Type III Pneumococcus, but also specific
precipitins for the globulin of horse serum.

The presence in immune rabbit serum of precipitating antibodies
for the foreign protein used in preparing the antigen is shown in
Table V. The antiprotein precipitins evoked by the combined anti-
gen may be attributed to one or both of two possibilities.  In the first
place, despite attempts to remove the excess of unbound globulin
from solution, it is possible that sufficient protein remains free and
uncombined with the polysaccharide to function independently as
antigen.  It is also possible, however, that even in the absence of any
free globulin, the combined antigen itself may give rise to two quali-
tatively different antibodies, each specifically related to the corre-
sponding component of the antigenic complex.  This explanation of
the concurrence of two distinct varieties of antibodies in the im-
mune serum involves the assumption, that while the carbohydrate


OSWALD T. AVERY AND WALTHER F. GOEBEL      445

has acquired antigenicity through combination with the globulin, the
protein molecule itself has not lost its own specific antigenic property.
According to this view, a single compound of carbohydrate and pro-
tein may possess the dual Sntigenic property of stimulating two
separate and specific antibodies; one evoked by the newly acquired
antigenicity of the polysaccharide in union with globulin, and the other
stimulated by the protein molecule itself in which the chemo-specific
groups essential for antigenicity have not been masked in the linkage
with the carbohydrate.

TABLE V

Antiglobulin Precipitins in Sera of Rabbits Immunized with Ty#e III Synthetic
                 Antigen

                        Globulin (horse serum)*
Immune sera
           1:20,000        1:4o,lwo        1:8O,ooo       1: 160,000
II;     +++ `,    +-f-+       ++           f
         ++-t*      +-I-+       ++        +
g;      ++++      +++*       +*          f
         ++++      +++        +A         i-
  (4      ++++      ++4        +           f

++++ = flocculent precipitation, clear supematant.
+ = slight, diffuse turbidity.
* The diazonium derivative of the capsular polysaccharide of Type III Pneu-
mococcus was coupled with globulin from horse serum.

Of the two explanations, the first, namely that the free globulin in
the antigenic mixture accounts for the antiprotein antibodies, is the
simpler and perhaps the more likely one.  However, the second con-
cept, which ascribes a dual antigenicity to the sugar-protein, affords
an immunologically interesting and theoretically possible explanation.

DISCUSSION

In the present state of our knowledge it would be hazardous to pre-
dict the precise. conditions under which complex carbohydrates by
themselves may function as antigens. The present study simply
def%es certain experimental conditions under which the capsular
polysaccharide of Type III Pneumococcus, in chemical union with a


446       CONJUGATED CARBOHYDRATE-PROTEINS. V

foreign protein, is rendered specifically antigenic in a particular species
of animals, in which the carbohydrate alone has never been found to
incite antibody formation.  The `%ynthetic antigen" elicits in rabbits
a type-specific antipneumococcus response, which neither one of its
constituents alone is capable of inciting when injected singly into
these animals.

In order to effect the chemical union, it was first necessary to syn-
thesize a derivative of the bacterial carbohydrate that could be diazo-
tized and coupled to protein by means of the diazo linkage.  This
was accomplished by forming the aminobenzyl ether of the polysac-
charide, a derivative which fulfills the chemical requirements while
still retaining the serological specificity of the original carbohydrate.

Antisera prepared by immunization .with the combined antigen con-
tain type-specific antibodies which precipitate the Type III capsular
polysaccharide, agglutinate pneumococci of the homologous type,
and protect mice against infection with virulent strains of Type III
Pneumococcus.  The fact that, in the present instance, the antipneu-
mococcus response is produced by an antigen known to contain but a
single component of the bacterial cell, indicates the unity of the anti-
bodies participating in the type-specific reactions of precipitation, ag-
glutination, and protection, and relates the specificity of these anti-
bodies to that of the capsular polysaccharide in the reactive part of
the antigenic molecule.

CONCLUSIONS

1. Type-specific antipneumococcus immunity has been induced in
rabbits by immunization with antigen prepared by combining a specific
derivative of the capsular polysaccharide of Type III Pneumococcus
with globulin from horse serum.

2. Rabbits immunized with this antigen acquire active immunity
against infection with virulent Type III pneumococci.
3. The sera of the immune rabbits contain type-specific antibodies
which precipitate the Type III capsular polysaccharide, agglutinate
Type III pneumococci, and specifically protect mice against Type III
infection.

4. The experimental data are discussed with reference to: (1) the
concurrence in the immune sera of type-specific antibodies for Pneu-


OSWALD T. AVERY AND WALTHER F. GOEBEL      4.47

mococcus and precipitins for horse globulin; (2) the determining influ-
ence of the capsular polysaccharide on the specificity of the antigen
as a whole; (3) the unity of the. type-specific precipitins, agglutinins,
and protective antibodies induced by a single component. of the pneu-
mococcus in chemical union with an unrelated, animal protein,

BIBLIOGRAPHY

1. Landsteiner, K., and Lampl. H., Biochem. Z., 1918,86, 343.
Landsteiner, K., Bimhem. Z., 1919,93,106; 1920,104,280.
Landsteiner, K., and van der Scheer, J., J. Exp. Med., 1927,45, 1045; 1928,
  48,315; 1929,50,407.

2. Goebel, W. F., and Avery, 0. T., J. Ezp. Med., 1929,50,521.
Avery 0. T., and Goebel, W. F., J. Exp. Med., 1929,50, 533.
Tillett, W. S., Avery, 0. T., and Goebel, W. F., J. Exp. Med., 1929,50, 551.
3. Goebel, W. F., and Avery, 0. T., J. Exp. Med., 1931,54,431.