Development of vaccines against shigellosis: Memorandum from a WHO Meeting*

Memoranda are state- Les Minoraxdums ments concerning the exposent les conclu- Mem1tl l"lnoranda/ conclusions or recom- sions et recomman- mendations of certain dations de certaines WHO scientific meet- reunions scientifiques or/ /syngs they are signed de !'OMS; ils sontMe~~,-,-zorandti,-i-zs~~~~~by the participants in signes par les partici- the meeting. pants d ces reunions. Bulletin of the World Health Organization, 65 (1): 17-25 (1987) © World Health Organization 1987 Development of vaccines against shigellosis: Memorandum from a WHO Meeting* Endemic shigellosis is a worldwide problem, with a high morbidity rate in most developing countries; substantial mortality may also occur, especially with disease caused by Shigella dysenteriae serotype 1. The limited efficacy of current measures to control this infection makes the development of vaccines for the prevention ofshigellosis particularly important. This Memorandum describes the clinical features of and immunity to shigellosis, summarizes the present status of efforts to develop suitable vaccines, and lists the topics that should be given priority in research. Dysentery and diarrhoea caused by Shigella are major public health problems in the developing countries. Shigella is one of the five most frequently identified pathogens in children with acute diarrhoea or dysentery (the others being rotavirus, enteropatho- genic Escherichia coli, enterotoxigenic E. coli and Campylobacter jejuni). Infection with Shigella may lead to a number of serious complications and mortality rates can be high. Severe epidemics may be caused by S. dysenteriae serotype 1 (Shiga's bacillus). The development of vaccines for the prevention of shigellosis is particularly important since the measures currently used to control this infection, par- ticularly those concerned with case management and the control of epidemics, are of limited efficacy. For example, strains of Shigella, especially epidemic strains of S. dysenteriae serotype 1, may be resistant to most available antibiotics, and even appropriate antibiotics may not produce a rapid clinical improve- ment or prevent death when the disease is severe. Oral rehydration therapy, also, is of little value for * This Memorandum is based on the report of a WHO meeting held at the National Institute of Cholera and Enteric Diseases, Calcutta, India, on 19-22 May 1986 (unpublished document WHO/ CDD/IMV/86.1). The meeting was organized in collaboration with that institute and the International Centre for Diarrhoeal Disease Research/Bangladesh, Dhaka, Bangladesh. A list of the participants is given on pages 24-25. Requests for reprints should be addressed to Diarrhoeal Diseases Control Programme, World Health Organiz- ation, 1211 Geneva 27, Switzerland. A French translation will appear in a later issue of the Bulletin. patients with dysentery, as dehydration is not necessarily an important feature of serious illness. Furthermore, treatment of serious episodes is expens- ive owing to the need for specialized care, which may require the use of new and costly antibiotics. Measures besides vaccines that are of value in pre- venting shigellosis involve the interruption of trans- mission of the pathogen by improved personal hygiene and sanitation, and the provision of adequate quantities of water for household use. It is also prob- able that the incidence or severity of illness can be diminished by non-specific interventions such as measles immunization, measures to improve nutri- tion, and possibly vitamin A supplementation. This report summarizes the present status of efforts to develop vaccines for shigellosis and lists the recommended topics for priority in research. It is hoped that these recommendations will serve to stimulate further research which will ultimately lead to the development of efficacious anti-shigellosis vaccines. EPIDEMIOLOGY AND CLINICAL FEATURES Endemic shigellosis Endemic shigellosis, due mostly to S. flexneri (in developing countries) and S. sonnei (in developed countries) is a worldwide problem. In most develop- 4746 -17- MEMORANDUM ing countries, shigellosis has a high morbidity rate, and in some there is also substantial mortality. Children between 1 and 5 years old are especially affected. Transmission is primarily from person to person and is greatest when personal cleanliness and domestic hygiene are very poor; food and water may also be sources of infection. Secondary infection rates within affected families may be as high as 30-50%. Natural animal hosts (excluding rhesus monkeys in captivity) or environmental reservoirs have not been described. Epidemic and pandemic shigellosis A pandemic of shigellosis due to S. dysenteriae serotype I began in 1969 in Central America and now encompasses a large area of Central Africa and countries of, and adjacent to, the Indian subconti- nent. Plasmid analysis has shown that the pandemic is not due to the spread of a single clone of this organ- ism and the reasons for the occurrence of large out- breaks at separate geographical sites are unclear. In each location, however, the strain has been resistant to multiple antibiotics, including those most fre- quently used to treat shigellosis; for example, the strain currently prevalent in Bangladesh is resistant to both trimethoprim-sulfamethoxazole and ampicillin. Control strategies At present, the only strategies that can prevent infection are improved sanitation and increased water supply, which act by reducing faecal-oral transmission. Specific effective measures include handwashing with soap and water after defecating and before handling food, hygienic practices in the preparation and storage of food, and safe disposal of faeces. Breast-feeding also appears to be effective in reducing the severity of Shigella infections in infants and young children up to 3 years of age. Pathogenesis Shigellosis is the most communicable of all bac- terial enteric diseases: as few as 10 live organisms can cause disease in healthy adults. Little is known about the ways in which Shigella (which are acid-sensitive) manage to survive passage through the stomach and cause watery diarrhoea, which is a feature of some cases, or migrate from the lumen to the epithelial surface of the large bowel. It is certain, however, that to cause dysentery, Shigella must penetrate and multiply within the epithelial cells of the colon. This process leads to epithelial cell death, mucosal inflammation, and epithelial ulceration and haemor- rhage, which are the pathological hallmarks of the disease. Clinical features The clinical manifestations of shigellosis generally include frequent passage of stools containing blood and mucus, fever, abdominal pain, and tenesmus. The illness usually starts with watery diarrhoea followed after 24-48 hours by the appearance of blood and mucus in the stools. Dehydration may occur, but only in a small proportion of cases. Various complications, which may occur in 15-30% of hospitalized cases, and especially in children, include prolonged anorexia, nutritional deterior- ation, protein-losing enteropathy, the haemolytic uraemic syndrome, leukaemoid reaction, pneu- monia, conjunctivitis, arthritis, paralytic ileus, toxic megacolon, colonic perforation, rectal prolapse, and persistent diarrhoea. Mortality rates in hospitalized cases due to S. dysenteriae serotype 1 can exceed 10% despite treatment by recommended methods. The disease is most severe, and mortality highest, when it occurs following measles or when there is pre-existing malnutrition. IMMUNITY TO SHIGELLOSIS Protection due to previous infection Several lines of evidence indicate that shigellosis is an immunizing disease. These include epidemio- logical data, observations in volunteers, and studies in animals. Studies in a home for severely retarded children where infections with S. sonnei and S. flex- neri serotype 2a were endemic showed that children with very poor personal hygiene experienced a high attack rate of clinical shigellosis during the first 12-24 months after admission. Thereafter, the attack rates fell sharply and remained low for the duration of the stay in the institution despite continued frequent exposure to infected and ill children. This pattern seemed to indicate that after one or more clinical infections with Shigella the children became at least partially immune to reinfection. Likewise, the occur- rence of shigellosis predominantly in young children in endemic areas suggests an age-related acquisition of immunity. Studies on volunteers demonstrated that young adults who developed shigellosis after an initial experimental challenge were protected against illness when subsequently challenged with the homologous strain (S. flexneri serotype 2a), the protective efficacy of previous infection being 64%0. However, there was no difference between rechallenged and control volunteers in the excretion rate for Shigella; it is possible that more than one exposure to Shigella is needed to evoke high-level immunity. Finally, studies in both monkeys and rabbits have shown solid protection against homo- logous rechallenge 3 to 4 weeks after an initial virulent challenge. 18 DEVELOPMENT OF VACCINES AGAINST SHIGELLOSIS Mechanisms of immunity to shigellosis Immunity to shigellosis appears to depend largely or entirely upon local immune mechanisms. Paren- teral immunization leads to high titres of circulating antibodies but is not usually protective. Most examples of protection by immunization involve the presentation of antigens by the oral or enteric route. The exact mechanisms of protection are not known and may involve local antibodies, mucosal cell- mediated immunity, or both. It is likely that these interfere with multiplication of the small inoculum initially ingested, although it is not known at what site this effect occurs. Whether antibody to Shiga toxin plays a protective role, especially against infec- tion due to S. dysenteriae serotype 1, is not certain. Animal models for study of immune protection Animal models that have been used to study pro- tective immunity include the guinea-pig kerato- conjunctivitis model (Sereny test), orally challenged rhesus monkeys, and a recently developed oral challenge model in rabbits. The two oral challenge models appear to be preferable because they involve enteric infection. The monkey model has been used most extensively in vaccine studies. Its advantages include the similarity of the disease in monkeys to that seen in man, the fact that captive monkeys are highly susceptible to infection with Shigella, and the capacity of the model to demonstrate vaccine- induced protection. Disadvantages include the high cost of rhesus monkeys, technical problems in handling them, and the possible confounding effect of previous unrecognized infection with Shigella. An adult rabbit model has been developed for S. flexneri infection which involves pretreatment of the animals with tetracycline, oral administration of tetracycline-resistant bacteria after neutralization of gastric acid, and a single intraperitoneal dose of opium. With this model, small bowel colonization can be documented following inoculation with 106-109 viable bacteria and death occurs following inoculation with 1010 bacteria. An initial colonization provides solid protection against a subsequent colon- izing or lethal challenge. This model should be useful for further studies of immunity to Shigella flexneri; it has not, however, proved suitable for studying immunity to S. dysenteriae serotype 1, as the strains tested colonize poorly and do not cause illness. Serology of infection with Shigella Shigellosis may evoke both humoral and cell- mediated immune responses and these may be either mucosal or systemic. So far, only systemic humoral responses have been studied in depth. Antibodies measured have been those directed against the lipopolysaccharide (0-antigen) and Shiga toxin. Responses to outer membrane proteins (encoded by the virulence plasmid of relative molecular mass (Mr) 140 x 103) have been studied only to a limited extent, using the Western blot method. Assay ofantibodies to Shigella lipopolysaccharide. Lipopolysaccharide (LPS) antigens can be prepared in pure form and used in enzyme immunoassays (EIA) for sensitive, class-specific antibody determin- ations. However, most Shigella 0-antigens, except that of S. sonnei, cross react with those of certain E. coli, which reduces the specificity of the assay. The 0-antigen of S. dysenteriae serotype 1 is related to E. coli 0 group 1, but has little known relationship with other E. coli or other entero- bacteria. A sensitive and specific enzyme immuno- assay has been developed which can be used for sero- epidemiological purposes and to study the immune response to candidate vaccines. S. flexneri have some 0-antigenic relatedness with other enterobacteria, but there is also extensive cross- reactivity between serotypes I to 5. Consequently, serotype-specific antibody assays based on LPS antigens are, with a few exceptions, not feasible. However, an S. flexneri enzyme immunoassay has been developed which is sensitive and readily detects antibody responses after natural infections. This assay may be useful for assessing the immunogenicity of candidate S. flexneri vaccines. S. sonnei has a unique 0-antigen which is so far known to be shared only by one serotype of Plesio- monas shigelloides. A sensitive and specific enzyme immunoassay exists which is suitable for assessing the immunogenicity of vaccine candidates and for sero- epidemiological studies. Systemic and intestinal antibody responses. Studies of the Ig class of serum antibody to Shigella LPS in man show that the IgA response peaks after 2-3 weeks and remains elevated for about 2 months; the IgM response follows a similar pattern. In contrast, the IgG response peaks after 3-6 weeks and remains elevated for 6-12 months after the diarrhoeal episode. Intestinal antibody responses to Shigella have not been extensively studied in man. Studies in rabbits, however, have shown that the inoculation of live S. flexneri or E. coli/S. flexneri hybrids into jejunal Thiry-Vella loops elicits a pronounced sIgA anti-LPS response. The observation that this response can be rapidly boosted by reinoculation after several months supports other evidence that memory exists in the intestinal sIgA system. Few studies have been done on the Shiga antitoxin response following Shigella infections. Preliminary investigations using an enzyme immunoassay to MEMORANDUM detect Shiga antitoxin revealed antibody responses in the sera of patients infected with S. dysenteriae sero- type 1 but not in the sera of patients infected with S. flexneri or S. sonnei. However, another study using a sensitive and specific antitoxin assay, based on neutralization of toxin-induced damage to HeLa cell monolayers, detected toxin-neutralizing anti- bodies in the sera of patients convalescent from infec- tions due to S. flexneri and S. sonnei. The reason for this discrepancy is not known and further studies are necessary to evaluate the usefulness of these different assays. VIRULENCE DETERMINANTS OF POSSIBLE RELEVANCE TO VACCINE DEVELOPMENT A detailed understanding of the virulence mechan- isms of Shigella and of the specific antigens involved in virulence should facilitate the rational develop- ment of Shigella vaccines. Key events in the patho- genesis of shigellosis include epithelial cell invasion, intracellular multiplication of shigellae, epithelial death followed by bacterial spread within the mucosa, and finally, tissue destruction associated with locally severe inflammation. Current knowledge of these events and of the possible pathogenic role of Shiga toxin is summarized below. Epithelial cell invasion Available data indicate that a variety of plasmid and chromosomal genes mediate the ability of Shigella to invade, multiply in, and eventually destroy intestinal epithelial cells. A large plasmid (of relative molecular mass, Mr, 120-140 x 103), referred to as the virulence plasmid, is essential to all Shigella serotypes for inducing their own phagocytes by epi- thelial cells and for promoting the rapid intracellular growth of bacteria. The molecular mechanisms in- volved in the phagocytosis of Shigella by epithelial cells are not yet understood; however, several outer- membrane proteins encoded by the virulence plasmid have been identified and may play key roles in the process. Recent evidence indicates that the same plasmid encodes a contact haemolytic activity which causes lysis of the membrane of the phagocytic vacuole, thus releasing bacteria into the cytoplasm where they are able to multiply rapidly and escape attack by lysosomal enzymes. Bacterial spread within the mucosa The spread of shigellae within the mucosa requires that the bacteria survive and multiply within the lamina propria, a process which leads to acute in- flammation and tissue destruction. Several chromo- somal segments have been identified that specify bacterial functions thought to be involved in these processes. These regions have been associated with: (i) 0-antigen formation, (ii) high-affinity iron uptake systems, or (iii) the capacity to provoke kerato- conjunctivitis in guinea-pigs. Two chromosomal loci specify complete 0-antigen biosynthesis in S. flexneri. On the other hand, S. sonnei 0-antigen is encoded by the large virulence plasmid. In S. dysenteriae serotype 1, complete expression of 0-antigen requires both a small 6 x 103 Mr plasmid and a chromosomal locus. The role of Shigella 0-antigen in bacterial virulence has not been fully defined, but smooth lipopolysaccharide may be important both for intraluminal survival of Shigella and for protection of the bacteria in tissue from the bactericidal activity of serum. Shiga toxin S. dysenteriae serotype 1 tends to cause more severe dysentery than other serotypes of Shigella and also produces high levels of Shiga toxin; whether or not these two features of S. dysenteriae serotype 1 are causally related is not known. Shiga toxin production has not been consistently demonstrated among other Shigella, and where it has been detected (some strains of S. flexneri serotype 2a and S. sonnei) the level of toxin was 10 000-fold to 100 000-fold less than that measured with S. dysenteriae serotype 1. Shiga toxin produced by S. dysenteriae serotype 1, which inhibits protein synthesis in eukaryotic cells, has been purified to homogeneity and partially characterized. It is composed of one A subunit con- taining the enzymatically active component and 5 or 6 copies of a receptor-binding B subunit, and has three biological activities: (i) cytotoxicity for certain eukaryotic cells, (ii) paralytic activity or lethality for various animal species, and (iii) enterotoxicity in the rabbit small intestine. These biological activities could explain the greater severity of disease caused by S. dysenteriae serotype 1, the watery diarrhoea that may occur during shigellosis, and the lesions in the vascular endothelium of the kidney that characterize the haemolytic uraemic syndrome which sometimes complicates shigellosis. SHIGELLA VACCINES Background In studies carried out in the 1940s and 1950s, paren- teral killed whole-cell Shigella vaccines failed to pro- vide significant protection either in experimental challenges of volunteers or in controlled field trials in endemic settings; a few attempts to immunize monkeys orally with killed Shigella also yielded equi- 20 DEVELOPMENT OF VACCINES AGAINST SHIGELLOSIS vocal results. Since the mid-1960s, research has focused largely on the development of live oral Shigella vaccines and several have been prepared that have proved to be safe in clinical studies; some of these also prevented shigellosis in experimental challenge studies in volunteers or in controlled field trials, or both. However, none was ideal: too many doses were required, occasional genetic revertants arose, and in certain populations side-effects (e.g., vomiting) were encountered. The advent of recombinant DNA technology now makes it possible to analyse with precision the impor- tant determinants of bacterial pathogenicity and immunogenicity at both the molecular and genetic levels. This should facilitate the construction of defined bacterial strains with all the properties con- sidered important in a vaccine for shigellosis, in- cluding safety, genetic stability, immunogenicity with a minimum number of doses, and amenability to large-scale production and lyophilization. An import- ant requirement is that such strains should efficiently colonize or invade the intestinal epithelium so that effective delivery of antigens to the enteric lymphoid tissue is assured. Vaccine strains developed by these methods for other enteric diseases may also prove suitable as carriers for delivering the critical pro- tective antigens of Shigella, thus generating bivalent or possibly multivalent vaccines. Currently, re- searchers are using genetic engineering techniques to develop several types of Shigella vaccines. Previous and current live oral Shigella vaccine can- didates can be divided into four broad classes: -attenuated Shigella mutants; -"mutant hybrids" (Shigella attenuated by the in- corporation of E. coli gene segments); -E. coli that contain introduced Shigella genes; and -other carrier bacteria (such as attenuated Sal- monella typhi) that contain genes encoding synthesis of critical Shigella antigens. The most noteworthy of these candidate vaccines are briefly reviewed below. Attenuated Shigella mutants T32 Istrati. This strain is a mutant developed in Romania by repeated passage of a S. flexneri sero- type 2a strain on agar; it lacks the plasmid that encodes invasiveness and appears to be genetically stable. Large controlled field trials in Romania and China using an immunization schedule involving 4 or 5 doses of up to 3 x 1011 live organisms per dose have shown this vaccine to be safe and effective in prevent- ing clinical shigellosis; it is not known, however, whether the vaccine would be effective after lyophil- ization. In some field trials, significant protection was also observed against serotypes other than S. flexneri serotype 2a, i.e., other serotypes of S. flexneri and S. sonnei. This attenuated mutant vaccine strain is in widespread use in Romania, where experience suggests that its efficacy can be prolonged with biannual booster doses. Its major shortcoming is the requirement for multiple large doses and frequent boosting. Streptomycin-dependent mutants. Non-invasive streptomycin-dependent (SmD) strains of several S. flexneri and S. sonnei serotypes were developed in Yugoslavia and found to be safe and protective when used as oral vaccines in volunteers and in large-scale controlled field trials in that country. After 4 or 5 doses of up to 5 x 1010 live bacteria per dose, these vaccines evoked serotype-specific protection for at least 6 months, but less than one year; a single oral booster dose after one year maintained protection for an additional year. Limited field trials with SmD vaccines were also carried out in the USA in the early 1970s. In one trial involving institutionalized children who were intensely exposed to Shigella, the vaccines were ineffective. Moreover, in some trials, reversion to streptomycin independence occurred, especially with the S. sonnei strain. No further studies of these strains have been performed. New attenuated mutants. New attenuated Shigella mutants are under development, including aromatic (aro-) auxotrophic mutants and galactose epimerase- less (gal E) mutants. These vaccines would differ from the T32 and SmD mutants in being genetically defined and capable of invading epithelial cells. The basis for their attenuation would be their inability to sustain growth within host tissue. Another possible approach to preparing attenuated Shigella mutants would be to delete the genes encoding the production of Shiga toxin, high affinity iron uptake systems, or both, while retaining the plasmid that encodes epi- thelial invasion. Mutant hybrid vaccines The introduction of specific E. coli K-12 chromo- some segments (e.g., the xylose-rhamnose region) into pathogenic Shigella drastically reduces their viru- lence. Transfer of these segments into attenuated colonial mutants of Shigella produced a series of relatively stable, non-invasive hybrid derivatives of S. flexneri serotype 2a and S. dysenteriae serotype 1 which protected monkeys against experimental shigellosis. The S. flexneri serotype 2a hybrid was shown to be genetically stable and safe in both healthy adults and healthy institutionalized children. However, the extent of protection observed in volun- teers did not reach statistical significance and it was noted that the strain did not proliferate well in vivo. The S. dysenteriae serotype 1 hybrid, although MEMORANDUM genetically unstable in one out of 145 volunteers, was notable in that it exhibited good intestinal coloniz- ation after a single oral dose. No field trials were undertaken to test the efficacy of these vaccine strains. E. coli with genes encoding Shigella antigens In the mid-1970s, loci specifying S. flexneri sero- type 2a group- and type-specific somatic antigens were transferred into an E. coli 08 strain and the re- sultant genetically stable, non-invasive hybrid, which expressed the Shigella 0-antigen, was tested as a live oral vaccine. Although protective in monkeys and safe in adults, this candidate vaccine did not protect adult volunteers in experimental challenge studies. More recently, the 140 x 103 Mr plasmid that encodes epithelial cell invasion has been transferred into E. coli K-12, together with chromosomal genes encoding the group- and type-specific 0-antigens of S. flexneri serotype 2a. The resultant hybrid E. coli expresses smooth S. flexneri serotype 2a 0-antigen and invades epithelial cells, but does not cause fluid secretion in ligated segments of rabbit intestine. This vaccine is both safe and protective in monkeys. Clinical studies of vaccine safety and efficacy in healthy adult volunteers are under way. Analogous E. coli K-12 strains expressing other S. flexneri 0- antigens or S. dysenteriae serotype 1 0-antigen have also been prepared and studies in volunteers are planned. Other carrier bacteria that express Shigella antigens Salmonella typhi hybrids that express Shigella anti- gens. Attenuated S. typhi strains developed as live oral vaccines for typhoid fever are potential carriers for the delivery of selected Shigella antigens to host lymphoid tissue; strains of this type might provide protection against both typhoid fever and shigellosis. Such attenuated S. typhi strains, which include Ty2la (a chemically-induced gal E mutant) and 541Ty (a genetically-defined aro , purC mutant), apparently reach intestinal lymphoid tissue where they stimulate cell-mediated immune mechanisms as well as, to a variable extent, circulating and local intestinal antibody responses. One candidate vaccine (5076-IC) consists of Ty2la into which has been incorporated the 140 x 103 Mr plasmid of S. sonnei and which expresses S. sonnei (as well as S. typhi) 0-antigen. In volunteers, this bivalent vaccine is both safe and effective against challenge with S. sonnei. However, variability in the efficacy of different vaccine lots has delayed the initi- ation of field trials. If other effective live oral bacterial vaccines are developed, for example, attenuated Vibrio cholerae, these might also prove useful as carriers of Shigella antigens. General considerations regarding the effective expression of Shigella antigens by carrier organisms. As noted above, it is likely that only one or at most a few Shigella antigens, such as the 0-antigen, specific outer-membrane proteins, and perhaps a Shiga toxoid, may be required to evoke protection. It is, therefore, feasible to construct a series of hybrid plasmid "cassettes" or modules encoding these anti- gens which could be inserted into selected "antigen delivery systems" (e.g., E. coli or heterologous live bacterial vaccines). For example, the S. dysenteriae serotype 1 chromosomal and plasmid genes encoding 0-antigen synthesis have been cloned and combined into a single hybrid plasmid, thus creating a con- venient vehicle for transfer of the determinants of this antigen to a variety of carrier bacteria. Further research will be required, however, to define the requirements for efficient expression of these antigens in an optimally immunogenic form. For example, transcriptional and translational prob- lems may be encountered, which may require that the relevant genetic regulatory mechanisms be analysed and modified. Similarly, carrier organisms may not assemble and present heterologous antigens in an optimally immunogenic form. In particular, the assembly of functional heterologous 0-antigen may require additional genetic manipulations that provide an alternative LPS core upon which the new 0-side chain can be built. It may also be appropriate to eliminate or modify the expression of homologous 0- antigen so that it does not compete with the hetero- logous moiety for attachment to the core. A better understanding of the interactions of proteins with other components of the bacterial membrane, such as LPS and peptidoglycan, is required, as these inter- actions may have a considerable influence on the ways in which such antigens are assembled at the bac- terial surface and interact with the mucosal immune system. SHIGELLA VACCINE FIELD TRIALS Field trials are being contemplated for candidate Shigella vaccines to determine their efficacy under probable conditions of vaccine use. The general con- cepts of clinical trials (e.g., the need for a sufficiently large sample, an adequate control population, and unbiased case detection mechanisms) apply to these trials as to any others. Additional features of particu- lar importance for a Shigella vaccine trial must also be included. Some of these general and specific features are described below. 22 DEVELOPMENT OF VACCINES AGAINST SHIGELLOSIS 23 Field trial population The field trial should involve populations similar to those in which the vaccine would eventually be used. The immunized and unimmunized groups should be comparable with regard to age, sex, nutritional status, and incidence of previous infection with Shigella. The population should be defined with regard to general demographic characteristics and the presence of risk factors particularly pertinent to shigellosis (e.g., malnutrition, measles, and possibly vitamin A deficiency). Randomization Generally, random assignment of vaccine or placebo to individuals is preferred; however, some live Shigella vaccines may be excreted in faeces and transmitted to others in the family. By this means, members of a vaccinee's family may be unintention- ally immunized. For potentially transmissible vaccine strains, randomization should be by household and the same preparation should be given to all eligible members of a household. Outcome events Shigella vaccines are intended to protect individuals from clinical shigellosis; however, the possibility that immunization might diminish the incidence of asymptomatic infection should also be considered. Among outcome events, diarrhoea or dysentery associated with a positive stool culture for Shigella is the most important, although the incidence of asymptomatic infection should also be determined. Cases may be detected among patients attending hospitals or clinics, or by frequent (e.g., twice or thrice weekly) active surveillance in the community. Cases detected by active surveillance are likely to be relatively mild, whereas those detected in hospitals and clinics are likely to be more severe. The possi- bility that immunization may be most effective in reducing the incidence of severe, rather than mild or asymptomatic, infection should be considered. This may require that case detection methods emphasize surveillance of patients presenting to clinics and hospitals. On the other hand, active, community- based surveillance will be especially important when evaluating a S. sonnei vaccine, as disease caused by this serotype tends to be mild. Asymptomatic infec- tions should be detected by periodic culturing of a random sample of healthy vaccinees and controls. Case definitions must be developed that are precise, generally applicable, and can easily be used by field workers in an unbiased way. Specifically, definitions are needed for the following: "a vaccinated individ- ual", "diarrhoea", "dysentery", "shigellosis", "mild, moderate, and severe disease", "persistent diarrhoea" and "asymptomatic infection". Defin- itions may combine both clinical and laboratory find- ings and should distinguish cases in which Shigella is the only isolated pathogen from mixed infections due to Shigella and another enteric pathogen. Sample size calculations should be based on established rates of specific outcome events in the community under study (e.g., dysentery or diarrhoea due to the Shigella serotype present in the vaccine). Laboratory methods Testsforfaecal leukocytes and blood. To differen- tiate dysentery from diarrhoea, standardized pro- cedures should be used to document the presence of faecal leukocytes and blood. A chemical test for blood plus a microscopic examination for faecal leukocytes on each specimen may be required. Microbiological methods. Whenever possible, stool or rectal swab specimens should be plated onto solid media immediately after collection. When this is not practical, specimens should be transported to the laboratory within 24 hours (preferably less) using buffered glycerol saline (chilled, if possible) as the transport medium. Cary-Blair medium is not ideal for the transport of Shigella but should be used when other enteric pathogens will be sought. Primary isolation should include at least two culture media. The choice depends on the serotypes being sought; however, xylose-lysine-desoxycholate (XLD) agar would usually be one choice, the second being either MacConkey or SS agar. A detailed description of methods for isolating and identifying Shigella is available.a RECOMMENDATIONS FOR RESEARCH Animal models A convenient small-animal model is required that would be suitable for detailed, quantitative studies on the pathogenesis of infection with live Shigella (including S. sonnei, S. flexneri and S. dysenteriae serotype 1), the efficacy of candidate vaccines, and mechanisms of immunity to infection. Where possible, isogenic strains of Shigella with specific genetic modifications or deletions should be used for studies of mechanisms of pathogenesis and immunity. Virulence and immune mechanisms (1) The cell surface antigens of Shigella should be more extensively characterized and their role in a Manualfor laboratory investigations ofacute enteric infections (unpublished WHO document CDD/83.3 Rev. 1, 1986). 24 MEMORANDUM pathogenesis and immunity defined. Minor and as yet unidentified antigens should also be investigated, including those expressed under conditions prevalent in the bowel. Monoclonal antibodies to such antigens will probably be required. (2) The role of Shiga toxin in the virulence of S. dysenteriae serotype 1 (and other shigellae) should be determined and the protective role of mucosal or systemic antitoxin, if any, defined. The generation of toxin-negative mutants of S. dysenteriae serotype 1 is critical to this analysis. Such strains should be evaluated by standardized methods, preferably at a single reference laboratory, to confirm the toxin- negative phenotype (or genotype). Sequelae of shigel- losis that may be toxin-mediated, such as the haemo- lytic-uraemic syndrome, should be included in these studies. (3) The process and mechanism of mucosal colon- ization by Shigella should be defined, and the anti- gens involved in this process identified. The uptake and fate of Shigella in epithelial M cells should be investigated and correlated with the immunogenicity of individual strains. Isogenic virulent and avirulent mutants should be used in these studies. Immunity (1) The extent to which protection against shigellosis induced by previous infection is serotype- specific, or extends to heterologous serotypes, should be evaluated in animal models, studies on volunteers, longitudinal population-based studies, and vaccine field trials. Attention should be given to possible cross protection between S. flexneri serotypes, and between S. flexneri and S. dysenteriae or S. sonnei. (2) The roles of humoral and cell-mediated im- mune mechanisms, especially those operative within the bowel mucosa or at its surface, should be defined. The antigens (or antigen combinations) responsible for these responses should be identified. (3) It should be determined whether antibodies in milk or colostrum provide protection against shigel- losis; if this is so, the antigens and antibodies involved, and the extent of this protection, should be defined. (4) The systemic and mucosal immune responses in shigellosis should be defined. These include responses to 0-antigens, outer-membrane proteins, Shiga toxin, and other antigens of possible patho- genic importance. Vaccine development and evaluation (1) For public health use in developing countries, research efforts should focus on the development of vaccines for S. dysenteriae serotype I and the most prevalent serotypes of S. flexneri (lb, 2a, 3a, 4a). Polyvalent vaccines may be required. (2) Vaccine development should focus on live vaccines for oral use, including both avirulent mutants of Shigella and hybrid strains (heterologous vectors expressing Shigella antigens). (3) Optimal carriers for hybrid vaccines should be defined. These may include live S. typhi vaccine strains, avirulent E. coli, and live, avirulent V. cholerae vaccines. The features that determine the efficacy of a carrier should be defined. (4) Optimal methods for the preparation and administration of live oral Shigella vaccines should be defined. This would involve studies to determine means of formulation that maximize bacterial re- covery and growth in an immunogenic form when re- constituted. It should also be determined whether or not the inoculum needs to be protected from gastric acidity. (5) Shigella isolated from field trial participants should be carefully preserved to permit their sub- sequent evaluation for possible antigenic differences between isolates from vaccinees and controls. (6) Vaccine efficacy should be assessed with regard to the duration of protection, use of booster im- munizations, age of vaccinees (especially below the age of 3 years), and disease severity, as well as the nutritional status of the vaccinees. (7) Standard criteria should be developed for the evaluation of Shigella vaccine efficacy. These should include definitions of Shigella diarrhoea and criteria for assessing the severity of illness. Standard labora- tory diagnostic methods should also be recommended. (8) Vaccine development should seek a product that would be effective and safe in infants below 1 year of age and could be incorporated into the delivery system of national expanded programmes on immunization. LIST OF PARTICIPANTS C. Ferreccio, Department of Programme Support, Ministry of Health, Santiago, Chile S. Formal, Department of Bacterial Diseases, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC, USA M. M. Levine, Center for Vaccine Development, University of Maryland School of Medicine, Balti- more, MD, USA A. A. Lindberg, Department of Clinical Bacteriology, Karolinska Institute, Huddinge University Hos- pital, Huddinge, Sweden (Chairman) P. A. Manning, Department of Microbiology and Immunology, University of Adelaide, Adelaide, Australia DEVELOPMENT OF VACCINES AGAINST SHIGELLOSIS 25 T. Meitert, Cantacuzino Institute, Bucharest, Romania A. D. O'Brien, Department of Microbiology, Uni- formed Services University of the Health Sciences, Bethesda, MD, USA S. C. Pal, National Institute of Cholera and Enteric Diseases, Calcutta, India D. A. Sack, International Centre for Diarrhoeal Disease Research/Bangladesh, Dhaka, Bangladesh (Rapporteur) P. Sansonetti, Service des Enterobacteries, Institut Pasteur, Paris, France Y. Takeda, Department of Bacterial Infections, Insti- tute of Medical Science, University of Tokyo, Tokyo, Japan D. Taylor, United States Armed Forces Research In- stitute of Medical Sciences, Bangkok, Thailand K. Timmis, Department of Medical Biochemistry, University Medical Centre, Geneva, Switzerland Observers: M. Bennish, International Centre for Diarrhoeal Disease Research/ Bangladesh, Dhaka, Bangladesh I. Ciznar, International Centre for Diarrhoeal Disease Research/Bangladesh, Dhaka, Bangladesh A. Salam, International Centre for Diarrhoeal Disease Research/ Bangladesh, Dhaka, Bangladesh F. Tron, Clinical Research Department, Pasteur Vaccins, Marnes-la-Coquette, France WHO Secretariat: B. B. Gaitonde, WHO Regional Office for South- East Asia, New Delhi, India N. F. Pierce, Diarrhoeal Diseases Control Pro- gramme, World Health Organization, Geneva, Switzerland (Secretary)