BACTrIOLOGICAL REVIEWS, Dec. 1974, p. 371-402Copyright 0 1974 American Society for Microbiology

Vol. 38, No. 4Printed in U.S.A.

Vaccines and Cell-Mediated ImmunityFRANK M. COLLINS

Trudeau Institute, Inc., Saranac Lake, New York 12983

INTRODUCTION.371CELLULAR IMMUNITY TO INTRACELLULAR PARASITES.372ROLE OF "VIRULENCE" FACTORS IN THE DEVELOPMENT OF CELLU-

LAR IMMUNITY.373TRANSFER OF ACQUIRED RESISTANCE FROM VACCINATED TO NOR-

MALRECIPIENTS.375MEDIATORS OF CELLULAR IMMUNITY.376Mononuclear Phagocytes.376"Activated" Macrophages.378T-Lymphocytes.381

RELATIONSHIP OF DTH TO CELL-MEDIATED IMMUNITY.387INDUCTION OF CELL-MEDIATED IMMUNITY WITH VARIOUS TYPES OF

VACCINE.388Living Vaccines.388Inactivated Whole Cells.389CellComponents. 390

ROLE OF ADJUVANTS IN THE DEVELOPMENT OF CELL-MEDIATED IM-MUNITY.392

LITERATURECITED.394

INTRODUCTIONThe mechanism by which the normal host

overcomes a microbial infection and the reasonsfor the accelerated secondary response followingreinfection with the same microorganism hasintrigued microbiologists, immunologists, andepidemiologists for almost a century. The twoprimary arms of the host defense system werefirst described about the turn of this century,but the manner of their interaction with oneanother and with the parasite during the devel-opment of acquired resistance by the hostcontinues to be a matter of some controversy(125, 159, 164, 194, 247). The classical roles forspecific antitoxic, opsonic, and bactericidalantibodies in preventing many microbial infec-tions present an intellectually satisfying expla-nation for the humoral response almost invaria-bly seen in the convalescing host (195), butattempts to explain antimicrobial resistancesolely in terms of such immunoglobulin-mediated host responses (213) now seem toosimplistic (53).

Circulating specific immunoglobulins un-doubtedly do protect the host effectively againstmany infectious agents, especially those causedby the toxigenic microorganisms, some of theviruses, and the obligate extracellular parasitessuch as Diplococcus pneumoniae (75) and Pas-teurella multocida (17, 58). However, there is noindication that humoral factors alone can pre-vent such diseases as listeriosis, tuberculosis,brucellosis, or salmonellosis, in either actively

or passively protected animals (152, 154, 165,264). The inability of passively introduced anti-body to protect mice against salmonellosis hasbeen ascribed to the use of insufficient amountsof hyperimmune serum (127). However, enu-meration of the systemic bacterial population insuch animals indicates that almost as muchgrowth occurs within the tissues of the serum-protected animal as in the controls, despite thefact that the survival rates for the former aresignificantly increased (49). Such serum treat-ment increased the early inactivation of thechallenge population 10- to 100-fold (50, 115)but then, despite the continuing presence ofbactericidal antibodies in the circulation (135),the survivors multiplied extensively in vivo sothat the systemic population eventuallyreached near lethal proportions (32, 50), with anongoing bacteremia (135, 165) and an extensivedegree of multiplication by the pathogenic orga-nisms within the host's phagocytic cells (32, 67).The elimination of this systemic populationrequires the presence of activated or "angry"macrophages within the infected tissues, and inthe passively immunized host these must beproduced de novo, as a result of the challengeinfection itself (161). The specific humoral fac-tors in the circulation contribute very little tothis later intracellular bactericidal process(102). Largely as a result of this type of study,few immunologists would now espouse a purelyhumoral mechanism to explain acquired anti-bacterial resistance, although it is interesting to

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note that the immunological literature stillcontains numerous references to the inductionof "immunity" and "immune responses" in anexperimental context in which "humoral anti-body production," "plaque-forming cellcounts," or "blood clearance indices" mightperhaps be more properly used.The present review will be primarily con-

cerned with those aspects of cellular resistanceinvolved in the innate and acquired antimi-crobial activity of the activated macrophage.The immunobiology of T- and B-lymphocytesand their interactions during the developmentof cellular hypersensitivity by the appropriatelyimmunized host constitute one of the mostexciting areas in contemporary immunology,but this aspect has been the subject of severalrecent reviews (30, 189, 194) and will not bedealt with in great detail in the present paper.

CELLULAR IMMUNITY TOINTRACELLULAR PARASITES

Much of our present knowledge regardingcell-mediated immunity stems from the pion-eering studies of Lurie (146) carried out intuberculous rabbits. In a series of classic experi-ments, he demonstrated the need for activatedor "immune" macrophages if the growth ofvirulent tubercle bacilli was to be inhibited invivo (145). Such active cellular resistance wasassociated with the development of a tuberculinskin hypersensitivity by the host (153, 164);both of these responses seem to require thepresence of specifically sensitized T-lym-phocytes (158). At first, this close association ofthe two cellular responses to the mycobacterialantigens seemed to place the tubercle bacillusin a unique position as an infectious agent (154),but it is now realized that many other microor-ganisms can induce specific states of delayedskin hypersensitivity in the infected host (159).The term "facultative intracellular parasite"has been coined to describe such organisms, allof which are able to multiply actively withinnormal host phagocytes (239). Highly virulentstrains of Mycobacterium tuberculosis, Bru-cella abortus, Salmonella typhimurium, andmany other organisms are able to multiplyfreely within the phagocytic cells of the normalhost and may eventually kill the involved phag-ocytes (167, 218). Inactivation of such orga-nisms requires the entry of "activated" or"angry" macrophages into the lesion; the gener-ation of this specialized population of mononu-clear cells on the part of the infected hostinvolves a process referred to as "cell-mediatedimmunity" (157).

The above discussion should not be taken toexclude an important role for specific humoralfactors in the overall defense of the host. Therehas been a tendency in the past to allot apredominating role to either humoral or cellularfactors, often to the virtual exclusion of theother arm of the defense system (53). As aresult, a long-standing debate has developed inthe literature regarding the relative importanceof humoral versus cell-mediated defenses inprotection against many of these diseases (125,211, 247). Much of this controversy stems fromthe fact that the contribution made by the twodefense systems can vary extensively dependingupon the strain of organism, the host, and theexperimental conditions selected by the indi-vidual investigator for a particular study (11,32, 126). For instance, some investigators rou-tinely use an intraperitoneal route of challenge(125) and may even add gastric mucin toincrease the virulence of the challenge organism(234), whereas others recommend an intrave-nous (32), an intragastric (51, 62), or a subcuta-neous (57) route of challenge. At the same time,"immunity" may be assessed in terms of anincreased survival rate by the vaccinated group(73, 80, 132, 199), an increase in mean survivaltime (208, 212, 267), or a change in the size ofthe median lethal challenge dose (114, 169).Many of these experimental protocols indicate adegree of protection without giving a clue as tothe precise nature of the defense mechanism(s)involved. This may be particularly importantwhen nonspecific resistance factors are alsoinvolved (209). For instance, the increased rateof phagocytosis seen in the lipopolysaccharide-stimulated mouse peritoneal cavity may resultin sufficient inactivation of a lethal challengedose of unrelated microorganisms to permit thehost to survive. However, the actual defensemechanism involved may not depend upon thespecificity of the antigenic groups in the stimu-lating preparation (46, 212).As a result of numerous in vivo studies, it

seems that the best way to study the immuneresponse in a vaccinated animal is to comparethe rate of growth or inactivation of a sublethalchallenge population of bacilli in test and con-trol groups throughout the entire infectiousperiod (67, 165, 248). Serial bacterial countscarried out on liver and spleen homogenatesprepared from intravenously (165) or orallychallenged mice (51) have demonstrated un-equivocally that the effectively vaccinated hostcan express an accelerated antibacterial re-sponse to the challenge population from thetime of infection (69). This will ideally beexpressed as an immediate sharp decline in the

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bacterial counts in the liver and spleen follow-ing challenge. Thus, to be fully effective, avaccine must not only increase the early inacti-vation of the challenge population in the tissuesbut there must also be an enhanced activeantibacterial response capable of eliminatingthe entire bacterial population from the tissues.Only in this way can a clinical attack of thedisease be completely aborted. Thus, any ra-tional study of the role of vaccination in theprevention of infections caused by intracellularmicrobial parasites must be primarily con-cerned with the development and maintenanceof this state of cell-mediated immunity by thehost (160).

ROLE OF "VIRULENCE" FACTORS INTHE DEVELOPMENT OF CELLULAR

IMMUNITYVirulence seems to depend primarily upon

the rate of growth by a given strain of organismwithin the tissues of the susceptible host (38, 49,53). Despite considerable effort, no direct corre-lation has yet been shown to exist between theendotoxic content of the cell walls of varioussalmonellae and their virulence for experimen-tal animals (69, 147, 168, 178, 210, 249). Al-though most virulent salmonellae are antigeni-cally smooth and the transition from smooth torough is associated with a sharp loss of virulence(206), the reasons for this effect are still notclear. Immunochemical studies of the lipopoly-saccharides of smooth and rough salmonellaeindicate structural changes (144), but there isno explanation as to how these result in a loss ofvirulence, since endotoxins isolated from roughorganisms are still highly toxic for the host (38).Endotoxins have a variety of potent pharmaco-logical effects in the intact animal (176), but itis not clear what role this toxicity plays in thepathogenesis of the resulting disease (206). Infact, the mechanism by which virulentSalmonellae actually kill the host is still notclear; death does not seem to be due to a simpleendotoxemia since the total population of viru-lent S. typhimurium or S. enteritidis within thecarcasses of moribund mice seldom exceeds 10'viable bacilli, and perhaps 50 times this numberof heat-killed bacilli are needed to kill theanimal. Furthermore, BCG-infected mice areexquisitely sensitive to endotoxic challenge(240), but seem to be very little more suscepti-ble to living S. typhimurium challenge thannormal mice (122). There is considerable evi-dence that actively infected individuals becometolerant to the endotoxins of the causativeorganism (109, 184), but still the host succumbsto the infectious agent once the viable popula-

tion reaches 10' to 109 bacilli in the liver andspleen (117).

Despite these largely incompatible data,much of the rationale underlying recent studiesof the immunochemistry of the surface antigensof the Enterobacteriaceae and the role of thecorresponding humoral antibodies in the ex-pression of immunity to mouse typhoid fever ispredicated upon the assumption that thesmooth somatic and/or capsular antigens areprimarily involved in the pathogenesis of en-teric disease. The mechanism by which thesmooth somatic antigen antagonizes the normalhost defenses is still unclear (206). The long-chain smooth lipopolysaccharides may welllimit the access of lysosomal enzymes to theunderlying structural cell wall elements, so thatbacteriolysis is slowed or prevented. Such apostulate is almost certainly naive, however,since quantitative differences are known to existbetween the median lethal dose values for fullysmooth salmonellae bearing different smoothsomatic antigens (169). However, within a sin-gle group, the amount of protective surfaceantigen in strains of high and low virulencewould have to play an important role in slowingor preventing intracellular digestion of the for-mer. Such a thesis would be compatible withreports that dead Salmonella vaccines preparedfrom highly virulent strains (especially whengrown in vivo; 192) may be more protective thanthose prepared from attenuated organismsgrown in vitro (11, 126). However, such a directrelationship between the amount of smoothsomatic antigen on the Salmonella cell wall andthe immunogenicity of that organism cannotexplain the ability of many antigenically roughsalmonellae to protect mice and guinea pigsagainst challenge with the fully smooth paren-tal strain (104, 229). In at least one case, thisseems to be due to the ability of a gal-Edeficient mutant to synthesize the smooth so-matic antigen when growing in an intracellularenvironment (104). Other rough strains may beable somehow to persist in vivo long enough toinduce sufficient cellular immunity to nonspe-cifically protect the host against the later chal-lenge with the smooth parental strain (105,131). Where growth data show that the roughstrains cannot survive in vivo or when heat-killed suspensions of these organisms are used,all protective value disappears (24, 104, 229).On balance, the above findings seem to beconsistent with the thesis that the lipopolysac-charide-containing cell wall antigens are notdirectly involved in the development of cell-mediated immunity (64).Even closely related smooth strains of

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Salmonella may differ enormously in their viru-lence for mice (67, 100) and for chickens (129,228, 233), despite close similarities in the lipo-polysaccharide content of the organisms (47).Growth rates determined in vivo for a number ofgroup D salmonellae in mice and chickens differenormously, and it is this parameter of thehost-parasite interaction which seems to pro-vide the most accurate index of the virulence ofthe organism for a given experimental host (38,53). Blood clearance rates, sensitivity or resist-ance to the bactericidal action of specific anti-bodies in vitro, and percent inactivation bynormal peritoneal macrophages (32) may bearlittle relationship to the virulence of the orga-nism in the naturally infected host. If thisoverall growth rate by the organism within thereticuloendothelial organs of the host is theultimate controlling factor for Salmonella viru-lence, then the nature of the litniting metabolic,nutritional, or transport steps responsible forthese differences is still not at all clear (114,138) and further work in this area is clearlyneeded.Many of the "protection" studies carried out

with killed Salmonella vaccines have been pri-marily concerned with the toxigenic responsesby the challenged host, or with changes in therate of blood clearance or intracellular inactiva-tion of the challenge population by peritonealcells taken from vaccinated mice (15, 179).Relatively few studies have concentrated on thelevel of antimicrobial resistance (53, 56), andmany of these have used the intraperitonealroute of infection (32, 125, 208), despite prob-lems associated with the interpretation of suchdata (32). This has resulted in some semanticproblems regarding the use of the term "protec-tion" (211). Obviously, the survival of 100% of agroup of challenged, vaccinated mice in the faceof 100% mortality in the normal controls repre-sents a very real degree of protection (17).However, it is more difficult to assess thebiological significance of a 50% increase inoverall survival, regardless of the fact thatstatistical significance may have been demon-strated. This is especially the case when aunique inoculation method or some form ofprior immunosuppression must be used to en-hance the virulence of the challenge organism(126, 171, 181). In this context, increases in themean time to death may have very little rele-vance if the majority of both vaccinated andcontrol groups eventually succumb to the chal-lenge infection (270). In an attempt to obviatethis type of criticism, we have used serialbacterial enumeration of the in vivo bacterialpopulations to compare the fate of a sublethal

challenge population in the vaccinated andcontrol animals (165). Under such circum-stances, death or survival of the host no longerconstitutes a usable parameter of the hostresponse; the development of a protective ac-quired resistance can only be inferred from thepresence of a significant shift in the systemicbacterial growth rate at an earlier stage of thechallenge infection. Such an accelerated anti-bacterial response must be sufficient to preventcompletely the development of a clinical attackof the disease (32, 50), a criterion widely used infield trials of human typhoid vaccines (77). Theeffective vaccine will so sensitize the host thatan accelerated recall of the earlier inducedcell-mediated immunity will occur almost im-mediately following re-exposure to the parasite.The time required to regenerate this type ofprogressive antibacterial response is perhapsone of the most important parameters of theimmune reaction (48). The above discussionshould not be taken to mean that mortality dataare of no further use; death or survival should,however, be combined with an assessment ofthe bacterial growth rate in vivo and the demon-stration of a cell-mediated type of immuneresponse. In this way, the most comprehensiveassessment of the immunogenicity of differentvaccine strains may be made. This situation canperhaps best be illustrated in the followingexperiments.Groups of specific pathogen-free CD-1 mice

were infected with sublethal doses of the highlyvirulent S. enteritidis 5694 (intravenous LD50 =5 x 103 organisms), the mouse attenuated S.gallinarum 9240 (intravenous LD50 = 8 x 106),and the avirulent strain S. pullorum 223 (intra-venous LD50 > 108). The S. enteritidis popula-tion increased almost 1,000-fold over the first4-day period of the infection, whereas the S.gallinarum population remained relatively con-stant and the S. pullorum population declinedsharply from the time of inoculation (Fig. 1).Challenge of these three groups of "vaccinated"mice with a lethal dose of a drug-resistant strainof S. enteritidis SMR (intravenous LD60 = 104organisms) clearly indicated that both the S.enteritidis- and S. gallinarum-vaccinated micewere fully protected against the challenge infec-tion. There was, however, no progressive inacti-vation of the S. enteritidis SMR population inthe S. pullorum-vaccinated mice, at least overthe first 7-day period (Fig. 1). However, fromthe number of deaths observed up until day 7(Table 1), the S. pullorum-vaccinated miceseemed to be better protected from the lethaleffects of the challenge than the normal con-trols. By day 14, this "protection" had van-

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ished, constituting only a small increase in themean time to death, with both groups of miceshowing a mortality near 100%. Protection maybe made to appear greater (at least so far as

mortality is concerned) by using smaller chal-lenge doses (1 to 2 LD5O) or a strain with

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FIG. 1. Growth of S. enteritidis 5694 (top), S.gallinarum 9240 (middle), and S. pullorum 223 (bot-tom) in specific pathogen-free CD-I mice after intra-venous inoculation. Lr, liver; Sp, spleen. The brokenlines refer to the growth of a challenge inoculum of S.enteritidis SMR injected intravenously on day 7 of theprimary infection. The arrows indicate the size of theinocula.

reduced virulence (54), or by the use of alterna-tive inoculation routes (57). However, the objectof any vaccination regimen should be the com-

plete protection of 100% of the vaccinees againsta biologically realistic challenge infection, usingthe most virulent organism available, given by a

natural route of inoculation (61). Under suchconditions, the live S. pullorum vaccine clearlyfails dramatically (67).

TRANSFER OF ACQUIREDRESISTANCE FROM VACCINATED TO

NORMAL RECIPIENTSIt has been postulated that live salmonellae

induce the production of cytophilic antibodieswhich then mediate the resulting immune re-

sponse (125). If this were so, then the lack ofimmunogenicity shown by the live S. pullorumvaccine could be due to its inability to inducethe production of such antibodies. The possibil-ity that specific cytophilic antibodies providethe arming mechanism for the immune macro-

phage has been explored experimentally, withvariable results (107, 213). Although there is noquestion that specific anti-Salmonella immuno-globulins can be recovered from the surfacemembranes of peritoneal macrophages har-vested from immune mice (213), the criticalinvolvement of cytophilic antibodies in the ex-

pression of immunity to the naturally acquireddisease is still open to doubt (35, 247). If thesespecific antibodies are adsorbed onto the cellsurface from the serum, then it should bepossible to protect mice passively with largeenough doses of hyperimmune mouse serum

(210). Such transfer studies are important sincethey constitute the only definitive method fordetermining the real role of humoral versus

cellular factors in the expression of anti-Salmonella immunity in a fashion analogous tothat reported earlier for Listeria monocytogenes(155), B. abortus (118), and M. tuberculosis

TABLE 1. Mortality data for CD-I mice infected intravenously with S. enteritidis, S. gallinarum, or S.pulloruma

Time in daysVaccine Challenge

5 6 7 8 9 10 11 12 14 21 28

S. enteritidis 1 2 2 2 2 2/101S. gallinarum - 1 1 1/10S. pullorum 0/10S. enteritidis S. enteritidis SMR 0/10S. gallinarum S. enteritidis SMR 1 1 1/10S. pullorum S. enteritidis SMR 1 1 3 3 5 6 8 9 9 10/10

S. enteritidis SMR 1 2 4 4 4 6 9 9 10 10 10/10

a Animals from each group were superinfected with S. enteritidis SMR on day 7 of the primary infection.b Dead/total.

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infections (141). Normal CD-1 mice were in-jected with 0.25 ml of hyperimmune mouseserum (50) at the time of challenge with 8 x 10"S. enteritidis cells (2 LD5O). Two more injec-tions of 0.25 ml of serum were given intrave-nously on days 1 and 2 of the infection. Theresultant growth curves are shown in Fig. 2.There was an increase in blood clearance and anenhanced early inactivation of the challengepopulation in the serum-treated mice, but by 24h growth of the surviving bacteria occurred invivo at the same rate as in the controls, despitethe further serum injections (49). The passiveserum treatment had a definite protective valuein terms of overall survival (Fig. 2), but was notable to prevent the extensive growth of thechallenge organism in vivo, despite the fact thatthe original serum donors were highly resistantto such a challenge (49). The inadequacy of theresponse to the serum treatment is furtherhighlighted by the behavior of the superinfect-ing S. enteritidis SMR population injected 7days after the primary challenge (Fig. 2). Theimmediate and progressive decline in viabilityby both the liver and spleen drug-resistantpopulations, in the absence of any initial growthperiod, is one of the main characteristics of thecell-mediated immune response (64).Normal mice were adoptively immunized

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FIG. 2. Growth of S. enteritidis 5694 insively immunized with three doses ofhyperimmune serum (solid lines) or nor

serum (broken lines) injected intravenouslJdays. The dotted lines refer to the behcintravenous inoculum of S. enteritidis S2into the hyperimmune serum-treated micethe primary challenge infection. The figtgraph refer to the number of deaths perthe 14-day period of the study.

with 2 x 108 filtered spleen cells (155) preparedfrom actively immune donors used as a sourceof the hyperimmune serum. The mice showedan accelerated immune response 24 h afterchallenge with the live S. enteritidis SMR (Fig.3). Although the level of immunity was inferiorto that observed in the original spleen celldonors, it was clearly superior to that seen in theserum-protected animals (Fig. 2). Small num-bers (103 to 5 x 103) of viable salmonellae werealso transferred to the recipients with the fil-tered spleen cell suspension, and these host-adapted bacteria were paradoxically able tomultiply in vivo, even in the face of an immuneresponse against the drug-resistant challengepopulation. However, the transferred bacterialpopulation was too small to induce significantlevels of immunity de novo during the shortperiod of the transfer experiment. The generalconclusion to be drawn from both in vivo and invitro experiments of this type (99, 215-217)must be that specific humoral factors make animportant contribution during the early stagesof the immune response, but that virulentstrains of Salmonella then become establishedin the lymphoreticular organs of the host andthat the rapidly dividing bacteria seem to beprotected from the further bactericidal action ofcirculating serum factors (102, 222, 227). It isonly the emerging cellular immune responsedeveloped against the infecting population itselfwhich is finally able to eliminate the parasitefrom the tissues (160).

MEDIATORS OF CELLULAR IMMUNITY

Mononuclear Phagocytes)M Normal mouse peritoneal macrophages areTD known to inactivate a considerable portion of an2/ldied infecting Salmonella population, often within

minutes (32), but, if the challenge organism ishighly virulent, the survivors of this initialinactivation phase begin to multiply and mayeventually overwhelm the normal host defenses.Presumably the same is true for organismsleaving the gut and passing through the

.._ ddraining lymph nodes to reach the fixed12 14 phagocytes of the liver and spleen. There the

organisms multiply extensively and thei mice pas- systemic population eventually assumes lethal0.25 ml of proportions. The more attenuated the challenge,mal mouse organism, the smaller this secondary systemicyvdaily for 3 growth phase tends to be (55). Furthermore,iR injected many of the bacilli penetrating the gut mucosaon day 8 of will be inactivated by the normal hostures on the phagocytes within the lamina propria (241). As0 mice over a result, the size of the systemic bacterial

population will be greatly reduced and a

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FIG. 3. Adoptive transfer of anti-Salmonella im-munity to normal CD-I mice infused with 10 filteredspleen cells from S. gallinarum-vaccinated donors.The growth behavior of the S. enteritidis SMR chal-lenge injected 24 h after the immune spleen cells(bottom) or normal spleen cells (top) can be comparedwith the same challenge inoculum injected into a

group of mice used as the source of immune spleencells (middle).

sublethal infection is more likely to result (54).On the other hand, a completely avirulentorganism will be wholly inactivated within thelamina propria and should not gain entry to themore peripheral tissues at all (55). Thus,"protection" can vary considerably dependingupon the dose and the virulence of the challengeorganism. By such manipulations, a killedSalmonella vaccine can give rise to apparentlywidely differing levels of protection, depending

upon the experimental conditions selected (54).The fact remains, however, that a challengewith a highly virulent Salmonella strain canonly be controlled effectively by a cell-mediatedtype of immune response (56).

Parenteral infection of normal mice andguinea pigs with a variety of intracellularparasites has been criticized on the groundsthat the route of inoculation is an artificial oneand the protection data may bear littlerelevance to the naturally acquired disease (6,133). Recent studies have been designed tomimic the naturally acquired infection asclosely as possible (6, 260). However, oralinoculation of normal mice with entericpathogens is associated with a number oftechnical difficulties (174), and starvation, bi-carbonate treatment, massive oral pretreat-ment with antibiotics, or intraperitoneal opiuminjections (to inhibit normal gut mobility) haveall been used to increase host susceptibility tothe challenge infection. Presumably, such pre-treatments permit a local concentration of vi-able salmonellae adjacent to the gut mucosa,thus resulting in focal infections within thelamina propria of the small intestine (241). Be-cause of the nonbiological nature of such pre-treatments, the resulting infection data stillbear little more relevance to the naturally ac-quired disease than the earlier intraperitonealchallenges.As a result of these technical difficulties, very

few investigators have studied the progress ofSalmonella infections within the normalundisturbed intestinal tract (61). Such studiesare possible with B6D2 hybrid mice, which are100 to 1,000 times more sensitive tosalmonellosis than CD-1 mice (55). Whenanimals were infected intragastrically with 106viable S. enteritidis 5694 cells (40), within hourssmall numbers of salmonellae appeared in thePeyer's patches associated with the distalileum. Later, viable organisms were alsoisolated from the draining mesenteric lymphnode. No other Peyer's patches seemed to beinvolved at this stage of the infection (62), butwithin 48 h an extensive systemic infection hadbecome established (61). The subsequentamount of growth in the livers and spleens ofthese mice was little different from thatreported earlier for sublethally infected micewhich had received an intravenous challenge(49, 67). The immune mechanism involved incombating both types of experimental infectionseemed to be very similar (50, 51), although theevolution of the immune response in the orallyinfected animals was noticeably slower than inthe intravenous group, and this was related to

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the slower rate of spread by the oral infection tothe liver and spleen (41, 62).

Regardless of the route by which the virulentSalmonella challenge population enters thetissues, extensive growth will occur withinmany of the.unstimulated macrophages, butonce these cells become activated or "angry"they are able to inactivate even the mostvirulent bacilli (156). The nature of the changesin lysosomal activity associated with this abilityare probably both quantitative and qualitativein nature, although we still know relatively littleabout the actual bactericidal mechanismsinvolved (29). The activated cells are certainlylarger, with an increased number of cytoplasmicgranules (156), are able to spread on glasssurfaces faster (78, 185), and can engulf andinactivate foreign particles at an enhanced ratecompared with normal cells (153). As a result ofthese changes, both specific and nonspecificantibacterial activity is markedly increased (72,214). In the context of the present discussion,the specific aspects of these changes are ofprimary importance, but some workers havechosen to emphasize the nonspecificantibacterial activity of these cells (271).Although it is true that activated macrophagescan kill unrelated microorganisms (33, 60) at afaster rate than normal (considerableexperimental advantage has accrued from thisproperty; 31), the fact remains that specificinactivation rates are nearly always quantita-tively the greater. As an example, macrophagesharvested from L. monocytogenes-infected micetake up and inactivate Listeria cells far morerapidly than is the case for BCG-stimulatedcells (238). Furthermore, only the specific orga-nism can induce an anamestic recall of the ear-lier cellular immunity (111, 152); heterologousorganisms cannot achieve this effect (48). Thus,the widely believed misconception that cellularimmunity is essentially nonspecific in nature(14) arose from an uncritical evaluation of theinactivation of adventitious organisms alsopresent in the infected tissue. This latter effectmust not be allowed to detract from the specificnature of the induction mechanism or the factthat only the original infecting strain can triggerthe production of more activated cells. Once theprimary infecting organism has been eliminatedfrom the tissues, the number, of specificallyactivated cells tends to decrease sharply (186).The antigens involved are highly' specific andmay not be present even in strains from thesame Salmonella "O" group (48, 64). Thesensitized host retains an immunological"memory" for these sensitizing antigens whichwill enable it to mount the enhanced immune

response against the same organism months oreven years later (154, 161). However, it is notclear whether this Salmonella "memory" is dueto a persisting population of specificallysensitized small lymphocytes analogous to thatdescribed for humoral antibody production(124) or to the persistence of antigen followingthe establishment of a carrier state (117), oreven to an unsuspected latent infection (149).The latter two possibilities cannot bediscounted since detection of small numbers ofbacilli in the tissues can be extremely difficult(148). Thus, the problems associated with theelucidation of immunological memory underthe present circumstances are considerable, butthe mechanism(s) involved is clearlyfundamental to our understanding of thisextremely important recall phenomenon andthis work should be continued.

"Activated" Macrophages"Activated" macrophages are normally in-

duced by the interaction of immunocompetentT-lymphocytes and blood-borne monocytes as aresult of the release of specific sensitizing anti-gens by the actively multiplying bacterial cellswithin the tissues. This process has been thesubject of several recent reviews (79, 158, 161,189). However, there are other experimentalmeans of achieving lymphoreticular stimulation.The majority of killed bacterial vaccines havelittle or no ability to induce cellular activation(31, 66), although endotoxins, polyinosinic-poly-cytidylic acid, corticosteroids, and Bordetellapertussis or Corynebacterium parvum areknown to increase greatly the bactericidal activ-ity of spleen and liver macrophages (91). C. par-vum has been used successfully in the treat-ment of some tumors, in both humans and ex-perimental animals (232), and increases host re-sistance to a number of microbial infections (2).This latter effect is somewhat unexpected sinceC. parvum is known to inhibit some T-cell func-tions (221) and anti-Salmonella immunity seemsto be a T-cell-mediated host response. It wastherefore of some interest to see whether C.parvum-treatment quantitatively affected thegrowth of S. enteritidis in vivo. Groups ofnormal CD-1 mice were injected intravenouslywith 700, 350, 175, or 88 ug of heat-killed C.parvum, and 10 days later they were challengedintravenously with 5 x 103 viable S. enteritidiscells (1 LDO). The growth of the challengeorganism in both the livers and spleens of the C.parvum-treated mice (Fig. 4) should becompared with that seen in untreated animals(Fig. 1). The cellular responses in the C.parvum-treated mice slowed the growth of S.

COLLINS378

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VACCINES AND CELL-MEDIATED IMMUNITY 379

5

88 mcg C. PARVUM IV.

4

4

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FIG. 4. Effect of increasing amounts of heat-killed C. parvum vaccine injected intravenously into CD-I mice

7 days prior to their intravenous challenge with 3 x 103 to 5 x 103 viable S. enteritidis 5694 cells. Mice receiving

the 88-ag dose and the normal controls gave almost identical growth curves. The histograms refer to the

increase in foot thickness 24 h after injection of 5 lag of Salmonella test antigen into one hind foot, compared

with the contralateral foot which received only saline. An increase of 2 units (0.2 mm) or more was significant at

the 1% level.

enteritidis in both the liver and spleen, but parvum did develop significant levels of DTH,

there was no antibacterial immune response and there was a late cell-mediated immune

and there was an almost complete absence of response against the S. enteritidis infection

DTH to the Salmonella test antigen (71). (Fig. 4).

However, mice receiving 88CAg or less of C. C. parvum mobilizes the production of large

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numbers of monocytes within the spleens andlivers of intravenously treated mice. Protectionagainst the lethal effects of the S. enteritidischallenge could thus have been due to theincreased early nonspecific inactivation of thechallenge inoculum by the stimulatedmacrophages (91). There was a 10-fold increasein splenic weight in the C. parvum-treated micebut a 50% decrease in cellular proliferation(measured as tritiated thymidine [3H-TdR]incorporation by the dividing spleen cells; 190)due to the S. enteritidis infection, comparedwith that seen in the control animals (Fig. 5).Thus, in terms of tritium uptake per unit weightof splenic tissue, the 3H-deoxyribonucleic acid(DNA) content for the C. parvum-treated S.enteritidis-infected spleens was less than 25% of

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the corresponding control values, and thisdifference was quite significant (P < 0.02). Thepeak 3H-DNA value in the spleens of the controlmice (day 5) coincided with the peak DTHresponse, as well as with the appearance of theimmune response. Although the C.parvum-stimulated spleen 3H-DNA contentreached a lesser peak on day 5, this was notassociated with either DTH or a cell-mediatedtype of immunity, but seemed to be caused by arapidly expanding hematogenous populationwithin the splenic tissue. Histologically, theseSalmonella-infected, C. parvum-treatedspleens contained very few lymphocyte-likecells, but the lesions consisted of enormousaggregations of mononuclear cells seen in large,poorly organized granulomata throughout the

NORMAL CONTRO K

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FIG. 5. Effect of 700 fig of heat-killed C. parvum intravenously on growth of S. enteritidis 5694 in the spleenfollowing intravenous inoculation 14 days later. The normal control curve is shown on the right. The brokenhistogram refers to the 3-h foot swelling, and the solid bar, the 24-h footpad swelling. The bottom curves refer tothe uptake of 3H-TdR by the spleen cells, either per whole spleen (broken lines) or per 100 mg of splenic tissue(dotted lines). The weight of the spleen (mg) is represented by the solid lines.

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infected tissue. On the other hand, sectionsprepared from control spleens on day 5contained large numbers of lymphoid cells, aswell as many mature macrophages organizedwithin well-defined lymphoid follicles. As theinfection progressed further, these granulomastended to resolve. Presumably, it was themassive mononuclear infiltrates seen in the C.parvum-stimulated spleens and livers whichresulted in the depressed rate of growth shownby the infecting population (Fig. 4). Thereduced T-cell reactivity seen in these mice(note the virtual absence of DTH in Fig. 5) wasalso associated with an inability on the part ofthe infected host to mount a cell-mediatedimmune response analogous to that seen in thecontrols about day 5.The corresponding host responses to massive

intravenous doses of heat-killed M. tuberculosis(a nonspecific stimulus), S. enteritidis (specificstimulus), or Bordetella pertussis (histamine-sensitizing stimulus) were quite different fromthat seen with C. parvum. The Salmonellagrowth curves shown in Fig. 6 indicate that thestimulated mice were still able to mount a nor-mal immune response to the Salmonella chal-lenge. Both the heat-killed H87R, and S. en-teritidis suspensions increased host resistanceto the lethal effects of the virulent Salmonellachallenge (Fig. 7), but much of this protectionwas due to the increased early inactivation ofthe challenge population seen in these mice.Near normal growth rates and a cell-mediatedimmune response developed in all of these miceby day 5 or 6 (Fig. 6). Only the B. pertussis-treated mice showed an enhanced susceptibilityto the lethal effects of the Salmonella challenge(Fig. 7), and the mortality data correlate with anenhanced rate of growth seen in this group ofmice. This effect was presumably due to thehistamine-sensitizing action of the Bordetellasuspension (177). Thus, the present data seemto be in complete agreement with earlier reportsthat the C. parvum effect is almost uniquelylimited to this (and a few related anaerobic)species of Corynebacterium (3).There was still the possibility that C. parvum

was acting as an immunological adjuvant (123)which increased the humoral response toSalmonella antigens introduced with thechallenge inoculum. Attempts to demonstratespecific Salmonella hemagglutinins in the seraof C. parvum-treated mice, both before andafter Salmonella challenge, were largelyunsuccessful (71). When mice were pretreatedwith a mixture of 350 gg of heat-killed C.parvum and 200'g of dead S. enteritidis, thegrowth curves obtained after virulent challenge

(Fig. 8) make it clear that the mixture was nomore effective in inducing resistance to the S.enteritidis challenge than was C. parvum alone.Heat-killed S. enteritidis injected without theC. parvum was little better than theunvaccinated controls (Fig. 8). Thus, thecharacteristic slowed type of growth shown bythe S. enteritidis challenge population in thelivers and spleens of the C. parvum-treatedmice was not due to any adjuvant action on theSalmonella antigens by the C. parvum, at leastso far as conventional humoral responses wereconcerned.

T-LymphocytesThe striking advances in our recent

understanding of the intereactions betweenimmunocompetent T-lymphocytes andmononuclear phagocytes in the expression ofcell-mediated immunity to intracellularparasites (162, 189) represent one of the mostexciting areas in immunology today. The role ofthe thymus in the expression of immunologicalcompetence and cell-mediated immunity hasbeen the subject of numerous recent reviewsand needs little further discussion here (79, 175,189). However, it is interesting to note that,although DTH was early established as aT-cell-dependent reaction (12, 266), it has onlybeen in the last few years that this dependencehas been extended to include cell-mediatedimmunity to intracellular parasites (150, 188,242). The demonstration that adoptive transferof immunity to Listeria challenge can beachieved with anti-theta sensitive lymphocytes(139) confirms the involvement ofimmunocompetent T-lymphocytes in theexpression of antimicrobial immunity deducedfrom earlier studies using antilymphocyteserum (166) and the antimitotic drugvinblastine (244). Such findings have now beenextended to include antituberculous immunity(188, 238). It is interesting in this regard thatthe depressed state of cellular resistance seen inthe T-cell depleted host (THXB) wascapitalized upon by Rees (200) for increasingyields of M. leprae in the mouse footpad (224)several years before the involvement ofT-lymphocytes in leprosy immunity had beenformally demonstrated (106).Although T-lymphocytes are essential for the

expression of immunocompetence, they areunable alone to express immunity whenadoptively transferred to heavily irradiatedrecipients (244). Both cell-mediated immunityand DTH were observed only whennonsensitized macrophages or normal bonemarrow cells were also introduced along with

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6

COLLINS BAcrERIOL. REV.

5

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TIME IN DAYS

FIG. 6. Effect of 700 ,g of heat-killed S. enteritidis 5694 (bottom), M. tuberculosis H37Rv (middle), or B.pertussis (top) vaccine injected 7 days prior to intravenous challenge of the mice with 2 x 103 to 6 x 103 viableS. enteritidis 5694 cells. Lr, liver; Sp, spleen; BI, blood.

the T-lymphocytes into the irradiated recipient(143, 255). This double cellular requirement isconsistent with the known radiosensitivity ofmacrophage precursors within normal bonemarrow (254); normal macrophages are alsoessential for the expression of skin or footpadDTH in sublethally irradiated mice and guineapigs (255).Animals subject to thymectomy and

irradiation during adult life may still retainsome T-cell function, presumably due to small

numbers of T-lymphocytes transferred to theTHXB host with the normal bone marrow cellsused during the reconstitution (81). In anyT-cell depletion regimen, a percentage of themice die during the first week or so afterirradiation, despite the use of an antibioticumbrella to reduce systemic infections.Presumably those animals which have beencompletely depleted of T-cells can no longerresist invasion by their own normal gut flora, acommon source of potentially lethal infections

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in individuals suffering from radiation sickness(26). If anti-Salmonella immunity is apredominantly cell-mediated response, then theT-cell-depleted mouse should be incapable ofresisting even a modest challenge with anattenuated strain of S. enteritidis Se 795(intravenous LD,0 in C57BL mice = 10' viableorganisms). However, after such a challenge,the systemic Salmonella population actuallygrew at a slower rate in the THXB mice than itdid in the irradiated reconstituted controls (Fig.9). There was, however, no sign of an immuneresponse to the systemic infection and none of

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FIG. 7. Mortality curves for mice pretreated withheat-killed S. enteritidis, M. tuberculosis H37Rv, B.pertussis, or C. parvum, or with saline, 7 days prior tointravenous challenge with 2 x 10' viable S. enteriti-dis cells (2 to 5 LDI.).

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-1MEDIATED IMMUNITY 383

the mice developed any specific DTH, althoughthere was some Arthus (3 h) reactivity. Some ofthe THXB mice eventually died as a result ofthis slowed continuing infection, but thesurvivors eventually exhibited low levels ofdelayed footpad sensitivity, suggesting thatsome cellular immunity had been developedagainst the parasite. The sham-thymectomizedcontrols behaved essentially the same as thenormal controls. There is a superficial similaritybetween the Salmonella growth curves seen inthe THXB mice (Fig. 9) and those for the C.parvum-treated animals (Fig. 4). This mayindicate a common cellular defect in bothgroups of animals, but the evidence is still onlysuggestive and further study on this point willclearly be necessary.

Infection of THXB mice with the attenuatedstrain of Mycobacterium bovis (BCG) alsoyielded interesting results. BCG normally givesrise to a self-limiting infection with the develop-ment of tuberculin hypersensitivity and anantimycobacterial resistance in 10 to 14 days(65). However, when 107 viable BCG wereinoculated into THXB mice, a severe mycobac-teriosis developed with a 95% mortality over a100-day period (Fig. 10). Death of uninoculatedTHXB mice was less than 10% over the sameperiod, and none of the normal BCG-infectedmice died as a result of the infection. Growth of

CONTROLS

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FIG. 8. Growth of 104 viable S. enteritidis 5694 cells injected intravenously into mice pretreated with 350 iigof C. parvum alone (C.P.), 350 jig of C. parvum + 200X lg of heat-killed S. enteritidis 5694 (C.P.-5694),heat-killed S. enteritidis 5694 alone (5694), or saline only (controls). Circles, liver; squares, spleen.

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responsible for the deaths observed. If death ofthe THXB mice was not due to a runawaysystemic infection analogous to that seen inmiliary tuberculosis, then the most likely expla-nation would seem to be an aberration in thecellular response in the T-cell depleted host inthe face of the ongoing infection. An attemptwas therefore made to follow the cellularchanges occurring within the lungs and spleensof the BCG-infected animals by means of pulselabeling with 8H-TdR (188). North (187) showedthat the amount of splenic 3H-TdR incorpora-tion by normal mice increased sharply followinga tuberculous challenge, to a peak coincidingwith the emergence of the T-cell-mediated im-mune response on the part of the host. Thisfinding was confirmed in the present study,since by day 20 the BCG curves had turneddown and the uptake of label by the spleen wasfour times the preinfection value (Fig. 11). Thispeak was partly due to the doubling in thespleen weight over this time, but even when thecounts were corrected to a per unit weight basisthe rate of incorporation of 3H-TdR was stilldouble the normal values. This enhanced incor-poration rate then fell towards preinfectionlevels over the next 40 to 50 days. The amountof incorporation by the uninfected lung cellswas only 10% of that shown by the spleen (Fig.

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TIME IN DAYS

FIG. 9. Growth of S. enteritidis Se 795 in normalC57Bl mice (top), sham-thymectomized lethally irra-diated, bone marrow-reconstituted (XB) controls(center), or adult thymectomized, irradiated, bonemarrow-reconstituted (THXB) mice (bottom) afterintravenous challenge. The open histograms refer tothe 3-h footpad swelling, and the solid bars, to the24-h swelling after injection of 1 fig of Salmonella testantigen.

the BCG population in the lung, liver, spleen,bone marrow, and a variety of lymph nodescontinued unchecked for 30 days, but then a

slow decline in viability occurred until day 90,which coincided with the mean survival time forthese animals (Fig. 10). A lung bacterial popu-lation of 2 x 106 viable bacilli (seen in theTHXB animals at this time) did not cause anyapparent distress in normal mice (65), and itwas not clear how the continued presence of thisnumber of bacteria in the lung could have been

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384 COLLINS

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VACCINES AND CELL-MEDIATED IMMUNITY

11), but this value also increased at leastthreefold, to a maximum about day 20, followedby a slow steady decline to preinfection valuesby day 65.The DNA synthesis by spleen cells was some-

what more complex in the THXB mice. Therewas an initial sharp drop in 3H-TdR uptake by

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the spleen after infection (Fig. 12) followed by areturn to about preinfection levels by day 20.The incorporation curve then remained at aboutthis level until day 50 when a later minor peakwas observed (Fig. 12). This peak seemed tocorrespond to one seen about this time also inthe controls (Fig. 11), but its significance is still

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THXB MICE2o r SPLEEN

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not clear. T-cell depletion eliminated the largesplenic peak seen about day 20 in the controls,and this was presumably responsible for thealmost complete absence of tuberculin hyper-sensitivity in these mice (63, 191). On the otherhand, T-cell depletion had little effect on theearly lung incorporation of 3H-TdR, and as theinfection progressed so the rate of incorporationof label also increased slowly; by day 50 it wasthree times that seen in the controls. Signifi-cantly, this was the time when the infectedTHXB mice were beginning to die.

Histologically, the lungs of the THXB miceshowed many nonresolving granulomas whichcontained very few lymphocyte-like cells. Therewere large numbers of macrophages present,many of which had incorporated label. Thecellular density within the lung tissue slowlyincreased until the organ completely lost itsbuoyancy in water, suggesting that many of theanimals were dying as a result of pulmonaryinsufficiency (63). A similar picture has been

described in normal mice lethally infected witha highly virulent strain of tubercle bacillus(108). It is evident that the enormous influx ofmononuclear cells into the lung was unable toeliminate the attenuated mycobacteria from thetissues, and the resulting, fruitless tissue re-

sponse to the infection eventually killed thehost. It was only when anti-theta-sensitive lym-phocytes were present within the lesion thatspecifically activated macrophages could beproduced in sufficient numbers to induce an

antibacterial response which then resulted in a

rapid resolution of the lesions within the lungand elsewhere (188).The BCG-infected THXB mice demonstrated

a high degree of tuberculin anergy (63) ratheranalogous to that shown by lepromatous leprosypatients to the lepromin skin test antigen (36,106). This latter state could be overcome, atleast in some cases, by the injection of specifictransfer factor (37) or by multiple injections ofnormal human blood leukocytes (142). In both

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VACCINES AND CELL-MEDIATED IMMUNITY

cases, the return of hypersensitivity was associ-ated with a dramatic regression of the clinicaldisease, and this study constitutes a very cleardemonstration of the practical advantageswhich may follow the direct application ofimmunological theory to clinical practice.

RELATIONSHIP OF DTH TOCELL-MEDIATED IMMUNITY

Most facultative intracellular parasites in-duce a state of delayed skin hypersensitivity toone or more microbial antigens in the infectedhost. Skin sensitivity usually appears about thetime when cell-mediated immunity is also firstobserved (65, 164), although there may be somequantitative differences in the size of the re-sponse seen in mice of varying susceptibility tothe infection (198). On the other hand, killedvaccines induce humoral rather than cellularresponses (34, 50), and the level of acquiredresistance to the naturally acquired disease ismuch lower (64, 264). There has been an ongo-ing debate for some years regarding the exactnature of the relationship between specific DTHand acquired antimicrobial resistance (153, 154,181). At least some of this controversy stemsfrom the fact that tuberculin hypersensitivitycan be detected in the apparent absence ofantituberculous resistance and vice versa (65,164). This may occur partly because of difficul-ties in demonstrating antituberculous immu-nity in BCG-vaccinated animals (56) and partlybecause peripheral tuberculin sensitivity is notalways a valid index of cellular hypersensitivity(65, 265). Large doses of viable tubercle bacillimay desensitize the host to tuberculin withoutaffecting its ability to mount an effective an-tituberculous response (56, 159). Spleen cellstaken from such an anergic host will stilladoptively sensitize normal animals, indicatingthe presence of tuberculin-sensitive lympho-cytes within the heavily infected spleen (65).Because of this fact, purified protein derivativeunresponsiveness, especially in heavily vac-cinated or infected mice, should not be taken asprima facie evidence for separate cellular mech-anisms for DTH and antituberculous immunity(180).

Different strains of mycobacteria differwidely in the level of cross-resistance that theycan induce against a subsequent challenge witha virulent strain of M. tuberculosis (52). Theimmunogenicity of the vaccinating organismseems to depend more upon its ability to persistwithin the normal host tissues than on thecloseness of any phylogenetic relationship exist-ing between the vaccine and challenge strains.

The size of the immune response also variesdepending upon the virulence of the vaccinatingstrain since this will determine the amount ofgrowth which occurs in vivo after inoculation(59). This is even the case with different sub-strains of BCG which can vary extensively intheir in vitro (83) and in vivo (68, 197) behavior.Commercially available preparations of BCGmay vary extensively in their immunogenicity,both for humans and for experimental animals(28, 84). These vary from the maximal re-sponses shown by BCG Pasteur (163, 225) to avirtually complete lack of immunogenicityshown by a streptomycin-resistant strain (68) ofBCG Tice (BCG SMR). If a large enough vac-cinating dose of the latter is used, even thisnonreplicating Mycobacterium may becomeimmunogenic, provided that the viable popula-tion is large enough to persist in vivo for a periodof several weeks (52, 56). The poor immunoge-nicity of BCG SMR has been ascribed to therapid decline in viability seen when 106 viablebacilli are introduced into normal mice (70). Asecond explanation for this phenomenon how-ever, could be that one or more critical sensitiz-ing antigens had been lost from the cell walls ofthe drug-resistant mutant. This possibility wasrendered very unlikely by the finding thatheat-killed BCG SMR induced an equally effec-tive level of antituberculous immunity in miceas the parent BCG Tice, provided that twodoses of the vaccine were used and the organismwas suspended in Freund's adjuvant. There wasa 100-fold difference in the lung and spleenbacterial counts 14 days after a virulent Erd-man challenge in both groups of vaccinatedmice compared with the controls (70), and thiswas highly significant (P < 0.01). Thus, thelack of immunogenicity shown by the livingBCG SMR vaccine did not seem to be due to aloss of sensitizing antigens but was rather due tothe rapid decline in viability of both liver andspleen populations below an arbitrarily desig-nated sensitizing threshold value. Weekly injec-tions of 10. viable BCG SM R or a single intrave-nous dose of 108 viable bacilli elevated theviable populations above this threshold, whichseemed to be about 105 viable organisms for theliver. This residual viable population was suffi-cient to trigger the necessary cell-mediatedimmune response by the host. Heat inactivationof the 108 BCG SMR population destroyed thisprotective value unless the cells were firstincorporated into Freund's adjuvant (66, 70).

Claims have been made that some inacti-vated antituberculous vaccines can induce im-mune responses equivalent to that observedwith live BCG preparations (204, 226). How-

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ever, quantitative comparisons of the cellularresponses to the live and dead vaccines (usingthe highly sensitive indicator strain L.monocytogenes; 37) indicate that the responseto the former is invariably superior (66). On theother hand, the host response to inactivatedpreparations presented in an appropriate oil-based adjuvant often outlasts that achievedwith the living preparation (8,203), and thisfact alone more than justifies the continuedsearch for improved inactivated immunogensfor use in the prevention of tuberculosis, entericfever, gastroenteritis, brucellosis, and perhapssome urogenital infections.

INDUCTION OF CELL-MEDIATEDIMMUNITY WITH VARIOUS TYPES OF

VACCINE

Living VaccinesAlthough it is generally agreed that live

attenuated vaccines induce a highly effectivetype of immune response (53, 116, 248, 264), thistype of preparation is not yet commerciallyavailable for the prevention of enteric disease inhumans. Several experimental vaccines havebeen developed recently (4, 76, 85) but are stillin the initial stages of assessment in humanpopulations (86). Inactivated vaccines againstenteric fever and cholera (16, 77, 98) mayincrease resistance to the naturally acquireddisease, but enthusiastic claims for their ef-ficacy must be evaluated in the light of thepredominantly IgG and IgM antibody responseswhich they induce (27, 43). In the experimentalsystem, neither live nor killed S. enteritidisvaccines can completely prevent the passage ofchallenge organisms across the intact intestinalmucosa (57, 61), although they may delay theappearance of systemic disease for as much as72 h (62). The live vaccine has the greatadvantage that entry of the challenge inoculuminto the tissues induces an accelerated recall ofthe earlier cell-mediated immunity which isable to control the further growth of the orga-nism before the infection can assume clinicallysignificant proportions (61). Killed vaccines arequite unable to achieve this type of response,and although all of the vaccinated mice maysurvive the challenge, viable counts carried outon the liver and spleen make it quite clear thatthey have all suffered a severe clinical attack ofmouse typhoid fever. This apparently poor levelof experimental protection seems to be at vari-ance with the large body of typhoid fever fieldtrial data (13, 112, 113, 119) showing significantprotection in appropriately vaccinated humanpopulations. However, it should be remembered

that such trials merely indicate a significantdecrease in the number of cases of clinicaltyphoid fever per 100,000 vaccinated individu-als compared with that observed for a similargroup given a suitable placebo (119, 136). Bothgroups of individuals will usually be exposed toa relatively low level of endemic infection whichwill result in a number of clinical cases oftyphoid fever in both, but with a reducednumber of patients requiring hospitalization inthe vaccinated group. Unless the overall inci-dence of endemic disease is very low (136),complete protection will not be achieved with akilled vaccine. Controlled experimental studiesusing human volunteers indicate that the inci-dence of disease and the apparent level ofprotection against a standardized oral dose ofvirulent S. typhi Ty2 varies with the size of thechallenge inoculum (120). An inoculum of morethan 107 viable bacilli will overwhelm anyimmunity induced by previous exposure tokilled typhoid vaccine. This is also consistentwith the clinical finding that typhoid fever mayoccur within weeks of administration of abooster dose of typhoid vaccine (9) and even inrecently convalescent individuals (172). As itstands at present, the statistics obtained fromthese human trials make it clear that the killedtyphoid vaccine is better than nothing in anendemic situation (234), but that the develop-ment of more effective attenuated living vac-cines for human use is a matter of some urgency,especially in light of the recent severe out-breaks of typhoid fever due to drug-resistantstrains of S. typhi of unusual virulence forhumans (258).Salmonella "carriers" have a high degree of

resistance to re-infection, and this state usuallylasts as long as the primary infection persists invivo (48, 116, 117). However, not all live sal-monellae are able to induce solid resistanceagainst reinfection. For instance, S. pullorumhas no immunogenic value against a subsequentchallenge with the antigenically related S.enteritidis SMR (Fig. 1). On the other hand, theantigenically related chicken pathogen (S.gallinarum) is highly protective under thesecircumstances (67). Although there are a num-ber of minor biochemical, physiological, andantigenic differences between the two avianpathogens (47), they are not sufficient to ex-plain the total lack of immunogenicity shown bythe live S. pullorum vaccine in mice (57, 64).Recent studies from this laboratory indicatethat the inability of S. pullorum to establish apersisting bacterial infection in the mouse issomehow associated with this phenomenon(Fig. 1). In the day-old chick, on the other hand,

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both strains of S. gallinarum-pullorum multiplyextensively in the liver and spleen (Collins,unpublished data), and at 5 weeks a high degreeof resistance to reinfection can still be demon-strated in both groups of birds (233). Thisresponse appears to be cellular rather thanhumoral in nature (228), although there is littledoubt that specific antibodies are also involved(129). Smith (228) reported good protection inchickens receiving a living but not a killed S.gallinarum vaccine prior to oral challenge. Sim-ilarly, in our own studies, live but not dead S.gallinarum vaccines increased resistance inchallenged mice (64) and in chickens receivingan intravenous challenge with the virulent S.gallinarum 9240 (Collins, unpublished data).Anti-0-9 and 0-12 antibody titers were elevatedin the sera of both groups of vaccinated animals,but no correlation could be shown betweenblood clearance values for S. gallinarum-pullorum in either mice or chickens and thelevel of virulence demonstrated by the infectingorganism for the corresponding host (53). Al-though these data do not exclude the involve-ment of opsonic antibodies in host protection(especially with regard to the behavior of thesefowl pathogens early in the infection; 130), thereseems little doubt that the overall defenseagainst systemic salmonellosis in both mice andchickens depends upon a cell-mediated immunemechanism (53, 162).

S. pullorum induces an excellent humoralresponse in vaccinated mice, and this is alsoseen from the ihimediate (3 h) type of hypersen-sitivity developed following the footpad injec-tion of a test antigen isolated from this orga-nism (64). There was little or no delayed hyper-sensitivity to the Salmonella sensitin (57) de-spite its known presence in S. pullorum culturefiltrates. Apparently the critical antigen(s) in S.pullorum is inactivated very quickly in vivo so

that the inability of the organism to multiply inthe mouse tissues results in an inadequateantigenic stimulus to the host defenses. As a

result, no DTH is developed even when re-

peated daily doses of viable S. pullorum are

administered to artificially maintain a sensitiz-ing population of organisms. Apparently, thesensitizing population within the liver andspleen must remain in a metabolically activestate (although it may not be necessary for theorganisms to divide during this time; 89, 90) inorder to induce the necessary cellular response.The S. pullorum population does not do so (Fig.1) and the vaccine is therefore nonimmunogenicfor mice. The reason for this exquisite sensitiv-ity to the bactericidal action of normal mouse

phagocytes is still a mystery. Rough salmonel-

lae are known to be inactivated very rapidly bynormal phagocytes (210), but S. pullorum isantigenically smooth (47) and seems to resistblood clearance as effectively as the highlyimmunogenic S. gallinarum (53). Subtle differ-ences obviously exist between the cell wallstructure of S. gallinarum and that of S. pul-lorum, and these differences presumably renderthe latter more sensitive to the bactericidalaction of the lysosomal enzymes of the normalmouse macrophage but not apparently to thecorresponding chicken phagocyte. Clearly, wehave a lot more to learn about this fascinatingprocess.

Inactivated Whole CellsAssessment of the immunogenicity of differ-

ent killed vaccines often depends upon thecriteria used to measure the resulting "protec-tion" expressed by the vaccinated host. Themere demonstration of survival may not giveany clue as to the true nature of the immunemechanism involved, and extrapolation of pro-tection data from one host species to anothermay be quite misleading (53). As an example ofthe latter, the widely accepted tenet that ace-tone-killed typhoid vaccines are superior intheir protective value to heat-killed prepara-tions (95) may be true for mice, but not forhumans (136). The mild chemical inactivationof typhoid vaccines was initiated after thedemonstration by Felix that a surface "Vi"antigen was present in most virulent strains ofS. typhi (94) and that this antigen was "lost"after heating, together with most of the mouseprotective value of the vaccine (93). Althoughthe interpretation of some of his protection datawas quickly disputed (193), the importance ofthe Vi antigen as an immunogen in humantyphoid vaccines was widely accepted, and thechemical method of inactivation for typhoidand TAB vaccines has been retained ever since(10, 234, 236). This is somewhat ironic in view ofthe repeated demonstration that purified Viantigen is quite heat stable (237), and can, infact, withstand boiling water temperatures.Boiling merely releases the intact Vi antigenfrom the cell surface, a fact that has been usedrepeatedly in its in vitro assay (262). Heatinactivation of the typhoid vaccine releases theVi antigen into the liquid phase from which itwill, of course, be lost if the cell suspension iswashed prior to use. There is in fact very littlequantitative data in the literature to indicatethat purified Vi antigen has any protectivevalue whatsoever in humans (120, 173), al-though there is no question that it is importantin the mouse protection test when the intraperi-

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toneal route of challenge is used (19, 95, 101).The mouse is not naturally susceptible to S.typhi infection, and death will occur only whenmassive inocula are injected directly into theperitoneal cavity (62), frequently with the addi-tion of hog gastric mucin to inhibit phagocytosis(234, 235). In the peritoneal cavity of thevaccinated mouse, anti-Vi antibodies do playan important protective role (128, 263). How-ever, the validity of the intraperitoneally in-fected mouse as a model of human typhoid feverhas been disputed by a number of workers (88,193) and at present, evidence for a protectiverole for Vi antigen in human typhoid fevervaccines is not strong (43). However, despitethis caveat, claims for increased protectionfollowing the use of acetone-killed vaccinescontinue to appear in the literature (15, 126),although a careful examination also reveals anumber of reports that heated phenol-killedsuspensions can be just as protective (61, 69, 82,113).Claims for increased immunogenicity by ace-

tone-killed suspensions of S. typhimurium dueto the preservation of a heat-labile "virulence"antigen 0-5 (126) should be interpreted in thelight of later reports that this antigen is alsoquite heat stable (44, 134). In fact, the wholequestion of the role of somatic antigens in thedevelopment of host resistance is still not at allclear (111), especially with respect to orallyinfected individuals (51, 61). The opsonic andbactericidal antibody titers induced by paren-teral Salmonella vaccines (mainly IgG and IgMimmunoglobulins; 43, 207, 243) show little cor-relation with the level of acquired resistance tothe naturally acquired disease (27, 62, 110).Even when the immunizing regimen is adaptedso as to induce a maximal IgA response (87, 98,243), the level of protection against an oral S.enteritidis challenge is still relatively low (61),and it is not presently clear just how thesesecretory IgA immunoglobulins protect the hostagainst this pathogen at the mucosal level (62).

Cell ComponentsSalmonella cell walls have not been tested

extensively for their immunogenicity in mice(39), although excellent specific protection hasbeen claimed with Listeria walls (137) andMycobacterium cell walls (202). The latterinduce both tuberculin hypersensitivity and aconsiderable level of antituberculous resistance,especially against an aerogenic challenge infec-tion with virulent M. tuberculosis H,,7R. To beprotective, the purified walls must be mixedinto a paste with a small amount of mineral oiland then injected into mice intravenously as an

oil-in-water emulsion (8, 205). This type ofvaccine has been used extensively in experimen-tal studies of antituberculous immunity in mice(204) and monkeys (203), but so far it has notbeen used clinically in humans. Recent studiesindicate that BCG cell walls also have signifi-cant antitumor activity (273), and it may wellbe that this antituberculous reagent will play animportant role in the future, not against thespecific infectious agent for which it was devel-oped, but rather as part of the developingimmunological armamentarium to be usedagainst some human cancers.Almost every mycobacterial product so far

tested has some antituberculous activity (18,217), especially when incorporated into a min-eral oil carrier, although many of such productsmay then be too toxic for human use. Onemycobacterial cell product which has receivedextensive study is the ribosomal antigen iso-lated from M. tuberculosis H,7Ra by Youmans(267). Analogous preparations have also beenmade from S. typhimurium by Venneman andBigley (253) and from Staphylococcus aureus byWinston and Berry (261). Vaccinated miceshowed increased resistance to challenge follow-ing intraperitoneal or subcutaneous injectionsof a ribonucleic acid (RNA)-containing compo-nent of the cell contents (253). Some protectionhas also been achieved by use of a syntheticnucleotide preparation (269). Studies with thesevaccines raise a number of important theoreti-cal immunological considerations, since You-mans (272) has repeatedly reported excellentantituberculous resistance in the absence ofdetectable levels of tuberculin hypersensitivity.This last point has been disputed, however (18,205). Some of the controversy regarding theimmunogenicity and/or allergenicity of ribo-somal and cell wall vaccines may well stem fromtechnical considerations involved in the demon-stration of peripheral hypersensitivity in heav-ily infected mice (63). Furthermore, there issome question as to whether the different exper-imental protocols used actually measure thesame parameters of the host response. Forinstance, Youmans employs a large intravenouschallenge inoculum which kills more than 90%of the controls within 30 days (270). Protectionis then expressed as the percentage increase insurvival at day 30 in a similarly challengedprevaccinated group. This protocol in effectdemonstrates an increased mean survival by thevaccinated animals since most of the mice willdie eventually as a result of the infection. Underthese conditions, "protection" may be moreantitoxic than antibacterial in nature, involvinghumoral and nonspecific factors (250, 271).

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Other investigators use a small aerogenic chal-lenge in the vaccinated host and compare theamount of growth and tubercle formation in thelungs of the sublethally infected test and controlanimals (23, 204). This method may also berecommended on the grounds that it bears acloser relationship to the natural human infec-tion (260).

In the corresponding studies using ribosomalantigens isolated from S. typhimurium, hostresistance to virulent challenge has also beenreported to be significantly increased (251).Unfortunately, the most commonly used testprotocols employ an intraperitoneal route ofinfection in which antibodies developed againstcontaminating cell wall antigens might be ex-pected to exert a maximal protective effect (32).As a result, there has been considerable debateregarding the nature of the protective moiety inthese ribosomal vaccines. Smith and Bigley(230) suggested that the active principle may bea cellular protein, or perhaps contaminatingsomatic antigens carried mechanically on theRNA moiety. The latter has no antigenic prop-erties per se but merely acts as an immunologi-cal adjuvant. This last possibility gains furthercredence from the report by Houchins andWright (121) that a specific immunogen can beisolated from their ribosomal antigen preparedfrom S. typhimurium and that this materialseems to be a high-molecular-weight glycopro-tein or mucopolysaccharide. The RNA portionin their preparation did not seem to be directlyinvolved in the host response. This would alsobe consistent with the findings of Vennemanand Bigley (253) that the immunogenicity oftheir ribosomal antigen was not destroyed byribonuclease treatment. This seems to be indirect contrast to the sensitivity shown by themycobacterial ribosomal antigens to this en-zyme (268). On balance, it seems possible thatthe RNA present in this type of vaccine mayfunction mainly as a carrier which perhapsamplifies the humoral response to the traces ofimmunogen carried on it. This conclusion wouldbe compatible with the fact that ribosomalvaccines without an adjuvant can also protectmice (250). A second possibility would be thatthe RNA nonspecifically increases host resist-ance to the challenge organism, since syntheticpolyinosinic-polycytidylic acid has recentlybeen shown to increase resistance to listeriosis(191) and to staphylococcal infections (257).The ribosomal particles may also increase mac-rophage activation since carbon clearance ratesare increased and both Listeria and Staphylo-coccus are inactivated more readily by ribo-some-stimulated mouse macrophages (201).

Claims have been made that resistance in-duced with adjuvant-containing ribosomal vac-cines can be adoptively transferred to normalrecipients with peritoneal exudate cells but notwith immune serum (252). Smith and Bigley(231) also reported that mice immunized withsuch a preparation developed a specific state ofDTH to the ribosomal antigens. This last find-ing is particularly important if a close relation-ship exists between DTH and cell-mediatedresistance to salmonellosis (53, 164, 183). How-ever, the exact nature of the immunogen in-jected may be less important than its composi-tion following processing by the phagocytic cellssurrounding an infectious lesion (196). The onlything that is known about such immunogens isthat they are not lipopolysaccharide in nature(64). With very few exceptions (74, 103), de-layed hypersensitivity has been demonstratedonly against protein antigens (246) and poly-peptides (45, 219). It is perhaps unfortunatethat the protein component of endotoxin, aswell as other soluble protein components ofthe bacterial cell, have received such scant at-tention in the past as possible immunogens(20). It is known that soluble proteins arereleased into the culture medium from activelygrowing cells (245). Presumably many of theseproteins are extracellular enzymes involved inthe digestion of macromolecules within theculture medium. At present, we know very littleabout their antigenic properties or functionswithin an intracellular environment (21, 22).Enzymes such as hyaluronidase and collagenasehave been studied extensively for their disease-enhancing properties, but there is no reason tosuppose that some of these proteins may notalso act as immunogens within the infected host(170).

In vitro cultures of Salmonella release a greatdeal of antigenic material into the medium asthe culture passes into the stationary phase andan increasing number of cells begin to autolyze(64). However, the sensitins isolated from suchculture filtrates are normally heavily contami-nated with endotoxin and nucleic acid. In theactively infected host, the logarithmically mul-tiplying organisms are all in a metabolicallyactive state and would release any criticalsensitins to the surrounding phagocytes as aslow, steady stimulus. It is known that livingorganisms in the tissues must be in directcontact with the host's inflammatory cells,since enclosure of the organisms in a peritonealdiffusion chamber induces a humoral ratherthan a cellular immune response (5). The sensi-tizing antigen(s) released from the activelygrowing organism into the surrounding tissues

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somehow induces a predominantly cellular typeof hypersensitivity (196). It is only with theevolution of the cellular immune response thatlarge numbers of bacilli will be destroyed,presumably releasing large amounts of lipopoly-saccharide and nucleoprotein into the tissues,and it is then that the characteristic antibodyresponse begins to develop (64, 110, 133).

Cellular proliferation within the infectiouslesion itself, and in the draining lymph nodes(62, 163) and the liver and spleen (187, 190),may peak at different times, depending upon

the route of infection involved (41). As a rule,the maximal systemic immune response corre-

lates best with the peak in tritiated thymidineincorporation by the spleen cells (186-188). Thisalso coincides with the time when the DTHresponse reaches a maximum (158, 191). Thispeak cellular division usually declines againwith time, and this is often associated with a

slowed rate of inactivation by the remainingbacterial population in vivo (56). Eventually a

stable balance will be struck between the inhib-itory effect of the host defenses and the rate ofbacterial multiplication in vivo, and a more or

less permanent carrier state evolves (117). Thesequence of cellular events during the infectionand convalescence of the host has perhaps beenmost thoroughly investigated in mice sufferingfrom listeriosis (151, 155). After a peak inbacterial counts on day 2 or 3, the level ofcellular immunity increases to a maximumabout day 6 and then declines again but re-

mains above normal for as long as the stimulat-ing infection persists in vivo. Incorporationrates for the liver and spleen also reach a peakabout day 6 and then rapidly decline towardsnormal values again (186). Adoptive transferstudies indicate that cellular immunity is pro-

portional to the number of actively dividingT-cells within the spleen (190). The systemicListeria population will be eliminated com-

pletely within 10 to 12 days, and the number ofspecifically sensitized T-cells also falls precipi-tously. By 4 weeks, the host's cellular defenseswill have essentially returned to normal, and by4 months the host will again be fully susceptibleto this infectious agent (155). Other bacteriatend to persist in vivo for longer periods, and,even after the organisms have apparently beencompletely eliminated from the liver andspleen, isolated abscesses may be detected inother organs, such as the lung or kidney. Forinstance, viable S. enteritidis can be recoveredfrom the kidneys of intravenously infected ratsup to 12 months after the infectious agent hasbeen apparently eliminated from the liver andspleen (256). Such a residual (frequently latent)

infection may have a considerable survivalvalue for the host by ensuring that an acceler-ated immune response occurs each time theindividual is reinfected. This may be particu-larly important for subjects living in an endemicdisease area.

Latent carrier states seem to be particularlyimportant in the case of M. tuberculosis (18)and Corynebacterium kutcheri (92) infectionswhere the infectious agent may persist in vivothroughout the life of the individual (149). Thisprobably explains the continuing tuberculinsensitivity seen in many patients who havemade an apparently complete recovery fromactive tuberculosis (56). The bacilli within thelatent focus of infection leak small amounts ofsensitizing antigen(s) into the host tissues,resulting in the continuing presence of specifi-cally sensitized T-lymphocytes within thedraining lymph node. The presence of thesecells within the tissues probably represents asufficient defensive "edge" to ensure an acceler-ated immune response at any time when theindividual is reinfected with the parasite. Theresulting infection will then be limited to sub-clinical proportions. Such immunity will neverbe absolute, however, since a large enough doseof highly virulent organisms may induce asecond attack of clinical disease before the hostdefenses can be mobilized sufficiently. Simi-larly, if the host defenses are temporarily re-duced by dietary or environmental factors(140), or by prior immunosuppressive therapy(220), then a second attack of the disease willvery likely occur. At present we know littjeabout the interaction of many of these variableson the host-parasite relationships involved inthe maintenance of acquired resistance, andfurther investigation of the relevant parametersis clearly warranted.

ROLE OF ADJUVANTS IN THEDEVELOPMENT OF CELL-MEDIATED

IMMUNITYFreund first described the striking enhance-

ment to the immunogenicity of antigens sus-pended in a water-in-oil emulsion containingwhole mycobacterial cells (96). The reagent isreferred to as Freund's complete adjuvant. Thewhole mycobacteria may be replaced by BCGcell walls, by the Wax D component of themycobacterial lipid fraction and even, in somecircumstances, by large quantities of gram-neg-ative lipopolysaccharide (7, 223, 259). Multipleinjections of Freund's complete adjuvant intonormal mice induce considerable levels of tu-berculin hypersensitivity and an enhanced an-tituberculous resistance (66, 70). Although the

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reason is still not clear, the addition of extrane-ous protein antigens to the emulsion (97) resultsin the induction of a state of delayed hypersen-sitivity to an added antigen(s). Nelson andMildenhall (183) reported observing some DTHin mice receiving a soluble protein incorporatedinto Freund's incomplete adjuvant. In the pres-ent study, no cellular hypersensitivity could bedetected when killed whole Salmonella cellswere presented to the host in this vehicle (57).This apparent conflict may be resolved in termsof the differing antigens used in the two studies.The mosaic of polysaccharide antigens presenton the Salmonella cell wall surface will inducestrong humoral responses resulting in Arthus- orimmediate-type hypersensitivity which couldeasily mask or suppress any delayed componentof the host response (62). In studies carried outin this laboratory, the addition of 200 gg ofkilled salmonellae to Freund's complete adju-vant increased the level of anti-Salmonellaimmunity against both intravenous (54) andoral challenges (57) with virulent S. enteritidis.At the highest level of killed S. enteritidis tested(500 gg of whole cells), the immune response tothe adjuvant-carried vaccine was equivalent tothat seen previously only in mice receiving aliving attenuated vaccine (54). On the otherhand, when the same amount of bacterial anti-gen was introduced into the tissues in Freund'sincomplete adjuvant, only an increased hu-moral but no cellular response could be ob-served (62). The level of specific resistance tothe virulent S. enteritidis challenge was thenlittle better than that seen in controls receivingFreund's adjuvant alone.The mode of action of the mycobacterial

component of Freund's complete adjuvant isstill unclear. The presence of the mycobacterialcells in the oil droplets (23) apparently increasesthe cellular response in the tissues surroundingthe antigen depot (25). This somehow results ina delayed type of hypersensitivity, in additionto the enhanced antibody production normallyseen when the water-in-oil emulsion is used(96). Such a response could not be observedwhen the whole mycobacteria (or purified cellwalls) were suspended in the aqueous phase ofthe emulsion (204, 205). The tissue response toFreund's complete adjuvant is particularly se-vere, so that this type of preparation cannotpresently be used in humans (16). In an attemptto overcome this toxicity, a water-soluble adju-vant has been isolated from mycobacterial cellwalls which has been claimed to be able toinduce high levels of DTH to added proteinantigens without being highly irritant to thetissues (1, 42). Although such a reagent un-

doubtedly increases the humoral response to theadded antigens, the data presented must beinterpreted very carefully, in the light of possi-ble Arthus or Jones-Mote type of reactions(182), and, in general, claims for marked adju-vant action in the absence of a severe granulo-matous tissue response should be regarded withsome reservation at present.

Mycobacterial enriched water-in-oil emul-sions are the only practical alternative to livinginfections in the induction of a specific cellularresponse to the microbial antigens. Living BCGinduces an almost pure T-cell response on thepart of the infected host (159), but salmonellaetend to induce a mixed B- and T-cell reaction.At the time the immune response to live S.enteritidis first becomes apparent (day 4 to 5),this humoral (B-cell) response is still too low forready detection (61, 64). On the other hand,dead S. enteritidis (or S. pullorum) induces apure humoral response in the vaccinated mouse(67). As was shown in Fig. 1, even living S.pullorum cells were unable to induce a cell-mediated immune response, and this organismappeared to be completely nonimmunogenic formice. However, when heat-killed suspensions ofS. pullorum (200 Mg) were suspended inFreund's complete adjuvant and two weeklyinjections were given subcutaneously, the cellu-lar immune response to the S. pullorum anti-gens was almost as good as that achieved withthe corresponding S. enteritidis preparations(57). Protection tests using decreasing amountsof the two bacterial suspensions indicated thatsome quantitative antigenic differences existedbetween them (61), but this did not seem to besufficient per se to account for the total lack ofimmunogenicity shown by the live S. pullorumvaccine (Fig. 1). The nature of the qualitativeand quantitative cellular changes which occuras the antigens are released by the live sal-monellae in vivo are clearly very different de-pending upon the strain and the circumstancesunder which the antigen is introduced into thetissues.

Unravelling the cellular interactions involvedin these two contrasting host responses consti-tutes one of the most provocative and intriguingproblems in comtemporary immunology. Sincethe host-parasite relationships involved in theearly phases of a systemic microbial infectionprobably play a crucial role in determining theultimate fate of the host, a better understandingof the relevant cellular interactions occurring invivo will not only influence the development ofimproved immunizing procedures, but may wellhave important implications in the control ofseveral human infections for which there is

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presently no effective prophylaxis, and even forthe immunotherapy of some human cancers.

ACKNOWLEDGMENTSWork described in this paper was supported by

grant AI 07809 from the U.S.-Japan CooperativeMedical Science Program administered by the Na-tional Institute of Allergy and Infectious Diseases andpartly by the U.S. Army Research and DevelopmentCommand, Washington, D.C., under contract DA-DA-17-71-C-1056.

I thank William Woodruff, Lorraine Schuler, Vin-cent Montalbine, and Linda Auclair for their excel-lent technical assistance during the studies outlined inthis review. The collaboration of A. Volkman, of thisInstitute, during the studies of the growth of S.enteritidis in the T-cell depleted mice is gratefullyacknowledged.

LITERATURE CITED1. Adam, A., Z. Ciorbaru, J.-F. Petit, and E.

Lederer. 1972. Isolation, properties of a macro-molecular, water-soluble, immune adjuvantfraction from the cell wall of Mycobacteriumsmegmatis. Proc. Nat. Acad. Sci. U.S.A.69:851-854.

2. Adlam, C., E. S. Broughton, and M. T. Scott.1972. Enhanced resistance of mice to infectionwith bacteria following pretreatment withCorynebacterium parvum. Nature. N. Biol.235:219-220.

3. Adlam, C., and M. T. Scott. 1973. Lym-phoreticular stimulatory properties of Coryne-bacterium parvum and related bacteria. J.Med. Microbiol. 6:261-274.

4. Agarwal, S. C., and N. K. Ganguly. 1972. Oralimmunization with L-forms of Vibrio choleraein human volunteers. Infect. Immunity6:17-20.

5. Akiyama, T., K. Maeda, G. Asaba, H. Naga-tomi, S. Watabili, and D. Ushiba. 1964. The useof diffusion chambers in investigating thecellular nature of immunity in experimentaltyphoid and tuberculosis. Jap. J. Microbiol.8:37-48.

6. Akiyama, T., K. Maeda, and D. Ushiba. 1962.Studies on immunity in experimental typhoid.Jap. J. Bacteriol. 17:829-834.

7. Anacker, R. L., W. R. Barclay, W. Brehmer, G.Goode, R. H. List, E. Ribi, and D. F. Tarmina.1969. Effectiveness of cell walls of Mycobacte-rium bovis strain BCG administered by vari-ous routes and in different adjuvants in pro-tecting mice against airborne infection withMycobacterium tuberculosis strain H37Rv.Amer. Rev. Resp. Dis. 99:242-248.

8. Anacker, R. L., W. R. Barclay, W. Brehmer, C.L. Larson, and E. Ribi. 1967. Duration ofimmunity to tuberculosis in mice vaccinatedintravenously with oil-treated cell walls ofMycobacterium bovis strain BCG. J. Immu-nol. 98:1265-1273.

9. Anderson, E. S., and H. G. H. Richards. 1948.An outbreak of typhoid fever in the MiddleEast. J. Hyg. 46:164-172.

10. Anonymous. 1964. Preparation of dried acetone-inactivated and heat-phenol inactivated ty-phoid vaccines. Bull. World Health Organ.30:635-646.

11. Archer, J. R., and D. Rowley. 1969. A quantita-tive comparison of the antigenic structure of avirulent and an avirulent strain of Salmonellatyphimurium. Immunology 17:551-558.

12. Arnason, B. G., B. D. Jankovic, B. H. Waksman,and C. Wennersten. 1962. Role of thymus inimmune reactions in rats. II. Suppressiveeffect of thymectomy at birth on reactions ofdelayed cellular hypersensitivity and the cir-culating small lymphocyte. J. Exp. Med.116:177-186.

13. Ashcroft, M. T., J. M. Ritchie, and C. C.Nicholson. 1964. Controlled field trial in Brit-ish Guiana school children of heat-killed phe-nolized and acetone-killed lyophilized typhoidvaccine. Amer. J. Hyg. 79:196-206.

14. Auzins, I., and D. Rowley. 1962. On the questionof specificity of cellular immunity. Aust. J.Exp. Biol. Med. Sci. 40:283-292.

15. Auzins, I., and D. Rowley. 1963. Factors involvedin the adherence of S. typhimurium C5 andmouse macrophages. Aust. J. Exp. Biol. Med.Sci. 41:539-546.

16. Azurin, J. C., A. Cruz, T. P. Pesigan, M. Alvero,T. Camerra, R. Suplido, L. Ledesma, and C.A. Gomez. 1967. A controlled field trial of theeffectiveness of cholera and cholera El Torvaccines in the Philippines. Bull. WorldHealth Organ. 37:703-727.

17. Bain, R. V. S. 1963. Hemorrhagic septacemia.Food Agricultural Organ. U.N. F.A.O. Agr.Stud. No. 62.

18. Baker, R. E., W. E. Hill, and C. L. Larson. 1972.Delayed hypersensitivity reactions provokedby ribosomes from acid-fast bacilli. I. Ribo-somal isolation, characterization, delayed hy-persensitivity and specificity. Infect. Immu-nity 6:258-265.

19. Baker, E. E., R. E. Whiteside, R. Basch, and M.A. Derow. 1959. The Vi antigens of Enterobac-teriaceae. I. Purification and chemical proper-ties. J. Immunol. 83:680-686.

20. Barber, C., and E. Eylan. 1972. Immunochemis-try of Salmonella typhimurium. Variations inthe proteinic moiety of the somatic antigen.Rev. Immunol. 36:1-13.

21. Barber, C., E. Eylan, and Y. Keydar. 1968.Contributions a l'etude des Salmonelles pro-teines communes des Salmonelles apparten-tatn auz groupes B, C, D et E, et leur relationavec Citrobacter ballerup. Pathol. Microbiol.31:165-174.

22. Barber, C., J. R. Vladoianu, and G. Dimache.1967. Contributions to the study ofSalmonella. 2. Protein determinants sharedby S. typhi, S. enteritidis and S.typhimurium. Immunology 12:411-416.

394 COLLINS

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

VACCINES AND CELL-MEDIATED IMMUNITY

23. Barclay, W. R., R. L. Anacker, W. Brehmer, andE. Ribi. 1967. Effects of oil-treated mycobac-terial cell walls on the organs of mice. J.Bacteriol. 94:1736-1745.

24. Baron, L. S., and S. B. Formal. 1960. Immuniza-tion studies with living vaccine of Salmonellatyphimurium. Proc. Soc. Exp. Biol. Med.104:565-567.

25. Bekierkunst, A., I. S. Levi, E. Yarkoni, E.Vilkas, A. Adam, and E. Lederer. 1969. Gran-uloma formation induced in mice by chemi-cally defined mycobacterial fractions. J. Bac-teriol. 100:95-102.

26. Benacerraf, B. 1960. Influence of irradiation onresistance to infection. Bacteriol. Rev.24:35-40.

27. Benenson, A. S. 1964. Serological responses ofman to typhoid vaccines. Bull. World HealthOrgan. 30:653-662.

28. Benoit, J.-C., and M. Panisset. 1963. Suvie etmultiplication du BCG et du Bacille tuber-culeux chez la souris. Acta Tuberc. Pneumol.Scand. 43:125-136.

29. Blanden, R. V. 1968. Modification of macro-phage function. J. Reticuloendothel. Soc.5:179-202.

30. Blanden, R. V. 1974. T cell responses to viral andbacterial infection. Transplant. Rev.19:56-88.

31. Blanden, R. V., M. J. Lefford, and G. B.Mackaness. 1969. The host response to Cal-mette-Guerin bacillus infection in mice. J.Exp. Med. 129:1079-1106.

32. Blanden, R. V., G. B. Mackaness, and F. M.Collins. 1966. Mechanisms of acquired resist-ance in mouse typhoid. J. Exp. Med.124:585-600.

33. Blanden, R. V., and C. A. Mims. 1973. Macro-phage activation in mice infected with Ectro-melia or lymphocytic choriomeningitis vi-ruses. Aust. J. Exp. Biol. Med. Sci.51:393-398.

34. Boyden, S. V., and W. E. Suter. 1952. Stimulat-ing effect of tuberculin upon production ofcirculating antibodies in guinea pigs infectedwith tubercle bacilli. J. Immunol. 68:577-589.

35. Boyle, W. 1967. An antigen common to mousecells and Salmonella typhimurium. J. Immu-nol. 98:256-259.

36. Bullock, W. E. 1968. Studies of immune mech-anisms in leprosy. I. Depression of delayedallergic response to skin test antigens. N.Engl. J. Med. 278:298-304.

37. Bullock, W. E., J. P. Fields, and M. W. Brandiss.1972. Transfer factor as immunotherapy forlepromatous leprosy. N. Engl. J. Med.287:1053-1058.

38. Cameron, J. A., D. F. Holtman, and C. D.Jeffries. 1960. The association of virulencewith endotoxin in Salmonella pullorum. J.Infec. Dis. 106:159-161.

39. Carey, W. F., and L. S. Baron. 1959. Compara-tive immunologic studies of cell structuresisolated from Salmonella typhosa. J. Immu-

nol. 83:517-520.40. Carter, P. B., and F. M. Collins. 1974. The route

of enteric infection in normal mice. J. Exp.Med. 139:1189-1203.

41. Carter, P. B., and F. M. Collins. 1975. Peyer'spatch responsiveness to Salmonella in mice.(Submitted for publication).

42. Chedid, L., M. Parant, F. Parant, R. H. Gustaf-son, and F. M. Berger. 1972. Biological studyof a non-toxic, water soluble immunoadjuvantfrom mycobacterial cell walls. Proc. Nat.Acad. Sci. U.S.A. 69:855-858.

43. Chernokhvostova, E., K. J. Luxemburg, V. Star-shinova, N. Androeva, and L. German. 1969.Study on the production of IgG, IgA andIgM-antibodies to somatic antigens of Salmo-nella typhi in humans. Clin. Exp. Immunol.4:407-421.

44. Ciinar, I., and J. W. Shands, Jr. 1970. Effect ofalkali on the immunological reactivity of lipo-polysaccharide from Salmonella typhimurium.Infect. Immunity 2:549-555.

45. Clark, C., and J. Munoz. 1970. Delayed hyper-sensitivity to insulin and its component poly-peptide chains. J. Immunol. 105:574-583.

46. Cluff, L. E. 1970. Effects of lipopolysaccharideson susceptibility to infections. J. Infec. Dis.122:205-216.

47. Collins, F. M. 1967. Serum mediated killing of 3group D Salmonellas. J. Gen. Microbiol.46:247-253.

48. Collins, F. M. 1968. Recall of immunity in micevaccinated with Salmonella enteritidis or Sal-monella typhimurium. J. Bacteriol.95:2014-2021.

49. Collins, F. M. 1969. Effect of specific immunemouse serum on the growth of Salmonellaenteritidis in nonvaccinated mice challengedby various routes. J. Bacteriol. 97:667-675.

50. Collins, F. M. 1969. Effect of specific immunemouse serum on the growth of Salmonellaenteritidis in mice preimmunized with livingor ethyl alcohol-killed vaccines. J. Bacteriol.97:676-683.

51. Collins, F. M. 1970. Immunity to enteric infec-tion in mice. Infect. Immunity 1:243-250.

52. Collins, F. M. 1971. Immunogenicity of variousMycobacteria and the corresponding levels ofcross-protection developed between species.Infect. Immunity 4:688-696.

53. Collins, F. M. 1971. Mechanisms in antimi-crobial immunity. J. Reticuloendothel. Soc.10:58-90.

54. Collins, F. M. 1972. Effect of adjuvant onimmunogenicity of a heat-killed Salmonellavaccine. J. Infec. Dis. 126:69-76.

55. Collins, F. M. 1972. Salmonellosis in orallyinfected specific pathogen-free C57B1 mice.Infect. Immunity 5:191-198.

56. Collins, F. M. 1972. Acquired resistance toMycobacterial infection. Advan. Tuberc. Res.18:1-30.

57. Collins, F. M. 1973. Immunogenicity of livingand heat-killed Salmonella pullorum vac-

395VOL. 38,1974

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

BACTERIOL. REV.

cines. Infect. Immunity 7:735-742.58. Collins, F. M. 1973. Growth of Pasteurella

multocida in vaccinated and normal mice.Infect. Immunity 8:868-875.

59. Collins, F. M. 1973. The relative immunogenic-ity of virulent and attenuated strains of tuber-cle bacilli. Amer. Rev. Resp. Dis.107:1030-1040.

60. Collins, F. M., D. L. Boros, and K. S. Warren.1972. The effect of Schistosoma mansoni in-fection on the response of mice to Salmonellaenteritidis and Listeria monocytogenes. J. In-fec. Dis. 125:249-256.

61. Collins, F. M., and P. B. Carter. 1972. Compara-tive immunogenicity of heat-killed and livingoral Salmonella vaccines. Infect. Immunity6:451-458.

62. Collins, F. M., and P. B. Carter. 1974. Cellularimmunity in enteric disease. Amer. J. Clin.Nutr., vol. 27 (Dec.).

63. Collins, F. M., C. C. Congdon, and N. E.Morrison. 1975. Growth of Mycobacteriumbovis (BCG) in T-lymphocyte depleted mice.Infect. Immunity 11:10-15.

64. Collins, F. M., and G. B. Mackaness. 1968.Delayed hypersensitivity and Arthus reactiv-ity in relation to host resistance in Salmonel-la-infected mice. J. Immunol. 101:830-845.

65. Collins, F. M., and G. B. Mackaness. 1970. Therelationship of delayed hypersensitivity toacquired antituberculous immunity. 1. Tuber-culin sensitivity and resistance to reinfectionin BCG vaccinated mice. Cell. Immunol.1:253-265.

66. Collins, F. M., and G. B. Mackaness. 1970. Therelationship of delayed hypersensitivity toacquired antituberculous immunity. II. Effectof adjuvant on the allergenicity and immuno-genicity of heat-killed tubercle bacilli. Cell.Immunol. 1:266-275.

67. Collins, F. M., G. B. Mackaness, and R. V.Blanden. 1966. Infection-immunity in experi-mental salmonellosis. J. Exp. Med.124:601-619.

68. Collins, F. M., and T. E. Miller. 1969. Growth ofa drug resistant strain of Mycobacteriumbovis (BCG) in normal and immunized mice.J. Infec. Dis. 120:517-533.

69. Collins, F. M., and M. Milne. 1966. Heat-labileantigens of Salmonella enteritidis. I. Mouseprotection studies. J. Bacteriol. 92:549-557.

70. Collins, F.M., and V. Montalbine. 1973. Relativeimmunigenicity of streptomycin-sensitiveand-resistant strains of BCG. Infect. Immu-nity 8:381-387.

71. Collins, F. M., and M. T. Scott. 1974. The effectof Corynebacterium parvum treatment on thegrowth of Salmonella enteritidis in mice. In-fect. Immunity 9:863-869.

72. Coppel, S., and G. P. Youmans. 1969. Specificityof the anamnestic response produced by List-eria monocytogenes or Mycobacteriumtuberculosis to challenge with Listeriamonocytogenes. J. Bacteriol. 97:127-133.

73. Cronley-Dillon, S. 1973. Comparison of the viru-lence for mice of S. typhimurium given by theintraperitoneal and subcutaneous routes. J.Hyg. 71:223-226.

74. Crowle, A. J., and C. C. Hu. 1967. Delayedhypersensitivity in mice to dextran. Int. Arch.Allergy Appl. Immunol. 31:123-144.

75. Curnen, E. C., and C. M. MacLeod. 1942. Theeffect of sulfapyridine upon the developmentof immunity to pneumococcus in rabbits. J.Exp. Med. 75:77-92.

76. Cvjetanovic, B., D. M. Mel, and 0. Felsenfeld.1970. Study of a live typhoid vaccine inchimpanzees. Bull. World Health Organ.42:499-507.

77. Cvjetanovic, B., and K. Uemura. 1965. Thepresent status of field and laboratory studiesof typhoid and paratyphoid vaccines. Bull.World Health Organ. 32:29-36.

78. Dannenberg, A. 1968. Cellular hypersensitivityand cellular immunity in the pathogenesis oftuberculosis: specificity, systemic and localnature, and associated macrophage enzymes.Bacteriol. Rev. 32:85-102.

79. Davies, A. J. S. 1969. The thymus and thecellular basis of immunity. Transplant. Rev.1:43-91.

80. Diena, B. B., E. M. Johnson, L. S. Baron, R.Wallace, and L. Greenberg. 1973. Assay oftyphoid vaccines with Salmonella typhosa-Salmonella typhimurium hybrids. Infect. Im-munity 7:5-8.

81. Doenhoff, M. J., A. J. S. Davies, E. Leuchars,and V. Wallis. 1971. The thymus and circulat-ing lymphocytes of mice. Proc. Roy. Soc. Ser.B 176:69-85.

82. Drysdale, A. 1947. A comparative study of phe-nolized and alcoholized TAB vaccines. J. Hyg.45:46-49.

83. Dubos, R., and C. H. Pierce. 1956. Differentialcharacteristics in vitro and in vivo of sub-strains of BCG. 1. Multiplication and survivalin vitro. Amer. Rev. Tuberc. 74:655-666.

84. Dubos, R., and C. H. Pierce. 1956. Differentialcharacteristics in vitro and in vivo of severalsubstrains of BCG. IV. Immunizing effective-ness. Amer. Rev. Tuberc. 74:699-717.

85. DuPont, H. L., R. B. Hornick, M. J. Snyder, J.P. Libonati, S. B. Formal, and E. J. Gan-garosa. 1972. Immunity in shigellosis. II. Pro-tection induced by oral live vaccine or primaryinfection. J. Infec. Dis. 125:12-16.

86. DuPont, H. L., R. B. Hornick, M. J. Synder, J.P. Libonati, and T. E. Woodward. 1971.Immunity in typhoid fever: evaluation of livestreptomycin-dependent vaccine. Antimicrob.Ag. Chemother. 1970, p. 236-239.

87. Eddie, D. S., M. L. Schulkind, and J. B.Robbins. 1971. The isolation and biologicactivities of purified secretory IgA and IgGanti-Salmonella typhimurium "O" antibodiesfrom rabbit intestinal fluid and colostrum. J.Immunol. 106:181-190.

88. Esposito, V. M., J. C. Feeley, W. D. Leeder, and

COLLINS396

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

VACCINES AND CELL-MEDIATED IMMUNITY

M. Pittman. 1969. Immunological response ofthree mouse strains to typhoid vaccine and Viantigen. J. Bacteriol. 99:8-12.

89. Fahey, K. J., and G. N. Cooper. 1970. Oralimmunization against experimental salmonel-losis. I. Development of temperature-sensitivemutant vaccines. Infect. Immunity 1:263-270.

90. Fahey, K. J., and G. N. Cooper. 1970. Oralimmunization to experimental salmonellosis.II. Characteristics of the immune response totemperature-sensitive mutants given by oraland parenteral routes. Infect. Immunity2:183-191.

91. Fauve, R. M., and M. B. Hevin. 1971. Pouvoirbactericide des macrophages spleniques ethepatiques de souris envers Listeriamonocytogenes. Ann. Inst. Pasteur Paris120:399-411.

92. Fauve, R. M., C. H. Pierce-Chase, and R. Dubos.1964. Corynebacterial pseudotuberculosis inmice. II. Activation of natural and experimen-tal latent infections. J. Exp. Med.120:283-304.

93. Felix, A. 1951. The preparation, testing andstandardization of typhoid vaccine. J. Hyg.49:268-287.

94. Felix, A., and M. Pitt. 1934. A new antigen of B.typhosus. Its relation to virulence and toactive and passive immunization. Lancet2:186-191.

95. Felix, A., and R. M. Pitt. 1951. The pathogenicand immunogenic activities of Salmonellatyphi in relation to its antigenic constituents.J. Hyg. 49:92-110.

96. Freund, J. 1956. The mode of action of immuno-logical adjuvants. Advan. Tuberc. Res.1:130-148.

97. Freund, J., J. Casals, and R. P. Hosiner. 1937.Sensitization and antibody formation afterinjection of tubercle bacilli and paraffin oil.Proc. Soc. Exp. Biol. Med. 37:509-513.

98. Fubara, E. S., and R. Freter. 1973. Protectionagainst enteric infection by secretory IgA anti-bodies. J. Immunol. 111:395-403.

99. Furness, G., and J. Ferriera. 1959. The role ofmacrophages in natural immunity toSalmonellae. J. Infec. Dis. 105:203-206.

100. Furness, G., and D. Rowley. 1956. Transductionof virulence within the species Salmonellatyphimurium. J. Gen. Microbiol. 15:140-145.

101. Gaines, S., J. G. Tulley, and W. D. Tigertt. 1961.Enhancement of the mouse virulence of a

non-Vi variant of S. typhi by Vi antigen. J.Immunol. 86:543-551.

102. Gelzer, J., and E. Suter. 1959. The effect ofantibody on intracellular parasitism of Salmo-nella typhimurium on mononuclear phago-cytes in vitro. Prolonged survival of infectedmonocytes in presence of antibody. J. Exp.Med. 110:715-730.

103. Gerety, R. J., R. W. Ferraresi, and S. Raffel.1970. Polysaccharide in delayed hypersensitiv-ity. 1. Pneumococcal polysaccharide as in-

ducer and elicitor of delayed hypersensitivityin guinea pigs. J. Exp. Med. 131:189-206.

104. Germanier, R. 1970. Immunity in experimentalsalmonellosis. 1. Protection induced by roughmutants of Salmonella typhimurium. Infect.Immunity 2:309-318.

105. Germanier, R. 1972. Immunity in experimentalsalmonellosis. mII. Comparative immunizationwith live and heat-inactivated cells of Salmo-nella typhimurium. Infect. Immunity5:792-797.

106. Godal, T., B. Myklestad, D. R. Samuel, and B.Myrvang. 1971. Characterization of the cellu-lar immune defect in lepromatous leprosy: Aspecific lack of circulating Mycobacteriumleprae-active lymphocytes. Clin. Exp. Immu-nol. 9:821-832.

107. Gordon, B. L. 1967. The case for cytophilicantibodies in cellular immunity. Ann. Allergy25:1-5.

108. Gray, D. F. 1958. Immunity, natural anergy andartificial desensitization in experimental tu-berculosis. Amer. Rev. Tuberc. 78:235-250.

109. Greisman, S. E., and W. E. Woodward. 1965.Mechanisms of endotoxin tolerance. III. Therefractory state during continuous infusions ofendotoxin. J. Exp. Med. 121:911-933.

110. Gupta, J. D., and C. E. Reed. 1971. Amount,class and specificity of antibody to the lipo-polysaccharide of Salmonella enteritidis afterimmunization by various schedules. Int. Arch.Allergy Appl. Immunol. 40:256-263.

111. Halliburton, B. L., and R. D. Hinsdill. 1972.Recall of acquired cellular resistance in miceby antigens from killed Brucella. Infect. Im-munity 5:42-47.

112. Hejfec, L. B. 1971. Size of the immunizing doseand post vaccination immunity to typhoid andparatyphoid B from the results of controlledfield trials. J. Hyg. Epidemiol. Microbiol.15:173-182.

113. Hejfec, L. G., L. A. Levina, M. L. Kuzminova,A. M. Slavina, A. K. Drozd, J. A. Tonojan,L. I. Koenman, N. E. Levites, L. A. Abidov,A. Z. Abidov, and L. F. Goncarova. 1969. Acontrolled field trial to evaluate the protectivecapacity of a single dose of acetone-killed agar-grown and heat-killed broth-grown typhoid vac-cines. Bull World Health Organ. 40:903-907.

114. Herzberg, M. 1962. Living organisms as immu-nizing agents against experimental salmonel-losis in mice. I. Virulence of autotrophicmutants. J. Infec. Dis. 111:192-203.

115. Herzberg, M., P. Nash, and S. Hino. 1972.Degree of immunity induced by killed vac-cines to experimental samonellosis in mice.Infect. Immunity 5:83-90.

116. Hobson, D. 1957. Resistance to reinfection inexperimental mouse typhoid. J. Hyg.55:334-343.

117. Hobson, D. 1957. Chronic bacterial carriage insurvivors of experimental mouse typhoid. J.Pathol. Bacteriol. 73:399-410.

397VOL. 38, 1974

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

BACTERIOL. REV.

118. Holland, J. J., and M. J. Pickett. 1958. A cellu-lar basis of immunity in experimentalBrucella infections. J. Exp. Med. 108:343-360.

119. Hornick, R. B., S. E. Greisman, T. E. Wood-ward, H. L. DuPont, A. T. Dawkins, and M. J.Snyder. 1970. Typhoid fever: pathogenesisand immunological control. N. Engl. J. Med.283:686-691.

120. Hornick, R. B., and T. E. Woodward. 1967.Appraisal of typhoid vaccine in experimen-tally infected human subjects. Trans. Amer.Clin. Climatol. Ass. 78:70-78.

121. Houchins, D. P., and G. L. Wright. 1973. Immu-nity to Salmonella typhimurium infection:characterization of antigens in active protec-tion by polyacrylamide gel electrophoresis.Infect. Immunity 7:507-511.

122. Howard, J. G., G. Biozzi, B. N. Halpern, C.Stiffel, and D. Mouton. 1959. The effect ofMycobacterium tuberculosis (BCG) infectionon the resistance of mice to bacterial endo-toxin and Salmonella enteritidis infection.Brit. J. Exp. Pathol. 40:281-290.

123. Howard, J. G., G. H. Christie, and M. T. Scott.1973. Biological effects of Corynebacteriumparvum. IV. Adjuvant and inhibitory activityon B lymphocytes. Cell. Immunol. 7:290-301.

124. Hunt, S. V., S. T. Ellis, and J. L. Gowans. 1972.The role of lymphocytes in antibody forma-tion. IV. Carriage of immunological memory

by lymphocyte fractions separated by velocitysedimentation and on glass bead columns.Proc. Roy. Soc. Ser. B 182:211-231.

125. Jenkin, C. R., and D. Rowley. 1963. Basis forimmunity to typhoid in mice and the questionof "cellular immunity." Bacteriol. Rev.27:391-404.

126. Jenkin, C. R, and D. Rowley. 1965. Partialpurification of the "protective" antigen ofSalmonella typhimurium and its distributionamongst various strains of bacteria. Aust. J.Exp. Biol. Med. Sci. 43:65-78.

127. Jenkin, C. R., D. Rowley, and I. Auzins. 1964.The basis for immunity to mouse typhoid. I.The carrier state. Aust. J. Exp. Biol. Med. Sci.42:215-228.

128. Joo, Z. P., and V. P. Juhasz. 1968. Mouseprotective ability of the international refer-ence preparation of typhoid vaccine. Z. Im-munitaetsforsch. 135:365-387.

129. Karthigasu, K., and C. R. Jenkin. 1963. Thefunctional development of the reticuloendo-thelial system of the chick embryo. Immunol-ogy 6:255-263.

130. Karthigasu, K., C. R. Jenkin, and K. J. Turner.1964. The nature of the opsonins in adult henserum and developing chick embryos to cer-

tain Gram negative bacteria. Aust. J. Exp.Biol. Med. Sci. 42:499-510.

131. Kawakami, M., H. Nakata, and S. Mitsuhashi.1959. Experimental salmonellosis. Immuniz-ing effect of live vaccine prepared from variousmutants of Salmonella having different cellwall polysaccharides. Jap. J. Microbiol.13:315-324.

132. Kawakami, M., N. Osawa, and S. Mitsuhashi.1966. Experimental salmonellosis. VII. Com-paripon of the immunizing effect of live vac-cine and materials extracted from Salmonellaenteritidis. J. Bacteriol. 92:1585-1589.

133. Kenny, K., and M. Herzberg. 1968. Antibodyresponse and protection induced by immuni-zation with smooth and rough strains in exper-imental salmonellosis. J. Bacteriol.95:406-417.

134. Kenny, K., S. Schlect, and 0. Westphal. 1970.Antibody response to various single-factor 0antigens of Salmonella. Infect. Immunity1:41-50.

135. Khairat, 0. 1946. The bactericidal power of theblood for the infecting organism in bactera-emia. J. Pathol. Bacteriol. 58:359-365.

136. Khasanov, M. I., L. B. Kheifets, and L. V.Salmin. 1962. A controlled field trial of thetyphoid component of polyvalent-enteric vac-cine. Bull. World Health Organ. 26:371-379.

137. Klasky, S., and M. J. Pickett. 1968. Immunoge-nicity and specificity of Listeriamonocytogenes cell walls. J. Infec. Dis.118:65-75.

138. Krishnapillai, V., and K. Karthigasu. 1969.Salmonella abony-Salmonella typhimuriumrecombinant nonvirulent for the mouse. J.Bacteriol. 97:1343-1351.

139. Lane, F. C., and E. R. Unanue. 1972. Require-ment of thymus (T) lymphocytes for resist-ance to listeriosis. J. Exp. Med.135:1104-1112.

140. Layton, H. W., and G. P. Youmans. 1965. Effectof dietary factors upon the resistance of albinomice to experimental infection with Mycobac-terium tuberculosis. J. Bacteriol. 90:958-964.

141. Lefford, M. J., D. D. McGregor, and G. B.Mackaness. 1973. Immune response to Myco-bacterium tuberculosis in rats. Infect. Immu-nity 8:182-189.

142. Lim, S. D., D. F. Kiszkiss, R. R. Jacoboan, Y. S.Choi, and R. A. Good. 1974. Thymus-depend-ent lymphocytes of peripheral blood in leprosypatients. Infect. Immunity 9:394-399.

143. Lubaroff, D. M., and B. H. Waksman. 1968.Bone marrow as a source of cells in reactions ofcellular hypersensitivity. J. Exp. Med.128:1425-1433.

144. Lideritz, O., A. M. Staub, and 0. Westphal.1966. Immunochemistry of 0 and R antigensof Salmonella and related Enterobacteriaceae.Bacteriol. Rev. 30:192-255.

145. Lurie, M. B. 1942. Studies on the mechanism ofimmunity in tuberculosis: the fate of tuberclebacilli ingested by mononuclear phagocytesderived from normal and immunized animals.J. Exp. Med. 75:247-268.

146. Lurie, M. B. 1964. Resistance to tuberculosis.Harvard Univ. Press, Boston.

147. Maaloe, 0. 1948. Pathogenic-apathogenic trans-formation of S. typhimurium. Acta Pathol.Microbiol. Scand. 25:414-430.

148. McCune, R. M., F. M. Feldman, and W. McDer-mott. 1966. Microbial persistence. II. Charac-

COLLINS398

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

VACCINES AND CELL-MEDIATED IMMUNITY

teristics of the sterile state of tubercle bacilli.J. Exp. Med. 123:469-486.

149. McDermott, W. 1958. Microbial persistence.Yale J. Biol. Med. 30:257-291.

150. McGregor, D. D., H. H. Hahn, and G. B.Mackaness. 1973. The mediator of cellularimmunity. V. Development of cellular resist-ance to infection in thymectomized irradiatedrats. Cell. Immunol. 6:186-199.

151. Mackaness, G. B. 1962. Cellular resistance toinfection. J. Exp. Med. 116:381-406.

152. Mackaness, G. B. 1964. The immunologicalbasis of acquired cellular resistance. J. Exp.Med. 120:105-120.

153. Mackaness, G. B. 1967. The relationship ofdelayed hypersensitivity to acquired cellularresistance. Brit. Med. Bull. 23:52-54.

154. Mackaness, G. B. 1968. The immunology ofantituberculous immunity. Amer. Rev. Resp.Dis. 97:337-344.

155. Mackaness, G. B. 1969. The influence of im-munologically committed lymphoid cells onmacrophage activity in vivo. J. Exp. Med.129:973-992.

156. Mackaness, G. B. 1970. The monocyte in cellularimmunity. Semin. Hematol. 7:172-184.

157. Mackaness, G. B. 1970. The monocyte in de-layed hypersensitivity, p. 461-477. In R. vanFurth (ed.), Mononuclear phagocytes. Black-well Scientific Publications, Oxford.

158. Mackaness, G. B. 1971. The induction andexpression of cell-mediated hypersensitivity inthe lung. Amer. Rev. Resp. Dis. 104:813-828.

159. Mackaness, G. B. 1971. Delayed hypersensitivityand the mechanism of cellular resistance toinfection. Progr. Allergy 15:413-424.

160. Mackaness, G. B. 1971. Cell-mediated immu-nity. 2nd Int. Convoc. Immunol., p. 241-248(Karger, Basel).

161. Mackaness, G. B. 1971. Resistance to intracellu-lar infection. J. Infec. Dis. 123:439-445.

162. Mackaness, G. B. 1971. Cellular immunity. Ann.Inst. Pasteur Paris 120:428-437.

163. Mackaness, G. B., D. J. Auclair, and P. H.Lagrange. 1973. Immunopotentiation withBCG. I. Immune response to different strainsand preparations. J. Nat. Cancer Inst.51:1655-1667.

164. Mackaness, G. B., and R. V. Blanden. 1967.Cellular immunity. Progr. Allergy 11:89-140.

165. Mackaness, G. B., R. V. Blanden, and F. M.Collins. 1966. Host-parasite relations in mousetyphoid. J. Exp. Med. 124:573-583.

166. Mackaness, G. B., and W. C. Hill. 1969. Theeffect of anti-lymphocyte globulin on cell-mediated resistance to infection. J. Exp. Med.129:983-1012.

167. Mackaness, G. B., N. Smith, and A. Q. Wells.1954. The growth of intracellular tuberclebacilli in relationship to their virulence. Amer.Rev. Tuberc. 69:469-494.

168. Mackenzie, G. M., R. M. Pike, and R. E.Swinney. 1940. Virulence of Salmonella typhi-murium. II. Studies of the polysaccharideantigens of virulent and avirulent strains. J.

Bacteriol. 40:197-214.169. Makela, P. H., V. V. Valtonen, and M. Valton-

en. 1973. Role of O-antigen (lipopolysaccha-ride) factors in the virulence of Salmonella. J.Infec. Dis. 128(Suppl.):81-85.

170. Malley, A., and L. Brecher. 1972. Hypersensitiv-ity to bacterial enzymes. 1. Atopic hypersensi-tivity induced in Rhesus monkeys. J. AllergyClin. Immunol. 49:36-42.

171. Marcus, S., D. M. Donaldson, and D. W. Esplin,1955. Role of host defenses in systemic Kleb-siella pneumoniae infection. Effect of antibi-otics in normal and X-irradiated mice. J.Immunol. 74:494-497.

172. Marmion, D. E., G. R. E. Naylor, and I. P.Steward. 1953. Second attacks of typhoidfever. J. Hyg: 51:260-267.

173. Melikova, Z. H. 1965. A comparison of themethods used in the laboratory assessment ofthe efficacy of dried typhoid WHO vaccines.Microbiol. Epidemiol. Immunol. 5:58-65.

174. Miller, C. P., and M. Bohnhoff. 1963. Changes inthe mouse's enteric microflora associated withenhanced susceptibility to Salmonella infec-tion following streptomycin treatment. J. In-fec. Dis. 113:59-66.

175. Miller, J. F. A. P. 1971. The thymus and theimmune system. Vox Sang. 20:481-491.

176. Milner, K. C., J. A. Rudbach, and E. Ribi. 1971.General characteristics, p. 1-65. In S. J.Ajl (ed.), Microbial toxins, Vol 4. AcademicPress Inc., New York.

177. Munoz, J. 1963. Symposium on the relationshipof structure of microorganisms to their immu-nological properties. 1. Immunological andother biological activities of Bordetellapertussis antigens. Bacteriol. Rev. 27:325-340.

178. Nakano, M., and K. Saito. 1969. Chemicalcomponents in the cell wall of S. typhimuriumaffecting its virulence and immunogenicity inmice. Nature (London) 222:1085-1086.

179. Nakano, M., and K. Saito. 1970. The chemicalcomposition of the cell wall of S. typhimuriumaffecting the clearance rate in mouse: influ-ence of specific antibody. Jap. J. Microbiol.14:451-461.

180. Neiburger, R. G., G. P. Youmans, and A. S.Youmans. 1973. Relationship between tuber-culin hypersensitivity and cellular immunityto infection in mice vaccinated with viableattenuated mycobacterial cells or with Myco-bacterial ribonucleic acid preparations. Infect.Immunity 8:42-47.

181. Nelson, E. L., and J. R. Becker. 1959. The effectof whole body X-irradiation on the bacteri-cidal activity of phagocytic cells. II. Survivalof Pseudomonas aeruginosa within the liversand spleens of mice. J. Infec. Dis. 104:20-23.

182. Nelson, D. S., and S. V. Boyden. 1964. Thecutaneous reactivity of guinea pigs to pureprotein antigens. I. A critical evaluation ofmethods for the induction of delayed-typehypersensitivity to pure proteins. Int. Arch.Allergy Appl. Immunol. 25:279-303.

183. Nelson, D. S., and P. Mildenhall. 1967. Studies

399VOL. 38, 1974

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

BACTERIOL. REV.

on cytophilic antibodies. 1. The production bymice of macrophage cytophilic antibodies tosheep erythrocytes: relationship to the pro-

duction of other antibodies and the develop-ment of delayed hypersensitivity. Aust. J.Exp. Biol. Med. Sci. 45:113-130.

184. Neva, F. A., and H. R. Morgan. 1950. Toleranceto the action of endotoxins of enteric bacilli inpatients convalescent from typhoid and para-

typhoid fevers. J. Lab. Clin. Med. 35:911-922.185. North, R. J. 1968. The uptake of particulate

antigens. J. Reticuloendothel. Soc. 5:203-229.186. North, R. J. 1970. The relative importance of

blood monocytes and fixed macrophages tothe expression of cell-mediated immunity toinfection. J. Exp. Med. 132:521-534.

187. North, R. J. 1973. Cellular mediators of anti-Listeria immunity as an enlarged populationof short-lived, replicating T-cells. J. Exp.Med. 138:342-355.

188. North, R. J. 1973. Importance of thymus-derivedlymphocytes in cell-mediated immunity toinfection. Cell. Immunol. 7:166-176.

189. North, R. J. 1974. Cell-mediated immunity andthe response to infection, p. 185-219. In Mc-Cluskey and Cohen (ed.), Mechanisms of cell-mediated immunity. John Wiley & Sons, Inc.,New York.

190. North, R. J., and G. B. Mackaness. 1973. Immu-nological control of macrophage proliferationin vivo. Infect. Immunity 8:68-73.

191. North, R. J., G. B. Mackaness, and R. W.Elliott. 1972. The histogenesis of immunologi-cally committed lymphocytes. Cell. Immunol.3:680-694.

192. Olitzki, A. L., and D. Godinger. 1963. Compara-tive studies on S. typhi grown in vivo and invitro. III. The immunizing potencies of ace-

tone-killed vaccines prepared from in vivo andin vitro grown bacteria and the immunizingpotency of substances isolated from infectedorgans. J. Hyg. 61:353-363.

193. Irskov, J., and F. Kauffmann. 1936. Studies on

the significance of "Vi antigen" in the mecha-nism of typhoid infection in mice. J. Hyg.36:514-524.

194. Parish, C. R. 1972. The relationship betweenhumoral and cell-mediated immunity. Trans-plant. Rev. 13:35-66.

195. Payling Wright, G. 1968. Protective immunity,p. 457-475. In P. G. Gell and R. R. Coombs(ed.), Clinical aspects of immunology, 2nded. Blackwell Scientific Publications, Oxford.

196. Pearson, M. N., and S. Raffel. 1971. Macro-phage-digested antigen as inducer of delayedhypersensitivity. J. Exp. Med. 133:494-505.

197. Pierce, C. H., R. Dubos, and W. B. Schaefer.1956. Differential characteristics in vitro andin vivo of several substrains of BCG. III.Multiplication and survival in vivo. Amer.Rev. Tuberc. 74:683-698.

198. Plant, J., and A. A. Glynn. 1974. Natural resist-ance to Salmonella infection, delayed hyper-sensitivity and Ir genes in different strains of

mice. Nature (London) 248:345-347.199. Raettig, H. 1961. Animal experiments about oral

immunization with inactivated antigen. Zen-tralbl. Bakteriol. Parasitenk. Infektionskr.Hyg. Abt. I Orig. 182:525-528.

200. Rees, R. J. W. 1969. New prospects for the studyof leprosy in the laboratory. Bull. WorldHealth Organ. 40:785-800.

201. Remington, J. S., and T. C. Merigan. 1970.Synthetic polyanions protect mice againstintracellular bacterial infections. Nature(London) 226:361-363.

202. Ribi, E. 1971. Currents in tuberculosis research.J. Infec. Dis. 123:562-565.

203. Ribi, E., R. L. Anacker, W. R. Barclay, W.Brehmer, S. C. Harris, W. R. Leif, and J.Simmons. 1971. Efficacy of mycobacterial cellwalls as a vaccine against airborne tuberculo-sis in the Rhesus monkey. J. Infec. Dis.123:527-538.

204. Ribi, E., R. L. Anacker, W. Brehmer, G. Goode,C. L. Larson, and R. H. List. 1966. Factorsinfluencing protection against experimentaltuberculosis in mice by heat-stable cell wallvaccines. J. Bacteriol. 92:869-879.

205. Ribi, E., C. Larson, W. Wicht, R. List, and G.Goode. 1966. Effective nonliving vaccineagainst experimental tuberculosis in mice. J.Bacteriol. 91:975-983.

206. Roantree, R. J. 1967. Salmonella 0 antigens andvirulence. Annu. Rev. Microbiol. 21:433-466.

207. Robbins, J. B., K. Kenny, and E. Suter. 1965.The isolation and biological properties ofrabbit -yM and -yG anti-Salmonella typhi-murium antibodies. J. Exp. Med. 122:385-402.

208. Robson, H. G., and S. J. Vas. 1972. Resistance ofinbred mice to Salmonella typhimurium. J.Infec. Dis. 126:378-386.

209. Rowley, D. 1956. Rapidly induced changes in thelevel of non-specific immunity in laboratoryanimals. Brit. J. Exp. Pathol. 37:223-234.

210. Rowley, D. 1971. Endotoxins and bacterial viru-lence. J. Infec. Dis. 123:317-327.

211. Rowley, D., I. Auzins, and C. R. Jenkin. 1968.Further studies regarding the question of cel-lular immunity in mouse typhoid. Aust. J.Exp. Biol. Med. Sci. 46:447-463.

212. Rowley, D., and C. R. Jenkin. 1962. Partialpurification of opsonins in pig serum to astrain of Salmonella typhimurium. Immunol-ogy 5:557-565.

213. Rowley, D., K. J. Turner, and C. R. Jenkin.1964. The basis for immunity to mouse ty-phoid. 3. Cell bound antibody. Aust. J. Exp.Biol. Med. Sci. 42:237-248.

214. Ruskin, J., J. McIntosh, and J. S. Remington.1969. Studies on the mechanism of resistanceto phylogenetically diverse organisms. J. Im-munol. 103:252-259.

215. Saito, K., T. Akiyama, M. Nakano, and D.Ushiba. 1960. Interaction between Salmonellaenteritidis and tissue cultured macrophagesderived from immunized animals. Jap. J.Microbiol. 4:395-407.

400 COLLINS

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

VACCINES AND CELL-MEDIATED IMMUNITY

216. Saito, K., and S. Mitsuhashi. 1965. Experimen-tal salmonellosis. VI. In vitro transfer of cellu-lar immunity by mouse mononuclear phago-cytes. J. Bacteriol. 90:629-634.

217.. Saito, K., M. Nakano, T. Akiyama, and D.Ushiba. 1962. Passive transfer of immunity totyphoid by macrophages. J. Bacteriol.84:500-507.

218. Sato, I., T. Tanaka, K. Saito, and S. Mitsuha-shi. 1962. Inhibition of Salmonella enteritidisingested in mononuclear phagocytes from liverand subcutaneous tissues of mice immunizedwith live vaccine. J. Bacteriol. 83:1306-1314.

219. Schlossman, S. F., and H. Levine. 1967. Immu-nochemical studies on delayed and Arthus-type hypersensitivity reactions. 1. The rela-tionship between antigenic determinant sizeand antibody combining site size. J. Immunol.98:211-219.

220. Schwartz, R. S. 1965. Immunosuppressive drugs.Progr. Allergy 9:246-303.

221. Scott, M. T. 1972. Biological effects of theadjuvant Corynebacterium parvum. II. Inhibi-tion of PHA, mixed lymphocyte and GVHreactivity. Cell. Immunol. 5:469-479.

222. Shaffer, J. M., C. J. Kucera, and W. W. Spink.1953. The protection of intracellular Brucellaagainst therapeutic agents and the bacteri-cidal action of serum. J. Exp. Med. 97:77-89.

223. Shaw, C.-M., E. C. Alvord, W. J. Fahlberg, andM. W. Kies. 1964. Substitutes for theMycobacteria in Freund's adjuvants in theproduction of experimental "allergic" enceph-alomyelitis in the guinea pig. J. Immunol.92:28-40.

224. Shepherd, C. C. 1960. The experimental diseasethat follows the injection of human leprosybacilli into footpads of mice. J. Exp. Med.112:445-454.

225. Sher, N. A., S. D. Chaparas, J. Pearson, and M.Chirigos. 1973. Virulence of six strains ofMycobacterium bovis (BCG) in mice. Infect.Immunity 8:736-742.

226. Smith, D. W., A. A. Grover, and E. Wiegeshaus.1968. Non-living immunogenic substances ofMycobacteria. Advan. Tuberc. Res. 16:1-30.

227. Smith, H., and R. B. FitzGeorge. 1964. Theeffect of opsonization with antiserum on thesurvival of Brucella abortus within bovinephagocytes. Brit. J. Exp. Pathol. 45:672-674.

228. Smith, H. W. 1956. The use of live vaccines inexperimental S. gallinarum infection inchickens with observations on their interfer-ence effect. J. Hyg. 54:419-432.

229. Smith, H. W., and S. Halls. 1966. The immunityproduced by a rough Salmonella dublin vari-ant against Salmonella typhimurium and Sal-monella cholerae-suis infection in guinea pigs.J. Hyg. 64:357-359.

230. Smith, R. A., and N. J. Bigley. 1972. Ribonucleicacid-protein fractions of virulent Salmonellatyphimurium as protective immunogens. In-fect. Immunity 6:377-383.

231. Smith, R. A., and N. J. Bigley. 1972. Detection

of delayed hypersensitivity in mice injectedwith ribonucleic acid-protein fractions of Sal-monella typhimurium. Infect. Immunity6:384-389.

232. Smith, S. E., and M. T. Scott. 1972. Biologicaleffects of Corynebacterium parvum. HI. Am-plification of resistance and impairment ofactive immunity to murine tumors. Brit. J.Cancer. 26:361-367.

233. Solomon, J. B. 1968. Immunity to Salmonellagallinarum during ontogeny of the chicken. 1.Onset of resistance to infection; the minor roleof opsonins. Immunology 15:197-206.

234. Spaun, J. 1957. Typhoid vaccine, Vi antigen andVi antibody. Acta Pathol. Microbiol. Scand.123:11-77.

235. Spaun, J. 1964. Studies on the influence of theroute of immunization in the active mouseprotection test with intraperitoneal challengefor potency assay of typhoid vaccines. Bull.World Health Organ. 31:793-798.

236. Spaun, J., and K. Uemura. 1964. Internationalreference preparations of typhoid vaccine.Bull. World Health Organ. 31:761-791.

237. Stuart, C. A. 1956. The unfortunate role ofprecedent in bacteriology. Bacteriol. Rev.20:203-206.

238. Stubbs, M., A. V. Kuhner, E. A. Glass, J. R.David, and M. L. Karnovsky. 1973. Metabolicand functional studies on activated mousemacrophages. J. Exp. Med. 137:537-542.

239. Suter, E. 1956. Interaction between phagocytesand pathogenic microorganisms. Bacteriol.Rev. 20:94-132.

240. Suter, E. 1962. Hyper-reactivity to eridotoxin inmice infected with BCG. Studies on the role ofconcomitant infection. J. Immunol.89:377-381.

241. Takeuchi, A. 1971. Penetration of the intestinalepithelium by various micro-organisms. Curr.Top. Pathol. 54:1-27.

242. Takeya, K., R. Mori, K. Nomato, and N. Na-kayama. 1967. Experimental mycobacterialinfections in neonatally thymectomized mice.Amer. Rev. Resp. Dis. 96:469-477.

243. Tomasi, T. B., and J. Bienenstock. 1968. Secre-tory immunoglobulins. Advan. Immunol.9:1-96.

244. Tripathy, S. P., and G. B. Mackaness. 1969. Theeffect of cytotoxic agents on the primaryimmune response to Listeria monocytogenes.J. Exp. Med. 130:1-16.

245. Turcotte, R. 1969. Yield of non-dialyzable Myco-bacterial constituents during the growth cycle.Can. J. Microbiol. 15:35-41.

246. Uhr, J. W. 1966. Delayed hypersensitivity. Phys-iol. Rev. 46:359-419.

247. Ushiba, D. 1965. Two types of immunity inexperimental typhoid; "cellular immunity"and "humoral immunity". Keio J. Med.14:45-61.

248. Ushiba, D., K. Saito, T. Akiyama, M. Nakano,T. Sugiyama, and S. Shirano. 1959. Studies onexperimental typhoid: bacterial multiplica-

401VOL. 38, 1974

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

BACTERIOL. REV.

tion and host cell response after infection withSalmonella enteritidis in mice immunizedwith live and killed vaccine. Jap. J. Microbiol.3:331-342.

249. Valtonen, V. V., and P. H. Makela. 1971. Theeffect of lipopolysaccharide modifications-antigenic factors, 1, 5, 12 and 27-on thevirulence of Salmonella strains for mice. J.Gen. Microbiol. 69:107-115.

250. Venneman, M. R., and L. J. Berry. 1971. Serum-mediated resistance induced with immuno-genic preparations of Salmonella typhimurium.Infect. Immunity 4:374-380.

251. Venneman, M. R., and L. J. Berry. 1971. Cell-mediated resistance induced with immuno-genic preparations of Salmonella typhimurium.Infect. Immunity 4:381-387.

252. Venneman, M. R., and L. J. Berry. 1971. Experi-mental salmonellosis: differential passivetransfer of immunity with serum and cellsobtained from ribosomal and RNA-immu-nized mice. J. Reticuloendothel. Soc.9:491-502.

253. Venneman, M. R., and M. J. Bigley. 1969.Isolation and partial characterization of animmunogenic moiety obtained from Salmo-nella typhimurium. J. Bacteriol. 100:140-148.

254. Volkman, A., and F. M. Collins. 1968. Recoveryof delayed-type hypersensitivity in mice fol-lowing suppressive doses of x-radiation. J.Immunol. 101:846-859.

255. Volkman, A., and F. M. Collins. 1971. Therestorative effect of peritoneal macrophageson delayed hypersensitivity following ionizingradiation. Cell. Immunol. 2:552-566.

256. Volkman, A., and F. M. Collins. 1973. Polyar-thritis associated with Salmonella infection inrats. Infect. Immunity 8:814-818.

257. Weinstein, M. J., J. A. Waitz, and P. D. Carne.1970. Induction of resistance to bacterial in-fections of mice with poly I:poly C. Nature(London) 226:170.

258. Weissman, J. B., P. A. Rice, D. J. Krogstad, W.B. Baine, and E. J. Gangarosa. 1973. Risk ofsevere intestinal infection to the traveler inMexico. J. Infec. Dis. 128:574-577.

259. White, R. G., P. Jolles, D. Samour, and E.Ledere. 1964. Correlation of adjuvant activityand chemical structure of Wax D fractions ofMycobacteria. Immunology 7:158-171.

260. Wiegeshaus, E. H., and D. W. Smith. 1968.Experimental models for study of immunity totuberculosis. Ann. N.Y. Acad. Sci.154:194-199.

261. Winston, S. H., and L. J. Berry. 1970. Immunityinduced by ribosomal extracts from Staphylo-

coccus aureus. J. Reticuloendothel. Soc.8:66-73.

262. Wong, K. H., and J. C. Feeley. 1972. Isolation ofVi antigen and a simple method for its mea-surement. Appl. Microbiol. 24:628-633.

263. Wong. K. H., J. C. Feeley, and M. Pittman.1972. Effect of a Vi-degrading enzyme onpotency of typhoid vaccines in mice. J. Infec.Dis. 125:360-366.

264. Woolcock, J. B. 1973. Resistance to microbialinfection-vaccines in theory and practice.Aust. Vet. J. 49:307-317.

265. Yamamoto, K., A. L. Anacker, and E. Ribi.1970. Macrophage migration inhibition stud-ies with cells from mice vaccinated with cellwalls of Mycobacterium bovis (BCG): rela-tionship between inhibitory activity of lungcells and resistance to airborne challengewith Mycobacterium tuberculosis H37Rv. In-fect. Immunity 1:595-599.

266. Youdim, S., 0. Stuntman, and R. A. Good. 1973.Thymus dependency of cells involved in trans-fer of delayed hypersensitivity to Listeriamonocytogenes in mice. Cell. Immunol.8:395-402.

267. Youmans, A. S., and G. P. Youmans. 1965.Immunogenic activity of a ribosomal fractionobtained from Mycobacterium tuberculosis. J.Bacteriol. 89:1291-1298.

268. Youmans, A. S., and G. P. Youmans. 1966.Effect of trypsin and ribonuclease on theimmunogenic activity of ribosomes and ribo-nucleic acid isolated from Mycobacteriumtuberculosis. J. Bacteriol. 91:2146-2154.

269. Youmans, A. S., and G. P. Youmans. 1970.Immunogenic mycobacterial ribosomal andribonucleic acid preparations: chemical andphysical characteristics. Infect. Immunity2:659-668.

270. Youmans, G. P., and A. S. Youmans. 1957. Themeasurement of the response of immunizedmice to infection with Mycobacteriumtuberculosis var hominis. J. Immunol.78:318-328.

271. Youmans, G. P., and A. S. Youmans. 1965.Nonspecific factors in resistance of mice toexperimental tuberculosis. J. Bacteriol.90:1675-1681.

272. Youmans, G. P., and A. S. Youmans. 1969.Allergenicity of mycobacterial ribosomal andribonucleic acid preparations in mice andguinea pigs. J. Bacteriol. 97:134-139.

273. Zbar, B., H. J. Rapp, and E. Ribi. 1972. Tumorsuppression by cell walls of Mycobacteriumbovis attached to oil droplets. J. Nat. CancerInst. 48:831-835.

402 COLLINS

on June 14, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from