ee ae ee ee Report of Dr. Avery (vith Drs. Stillman, Tillott, Julianclle, Gocbel, Dubos, Dawson and Francis). Studics on Pnoumococcus Infection and Immunity. I. Chemo-immunological Studics: l. Nature of the type-specific antigen. 2. Synthesis of carbvohydrate-protcin antigens. II. Reversion of "RY ——-—} "S" forms of Pneumococcus and the Inter- convertibility of the specific types. III. Cutancous reactions in pneumonia Natients to the protcin and specific curbohydrate of Pneumococcus. IV. Gutancous infection of normol ond immne rabbits with vR and «St pneumococci. V. Cutancous vaccination of rabbits with Pneumococcus; the character of thc antibody response, the resistance to infection and the hypersensitivencss induced in animals. VI. Reversion of "RY -----} "S" forms of Friedldnder bacilli and the interconvertibility of the specific types. VII. Nature and duration of the immmity produced in rabbits by the inhalation of pneumococci. VIII. Significence of oxidation-reduction processes in becterial growth. IX. Publications. In the vork on Pneumococcus infection and immunity cerried on in the bactcriological laboratories of the hospital during the past year in conjunction with the clinical study of pneumonia in the wards, the studies of special interest and those sufficiently supported by experimontel data to justify their inclusion in the annual report to agg eminem te i tte men + 205 193 the Bonrd of Scientific Dir.ctors rc, the chcmo-immunologicsl investi- gation corcerning the specific »ntigens of Pneumococcus, the rcsults of which arc alrendy yielding morc precis¢ lmowlodge of the chemicnl and biologicnrl propertizs, vad of the couses ond the preveution of the dissociation and consequent inasctivetion of thes: complsx xntigens, pofore cud after injection into the animel tody; the successful rad origin-1 synthesis of sugar-prot.ins by thc coupling of a enrbohydr«te radicle in glucosidic union vith serum globulin, undertaken vith the hope of thus oricntating the antigenic snecificity of the protcin molecule by the introduction of an antigenically inert glucoside; the imvorta:t confirm-tion end elaboration of the original observations of Griffith on the intcrconvertibility of specific types of Pncoumococcus, establishing the occurrence of fundomental variations in virulent pathogens, dofining conditioas under which avirulent oranisms may become virulent 21d indicating the possible gignificence of these adsptive changes in the course of infection -nd in the epidemiology of the disense; the discovery of the {moortant fact that in pneumonia patients, tro chemic-lly differant constituents of Pneumococcus may give rise to two distinct kinds of skin reaction, namely the immediate vheal and erythemn type of anaphylactic response illicited by the intracutaneous injection of a fraction of a milligram of the specific polysaccharide, and the Aelnyed tuberculin-like tyne of inflammatory reaction produced by minute amounts of the bacterinl protein, each of these reactions exhibiting their characteristic specificity and both suggesting the relationshin of hypersensitiveness to the processes and course of the disease; the observations made on pneumococcus infection of the skin of normal rabbits and of rabbits immunized with "R" and nS" strains end the correlation betvecn the kinds of lesions produced A een on rete imtlheR ROE So ~— sa 206 194 by theee virulent cnd ovirvlcat org»nisms, thus providing -n cxncri- ment*l method of determining: more saccurntely th: mechanism underlying the differences betvecn natural resistence md acquired immunity in terms of enccific antibodies ond cellulrr reactions; the observstion of significant diffecrouccs i. the charnetcr of the a.tibody response of rabbits to iutravenous «nd intracutiancous injection of pneumococci, demonstrating the fact that after introduction into the animal body type-specific strains ("S" forms) under certein circumstances may lose morc or less completcly the property of stimulnting the type-specific antibodics although still rot-ining unimpaired th: property of 11- liciting th. antivorotcin -ntibodics commonly provoked by all "R" cells, - afnet of significrice in understanding the phenomenon of antigenic dissociation and in intcrorcting the charcetcr of the immunc response to the complex vntigens of Preumococcus; the finding that folloving intracutancous v-ccination with heat-killed "R" and "S$" orgonisms, rabbits acquire a solid immunity aginst infection with virulent "S$" strains of homologous -nd hcterologous types, and the observtion that rabbits trcated with "R" cells become rcsisto..t to infection in the total absence of demonstrable type-specific nitibodies; the obsorva- tion thot intracataneous vaccin tion in rabbits mey lesd to the de- velopment of hyperscnsitivoness to the bicterinl protcin 7s shown by positive skin and eye renctions; tho demonstration of the reversion of avirulont organisms to the virulent forms and of the interconvertibility of thc specific types as indicated by the fact that Gram negative bacil- li of the Friedl4ndcr group may be caused to undergo these tronsforma- tions by the methods successfully employed in the case of Pueumococci; the production of an immunity of long duration in rabbits by the iuho- lation method demonstrating the possible importance of the resniratory 207 195 route in the requisition of specific resistance and illustrating the significoue: “hich the pecteri-l flor-. of the upver cir pssenges my heve in determining the rorctiors of immunity “nd hypers:nsitiveness to infection; cud finally the -pplicrtion of the principles of oxidation-ri.ductio. processes to the study of pncoumococcus, particular- ly with refercsec to the collul«r functions of grovth, thus bringing to light uc” frets which arc of generol biological significance as well as of practice nl use in the improvement of culture media. I. Chemo-immunological Studics on Pneumococcus.s Pneumococcus Antigen and Synthctic Carbohydrate Antigens. (Dr. Gocbel). Thc problems of undsrstandins the rclationship betre.n chemical con- stitution, physiolo;ic~l ¢ffcct and biological snecificity, yaich probably founc their oxi,in in the study of the activ: vrincinles of certnin noturnal drugs, hove dbccome so absorbing 71d so ombr-cing thet they hove attracted snd held the interest of the chemist, the pharms- cologist nd immmnologist rlixc. The question of protein specificity in prrticular, and the possibility of chonging specificity by altering the protcin molecule through chemicol menus, have engaged the minds of many investigators. On the other hand, thc réle which carbohydrates lay in th: phenomen» of immunity has oniy recently been disclosed, despite the foct that the presence of these substances in boeteria ond yeast has long becn ‘mowns Some venrs »go, Dochez and Avery observed that filtrates of pneumococcus cultures contained a stable substance which reacted specificelly with nntipneumococcus serum of the homologous type. This observetion lcd to the isolation and identification of specifically rencting substances from the three specific types of Pnoumococcus and from the three types of Friedl¥nder's bacillus. Since then other sae, A paid 208 196 investigators have isolated similar substances from various other species of bactcria. These type-specific substances fall into the class of carbohy- drates; they are unusual carbohydrates in that each contains a sugar acid as an integral part of its complex moleculee JImmunologically they belong to that important group of specifically reactive but non-anti- genic substances which Landsteiner has named haptens. One of the striking characteristics of these bacterial carbo- hydrates is their failure to produce antibodies when injected into the animal organism, though in the state in which they occur in the bac- terial cell they are not only type-specific but also effectively anti- genic as well. When the pneumococcus cell is permitted to autolyse under sterile conditions it has been found that the autolytic process is accompanied by an increase in amino and non-coagulable nitrogen and in ether-soluble fatty acids. This change is due to the action of liberated intracellular enzymes, protease and lipase, upon the native substances of the cell, When extracts of pneumococci containing the active intracellular enzymes are added to heat-killed pneumococci, lysis of the cells occurs and there is an increase in the non-coagu- lable and amino nitrogen comparable to the changes accompanying spontaneous autolysis. Furthermore, when extracts containing the active intracellular enzymes are added to emulsions of the alcohol- soluble lipoids extracted from pneumococci, an increase in the ether- soluble fatty acids occurs. When living pneumococci are dissolved by means of sodium desoxycholate and such solutions are incubated at 37°9G., an excess of the bile salt has been found to inhibit the action of the pneumococcus protease, but not the action of the lipase. Such solutions, however, no longer yield type-specific antibodies when 209 197 injected into animals, It seems, therefore, that the specific immu- nizing antigen of pneumococcus is impaired by the action of its own lipase. In order that the bacterial polysaccharide may be effective as an antigen it is believed that it must be combined with another cellular constituent, possibly a protein, in ester linkage to form an easily dissociable, comblex antigen. The products of hydrolysis of the specific carbohydrates of — Pneumococcus and Friedlander's bacillus have been studied in detail. All except the Type I Pneumococcus carbohydrate yield glucose on hydrolysis, The complex sugars of Pneumococcus Type III and Fried- launder bacillus Tyne A yield on hydrolysis isomeric aldobionic acids ( glucose-glucuronic acid) in addition to glucose. Evidence has also been secured which indicates that the specific polysaccharides of Type II Pneumococcus and of Type B and C Friedlander bacillus also contain other aldobionic acids within their molecules. The selective specificity of encapsulated organisms, such as Pneumococcus and Wriedlsnder's bacillus, seems to depend primarily on the hapten part of the hypothetical complex antigen. In all of the haptens studied thus far, the invariable presence of isomeric aldo- bionic acids seems to indicate that particularly the acid group (carboxyl group) and its stereo-chemical relationship to other groups of the intact polysaccharide molecule, which in each instance must neces- sarily be different, has a profound influence in orienting specific antibody response. In order to understand more fully the réle of carbohydrates in immune phenomena we turned to the group of mucoproteins, the naturally occurring conjoined carbohydrate-proteins, but without avail, for we did not find the carbohydrates either of ovomucoid or of tendomucoid iO 198 to behave as haptens. Nor did mixtures of purified specific soluble substance and protcin yield, on immunization, antitodies reactive with the specific carbohydrates. Thereforc, for the purpose of studying the role which simple sugars, end in particular sugar acids, of different } spacial configuration, such as those which are found in specific poly- saccharides, might play in altering the svccificity of proteins, it was thought that it might be possible to combine diffcrent sugars and their acid derivatives with a given prot.in and to observe any specific dif- ferences in antigenic properties that might result. The problem thus becomes two-fold, first to synthesize the desired sugar derivative, and second to devise some means of combining the sugar with thc protein. Some years ago, Psuly described a tcst for tyrosin and histidin in proteins by adding diazobenzene sulphonic acid to an alkaline solution of protein. The diazobenzene sulphonic acid combines with the protein, and on acidification the complex is pre- cipitated. This fundemental principle was made use of in combining glucose and galactose to serum globulin. The second problem, that of preparing a sugar derivative adaptable to this principle, was solved by the following synthesis: H / H GH,OH CH - (CHOH), a ( CH300)20 CH,OCOCH, - CH - (CHOCOCH,), - tL o»__|™ Lo ____| 00s Glucose, Pentaacetyl glucose. "y H H Br ; : / 5 CHZOCOCH, - CE - (CHOCOCH, ),- C_ + _ _ -, [| gp BTL, AB) «(OCG HNO Te tra-acetyl-bromo-glucose. . Silver paranitrovhenolate. ye ————> CH,0COCH, - CH - (CHOCOCH 3), - CL Ba (OH). O | OCcH4N0n = ——____-5 Tetra-acetyl-paranitrophenol B-elvcoside. 241 199 Ri: CH2OH - CH - (CHOH), - 6 Es CHaOH - CE ~- (CHOH)g - Co | OC.H,NC, —? 1 | OCgH NH. 0 | 0 Paranitrophenol - / ~glucoside. Paraaminophenol - f/f -elucoside. The end product of this series of reactions may then be converted into the corresponding diazonium derivative and coupled with protein in the presence of dilute alkali: H / CE,0H -CH - (CHOH), - C. HNO, 0 | OCgHNHRHC1 Sverre aaNEE H CH20H - CH ~ (CHOH), - 0” | | O- CgH,y - N= N- Cl 4+ Protein Vee C) need H CH,OH - CH - (CHOH), C7 ‘0 - Ogh, - N= N- Protein Thus one may couple practicolly any sugar (aldose) or its derivative with ony protein. We have thus for synthesized, in addition to intermediate glucosides, parnaminophenol f -elucoside, and paraaminophenol f-galoctoside. These hexosides have been coupled to pure serum globulin under conditions which do not alter, by denaturation, the protein molecule. Two different synthetic sugar proteins have thus been prepared. They exhibit different chemical properties, and although thé protein substrate is in each in- stance the same, they appesr to be different compounds. Animals are being immunized vith these synthetic sugnr protein antigens. Attempts are also being mrde to prepare the glucosides of aldobionic scids, the sugar acids found in the specific soluble substances of Pneumococcus, and to combine them with the bacterinl protein and to study the antigenic properties of these complex substances, 4 rir 200 II. Studies on the Interconvertibility of "R" and "S" Forms of Pneumo- coccus -nd the Interconvertibility of Pneumococcal Types. (Dr. Danson). In the January (1928) number of the Journ-l of Hygiene thcre appenred an article by F. Griffith in which he claimed to have domonstrated the fol lowiag points: (1) That uRe wneumococci could be reverted into the homologous *§" tynes by th: subcutancous injcction in mice of large amounts of live organisms; (2) thot "R" pneumococci could be reverted into the homologous "S" type bv the subcutaneous injection in mice of small nmounts of live "R" cultures together vith the hert-killed deposits of large mounts of the corresvonding ngu cultures; and (2) that "R" forms could be trans- formed into hetsrologous "S$" types by the subcuteneous injection in mice of smxll »mounts of live "RY organisms together vith the hevt-killed dcposits of lnrge smounts of heterologous "S" culturcs, In view of the significance of these findings, experiments were qt once undertaken to verify Griffith's results. (1) The reversion of "R" forms into the homologous "S" typc by the subcutsneous injection in mice of lerge omounts of living "R" cultures. It wis found thet this was na most cffective mcthod of causing "R" forms to revert to the "S" type, Different "R" straine varicd in the esmount of culture required end one strain (1/192/R) uniformly failed to revert. The results obtained were entirely in -ccord with those previously obtained by other methods. In 111 instences in which revorsion occurred thc "RM" forms invariably reverted to the snme spocific type from which they were originally derived. (2) The reversion of "R" forms into th: homologous "S" types by the subcutaneous injection in mice of small amounts of live "R" cultures together with th: heat-killed deposits of large amounts of the corres- ponding "S" culture. This method was found to be more effective than any ‘(iy Fe 243 201 other previously employed in producing the R ———}3 § change. Success wis attained vith onc culture (1/192 /R) which had resisted 211 previous cfforts to effect the transformation. This particulor culture had previously been passed intraperitonenlly through 105 consecutive mice 1nd still maintaine? all of its "R" characteristics. (The questiona@ the viability of any orgnnisms in the heat-killed deposits is corefully considered in the next part of the report.) (3) The transformation of "R" forms into heterologous "S" types by the subcutaneous injection in mice of small smounts (0.25 cc.) of living "R" organisms togethcr with the heat-killed deposits of large amounts (50-100 cc.) of "S" vaccines of heterologous types. Since the question of the viability of the organism in the vaccines is an all important consideration, the procedures employed in the preparation of the vnecince and the controls used in testing for stcrility are given in detail, The vaccines vere heat-killed in scaled »mpules totally immersed in water. Fifteen minutes heating .t 60° has invariably sufficed to kill all pneumococci but longer periods and higher temperatures have also been used. In Vitro Oontrols; (1) All vaccines havo been cultured on vlood agar plates and in blood broth. (2) Cultures have been made aero bic- ally and anrerobically in blood broth nd blood extract dextrose broth. (3) Vaccines have been cultured serially in 10 per cent serum broth for 20 transfers. In Vivo Controls: (1) An equal number of control mice have been inoculated with the v>ccine alone and later sacrificed and cul tured at varying intervals. (2) Control »nimals have been inoculated vith the vaccine together with other organisms, such as B influenzae and 214 a 202 aq streptococcus, (3) Mice intoxicated with alcohol hnve been injected vith large nmounts of vaccine. (4) In the majority of expcriments there sur- vived many test -nimals in which the transformation did not occur. In the course of thc work additional, and even more convincing, controls were afforded by certain observations which will be later reported. In a large series of expcriments the following points have been | established: - a) The subcutaneous injection into mice of 1 large amount (50 - 100 cc.) of a vaccine of an "S" culture, (heat-killed at temperatures varying from 60°C up to ond including go°c for periods of from 30 minutes to 2 hours), together with an "R" culturc derived from 4 heterologous type, results in the formation of virulent "S" pneumococci of the samc type as thet of the vaccine cmploved. (bv) If the vaccine is heated to a higher temperaturc than 80°C., the "R culture frequently reverts to the snme type os thit from which it was origin-lly derived, not to the type of the vaccine. (c) Any "R" orgonism rogardless of derivation potentially h-s the capacity of claborating the specific sol- uble substance of ony specific type of Pneumococcus, and by the ~rocedure . described can be transformed into "S" organisms of » heterologous typc. (d) All attempts to effect heterologous transformation "in vitro" have failed. (6) ‘The following conditions arc necessiryi- @ Large amounts of "S" yaccine ore necessary (50-100 cc.). b The vaccine mst not be allowed to autolyse before being heat- killed. c¢ The "R" culture mst be well "degraded" else it reverts to its own type. III. Cutaneous Reactions in Pneumonia Patients to Cell Constituents of the Pneumococcus. (Dr. Tillett -nd Dr. Francis). Potients acutely 411 with and convalescent from pnoumonin are being tested by intra- fy co cutancous inocul«tion to determine their reactivity to the protein nnd carbohydrate constituents of Pneumococcus, The cell fractions used for injection are (1) soluble svecific substances (polysaccharides) derived from Types I, II and III, in purified state, and (2) the socnlled nucleo- protein fraction, which cxhibits the protein-specificity of the species, The polysaccharides and the protein are injected in »mounts of 0.01 mem . On admission to the hospital ench pneumonia patient receives 5 intradermal tests: 1) Type I "S" substance;, 2). Type I] "St" substance; 3) Type tit "S" substance; 4) Nucleo-protcin fraction;. 5) Salt solution control, At the some time blood is vithdrawn in order to determine the presence or absence in the sera of »ntibodies reactive with the substances injected, These tests are repented every few dnys during the potient's stay in the hospital., Following injection, the patients are observed both for an immedinte resction and for a delayed response which appears after 24 to 48 hours,. Two independent and distinct forms of reactians are noted, The first of these is produced by the protein fraction. ‘Shortly after the in- jection of the material a slight reddening occurs, end occasionally a slight edema, This subsides rapidly, Beginyning at about the eighth hour, erythema, edema and tenderness, in varying degrees, appdear at the site of inoculation and then increase up to 20-24 hours when the maximum is renched. In general, the ne of this form of akin reaction to protein substance is similzr to the tuberculin reaction, and its oc- currence and course are briefly as follows;-. During the acute phase of the illness there is no response to the protein material, but following the return of the temperature to normal the reaction becomes positive, As convalescence proceeds the intensity of reaction becomes more marked, and the appearance more striking. Sufficient time has not elapsed since | | 216 | 204 ) the beginning of these experiments to determine hor long poticnts con- tinue tc exhibit this altcred reactivity to Pneumococcus protcin, The reaction of normnl individuals is nlso being studicd, but as yet suf- ficient dat». have not been accumulated to warrant any conclusions. The second form of renction has beon cbtained ith the type- specific crrbtohydrates. During the phase of acute jliness there is no reaction. However, coincident vith recovery a positive reaction may develop. The chnracter of this type-specific response offers a striking contrast to thot following protein injection. Fiftecn to 30 minutes following the intradermal inoculaticn, there xppears at the site of injection a whenl-like srelling with intense “white edemas Surrounding the wheal a zone of crythemn appesrs which becomes increasingly 1‘ rger and more intense. The height of renction occurs between 30 and 60 minutes, after which a gradual regression tkes place leaving a firm pale A edematous aren which may require 24 hours or longer to subside. At the site of injection of the carbohydrates of the heterclogous types and of the salt solution, which serve as controls, no reaction appears, This q form of typical "wheol and erythema" renction is of the immediate anaphy- lactic type and is in sharp contrast to the delayed protein response, which requires 24 hours for maximum reaction and is allergic in character. Positive results have been uniformly type-specific, that is, the reaction yr has occurred only with the specific carbohydrate homologous to the type of pneumococcus causing the discase. To date it has been strikingly noted in cases of Type I pneumonia treated with Type I antipneumococcus horse serum. In these treated cases, large amounts of Type I antibodies are i introduced intravenously. In serum-treated patients the occurrence of a positive reaction to the Type I "St substance appears to be related to ! recovery. It seems possible that a positive test might indicate an excess EET 217 205 of sntibodies in the blood «nd thereby serve ns an index of the >mount of serum therapy required. These rerctions nre cf further intercst ns 1n exnmple cf the reretivity of the soluble svecific substances in hum ns. It his slso becn found that, in prticnts not tronted vith immune serum coincident ith spontrneous recovery, 2 positive reaction may follor the injecticn of the homologous "S" substence, In addition to observations on the skin rencticns, sera obtaincd at the same timc hive been tested for the presence or absence of anti- bodies reactive rith the test miterfal ~ i.e. pneumococcus nucleo-protcin and specific polysaccharides. From the data so for obtained, no significant difference hns been noted in the sern vith reference tc content of protein precipitine before or »fter crisis. In other words, there scemsto be no rel-tion betrcen the amount of antiprotein-precipitin in the sera and reactivity of skin to this substince. However, there does ~ppenr to be a dofinite correlation betveen the renction to "S" substances and the pre- sence of specific antibodies. although some cases possessing circulating anti~S antibodies do not react, no case has as yet been found which gives a positive reaction in the absence of anti-S antibodies. It is too enrly in the work to interpret the significance of these results. However, it is a striking fact that two distinct types of reaction to two chemically different constituents of Pneumococcus, may be clicited by skin tests in pneumonia patients; the one, produced by the corbohydrate is type- specific, immediate and anavhylactic in character; the other, produced by the protein, is independent of type-specificity and is »nslogous to the delayed reaction characteristic of allergic phenomena, IV. Natural and Acquired Resistances to "R" snd "S$" Porms of Pneumococcus,s (pr. Tillett). The experimental studies, reported in previous publications, on natural and acquired resistance of rabbits to fy R18 iw pneumococcus inf cticns have indicsted that immunity may exist in the ab- sence of demonstrable type-specific antibodies. The fact was established that normal rabbits are capable of withstanding lnrge doses of Type III pneumococci although antibodies reactive rith this organism could not be demonstrated in the serum of the normally resistant animal. It has 2lso been shown that rabbits immunized vith degraded ovirulent, non-type-specific pneumococci - so-called "R" strains - acquire a considernble degree of resistance against subsequent infection “ith virulent pneumococci of any of the three specific tynes. Active resistance was demonstrable under these conditions in spite of the fact that the sera of the immunized rabbits contained no detectable type-specific antibodies capable of aggliw tinating type-specific organisms, or of precipitating the soluble specific substances, or of conferring passive protection on mice against pneumo- coccus infections Furthermore, it has been shown that, although passive protection of mice is ineffectual, the whole blood or serum of rabbits immunized with "R" organisms is capable of conferring immunity on normal rabbits against any of the three specific types of Pneumococcus, The results of the ex- periments on active and passive immunity in rabbits immunized with "Re cells reveal characteristica which differ from type-specific immnity; it seems possible that the underlying mechanism is dependent upon factors other than those participating in type-specific pnoumococcal reactions. Moreover, the so-called "R" immunity - stimulated in rabbits by "R" cells and effective against virulent "§" pneumococci ~ has been shown to possess certain characteristics similar to the natural resistance which normal rabbits possess against most strains of Type III. This form of "RM" immunity appears to represent an enhancement of these factors which are concerned in the natural resistance of normal rabbits. oT a aE oan iiiy Y BEESETE. 2S BS 219 207 During the post yeorr cxzperiments heve becn corricd on in on attempt to determine more accurntcly the fectors involved in type-specific im- munity, in "RY imrunit;, ond in naturnl resistinec. For this purpose, robbits immunized vith tyoc-specific org-nisms, cthers immunized vith "R" organisms, together vith norm-1 control animals hevc been infected by intradermal incculstion. The character of the loc] lesions nnd the course of the resultant blood invasion hive been simultaneously followed. Experiments of this nxiture have afforded a means of compnring certain similsritics -nd differences “hich have been found to cxist in these forms of naturnl and acquired imuni ty « The aninnls used in theso experiments mniy be divided into three groups:- A. Normal rabbits. B. Rabbits immunized with "R" pneumococci, C. Rabbits immunized with type-specific pneumococci, The pneumococci employed for infcction have been as follovws:~ 1) "R strains. Degraded, avirulent, and non- type-specific forms of pneumococci. 2) "S" strains. Tro strains of Type III pneumococcus have been used: one, avirulent for rabbits (designated SA); the other, virulent for rabbits (designated SV). 3) "S" Pneumococci, Type I and II, virulent for rabbits. The results of these cxperiments may be summarized as follows:- Ae Intradermal infection of normal rabbits:- 1. "R" Pnoumococci. The skin lesion which develops is small, discrete, firm, slightly red, and nodular; it appears within the first 24 hours and disappears in three to four deya. Repeated blood-cultures remain sterile. Phagocytosis of "R" organisms by polynorphomatlear leucocytes is readily desonstrable. The infected cninals invariably recover. 2. "S" Pneumococci. Type Illy. & Strain avirulent for rabbits. The skin lesion developed is large, edematous, reddish-purple, and within 48 hours may extend to the midline ventrally; marked ecchymosis commonly occurs, after a fev days central necrosis and ulceration take place; several weeks is required for complete healing. Blood cultures taken at frequent intervals reveal the presence of organisms varying in number from time to time, and persisting for several days but ultimately disappearing. Phagocytosis of these "S$" organisms by volymorphonuelear leucocytes is not demonstrable. Recovery of the animal ensues, b. Strain virulent for rabbits. The skin lesion produced by these organisms is identical with that caused by the avirulent straim It is large, edematous, ecchymotic and extends ventrally tc the midline. Blood cultures taken at frequent intervals, show that organisms are present in the circulation.a few hours after inoculation; the septicapria rapidly increases in severity until death ensues from an overwhelming blood infection. No phagocytosis is demonstrable. The infection is invariably fatal. 3. "8" Pneumococci. Type I and II, The skin lesion and blood infection resulting from the injection of the virulent strains is identical wi th that described for the virulent Type III, The animals die in 36-48 hours, B Intradermal inoculation. rabbits immnized with "R" Pneumococcl, 1. "S"-Pneumococeci. Type III; Virulent for normal rabbits, The skin lesion produced is a large, edematous, reddish-purple inflammation which spreads ventrally to the midline; as in normal rabbits inoculated with 'S" organisms ulceration and necrosis set in and several weeks are required for complete healing. Frequent blood cultures demonstrate the presence of organisms in varying numbers for several days. They finally disappear, however, and recovery of the animal ensues, The character of (V this lesion, as vell as the bacteremia, are similar to that occurring in normal rabbits injected with Type III "§" strain, avirulent for the svecies. 2. "S$" Pneumococci. Type I and IT; Virulent for normal rabbits:= The skin lesion and course of blood infection are similar to that described for virulent Type III. C. Intradermal Infection of Rabbits Immnized with Type-specific Pneumococci, I. "S Pneumococei:- In this group of en nrerrnremenanneels animals the organisms inoculated were always of a type homologous to the "S" strain used for immunization. The skin lesion produced is small, discrete, firm, slightly red, and nodular. It appears within the first 24 hours, and disapyears in three to four days. This lesion is similar to that produced in normal rabbits by the injection of "Rv pneumococci. Blood cultures always remain sterile. Phagocytosis of the sensitized type-specific organisms by polymorphonu¢kar leucocytes is readily demonstrable. In an analysis of these experiments the reactions to skin in- fection as observed in normal and immunized rabbits offer certain similarities and contrasts. There appears to be, in one group of animals, a skin infection followed by recovery which May be cited as follows: 1, Reaction and recovery of normal rabbits from infection with "Rt pneumococci. 2. Reaction and recovery of type-specifically immunized rabbits from infection with homologous type-specific organisms. The character of the skin lesion in theso two instances is the same. A smajl, insignificant nodule develops which disappears ina few days. Organisms are never present in the blood stream, and prompt (\ re 210 phagocytosis is demonstrable in cach instance. Recovery depends pri- marily upon phagocitic activity. The type-specific organisms being sensitized by type-specific antibodies are quickly disposed of. The "R" pneumococci possessing no capsule can be ingested as such. Natural and acquired resistance to pneumococcus infection, as exempli- fied in these two instances, indicates a close similarity in the mechanism of recovery. In another group of animals, the reaction and recovery differ from the first group and may be cited as follows:- 1, Reaction and recovery of normal rabbits from infection with "St pneumococci, Type III, avirulent for the species. 2. Reaction and recovery of "R" immunized rabbits from infection with virulent type-specific organisms I, II or III. Tie character of the skin lesion in these two instances is similar. A large, edematous, ecchymotic, purplish, spreading inflam matory reaction follows the intradermal inoculation of the organisms. Ulceration and slough ensue snd several weeks are required for complete disappearance of the reaction. Organisms may be present in the blood stream in varying numbers for several days before their final disappear- ance. In neither instance can phagocytosis by leucocytes be demonstratdl, Natural and acquired resistance to pneumococcus infection, as exempli-~ fied in these two instances, also indicates a close similarity in the mechanism of recovery. The degree of the inflammation, however, and the duration of the infection differ considerably from the mild lesion and transient infection described in the first group. Consequently, it seems not unlikely that the underlying mechanism is different in each instance, \ In the first groun, prompt phagocytic activity appears to be the r2 initinl renetion involved while in the sccond group the factors arc as yet not undcrstood. In so far as the work has progressed it scums unlikely that the primary reactions are duc solely cither to antibodics, as ordinarily understood, or to phagocytosis. However, the recent work of Griffith has shown that "R" pneumococci possess the crpacity to chang into any of the specific types. Consequently, it m-y not be impossible that "R" cells possess some incremcnt capable of inciting a response specific for each type of pneumococcus ond that this is responsible for the broad immunity resulting from immunization with "R" cells. The work hns not as yet progressed sufficiently to justify any con- clusions as to the naturs of the so-c>lled non-type-specific resistance. V¥. Intracutaneous Veccination of Rabbits with Pneumococcus. Js Antibody Response. (Dr. Julianelle). Suspensions of heat-killed pneumococci were injected into the skins of rabbits at intervals of seven days during a period of 10 to 14 weeks, in doses such that the total amount of bacterial substance injected was at least equivalent to that employed .in the routine intravenous immunization of rabbits, The antigens studied were Types I and III and a capsule-free "R" strain derived from a Type II organism. The sera of the rabbits immunized in this way were tested for the présence of agglutinins, precipitins and protective antibodies. The sera of rabbits immunized intradermally with Type I failed in more than 85 per cent of the animals to show any type-specific anti- bodies. They failed to agglutinate the corresponding type of pneumo~ coccus, and they failed to precipitate the homologous specific poly~ saccharide. In rare instances, passive protection was conferred upon white mice against infection by Type I, but in such cases the protec- tion afforded was not great, In the remaining rabbits, although type- specific antibodies were present, the titre as measured by agglutination \ (v wos low ranging from 1:1 to 1:20. On the other hand, none of the rabbits immunized with Type III by way of the skin, showcd any demonstrable type-specific antibodies. Thc ir sera showed 2 complete lack of type-specific agglutinins, precipitins, and protective santibodics, Although in the majority of instances, the type-specific pneumococcus failed to stimulate type-specific antibodies when intro- duced into the skin of rabbits, the serum of 211 the animals studied contained . high titre of the antiprotein, species-specific antibody, agglutinated "R" cells, and precipitated solutions of protein derived from »ny type of Pneumococcus. The intracutaneous inoculation of rabbits with "R" cells stimulated the produc tion of only the species- specific antibodies. In the majority of instances therefore, the results of immnization with type-specific Pncumococci cannot be distinguished from those obtained by immonization with "R" variants. Later rabbits were immunized with nucleovrotein solution and a purpura-producing extract in the manner described above. In no instance were type-specific agglutinins or precipitins demonstrated and no protective antibodies were found. The sers of these rabbits, however, possessed species-specific antibodies, II. Resistance to infection. It may be stated bricfly that following repeated skin inoculation of heat-killed suspensions of Pneumococecus, rabbits acquire a considerable degree of active immunity against infection tv virulent strains of both homologous and heterolo- gous typese For example, rabbits immunized intrecutaneously with Type I may survive the intravenous injection of two million lethal doses. Similarly, rabbits immunized with Type III mav survive a thou- sand or mor. minimal lethal doses. Moreover, animals inoculated {a 213 intracutancously vith Type III become 1s resistant to infcetion with Typc I as ~nimals similerly immunized with homologous organisms; and animals immunized with Type I possess 2 comprrable degre. of immunity to Type III infection. Even when on "3" strain derived from Type II is used for injection, robbits scquire . high deere: of active immu- nity to infection with virulent "S$" streins of hetcrologous type. Although rabbits tre-sted in this menncr develop 2 solid imm- nity ageinst infection with both homologous and hetverologous types, nevertheless the serum of these animals usu2lly confers upon white mice little or no vassive protection agrinst infection by even the homologous type. III. Hypersensitiveness. The intracutanecous injection in normal rabbits of 0.2 cc. of n hentcd vaccine, representing the bacteria from 2 cc. of broth culture is followed by a circumscribed, slightly raised and indurnted nodulc, reddish in color and measuring about 1 cm. in dinmeter. Upon repeated injection of the same amount of bacterial suspension at weekly intervals, the reaction changes in character; the size increases often reaching a maximum of 4 to 6 cm. in dinmeter, accompanied by a spreading edema and purplish discoloration. The re- actions persist longer, often breaking down with the discharge of sterile necrotic materi2l, and a maximum reactivity is reached after about 6 injections. Thereafter, the reactions are less intense and are more quickly resorbed. Two to three weeks after the final injection (8 to 12), the animals may be sensitive to the nucleoprotein and other protein derivatives of Pneumococcus when tested by the skin and ophthalmic reaction. In the doses employed, the nucleoprotein solution gave no local reaction when injected into the skin of normal rabbits, while extracts Cr ose lagpeppeiin mei = pete age pee rt cnt eet A SAR AERO ROR ote is Me een ER eee A Sa nanan nn Leche ithe meen op tes” ott gah A Sips ii Non Mina waa att containing purpura-producing. material caused » faint erythems in about half of the normsl controls. In skin-vaccineted rabbits, on the other hand, the protein substances induce an inflammatory reaction at the site of injection which my persist for threr to five days. In testing eye sensitivity in the skin-vaccinated rabbits, the cornea was anaesthetized and lightly scarified, and one drop of nucleo- protein or purpura-producing extract was then instilled into the con- junctival sac. In normal rabbits this procedure Causes no visible reaction. In rabbits sensitized by the intracutaneous method, a defin- ite reaction occurs within 24 hours which increases in intensity during the firat 24 to 48 hours and disapvears after 4 to 8 days. The reaction consists first of conjunctivitis with dilataticn of capillaries at the sclerocornenl junction, with clouding of the cornea end last of all development of panmts which occurs only occasionally. The ophthalmic reaction shows great varistion in severity in different rabbits and may not proceed beyond the stage of corneal turbidity. Of over 50 snimals immunized by the routine intravenous method, none have given a positive eye reaction, while 90 per cent or more of the rabbits immunized intra- cutancously reacted positively when later tested to protein derivatives of Pneumococcus, The hypersensitiveness is not elicited in sensitive animals by instillation in the eye of purified type-specific carbohydrates. The reaction therefore, is not type- but species-specific. The hypersensitive state may persist for at least four months. An interesting point in this connection is the fact that after the ophthalmic reaction has definitely diseppeared, the intravenous in- jection of nucleoprotein solution often causes ». reappearance of the eye reaction. VI. The Reversicn of "2" Priedlsnder bacilli to the "S" Forms, and the Interconvertibility of Specific Types. Numerous attemmts have been previcusly mode to cause reversion of "R" Friedlander bacilli to the "St variety by the various methods which have been effective in the case of Pncumococcus. The results of these attempts have been uniform- ly negative, and the reversion of an "R" form cof Friedlander's bacillus to its originel svecific tyne had never boen attained by "in vitro" methods, The recent studies of Griffith have demonstrated that, under propcr conditions, "R' pneumococci may be induced to revert not only to the "St antecedent, but even to the "S" form of a heterologous type. The method he employed consists of the simultancous injection subcu- taneously in white mice of the deposit of large quantities of heat- killed "S" orgonisms end small amountsof living "R" cells. The con- versicn to a different type, when it does occur, is to the type of the culture employed as vaccine. Subsequent confirmation of Griffith's results in this leboratory, suggested the possibility of the reversion of "R" forms of Friedlander bacillus by the newer technique. Up to the present time two sets of experiments have been comple- ted. On one occasion, of six mice injected with the deposit equivalent to 40 cc. of Type A culture heated for 70 minutes ot 5-60°C together with 0.25 cc. of "R" organisms derived from Type B or Type C, 7 mice yielded cultures containing both "R" and "S" colonies. the S- colonies proved to be of the Type A variety. Three mice which received similar quantities of heat-killed bacterie alone were sacrificed after 10 days, end in none of them were any bacteria cultivated from either the site of injection or the heart bloods On & second attempt, 4 mice each were injected with the deposit 228 216 of bacilli from ZO cc. of Type A culture previously heated (20 minutes at 56-60°C), together with 0.25 ec. of "R" culture dcorived from Type B, Type C and Grouv X. Tro mice of each group yielded cultures containing both "R' and "S" colonies; all the "S" colonies isolated, proved sero- logically to be of Type A. Four mice injected with the ssme quantity of Type A vaccine and 0.25 cc. of "R" cells derived from Tyve A, yielded cultures composed purely of "R" colonies. As controls, 4 mice received the heat-killed Type A devosit alone. They were killed after 10 days and cultures taken from the site of injection and from the heart bloou showed no growth. Since previous results from this laboratory showed that Type II Pneumococcus and Type B, Fricdlander's bacillus possess similar anti- genic properties, observations werc made on the reversibility cof "Rt Friedlander bacilli when injected subcutaneously into white mice together with heat-killed suspensions of pneumococcus Type II. Three different sets of experiments were crrried out, but in no instance was it found that Type II Fneumococcus exerted sny influence on the rever- sionof Friedl4nder's bacilli to either homologous or heterologous types. Tyo sets of oxporimente were performed to determine the effect of heat-killed cells of Type B, Friedlander's bacillus on the reversi- bility of "R" Pneumococci. In the first cxperiment, 3 mice each were injected with the deposit equivalent to 40 cc. of Type B bacilli, heated for 20 minutes at 56-60°C together with 0.25 cc. of "R" forms derived in one case from Type II and in the other from Type III Pneumococcus. Three of the six mice yielded cultures of mixed "R" and "S" pneumococcus colonies and in e-ch instance the "S" colony corresponded to the type from which the "X!t strain had been derived. On a second occasicn, 4 mice each were injected with 0.25 cc. of a culture of "RB" colls derived respectively from ercn cf th: three fixed types of Pneumococeus together “ith the devosit of hertcd bretcria (56 - 60°C for 30 minutes) ef 70 cc. culturc of Tyne B, Friedl#ader's bocillus. In no c-s., homever, 7s revorsion of the Pneumococcus variants okserved. Although the ditn from these observations are insufficicnt to warrant ony conclusicn, ther indicate that (1) "R" streins of Pricdlindr's brcillus m-y be converted to the "S" form of a different type; (2) "R" pneumccocci may be reverted to their homologous "S" type then injected subcutancously in mice with heat-killed suspensions of Type B, Fricdlsnder b-cillus; and (3) under the circumstances stated, URW gptrains of th. Friedl4nder becillus have never besn observed to Type II Pneumococcus,. VII. Immunity Induced by the Inhalation cf Virulcent Pneumococci. (Dr. Stillman). Studics of the inmunc response of rnrbbits to repeated inhslaticns of living pneumococci have been continued. The character and duration of immunity induced by inhnlaticn varies with the type of pneumococci used. It hes been demonstrated that rabbits chich have been repeatedly sprayed with Type I pneumococci develop in their serum type-specific agglutinins “nd protective antibodies. After the inhnlations of pneumococci are discontinucd, the agglutinins rapidly disapneor. The protective antibodies persist for long periods. The sera of to rabbits which have survived treatment and are now living, »lmost three years (9990 -nd 1020 deys) after ‘heir last exposure to sprayed pneumococci, still protect mice .vgainst infection with large doses (0.01 cc.) of a virulent culture of the seme tyve as that with which thoy wore original-~ ly sprayed. 230 wes The sera of rabbits sprayed 10 times with » strain of Rr pnceumo- coccus, derived from Tyve II organisms contrined no type-specific anti- bodies, The sere cf rabbits repentedly exvosed to sprayed mousc- virulent but not rabbit-virulent Type II pneumococci contained vrotective ontibodics but no dexonstreble agglutinins. The some strain of Type II was rendered rabbit-virulent by pnssage through » series of rabbits. The rabbits sprayed vith this strain s-oved the presence of both pro- tective antibodies -nd agglutinins in their serum. Two groups of rabbits have bcen repeatedly sprayed with a rabbit- avirulent and a virulent Tyne IIT pneumococcus. The sera of the rabbits exposed to »-n atmosphere cf avirulent Type JII orgsnism contained no type-specific antibodies. Although a large number of the cnimals dicd of pneumococcus septicemia following the firet exposure to the rabbit- virulent strain, those which have survived repented exposures have, 50 far, failed to develop any detectible type-specific antibodies. A series of rabbits were intravenously injected with a heat-killed sus- pensicn of the same rabbit-virulent strain. ‘The sera of a number of these »nimals contained both agglutinins and protective antibodies. The duration of the immunity in rabbits actively immunized by the inhalation method and by inoculation is being followed, VIII. Significance of oxidation-reduction processes in bacterial growth. (Dr. Dubos). It has been shown by previous work from this depart- ment that Pneumococcus cultures respond very readily to changes in the conditions of oxidation-reduction of the medium and environment in which they are placed. An attempt is being made to analyze the influence of such factors on the growth of Pneumococcus. ‘The following points are being studied;- Initiation of growth, as measured by the minimum number of cells (used . O41 219 1s inoculum) requir:d to initiate erowth; the growth curves and lag period; the density of growth, and thc number of cells developing per anit volume of medium; snd the vinbility of the cultures. It has been found that on understanding cf the problem could not be obtaincd without some kmovlcdge of the oxidnaticn~reducticn proper- ties of sterile media. When sterile plain broth is kept in the abseico of oxygen, this medium exhibits a progressive drift tovard highly reducing potentinls. This drift has been followed electrometrically and by the reduction of indicators of oxidation-reduction potentials. These indicators are progressively reduced by sterile broth in the order of the electromotive series; the indophcnols being reduced most rapidly (in a few hours) and indigo disulfonate more slowly (in several doys or weeks). Indicators with an Ei, negative to thet of indigo di- sulfonate were not reduced under any circumstances. These studies also indicate that plain broth contains substances which combine with oxygen to form toxic products ond ghat these oxidized substances may be decomposed by heat. 1) Initiation of growth. The number of cells of Pneumococcus necessary to initiate growth may vary a great deal with the age and condition of the broth. It appears that, in the presence cf oxygen, there is formed in plain broth a toxic substence which has a bacteriostatic - and even bactericidal - action on Pneumococcus and related organisms, This action may be checked by addition of certain reducing substances, by incubation of the culture under anaerobic conditions, by addition of blood and by heating the broth previous to inoculation. The action of these procedures may be explained by a reduction or breaking down of the toxic substance. The use of a large inoculum serves the same purpose. It was possible to trace the origin of this toxic substance oa) a 220 tc the peptone uscd in the pruprration of tho medium. Difforent peptenes behave differently in this respect, Wittels and Difec-pretccse peptoncs exhibiting to the smollast degree the ability to fcrm texic substances in the vresence of nir. “hen precauti-ns are ta'en to prevent the formiticn of th se texic cubstances, or when they are remcvcd by proper means, growth of Pneumococcus 1nd related organisms cin be obtained even when one or very few cells are used as incculun. 2) Bacteriostatic ncticn cf cxidizod dycs. Another evidence of the bacteriostatic action «f cxidized substoncces has bern obtoined from the study of the section of certain dves on bect.riel grorth. Wherens, the growth of many orgenisms is checked b the presence in the medium of the oxidized forms of certain dyes (indonh. nels ~nd methylene blue fcr instance), the reduced forms of the same dycs - even in lorge concentrations - sre without eny inhibitory action cn grevth. There arc also indications that the place of the reversible dyes in the oxids- tion-rcduction scale benrs 1 definite relation to their bacteriostatic action for diffcrent crgnnisms, 3) Growth curves snd lig period, Scme of Chceney's cbservations on the growth curve of Pneumoccccus can be partly accounted fer by the ecnditions of oxid-ticn-reduction prevailing in the medium. The course of development of the culture msy be very much accclerated by the pres- ence of reducing substances in thc medium or the meintenance of reducing conditions. The lag pericd probably corresvonds to the tine during which the cells are reducing the toxic substances of the :cdium, in which process some of the organisms are injured and succumb. 4) Viability. It hos been shovn in - previcus riveit that the processes of cellular oxidaticn markedly influeice the denth rate of pucumococeci. Under conditions of active aersticn, preuccccccus ce 233 esl cultures form large «mounts sf peroxide. It was founda that the viability of the cells is lessened when the peroxide production is increased, while the cells remnin »live longer then the conditions are such that the formition or accunulation of peroxide is prevented. The oxidation-reducti’n systoa.. of pneumococcus cells consist cr two ossentinl components. Cne of them can be revoved by washing the cells repentedly in salinc soluticn; this prccess results in 1 complete inactivetion of the oxidatinn-reduction system. It hrs beon observed that pneumococci mhich hove been deprived ef their oxidotion-reducticn activities by washing in snline solution remin viable lenger than the originnl unwashed cells. In the course of this werk, it has become morc and wore evident that an analysis of the oxidaticn-reducticn properties of Pneumoccecus cultures is renderod esvecinlly difficult by the fact tnat plain breth itself is en active and dynamic systca fro this point of view. York is in progress to develop n synthetic medium the behavior of which can be interpreted more nccurntely. Publications. Avery, 0.T., and Tillett, W.S., Anophylaxis mith the type-specific carbo- hydratee of pneumoccccus. J.Exp.Med., 1929, xlix, 251. Dawson, MeHe, The interconvertibility of "R" ond nSu ferns cf pneumo- ccccus. J-Exp.Med-, 1928, xlvii, 5770 Dubos, ReJ., Observaticns cn the exidnticn-reduction properties cf sterile brcteriologic;l medin. JeExp.Med., 1929, xlix, 507. Dutcs, Red., The initiation of growth cf certein facultative annero bes as related tc cxidation-reducticn processes in the wediun. J.Exp.Med., 1929, xlix, 559. Dubos, Red., The relation of the pactoricstatic action cf certain dyes to cxidaticn-reduction processes. J.Exp.Med., 1929, xlix,575. Goebel, WeF., and Avery, O-T., A study cf pneumococcus autolysis. J.-Exp.Ned., 1929, xlix, 267. sod 222 Julianelle, LAs, Bocterinl varinticn in culturcs of Fricdlander's bacillus. J.Bxp.lied., 1928, xlvii, 889, Julinnelle, LeAe, and Avery, 0.7., Intracutancous voecination cf rabbits with pneuroccecus. I. Antibody resnonsc. II. Resistance to infection. III. Hypersensitivencss. Proc.Soc.Exp.Biol.and Med., 1928, xxvi, 224. Tillett, 7.S., Active and wossive immunity to pneurococcus infocticn induced in rnboits by ivmunizaticn with "aR" oneumococcl. J.Exp.bed., 1928, xlviii, 791. Avery, O-T., and Dubdcs, R.J., The relation of oxidaticn-rivduction processes tc bicterinl antogonisn. (in rnenuscript). Avery, 0.T., ond Julianelle, LeA., The immune responses of ~hite .ce to injecticns of purificd soludle specific substance of Pneumococcus. (in monuscrint). Avery, 0.T., Geotel, W.F., ond Heidelberger, W., A scluble svecific substance fio gum arnbic. J.Exp.Med., 1929, (in press). Goebel, W.F., Dinzco Hexoside Preteins. The synthcsis of narnvasinophenel -glucoside and galactoside onc their counling “ith serum globulin. (in manuscrinvt). Stillman, Ernest., The eff.ct cf the reute« of irrunigaticn in the imeuniy response te pneumecoccus Tync I. J.Exp.hed., 1929, (in pross), Stillman, E., Production cf oneumococcus “neumenin in psartiolly immunized mice by the inhalaticn method. J-EapeNed., 1929, (in press). Stillmen, E., Experimental pneuriococcus pnoumcnin in the guiner pig. J.Bxp.Med., 1929, (in press). Stillmen, E., Early pulmonary lesicas in partially irrmne alccohcliged nice following inhnlation of virulent pnoumocecci. J.Exp.Med., 1929, (in ress).