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MICROBIOLOGICAL REVIEWS, Dec. 1983, p. 510-550 Vol. 47, No. 40146-0749/83/040510-41$02.00/0Copyright © 1983, American Society for Microbiology

New Knowledge on Pathogenesis of Bacterial EntericInfections as Applied to Vaccine Development

MYRON M. LEVINE,* JAMES B. KAPER, ROBERT E. BLACK, AND MARY LOU CLEMENTS

Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201

INTRODUCTION......................................... 511CHOLERA ......................................... 512

Motility and Chemotaxis ......................................... 512Mucosal Adhesion......................................... 512LPS ......................................... 512Flagellar sheath protein ........... .............................. 513Hemagglutinins ......................................... 513Outer membrane proteins ............. ............................ 514

Enterotoxin Production .......... ............................... 514Quality and Duration of Infection-Derived Immunity ............. .................. 514Immune Response in Humans ................. ........................ 515LPS ......................................... 516Flagellar sheath protein ........... .............................. 516Cholera lectin ......................................... 516Other cholera hemagglutinins......................................... 516Outer membrane protein......................................... 516Cholera toxin......................................... 516

Previous Cholera Vaccines ............ ............................. 516Killed Whole-Cell Vaccines......................................... 516

Parenteral whole-cell vaccines ..................... .................... 516Oral whole-cell vaccines......................................... 517

Toxoids......................................... 517Formaldehyde cholera toxoid .................. ....................... 517Glutaraldehyde cholera toxoid.......................................... 517Purified B subunit......................................... 518Procholeragenoid ......................................... 518

Combination Vaccines ......... ................................ 518Attenuated V. cholerae Vaccines.......................................... 519

Naturally occurring strains ......................................... 519Chemically mutagenized attenuated strains ...................................... 519Vibriophage-induced mutants .................. ....................... 521Genetically engineered mutants.......................................... 521

ETEC ......................................... 523Enterotoxins......................................... 523O:H Serotypes and Enterotoxin Phenotypes........................................ 524Colonization Factors......................................... 524Immune Response in Humans ............... .......................... 526Vaccines Against ETEC......................................... 526

Purified CFA fimbriae vaccines.......................................... 526Purified type 1 somatic pili (fimbriae) vaccine.................................... 527Summary of killed antigen vaccine ......................................... 528Live vaccines ......................................... 528

Toxoids.......................................... 529Procholeragenoid .......................................... 529ST toxoids .......................................... 529

EPEC .......................................... 530Vaccines Against EPEC.......................................... 533

SHIGELLA .......................................... 533Smooth LPS 0 Antigen .......................................... 533Epithelial Cell Invasiveness.......................................... 534Shigella Toxin .......................................... 535Shigella Vaccines.......................................... 535

TYPHOID FEVER.......................................... 536Vaccines Against Typhoid Fever........................................... 537

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ENTERIC INFECTIONS AND VACCINE DEVELOPMENT 511

VACCINES AGAINST NONTYPHOIDAL SALMONELLA, CAMPYLOBACTER, ANDYERSINIA ORGANISMS ............ ..................................... 539

LITERATURE CITED........................................................... 539

INTRODUCTIONEnteric bacterial infections, causing diarrhea,

dysentery, and enteric fevers, are importanthealth problems throughout the world (7, 18, 19,77, 146, 148, 200, 246, 265, 345). These bacterialinfections represent a notable burden, in particu-lar, for children living in less-developed regionsof the world (7, 18, 19, 77, 146, 148, 200, 246,265, 345); they also pose a risk for travelers fromindustrialized countries who visit less-developedareas (72, 74, 147, 278, 354). Among the mostimportant enteric bacterial pathogens recog-nized are the following: (i) enterotoxigenic Esch-erichia coli (ETEC) as a cause of diarrhea intravelers and in infants in less-developed coun-tries; (ii) Vibrio cholerae 01, responsible forendemic and epidemic cholera; (iii) Campylo-bacter jejuni, an important cause of diarrhea inyoung children throughout the world, as well asan agent of diarrhea in travelers; (iv) Shigellaspecies as a cause of both watery diarrhea anddysentery (loose stools with blood and mucus),particularly in less-developed areas; (v) Salmo-nella typhi, the etiological agent of typhoid fe-ver, the most common enteric fever (a general-ized infection of the reticuloendothelial systemand intestinal lymphoid tissue accompanied bysustained fever and bacteremia); and (vi) nonty-phoidal Salmonella sp., an important cause ofacute gastroenteritis in individuals in both devel-oping and industrialized countries.For many years attempts have been made to

prepare immunizing agents against some ofthese infections, with variable results. In manyinstances the vaccines prepared represented em-pirical approaches with no recognizable scien-tific basis for following that approach (e.g.,phenolized heat-killed V. cholerae used as aparenteral vaccine).More recently, however, there have occurred

great advances in our knowledge of the patho-genesis of infections due to these bacterialenteropathogens. This new information has beenapplied toward vaccine development, resultingin fresh evaluations of older vaccines as well asinnovative new approaches.

In this review, we attempt to synthesize thenew knowledge of pathogenesis of bacterial en-teric infections and relate this information tovaccine development. We restrict ourselves tohuman infections and to those in which signifi-cant strides have been made.As a general theme in the pathogenesis of

bacterial enteric infections, we can categorizepathogens into five groups on the basis of theirdegree of ultimate invasiveness after ingestion

by a susceptible host. These five groups are asfollows.

(i) Mucosal Adherence and Enterotoxin Pro-duction-The organisms in this group, exempli-fied by V. cholerae and ETEC attach to mucosaof the proximal small intestine as a preliminaryprerequisite to production of enterotoxinswhose effect on the mucosa leads to secretion.These organisms do not destroy the brush bor-der, invade the mucosa, or cause recognizablehistopathological lesions (32, 68, 230, 299, 365).

(ii) Mucosal Adherence and Brush BorderDissolution-Enteropathogenic E. coli (EPEC)of "classical" serotypes (as described in the1940s and 1950s; 78, 87, 2%) tightly adhere tomucosa of both the small and large intestine (78,296, 337, 389). These organisms cause a patho-gnomonic histopathological lesion, evident onelectron microscopy, characterized by dissolu-tion of the brush border of the enterocyte at thesite of attachment of the bacterium (286, 322,337, 389). Further invasion does not occur.

(iii) Mucosal Invasion and Intraepithelial CellProliferation-This pathogenic pattern is char-acterized by Shigella species which invade en-terocytes of the distal small intestine and thecolon, multiply therein, and cause cell dysfunc-tion and cell death (111, 231, 365, 382, 383, 385).Typically, Shigella organisms remain localizedwithin the epithelial cells; whereas some escapeinto the lamina propria, they rarely spread be-yond. Mesenteric adenitis and bacteremia arerare events in shigellosis (15, 231).

(iv) Mucosal Translocation Followed by Bac-terial Proliferation in the Lamina Propria andMesenteric Lymph Nodes-Salmonella species,C. jejuni, and Yersinia enterocolitica appear tofit into this category. Salmonella spp. other thanSalmonella typhi (and S. paratyphi A and B)invade the enterocytes and pass through thecells in pinocytotic vesicles to exit into thelamina propria, where they elicit a chemotacticresponse resulting in an influx of polymorphonu-clear leukocytes (139, 140, 205, 206, 365, 381,382, 384, 385). Salmonella spp., C. jejuni, andYersinia enterocolitica all appear to readilydrain into mesenteric lymph nodes (3, 24, 31,139, 205, 206, 218, 357). Further invasion result-ing in systemic infection and bacteremia canoccur but appears to be uncommon except incompromised hosts (in contrast with Salmonellatyphi and Salmonella paratyphi A and B; seebelow).

(v) Mucosal Translocation Followed by Gen-eralized Infection-Salmonella typhi and Sal-monella paratyphi A and B pass through the

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mucosa like other salmonellae. However, whenthese bacteria reach the lamina propria theyelicit a chemotactic response characterized by aninflux of macrophages which ingest the Salmo-nella spp. (366). These bacteria are drained intothe mesenteric lymph nodes. Some reach thebloodstream by way of lymphatic drainagethrough the thoracic duct; in the course of thisearly bacteremia these Salmonella spp. are re-moved by phagocytic cells of the reticuloendo-thelial system, particularly in the spleen, liver,and bone marrow (79, 126, 366). In the nonim-mune host the Salmonella spp. remain viablewithin these cells, and after an incubation periodof 10 to 14 days, the clinical syndrome known asenteric fever (typhoid or paratyphoid fever) be-gins, accompanied by bacteremia.

CHOLERAEpidemic and pandemic cholera is caused by

V. cholerae 01. Two biotypes exist, classicaland El Tor. Between the early 1960s and 1982the El Tor biotype spread throughout the worldto the virtual exclusion of classical biotype or-ganisms (146, 200). However, in 1982 diarrheadue to the classical biotype reappeared in epi-demic form in Bangladesh (345); it remains to beseen whether classical cholera will again pre-dominate in the Gangetic delta and whether itwill spread elsewhere in Asia or Africa. Withineach biotype two major serotypes exist, Ogawaand Inaba. Humans are the only known naturalhosts of V. cholerae 01 infection, and in naturethey usually ingest these organisms by means ofcontaminated water (21, 186, 252) or food (typi-cally seafood) (13, 22, 73, 267, 277, 344). Theprobable inoculum size in nature is believed tobe 102 to 103 organisms (364). This is corroborat-ed by volunteer studies wherein ingestion of 103V. cholerae El Tor Inaba with 2.0 g of NaHCO3by North American volunteers resulted in milddiarrheal illness in four of six individuals (237).Gastric acid in normochlorhydric persons servesas a very competent nonspecific defense barrier(237, 293). When seven fasting, healthy, youngadult North American volunteers ingested 106 V.cholerae El Tor Inaba in 300 ml of water (with-out NaHC03), neither infection nor illness en-sued (237). However, 9 of 10 volunteers whoingested the same inoculum with 2.0 g ofNaHCO3 and six of six who took the inoculumwith food developed cholera. These observa-tions in volunteers emphasize the importance ofthe gastric acid barrier and also show how V.cholerae can safely transit through it.When V. cholerae 01 organisms successfully

pass through the pylorus in a viable state, theyreach the critical anatomic site of host-bacteri-um interaction, the proximal small intestine.Here a complex series of events transpires in

which the vibrio, using an array of virulenceproperties, attempts to overcome a number ofnonspecific defense mechanisms possessed bythe host. The small intestine of the healthy adultliving in a developed area of the world is usuallyfree of coliforms (67). Two nonspecific defensemechanisms, small intestinal peristalsis and amucus layer coating the small intestine, appar-ently work in unison to accomplish this.

Motility and ChemotaxisV. cholerae 01 organisms must penetrate the

mucus layer and rapidly reach the enterocytes ofthe small intestine to which they must attach. Inthis manner they can overcome the normal de-fenses and colonize the small intestinal mucosa.V. cholerae can elaborate a potent mucinasewhich is believed to assist their entrance into themucus gel covering the mucosa (28, 29, 116, 226,351). Motility provided by the vibrio's unipolarflagellum is an important virulence property (11,12, 152, 154, 352). Freter and co-workers (118,121-123) have shown that motile V. choleraedirect themselves to the mucosal surface inresponse to chemotaxins. Substances that at-tract motile V. cholerae include L-amino acids, anumber of monosaccharides, and pepsin-digest-ed mucosal extracts (118). Many workers havedemonstrated that nonmotile, fully enterotoxi-genic mutants exhibit notably diminished viru-lence (11, 152-154, 352).

In several animal and in vitro models it hasbeen shown that motile V. cholerae rapidly enterthe mucus gel and can be found in intervillousspaces within minutes to a few hours (118, 121,122, 153, 295, 352). In animal models V. chol-erae appear in greatest numbers in intervillousspaces and in the crypts.

Mucosal AdhesionThe precise mechanism by which V. cholerae

adheres to the enterocytes of the crypts and villiand the specific antigens responsible are notentirely clear; this is currently the subject ofintense research since it has great implicationsfor vaccine development. Among the V. chol-erae surface antigens that have been implicatedas being involved in adhesion to the mucosa ofthe proximal small intestine are lipopolysaccha-ride (LPS) (39, 178), flagellar sheath protein andother flagellar antigens (11, 12, 80, 184), "chol-era lectin" (102), various hemagglutinins (17, 36,89, 102, 165, 179, 192, 193, 199), and the majorouter membrane protein (196, 198, 199, 203).LPS. The LPS of Inaba and Ogawa serotypes

have been chemically defined with some preci-sion (171, 197, 204, 327). V. cholerae LPS re-sembles that of other gram-negative organismsin having a lipid A moiety (responsible for mostof the endotoxic biological activity) and an 0-antigenic polysaccharide. In most other gram-

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negative LPS these components are linked bymeans of 2-keto-3-deoxyoctonate (KDO) acid-labile core sugar. Ogawa and Inaba LPS lack 2-keto-3-deoxyoctonate; rather, they possess theacid-labile sugar fructose, which may serve thesame linkage function as 2-keto-3-deoxyocton-ate.Two pieces of evidence provide the basis for

the contention that LPS is involved in adhesionof V. cholerae 01 to intestinal mucosa. (i) In arabbit loop model, purified Inaba LPS signifi-cantly inhibited attachment of V. cholerae Inabato rabbit mucosa, whereas E. coli LPS had noeffect (39). (ii) In several animal and in vitromodels, antibody against Ogawa or Inaba LPSprevented adhesion of V. cholerae to intestinalmucosa (39, 120).

Flagellar sheath protein. Much evidence alsosupports the hypothesis that a component of theV. cholerae 01 flagellum is involved in theprocess of adhesion to mucosa (11, 12, 80, 184).As mentioned previously, nonmotile mutantsare less pathogenic. Furthermore, loss of motil-ity correlated with loss of adhesive factors (ad-hesins) from the surface of the vibrio. Nonmotilemutants fail to adhere to intestinal brush bor-ders, to hemagglutinate erythrocytes, or to asso-ciate with mucosa of intestinal slices, in contrastwith the motile parent vibrios from which theywere derived and in contrast with motile rever-tant organisms (120, 121).

Several investigators have described a flagel-lar sheath protein (108, 184). Antibody directedagainst this protein appears protective againstcholera in one animal model, and it has beensuggested that this protein serves as an adhesin(80). Hranitzky et al. (184) have begun purifica-tion and characterization of this protein.

Hemagglutinins. In 1961, Bales and Lankford(Bacteriol. Proc., p. 118, 1961) noted that V.cholerae attach to erythrocytes and hypothe-sized that the erythrocyte-vibrio interaction mayresemble that which occurs between V. choleraeand intestinal mucosa. Finkelstein and Mukerjee(102) observed that El Tor vibrios (but notclassical vibrios) agglutinated chicken erythro-cytes after cultivation on solid media. Since 1976there have been numerous reports of investiga-tions of the hemagglutinating properties of bothEl Tor and classical vibrios (17, 36, 89, 102, 165,179, 192, 193, 199). These investigations havereceived increased attention in recent years be-cause analogous studies with ETEC have shownthat hemagglutination reactions can serve asscreening tests to establish the presence of adhe-sions that attach to receptors on small intestinalmucosa.Hanne and Finkelstein (165) have recently

described the hemagglutinating patterns exhibit-ed by classical and El Tor biotypes of V. chol-

erae. These workers describe a total of fourapparently distinct hemagglutinins of which twoare exclusive to El Tor strains, whereas two, theL-fucose-sensitive hemagglutinin and the solublehemagglutinin (cholera lectin), are found in bothEl Tor and classical vibrios. According to Hanneand Finkelstein (165), depending on growth con-ditions, all four of the hemagglutinins can befound in El Tor strains, whereas two have beenidentified in classical strains.

El Tor vibrios elaborate a cell-associated hem-agglutinin that reacts with chicken (and human)erythrocytes. The reaction is inhibited by D-mannose and D-fructose. This is the characteris-tic El Tor hemagglutinin utilized in biotyping ofV. cholerae.

Classical vibrios elaborate a distinct cell-asso-ciated hemagglutinin that is inhibited by L-fu-cose. This hemagglutinin, which is preferentiallyexpressed in the logarithmic phase of cultures, isbest detected with human erythrocytes. Hanneand Finkelstein (165) found that log-phase cul-tures of El Tor strains also express an L-fucose-sensitive hemagglutinin which is ordinarilymasked by the distinctive El Tor hemagglutinin;they discovered this L-fucose hemagglutinin inEl Tor by using mutants of El Tor strains thatlacked the cell-associated mannose-sensitivehemagglutinin of chicken erythrocytes.Hanne and Finkelstein (165) described a third

cell-associated hemagglutinin which appeared instationary-phase cultures of El Tor vibrios. Thishemagglutinin was not inhibited by L-fucose, D-mannose, or any other sugars tested.The fourth hemagglutinin described by Hanne

and Finkelstein (165) is a soluble hemagglutinindetected in late-log-phase cultures of all strainstested. This soluble hemagglutinin was not inhi-bitable by any sugars tested but required Ca2+for maximal activity. Finkelstein and co-work-ers (101, 104) had previously referred to thesoluble hemagglutinin as cholera lectin. Choleralectin has been purified to apparent homogeneityby Finkelstein and Hanne (104). The purifiedprotein exhibits many biological activities be-sides hemagglutination (102). In particular, chol-era lectin manifests protease activity; it hydro-lyzes fibronectin and mucin, cleaves lactoferrin,and nicks the A subunit of heat-labile enterotox-in (LT) of E. coli (102). Purified cholera lectininhibits attachment of V. cholerae (of eitherbiotype and serotype) to small intestinal mucosaof infant rabbits. Finkelstein et al. (102) reportthat cholera lectin, which exhibits so manydiverse biological activities, is apparently identi-cal to the V. cholerae mucinase discovered byBurnet in 1947 (28-30). These workers believethat cholera lectin plays a critical role in thepathogenesis of cholera.Holmgren et al. (179) have also investigated

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the hemagglutinins of V. cholerae but have paidcloser attention to the cell-associated antigens.By manipulating culture and handling proce-dures, these workers were able to maximizeproduction of cell-associated hemagglutininwhile suppressing production of the solublehemagglutinin. They corroborated that El Torstrains manifest mannose-sensitive hemaggluti-nation of chicken erythrocytes, whereas classi-cal strains agglutinate human erythrocytes in areaction that is inhibited by L-fucose. Holmgrenet al. (179) found that milk from Swedish womencontains materials that inhibit the hemagglutina-tion of V. cholerae; they include high-molecular-weight glycoproteins that interfere with El Torstrains and glycoprotein and oligosaccharidesthat inhibit classical strains. Holmgren et al.(179) believe that these glycoproteins found inhuman milk resemble (or are structurally identi-cal to) the intestinal cell receptors for V. chol-erae adhesins.Outer membrane proteins. Increasingly, with

certain other gram-negative bacterial pathogens,outer membrane proteins have become recog-nized as playing a role in pathogenesis and asantigens against which the host immune re-sponse is directed (26, 50, 96, 158, 166, 259, 377,378). Several lines of evidence suggested thatouter membrane proteins may play a role in thepathogenesis of cholera, including the following.(i) Outer membrane proteins appear to be re-sponsible for the high surface hydrophobicity ofV. cholerae (89, 199). Hydrophobic bondingmay be one mechanism by which vibrios adhereto target cells. (ii) The major outer membraneprotein of V. cholerae is apparently identical,irrespective of biotype and serotype (196, 198,203). This would explain the observationswherein infection with one serotype conferssolid immunity against later challenge with theheterologous serotype (229, 235, 237, 251). Ofcourse, another explanation can be based on theshared group 01-specific "A" antigen of LPS.

Enterotoxin ProductionAs V. cholerae organisms successfully asso-

ciate with the mucosa of the proximal smallintestine, they elaborate cholera enterotoxin(also called choleragen or cholera toxin). This isone of the best-studied proteins with respect tostructure, function, and biological activity andreaders are referred to several reviews (97, 100,141, 176, 390). Briefly, cholera toxin (molecularweight, 84,000) is composed of five B subunits(molecular weight, 11,000 each) and one A sub-unit which is further divided into A1 (molecularweight, 24,000) and A2 (molecular weight, 5,000)subunits. The B subunits bind the toxin toreceptors (GM, ganglioside) present on mucosalenterocytes. The A2 subunit is believed to serve

in holding the Al and B subunits together inproper tertiary configuration. As the B subunitsbind to GM, receptors, a conformational changeis believed to occur by which the Al subunitgains entry into the enterocyte. Al is the enzy-matically active ADP-ribosylating subunit whichirreversibly activates the adenylate cyclase sys-tem of the enterocyte, resulting in an intracellu-lar accumulation of cyclic AMP. Elevated levelsof cyclic AMP modify the nature of the cellmembrane, resulting in outright secretion bycrypt cells and cells of the sides of villi anddecreased absorption by villus tip cells. Thesecreted fluid is low in protein and rich inelectrolytes, including Na+, K+, Cl-, andHCO3 . At peak purge, the rice water stools ofadult cholera patients contain ca. 135 meq ofNa+ per liter, whereas stools of young childrenwith cholera have approximately 100 meq ofNa+ per liter. If copious stool losses continuewithout proper therapy, severe dehydration andacidosis followed by renal shut down, shock,and death can ensue. Since stool losses in adultscan increase to a rate of 1.0 to 1.25 liters/hshortly after onset, this chain of events canprogress rapidly.

Recently it was shown that the oral adminis-tration of purified cholera toxin to adult volun-teers resulted in a clinical syndrome virtuallyidentical to cholera (M. M. Levine et al., unpub-lished data). Six to 14 h after ingesting a mere 5.0jig of purified cholera toxin (with NaHCO3 toneutralize gastric acid), four of five individualsdeveloped diarrhea (Table 1). Four volunteerspassed more than 1.0 liter of diarrheal stool,whereas one individual purged more than 6liters. Of six volunteers who ingested 0.5 [Lg oftoxin, none developed clinical reactions, where-as two volunteers who ingested 25 pLg eachpurged approximately 20 liters of rice waterstool. Thus, the dose response of humans tocholera toxin exhibits a notably steep slope overa narrow range of toxin doses. Based on theseobservations, one can regard V. cholerae as anelaborate delivery system for cholera toxin.

Quality and Duration of Infection-DerivedImmunity

In endemic areas such as Bangladesh, theincidence of cholera is highest in children 2 to 4years of age and incidence rates diminish there-after with increasing age (144, 275, 290, 291). Atthe same time, the prevalence of vibriocidalantibody in serum increases with age (290, 291).These epidemiological observations point to thedevelopment of acquired immunity from clinicaland subclinical infection.The quality and duration of infection-derived

immunity have been convincingly demonstratedin volunteer studies. Cash et al. (34) showed that

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TABLE 1. Response of healthy adult volunteers after ingestion of various doses of cholera toxin"

Total diar- No. of DurationVolunteer Cholera toxin Incuba rheal stool diarrheal of diar-dose(p.g) tion (h) ~Vol (Ml) stools rhea (h)

6004-1 0.56004-3 0.56004-7 0.5

6006-2 2.56006-3 2.56006-4 2.56006-5 2.56006-6 2.5

6008-1 5.0 13.5 1,695 5 31.56008-2 5.0 9.5 1,281 5 36.06008-3 5.06008-5 5.0 6 6,023 33 89.06008-6 5.0 5.5 1,020 6 41.5

6004-5 25 5 21,649 50 946004-9 25 7 22,074 47 91.5a To diminish gastric acidity and protect toxin from digestion during its transit through the stomach, volunteers

received cimetidine (300 mg) 3 h before and 2.0 g of NaHCO3 concomitant with ingestion of cholera toxin.

an initial clinical cholera infection due to classi-cal Inaba vibrios confers solid immunity to re-challenge with the homologous organism. Le-vine et al. (229, 251) subsequently showed thatan initial clinical infection due to classical vib-rios of either serotype led to complete protoctionupon subsequent challenge with classical vibriosof the heterologous as well as the homologousserotypes (Table 2). Furthermore, not only wererechallenged veteran volunteers clinically pro-

tected, but also classical vibrios were neverrecovered by direct culture of stools (Table 2).These workers also showed that the solid immu-nity lasted at least 3 years, the longest intervaltested (235).Most recently, Levine et al. have examined

the immunity conferred by clinical infection withEl Tor vibrios (Table 2) (237). An initial clinicalinfection due to V. cholerae El Tor was found toprovide significant protection against rechal-lenge with El Tor vibrios of either the homolo-

gous or the heterologous serotype. In total, the'vaccine efficacy" of El Tor vibrios was 90%(versus 100% for classical vibrios). In contrast tothe experience with classical vibrios in volun-teers, individuals rechallenged with El Tor vib-fios often had positive direct coprocultures. It isnot clear whether this is due to an enhancedcapacity of El Tor vibrios to survive in nondiar-rheal stool or whether El Tor vibrios stimulateless potent intestinal immunity than classicalvibrios. Serotype-homologous, biotype-heter-ologous rechallenge studies in volunteers shouldgive an answer to this question.

Immune Response in HumansMultiple antigens elaborated by V. cholerae

were discussed above as possibly playing acritical role in pathogenesis. Presumably, then,host-immune responses directed against theseantigens serve as mediators of the potent immu-nity observed in volunteer studies. Information

TABLE 2. Protective efficacy conferred by prior infection with pathogenic V. cholerae of classical or El Torbiotype or attenuated V. cholerae El Tor

Attack rate for diarrhea' Isolation of V.c/holerae from

Immunizing V. cholerae strains Controls Vaccin- Vaccine ef- vaccinees (di-(%) eesb (%) ficacy (%) rect coprocul-

tures)

Pathogenic classical vibrios 24/27 (89) 0/16 100 0/16Pathogenic El Tor vibrios 32/37 (86) 2/22 (9) 90 8/22Texas Star attenuated El Tor 18/25 (72) 7/25 (28) 61 17/25

a Challenge with 106 pathogenic V. cholerae.b Includes both serotype-homologous and heterologous challenges.

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on immune response is crucial in vaccine devel-opment to help determine what antigens areparticularly desirable for inclusion in immuniz-ing agents. Therefore, it is helpful to reviewwhat information is available to document theoccurrence of immune responses to the variousantigens discussed above.LPS. After cholera infection, serum and intes-

tinal or milk secretory immunoglobulin A (SIgA)antibody responses can be shown by enzyme-linked immunosorbent assay (ELISA) againstpurified LPS antigen (262, 263; M. M. Levineand C. R. Young, unpublished data). Serumvibriocidal antibody (which is assayed againstlive V. cholerae in the presence of complement)is directed mainly (but not entirely) against LPSantigen. Serum vibriocidal antibody levels rosesignificantly in more than 90% of North Ameri-can volunteers who participated in cholera chal-lenges (45).

Flagellar sheath protein. Heretofore, therehave been no reports of the serum or localintestinal antibody response to flagellar sheathprotein.

Cholera lectin. There have been no publishedreports yet of the immune response of humans tocholera lectin, the soluble hemagglutinin of V.cholerae, after cholera infection.Other cholera hemagglutinins. Studies are just

now beginning in some laboratories to look atthe immune response in humans to the cell-associated hemagglutinins of V. cholerae afterinfection. No reports have yet been published.Outer membrane protein. Sears et al. (S. D.

Sears, M. M. Levine, C. R. Young, and K.Richardson, Abstr. Annu. Meet. Am. Soc. Mi-crobiol. 1983, E90, p. 91) have characterized thefrequency, magnitude, and kinetics of the serumantibody response of humans to the major outermembrane protein of V. cholerae after experi-mental cholera infection in volunteers. Approxi-mately 50% of individuals with induced cholera,irrespective of biotype or serotype of the chal-lenge organism, manifested an immune re-sponse, mainly in the IgG class of antibody.With an ELISA, these workers found that themagnitude of responses was very similar wheth-er the outer membrane protein antigen wasderived from the homologous strain or fromstrains of differing serotype and biotype. Inpreliminary studies, these workers showed thatsome volunteers manifest a brisk intestinal SIgAresponse to the outer membrane protein. Thestudies of Sears et al. demonstrate that the majorouter membrane proteins of V. cholerae interactwith the host, resulting in a measurable immuneresponse in many individuals in the course ofinfection.

Cholera toxin. Cholera toxin is a potent anti-gen, the resultant immune response being direct-

ed almost entirely against the B subunit (176,315, 370). Virtually all neutralizing activity ofcholera antitoxin can be absorbed with a purifiedB subunit.More than 90% of North American volunteers

who ingested V. cholerae while serving as con-trols in challenge studies to assess vaccine effi-cacy developed rises in serum antitoxin which ismeasurable in either toxin-neutralizing assays orby IgG-ELISA (256, 336, 398). Serum-neutraliz-ing antibody resides in the IgG class and anti-body measured by IgG-ELISA correlates close-ly with neutralization assays. Serum IgGantitoxin is extremely long-lived, elevated levelsbeing demonstrable in most North Americans atleast 2 years after experimental cholera infection(256).Approximately 60% of volunteers who partici-

pated in experimental cholera studies manifestrises in SIgA antitoxin in intestinal fluid mea-sured before and 9 days after challenge (235;Levine and Young, unpublished data). The localantitoxin response tends to be relatively short-lived, with antibody levels usually dropping by 1month postchallenge.

Previous Cholera VaccinesVirtually within 1 year of discovery by Koch

in 1883 of the bacterium that causes cholera(217), attempts began to prepare vaccines toprevent the disease. That search has continueduntil today, with a series of immunizing agentshaving been produced along the way, each suf-fering one or another significant drawback.The cholera vaccines developed between 1883

and 1982 can be broadly divided into two catego-ries, those that aim to stimulate antitoxic immu-nity and those intending to induce antibacterialimmunity. Experiments with animal models canbe cited to support a protective role for eitherantitoxic or antibacterial immunity (80, 124, 178,227, 314, 317, 319, 320, 331, 373) and to suggestthat with both types of antibodies working inunison the result is a synergistic effect (178, 314,331, 373). However, cholera is an infectionrestricted to the human host, and the relevanceof the various animal models with respect tohuman infection is not entirely clear. Thus wewill restrict our discussion to those immunizingagents that have been, are presently being, orwill shortly be tested in humans. Since V. chol-erae is a noninvasive enteropathogen of thesmall bowel that does not penetrate the mucosa,one may surmise in this discussion the impor-tance of local immunity at the mucosal surface,particularly mediated by SIgA.

Killed Whole-Cell VaccinesParenteral whole-cell vaccines. Since the end

of the 19th century, killed whole V. cholerae

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organisms have been used as parenteral vac-cines; these vaccines are still commerciallyavailable. Experience with the parenteral whole-cell vaccines has been reviewed recently by Joo(194) and by Feeley and Gangarosa (90). Suchvaccines stimulate high titers of serum vibrioci-dal antibodies. They also stimulate increases inintestinal SIgA antibody to V. cholerae somaticO antigen when given to recipients who arealready immunologically primed from prior anti-genic contact, but not in unprimed persons livingin a nonendemic area (Sweden) (372, 374). Infield trials, parenteral killed whole-cell vaccineshave been shown to confer significant protectionagainst the homologous V. cholerae serotype,usually for a period of less than 1 year (90, 194,290, 316). They have also protected volunteersagainst experimental infection (34). There issome evidence to suggest that parenteral whole-cell Inaba vaccine provides good short-termprotection against Ogawa as well as againstInaba cholera (290), whereas Ogawa vaccine iseffective only against Ogawa (316).By use of adjuvants, it has been possible to

maintain a vaccine efficacy of approximately70% for up to 12 to 18 months with parenteralvaccine (90, 194, 316).

Oral whole-cell vaccines. Killed vibrios havealso been used as oral vaccines. Freter andGangarosa (117, 118) and Ganguly et al. (127)have shown that killed whole vibrios adminis-tered orally stimulate the appearance of localintestinal antivibrio antibody.Cash et al. (34) fed commercial whole-cell

vaccine to volunteers who ingested 1.6 x 1010classical Inaba vibrios daily for 10 days; somevaccinees received booster doses 1 month afterprimary immunization which consisted of 1.6 x1010 vibrios daily for 5 days. When challengedwith 106 pathogenic classical Inaba 569B vibrios,only 5 of 16 vaccinees (31%) developed diarrheaversus 47 of 58 controls (81%), giving a vaccineefficacy of 62%. In the same volunteer model,two doses of parenteral Inaba whole-cell vaccineprovided a vaccine efficacy of 81% against ex-perimental challenge with classical Inaba 569B(34). These data show that killed V. choleraeadministered parenterally or orally to humansprovide a demonstrable degree of protectionagainst challenge with pathogenic V. cholerae.

ToxoidsImmunizing agents intended to prevent chol-

era by means of stimulating antitoxic immunityinclude (i) formaldehyde-treated cholera toxin(i.e., formaldehyde cholera toxoid), (ii) glutar-aldehyde-treated cholera toxin (i.e., glutaralde-hyde cholera toxoid), (iii) purified B subunit,and (iv) procholeragenoid (with or without form-aldehyde treatment).

Formaldehyde cholera toxoid. It was foundthat treatment of purified cholera toxin in vitrowith formaldehyde eradicated its toxicity, re-sulting in a toxoid that exhibited little toxicbiological activity but that stimulated antitoxicantibodies after parenteral immunization of ani-mals (300). Unfortunately, when the first toxoidof this type was administered to monkeys andhumans as a parenteral vaccine, the toxoid re-verted to partial toxicity, causing unacceptablelocal adverse reactions at the site of inoculation(300). Consequently, further studies with thisparticular toxoid were abandoned. Investigatorsin Bangladesh administered an aluminum-adju-vanted formalinized cholera toxoid parenterallyto Bangladeshi volunteers, including lactatingmothers (276). This formalinized toxoid, whichwas prepared by Wellcome Research Labora-tories, United Kingdom, stimulated increases inserum IgG and breast milk SIgA antitoxin with-out eliciting notable local reactions. No fieldtrials with this vaccine were undertaken.Ohtomo (304) used Formalin in the presence

of glycine to prepare a cholera toxoid (lot 11)that was immunogenic in humans when givenparenterally and did not cause local reactions atthe site of inoculation. A large-scale field trialwith this parenteral toxoid vaccine was carriedout in the Philippines but the vaccine showed noevidence of efficacy (298).

Glutaraldehyde cholera toxoid. Rappaport andco-workers (324) described methods for large-scale preparation of a glutaraldehyde choleratoxoid that was essentially free of contaminatingsomatic antigen. It was hoped that this antigencould be used to assess in a "pure' manner theprotective role of antitoxic immunity alone. Alarge-scale field trial of this toxoid given as aparenteral vaccine was carried out in Bangla-desh in 1974 (53). The toxoid stimulated hightiters of circulating antitoxin in Bangladeshi re-cipients. Two waves of cholera, El Tor Inabafollowed by El Tor Ogawa, struck the field area,allowing a fair evaluation of vaccine efficacy. Aprotective effect could be demonstrated in onlyone age group and was restricted to the period ofthe Inaba epidemic (53). Thus, glutaraldehydecholera toxoid given alone as a parenteral vac-cine provided little protection and was notablyinferior to similar field trials in the same popula-tion with parenteral killed whole-cell vaccines.

Levine et al. (251) investigated glutaraldehydecholera toxoid as an oral vaccine on the assump-tion that toxoid given by this route might bemore efficient by stimulating intestinal antitoxin.Two groups of volunteers were immunized withthree 2.0-mg or three 8.0-mg doses of toxoidgiven directly into the small intestinal lumen (viaintestinal tube) at monthly intervals. The vaccin-ees and unimmunized controls then participated

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in experimental cholera challenge studies. Inneither challenge study was the attack rate orseverity of diarrhea significantly diminished inthe vaccinees in comparison with controls (251).In part the lack of efficacy of oral glutaraldehydecholera toxoid may be due to the fact that thecapacity of B subunits to bind to GM, ganglio-side is greatly diminished as a consequence oftoxoiding with glutaraldehyde.

Purified B subunit. Purified B subunit hasbeen given as an oral or intramuscular vaccine tolarge numbers of persons living in endemic (Ban-gladesh) and nonendemic (Sweden) areas; nountoward reactions have been encountered(375). It has been shown that purified B subunitgiven either orally or parenterally to Banglade-shis stimulates the appearance of SIgA antitoxinin intestinal fluid (375). It is believed that theseBangladeshi recipients of B-subunit vaccine areimmunologically primed to recognize this anti-gen due to prior natural exposure consequent totheir living in a cholera-endemic area. Immuno-logical priming explains the appearance of intes-tinal SIgA antitoxin after parenteral immuniza-tion. The duration of intestinal SIgA antitoxin,however, was much greater after oral than afterparenteral immunization. Oral immunization isclearly the more attractive route because ofrelative simplicity of application and because ofincreased longevity of the intestinal antitoxinresponse. In studies reported so far, Svenner-holm et al. (375) have given one or two 0.5-mgoral doses of purified B subunit 25 days apart.The major advantages of using B-subunit oral

vaccine to stimulate antitoxic immunity includeits complete safety (there is no potential forreversion to toxin as exists with toxoids) andretention of its capacity to adhere to toxinreceptors on enterocytes. Animal studies sug-gest that it is less potent than native holotoxin instimulating antitoxin (317). The relevance of thisobservation with respect to humans is notknown.

It is likely that purified B subunit would beused in conjunction with oral killed vibrios as acombination oral vaccine intended to stimulateboth antibacterial and antitoxic antibodies.

Procholeragenoid. Procholeragenoid is thehigh-molecular-weight (ca. 106) toxoid that re-sults when cholera enterotoxin is heated at 65°Cfor at least 5 min, as described by Finkelstein etal. (103). Procholeragenoid is immunogenicwhile retaining less than 5% of the biologicaltoxic activity of the parent toxin (103). German-ier et al. (135) prepared procholeragenoid byheating cholera toxin at 65°C for 25 min. Theresultant procholeragenoid preparation exhibit-ed less than 1% of the biologically active toxicactivity of the parent toxin. This minimal residu-al toxic activity could be completely abolished

by treating procholeragenoid with formalde-hyde. The resultant formaldehyde-treated pro-choleragenoid was shown to be at least as potentas the parent toxin in stimulating serum antitox-in after immunization of rabbits (135). Swissvolunteers developed brisk serum antitoxin re-sponses after parenteral immunization with 10-,30-, or 100-[Lg doses of formaldehyde-treatedprocholeragenoid (136). No notable adverse re-actions were encountered.As an oral antigen it has been found that

procholeragenoid is more immunogenic whengiven in the form without formaldehyde treat-ment. Working with dogs, Pierce et al. (318)found untreated procholeragenoid to be welltolerated as an oral vaccine; oral doses up to 500,ug (with NaHCO3) did not cause diarrhea. Five500-jLg doses spaced over 42 days stimulatedsignificant protection in dogs against oral chal-lenge with pathogenic V. cholerae.Doses of 50 and 200 jg with NaHCO3 have

been given to groups of six and four adultvolunteers, respectively, without eliciting ad-verse reactions (Levine et al., unpublisheddata). It is likely that procholeragenoid wouldeventually be used in conjuncton with killedvibrios or other relevant antigens capable ofstimulating antibacterial immunity to enhancethe antitoxic immunity induced by procholera-genoid.

Combination Vaccines

The major attraction of a nonliving oral chol-era vaccine is its safety. A review of availableinformation leads one to conclude that an oralvaccine consisting of a combination of antigens,intending to stimulate both antibacterial andantitoxic immunity, would be most likely tosucceed. This conclusion stems from the follow-ing observations.

(i) Toxoid vaccines that stimulate purely anti-toxic immunity have not been shown to beefficacious in protecting humans against cholera(53, 251, 298), although they may protect animalmodels (124, 178, 314, 317-320).

(ii) Oral or parenteral killed whole-cell vac-cines that stimulate no antitoxic immunity pro-vide significant protection against cholera inhumans, albeit for a short period of time (34, 90,194).

(iii) Combinations of antigens (such as crudecholera toxin or toxin plus LPS) that stimulateboth antitoxic and antibacterial immunity givesynergistic protection over what each antigenalone provides (178, 314, 331, 373).Three studies so far have been carried out in

humans with combination oral vaccines. In thefirst, nine volunteers who ingested glutaralde-hyde cholera toxoid (2 mg weekly for 4 weeks)

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plus killed El Tor Inaba vibrios (10't) vibriostwice weekly for 4 weeks) were challenged after1 month with 106 pathogenic El Tor Inaba vib-rios, along with six unimmunized controls (Ta-ble 3). Diarrhea occurred in only two of ninevaccinees versus four of six controls (vaccineefficacy, 67%), and illness was clearly attenuat-ed in the 2 ill vaccinees. More pertinent, per-haps, is the observation that V. cholerae couldbe directly cultured from stools of only two ofnine vaccinees versus six of six controls; thisdemonstrates that immunological mechanismsimpeded the proliferation of vibrios.More recently, three oral doses of B-subunit/

killed whole-cell vaccine (provided by J. Holm-gren, Gothenburg, Sweden) were given to adultvolunteers who participated in a vaccine efficacychallenge study (R. E. Black et al., unpublisheddata). The combination vaccine was given ondays 0, 14, and 28. Each of the three doses ofvaccine contained 5.0 mg of purified B subunitand 2 x 1011 killed V. cholerae (5 x 1010classical Inaba, 5 x 1010 classical Ogawa, and1011 El Tor Inaba). A group of 11 volunteersimmunized with this combination vaccine werechallenged 1 month after their last dose with 106pathogenic V. cholerae El Tor Inaba, along with7 control volunteers. Diarrhea occurred in 7 of 7controls but in only 4 of 11 vaccinees (P = 0.01).The illness in the four vaccinees was significant-ly milder as quantified by total diarrheal stoolvolume and total number of loose stools.

In a third study a group of 15 volunteersreceived three oral doses (days 0, 14, and 28) ofa procholeragenoid/killed whole-cell vaccinecombination (prepared by R. Germanier, SwissSerum and Vaccine Institute, Berne). Thewhole-cell component contained both Ogawaand Inaba serotypes of both biotypes, 5 x 10'0organisms each. The bacteria were heat treatedin the presence of Formalin. The first two dosesof procholeragenoid contained 50 p.g, whereasthe third dose had 200 [Lg. These 15 immunizedvolunteers and 6 controls participated in a chal-lenge study to assess vaccine efficacy. Attackrates were not significantly different between thevaccinees (11 of 15) and controls (6 of 6), and bythis parameter vaccine efficacy was only 27%.However, this particular challenge turned out tobe rather "hot." By other parameters, such astotal diarrheal stool volume (1.6 versus 9.4 li-ters; P < 0.05) and total number of loose stools(6.5 versus 22.0; P < 0.05), it was apparent thatdespite the high attack rate illness was signifi-cantly attenuated in vaccinees versus controls.

Thus, results of studies with oral toxoid/killedwhole-cell vaccine combinations demonstratecomplete safety and a clear-cut, measurabledegree of efficacy. The protective vaccine effica-cy, however, is only moderate, 27 to 67%, and

multiple doses are required to induce the protec-tion.

Attenuated V. cholerae VaccinesThe observation that both classical and El Tor

clinical cholera infections in North Americanvolunteers stimulate a high degree of protectiveimmunity for at least 3 years (34, 229, 235, 237,251) intensified research to develop new choleravaccines. Based on these observations in volun-teers, perhaps the most promising approachtoward immunological control of cholera may beby means of attenuated non-enterotoxigenic V.choler-ae strains used as oral vaccines.

Naturally occurring strains. NontoxinogenicV. cholerae 01 strains isolated from environ-mental sources in India and Brazil have beenevaluated in volunteers as potential vaccine can-didates with disappointing results (35, 236).They either failed to colonize the intestines ofhumans or did so minimally; vibriocidal anti-body responses were meager; and they failed toprovide protection in experimental challengestudies (35, 236).

Chemically mutagenized attenuated strains.Finkelstein et al. (106, 174) mutagenized classi-cal Inaba 569B with nitrosoguanidine, leading toisolation of a hypotoxinogenic mutant. Thisstrain, M13, was fed to volunteers. Diarrhea didnot occur but the strain colonized poorly. Chal-lenge studies demonstrated that some protectiveefficacy was conferred by immunization withmultiple doses (396).Honda and Finkelstein (180) mutagenized El

Tor Ogawa 3083 with nitrosoguanidine andpainstakingly analyzed thousands of colonies todetect one that continued to produce the immu-nogenic B subunit while failing to produce de-tectable A subunit or holotoxin. One isolate,Texas Star-SR, fulfilled these criteria. TexasStar-SR produces normal or increased amountsof B subunit but is negative in assays for holo-toxin activity or A subunit activity (180).Texas Star-SR has been extensively evaluated

in volunteers (237, 239, 240). Groups of 5 to 14volunteers ingested 105 to 5 x 1010 Texas Star-SR organisms in a single dose; 8 other volun-teers received two 109 organism doses 1 weekapart and 18 more ingested two 2 x 1010 orga-nism doses 1 week apart. Some degree of diar-rhea was seen in 16 of the 68 vaccinees (24%). Inonly one individual did the total stool volumeexceed 1.0 liter (1,464 ml). Typically, the vac-cine-induced diarrhea consisted of two or threesmall loose stools totalling less than 400 ml involume. Vaccine organisms were recoveredfrom coprocultures of approximately one-half ofthe vaccine recipients. Where jejunal fluid wascultured (recipients of doses of 108 or morevaccine organisms), cultures were positive in 35

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of 46 vaccinees (76%). Hundreds of Texas Starclones recovered from coprocultures and jejunalfluid cultures were examined for cholera holo-toxin by the sensitive Y-1 adrenal cell assay;

none were positive.Significant rises in serum antitoxin were de-

tected in only 29% of the vaccinees; however,93% manifested significant rises in serum vibrio-cidal antibody and the titers approached thoseencountered after infection with pathogenic V.cholerae. In experimental challenge studies involunteers, one or two doses of Texas Star-SRwere found to confer significant protectionagainst challenge with both El Tor Ogawa and ElTor Inaba vibrios (Tables 2 and 4).

Although the clinical, immunological, andbacteriological studies with Texas Star haveprovided invaluable data to support the conceptof using attenuated strains to mimic infection-derived immunity to cholera, the Texas Starstrain itself suffers from certain drawbacks, asfollows. (i) The method of attenuation, mutagen-esis with nitrosoguanidine, induces multiple mu-

tations, not all of which are necessarily recog-nized. (ii) The precise genetic lesion presumedto be responsible for the attenuation of TexasStar is not known. Therefore, until this is clari-fied there always remains the theoretical possi-bility of reversion to virulence.

Vibriophage-induced mutants. One method toovercome the possibility of reversion to toxige-nicity was reported by Mekelanos et al. (270),who used mutagenic bacteriophages to deleteDNA sequences encoding cholera toxin. Dele-tion of such sizable amounts of DNA eliminatesthe possibility of reversion and yields stable,nontoxinogenic strains of V. cholerae. So far, nomutants derived by this method have been eval-uated in volunteers, nor have reports of efficacystudies in animal models been published.

Genetically engineered mutants. Because ofthe promising clinical results with Texas Star,Kaper et al. (201; J. B. Kaper, M. M. Baldini,H. A. Lockman, and M. M. Levine, Abstr.Annu. Meet. Am. Soc. Microbiol. 1983, B55, p.

32) proceeded to develop an attenuated V. cho-lerae strain by techniques of recombinant DNA.In this manner it was hoped to construct a strainhaving none of the drawbacks of a chemicallymutagenized strain such as Texas Star.The attenuated V. cholerae vaccine construct-

ed at the Center for Vaccine Development elimi-nates the disadvantages of the previously testedattenuated strain, Texas Star, because of themethod of attenuation. A very specific methodof mutagenesis was used so that no unexpectedsecondary mutations were present and the pre-cise genetic lesion was known and was such thatno possibility of reversion to virulence existed.The first step in construction of the vaccinestrain entailed the cloning in E. coli of genesencoding cholera toxin (201). The genes encod-ing the A and B subunits were mapped, andDNA sequencing studies were performed todetermine the amino acid sequence of the sub-units (H. Lockman and J. Kaper, Abstr. Annu.Meet. Am. Soc. Microbiol. 1983, B44, p. 30; J.Biol. Chem., in press). The cloned genes were

then treated in vitro with restriction endonucle-ases to precisely delete the DNA sequences forboth subunits. In place of the toxin genes, a

fragment of DNA encoding resistance to mercu-ry was cloned. Subsequent manipulations result-ed in a recombinant plasmid consisting of mer-

cury resistance genes in place of the toxin genesand surrounded by flanking DNA sequences ofthe V. cholerae chromosome approximately 7kilobases in length. The vibrio and mercuryresistance DNA sequences were conjugallytransferred from E. coli to V. cholerae on a

plasmid belonging to incompatibility (Inc) groupP. This plasmid replicated extrachromosomallyin V. cholerae El Tor Inaba strain N 16961where, at a very low frequency, the homologoussequences flanking the toxin and mercury resist-ance genes recombined (Fig. 1). This recombina-tional event resulted in the mercury resistancegenes replacing the toxin genes on the chromo-some. The specificity of this substitution anddeletion of the toxin genes was dictated by the

TABLE 4. Protective efficacy of Texas Star attenuated V. cholerae El Tor Ogawa oral vaccine againstchallenge with pathogenic V. cholerae El Tor of Ogawa and Inaba serotypes

Challenge strain Group Attack rate for di- Mean diarrheal stool vol (ml) perarrhea ill volunteer

El Tor Ogawa Controls 7/10a 2,100 (700-3,400)bP = 0.15 P < 0.01

Vaccinees 2/7 300 (200-400)

El Tor Inaba Controls 11/15 4,400 (400-10,300)iP < 0.01 P < 0.01

Vaccinees 5/18 J 800 (300-1,200)

aNumber ill/ number of volunteers challenged.b Range.

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Su

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FIG. 1. Construction of attenuated V. cholerae vaccine candidate, JBK70. (A) V. cholerae strain N16961contains one chromosomal copy of cholera enterotoxin genes (tox) which are flanked by sites for restrictionendonucleases HindIll (H), BgIII (B), and PstI. A recombinant plasmid (pJBK69) containing the chromosomalregion surrounding the toxin genes but with mercury resistance genes substituted for the toxin genes wasintroduced into N16961. pJBK69 belongs to plasmid incompatibility group P (IncP). (B) At a rare frequency, thehomologous sequences flanking the chromosomal toxin genes and the plasmid mercury resistance genes willrecombine, resulting in the displacement of the chromosomal toxin genes by the mercury resistance genes. Thisevent was detected in a population of V. cholerae containing pJBK69 by introduction of a second IncP groupplasmid which is incompatible, i.e.. cannot be stably maintained with pJBK69 and encodes sulfonamideresistance. (C) Resulting mercury- and sulfonamide-resistant colonies represent those cells in which the IncPplasmid encoding sulfonamide resistance is stably maintained extrachromosomally, the mercury resistancedeterminant is integrated into the chromosome, and the toxin genes and the pJBK69 replicon are lost.

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homologous flanking chromosomal sequences.To detect this rare event, the plasmid incompati-bility phenomenon was exploited in which asecond Inc P plasmid was introduced. Since twoplasmids of the same incompatibility cannot bestably maintained in the same cell, simultaneousselection for sulfonamide resistance on the in-coming group P plasmid and mercury resistancein place of the toxin genes selects for the mutantin which the mercury resistance determinant isintegrated into the chromosome and the sulfon-amide resistance gene resides on the incominggroup P plasmid (Fig. 1). Such a mutant wasrecovered (JBK70) and, when tested by DNAhybridization with cholera toxin genes asprobes, found to be completely deleted for thetoxin genes. In addition, a plasmid was con-structed which contained the B-subunit geneunder the control of the strong trp promoter.This plasmid, which directs production of onlythe B subunit, was introduced into the nontoxin-ogenic JBK70 mutant to produce an attenuatedstrain of V. cholerae, producing an immunogen-ic B subunit of cholera toxin. In addition, otherparent strains of the Ogawa serotype have beentreated in a similar manner to attenuate them bydeletion of the toxin genes. Volunteer studiesare now under way to evaluate the safety andefficacy of vaccine candidates produced by thismethod.

ETEC

With respect to pathogenesis, ETEC resembleV. cholerae 01. After successful passagethrough the hostile gastric environment, ETECmust penetrate the mucus layer to reach themucosal cells of the proximal small intestinewhere they adhere and elaborate LT or heat-stable (ST) enterotoxins. Whereas motility is aprerequisite for expression of full virulence of V.cholerae 01, human ETEC pathogens are oftenH-, nonmotile strains.

Enterotoxins

The genes in E. coli encoding the synthesis ofLT and ST are found in transferable plasmids(161, 356, 358). A comparison of the biologicalproperties of LT and ST is summarized in Table5. LT is a high-molecular-weight (ca. 86,500)protein that resembles cholera toxin in struc-ture, function. and mechanism of action (42, 58,142, 224, 289, 335, 363, 380). Both LT andcholera toxin are composed of one enzymatical-ly active (ADP-ribosylating) A subunit joined tofive binding B subunits. The receptors for LT Bsubunit found on enterocytes include GM, gan-glioside and a recently described glycoprotein(175, 177). After binding of LT to an enterocyteby its B subunits, the A subunit in some manner

TABLE 5. Comparison of the biological,pharmacological, and immunological properties of

LT and ST enterotoxins of E. coli

Property LT ST

Mol wt 86,500 1,000-6,000Inactivated by boil- Yes No

ingDelay before onset Yes No

of secretion bymucosa

Antitoxin appears Yes Noafter natural in-fection

Enterocyte enzyme Adenylate Guanylatestimulated cyclase cyclase

Genes usually in Yes Yesplasmids

gains entrance to the cell. LT irreversibly acti-vates adenylate cyclase in the enterocyte, lead-ing to an accumulation of cyclic AMP. Theintracellular accumulation of this cyclic nucleo-tide causes overt secretion by crypt cells anddecreased absorption by villus tip cells. Theresult is net secretion into the lumen of theproximal small intestine of electrolyte-rich fluid,which will appear clinically as watery diarrhea.LT from human ETEC pathogens is closely

related to but distinct from LT found in porcineETEC strains (128, 182). Both human and por-cine LTs are immunologically related to choleratoxin (43).ST found in ETEC pathogenic for humans is a

small polypeptide that is soluble in methanol.Whereas it is not entirely clear how ST binds toor enters enterocytes (386), it is known that thispolypeptide activates guanylate cyclase activity,leading to an intracellular accumulation of cyclicGMP (98, 155). This cyclic nucleotide alters theenterocyte membrane function, resulting in netsecretion (323). ST found in human pathogenscauses fluid accumulation in the intestine ofinfant mice and has been given the designationST A or ST I to differentiate it from a methanol-insoluble ST found in some ETEC pathogenicfor pigs (27); the latter ST, which does not causefluid accumulation in the intestine of infant mice(but does in infant pigs), has been referred to asST B or ST II. Only ST A is of importance inhuman ETEC pathogens. By means of DNAhybridization techniques, using cloned ST Agenes from different sources as probes (288), ithas been shown that at least two distinct sub-classes of ST A exist. These two classes, whichhave been referred to as ST human and STporcine (or bovine), have been shown by DNAsequencing studies to share 69% DNA homology(289a, 361a). One of these ST A genes resides ona bacterial transposon (361). Genes encoding the

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ST B toxin have been cloned and found to sharelittle homology to ST A genes (C. H. Lee, M.So, S. L. Moseley, and H. W. Moon, Abstr.Annu. Meet. Am. Soc. Microbiol. 1983, B47, p.31). Natural ST A elaborated in the course ofinfection is apparently not immunogenic.

O:H Serotypes and Enterotoxin Phenotypes

The ETEC strains that cause diarrhea in hu-mans comprise a heterogeneous array of O:K:Hserotypes and enterotoxin phenotypes. In gener-al, strains that produce both LT and ST (LT'/ST') tend to cause more severe diarrhea. Thus,in studies involving patients identified in hospi-tals and treatment centers, most strains wereLT+/ST+; furthermore, these strains were foundto commonly fall within a small number of 0serogroups including 06, 08, 015, 020, 025,063, 078, 0115, 0128, 0148, and 0159 (20, 76,279-281, 306, 308, 329, 340). In each 0 sero-group the number of H types was also limited.For example, H16 usually occurred with 06;Hll and H12, with 078; and H28, with 0148.Evans et al. (86) noted that the stability in ETECstrains of plasmids containing genes for theproduction of LT and ST was related to O:Hserotype, with certain combinations displayingextreme stability. The precise biochemical orgenetic explanation for this relationship has notyet been clarified. As will be seen below, manyof the LT+/ST' strains associated with certainO:H serotypes are also correlated with the pres-ence of certain specific colonization factors.

In later epidemiological studies, it came to berecognized that ETEC were very frequentcauses of diarrheal disease in nonhospitalizedpersons, including infants in less-developed ar-eas and travelers. When prospective studies ofdiarrheal disease were carried out in villages inendemic areas and in travelers, it was found thatLT-/ST+ strains, in particular, and LT+/ST-strains to a lesser extent, were important (18, 19,156, 278, 339). In such studies approximately 40to 50% of strains isolated from cases of diarrheaproduce only ST, 30 to 40% are LT'/ST+, andthe remaining 20 to 30% elaborate only LT. Incontrast with LT+/ST+ strains, LT-/ST+ andLT+/ST- phenotypes include a much wider ar-ray of 0 serogroups.ETEC are also a major cause of diarrheal

disease in neonatal herd animals. The animalserotypes are distinct from those causing diseasein humans and tend to be species specific as well(157, 160, 362).

Colonization Factors

ETEC must possess accessory virulenceproperties in addition to LT or ST to causediarrheal disease in humans. The best-character-

ized accessory virulence properties are coloni-zation factors which allow ETEC to adhere tospecific receptors on enterocytes of the proxi-mal small intestinal mucosa, thereby overcom-ing the potent peristaltic defense mechanism(230). All specific colonization factors identifiedso far in either animal or human ETEC strainshave turned out to be fimbriae (also referred toas pili); these are hairlike protein organellesfound on the surface of the bacteria (Fig. 2)(108).Adhesion pili which serve as colonization

factors on ETEC were first identified in animalstrains. The heat-labile K88 antigen, found inETEC strains that cause diarrhea in neonatalpiglets, was shown to be protein in nature and toexhibit morphology of thin (3 to 4 nm) hairscovering the surface of the bacteria (307, 359,367). In their classic studies in piglets, Smith etal. (359) showed that strains possessing plasmidsencoding both enterotoxin and K88 were fullypathogenic, whereas strains having enterotoxinbut not K88 did not cause illness. A derivativepossessing K88 but no enterotoxin caused milddiarrhea in some piglets.Adhesion fimbriae tend to be rather species

specific. Thus, the K88 antigen fimbriae areencountered only in strains pathogenic for pig-lets (359), K99 is associated with calf, lamb, andpiglet pathogens (285, 309), and type 987 fimbri-ae are found only in porcine strains (188).

Heretofore, at least three distinct types ofputative adhesion fimbriae have been detectedin human ETEC pathogens, including coloniza-tion factor antigen I (CFA/I), CFA/II, and E8775fimbriae (82, 83, 85, 387). Several other distinctfimbriae that may also serve as colonizationfactors in E. coli have also been described (60,63, 181).Many of the above fimbriae share common

properties, particularly K88, K99, CFA/I, andCFA/II. Genes for these fimbriae are encoded intransmissible plasmids which frequently encodeST or LT as well. These fimbriae are preferen-tially expressed when the strains are grown onsolid agar rather than in broth; they are notexpressed when cultures are incubated at 18 to22°C. Bacteria bearing these fimbriae causemannose-resistant hemagglutination of erythro-cytes of certain animal species, providing asimple screening test to detect their presence.Known adhesion fimbriae are encountered inapproximately 65 to 75% of LT'/ST' strains, in20 to 25% of LT-/ST' strains, and rarely inLT+/ST- isolates (254). Intensive investigationsare currently under way to identify the adhesionfimbriae or other colonization factors present instrains lacking CFA/I and II and E8775 fimbriae(60, 63, 181, 254). This is critical because thefeasibility of immunoprophylaxis of ETEC diar-

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FIG't-op of tso,, i CA s ,

FIG. 2. Electron photomicrograph of ETEC bacteria showing CFA/I fimbriae on their surface. x28,000.

rhea in humans by means of purified fimbriaevaccines will depend on identifying the CFAscommon to the vast majority of human ETECpathogens. Only in this manner can broad-spec-trum protection be expected.

It is known that with repeated passage onagar, LT'/ST' strains possessing CFA/I orCFA/II can lose the plasmid encoding the CFAand often toxin genes as well. CFA/I plasmids,for example, also encode ST; indeed, the ST andCFA/I genes appear to be adjacent. Some work-ers have implied that LT-/ST' and LT+/ST-strains isolated from cases of diarrhea do notrepresent true pathogens but rather are strainsthat have lost plasmids from a previous state inwhich they were LT'/ST' and CFA positive. Insupport of this theory, it was shown that a CFA-negative LT+/ST- strain (H10407P) derived inthe laboratory from an LT'/ST' CFA/I strain(H10407) did not cause diarrhea, colonize theintestine, or stimulate antibody responses whenfed to humans (84, 350). This theory is contra-dicted, however, by other volunteer studieswhich show that naturally occurring LT-/ST'and LT+/ST- strains isolated from cases ofdiarrhea are indeed pathogenic (253). Theycause diarrhea, colonize the intestine, and stim-ulate antibody responses when fed to volun-teers. These observations are not really contra-

dictory. LT'/ST' strains that possess CFA/I orCFA/II need this full armamentarium of viru-lence properties to cause diarrhea. Thus, a de-rivative of such a strain lacking a critical plasmidis nonpathogenic. In contrast, other strains (253,260) that circulate in nature are pathogenic withonly one enterotoxin and in the absence of thefew colonization factors we recognize so far.No heterogeneity is known to exist within

those adhesion fimbriae referred to as CFA/I. Incontrast, both Cravioto et al. (51) and Smyth(360) have described antigenic heterogeneitywithin the entity referred to as CFA/II. It ap-pears that there exists a common antigen instrains that possess CFA/II that was originallydetected in immunodiffusion studies; this com-mon antigen has been referred to as component3 (51) or coli surface antigen 3 (CS3) (360). It isknown that CS3 is composed of subunits ap-proximately 15.3 kilodaltons in size. The exactmorphological nature of CS3 is under investiga-tion; it is not a fimbrial structure 6 to 7 nm inwidth like some fimbriae but it may consist ofextremely fine (2 to 3 nm) fibrils (M. M. Levine,P. Ristaino, G. Marley, C. Smyth, and S. Knut-ton, unpublished data). Many strains possessing"CFA/II" have additional antigens which areclearly fimbrial in morphology. Two such anti-gens are CS1 and CS2 described by Smyth (360)

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which appear to be identical to components 1and 2 of Cravioto et al. (51). Cravioto et al. (51)noted that whether 06:H16 strains (the mostcommon serotype associated with CFA/II) havecomponent 1 or 2 in addition to component 3 is afunction of biotype.There exists a close correlation between pres-

ence of specific adhesion fimbriae and certain 0serogroups. CFA/I is found in 015, 025, 063,078, and 0128 strains, whereas CFA/II is seenin 06, 08, 080, and 085 strains and E8775fimbriae are found in 025, 0115, and 0167strains. Some commonly encountered LT'/ST'serotypes never have currently recognizedCFAs. For example, 0148:H28 and 0159:H4have been repeatedly isolated throughout theworld as LT'/ST' strains but they never pos-sess CFA/I, CFA/II, or E8775 fimbriae. CFA/I,CFA/II, and type E8775 fimbriae are virtuallynever encountered in non-enterotoxigenic nor-mal flora strains.Many ETEC (50 to 75%) elaborate type 1

somatic pili (16, 253, 254). However, this is thesame frequency with which these pili are foundin nonpathogenic colonic flora E. coli (253, 254).Thus, it is not clear that they play a role in thepathogenesis ofETEC diarrhea. Indeed, individ-uals convalescent from experimental ETEC di-arrhea make serum and local intestinal antibodyto many antigens but not to type 1 somatic pili(234, 238), thereby providing evidence for thecontention that these fimbriae are not involvedin pathogenesis of ETEC diarrhea.

Immune Response in HumansBoth serum and intestinal SIgA antibody re-

sponses to the homologous 0 antigen occur inapproximately 90% of persons with clinical in-fections due to ETEC (61, 84, 234, 238, 241,253). The serum 0 antibody is predominantly inthe IgM class and peaks 8 to 10 days after onsetof infection.Appearance of neutralizing or binding antitox-

in to ST after ETEC infection in humans has notbeen reported. In contrast, most persons whoexperience diarrhea due to LT-producing strainsof E. coli manifest significant rises in serum LTantitoxin (84, 149, 234, 238, 253). In experimen-tal challenge studies in volunteers, rises in se-rum IgG antitoxin measured by ELISA afteringestion of LT-producing E. coli were detectedin 77% of ill persons with purified human LT asantigen and in 34% with cholera enterotoxin(Levine et al., unpublished data). The serumantibody that appears after LT infection exhibitsneutralizing activity. Rises in levels in SIgAantitoxin in intestinal fluid have also been de-scribed after experimental infection with LT-producing E. coli (234, 238).

Rises in serum IgG and intestinal SIgA anti-

body to CFA/I have been documented afterinfection with CFA/I-bearing strains (41, 61, 84,234, 238). Levine et al. (234, 238) detected risesin serum IgG ELISA antibody to CFA/I in 67% ofindividuals infected with ETEC bearing CFA/L.In contrast, only one of 78 persons exhibited arise in serum IgG ELISA antibody to type 1somatic pili in the course of infection (238).

Vaccines Against ETEC

Current approaches to immunoprophylaxis ofETEC infection involve vaccines that stimulateantitoxic or antiadhesion immunity or both bymeans of killed antigens or attenuated strains. Itis likely that the most effective vaccines willcontain appropriate antigens intended to simul-taneously stimulate both antibacterial and anti-toxic immunity, thereby leading to a synergisticprotective effect. Since ETEC are noninvasiveenteropathogens that do not penetrate the intes-tinal mucosa, it is believed that the critical site ofimmunity is the mucosal surface and almostcertainly involves SIgA antibody. Accordingly,immunization regimens in humans must be di-rected towards stimulation of intestinal mucosalimmunity.

Purified CFA fimbriae vaccines. Extremelypromising results have been obtained in veteri-nary medicine in preventing neonatal deathsfrom diarrheal disease in herd animals by use ofpurified fimbriae as vaccines (1, 287, 292, 338).Pregnant sows and cows were inoculated paren-terally with purified K88, K99, or 987-type fim-briae. Newborn piglets and calves suckled onimmunized mothers were significantly protectedagainst death from diarrhea when challengedwith ETEC bearing the homologous fimbrae, incomparison with animals suckled on nonvacci-nated mothers. Protection of the newborn ani-mals is mediated by antifimbrial antibody inmaternal colostrum. These observations in vet-erinary medicine have generated considerableoptimism for the use of purified fimbriae asvaccines to prevent ETEC diarrhea in humans.

Considerable data from animal models havebeen generated recently to establish that purifiedCFA/I and CFA/II fimbrial vaccines adminis-tered orally or enterally can stimulate intestinalSIgA anti-CFA antibody and protective immuni-ty. De la Cabada et al. (62) and Evans et al. (81)have shown that rabbits immunized orally withtwo 1.0-mg or two 5.0-mg oral doses of purifiedCFA/I fimbrial vaccine developed significantincreases in the number of anti-CFA/I-producingcells present in the intestinal mucosa. Whenchallenged enterally with a CFA/I-positiveETEC strain, the immunized rabbits were signif-icantly protected in comparison with unimmu-nized control rabbits. Protection was not seen in

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other immunized rabbits challenged with a CFA/II-positive ETEC strain, demonstrating that im-munity is restricted to ETEC bearing the ho-mologous fimbrial antigen.

Similarly, Boedeker and collaborators (23)surgically prepared chronic, externalized intesti-nal loops in rabbits to study the effects ofmucosal immunization with purified CFA/II fim-briae. Rabbits immunized with three 0.1-mgweekly doses of CFA/II did not manifest rises inSIgA anti-CFA/II, whereas rabbits that receivedthree weekly 1.0- or 2.0-mg doses had prominentSIgA anti-CFA/II responses in their intestinalwashings. Rabbits immunized with eight enteral2.0-mg doses of purified CFA/II fimbriae devel-oped marked and long-lasting increases in SIgAanti-CFA/II in intestinal washes. It is importantto note that repeated mucosal immunizationwith purified CFA/II fimbriae did not induce astate of immune unresponsiveness (tolerance) tosubsequent parenteral inoculation with E. colibearing the homologous fimbriae (238). This isan important observation that has paved the wayfor studies to begin in humans with purifiedfimbriae as an oral vaccine. It is known thatrepeated oral immunization with certain anti-gens, while stimulating an intestinal SIgA muco-sal response, can lead to proliferation of sup-pressor T cells that severely depress the serumIgM and IgG response to the same antigen whenit is inoculated parenterally (4, 5, 37, 378, 388).Some concern had been expressed that repeatedoral immunization with E. coli surface antigensby inducing tolerance might impair an individ-ual's ability to successfully respond to systemicinfection with other E. coli, as in the course ofpyelonephritis. Therefore, it was critical to in-vestigate in animal models whether this phenom-enon occurred with CFA antigens before initiat-ing oral immunization in humans. Fortunately,rabbits developed serum IgG anti-CFA/II as wellas SIgA intestinal anti-CFA/II after enteral im-munization with eight spaced doses of antigen.Furthermore, significant increases in serum IgGanti-CFA/II occurred when the same rabbitswere inoculated parenterally weeks later with E.coli bearing CFA/II (238), showing that toler-ance was not present. With the successful com-pletion of these safety and immunogenicity stud-ies in animal models, reactogenicity,immunogenicity, and efficacy studies of purifiedCFA/II fimbrial vaccine in humans are begin-ning. It is hoped that such studies will serve asan assessment of the effectiveness of a proto-type-purified fimbrial oral vaccine in stimulatingantiadhesion immunity. Should the monovalentCFA/II vaccine prove promising, an intensivesearch will have to be made to identify othercolonization factors in ETEC, in addition toCFA/I, CFA/II, and E8775 fimbriae. These anti-

gens would be included in a polyvalent fimbrialvaccine to ensure broad-spectrum protection.

Purified type 1 somatic pili (fimbriae) vaccine.Only a minority of the ETEC strains from largesurveys of infant diarrhea in less-developedcountries or from cases of travelers diarrheapossess CFA/I or CFA/II (150, 254, 308, 329).Furthermore, these fimbriae occur predominate-ly in LT'/ST' strains within a restricted set ofO:H serotypes. Therefore, antigens are neededto include in a polyvalent fimbrial vaccine thatwill provide protection against ETEC pathogensthat lack the currently recognized adhesins suchas CFA/I and CFA/II (254). This particularlyapplies to the commonly encountered LT-/ST'strains.Many of the ETEC strains that lack known

mannose-resistant hemagglutination adhesinssuch as CFA/I and II nevertheless posses type 1somatic pili which are associated with mannose-sensitive hemagglutination of guinea pig erythro-cytes (253, 254). Type 1 somatic pili, however,are found with equal frequency among ETECand nonpathogenic normal flora isolates (+70%of strains), and their role, if any, in the patho-genesis of ETEC diarrhea is unknown. Never-theless, they have been shown to bind E. coli tointestinal epithelial cells in vitro and such bind-ing can be inhibited by monospecific antipilusantibody (187). Accordingly, Levine and collab-orators (234, 238) undertook to assess type 1somatic pili as a potential immunizing agent inthe hope that inclusion of this antigen mightbroaden the spectrum of protection of a futurepolyvalent fimbrial antigen vaccine, particularlyvis-a-vis ETEC strains that lack CFAs but pos-sess type 1 somatic pili.

Purified type 1 somatic pili were preparedfrom E. coli H10407 by Brinton (25) by a modifi-cation of the pilus crystal solubilization-recrys-tallization method. Purified type 1 somatic pilivaccine was administered parenterally to a totalof 100 healthy young adults in doses of 45, 90,180, 450, 900, or 1,800 ,ug. The type 1 somaticpili vaccine was demonstrated to be safe in 100volunteers. However, a highly immunogenic,nonreactogenic immunization schedule was de-vised involving 1,800-,ug primary and 450-,ugbooster doses. Recipients of the vaccine did notmanifest significant alterations in intestinal tran-sit time, absorptive capacity of the bowel, or theprevalence of colonic E. coli bearing type 1somatic pili of the antigenic variety contained inthe vaccine.

Reports from Swedish workers (372, 374) hadpreviously shown that parenteral immunizationcan boost levels of intestinal secretory antibodyto specific antigens in immunologically primedpersons; the Swedish investigators were work-ing with parenteral whole-cell cholera vaccine in

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Pakistanis and cholera B-subunit vaccine inBangladeshis. In the type 1 pili studies it was

assumed that most persons are immunologicallyprimed to recognize several different antigenicvariants of type 1 somatic pili consequent to thepresence of E. coli flora in the colon. Thus, itwas not unreasonable to expect that parenteraltype 1 somatic pili vaccine might boost levels ofintestinal IgA as well as serum IgG antibody tothis antigen. This in fact proved correct. Individ-uals who received two parenteral inoculations ofpurified type 1 somatic pili developed significantrises in intestinal SIgA antibody to this antigen(238).

In total, five challenge studies were carriedout in volunteers to assess vaccine efficacy.Consistent evidence of protection was not ob-served.Some insight into the role of type 1 somatic

pili as a surface antigen and the interaction of piliwith the host mucosa and immune system can begleaned from an analysis of antibodies afterinfection. It is of interest to note that almost allpersons manifested significant antibody rises tothe homologous 0 antigen; most challenged withE. coli H10407 had rises in antibody to LT andCFA/I. Of the 38 vaccinees and 40 controlvolunteers who participated in vaccine efficacystudies, 34 vaccinees (89%) and 35 controls(88%) manifested significant rises in serum anti-body to 0 antigen of the challenge strain, where-as only 1 of these 78 volunteers (a control) had a

significant rise in antibody to type 1 somatic pili(238). These serological observations call intoquestion whether type 1 somatic pili are ex-

pressed in vitro in the small intestine as theyinteract with the host's mucosal surface.Summary of killed antigen vaccines. The likely

nonliving vaccine of the future will probablycontain components to stimulate both antibac-terial and antitoxic immunity (371). The majorantibacterial antigens will probably consist of a

pool of colonization factor fimbriae (230). Thetoxoid immunogen should stimulate anti-ST as

well as anti-LT antibodies, both capable of toxinneutralization. Candidate proteins to which STmight be covalently linked include LT, procho-leragenoid or "procoligenoid" (see below), andcertain CFA fimbriae. The major drawback tononliving oral antigens is that multiple doses are

required to prime the secretory immune systemand assure a good local antibody response. Newdevelopments in adjuvants may make the task oforal immunization much easier.

Live vaccines. Another approach toward pre-vention of ETEC diarrhea in humans involvesthe use of attenuated strains of E. coli bearingseveral critical antigens (232, 238). In theory,such a strain would colonize the small intestineof humans after ingestion of a single dose and

would stimulate a potent immune response tothe critical antigens without causing significantuntoward reactions. After examining the anti-genic composition of ETEC strains isolated innature, one might conclude that the likelihood ofsuccessful development of such attenuated vac-cine strains is small. For example, in natureCFA/I and CFA/II are never found in the samestrain nor does one ordinarily encounter morethan one 0 antigen in a single strain. The adventof recombinant DNA technology, however,brings into the realm of reality the constructionof such strains by genetic engineering. Already,the genes encoding porcine and human LT (44,56, 57, 59, 397) and their subunits have beencloned into high-copy plasmid vectors; E. coliK-12 bearing such plasmid vectors elaboratescopious quantities of LT or B subunit. In theory,one envisions a single smooth E. coli vaccinestrain genetically engineered to produce largequantities of CFA/I, CFA/II, other CFA anti-gens, B subunit, and perhaps an ST toxoid.Before this goal can be realized the genes encod-ing the CFAs must be successfully cloned. Here-tofore, this has proven frustrating. For example,the genes encoding CFA/I are intimately associ-ated with the genes for ST, and it has not yetbeen possible to successfully clone the CFA/Igenes on a single plasmid free of ST (266, 328,394).The spontaneous occurrence in the laboratory

of an LT-/ST- CFA/II-positive variant of apreviously enterotoxigenic 06:H16 LT'/ST'strain offered the opportunity to investigate thebehavior of an attenuated E. coli vaccine proto-

TABLE 6. Microbiological and geneticcharacteristics of E. coli strain E1392/75-2A

(06:H16)" candidate vaccine strain

Test/characteristic Result

Mannose-resistant hemagglutinationBovine erythrocytes +Human erythrocytes

Agglutination with CFA/II antiserum +Test for LT

Y-1 adrenalELISA -

Test for STInfant mouse

Detection of enterotoxin gene sequenceswith genetic probesb

LT -

Porcine STHuman ST' Spontaneously derived laboratory derivative of a

previously enterotoxigenic (LT'/ST') strain; kindlyprovided by Bernard Rowe, Central Public HealthLaboratory, Colindale, England.

h DNA hybridizations with cloned LT and ST genesto detect homologous sequence.

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TABLE 7. Clinical, bacteriological and immunological response of volunteers after ingestion of non-enterotoxigenic CFA-II-positive E. coli vaccine candidate E1392/75 2A

Vaccine dose Positive cultures Significant serum antibody rises to:(viable organ- Diarrhea Jejunal 06 CFA/II LT

isms) Stool fluid 0 F/1L

109 0/4" 4/4 1/4 2/4 0/4 0/41010 1/4b 4/4 2/4 3/4 2/4 0/46 x 1010 1/11C 11/11 11/11 7/11 5/11 0/11

a Number positive/number of volunteers vaccinated.b This individual passed four loose stools totalling 243 ml in volume.' This individual passed nine loose stools over 3 days totalling 1,197 ml in volume.

type in humans. The properties of this strain, E.coli 1392/75-2A, which was provided by BernardRowe of the Central Public Health Laboratory,Colindale, U.K., are summarized in Table 6.This prototype vaccine strain was given to adultvolunteers in doses of 109, 1010, or 6 x 1010organisms with NaHCO3 (232, 238). All recipi-ents excreted the strain, most had serologicalresponses, and the strain was recovered fromcultures of jejunal fluid of 14 of 19 vaccinees(Table 7). Two vaccinees, however, developedmild diarrhea, presumably as a consequence ofcolonization of the proximal small intestine.Strain E1392/75-2A has the potential to serve asa recipient strain into which can be insertedhigh-copy plasmid vectors containing recombi-nant DNA encoding CFA/I, LT B subunit, andother relevant antigens. Based on experiences inhumans and piglets after oral inoculation withnon-enterotoxinogenic adhesive variants of E.coli (359) and V. cholerae (240), it appears thatthe occurrence of mild diarrhea in approximate-ly 20% of recipients may be the price to be paidfor immunization with attenuated strains thatadhere to and colonize the proximal small intes-tine.

Toxoids

Pregnant sows immunized parenterally withporcine LT manifested significant rises in LTantitoxin in their milk, and piglets suckled on theimmunized sows were significantly protectedagainst diarrhea due to ETEC (64, 66). Thesedata from veterinary field trials suggest thatvaccines that stimulate a vigorous antitoxin re-sponse may have a role in protecting humansagainst LT-producing E. coli. Such toxoidsmight be used either alone or in conjunction withother oral killed vaccines such as purified pili.

It is possible to alter the toxicity of LT andcholera enterotoxin by treatment with formalde-hyde or glutaraldehyde. This is not, however,being pursued with vigor as a likely successfulimmunoprophylactic preparation because of thedisappointing results obtained with glutaralde-hyde cholera toxoid (53, 251). Neither parenteral

(53) nor enteral (251) glutaraldehyde choleratoxoid gave notable vaccine efficacy.

Candidate vaccines intended to enhance pro-tection by stimulating neutralizing antitoxin toLT or ST include purified LT B subunit, procho-leragenoid, ST-carrier protein toxoids, and STconjugated to LT B subunit.

Klipstein and Engert (209) have carried out aseries of experiments involving immunization ofrats with B subunit from porcine LT or LTholotoxin. Rats were immunized orally, paren-terally, or in combination, and vaccine efficacywas assessed by the degree of intestinal fluidaccumulation in ligated intestinal loops of theimmunized and control rats challenged with LTor viable LT+/ST- and LT'/ST' strains. Ratsimmunized by the combined parenteral/oralroute with either LT B subunit or holotoxindemonstrated a protective effect, although holo-toxin was the superior immunogen. The technol-ogy for large-scale production of E. coli LT andB subunit has not yet been described but isprobably not long in coming. The successfulcloning of LT genes from a human pathogen intoa high-copy plasmid vector (56, 397) shouldgreatly increase yields of human LT from cul-tures.

Procholeragenoid. Heat treatment of choleraenterotoxin leads to a high-molecular-weight(ca. 900,000) toxoid, procholeragenoid, which iscomparable in immunogenicity to the parenttoxin but has only 1% of the parent's toxicity(103, 135). Procholeragenoid has been used toparenterally immunize pregnant sows in fieldtrials (125). Piglets born to and suckled on theimmunized sows were significantly protectedagainst diarrhea and death due to infection withETEC. These observations suggest that procho-leragenoid, since it is such a potent antigen,might serve as an oral vaccine to enhance pro-tection against ETEC as well as V. cholerae.Once the technology exists to prepare and purifyhuman LT in large quantities, it may be possibleby heating to prepare an LT procoligenoid anal-ogous to procholeragenoid.ST toxoids. Since three-fourths of the ETEC

strains that cause travelers diarrhea in humans

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elaborate ST (with or without LT), stimulationof antibody capable of neturalizing ST is aworthy goal. Klipstein and co-workers (210)have obtained encouraging results by immuniz-ing rats with ST conjugated to porcine IgG. Ratsimmunized with this antigen manifested neutral-izing activity demonstrable when ligated intesti-nal loops were challenged with ST or viableLT-/ST' E. coli. No neutralizing activity wasencountered when loops were challenged withLT or viable LT+/ST- or LT'/ST' strains.

It is not clear whether the encouraging resultswith ST toxoid obtained by Klipstein et al. (210)with rat closed-loop challenges can be extendedif whole animal models are used. The onlypertinent data obtained so far result from studiesin pigs carried out by Moon et al. (284). Pregnantsows were immunized with Giannella's (138) ST-bovine IgG conjugate. Piglets who suckled onimmunized mothers had almost the identicalattack rate for diarrhea as piglets suckled oncontrol sows. However, there was some sugges-tion of a decrease in mortality in the piglets fedfrom vaccinated sows (284). Since the ST toxoidutilized by Moon et al. (284) was not identical toKlipstein's, it is difficult to draw conclusionsbased on a comparison of two similar but dis-tinct toxoids tested in two very different sys-tems.

Subsequently, Klipstein et al. (211) prepared abivalent LT/ST toxoid by cross-linking ST toporcine LT. The resultant toxoid retained lessthan 0.15% of the biologically active toxigenicityof the parent LT and ST components. Rats givenintraperitoneal primary immunization followedby boosters per os with the LT/ST toxoidshowed notable protection when intestinal loopswere challenged with LT, ST, or viable ETEC ofLT+/ST+, LT+/ST-, or LT-/ST+ phenotype.More recently, these workers have begun

testing in the same rat model a toxoid consistingof laboratory-synthesized ST (214) conjugated tothe B subunit of porcine LT (212, 213, 215). Thisconjugate of ST is apparently devoid of entero-toxic activity in the infant mouse assay andstimulates neutralizing antibody to both LT andST. Rats immunized with parenteral primingdoses followed by per os booster doses of theST/LT-B toxoid conjugate developed significantrises in mucosal IgA antitoxin against both LT-Band ST. Ligated intestinal loops of immunizedand control rats were challenged with heterolo-gous serotypes of human or porcine E. coli thatelaborate LT, ST, or both. In comparison withcontrols, secretion was diminished by 61 to 81%in intestinal loops of immunized rats (213). Com-parable evidence of diminished secretion wasobtained when immunized rats and controlswere comparably challenged with porcine LT orST.

Rabbits and rats were immunized orally withthree doses of ST/LT B toxoid. Immunizedrabbits showed diminished secretion in compari-son with controls when challenged by the ligatedloop method (215).

EPECIn the 1940s and 1950s investigators working

in the United Kingdom, Europe, and the UnitedStates incriminated E. coli of certain 0:H sero-types within certain 0 serogroups as importantcauses of infant diarrhea (87, 202). These sero-types were commonly isolated from infants withsummer diarrhea and from affected babies innursery outbreaks, but they were rarely recov-ered from healthy control infants (24a). Neter(296) coined the term "enteropathogenic E.coli" (EPEC) to refer to the serotypes of E. coliassociated with infant diarrhea between 1945and 1953. By the early 1950s Ewing and co-workers (87) showed that strains within sero-groups 026, 055, 086, 0111, 0125, 0126, 0127,and 0128 were commonly isolated from patientswith diarrheal disease.

In the early 1950s serotype represented theonly marker for these diarrheagenic strains. Noanimal models existed to demonstrate pathoge-nicity and no other recognized virulence proper-ties were identifiable by which to differentiatethe diarrhea-associated strains from other E.coli. Accordingly, in the early 1950s severalgroups carried out challenge studies in humansinvolving oral feeding of EPEC serotypes (95,195, 220, 221, 297, 393).Four groups of investigators reported feeding

studies in humans with EPEC strains of sero-group 0111; these are summarized in Table 8.Neter and Shumway (297) fed 0111 to a 2-month-old infant and induced diarrhea with anincubation period of less than 24 h. The remain-ing challenge studies involved healthy adults inwhom diarrhea also occurred after ingestion of108 to 9 x 109 organisms. Among the mostinteresting clinical studies were those of Koya etal. (220, 221) in Japan. These investigators re-ported high concentrations of 0111 in the smallintestine of the volunteers and surmised that thisrepresented the critical anatomic site of host-parasite interaction. They confirmed this bybypassing the small intestine and inoculating 1090111 organisms directly into the colon by meansof a Miller-Abbot intestinal tube or by sigmoid-oscope. Colonization of the colon occurred butno diarrhea ensued. Similar volunteer studiesinvolving healthy adults were reported by Juneet al. (195) and Wentworth et al. (393) in whichEPEC strains within 055 and 0127 serogroupscaused diarrhea (Table 8).These studies established unequivocally that

EPEC recovered from patients with diarrhea

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TABLE 8. Clinical response of volunteers after oral or intracolonic administration of EPEC strains ofserogroups 0111, 055, and 0127

Study Subjects Challenge organism Incuba- Illness(inoculum) tion (h)

Neter and Shumway, 2-mo-old in- 0111 (108) <24 Diarrhea1950 (297) fant

Kirby et al., 1950 Healthy adults 0111 (2 x 109) <24 Diarrhea(208)

Ferguson and June, Healthy adults 0111 (7 x 106-9 x 109) 10 Diarrhea, abdominal1952 (95) cramps, fever,

vomiting

Koya et al., 1954 Healthy adults 0111 (107-109) 10-16 Diarrhea (high counts(220) of 0111 in small in-

testine)

Koya et al., 1954 Healthy adults 0111 (109 given into the None(221) colon by Miller-Ab-

bott tube or by sig-moidoscope)

June et al., 1953 Healthy adults 055 (108-10Io) 6-16 Diarrhea, fever, ab-(194) dominal cramps

Wentworth et al., Healthy adults 0127 (4 x 104-8 x 106) 7-17 Diarrhea, fever1956 (393)

and selected solely by means of serotype wereindeed capable of causing diarrhea when fed tovolunteers. In the late 1960s and early 1970s twonew classes of E. coli capable of causing diar-rhea came to be recognized and described,ETEC and enteroinvasive E. coli. In each in-stance a discrete virulence property was de-monstrable, including Shigella-like epithelialcell invasiveness for enteroinvasive E. coli andproduction of LT or ST for ETEC. Between1971 and 1978 many investigators tested classi-cal serotype EPEC strains from patients withdiarrhea for their ability to invade epithelial cellsand for the presence of LT and ST. With rareexceptions EPEC strains were found to lackthese virulence properties (75, 145, 150, 159,341, 342). These oft-repeated observations stim-ulated acrimonious debate among microbiolo-gists, leading to polarization into two factions.One group claimed that EPEC strains possessLT and ST plasmids when they cause diarrheabut that the plasmids are easily lost on subcul-ture and storage, thereby explaining the lack ofenterotoxigenicity in the EPEC strains tested.Others argued that EPEC were indeed capableof causing diarrhea but by a mechansim distinctfrom Shigella-like invasiveness or production ofLT or ST. This debate was resolved by a seriesof volunteer studies carried out by Levine andco-workers (233). These investigators fed togroups of volunteers various doses of one ofthree EPEC strains that were isolated years

earlier in outbreaks of infant diarrhea. Thesestrains, which had been stored for up to 7 years,were shown to be negative for LT, ST, andShigella-like invasiveness. Nevertheless, twostrains, E2348/69 (0127:H6) and E851/71(0142:H6), caused clear-cut diarrhea when fedto volunteers (Table 9). The short incubationperiod mimicked observations made in volun-teer studies in the 1950s. The clinical syndromeincluded abdominal cramps, diarrhea, nausea,vomiting, and low-grade fever. One volunteerhad copious diarrhea, purged 5.6 liters of ricewater stools, and required intravenous fluids tomaintain hydration. Most of the volunteers de-veloped rises in serum antibody to the homolo-gous E. coli 0 antigen but none manifested risesin LT antitoxin. Isolates recovered from copro-cultures of ill volunteers were again tested forinvasiveness, LT, and ST and were shown to benegative. More recently, these workers (Blackand Levine, unpublished data) fed to volunteersan EPEC strain (0114:H2) isolated from a caseof sporadic infantile diarrhea (Table 9). Thisnoninvasive, LT- and ST-negative strain alsocaused unequivocal diarrhea. These observa-tions conclusively establish that classical sero-type EPEC are indeed capable of inducing diar-rhea but by mechanisms distinct fromproduction of conventional enterotoxins or Shi-gella-like invasiveness.The above volunteer studies stimulated re-

newed interest in EPEC and in searching for the

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TABLE 9. Clinical bacteriological, and serological response of volunteers after ingestion of EPEC associatedwith sporadic cases or outbreaks of infant diarrhea

Tests for:

Shigella- Mean incuba- Diarrhea Mean diarrheal stoolStrain (serotype) Source like in- Inocula tion (h) attack vol (ml) per ill

LT ST vasive- rate volunteerness

E851 (0142:H6) Epidemic Glasgow - - - 106-10Io 18.7 (9.63)b 7/15 a 843 (437-1,403)bE2348 (0127:H6) Epidemic Taunton - - - 106, 1010 9 (9-12) 3/9 2,700 (894-5,612)E74/68 (0128:H2) Epidemic Teeside - - - 106-10° 0/5E128010 (0114:H2) Sporadic case, - - - 108, 1010 12.2 6/11 1,130 (328-2,045)

BangladeshHS (09:H4) Nonpathogenic - - - 1010 0/4

normal floracontrol strain

' Number with diarrhea/number of volunteers challenjb Range.

mechanisms by which they cause diarrhea. Atabout this time, three critical pieces of informa-tion became available, (i) Polotsky et al. (322)examined by light and electron microscopy thehistopathological lesions evident when rabbitligated intestinal loops were infected with classi-cal EPEC strains. (ii) Ulshen and Rollo (389)described the histopathological lesion present inthe intestinal biopsy from an infant with diarrheadue to classic EPEC serotype 0125ac:H21. (iii)Rothbaum et al. (337) described the histopatho-logical lesions that existed in light and electronmicroscopic examination of intestinal biopsiesfrom infants infected with EPEC of serogroup0119 in the course of a protracted communityoutbreak. In each of these reports, light micros-copy revealed microcolonies of E. coli tightlyadherent to villus tip cells with effacement of theepithelial cells. On electron microscopy a char-acteristic histopathological lesion was noted.EPEC were seen tightly adherent to epithelialcells with destruction of the brush border butwithout overt invasion. Often the bacteria werefound perched atop a pedestal of destroyedmicrovillous material. Recently this characteris-tic histopathology has been reproduced byMoon et al. (286) in gnotobiotic and colostrum-deprived piglets infected with human EPEC.Cravioto et al. (51) noted that 80% of a largecollection of EPEC strains adhered to HEp-2cells in tissue culture in the presence of man-nose. Normal-flora E. coli and ETEC strainsrarely showed adhesiveness for HEp-2 cells.

Baldini and co-workers (14) applied the tech-niques of molecular genetics to the study of theadhesive phenomenon of EPEC. These workersanalyzed the plasmid content of 32 EPEC strainsand 32 normal-flora E. coli. Thirty-one of 32EPEC were found to harbor a plasmid 55 to 72megadaltons (Mdal) in size, whereas such plas-mids were found in only 19 of 32 (60%) normal-flora E. coli (P < 0.0001). One of the strains fedto volunteers by Levine et al. (233) (E2348/69)

was found to possess only one plasmid in thissize range. E2348/69 was cured of this plasmidand then assayed for HEp-2 adhesiveness.E2348/69 containing the 55-Mdal plasmid ad-hered to HEp-2 cells, whereas the cured deriva-tive was not adherent. This plasmid was markedwith a transposon encoding ampicillin resistanceand transferred into E. coli K-12. E. coli K-12does not adhere to HEp-2 cells, but the trans-conjugant bearing the plasmid from E2348/69became adherent for HEp-2 cells. HEp-2 adhe-siveness was shown to correlate with ability toattach to intestinal mucosa. Colostrum-deprivedpiglets fed the parent E2348/69 developed patho-gnomonic EPEC histopathological lesions,whereas piglets fed the plasmid-free derivativedid not. These workers are presently mappingand cloning the genes involved with EPEC adhe-sion. When labeled with 32p, the cloned adhe-sion genes could provide a DNA probe to detecthomologous gene sequences in other E. coli,thereby serving as a diagnostic tool to detectEPEC.While studies of EPEC enteroadhesiveness

were under way, others were examining EPECfor the presence of unconventional enterotoxins.In the 1978 report of volunteer studies withEPEC (233), it was also noted that cell-freeconcentrated supernatants prepared from thetwo diarrheagenic strains manifested entero-toxic activity in acute canine jejunal loops.

Independently, Klipstein et al. (216), usingbacteria-free culture supernatants in a rat perfu-sion model, showed that EPEC strains (includ-ing E851/71 and E2348/69 that caused diarrhea involunteers) elaborated an enterotoxic materialthat caused net secretion of the rat small intes-tine.The most extensive and exciting investiga-

tions in this area have been the studies ofO'Brien et al. (301). These workers have shownthat EPEC strains indeed produce a cell-associ-ated protein toxin that appears to be identical to

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the enterotoxin-cytotoxin-neurotoxin elaboratedby Shigella dysenteriae 1 and other Shigellaserotypes. O'Brien's EPEC toxin mimics Shigatoxin in being lethal and paralytic for mice andcytotoxic for HeLa cells and in causing fluidsecretion when inoculated into ligated rabbitileal loops. The enterotoxic activity in rabbitloops and HeLa cell cytotoxicity can be neutral-ized by Shiga antitoxin. In immunodiffusiontests in agarose, EPEC toxin is indistinguishablefrom Shiga toxin and appears to have a similarsubunit structure.

In synthesizing these various observations onthe pathogenesis of EPEC we can now hypothe-size the series of events leading to EPEC diar-rhea. A susceptible infant ingests an inoculum ofEPEC. The organisms reach the small intestinewhere they intimately adhere to epithelial cells.They elaborate a cytotoxin-enterotoxin whichdestroys the brush border of the enterocyte. Thecombination of altered brush border and effectsof the toxin initiate diarrhea. The precise bio-chemical events by which Shiga toxin might leadto diarrhea are not clear; there is conflictingevidence as to whether this is mediated bystimulation of adenylate cyclase (38, 64, 107).

Vaccines Against EPECIn Germany (258, 283) and Hungary (222, 223,

325, 326) during the late 1960s and early 1970s,considerable work was carried out investigatingoral vaccines to protect against EPEC. Duringthis period nosocomial infection was a seriousproblem for very young infants who were admit-ted to hospital. EPEC 0111 and 055 were thepredominant pathogens, and the rise of nosoco-mial infection increased with the duration ofhospital stay.The oral vaccines against EPEC that were

shown to be safe in infants include: (i) a sodiumdeoxycholate Boivin extract vaccine preparedby Mochmann and co-workers (283) from 0111and 055 EPEC strains; (ii) a similar deoxycho-late Boivin preparation produced from 0111,055, and 086 by Rauss and co-workers (325,326); (iii) a vaccine consisting of formaldehyde-killed whole-cell E. coli 0111, 055, and 086 (325);and (iv) attenuated streptomycin-dependent0111 organisms (258).The formaldehyde-killed and streptomycin-

dependent vaccines were not submitted to fieldtrials of efficacy. The Boivin extract vaccineswere evaluated to some extent for efficacy infield trials with variable success. These vaccinesare not presently in routine use anywhere in theworld.Now that the special enteroadhesive proper-

ties of EPEC have been characterized andshown to be associated with a plasmid (14), itshould be possible to identify the phenotypic

gene products responsible for this phenomenon.It is very likely that fimbriae or outer membraneproteins will prove to be the organelle of adhe-sion. When such information becomes available,it should be possible to prepare oral vaccinesconsisting of the purified antigen.

SHIGELLA

Only humans and higher apes serve as thenatural host and reservoir of Shigella organisms.As few as 10 viable organisms can lead toclinical infection in a well-nourished healthyadult (245). Shigella organisms appear to beparticularly adept at surviving the gastric envi-ronment to arrive in the small intestine in aviable state, since the inoculum size required tocause shigellosis is minute and it is not neces-sary to neutralize gastric acid with sodium bicar-bonate to experimentally infect humans. This issomewhat surprising since in vitro Shigella or-ganisms are sensitive to hydrochloric acid inconcentrations equivalent to those found in thefasting stomach.To manifest pathogenicity Shigella must (i)

possess smooth LPS 0 antigen, (ii) have genesencoding the ability to invade epithelial cells andproliferate therein, and (iii) elaborate a toxinafter cell invasion. Each of these virulence prop-erties is described in further detail below.

Smooth LPS 0 Antigen

The importance of smooth LPS as one of thevirulence properties of Shigella is best illustrat-ed by the phase variation exhibited by Shigellasonnei (219, 346). Phase I S. sonnei possess asmooth, complete LPS structure with 2-amino-2-deoxy-L-altruonic acid as a component of theoligosaccharide repeat unit; phase I organismsare usually virulent. Phase II S. sonnei arerough, do not manifest the 0-repeat polymer,and are nonpathogenic. Loss of a 120-Mdalplasmid is associated with the alteration fromphase I to phase II (219, 346). Reintroduction ofthis large plasmid into phase II S. sonnei re-stores them to phase I and regenerates theirepithelial cell invasiveness. Although avirulentsmooth LPS phase I S. sonnei are occasionallyencountered, no virulent phase II S. sonnei havebeen described.

S. dysenteriae and S. flexneri are known toexhibit colonial variation wherein some translu-cent, virulent colonies become opaque and avir-ulent (225). This phenomenon is distinct fromphase I/II variation seen in S. sonnei since boththe translucent and opaque colonies manifestsmooth complete LPS.

Shigella-E. coli genetic hybrids in whichgenes encoding production of E. coli 08 or 025LPS were transferred to S. flexneri 2a recipients

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were prepared by Gemski et al. (129). Thetransconjugant Shigella sp. strains expressing E.coli 08 smooth LPS surface antigen were allavirulent. In contrast, some Shigella sp. trans-conjugants bearing E. coli 025 LPS remainedvirulent, invaded guinea pig intestinal mucosa,and caused kertoconjunctivitis when inoculatedinto the conjunctival sac. The immunodominantrepeating sugar of the 08 LPS is D-mannose.This is distinct from LPS of S. flexneri 2awherein the structure providing type specificityinvolves attachment of glucosyl secondary sidechains to rhamnose of an N-acetylglucosamine-rhamnose-rhamnose repeat unit. Although theprecise chemical identity of E. coli 025 LPS hasnot yet been elucidated, it is recognized thatrhamnose is present in its 0 repeat unit. Theseobservations emphasize the role of specific LPSas one of the virulence properties of Shigellaspp. and suggest that the chemical structure ofthe 0 repeat sugar unit is one factor in Shigellasp.-host cell interaction.The role of smooth LPS in the pathogenesis of

Shigella sp. infection has been further studiedby Okamura et al. (305). These workers notedthat some rough mutants derived from a virulentsmooth S. flexneri 2a strain can successfullyinvade HeLa cells but they were incapable ofcausing keratoconjunctivitis in guinea pigs (theability to cause keratoconjunctivitis in guineapigs is a classic test of the virulence of Shigellasp. strains). When 0-antigen genes from asmooth avirulent S. flexneri Hfr strain weretransferred to the HeLa cell invasive roughmutant that was unable to cause keratojunctivi-tis, the transconjugants regained the ability tocause keratoconjunctivitis in guinea pigs.

Epithelial Cell InvasivenessStudies carried out over a period of more than

25 years by S. B. Formal and co-workers at theWalter Reed Army Institute of Research haveinvestigated the relationship between the abilityof Shigella spp. to invade and multiply withinepithelial cells and their potential to cause dis-ease. These workers showed that virulentstrains of S. flexneri 2a that caused enteritis inguinea pigs and dysentery in monkeys after oralchallenge manifested the ability to invade andproliferate within epithelial cells of the intestinalmucosa (109, 225). Virulent strains also causedpurulent keratoconjunctivitis in guinea pigs afterinoculation into the conjunctival sac, and theulcerative lesions of corneal epithelium wereanalogous to those seen in intestinal mucosa(225). These virulent strains also readily infectedHeLa cells in tissue culture (225). In contrast, anopaque colonial variant of S. flexneri 2a thatfailed to cause disease in guinea pigs and mon-keys after oral challenge was found to be unable

to invade HeLa cells, guinea pig corneal epithe-lium, and monkey or guinea pig intestinal muco-sa. This avirulent mutant was nevertheless ableto proliferate within the intestinal lumen.DuPont et al. (69) and Levine et al. (243)

carried out studies in humans that further docu-mented the correlation between the ability ofShigella organisms to manifest epithelial cellinvasiveness and their capacity to cause diarrhe-al illness.

In elegant and precise electron microscopicstudies of Shigella sp. infection in guinea pigsand monkeys, Takeuchi et al. (382, 383, 385)described the host cell-bacteria interaction at theultrastructural level. They noted that bacteriawithin colonic epithelial cells were usually en-closed by a single or double membrane-limitedvacuole.

In recent years both bacterial and epithelialcell components of the process of cell invasionhave been studied. Japanese workers (302, 303),using cinemicrographic techniques, noted thatwhen pathogenic Shigella strains attached toHeLa cells in tissue culture they stimulated aruffling movement of the cell membrane thatinitiated a pinocytotic process by which theadherent bacterial became incorporated into theHeLa cell. They also showed that the process ofHeLa cell invasion was enhanced by the pres-ence of certain divalent cations in the culturemedium including Ca2+, Mg2 , and Fe21 (312,313). Addition of EDTA to the growth medium(resulting in binding of divalent cations) caused anotable reduction in cell infectivity of Shigellaspp.The Japanese workers (310, 311) also demon-

strated that the phagocytic depression that ap-pears in the cytoplasmic membrane after thetransitory ruffling of the HeLa cell membrane byadhering Shigella organisms may be induced bya low-molecular-weight extracellular productelaborated by pathogenic Shigella spp. culturedin the presence of Mg2+. This product notablyincreased the infectivity of S. flexneri 2a in theHeLa cell culture system but had no enhancingeffect on infectivity of S. sonnei for HeLa cells.The cell-free product also enhanced the pinocy-totic engulfment by HeLa cells of Formalin-killed but not heat-killed S. flexneri 2a.Hale and Bonventre (162) further explored the

mechanism of cell invasiveness by S. flexneri 2a,using Henle 407 human intestinal epithelial cellsin culture. These workers noted that virulentShigella strains participate in an active fashionin the infection of cells in tissue culture. Theyfound that heating Shigella organisms at 50°C for2 min almost completely ablated the infectivityof S. flexneri 2a for Henle cells and that asublethal pulse of UV irradiation also dimin-ished infectivity.

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Hale et al. (163) noted that entry of pathogenicS. flexneri 2a into Henle cells was suppressed bycompounds that interfere with the uptake ofparticulate matter by phagocytic cells. Suchreagents included cytochalasin B, dibutyryl-cy-clic AMP, cholera enterotoxin, iodoacetate anddinitrophenol. By using transmission electronmicroscopy to visualize the Shigella organismswithin Henle cells, many intracellular bacteriawere seen to be membrane bound in structuresresembling phagosomes. The Shigella sp.-infect-ed Henle cells also manifested degenerative ul-trastructural alterations similar to those de-scribed by Takeuchi et al. (382, 383, 385) inShigella sp.-infected intestinal cells of guineapigs and monkey. These alterations, indicativeof cell injury, included surface blebs, absence ofmitochondrial cisternae, multiple bloated mem-brane-bound vesicles, and accumulation of lip-ids. The Henle cell nucleus was pyknotic withkaryolysis of the nucleolus.

Infection of cells by pathogenic Shigella or-ganisms requires active metabolic events initiat-ed by both the bacterium and the cell. Pathogen-ic, but not nonpathogenic, Shigella strains arecapable of initiating an endocytotic process inepithelial cells by which those normally nonpha-gocytic cells proceed to engulf and internalizethe bacteria. The precise identity of the bacterialproducts that stimulate changes in the epithelialcell membrane is presently under study; evi-dence points to one component as being cellfree, heat labile, and enhanced by divalent cat-ions.There appear to be multiple genes encoding

the properties of epithelial cell invasiveness,ability to proliferate within epithelial cells, andcapacity to cause keratoconjunctivitis in guineapigs. Formal et al. (112) identified one locus nearthe purine E region of the Shigella sp. chromo-some that represents a gene locus associatedwith the ability to provoke keratoconjunctivitisin a guinea pig's eye. Virulent S. flexneri alsopossess large 120- to 140-Mdal plasmids that,according to Sansonetti and co-workers (348),contain genetic material relating to invasivenessfor epithelial cells. Loss of this plasmid results inavirulence. Genetic transfer of the 140-Mdalplasmid back into avirulent, plasmid-free strainsresulted in reacquisition of epithelial cell inva-siveness by the recipient strains. Silva et al.(355) have documented the presence of suchlarge plasmids in all species of Shigella and haveshown that only strains possessing the largeplasmid can evoke keratoconjunctivitis in theguinea pig eye test.Hale et al. (164) has shown that the 140-Mdal

plasmid in S. flexneri encodes the production ofcertain outer membrane proteins. It appears thatthese proteins are involved in the process by

which Shigella organisms invade epithelial cells.Shigella minicells which harbor the 140-Mdalplasmid were found to be able to invade HeLacells, whereas syngenetic plasmid-free minicellscould not. Hale et al. (164) have hypothesizedthat the outer membrane proteins encoded bythe virulence plasmid represent bacterial adhe-sins which bind to as yet undetermined recep-tors on the surface of host enterocytes.Some years ago Formal et al. (113) and Fal-

kow et al. (88) noted that genes in the xylose-rhamnose region of the Shigella sp. chromo-some appear to be involved with the ability ofShigella organisms to proliferate within epitheli-al cells after cell invasion. Recently, Sansonettiet al. (347) were able, by the stepwise conjugaltransfer of a large plasmid and three chromo-somal segments from S. flexneri 2a to E. coli K-12, to construct a strain possessing the prerequi-sites for expression of full virulence. In additionto the 140-Mdal plasmid, these workers trans-ferred into the K-12 strain: (i) the S. flexneri 2achromosomal his region (encoding genes forsmooth LPS 0-antigen synthesis); (ii) the kcplocus (linked to the lac-gal region); and (iii) achromosomal portion linked to the arg and mtlloci (necessary to elicit an intestinal secretoryresponse). The resultant modified E. coli K-12strain caused keratoconjunctivitis in guinea pigs,invaded rabbit ileal mucosa, and caused fluidsecretion in rabbit ileal loops.

Shigella ToxinSince the turn of the century it has been

known that S. dysenteriae I elaborates an exo-toxin that causes limb paralysis when inoculatedinto small animals (neurotoxin) (49a, 390a). Lat-er it was shown that this toxin caused death ofhuman cells in tissue culture (cytotoxin) (390b).More recently, Keusch et al. (206a, 207) demon-strated that this Shigella toxin was capable ofstimulating intestinal secretion (enterotoxin) andthat the same toxin was responsible for all threebiological activities (207). Keusch and Jacewicz(207a) and O'Brien et al. (300a) found thatserotypes other than S. dysenteriae I also pro-duce the toxin. Recent reviews on the neuro-/cyto-/enterotoxin eleborated by Shigella havebeen published by Levine (231) and Keusch etal. (206a) and the reader is referred to them.

Shigella VaccinesKilled parenteral vaccines consisting of whole

Shigella organisms (115, 167, 170, 353) or For-malin-treated Shigella toxoid (268) have provenineffective in experimental challenge studies andin field trials. In contrast, efficacy has beenshown for attenuated Shigella strains utilized asoral vaccines. Among the attenuated strains

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tested in humans in the 1960s and 1970s werecolonial mutants (69, 189-191, 269), streptomy-cin-dependent organisms (69, 70, 242, 244, 247-249, 272-274), mutant hybrids (noninvasive Shi-gella mutants bearing portions of the E. coligenome) (69, 244), and E. coli expressing Shigel-la 0 antigens (255). Experience with these oldervaccines has been reviewed by Levine and Hor-nick (250) and Formal and Levine (114).New knowledge of the pathogenetic steps

involved in invasion of epithelial cells by Shigel-la strains and of the role of large plasmids hasbeen applied in the development of experimentalvaccines. Adamus et al. (2), for example, immu-nized guinea pigs and rabbits subcutaneouslywith outer membrane proteins of S. flexneri 3aand S. sonnei phase I. Immunized animals aswell as controls were challenged by inoculationinto the conjunctival sac of 5 x 108 to 1 x 109pathogenic organisms of the homologous strain.In this homologous system, controls developedsevere keratoconjunctivitis, whereas in immu-nized animals no changes or very mild eyechanges were observed. In a heterologous chal-lenge, rabbits immunized with S. sonnei outermembrane protein were completely protectedfrom keratoconjunctivitis due to challenge withS. flexneri 3a. In a similar heterologous experi-ment in guinea pigs, vaccinated animals devel-oped mild eye infections, whereas controls hadsevere keratoconjunctivitis. These preliminaryresults in animal models suggest that the outermembrane proteins of pathogenic Shigellastrains may indeed be important immunogens tobe found in vaccines.Formal et al. (110) used the observation that a

120-Mdal plasmid confers phase I properties onS. sonnei to prepare a Salmonella typhi-Shigellasonnei hybrid vaccine. Ty2la attenuated Salmo-nella typhi oral vaccine (whose safety and effica-cy have been demonstrated in humans) servedas the recipient into which the phase I S. sonneiplasmid was conjugally transferred. The result-ant transconjugant strain, Salmonella typhi5076-1C, was found to manifest both Salmonellatyphi and S. sonnei 0 antigens, and animalsimmunized with this hybrid strain developedantibodies against both 0 antigens. Preliminaryfeeding studies in several dozen volunteers haveshown that this vaccine strain causes no adversereactions in doses as high as 5 x 1010 viableorganisms (R. Black, M. Levine, M. Clements,and S. Formal, unpublished data).

TYPHOID FEVERSalmonella typhi and Salmonella paratyphi A

and B, the causative agents of typhoid andparatyphoid fever, represent highly invasive or-ganisms that rapidly and efficiently penetrateintestinal mucosa and eventually make their way

throughout the reticuloendothelial system to re-sult in a systemic infection. Typhoid fever is farmore frequent than paratyphoid fever and inmany parts of the world S. typhi infectionsremain a major public health problem. In thepreantibiotic era, case fatality rates of 10 to 20%were typical in patients with typhoid fever.

S. typhi is a highly host-adapted pathogen;humans comprise the sole natural host as well asthe epidemiological reservoir for this organism.Our knowledge of the pathogeneis of typhoidfever comes from four sources: (i) clinicopatho-logical observations in humans (185, 264, 343,369), (ii) volunteer studies (183, 366), (iii) studiesin a chimpanzee model (79, 126, 282), and (iv)analogies drawn from Salmonella typhimuriumand Salmonella enteritidis infection in mice, the"mouse typhoid" model (33, 46, 47, 49, 261).The probable steps involved in pathogenesis ofS. typhi infection are summarized below.Humans ingest the causative organisms in

contaminated food or water; contact transmis-sion is believed to be uncommon. The inoculumsize ingested greatly determines the clinical at-tack rate and also affects the incubation period(183, 294). Doses of 109 and 108 pathogenic S.typhi ingested by volunteers led to clinical ill-ness in 98 and 89% of individuals, respectively;doses of 105 caused typhoid fever in 28 to 55% ofvolunteers, whereas none of 14 persons whoingested 103 developed clinical illness.Upon arrival in the small intestine, S. typhi

cells rapidly penetrate the mucosal epithelium toarrive in the lamina propria. Gerichter (130) hasisolated S. typhi from the blood of mice minutesafter an oral challenge. An elegant electronphotomicrographic documentation of the stepsinvolved in the passage of S. typhimuriumthrough intestinal mucosa is contained in theclassic reports of Takeuchi (381, 382, 384).When they reach the lamina propria in thenonimmune host, S. typhi cells elicit an influx ofmacrophages which ingest but are generally un-able to kill the organisms. Some S. typhi appar-ently remain in macrophages in the lymphoidtissue of the small intestinal mucosa, whereasother bacteria are drained into mesenteric lymphnodes where further multiplication and ingestionby macrophages takes place. Shortly after inva-sion of the intestinal mucosa, a primary bacter-emia is believed to take place in which S. typhicells are filtered from the circulation by phago-cytic cells of the reticuloendothelial system. It isbelieved that the main route by which S. typhicells reach the bloodstream in this early stage isby lymph drainage from mesenteric nodes enter-ing the thoracic duct and then the general circu-lation. It is possible that ingestion of a massiveinoculum followed by widespread invasion ofthe intestinal mucosa could result in direct

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bloodstream invasion. As a consequence of thisearly bacteremia, the pathogen attains an intra-cellular haven throughout the organs of thereticuloendothelial system wherein it residesduring the incubation period (usually 10 to 14days) until the onset of clinical typhoid fever.Illness is manifested by increasing fever, head-ache, abdominal pain, and a fairly sustained"secondary" bacteremia. Hornick et al. (183)noted that one volunteer who began receiving a

7-day course of oral chloramphenicol only 1 dayafter ingesting S. typhi developed clinical ty-phoid fever 9 days after the antibiotic. Thisdemonstrates that within 24 h of ingestion of S.typhi, the organisms have attained their intracel-lular haven.Most S. typhi strains isolated from patients

with typhoid fever possess a polysaccharideantigen (Vi) which covers the LPS 0 antigen(40). Felix and Pitt (92-94), who discovered Viantigen, showed that it enhanced the pathoge-nicity of S. typhi for mice. Vi antigen may play a

role in protecting S. typhi from the adverseeffects of antibody directed against 0 antigenduring the bacteremic phase of clinical typhoidfever (92-94, 95). Both epidemiological and vol-unteer studies support the contention that S.typhi strains that possess Vi antigen are morevirulent than strains lacking this antigen (91, 99,183).

Vaccines Against Typhoid Fever

Studies in mice with S. typhimurium showthat both killed antigen and live bacterial vac-cines can provide significant protection (6, 46,48, 131-135, 172, 376, 377). However, moststudies show that the magnitude and duration ofimmunity conferred are superior with live oralvaccines. The operative mechanism of immunityconferred by live vaccines is believed to bemainly cell mediated (T-lymphocyte-directedstimulation of macrophages), with lesser butmeasurable roles played by circulating and intes-tinal secretory antibodies.

Several varieties of killed whole-cell parenter-al S. typhi vaccine have been subjected to rigor-ous field trials to evaluate safety and efficacy inhumans (8-10, 54, 55, 169, 250, 321, 399, 400).The acetone-killed vaccine in particular showedconsiderable efficacy (70 to 90%) for up to 7years. However, these parenteral whole-cellvaccines caused significant adverse reactions(fever, malaise, severe local reactions) so com-

monly (ca. 20 to 25% of recipients) that theyhave not been considered useful public healthtools (10, 169, 321).Thus, the major thrust in development of new

immunizing agents against typhoid fever is toidentify immunizing agents at least equal in

efficacy to the parenteral acetone-killed vaccinebut which cause no adverse reactions. One suchvaccine candidate that showed much promiseinitially was the streptomycin-dependent S. ty-phi mutant developed by Reitman (330). In ex-tensive studies in volunteers it was shown thatthis oral attenuated vaccine was extremely welltolerated, and freshly harvested vaccine washighly protective against experimental challenge(71, 245). However, when lyophilized vaccinewas given no protection was conferred (245).Since a lyophilized vaccine formulation is re-quired for field studies and for eventual use enmasse, further studies with the attenuated strep-tomycin-dependent S. typhi vaccine were aban-doned.An important advance in the area of typhoid

vaccine is the attenuated galE mutant S. typhistrain Ty2la developed by Germanier and Furer(134). This mutant has a complete absence of theenzyme UDP-galactose-4-epimerase, and the ac-tivity of the two other Leloir enzymes, galacto-kinase and galactose-1-phosphate uridyl trans-ferase, is diminished by approximately 80% incomparison with the parent S. typhi. In wild-type cells the galactose of LPS comes fromUDP-galactose, which is made from UDP-glu-cose by action of the epimerase enzyme of theLeloir pathway, specified by gene gaIE+; ingalE mutants lacking the epimerase, exogenousgalactose can be assimilated and converted toUDP-galactose (and thereby utilized for LPSsynthesis) by the action of the other two Leloirpathway enzymes. When grown in the absenceof galactose, Ty2la does not possess a smooth 0antigen; in this state it is not very immunogenicand does not confer protection (143). Grown inthe presence of galactose, smooth LPS 0 anti-gen is produced. However, because of its lack ofepimerase, strain Ty2la (like other galE mu-tants) accumulates galactose-l-phosphate andUDP-galactose when grown in the presence ofgalactose; such accumulation may be the causeof bacterial death, by lysis, which follows theaddition of galactose to growing cultures ofstrain Ty2la or other galE mutants.

Initial studies in 155 adult North Americanvolunteers showed that this vaccine was safewhen multiple oral doses of 3 x 1010 to 10 x 1010viable organisms were administered; no adversereactions were encountered (143). Galactose-fermenting organisms were not identified amongthe 958 coproculture isolates that were recov-ered for testing, thereby demonstrating the ge-netic stability of the strain. Vaccine organismsgrown in the presence of galactose conferredsignificant protection (vaccine efficacy, 87%) inthe face of challenges with inocula that inducedtyphoid fever in 38 to 53% of unimmunizedcontrols (143).

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Stimulated by the exciting results in volunteerstudies, Wahdan et al. (391, 392) carried out a

field trial of efficacy in 6- and 7-year-old schoolchildren in Alexandria, Egypt, where typhoidfever is endemic. In this trial, 14,735 childrenreceived a total of three 109 organism doses ofvaccine given on Monday, Wednesday, and Fri-day of 1 week, whereas 14,557 schoolchildrenwere randomized to receive placebo. A smallernumber of children received only two doses ofvaccine (1,081) or placebo (622). Lyophilizedvaccine or placebo was reconstituted with dilu-ent and administered after the children chewed atablet containing 1.0 g of NaHCO3 to neutralizegastric acidity.

During the first 12 months of surveillance,seven cases were detected in the placebo groupbut none among the vaccinees (392). Over 3years of surveillance a total of 22 cases wereconfirmed in the placebo groups versus only 1case in the vaccinees (391); this case occurred ina child who received two doses of vaccine.These results in a preliminary field trial wereextremely encouraging. There was, however,need for further study. The excellent efficacyobserved in Egypt was in the face of an annualincidence rate of only 44 per 105 schoolchildren.In many other endemic areas of the world theincidence of typhoid fever in schoolchildren isseveralfold higher. Furthermore, the formula-tion utilized in Egypt was not very practical formass immunization, ahd it was important tolearn whether fewer than three doses of vaccinecould provide protection.

Accordingly, in 1982-1983 a second random-ized, double-blind field trial of efficacy wasundertaken in another endemic area where a

highly practical enteric-coated formulation ofvaccine was tested; children 6 to 18 years of agewho ingested one or two doses of vaccine werecompared with others who received placebo.The site selected for this trial, Area Norte,Santiago, Chile, has one of the highest recordedincidences of typhoid fever anywhere in the

world, so the vaccine would be tested underconditions where the epidemiological force ofinfection was very high. The enteric-coated for-mulation was found to be very practical forlarge-scale vaccination (91,900 participatingschoolchildren received vaccine or placebo) andcaused no notable adverse reactions. Prelimi-nary results from the first 10 months of surveil-lance are summarized in Table 10 (Black et al.,unpublished data). Overall vaccine efficacy was50% with two doses of enteric-coated vaccinegiven 1 week apart and 30% for one dose ofvaccine. It is not clear whether the lesser effica-cy encountered in the Chile trial, as comparedwith Egypt, was due to the change in formula-tion, fewer doses, or the much higher force ofinfection (the annual incidence rate in Chile inthe placebo group was five times higher than inthe Egyptian trial). Another field trial in a differ-ent area of Santiago is being carried out in 1983-1984 to ascertain answers to these questions.The precise mechanisms of immunity stimu-

lated by Ty2la attenuated S. typhi oral vaccineare not well studied. Analogous studies in micewith a galE mutant of S. tyhimurium suggest thatcell-mediated immune mechanisms are the pre-dominant effectors of protection (131-133). Pres-ently there exists no simple test to measuresuccessful vaccine "takes" with Ty2la. As aconsequence, large-scale field trials have to beundertaken to assess modifications in formula-tion, dosage schedule, etc. Recently, Roberts-son et al. (332, 334) described studies of cell-mediated immunity to S. typhimurium infectionin calves, including both T-lymphocyte stimula-tion studies with various antigens and delayedhypersensitivity skin tests. They found that lym-phocytes of S. typhimurium-infected calveswere significantly stimulated by the 0-antigenicpolysaccharide (0,4,5,12) of S. typhimurium aswell as by a Salmonella outer membrane protein(porin) antigen. These workers created artificialglycolipids by convalently linking an octasac-charide prepared from the 0 polysaccharide to a

TABLE 10. Results of a field trial of Ty21A Salmonella typhi vaccinea in 137,697 schoolchildren in thenorthern area of Santiago, Chile, for the first 10 months (July 1982 to April 1983) after vaccination

No. of No. of ca- Rate of ty- Vaccine ef-Test group childosesb of typhoid fever ficacy (%)children ~ phoid fever per 100,000 fcc

Vaccine groupTwo doses 27,485 23 83.7 53One dose 32,707 40 122.3 31Placebo 31,762 56 176.3'

Refused vaccination 45,743 81 177.1

a 109 Ty2la S. typhi in enteric-coated capsules.b Cases confirmed by isolation of S. typhi from blood, bone marrow, or bile-stained duodenal fluid cultures.c Difference in rates, P < 0.003.

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12-carbon aliphatic acid chain. The artificialglycolipids were successfully utilized as skin testantigens in classical tests of delayed hypersensi-tivity. Studies are currently under way to adaptcomparable in vitro and in vivo tests to assesscell-mediated immune responses in human re-cipients of Ty2la oral vaccine.Another method of mutagenesis has been used

by Hoiseth and Stocker (173, 368) to derivearomatic amino acid dependent strains of S.typhimurium for use as vaccines. Some auxotro-phic mutants which require a metabolite notavailable in vertebrate tissues would be unableto grow in such tissues and thus be nonvirulent.These workers utilized the tetracycline resist-ance transposon TnlO to derive insertion mu-tants requiring tryptophan, tyrosine, phenylala-nine, p-aminobenzoic acid, and 2,3-dihydroxybenzoate. By exploiting the propertyof TnJO to make deletions in adjacent genes,stable, nonreverting aro mutants were selectedwhich were nonvirulent for mice. BALB/c miceimmunized by intraperitoneal injection of livearo-S. typhimurium were completely protectedagainst subsequent challenge with virulent S.typhimurium.Robertsson et al. (333) have recently exam-

ined in calves the safety and efficacy of thearomatic-dependent mutant of Stocker et al.(368). The vaccine was given orally or parenter-ally to 4- to 5-week-old calves. Twenty-one dayslater, vaccinated and control calves were chal-lenged orally with pathogenic S. typhimurium.The oral attenuated vaccine gave significantlybetter protection than the parenteral killed vac-cine. These results are sufficiently promising toevoke interest in an analogous aromatic-depen-dent S. typhi oral vaccine strain for use inhumans. Such a method of attenuation could beapplied to a variety of animal and human patho-gens for vaccine development.A different modern approach in vaccine devel-

opment against typhoid fever involves the use ofpurified Vi antigen as a parenteral vaccine (228,395). This parenteral vaccine does not elicitsevere adverse reactions (as do killed whole-cellvaccines) and high titers of circulating IgG Viantibody are stimulated. It is hoped that suchantibody would prevent the primary bacteremiaduring which the reticuloendothelial system be-comes seeded with S. typhi after penetration ofthe intestine. Highly purified Vi vaccine is welltolerated and immunogenic but has not beenevaluated for efficacy (228). A partially purifiedVi parenteral vaccine tested by Hornick et al.(183) did not provide notable protection againstexperimental challenge in volunteer studies. It isof interest to note that Ty2la attenuated S. typhioral vaccine, which was highly protective involunteer studies, does not possess Vi antigen.

VACCINES AGAINST NONTYPHOIDALSALMONELLA, CAMPYLOBACTER, AND

YERSINIA ORGANISMSSerious efforts to develop vaccines against

Campylobacter organisms have not yet begunsince research is just beginning to more fullyunderstand pathogenesis, antigenic composi-tion, and whether immunity follows infection.Similarly, there exist no vaccine candidates asyet to protect humans against Yersinia enteroco-litica. Vaccine development efforts against S.typhimurium and other nontyphoidal serotypesare mainly intended for veterinary rather thanhuman use (333). For these reasons these areaswill not be discussed further.

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