IMMUNOLOGICAL RELATIONSHIPS OF CELL CONSTITU-
         ENTS OF PNEUMOCOCCUS.

SECOND PAPER.

BY OSWALD T. .4VERY, M.D., AND MICHAEL HEIDELBERGER, PH.D.
(From the Hosfiital of The Rockjeller Institute for Medical Research.)

(Received for publication, June 8, 1925.)

In the course of studies carried on in this laboratory during the past
3 years on the chemical and immunological properties of the cell con-
stituents of Pneumococcus, certain facts have been acquired which
indicate a significant relationship between the chemical constitution
and biological specificity of the bacterial celLIn  It seems desirable at
this time to bring together in summary form these facts in order to
relate them to the problem as a whole.

In the work thus far, two of the cellular constituents of Pneumococ-
cus have been studied. One of these is the substance precipitated
from solutions of Pneumococcus by dilute acetic acid, which, although
comprising a mixture of proteins, may for the purposes of the present
discussion be referred to as bacterial nudeoprotein.  The other con-
stituent, non-protein in character, is the so called soluble specXc
substance, which is now considered to be a carbohydrate of the poly-
saccharide type. While it is realized that these two substances,
important as they are, do not comprise the whole antigenic mosaic of
the cell, they are emphasized at this time because they happen to be
the first chosen for investigation, and the only ones thus far isolated.
Studies of the antigenic and serological. properties of these two
chemically diverse substances as they exist together in the intact cell
and free in a dissociated state, have yielded certain facts which con-
tribute to a better understanding of the differential specificity of the

1 For the first paper see Avery, 0. T., and Heidelberger, M., I. Exp. Med., 1923,
xxxviii, 81.

* Cj. in part the two preceding papers, also the chemical studies from this
laboratory referred to in these.
                      367


368       CELL CONSTITUENTS OF PNEUMOCOCCUS. II

various types of pneumococci.  For not only do these cellular com-
pounds belong to two wholly different classes of chemical substances
namely protein and carbohydrate, but the carbohydrate constituent
of each of the three tied types of pneumococci has been found to be
a chemically different polysaccharide. These substances possess
the unique distinction of reacting specifically with antibacterial serum
of the homologous type, although in the free state, dissociated from
the cell, they are devoid of the power of inciting the formation of anti-
bodies upon injection into animals.  The protein on the other hand,
is antigenic; the serum of an animal immunized with this substance
reacts with protein derived from any type of Pneumococcus.  It at
once becomes obvious that as these two components exist in the cell,
they enter into an antigenic complex which has different fmmunologi-
cal properties from those exhibited by either substance alone.  The
serological differentiation of the various types of pneumococcf is
dependent, therefore, not only on the chemical and antigenic differ-
ences in these component substances, but is also intimately related to
the structural character and morphological integrity of the bacterial
cell.

That these principles, so strikingly exemplified in the immunological
reactions of Pneumococcus, are of more general biological signiticance
is illustrated in the somewhat analogous relationships existing within
other groups of bacteria and of yeasts.  The work of Zinsser, Mueller,
and their associates (1) indicates that from a number of microorgan-
isms, such as staphylococci, meningococci, and Friedhinder, typhoid,
and tubercle bacilli, a non-protein residue substance may be extracted
which bears a definite relation to the specific character of the bacteria-
cell.  Smith and Reagh (2) in 1903 described ditferences in the serol
logical behavior of motile and non-motile bacilli of the hog-cholera
group. Recently Orcutt has shown that these differences are depend-
ent upon two separate agglutinogens corresponding to the ffagellar
and somatic substances of the cell; the presence of both of these fat-,
tors in the motile forms and the absence of the flagellar antigen in
non-motile types account for the variations observed in the immuno-
logical behavior of these bacilli.  Felix (3) and others have found that
the differences in agglutination of the 0 and H forms of ~WO~~US bacil-
lus are related to the occurrence in these organisms of one or both of


OSWALD T. AVERY AND MICHAEL HEIDELBERGEB     369

two separate substances, involving the endo- and ectoplasm of the
cell.  These analogies while close are not absolute; the ffagelar sub-
stance of motile bacilli is in itself antigenic, while the soluble specific
substance of Pneumococcus is a non-antigenic carbohydrate. The
significant fact, however, is that in these widely divergent groups of
microorganisms two distinct cellular substances are determinative
factors in bacterial specificity.

Structure of I& Cell.-Before considering in detail the immunoIogical
characters of these two cellular constituents of Pneumococcus it may
add to the clearness of the discussion to picture the form or pattern
of the cell as it relates to the disposition of these substances.  For
undoubtedly cell configuration refiects in some measure the ease with
which this organism participates in immunity reactions and the avidity
with which it interacts with antibody.  Many of these reactions are
presumably surface phenomena, and the nature of the reactive material
at the periphery of the cell may determine the readiness of response
and even the specificity of reaction.  Pneumococcus is an encapsulated
organism, and there are grounds for the belief that the ectoplasmic
layer of the cell is composed of carbohydrate material which is identi-
cal in all its biological characters with the type-specific substance of
Pneumococcus. On the other hand, the endoplasm, or somatic
substance, consists largely of protein which, as previously pointed out,
is species- and not type-specific.  This protein is possessed in common
by all pneumococci while the carbohydrate is chemically distinct and
serologically specific for each of the three fixed types. The cell, there-
fore, may be conceived of as so constituted that there is disposed at its
periphery a highly reactive substance upon which type specificity
depends.  The structure and, as will be pointed out later, the morpho-
logical integrity of the cell are determinative factors in bacterial
specificity.

Chemistry of the Soluble Specific Substance.-The following data
briefly summarize our knowledge of the soluble specific substance of
the three fixed types of Pneumococcus at the stage.of purification so
far attained (4).

The soluble specific substance of Type II pneumococcus appears to
be a weakly acidic, nitrogen-free polysaccharide made up chiefly of
glucose units. Its specific optical rotation is about P/US 74". It


370       CELL CONSTITUENTS OF PNETJMOCOCCUS. II

reacts at a dilution of 1:5,000,000 with antibacterial serum of Type II
pneumococcus, and does not react with Type I or Type III antisera.
The soluble specific substance of Type III pneumococcus, while
also apparently a nitrogen-free polysaccharide, differs from the Type
II derivative in many particulars.  It rotates the plane of polarized
light about 33" to the Zeft. It is a strMtg acid and is made up of
units of glucose and either glucuronic acid or some derivative of this
acid. It also separates in insoluble form from solutions strongly
acidified with hydrochloric acid. In as high a dilution as 1: 6,000,OOO
it still reacts with Type III antipneumococcus serum.

TABLE I.

Chemical Characteristics of the Soluble Specifc Substances oj Ty#es I, II, ad III
                   P~UTFWCOCCUS.

Type.

I


II
III

                  ..i
            1                  Highqtpilution
OPti-1 c H N g
rotation.                Reducing sugars on bydmlpir.  preci~?tith
                 rr.;                      homologou
          ze                       immuneserum.
__----
           N$
+300o43.3* 5.8 5.0t    28 (Galacturonic acid.) (Am- 1:6,000,000
                 ino sugar derivative.)

   +74"45.8 6.4 0.0 1250 70     Glucose.       1:5,ooo,ooo
   -33O42.6 5.6 0.0  340 75 Glucose (glucuronic acid). 1:6,000,000

* Theory for (C&&~O&: C = 44.4 per cent; H = 6.2 per cent.
t Amino N: 2.5 per cent.

The Type I soluble specitic substance (4), on the other hand, while
also polysaccharide in nature, differs from the other two type-specific
substances in containing nitrogen as an apparently essential compo-
nent. In spite of a nitrogen content of 5.0 per cent the substance
gives none of the usual protein colgr tests.  One-half of the nitrogen is
liberated when the substance is treated with nitrous acid.  Reducing
sugars appear at the same time and the specific reaction vanishes, and
since the carbon and hydrogen content are close to the theoretical
values for polysaccharides it appears likely that a nitrogenous sugar
derivative is involved. It is a strong acid and a weak base, and is very
sparingly soluble in water at the isoelectric point.  Its specific opti-
cal rotation is +300", and on oxidation with nitric acid it yields mucic


OSWALD T. AVERY AND MICHAEL BEIDELBERGER    371

acid. In the specific precipitin reaction with homologous antipneu-
mococcus serum it can be detected in dilutions as great as 1: 6,000,000.

In Table I are summarized the available data concerning the chemi-
cal differences in the polysaccharide derivatives of pneumococci of
Types I, II, and III.

Although the specific polysaccharide by itself evokes no antibodies
upon injection into rabbits it is specifically reactive with antibody
induced by immunization with intact cells.  The fact that this specifi-
cally reactive carbohydrate is non-antigenic when dissociated from
the other cellular constituents and is capable of inciting antibody
formation only in the form in which it is present in the intact cell,
forces the conclusion that in the latter instance it exists not merely
as free carbohydrate but also in combination with some other sub-
stance which confers upon it specitic antigenic properties.  Immuniza-
tion with intact bacteria containing this carbohydrate complex elicits
antibodies which not only agglutinate the formed cells but precipitate
solutions of the carbohydrate isolated from pneumococci of the homol-
ogous type. How the specific polysaccharide is combined in the cell,
whether with protein or some other constituent is not yet clear, but
it is evident that the compound thus formed is the dominant and essen-
tial antigen of the cell, and the one responsible for type specificity.

The immunological relationships of the protein and carbohydrate
fractions of the cell are graphically presented in Fig. 1, in which S
represents the specific soluble substance (carbohydrate) and P the
protein of the Pneumococcus. The symbols used are in no sense to
be construed as interpretive of the mechanism involved, but serve
simply to visualize the interaction between these cell constituents
and their respective antibodies.  The reactions illustrated may be
briefly summarized as follows:

A (Fig. 1, A).-Immunization with intact. cells of a given type of
Pneumococcus gives rise in the serum to the presence of antibodies
which are type-specific; such sera specifica% agglutinate the homolo-
gous type of Pneumococcus, protect m&e against virulent organisms,
of the same type, and precipitate solutions of the corresponding puri-
fied carbohydrate (Table II). While'this type-specific carbohydrate
antibody is provoked only in response to the antigenic stimulus of the
whole cell, a serum containing this antibody alone without a trace of


          A. Intact ceil [PS]
        21      Atii-3 sQr=um Type I specific
`?lW    g ""..P"

.  m p-xt(xjj    g-J ,,"""H"

B. Isolated constituetitts -OF celi [PI, IS]

Ppatein Ip<

cl

Anti-P aerum,not +qpe-qecific

(s, -

Caracmydrvse (sa
    Ga

     No antihdy veeponsr.
-

C. Solutions of pneumococci

in wbicn complete Qi33ociation of pPotein and cwbc~~te cccws [p].[S]

a

AnttP3eeum,not typespecifk

  D. SuSp6rmons of pneumococci
containmQ intact cell8 and dissociated cell
   constituents [PSI [Pl.Is]

Anti-S Type I speafic
o P not type m

Anti-5 Type P specific
- Pm? type *

I Jp,g--j    Am-SType BL epecifio
I, 43    cl    `1) P not type .
   @ -
FIG. 1. Immunological relationships of protein and soluble specific sub-
stance (carbohydrate) of Pneumococcus.
                      372


OSWALD T. AVERY AND MICFUEL IIEIDELBEBGER    373

protein antibody is rarely obtained for reasons which will be discussed
later. Whether singly or in conjunction with other antibodies this
specific carbohydrate antibody has been observed only under con-
ditions in which the intact bacterial cell has been employed for
immunization.

B (Fig. 1, B).-The specific polysaccharide (S) when isolated from
the cell is incapable of inciting antibody formation upon injection into

TABLE II.

   Protein
   pa         P --t-+-t

Soluliona, exlmcts
  Containir$      P - - t - + - t
fl%eSandfreeP
Suspension of intact

teSP]4k%ohydrate. and protein combined
  aotigzn of cetl
; S -Eke caFbohydrate,the soluble specifi
  substhnce of ceU
fP - rpee protein of cell

*E'mx 5, as ant$p, does not fix
complement wit immune horse
serum; is active with immune
rabbit SerUm"

animals.  The isolated protein (P), on the other hand, is antigenic
and gives rise to an immune serum which reacts with pneumococcus
protein regardless of the type from +ich it is derived.  Antiprotein
sera do not agglutinate type-specific strains of pneumococci and do not
precipitate solutions of the soluble specific substance (3) (Table II).
C (Fig. 1, C).-Solutions and extracts of pneumococci behave anti-
genitally precisely as do solutions of pneumococcus protein; the disso-

8 Cf. VoUmond, E. (in press).


374       CELL CONSTITUENTS OF PNEUMOCOCCUS. II

ciation of the antigenic complex which occurs whenever the cell is
dissolved results in the liberation of free carbohydrate (S) and free
protein (P) in solution.  In such solutions and extracts the only
constituent which functions as antigen is the protein, for free S is
non-antigenic. The sera of animals immunized with solutions of
pneumococci, in which complete dissociation of these cell constituents
has taken place, contain only antibodies reactive with the protein.
Such sera exhibit the same reactions as do those prepared by immuniza-
tion with protein alone (Table II).

It is evident from these facts that morphological dissolution of pneu-
mococci is accompanied by antigenic dissociation, for sera prepared
from filtered solutions of disintegrated cells free of formed elements
fail to exhibit any of the dominant type-specific properties which
characterize sera obtained by immunization with whole bacteria (com-
pare A and C, Fig. 1). Morphological integrity of the bacterial
cell, therefore, is requisite for the expression of its full antigenic power.

D (Fig. I, @.-It becomes obvious, therefore, that the character of
the antibody response is determined by the nature of the cell material
used for immunization. The injection of suspensions of pneumo-
cocci into animals induces the formation of antibodies against S alone
or against both S and P separately, depending upon whether or not
these suspensions contain only intact cells or a mixture of both intact
and dissolved cell bodies.  Since pneumococci readily undergo autoly-
sis and dissolution, suspensions and indeed cultures of these organisms
almost invariably contain not only formed elements, but also more or
less of dissociated cell constituents in solution.  The predominance of
the former in such suspensions stimulates the production of the type
specific S antibodies, while the occurrence in these same suspensions of
dissociated cell protein provokes the formation of the species-specific
protein antibodies.  The more common result of immunization with
cell suspensions is the occurrence in the serum of both the carbohydrate
and protein antibodies.  The former invariably predominate, the
latter are present in varying concentration depending upon the
amount of cell dissolution which has taken place in the material pre-
vious to or after injection into the animal body.  Therefore, use of
suspensions of pneumococci containing both intact cells and the soluble
products of cell disintegration yields on immunization not only type-


OSWALD T. AVERY AND YICFNEL HFJDELBERGER    37.5

spec&c antibodies but antibodies reacting with the protein substance
which is common to all pneumococci.  It is the presence of this protein
antibody with its broader zone of activity which is responsible for the
confusing cross-immunity reactions occasionally encountered in sup-
posedly type-specific sera.  That these two antibodies are separate
and distinct is shown by absorption tests; the antiprotein reacting
bodies in such sera can be removed by absorption with the protein of a
heterologous type without diminishing the titer of specific agglutinins
for the homologous culture or the precipitins for the specitic polysac-
charide of the corresponding type.

The relationship of the carbohydrate constituent of Pneumococcus
to type specificity, and the establishment of its identity with the
soluble specsc substance previously discovered (5) in culture fluids
and in the urine and blood of patients suffering from pneumococcus
pneumonia are facts indicative of the biological importance of this
substance both to the bacterial and animal organisms.

The elaboration of this specific carbohydrate is a specialized func-
tion most active in pneumococci rapidly multiplying in the animal
body or in suitable culture media.  The stimulus to the full expression
of this function is found under circumstances providing optimal
growth conditions.  Under these copditions pneumococci exhibit
maximal capsular development, exalted virulence, and distinct type
specificity. Conversely, when optimal conditions are lacking the
organisms may become degraded, lacking capsules, of little or no viru-
lence, and devoid of type distinction. Without further supporting
evidence it would of course be hazardous to venture the assertion that
all three of these characters are necessarily and causally related to the
S-producing function of the cell.  However, the earlier work of Stry-
ker (6) and the more recent studies of Griffith (7), Reimann (8), and
Amoss (9), as well as our own observations, indicate that under cer-
tain cultural environments pneumococci lose their type spec%city
and become avirulent.  Moreover, these changes are now known to
be accompanied by a loss of the capacity to elaborate this soluble
spe&ic substance.

In 1917 Cole (10) observed that infected exudates and sera contain-
ing these soluble reactive substances of pneumococci possessed the
property of neutralizing pneumococcus antibodies, and pointed out the


376      CELL CONSTITUENTS OF PNEUMOCOCCUS. II

significance of this fact in relation to the therapeutic administration
of immune serum. Moreover, the work of Sia (11) on the effect of
this carbohydrate substance on the growth of pneumococci in normal
serum-leucocyte mixtures indicates that the addition of this substance
in very small amounts exerts a definite and specific efIect in annulling
the inhibitory action of the sera of naturally resistant animals.

If final proof be brought for the conception that the capsular zone
of the organism is largely composed of this carbohydrate substance, is
part of the defense mechanism of the cell, and is the site of its initial
contact with antibody, then these soluble bacterial polysaccharides
acquire new significance not only in the serological reactions of the cell,
but in the actual processes of infection and immunity in the host.

SUMMARY.

In this paper the general immunological significance of the intact
pneurnococcus cell and of its protein and carbohydrate components
is discussed.

BIBLIOGRAPHY.

1. Zinsser, H., and Parker, J. T., J. Exp. Med., 1923, xxxvii, 275. Mueller,
J. H., and Tom&k, J., J. hp. Med., 1924, xl, 343. Zmsser, H., and
Mueller, J. H., J. &p. Med., 1925, xli, 159. Mueller, J. H., Proc.Soc.
  Exp. Biol. and Med., 1924-25, xxii, 209. Mueller, J, H., Smith, D. E., and
Litarczek, S., Proc. Sot. &p. Biol. and Med., 1924-25, xxii, 373.
2. Smith, T., and Reagh, A. R., J. Med. Research, 1903, ix, 270.
3. Felix, A., J. Immunol., 1924, ix, 115.
4. Heidelberger, M., Goebel, W. F., and Avery, 0. T., J. Exp. Med., 1925,
  xlii (in press).

5. Dochea, A. R., and Avery, 0. T., J. Exp. Med., 1917, xxvj, 477.
6. Stryker, L. M., I. hp. Med., 1916, xxiv, 49.
7. Griflith, F., Rep. Pub. Eealth and Med. Subj., No. 18,1923,1.
8. Reimann, H. A., J. Exp. Med., 1925, xii, 587.
9. Amoss, H. L., I. Ezp. Med., 1925, xii, 649.
10. Cble, R., J. Exp. Med., 3917, xxvi, 453.
11. Sia, R. H. P., Proc. Sot. Exp. Biol. and Med., 1924-25, xxii, 262.