Vaccination against diarrheal disease

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29. Schrader JA, Peck HV, Notis WM, et al.: A role for culture in diagnosis of Helicobacter pylori related gastric disease. Am J Gastroenterol, in press.

30. Sjnstedt S, Kager L, Veress B, et al.: Campylo- bacter pylori in relation to other microorganisms on gastric mucosa. In: Megraud F, Lamouliatte H (eds): Gastroduodenal Pathology and Campylo- bacter pylori. New York, Elsevier Science Pub- lishers, 1989, pp. 35-38.

31. Soltesz V, Zeeberg B, Wadstrom T: 1992. Opti- mal survival of Helicobacter pylori under various transport conditions. Clin Microbiol 30:1453-1456, 1992.

32. Strickland RG, Fenoglio-Preiser CM: 1991 Gas- tritis: Classification and histology then and now. In: Marshall B J, McCallum RW, Gwerrant RL, et al. (eds): Helicobacter pylori in Peptic Ulcera-

tion and Gastritis. Boston, Blackwell Scientific, 1991, pp. 1-18.

33. Talley NJ, Kost LA, Haddad AR, et al.: Compari- son of commercial serological tests for detection of Helicobacter pylori antibodies. JCM 30:3146- 3150, 1992.

34. Taylor DN, Blaser M J: The epidemiology of Helicobacter pylori infection. Epidemiol Rev 13:42-59, 1992.

35. Tummum NKR, Cover TL, Blaser M J: Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: Evidence of link- age to cytotoxin production. Infec Immunol 61:1799-1809, 1993.

36. Vaira D, Holton J, Calms S, et al.: Urease tests for Campylobacter pylori: Care in interpretation. J Clin Pathol 41:812---813, 1988.

37. Vandenplas Y, Blecker U, Devreker T, et al.:

Contribution of the 13C-urea breath test to the detection of Helicobacterpylori gastritis in children. Pediatrics 90:608-611, 1992.

38. Von Wulffen H, Grote ILl, Gterman S, et al.: Im- munoblot analysis of immune response to Cam- pylobacter pylori and its clinical associations. J Clin Pathol 41:653--659, 1988.

39. Warren JR, Marshall BJ: Unidentified curved baccilli on gastric epithelium in chronic active gastritis. Lancet 1:1273-1275, 1983.

40. Westblom TU, Madan E, Gudipati S, et al.: Diag- nosis of Helicobacter pylori infection in adult and pediatric patients by using Pyloriset, a rapid latex agglutination test. J Clin Microbiol 30:96- 98, 1992.

V ac c ina t ion Aga ins t Diarrhea l Disease Thomas L. Hale Walter Reed Army Institute of Research, Washington, D.C.

O n a global basis, infectious diarrheal disease is estimated to cause up to 5 million deaths annually,

exceeding pneumonia and tuberculosis as the most deadly of infectious diseases. In addition to the occasional fatal infection, debilitating diarrheal episodes average up to 10 per year during the first 2 years of life in many areas of Africa, Latin Amer- ica, Asia, and Oceania. Even adults living in endemic locales can be stricken with serious or fatal infections during epidemics of Vibrio cholerae or Shigella dysenteriae type 1,1 With approximately 20 million travelers from industrialized countries visit- ing developing countries every year, the 30%-50% diarrheal attack rate in these immunologically naive visitors has a serious impact on health-care utilization in both the developing countries and the countries of origin. 2 Thus, diarrheal disease impedes both individual and national development while extracting a huge cost in treatment and control programs. The obvious public-

health solutions of reticulated water and safe sewage disposal are utopian consider- ing the daunting costs and the indifference of industrialized countries. Administration of vaccines that alleviate diarrheal symptoms in native children and in foreign visitors remains the most realistic current approach.

Endemic diarrheal disease in nonindus- trialized countries is associated with rota- virus, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, Shigella flexneri, Campylobacter jejuni, and non- typhoidal Salmonella species, typically in descending order of prevalence. However, epidemiological surveys of diarrheal etiol- ogy in household settings are complicated by a substantial incidence of putative enteric pathogens isolated from apparently healthy individuals. For example, only about one of three infections with ETEC and one in four infections with V. cholerae E1 Tor are associated with disease. 2 These observations suggest that nonspecific host-

defense mechanisms, or even actively acquired immunity to commonly encountered intestinal pathogens, does not necessarily prevent infection but rather re- duces the infectious dose to a subclinical level. In this balance, the total number of infectious organisms that survive gastric transit and arrive at the intestinal niche is a key factor in determining the clinical mani- festation of the infection (i.e., subclinical colonization or diarrhea). Thus, the most realistic goal of vaccination is to reduce the infectious load of a diarrheal agent to a subclinical level. Intuition and experience suggest that the difficulty of achieving this goal is a function of the minimum inoculum size that elicits clinical disease in a substantial proportion of individuals. For example, the infective dose of V. cholera or ETEC in volunteers is in 10 6~ colony- forming units (CFU), 3-s and immune protection against these noninvasive organisms is more readily achieved than is protection against enteroinvasive Shigella

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130 CLINICAL IMMUNOLOGY Newsletter Vol. 13, No. 9/10, 1993

species with an infective dose of 101-10: CFU.~

The challenge of vaccinology is to supplement nonspecific intestinal defense mechanisms (e.g., gastric acidity, intestinal mucus and peristalsis, and competition from normal flora), lgA-mediated immune exclusion (i.e., prevention of binding of potential pathogens or bacterial toxins to the intestinal epithelium), IgG- and IgA-mediated bactericidal mechanisms (e.g., opsonization or antibody-dependent T-cell- mediated cytotoxicity), and cell-mediated immunity involving cytotoxic T cells are protective immune responses that can be specifically enhanced. These responses can be elicited by whole bacterial cells, which are effective vehicles for presentation of antigen when phagocytosed by macrophages in vitro. E. coli expressing cloned T-cell epitopes that are presented either by the class 17 or the class II major histocom- patibility complex 8 effectively elicit T-cell interleukin-2 (IL-2) responses when ingested by macrophages. Antigen presentation occurs even when the T-cell epitopes are confined to the bacterial cytoplasm. Thus, whole-cell vaccines elicit both CD4 and CD8 T-cell responses, and these vaccines stimulate protective mucosal immunity.

Efforts to generate immune responses protecting against diarrheal disease by vaccination can be categorized according to etiological agent: (1) V. cholerae vaccines that prevent colonization and/or intoxica- tion; (2) ETEC vaccines presenting colonization factor antigens and/or enterotoxin toxin epitopes that will elicit protective immunity against a plethora of ETEC serotypes; and (3) Shigella vaccines protecting against a limited number of serotypes by preventing intestinal invasion or by preventing the intercellular spread of intracellular organisms. Progress in these areas will be summarized.

Vibrio cholerae

Pathogenesis

Cholera is usually contracted after ingestion of food or water that has been con- tained with fecal material. Transit of a critical inoculum of V. cholerae through

the stomach is enhanced by neutralization of gastric acidity. 5 Virulent organisms penetrate intestinal mucus by chemotactic motility and colonize the small intestine. Colonization is effected by a number of factors including the "toxin coregulated pili" (TCP) of classic V. cholerae 9 and the hemolysin (encoded by the hlyA locus) of the El Tor biotype. '° Although heavy colo-

T h e challenge of vaccinology is to supplement nonspecific

intestinal defense mechanisms (e.g., gastric acidity, intestinal

mucus and peristalsis, and competition from normal flora). lgA-mediated immune exclusion

(i.e., prevention of binding of potential pathogens or bacterial

toxins to the intestinal epithelium), lgG- and

lgA-mediated bactericidal mechanisms (e.g., opsonization or antibody-dependent T-cell-

mediated cytotoxicity ), and cell-mediated immunity involving

cytotoxic T cells are protective immune responses that can be

specifically enhanced.

nization per se apparently elicits mild diarrhea, ~1 the voluminous diarrhea that charac- terizes cholera gravis is caused by the cytotonic cholera toxin (CT), which is encoded by the chromosomal ctx locus. This locus is linked by two or more copies of a repeated sequence, termed RS 1, that com- prises a site-specific recombination sys- temJ 2 Genes encoding an additional cytotoxic toxin designated "zonula oc- cludens toxin" (ZOT) 13 and accessory cholera enterotoxin (ACE) are also located on the core virulence region flanked by the RS 1 repeats? 3

Acquired Immunity

Volunteer studies have shown that an initial infection with virulent V. cholerae stimulates an immune response that protects 90%-100% of subjects against rechallenge with the homologous biotype. ]° Protection is associated with both antibacterial immunity (i.e., secretory IgA recognizing the lipopolysacchafide [LPS] somatic antigen), s'1~'~4,15 and secretory IgA neutralizing CT? ,15J6 In contrast to LPS and CT, TCP is poorly immunogenic, and secretory IgA recognizing this pitus is not required for protection? s V. cholerae is an excellent colonizer of the intestinal epithelium, and these organisms are readily ingested by the M cells that introduce antigens to the intestinal lymphoid follicles? 7 CT is also one of the most effective immunogens known, and this lectinlike protein has immunopotentiating adjuvant activity. The powerful mucosal immune response elicited by key determinants of pathogenesis, :dong with the availability of neutralizing epitopes recognized by mucosal antibody during V. cholerae infection, have encouraged cholera vaccine development efforts for a number of years.S.t0, ~

Cholera Vaccines

Although a killed, whole-cell parenteral V. cholerae vaccine has sometimes been rec- ommended to travelers before travel to endemic areas, this vaccine is both reactogenic and limited in efficacy. Oral immunization with heat or formalin-killed cholera vibrios (WC vaccine) in combination with the nontoxigenic binding (B) subunit of CT elicits significant protection against diarrheal disease in immunologt- cally naive volunteers Is and in field trials? Studies of the effectiveness of these vaccines have illustrated the importance of secretory IgA recognizing the vibrio somatic antigen. Addition of the CT B subunit slightly enhanced the efficacy of the WC vaccine in a Bangladeshi field trial (85% vs. 58% protection). However. the enhanced efficacy was short-lived, and both vaccines had approximately 60% efficacy at the end of 3 years of diarrheal disease survey.

The advantages of killed vaccines in-

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clude safety for children and immunosup- pressed individuals, absence of environ- mental impact, convenience in transport and delivery, and flexibility in the formulation of a cocktail of WC serotypes that can counter antigenic selection during an epidemic. Examples of the latter include the immune selection of the Inaba mutant serotype during the course of epidemics with serotype O10gawa n and the recent emergence of a pathogenic vibrio of non-O1 serotype in India and Bangladesh. The latter exotic vibrio is immunologically distinct from classic Inaba or Ogawa strains, and it has practically replaced the classic O1 serotype in southern Bangladesh within less than 1 year. The total number of cholera cases has more than doubled since the ap- pearance of this strain. TM

Two disadvantages inherent in killed WC-CT B vaccines are the requirement for two oral doses and the production costs for large quantities of a vaccine that requires 10 u organisms per dose. Interestingly, recombinant DNA technology has made production of the CT-B subunit more economical than production of the WC component. WC-CT B vaccines may be useful for travelers or for military troops deployed into theaters with epidemic cholera or with potential biological warfare threats. However, living, attenuated V. cholerae vaccines that could be given in a single dose of approximately 10 s may be more practical for diarrheal disease conlrol programs in developing countries.

Currently, the only live cholera vaccine candidate that has advanced to phase-III field trials is the CVD103-HgR slrain? ° The parent strain of this genetically engineered vaccine is Inaba 569B, a laboratory strain of V. cholerae with undefined mutant phenotypes) 9 This strain was empiri- cally chosen on the basis of decreased colonization and the absence of Shiga-like toxin activity that is detectable in some V. cholerae swains. Expression of the enzy- matically active A subunit of CT was eliminated from 569B by homologous recombination with a ctx gene) ° It should be noted that the RS 1 site-specific recombination site is presumably retained by CVD103, and reintegration of ctx by gene transfer from virulent V. cholae is a theo-

retical possibility. A mercury resistance transposon was inserted into the hlyA gene so that the vaccine sir, fin could be readily differentiated from wild-type Inaba in field settings. Unexpectedly, it was discovered that the CVD103-HgR version was ex- creted by only 28% of volunteers ingesting 10 s organisms versus 87% of volunteers ingesting the CVD103 hlyA ÷ parent, m

Unlike ear!ier versions of ctxA V. cholerae mutants, CVD103-HgR does not elicit mild diarrheal symptoms when ingested by volunteers, and a single dose of 108 organisms gives complete protection against an experimental challenge with virulent Inaba. 2° The impressive stimula- tion of the mucosal immune system by the poorly colonizing, attenuated CVD 103- HgR vaccine is apparent in the number of IgA antibody-secreting cells (ASC) specific for Inaba LPS or CT that are detectable in the peripheral blood of volunteers after a single vaccination. 14 The numbers of ASC recognizing these antigens is com- parable with those detected after clinical cholera.

CVD103-HgR may soon be the first living cholera vaccine licensed. In this event, C VD 103-HgR could not only be an effective live cholera vaccine but also a vector for expression of foreign genes and heterologous antigens. Nonetheless, it should be noted that continuing basic research on the pathogenesis of V. cholerae may even- tually yield vaccine candidates with more def'med attenuating features (e.g., deletion of the entire toxin core region along with the RS 1 recombination sites). This cogni- tive approach may further improve the safety, immunogenicity, and stability of live cholera vaccines.

Enterotox igen ic Escherichia coli

Pathogenesis

ETEC strains are implicated in approximately 20% of acute diarrhea occurring in developing countries and in up to 50% of traveler's diarrhea. 21a2 The most common source of these infections is food, espe- cially uncooked vegetables. Approxi- mately 60% of enterotoxigenic strains express a heat-stabile, enterotoxic peptide (LT) that is structurally and immunologi-

cally similar to CT. About 40% of the L'P strains also express a heat-stable, enterotoxic peptide (ST). This toxin is the only diarrheagenic factor identified in approximately 40% of disease isolates. 4;~m Be- cause ingestion of an LT-/ST ETEC mutant caused mild diarrhea in 15% of volunteers, u it is suspected that colonization factors or unidentified enterotoxius also contribute to ETEC pathogenesis. ETEC express antigenically distinct fimbrial colonization factors that mediate adherence to the intestinal epithelium. ETEC isolates can be characterized serologically into colonization factor antigen (CFA) families. For example, the CFAII family expresses coil surface antigens (CS) 1, 2, and 3 and the CFAIV family expresses CS4, 5, and 6. These families typically represent 20%--40% of diarrheal isolates, respec- tively. The homogeneous CFAI is found on 5 %--10% of isolates, and approximately 30% of isolates express uncharacterized colonization factors. 4m,u

Acquired Immunity

Volunteer studies indicate that a single epi- sode of experimentally induced ETEC diarrhea elicits 78% protection against subsequent challenge with the same etiologic agent. Upon rechallenge, previously infected "veterans" excrete ETEC, but the organisms are detected 24 hr later than in naive controls) Thus, the protective immune response elicited by prior ETEC infection appears to decrease the infectious dose to a subclinical level. When veterans were rechallenged with an LT + ETEC strain expressing heterologous O antigen and CFA, no protective immunity was demonstrated. 3 Community-based studies in Bangladesh and Mexico t also indicate that antitoxic protection is not elicited by natural infectious. However, Bangladesh field trials employing the WC-CT B cholera vaccines indicate that the CT B toxin subunit elicits 86% cross- protection against LT*ETEC diarrhea for a short period of time. ~8 In sub- stance, these data suggest that clinical infection elicits antibacterial immunity specific for ETEC O antigen or colonization-factor antigens (CFA) whereas multiple vaccinations with CT B can elicit

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132 CLINICAL IMMUNOLOGY Newsletter Vol. 13, No. 9/10; 1993

supervenient antitoxic immunity.

ETEC Vaccines

LT*S'I'* ETF_~ strains fall into at least 17 common O-antigen serogroops, and LT*/ST and LT/ST fall in to a much broader array of sexotypes. This heteroge- neity of O serotypes greatly complicates design and manufacture of multivalent ETEC vaccines based on serotype. None- theless, a vaccine eliciting protective immune responses against LT B in addition to CFA, CFAII, and CFAIV should have efficacy against 50%--70% of ETEC isolates. CFA subunits present multiple B-cell epitopes, 25 but vaccination of volunteers with isolated CFA fimbriae is difficult he- cause of proteolysis by gastric pepsin. ~ In- terestingly, CFA carried on formalin- inactivated ETEC is stable in gastric fluid." Thus, two oral doses of formalin- inactivated ~ vaccine elicit CFA-specific, IgA-ASC indicative of a mucosal IgA response in almost 90% of volunteers. 2~ A formalin-inactivated cocktail of ETEC O-antigen serotypes expressing CFAI and CFAII along the purified CT B is being tested as a prototype oral vaccine. 2z26 Two doses of this inactivated whole-cell vaccine elicited significant intestinal IgA responses against the CFA component in approximately 80% of volunteers, and 90% of volunteers also re- sponded to CT B.27 These data have encouraged continued development of in- dustrial-scale preparations of formalin- killed ETEC-CT B vaccine containing CFAIV as well as CFAI and CFAII. An alternative method for inactivating ETEC in- volves treatment with colicin E2 to digest the bacterial DNA without damaging protein or polysaccharide antigens. In volunteer studies, two oral doses of colicin- inactivated E'I'F.C (LT*/ST*, CFAI) gave 75% protection against challenge with either homologous or heterologons L'I'*/ST ~ CFAI serotypes. ~

S/a..~//a Species

Pathogenesis

Shigellosis or bacillary dysentery is a highly contagious infection that is gener- ally spread by the excreta of infected individuals either directly by the fecal--oral

route or by contaminated food, flies, or water. Under experimental conditions, ingestion of as few as 10 organisms can cause disease in 10% of volunteers and 500 organisms can elicit disease in approximately 50% of volunteers. 6 Unlike V. cholerae or E. coli, shigellae are nonmotile and have no identified coloniT_~on factors. Therefore, these organisms do not colonize the small intestine, and there is probably little colonization of the absorptive epithelium of the large intestine during the initial stages of infection. Experimental fmdings

T h e genetic analysis of Shigella pathogenesis, along with

immunoblot analysis of the humoral immune response to

infection, has allowed the identification of antigenic

virulence determinants with vaccine potential.

from ligated rabbit ileal loops 28 or from en- doscopy of monkeys suggest that shigellae initiate infection of the intestinal epithelium after being ingested by the special- ized follicle-associated epithelial cells (M cells). Significantly more intracellular shigeU~ are seen in ileal M cells when rabbit intestinal loops are inoculated with virulent shigellae as opposed to heat-killed or noninvasive strains. 2s

Infection of rabbit Peyer's patch M cells with virulent S. flexneri 2a for 18 hr results in ulceration of the dome surface, extensive spread of shigellae to villus epithelium, acute inflammation throughout the lamina propri& and accumulation of bloody, mucoid fluid within the intestinal lumen. Similar pathologic changes are

seen in the colonic epithelium of humans or primates with naturally acquired or experimentally induced bacillary dysentery. Presumably, these changes are initiated by the infection of M cells overlying diffuse lymphoid follicles of the large intestine and colon with subsequent spread of shigellae into the underlying lymphoid tissue and into the adjacent epithefium. Be- cause the resulting colitis is accompanied by malabsorption of water, the volume of stool is dependent upon ileocec~ flow and the infected individual passes frequent, scanty, dysenteric stools. 29

Studies with cultured polarized colonic epithelial cell monolayers have differentiated a number of stages in the intercellular life cycle of shigellae that underlie the pathogenic manifestations of bacillary dys- eatery. These organisms invade by induced endocytosis at the basolateral surface of epithelial cells. Presumably, the shigellae gain access to the basolateral surface of absorptive epithelial cells in the colon after being transcytosed through follicle-associated M cells. Shortly after invasion of the epithelial cell, the endocytic vacuole lyses and shigellae multiply freely in the host-cell cytoplasm. The organisms then associate with preformed actin ilia. ments in the perijunctional rings that line the internal aspect of polarized cells at the zonula adherens. 3° Exhibiting organ- ellelike movement (Olin), the bacteria move unidirectionally along the actin filaments until they are distributed uniformly around the inside of the host cell at the perijunctional area. During the process of multiplication, an occasional bacterial cell exhibits the tmipolar deposition of F-actin in a tail formation. This bacterium is then propelled by the constricting actin tail into a protrusion of the host-cell plasma mem- brane, and this protrusion is endocytosed at the basolateral surface of the adjacent epithelial cellfl The resulting double-mem- brane endosome in the adjacent cell is then lysed, and the process of Oim and intercellular spread are reinitiated.

The genetic analysis of Shigella pathogenesis, along with immunoblot analysis of the humo~ immune response to infection, has allowed the identification of antigenic virulence determinants with vaccine

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potential. 32 These determinants are encoded by genes carried on large plasmids that are interchangeable and functionally homologous in the Shigella genus. For example, site-specific mutagenesis of plasmid genes has shown that "invasion plasmid antigen" B (IpaB) mediates the uptake of shigellae by epithelial cells in vitro. This 62-kDa protein also causes the disso- lution of endocytic vacuoles in epithelial cells, and it is responsible for lysis of phagocytic vacuoles and ultimately for apotheosis of cultured macrophages. 33 Two other virulence-associated, plasmid-encoded proteins, IpaC and IpaD, are also required for uptake of shigellae by tissue culture cells. The determinant responsible for Olm has not been identified, but the intercellular-spreading (Ics) phenotype is clearly associated with a 120-kDa antigenic protein, designated IcsA or VirG. 34 A cleaved form of this plasmid-encoded protein has ATPase activity, and IcsA se- creted in a unipolar pattern is detectable throughout the length of the polymerized actin tail that propels shigellae into adjacent epithelial cells,

Acquired Immunity

The unique ability of Shigella species to grow freely in the cytosol of colonic epithelial cells is an exquisite adaptation for survival in the human intestine. The intracellular environment is an immunologically privileged site with an absence of competing bacteria and an unlimited sup- ply of nutrients. Thus, immune protection against shigellosis is more difficult to achieve than is protection against noninvasive enteric pathogens such as V. cholerae or ETEC. For example, community-based studies in Bangladeshi children suggested only a ,t0% decrease in the incidence rate of second and third episodes of S. flexneri infection) Acquired immunity appears to be serotype specific because the incidence rate of second and third episodes with heterologous Shigella species was unchanged in this study. In volunteer studies, prior clinical disease elicited 60% protection from a second challenge with S. flexneriY Other volunteer studies with S. sonnei indi- cated 75% protection from disease upon rechallenge.

Epidemiological studies in military populations suggest that pre-existing serum antibody recognizing a Shigella serotype correlates with protection against homologous infections. 36 Although heterologous rechallenge studies have not been conducted in volunteers, rechallenge experiments in monkeys indicate no cross- protective immunity between S. flexneri and S. sonnei f l These observations suggest that the extensive repertoire of shared B-cell epitopes on the IpaB, IpaC, ~,39 and IcsA do not elicit protective levels of antibody during clinical infection. On the other hand, antibody recognizing the S. flexneri 2a somatic antigen has a protective effect in volunteers challenged with the homologous serotype even when ingested in the form of immune bovine colos- trum. 4° Because bovine IgG in the lumen of the bowel would not be expected to penetrate the intra- or interepithelial spaces, it could be speculated that this antibody functions by immune exclusion or by inhibition of the initial intracellular bacterial growth within M cells.

In vitro experiments have shown that serum antibody from patients that are conva- lescing from shigellosis opsonizes homologous shigellae for granulocyte phagocytosis. This antiserum also mediates antibody-dependent, cell-mediated (ADC) bactericidal activity of lymphocytes and monocytes isolated from peripheral bloodfl Secretory IgA purified from ileal loops of rabbits following vaccination with invasive shigellae mediates ADC bactericidal activity of mouse lymphocytes isolated from Peyer's patches, lamina prop- fia, and the intestinal epithelium? 2 Thus, IgG- or IgA-recognizing somatic antigen can confer immune protection by coating shigellae in the intestinal lumen, by opsonization, or by arming lymphocytes in the interstitial spaces of the lamina propria or epithelium.

Although antibody is undoubtedly a key host defense mechanism against shigellosis, intracellular shigellae can spread from cell to cell in tissue culture monolayers without exposure to the interstitial spaces. 43 Thus, it must be assumed that these organisms are also sequestered from the humoral immune response during

much of their in vivo life cycle. Natural killer (NK) cells that lyse Shigella-infected tissue culture cells are present in the peripheral blood lymphocytes of immunologically naive donors, and their cytotoxic activity is potentiated by ̀ /-interferon or IL-2. u Because "/-interferon can also inhibit Shigella invasion of tissue culture cells, 45 cytokines and cytotoxic cellular mechanisms may augment the protective humoral immune response to Shigella infection.

The plethora of commonly occurring ETEC serotypes and the immune selection of exotic V. cholerae serotypes add ele- ments of uncertainty to any vaccine against these diarrheal agents that is based solely on somatic antigen. In contrast, only two or three serotypes of Shigella usually predominate in any given geographic area. Induction of humoral immunity against plasmid-encoded Ipa proteins and/or cell- mediated immunity against unknown protein determinants may be necessary to augment somatic antigen vaccines; however, vaccines delivering LPS antigen to B cells appears to be the key to immunization against shigellosis. Because S. flexneri 2a, S. flexneri 3, S. sonnei, and S. dysenteriae 1 are responsible for the major portion of endemic and epidemic shigeilosis, an effective multivalent vaccine con- sisting of these component serotypes would be a useful public-health tool. The unmet challenge of Shigella vaccine development is to deliver these antigens in a safe and effective formulation.

Parenteral Vaccines

Parenteral infection of killed, whole-cell Shigella vaccines does not protect against shigellosis, but the emergence of two types of nonreactogenic, somatic antigen vaccines has rekindled interest in inject- able preparations. The first of these con- sists of ribosomal subunits separated by polyethylene glycol precipitation from sonically disrupted shigellae. The serotype- specific immunizing component of ribosomal vaccines is the cytoplasmic form of O-polysaccharide (i.e., L-hapten) that is de- void of toxic lipid A and KDO. *s Two sub- cutaneous injections of a ribosomal vaccine prepared from S. sonnei elicit se-

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cretory IgA recognizing the group-D somatic antigen and confers better than 85% protection against oral challenge in the rhesus monkey model. 47 Clinical studies of Shigella ribosomal vaccines may begin in the near future.

Acid-hydrolyzed (detoxified) O-specific polysaccharide (O-SP) protein conjugates are the second type of parenteral vaccine that is currently under investigation. 4s Con- sistent with the premise that a protein car- rier can elicit T-cell help and enhance the immune response against a T-independent polysaccharide antigen, these vaccines elicit strong serum IgG and IgM responses against thesomatic antigen componeni in volunteers after a single injection. Thus far, covalent conjugates of S. dysenteriae 1 O-SP with tetanus toxoid and S. flexneri 2a or Plesiomonas shigeiloides (i.e., S. sonnei) O-SP conjugated with recombinant Pseudomonas aeruginosa exoprotein A (rEPA) have been evaluated for safety and immunogenicity in volunteers? s Serum immune responses against P. shigelloides O- SP have been particularly strong in Israeli volunteers who may have had previous subclinical exposure to S. sonnei. Al- though the efficacy of these parenteral vaccines has not been demonstrated in animal models or in volunteer challenge studies, they are considered attractive candidates for testing in endemic areas because they are conveniently delivered and nonreactogenic.

Live Oral Vaccines

In endemic areas, the peak incidence of clinical shigellosis occurs during the first 4 years of life, and the incidence decreases to approximately 25% of the maximal rate in older children and adultsfl These epidemiologic data, along with the volunteer challenge studies suggesting that a significant degree of protective immunity is elicited by prior clinical disease, indicate that orally administered, living, attenuated vaccines could protect against bacillary dysentery. The recent genetic studies eluci- dating key aspects of Shigella pathogenesis have encouraged the development of genetically engineered vaccine candidates) 2 Examples include (1) an S. flexneri strain with deletions in the icsA plasmid

gene and in the iucA (aerobactin) chromosomal locusS°; (2) aroD auxotropic mutants of S. flexneri that are unable to grow in the cytoplasm of mammalian cells, st and (3) an E. coil K-12 strain carrying the invasion plasmid of S. flexneri in addition to the chromosomal genes encoding the 2a somatic antigen, s2

A prototype icsA-iucA S. flexneri 5 vaccine (SC570) was designed to take advan- tage of the Shigella invasive phenotype,

T h e experience with EcSf2a-2 illustrates the basic dilemma o f Shigella vaccine development: achieving a balance between

reactogenicity and immunogenicity that results in

protection f rom the inoculum of shigellae commonly encountered

in endemic environments.

which enhances the colonization of antigen-sampling M cells in the intestinal epithelium,2S facilitating delivery of bacterial antigens to the gut-associated lymphoid tissue. The SC570 strain is attenuated by virtue of the icsA mutation (inhibiting the intercellular spread of shigeUae to the adjacent colonic epithelium) s3 and the iucA mutation (inhibiting intercellular bacterial multiplication), s4 Three doses of SC570 gives significant protection against challenge with the wild-type S. flexneri parent in rhesus monkeys. 5° An S. ftexneri 2a version of the icsA-iucA double-deletion mu- tam without residual antibiotic markers has been constructed for safety trials in volunteers.

Wild-type shigeUae attenuated by virtue of an aroD mutation are unable to multiply in the cytoplasm of mammalian cells be-

cause these organisms cannot carry out the biosynthesis of aromatic metabolites such as chorismic acid, a precursor of p-ami- nobenzoic acid (PABA). This product is required for the biosynthesis of tetrahydrofolate, a donor of the N-formyl group of N- formylmethionyl-tRNA. Mammals acquire the folic acid precursor of tetrahydrofolate through dietary nutrients, and intermediary metabolism does not include a PABA bio- synthetic pathway. Because bacteria cannot assimilate the folic acid that is available in the mammalian cytoplasm, multiplication of intracellular aro mutants is limited by a gradual depletion of N-formylmethionyl-tRNA with the resultant inhibition of protein synthesis. A S. flexneri 2a aroD deletion mutant (SFLI070) pro- duced significant protection in fascicularis monkeys after four oral doses, st Safety tri- Ms of SFL1070 in volunteers are planned.

The genes encoding the Shigetla invasive phenotype are carried on mobilizable plasmids, and conjugal transfer of the virulence plasmid of S. flexneri into E. coil K- 12 confers the ability to invade the intestinal epithelium, s5 Therefore, an invasive E. coli K-12 hybrid vaccine was constructed by conjugal transfer of the SI flexneri 5 virulence plasmid along with the chromosomal loci encoding the serotype 2a somatic antigen, s2 Due to an anomaly of the E. coli chromosome, the resulting hybrid is deficient in expression of the icsA plasmid gene. In addition, aroD was deleted from the hybrid Chromosome so that the final Construct (EcSf2a-2) is inhib- ited in both intercellular spread and intracellular multiplication. Three doses of EcSf2a-2 protects rhesus monkeys from challenge with S. flexneri 2a. 56 Three- and four-dose regimens of EcSf2a-2 have been evaluated in three volunteer studies, s6 but significant immunity against S. flexneri 2a has not been demonstrated, Nonetheless, these challenge studies and additional safety trials in over 300 volunteers have es- tablished the safety and immunogenicity of the four-dose regimen of EcSf2a-2, and a field trial of this regimen is currently being conducted in Israel.

The experience with EcSf2a-2 illustrates the basic dilemma of Shigella vaccine development: achieving a balance

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Vol. 13, No. 9/10, 1993 CLINICAL IMMUNOLOGY Newsletter 135

between reactogenicity and immunogenicity that results in protection from the inoculum of shigellae commonly encountered in endemic environments. The current 8x 108- CFU dose of EcS f2a-2 is the maximum that can be safely administered to large numbers of volunteers, but this inoculum elicits IgA ASC recognizing the S. flexneri 2a somatic antigen at approximately one- fourth the levels detected after infection with wild-type organisms. This level of immune response is apparently insufficient to demonstrate protection against an experimental Shigella challenge, but the possibility remains that it may be sufficient to prevent naturally acquired infection. It is also possible that the icsA-iucA and/or the aroD strains ofS. flexneri 2a will elicit more effective immune protection than the EcSf2a-2 E. coli-Shigella hybrid strain, but the residual reactogenicity of these mutants may also prove problematic. A combination of parenteral vaccination with O-SP or ribosomal products followed by one or two oral inoculations with an invasive, attenuated live vaccine is an attractive alternative to the regimens now being t e s ted . 57

Acknowledgments

The views presented here do not purport to reflect the position of the Department of the Army of the Department of Defense (para. 4-3, AR 360-5). cIN

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Editors: Alan L. Landay, Ph.D. Henry A, Homtmrger, M.D.

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