61

    Black tongue in dogs and its relation to spree, pellagra, and per-

nicious anemia. The studies of tropical spree in Porto Rico made it clear
that an experimental method would be of great value in studying the nature
and treatment of that condition.  Moreover, it was thought that animal tests
might show a relationship between sprVe, pellagra, and pernicious anemia.
Several dogs kept on diets low in protein and Vitamin B developed a condition
characterized by stomatitis, diarrhea, and central nervous system disturbances

in from five to eight weeks. With the exception of the last , the condition
resembled strikingly acute tropical spree.  Preliminary observations indicate
that disturbances of the gastric function are associated with the other symp-

toms. It is proposed to test the activity of the gastric juice of these dogs
for power to relieve pernicious anemia in human beings.  As it has been found
possible to prevent the disturbances of the central nervous system, dogs may
be kept in a state of chronic illness similar to tropical spree, and thus it
may be ascertained whether this chronic disease leads to experimental perni-

cious anemia.

   (=s. Avery, Stillman, Goebel, Dubos, Francis, Pabers, Goodner,
Alloway, and Terrell). The special Bubjects dealt with in the report are:

The decomposition of the capsular polysaccharide of Type III
       pneumococcus by a bacterial enzyme,
Methods of purification and concentration of the specific

enzyme,

Protective action of the enzyme in dermal infection of rab-
      bits with Type III pneumococcus,
Production of pneumococcus pneumonia (Type III) in monkeys.


I

422

Isolation of microorganisms decomposing the capsular polysaccha-

       rides of other types of pneumococcus.

Chemo-ixnunological studies on conjugated sugar-proteins,
The specificity of antigens prepared by combining c;C, and ,&

       glucosides of glucose with protein,

The synthesis of glucosides of the disaccharides, cellobiose,

       and maltose.

Studies on the interconvertibility of the specific types of pneu-

mococcus,

The degree and duration of active and passive immunity to pneumo-

         coccus in rabbits.

There follows a general account on the immune-chemistry of the

pneumoco ecus , which is presented in place of the more detailed and technical
descriptions contained in the report to the Scientific Directors.

    Specific carbohydrates in pneumococcus infection (Oswald T. Avery).
"An important advance in the study of infectious diseases came with the know-

ledge that bacteria, though simple in fom and structure, exhibit differences
in biological specificity as sharply defined as are those characteristfc of

the more complex forms of life.  The study of the immmological specificity
of microorganisms is not only necessary in the elucidation of the biological
relationships existing between varieties of the same species of bacterium,
but is essential to the working out of epidemiological problems and to the de-
velonment of methods useful in the control of infectious diseases by specific

therapeutic and prophylactic measures.

"Leaving out of consideration the promising but difficult field of

chemotherapy, the problems of specific cure and prevention of infection lie


63

in the attempt to interpret and imitate by artificial procedures certain pro-
tective processes of nature which constitute that which we call imrmznity.  In

order to imitate successfully the natural processes involved in spontaneous

recovery from disease, it is necessary to know the nature of the specific re-
actions between the infecting agent and the body tissues of the host.  The

specificity of these biological processes is advantageously studied by means

of the so-called immunity reactions.  These serve as a measure of the capaci-
ty of the animal organism to produce protective substances, and they afford
a means of studying the interaction between these specific antibodies and the
infectious microorganism when both are brought together in the animal body or

in the test tube.

    "Investigations on the specificity of these reactions have added
much to our knowledge of the many and diverse problems of infection.  The

study, however, is one of varying complex1 ty;  the methods suitable in one in-

stance fail utterly when applied to another type of infection.  While the

mechanism of the interaction between host and parasite has to a certain ex-
tent been exposed, the mediate problem lies in reconstructing for each mi-
crobe a more precise knowledge of the biological properties peculiar to it
end the specific reactions which the body develops against it.  One approach
to this problem is the attempt to relate specific differences in biological
behavior to fundamental differences in the function and chemical coxqosition
of the component parts of the bacterial cell and to determine the character

of the tissue responses to these separate constituents.  The total 1-e

response of the host comprises not alone a reaction to the parasite as a
whole, but, in addition, `the specific and individualized responses to the
chemically distinct and irmminologically specific bacterial constituents.


424   `I
                 .I'

64

"For the past several ye ars we have been seeking to acquire a more

intimate knowledge of the relation between the immune-chemistry and the bio-

logical activities of pneumococcus, the most frequent and one of the most fa-

tal of the microbic incitants of pneumonia in man.
                        .

     "I shall review one phase of the studies, namely, the role of spe-
cific carbohydrates in nneumococcus infection and immunity. Renewed impetus

was given to the study of pneumonia by the working out of the biological clas-

sification of pneumbcocci which made possible the recognition of sharply de-
fined and specific types within this previously confused species of microor-

gani sms.  This has made possible the determination of the frequency of occur-

rence of specific types of pneumonia, and the recognition of differences in

the severity and mortal1 ty of the diseases they produce,  A study of the pres-

ence of pneumococci in the mouth secretions of healthy individuals proved the
                     .
dissemination of the disease-producing types by healthy carriers and convales-

cents end suggested a new interpretation of the epidemiology of the disease,
while knowledge of type-specificity among pneumococci provided the only ra-
tional basis for the possible development of specific treatment by immune
serum, which in the case of type I infections, at least, has proved of dis-

I ii

tinct value.

"Pnaumococcus is a unicellular microorganism which is surrounded
              .

by an envelope of materikl known' as the cell capsule.  This'capsular layer i's

                                                                    .
particularly well `d&eloped ii' the case of pneumko&i `&$&le" of grow&g .and
multiplying in the aninial body;  During growth these encapsulated cells.elabo-
rate in the medium of their environment a diffusible substance which in sokk

ble form retains the type-soecificity of the bacterial cells from which it is

derived.  This soluble specific substance is found not only in the filtrates

of young cultures, but also in the body flu1d.s of animals experimentally in-

---* ..-.._      -I___-                            - --- -~-.. -.                    .--.     &           &


65

fected, and in the blood and urine of patients during the course of pneumo-

coccus pneumonia.  The function of elaborating this specific material is most

highly developed in the most virulent organisms. There are grounds for the
belief that the capsule of these virulent cells is comnosed largely of this

soluble specific substance.  Thus, there is disposed oeripherally about the
cell an outer layer of capsular substance which reacts suecifically with the

serum of immune animals.  The reaction is remarkably specific, occurring only
when the antiserum and the reacting substance are both of the same specific

type.  These immonological reactions form the basis of the original classifi-
cation and were worked out before there was any knowledge of the chemical na-
ture of the substances upon which tyoe-specificity depends. The actual isola-
tion of these specific substances in purified form, the determination of their

chemical constitution and their relationship to the inmnsnologfcal properties
of the cell as a whole are problems to which I shall direct attention.
     "The type-suecif ic capsular substances of pneumococous, first

chemically isolated by Dr. Beidelberger , have been found in each instance to
belong to the class of sugar-like substancesr namely the carbohydrates. Xo
matter from what type of pneumococons these specific substances are reoover-
ed they all possess In copplon the chemical properties -of complsx sugars -

the nolysaccharides. :.3ut, `: interestingly, enough, the causular polysaccharids
                                                                           /

derived from each snecific type of organism is chemically distinct, eachrpos-      ~ ,.,
sessing unique chemical properties whioh serve to differentiate it sharply.., ;`-  - ;,

from the &hers.  Moreover, solutions of these capsular polysaccharides, in I

ohemically purified form,  e xhibit immunologically the same specificity as do
t&e bacteria of vhich they originally formed f~ part.  Some idea of how re-
markably reactive these sugars sre msy be judged from the fact that, by the

use of an anpropriate ser7xn, their presence may be detected in dilutions as


66

high as 1:5,000,000.

     "A study of the chemistry of the capsular polysaccherldes has

shown that these substances are unusual compounds of simple sugars and uranic

acids.  Although possessing many properties in common, they exhibit character-

istic differences.  For example, of the specific substances of the first

three types of pneumococcus, the type I polysaccharide differs sharply from

the other two in containing nitrogen as an integral part of the molecule, and

in possessing basic as well as acidic properties;  on the other hand the type

II nolysaccharide is a dextrorotatory weak acid and the type III a levorota-
tory strong acid, neither of which contains any nitrogen in the molecule. The
fact that the particular constituent determining type-suecificity is chemical-
ly a carbohydrate is the more striking since iumunity reactions have hitherto

been considered exclusively the function of proteins. Of equal importance is

the fact that this selective specificity la in each instance determined by

the chemical constitution of the particular polysaccharide in the capsule and

that the presence of this morphological structure conditions both the inva-

siveness of the parasite and the immune response of the host.

     "The fact that polysaccharides elaborated by bacteria growing in

the focus of disease may be found in the blood and urine, unchanged in spe-
cificity, indtcates that the body possesses no enzymes capable of breaking

thn down into simpler sugars.  There is no evidence that these complex bao-

terial sugars as such are directly responsible for the intoxication accompany-

ing the infection.  So far as is known, they are not primarily toxic, at least

not in the sense of true bacterial toxins.  There are facts,, however, which

indicate that indirectly at least they may have a harmful effect upon the

natural processes of recovery.  Because of their qecific capacity to bind an-

tibodies, they tend to neutralize the immune substances in the blood and thus


427
67

prevent the protective antibodies from reaching the infected areas, Moreover,
the capsular polysaccharides are known to exert an inhibiting action on phago-

cytosis ( one of the most imnortant cellular defenses of the body against pneu-

mococcus infection.

"Theoretically, at least, there is `no apparent reason why these

complex sugars as isolated substances should not by themselves be capable of
stimulating the formation of antibodies in the animal body. Assuming that

they are of sufficient molecular size, they possess in complexity of structure

and colloidal behavior certain nroperties generally considered essential to

the antigenicity of proteins.  Indeed, Francis end Tillett have found that in

the present state of purity the capsular polysaccharides are capable of incit-
ing antibody production when iaected in minute amounts into the skin of con-

valescents and normal individuals.  However, with few exceptions all attempts

to evoke any immune response in animals with the highly purified polysaccha-

rides alone have been uniformly unsuccessful.  On the other hand, the more

these carbohydrates are chemically purified, the more reactive they become in

the specific serum of irauune animals.  Under these conditions it appears that,

removed from the bacterial cells, the capsular polysaccharides still retain un-

impaired the property of binding with antibodies, although in this form they
become quantitatively less active in stimulating antibody production in ani-

male.  In this respect they may rightly be included in the group of immono-

logically important substances which Landsteiner has called haptens, -- sub-
stances which have lost more or less completely their antibody-stimulating

function without impairment of the property of specifically combining with

antibodies.

     "The elaboration of the capsular uolysaccharide is an important

function of the cell.  When this function is suppressed or inhibited, as it


il

66

may be under certain experimental conditions of growth, the capsule is no

longer formed.  As a `result the organisms lose their type-specificity and ex-

hibi t only the common, undifferentiated characters of the species.  On the ba-

sis of colony differentiation these degraded organisms are spoken of as the

"R" or rough forms, and the original encapsulated types are referred to as the

smooth or `IS" organisms.  The unencapsulated R forms of pneumococci, irrespec-

tive of their type derivation, are no longer capable of invading the animal

body;  they have lost their virulence, and are readily taken up and destroyed

by the phagocytes of the host;  immunologically they exhibit only the species-
suecif icity common to all the degraded R forms of pneumococcus.  This trans-

formation resulting in a loss of specific characters may occur in the animal
                              . .

body as well as in the test tube. However, these degraded, avirulent variants
do not necessarily remain the harmless saprophytes they were originally

Caught to be, since it is now known that under suitable conditions they may
regain all the specific characteristics that distinguished the original para-

sitic type from which they came.

    "Of even greater biological interest is the phenomenon of the inter-
convertibility of the specific types of pneumococcus.  Griffith of Icndon first
showed experimentally by a special technic in mice that R forms derived from
one specific type of pneumococcus may be caused to acquire the characteristics

of enother specific type.  !Chi 8 Important fact has been confirmed by a nomber

of investigators.  In addition, Dawson and Sia by special cultural methods,
have found that the actual change from one specific type of pneumococcus to
another may be brought about in the test tube outside the animal body.

    I'The experimental evidence now available seems to indicate that any
R strain of pneumococcus has potentially the function of elaborating any one

of the specific capsular polysaccharides; -- the particular one being deter-

-.                                                                           .___


69

mined by a particular stimulus of a specific nature.  Alloway has recently
found that this potential function latent in the living R cells may be specif-
ically activated by the addition to an appropriate medium of a bacterial ex-
tract prepared from a given specific type of pneumococcus. Under these con-

ditions, the R forms irrespective of their type derivation again elaborate a
capsular material identical in specificity with that of the type of pneumo-

coccus from which the-extract was prepared.

     "There is at present no certain proof that transformations of this

kind ever occur spontaneously in nature.  Nor is there as yet any epidemio-
logical or clinical evidence that this form of reversible adaption is a factor

in the origin of human infection. However, the experimental evidence leaves

no doubt that the non-invasive, non-encapsulated R cells under favorable cir-
cumstances are potentially capable of again developing into highly virulent
organisms, and that the acquisition of virulence is invariably associated
with the restoration of the function of elaborating the specific capsular car-

bohydrates.  Indeed, it is most significant that no matter whether one con-
siders pneumococcus from the viewpoint of vircLlence, antigenicity, or its ca-

pacity to undergo vaxiat ion, the single determining factor associated with
all these characters is the function of synthesizing the specific capsular

polysaccharides.  Scarcely less important is the fact that the immunological
specificity of each of the specific types of pneumococcus depends upon the
chemical individuality of the particular carbohydrate in the cell capsule.

     "As chemical substances, separate and apart from the bacterial
cells, these carbohydrates have been found to incite specific reactions in the
tissues of sensitized animals and in the skin of patients convalescent from

pneumococcus pneumonia.  Guinea pigs passively sensitized with the precipitat-
ing serum of an imrmne rabbit suffer violent ana@ylactic shock and die within


"
d                     430 .?

70

three to four minutes following the intravenous injection of as little as ,055

mg. of the homologous polysaccharide.  The anaphylactic reactions are strictly

type-specific.  There is now ample evidence to support the view that protein-

free, even nitrogen-free, carbohydrates may induce acute anauhylaxis in specif-

ically sensitized animals.

     rrlillett and Francis have found that the injection of 0.01 mg. of
specific polysaccharide into the skin of patients recovering from pneumococcus
pneumonia may evoke an inxnediate local reaction in the form of a wheal sur-

rounded by a zone of erythema.  The cutaneous reactions develop rapidly within

fifteen minutes and subside completely in from one to two hours;  tileg are

elicited only by the specific polysaccharide derived from the same type of
pneumococcus as that causing the infection in the patient.  !Phe capacity of

the skin to react to the specific bacterial sugar is intimately associated
witA recovery and closely parallels the occurrence of tyoe-specific antibodies

in the patient's serum.  The results indicate that this specific skin test has

prognostic significance and may become of value in determining the therapeutic

dosage of antipneumococcus se-.

       "Studies on lsynthetic antigens' prepared by chemically combining
derivatives of glucose'and galactose with proteins have shown that even these
simple sugars exert a determining influence on the inxrnnological specificity

of compounds of which they form a part.  The newly acquired specificity of
these artificially conjugated sugar-proteins is in each instance determined
by the chemical structure of the carbohydrate irrespective of the protein to

which it is attached.  It is especially significant in the case of glucose
and galactose that the two sugar derivatives differ from each other only in
tine spatial arrangement of the hydrogen and hydroxyl grouns on a single carbon

atom.  It is a remarkable fact that the mere rotation of this carbon atom

- -i-G.-..  . .   .- ---_-._- ____ A-,;;;,   -*- ..-- --------~----
                                        -'

                                                    ~__~


through an angle of 180 0 suffices to change completely the antigenic specifici-
ty of two substances otherwise chemj.cally identical.  In the case of these ar-

tificially prepared sugar-proteins, the two isomeric sugar derivatives can be
selectively differentiated one from the other by serological methods.  These
observations on the kmnuno-chemistry of carbohydrates confirm the original
studies of Landsteiner on the specificity of axo-`proteins and furnish addition-
al evidence of the general dependence of immUnologica specif icfty upon the
chemical constitution of the reactive substances.  It is evident, therefore,

that simple sugars, which by themselves are non-antigenic, may, when coupled

to a protein, specifically determine the immune response of treated animals,
and that the antibodies thus engendered reflect the orienting influence of the
sugar radical on the specificity of the antigen as a whole.
    "From these results we were led to test the possibility of `synthe-
sizing' an artificial bacterial antigen.  For this purpose the capsular poly-
saccharide of type III pneumococcus was chosen, since it contains no nitrogen
and in its present state of purity may be regarded as a definite chemical en-

tity.  Moreover, if results were obtained with this particular polysaccharide
they would be the more significant , since the free substance by itself has

never been fcund.to elicit any ixunune response in rabbits, and even the ori-
ginal bacterial cells from which it is derived fail in a mejority of instances
to incite specific antibodies in these animals.  From a chemical point of view,
the difficulty lay in synthesizing the appropriate derivative of this complex

sugar.  It must be one capable of being coupled to protein and one in which
the chemo-specific groups of the polysaccharide are not masked by the chemical

procedures.  Dr. Goebel succeeded in synthesizing the amino-benzyl-ether of
the type III polysaccharide and in coupling the diazonium derivative with a
foreign protein, namely, the globulin from horse serum.  This soluble antigen


72

therefore has in common with type III pneumococcus only the specific capsular

polysaccharide.  Rabbits injected with this artificial antigen uniformly de-

veloped in their serum type-specific antibodies.  The antiserum thus produced

not only precipitates the original polysaccharide, but agglutinates living cul-
tures of type 11X pneumococcus and protects animals against infection with vir-

ulent organisms of the homologous type.

     "Knowledge of the chemical nature and significance of the capsular
polysaccharides in pneumoaoccus infection and ixmnrnity led us to search for en-
zymes capable of decomposing these' specific carbohydrates. A mxnber of en-
zymes of animal and plant origin as well as cultures of various bacteria,
yeasts, molds and soil actinomycetes, many of which were known to decompose

cellulose and other complex carbohydrates , were tested without success. b!y

associate, Dr. Ihzbos, isolated from peat soil a bacillus which possesses an
enzyme that acts specifically on the capsular polysaccharide of type TIf pneu-


mo co 03x3.  From these bacilli the active enzyme has been extracted in soluble

form.  By technical procedures, active preparations of the enzyme have been
purified and concentrated without appreciable lose in potency.

    "In view of the marked dffferencee in the chemical composition of

the various capsular polyaaccharides, it is not surprising to find that the    ,
enzyme decomposes only the type J!II substance and has no effect upon any Of

the other bacterial sugars thus far tested.  In this respect, the selective ac+
tfon of the enzyme is as specific as in the Immune reaction between the type
IS1 polysaccharide and its homologous antibody.  The polyaaccharide aoted upon
by the enzyme loses its serological specificity and is no longer precipitable

by type III antipneumococcus serum.  This enzyme not only acts on the chemi-
cally isolated sugar, but it specifically decomposes this substance in the na-
tive form in which it exists in the cepsulea of the living cells., When a


sterile solution of active enzyme is added to a growing culture of type III
pneumococci, the organisms lose their specific agglutinability and the soluble
capsular polysaccharide is serologically no longer demonstrable in the culture

fluid.  Under these conditions, the enzyme decomposes the capsular substance
as rapidly as it Is formed without impairing the viability of the decapsulated

organ1 sms.

    "The action of the enzyme does not result in a loss of the function

of elaborating the capsular substance, since pneumococci so treated promptly
regain their capsulea when transferred to an enzyme-free medium.  !t%e active


enzyme, therefore, represents a specific agent which by itself is neither bac-
tericidal nor bacteriolytic but which, by decomposing the capsular structure,
completely alters the biological behavior of the bacterial cell.

      "In view of these findings, experiments were carried out to deter-
mine whether the enzyme would favorably influence the course of experimental

infection in mice with type III pneumococcus.  It was found that a single in-

jection of an active preparation of enzyme protected mice against infection

with a million times the number of virulent organisms invariably fatal in the

untreated animals.  The protective action of the enzyme is type-specific; just
as in the teat tube .it deqomposes only the type III polysaccharlde, so in the

animal body it is effective only against infection with type III pneumouocous.

%xperimental evidence indicates that in mice the enzyme also has

a &%tlv~ action when administered in the course of an infection already"wel1
established'at-`the time of treatment.  The administration or the specific
agent as late as eighteen hours after the onset of infection has brought about
the recovery of mice infected with multiple lethal amounts of a virulent cul-

ture of type XI? pneumococcus.  Experiments carried out in collaboration with

Dr. Goodner and Dr. Dubos have shown that the enzyme has also a marked curative


*

a

  I



tib

434 '
74

action in the disease brought about by infecting rabbits lntradermally with a

highly virulent strain of type III pneumococcus.  The experimental disease is

characterized by the rapid development at the site of inoculation of an in-

tense inflammatory lesion with spreading edema, marked cellular infiltration

and hemorrhagic necrosis, accompanied by fever and the early invasion of the

blood stream with increasing numbers of pneumococci.   The infection ordinari-

ly terminates fatally within three to four days in 95 per cent of untreated

rabbits.  Following the intravenous InJection of an adequate amount of active


enzyme, the bat teremia promptly d1sapnear.s ;  the local lesion, freed of bac-

teria, undergoes the natural processes of healing, and recovery occurs in 95

per cent of the animals so treated.

     "The experimental results support the view that the primary action
of the enzyme lies in its capacity to decompose the capsular polysaccharlde

of the Invading pneumococcl.  The prpoess of decapsulation brought about by

the direct action of the enzyfne strips the bacteria of their capsular defenee

and thereby exposes their naked and unprotected bodies to direct atta& by

the phagocytes of the host.  Thus phagocytosls, Ineffective against the en-

CapUht8d forma, now becomes the important mechanism in the final d&struction

of organisms from which the capsular substance has been removed by the action

of the enzyme.

     "In this sense, the enzyme may be said t0 initiate a protective rb

action, the successful Issue of which depends upon the effective phagocytic

response of the host.  For these reasons, it at once becomes apparent that

the curative action of the enzyme is subject to the limitations iposed by the

variations that occur in the cellular defense of the infeoted animal.

      "These studies suggest that the capsule -- long recognized as a

defense mechanism on the part of virulent bacteria -- is a decisive factor `in


                                  435

                                                   75


determining the fate of pneumococci in the animal body and that this structure
is vulnerable to attack by agents other than specific antibodies. I'

     Studies on Hemonhilus influenzae (Drs. Cole and Pittman).   The

study of bacteria of the genus Hemonhilus described in the last report has been

continued.  Strains of all species isolated from the respiratory tract are

still being studied:  the greater emphasis, however, has been placed on an in-
vestigation of the immunological relationship of S and R variants which were

derived from these S strains, the production in a horse of a specific anti-

serum sgainst Type b bacilli,  an d a method of determining the protective PO-

tency of this antiserum.
     Type b H. influenzae antiserum has been produced in a horse.  Sev-

eral hospitals have been supplied with the serum, and a few patients suffering

with influenza1 meningitis have been treated with it.  Vhile the results have

thus far been unsatisfactory, It has not been used in a sufficient nuziber of

cases to form definite conclusions concerning its efficacy.  At present

strains from 22 cases of Influenza1 meningitis have been studied, and they
have all been of Type b with the exception of one, which was not type-speclf-
lc and which produced rough (R) colonies.
     That the Type b antisem contains a protective substance has been
definitely shown by the prevention of the lesion (injuries) which Type b or-

ganisms induce in .the skin of rabbits.  This protective S'libSt6UlC8 CaIl be quit8
accurately measured.  The smallest amounts of the sera from the three large
successive bleedings that would protect against the "standard skin lesion

dose" were, respectively, l/200, l/ 600 and l/ 800 cc.  The W standard skin le-

sion dose" of culture is ten times the smallest amount of culture which will

induce a lesion that remains positive for three days.  It is of interest to

note that the protective titre of the serum has steadily increased during a


`26

4

course of irauunia~tion of more than a year.  The serum is apparently type-

specific in its +tion, since it only prevents the skin lesion induced by

Type b organisms.  The development of the lesions following the inoculation
with other type-specific strains and the R variants is apparently not in-

fluenced by the immune serum.

\         (Dra. Rivers, Berry, Benjamin, Sprunt , and Schwentker) .   Patient8

  with a variety of infectious diseases have been admitted to the werd for clin-
  ical Studies, particularly in regard to the changes In the blood picture a8
  revealed by the supravital method of staining, end for the purpose of obtain-
  ing material with which animals and tissue cultures were inoculated. The case

of egranulocytosis proved to be of interest because it occurred

sillectomy and responded favorably to tntravenous injections of

phate.

following ton-

adenine sul-

     Cultivation of vaccine virus for use in mar,*  In a previous report
a simple medium for the artificial cultivatiop of vaccine virus was described.
The results of the work indicated that vaccine virus i8 capable of multiplica-
tion in the presence of minced chick embryo tissue suspended in Tyrodets solu-

tion, we experiments, however, were conduuted with a neurovaccine virus,
and, although the active agent obtained caused typical vaccinal lesions in

rabbits, it was deemed best not to test it in human beings. To obtain a c+-      :

ture virus for Jennerian prophylaxis in man, It seemed advisable to adapt a

dermal atrain of vaccine virus to our method o$ cultivation. The report deals

with the results of this work.
    Vaccine virus, Lot 611, prepared by the rJew York City Board of

Health was used to initiate the cultures. Having rid the dermalvaccine vi-

rus of bacteria it remained for us to determine whether it was possible to