Chemo-Imurenolo~ical Studies in Pneumococcus

     Infection and Immunity.

     The disease acute lobar pneumonia, which ranks high anor%

the leading causes of death in this country, is due in the great ma-

jority of instances to a microorganism commonly called the Fneumo-


coccus.   Studies of this disease carried out in wards end labora-

tories of the Hospital have been directed toward acquiring a better

understanding of the interactions between host end parasite upon

which the outcome of the infection depends.   To this end our efforts

have centered in the attempt to reconstruct for this particular micro-

organism a more precise knowledge of the biological properties pecul-

iar to it and the nature of the protective processes which the animal

body develops against it.

     It now appears that specific differences in the inmxunolog-

ical reactions of various typos of Pneumococcus are related to funda-

mental differences in the chemical nature of the separate cell constit-

uents.  The correlation between the chemistry of the bacterial cell

and its biological activities as a parasite is becoming increasingly

important.  Recent studies suggest athat in the past we mag have re-

garded bacteria too much as a whole without appreciating that they con-

sist of separate component parts of diverse Function and composition."

The total imaux~e response of the host comprises not alone a reaction

to the bacterial. cell as a whole, but in addition the specific and in-

dividualized responses of its chemically distinct and biologically

specific constituents.


                                                    10

     To illustrate the advance in our knonledge ahich has grorm

out of studies ia this newly doveloping field of chemo-irenunology, may

I recdl certain facts concerning CL single componcrt of Eneumococcus

and point out the intimato rolation which this substance boars to the

morphological structure and biologicel activities of the cell as a

rrholo.

     It nil1 not bo necessary here to recall in dotail tho oarli-

er studios of that large group of investigators ;7ho 30th in this coun-

try and abroad have contributed so largely to this problem, save in

so for as the facts they have revealed are related to and form the

basis of the more recent no&.  You are already f omiliar nith tho ro-

nomd impetus that aas given the study of pneumonia through the aorking

out of the biological classification of Pneumococcus which made possible
the recognition of sharply defined and specific types within this pre-

viously confused group of microorganisms.   You nil1 rocall that by the

application of this method it ha8 been possiblo to dstormine the fre-

quency of occurrence of these specific types in pneumonia and to rec-
ognize differe&e&in the severity a& mortality of the infections they

produce; that% at&y of the presence of' the classifiable types of ".
Pnoumococcus in the aiouth secretions of healthy individuels has 're-

veiled a mode of dissemination of the disease-producing varieties by

hezilthy cmriers and convalescents. md that this mechmism of sprod
'has suggested a new interpretation of the epidemiology of the,disoase;

and finally that the knonlodge of type-specificity among pneumococci

has furnished a rational basis for the development of en immune serum

which in the treatment of !Cype I infection at least has proved of dis-

tinct therapeutic value.


     The biological classification was worked out before we hod

any knowledge of the chemical nature of the substonce upon which these

: '






I! '

specific relationships are now known to depend.   In the course of              il !
                                 f

these studies we were naturally lod to inquire as to tho nature of

this extraordinary specificity;  upon what constituent of the cell it

depends and why organism which are morphologically so identical should

diffor so mrkodly in their immunological properties.   The answer to

these questions lies In the fact nom recognized that the selective spo-
cificity of these bacterial types depends upon tho presence in the cell

of a spacific substance which is distinctive for each type.   This sub-

stamo has bean isolated and chemically identified, its origin has been

traced to 3 definite structural part of tho bacterial cell `and its

elaborntion has been found to be a special function of organisms that

are especially adapted to growth in the animal body.

     Under suitable cultural conditions Md particularly when they

grm in the tissues of a susceptible host, pnoumcocci devolop about

them a peripheral layer of rmterial which ensheathos their bodies in en

enveloping structure known as the cell capsule.  During growth these

epcapsulated cells release into the nedlun of their environment a dlf-

fusible substance which in soluble fom rotains tho type-specificity

of the orgrrnisms from which it is derived.  This soluble specific mib-

stence Is found not only In the cell-free filtrates of young cultures

but also in the body fluids of experimental ani.mils and in the blood

and urine of patients during the course of pneunococcus pneumonia.
!i%o function of elaborating this specific nntcrial is nest highly de-

vcloped in tho most vimlent form.  There is ground for the beliof

that the capsules of the virulent ~011s axe couposed largely of this


12

soluble specific substance.  Thus, there is disposed peripherally

about the bacteria an ectoplasmic layer of capsular material which re-

acts in a specific manner with the serum of an immunized auimal.  This

reaction is remariz3bly specific, occurring only when ths autisem and

the reacting substcnce are both of the ssme specific type.   These im-

munological reactions form the basis of the original classifications.

But the actual isolation of these specific substances, the detomina-

tion of their chetical constitution, and their relationship to pneuino-

coccus infection and immnity ore rmre recant devoloproents in the solu-

tion of the problem.

It my'now be confidently stated that the substcncos which

distinguish the specific types of Pneunococcus &We chouically coXpl8x

suga.rs - polysaccharidos - and that they have their origin in the cap-

sular nechcudsrr of tho ~011.  l'?o nattor fron what typo of Pnounococcus

those substances are isolated, they all possoss in co-n the chonfcnl

properties of corqlex sugars.  3ut interestingly enough, the sug,ar de-

rived fron each specific type of Pnounococcus is cheticcilly distinct,
possosslng unique properties which serve to distinguish it fron the

others.  These sugars, when choticellg isolated and purified, still

retain the cqmcfty to react specifically in antipnounococcus sorm.
They axe cheslically different and serologically specific for cmch type
of meuEococc&  'Pgpe-specificity, therefore, is detomined by the
chotical indivichud.ity of the particular polysaccharido prcssnt in tho

cell capsule.

     The fact that immunological specificity is dotemined by a

carbohydrate is ali the core strikkg since proviously oilly proteins

mm2 thought to pzrticipato iii the iimunitg roactiom.

--.--._--


13

    The fact that polyscLccharides, elaborated by the bacteria at
the focus of disease, are found in the blood and are excreted in the
urine unckngod in specificity is cvidonce that they are diffusible sub-
stances for which the body possesses no enzyme capable of bro&*g the;:

down into siqlor and inert sugars.  Tho significmce of this fact
will bocoze mre evident when later OZA we shall discuss the protactivo
action of a bacterial enzyrze recently discovered which is capable of
docouposing the czpaular polysaccharido of Type III Pnour~~occus in

the animal body.

    Although thoro is no direct proof that the capsular polysc~c-
charides of Prmmococcus are primrily toxic and directly responsible
for tho intoxication that acco~ptiios tho discaso, thorn is ovidcnce
that indirectly at least they nay have a hamful effect on the natural

processes of recovery.  Because of the avidity with nhi& they unite
with antibodies, they tend to neutralize the imuno substmcos in the
blood and thus to prevent theso protective age&s fron roaching the in-

fected areas,  Moreover, the czqmu.lar polysaccharides are q~ovm to in-

hibit phogocytosis , a protective process by which the nhito blood cells
seek to destrdy and rar;ove the invadi~ bact'oria frm ths bddy.   %I- `

capsul&ed pnemococci surrounded by M ectoplasnic layer of carbo-

hydrate natorial resist phmgocytosis, while the sme orgmisns stripped
of the C~QSU&W sub&mm md existing MI n&~d ~011s m3 rodily on-

gulfed by the pho@xytes.  ~211s fxt ~onfims the old observation
that capsule fornation is a protective mchonis~~ on 42-m part of tho
bacterial co11 by neam of which it seeks to protect itself ogaiilst the

cellular dafense of the Lost.


Fully virulent and encapsulated pnexmococci, when subjected

to en unfavorable environment, may lose these specific characteristics

and become degraded into non-virulent forms, without capsules and with-

e

out the irmmological specificity that distinguished their parasitic

antecedents.  However, these relatively avirulent degraded form my

again revert to highly virulent encapsulated organism.   The fact that

the non-invasive varients of F+nemococcus are potentially capable of

regaining virulence under favorable conditions is a natter of consider-

able biological and epidemiological importance.  It suggests the pos-
sibility that the degraded and attenuated forms my not, under all Cir-

~stencesI remain the harmless saprophytes they are generally thought

to be.

The facts which I have thus briefly outlined will serve to

indicate the significance of specific carbohydrates in the structure

end function of the bacterial C811.  We have seen that the specificity

of the recognized types of Pnemococcus depends on the presence of a
pactictim carbohydrate in the cell capsule; that the elaboration of
this specific sugar is a function most highly developed in fom adapted

to growth in the anipal body;  that when this function.is inhibited Or       

suppressed, the bacteria lose their capnles and revert to the cormon

undifferentiated foms of the species;  that these degraded and rela-
tiv81y harnl8SS vmia?lta Ltce Capable Of E@Il d0velopi~ intO highly

virulent encapsulated organisms;  and that the acquisition of virulence

aud specificity is invariably associated with the regaining of the

function of elaborating the capsular polysaccharide.

When the capsular polysaccharidos are isolated as cheL?ically

pure substances, they still retain unimpaired the proportg of reacting


specifically in an immne senm of the honologou8 type, but they lose
nore or less completely the property of stixulating the fomatio2 of

antibodies when injected into enirmls.   Since in the native state in

which they exist as a part of the bacterial cell they function as true

,mtigens in producing autibodies, it appears that they rmst exist thore
not as free polysaccharides but in SOIX other fern possibly combined
with sane substance which confers upon then the properties of a true

antigen.

    It has 10% been recoeized that S~JZ-JAO sugars such as glucose
do not possess the property of on antigen, tho.t is. they are incz2able
of sttimloting the form&ion of antibodies in the mirml body. Hotrever,
it is nom known that If these sugars are combined by chetical 138ans with
a protein, that is, with a substance naturally ondoned with ar,tigenic
properties, the new sugar conpounds thus forned incite the fornation of
antibbdiea that are specific for the particular sugar usod.   The study
of synthetic aptigens prepared by combining a simple sugar with protein
has shown that the specificity of the newly forced ccqounds is deter-
mined by the cher&al fndividurrlity of the reactive carbohydrate ir-

respective of the protein to which it is attached.  Antisera produced

by izxnmization aith these conjugated mg~protefne immriably reflect

the controlling influence of the carbohydrate on the specificity of

the whole conpound.  The studies on the siqle non-bacterial sugars
qhasize again the significance of carbohydrates in orienting the,spe-
cific immne response of the body to substances of this Cla88.

     The results of this work led us to test the possibility of
smthesizing en artificial pneUIIOCOCCU8 antigen by combining the crq?-

sular polysacchoride with a foreign protein.  For this purpose the


16

polysaccheride of Type III was chosen since in its purified form it
contains no nitrogen and represents a definite chemical entity. Fur-
ther, if results were obtained with this particular 8ugar, they would
be all the more interesting since the isolated pure substance itself
has nover been found to elicit antibodies in rabbits and even the in-
tact cells from which it is derived frequently fail to incite antibody

formation in these animals.  By en intricate chemical synthesis, the
details of which need not concern us here, it was faund possible to
combine the Type III capsular polysaccharide in Stable chemical union
with an Unrelated protein of animal origin.  This artificial antigen
has in co-n with Type III Pneumococcvs only the capsular polysac-
charide, the protein with which it was combined being of nidely remote

biological origin.  Rabbits injected with this artificial a;ntigen were
actively izmxune to suhsepuent infection and their sorum specifically
agglutinated living cultures of Type III Pneumococci, precipitated SOlU-

tions of Type III polysaccharide, end protected mice against Type III

Pneumococcue infection.  In other vrords, this synthesized compound,
containing only a single component of the pnewnococcus cell, called
forth m imenme reeponrre a8 specific in nature a8 that induced by the

whole microorgaaistl,

Bavlng found 'that the capsule of the virulent type8 Of PWU-

.- .

mococc~s consists of a polysaccharide, it occUrred to us that there

might oxist in nature, as it were , aa enzyme caprible of decomposing

this ccmplex sugar.  For the past several years a systematic search
for en ,egent of this nature has boon carried on.   Various enzymes of
animal end plant tissue and a number of difforent species of bacteria,

nolds, yeasts, and Actinorqyccs, many of which were Norm to decompose


other complex carbohydrates, were tested without success.  Recently,

however, fron soil a bacillus has been isolated which' possesses an en-
zym capable of decomposing the crrpsular polysaccharide of Type III

Pnewococcus.  This enzyme can be extracted in soluble fern fron the

bacilli.  Vhen a sterile extract containing the active enzyne is added
to a solution of the Type III polyaaccharide, the sugar is rapidly de-

composed and loses all of its specific properties.  The enzyne is spe-

cific in its aotion, decorzposing only the capsular substance of Type

III;  it ha8 no effect on the capsular carbohydrates of Type I or II,
which, although belonging to the mm3 class of couplex sugars, are in-

dividually different in chemical constitution.
     The enzym acts not only on the chemically isolated polysac-

charide but it ha8 the sme destructive action on the capsular sub-
stance of living pneumcocci when growing in the culture tube or in the

tissues of infected aninals.  It8 action in combatting i.XfeCtiOn in

treated nice and rabbit8 is being carefully studied.  In repeated ex-

perimnts it ha8 been found that a single injection of active enzyme
protect8 nice against infection with a azillion times the fatal dose of

a vimlent culture of Type III Pmm~coccus.  The nschanisn underlying

this protective action appears to rest on the capacity of the enzyne

to decoqpose ,the cqmular substance Burrounding the living pneumococci,
thus exposing the n&d bacterial bodies, stripped of their cnpstiar i.

defense, to the direct attack of the phagocytic cells of the host,

    Recent experinents 8hOW that the specific onzym not only
protects aninals if adninietered at the tine of infection, but that it

also has a curative action on en infection a1reod.y well established

before treatmnt is be,-.  For em:ple :  a sLmJ.1 quantity of a viru-


18

`I

lent culture of Type III F%.mmococcu8 injected into the skin of a
rabbit produce8 CL mrked and progressive inflmratory lesion at tho

site of inoculation.  Fron this local focus of infection, pnewo-
cocci rapidly invade tile blood strea, setting up CL septicmia nhich
increase8 until death occurs, often nithin 72 hours.   Rabbits, in-
fected with much larger mounts of culture and treated only after tho

lqse of 24 hours, have recovered completely folloting a Single E-&-

quake dose of the active enzym.
    Before the thornpeutic limitations of this specific agent
can ba accurately defined, Q mh further study mst be made of its

US0 in Larger Miudl8, such as t2OIlkSY8, in which it ie possible to in-
duce a disease of the luags that in clinical course and pathology liore

closely resembles pneunococcus pnemonin in men.

   These studies exphnsiae the fact that the capsule - long
roco&zed a8 tl defense LXX&U-AEEI on tho past of viruloxt bactoricc -
is a decisive factor in dctercining the fate of pneumcocci in the
animxl body and that this structure is vulnerable to attack by bio-

logically specific agents.

    In this brief review I have attoqted to point out the sig-
nificace of couplex eugara in the chezicti structure and biological
activities of pn~coccl; bon the ~q8Uhr pOly8mChmidO8 detor-
nine the specificity md i.nva8iven~ss of these org8ni8na'md influence
the immnity reapoqaes of the host;  aad finally, the relation which
these chetical and iarmnological facts bee,r to the problem of pneu-

nococcus infection and imunity.

Osnald T, Avery.