Involvement of cell envelope components in the pathogenesis of Vibrio cholerae: targets for cholera vaccine development

Short Review Involvement of cell envelope components in the pathogenesis of Vibrio cholerae: targets for cholera vaccine development Paul A. Manning

Parenteral cholera vaccines have for some years been removed from the WHO recommendations. With a greater understanding of the immunology of gut infections has come the realization that oral vaccines are the most promising approach. There are several possible approaches: killed vaccines, attenuated Vibrio cholerae and true recombinants in which V. cholerae protective antigens are introduced into suitable carrier strains. This latter approach has perhaps greater potential as a carrier strain can be chosen~developed which most effectively stimulates the local immune response, in the gut. This necessitates the identification of the important protective antigens. These antigens are thought to be components of the cell envelope which are involved in adhesion and colonization. Keywords: Cholera; live oral vaccines; Salmonelh~ typhi carrier; outer membrane proteins: haemagglutinins; fimbriae; lipopolysaccharide: flagellum

properties of V. cholerae which may be important in the process will be briefly discussed. There are also extracellular proteins such as deoxyribonuclease 23, neuraminidase, protease 4, haemolysin 5"6, and cholera toxin 7,s which probably contribute to this process of colonization. Molecular cloning of the genes for these determinants could be useful in terms of vaccine development by constructing strains in which the ability to produce the property is eliminated. However, this approach to developing cholera vaccine strains by attenuation will not be discussed in detail here. Within V. cholerae 01, there are two biotypes, Classical and El Tor, which can be differentiated by a number of properties. Since there is apparently also a difference in the ability of the two organisms to cause disease then these differences need always to be taken into account when developing a vaccine strategy.

Fimbriae Introduction Cholera is an acute diarrhoeal disease of man which has occurred in pandemic proportions in recent history. The current pandemic commenced in about 196l in the Celebes and has spread world-wide from this initial focus. Vibrio cholerae of the O1 serotype is the causative agent of cholera. It is a Gram-negative non-invasive pathogen which elaborates a powerful enterotoxin responsible for the major effects of the disease I. Being non-invasive means that it remains in the gut lumen or attached to the gut mucosa during the course of the infection, and this requires it to be able to effectively colonize this surface. Thus, the cell-cell interactions, between V. cholerae on the one hand and the gut epithelium on the other, are of prime importance in the development of the disease. In this article, the surface

Department of Microbiology and Immunology, The University of Adelaide, Adelaide, SA 5001, Australia 0264-410X/87/020083-05 $03.00 ~) 1987 ButtePNOrth& Co. (Publishers) Ltd.

Fimbriae (or pill) have been observed on numerous adherent bacteria and particularly in the case of enterotoxigenic Escherichia coli ( E T E C ) have been clearly demonstrated to be responsible for adhesion to the gut epithelium (see Refs 9 and 10 for reviews). They provide a means of overcoming the close range electrostatic repulsion between cell surfaces by permitting adherence at a distance. The presence of fimbriae on F. cholerae cells has been the subject of much discussion. However, several groups have now clearly demonstrated fimbriae, although usually in low numbers IH3. Ehara and coworkers 14 have purified fimbriae and shown them to have a subunit molecular weight of about 16 000. This material also possesses haemagglutinating activity for human O-erythrocytes suggesting that fimbriae may play a real role in colonization. Certainly electron micrographs suggest adhesion of vibrios via material resembling bundles of fimbriae 13, Thus by analogy with E T E C it could be expected that antibodies to fimbriae would protect ')m. No evidence for this is at present available. Vaccine, Vol. 5, June 1987

83

Cell surface of Vibrio cholerae: P.A. Manning

Haemagglutinins Haemagglutination has classically been used as a tool for assessing the ability of cells to adhere. Unfortunately, the specificity of the erythrocytes which are able to be agglutinated often bears no relationship to the species which is the host of the disease organism. Thus, such results should always be taken with caution. However, haemagglutination is probably still a good indicator of the properties of the molecule being examined. V. cholerae has been shown to elucidate several haemagglutinins which differ in numerous ways: spectrum of erythrocyte activity, sugar sensitivity pattern, Ca 2+ requirement and phase of expression 15. Strains of both biotypes possess at least one cell associated haemagglutinin. The El Tor haemagglutinin is used in biotyping and is inhibited by D-mannose and D-fructose whereas the Classical haemagglutinin is inhibited by Lfucose. Soluble haemagglutinins have also been detected and they were not inhibited by the sugars tested. Franzon and Manning ~ have cloned the gene for a cell-associated haemagglutinin and determined its nucleotide sequence (manuscript in preparation). This hasnow been clearly distinguished from the soluble haemagglutinin described by Finkelstein ~5"~7 and Svennerholm ~. It is an outer membrane protein of 24 000 daltons which mediates haemagglutinin resistant to D-mannose, D-fructose, D-glucose, I>galactose and L-fucose. By Southern hybridizations, the gene has been demonstrated to be present in all biotype-serotype combinations and hybridization positive - - clones expressing the haemagglutinating activity can also be isolated from both biotypes/serotypes. However, van Dongen and de Graaf v~ who subsequently cloned this gene have detected some strains in which it is absent. Construction of specific mutants in this protein should provide a means of assessing its function in pathogenesis. The soluble-haemagglutinin is particularly interesting. The purified protein has proteolytic activity, is a metallo enzyme containing zinc and is activated by calcium 17'2°. Its activity extends to mucin, fibronectin and lactoferrin and may be responsible for the activation of the A subunit of cholera toxin 2~22. A possible indication of its significance is the observation that protease-deficient mutants, although genetically illdefined, were less virulent 4. A slime-agglutinin present on the surface of E1 Tor strains has been described 23. The role of this external layer in attachment has been studied 24. It was found that both E1 Tor and Classical strains adhered to the mucosal surface of adult mouse intestine, whereas only E1 Tor strains adhered as well to the serosal surface. These authors suggested that the slime agglutinin was identical to the brush border agglutinin described previously2~'2~', however, it was not thought to be at all critical for the pathogenesis of cholera. In a recent study, Booth and Finkelstein 27 have surveyed the presence of haemagglutinins in V. cholerae 01 and non-01. Basically they concluded that although the haemagglutinins may be necessary virulence determinants, they are insufficient to render a cell virulent. Little direct information is available on the significance of the haemagglutinins as protective antigens, but 84

Vaccine, Vol. 5, June 1987

it has been demonstrated that pre-treatment of rabbit gut with soluble haemagglutinin prevents adherence of vibrios ~7'2s. However, this could be a property of its proteolytic activity, resulting in the appropriate receptors being stripped from the gut surface.

Lipopolysaccharide V. cholerae 01 exists basically as two serotypes, Inaba and Ogawa, which share common antigens but can also be differentiated in that Ogawa strains possess a determinant not present on Inaba strains 2'~3°. The reverse does not apply since it appears that Ogawa strains possess small amounts of an antigen present in much greater amounts on Inaba strains. The lipopolysaccharide (LPS) has been implicated in adhesion 3~ and antibodies to the LPS are highly protective in animal models 3z (Guinee, P.A.M., Jansen, W.H., Gielen, H., Rijpkema, S.G.T. and Peters, P.W.J., manuscript in preparation). However, purified LPS does not possess any haemagglutinating activity3:~. Recently the genes for the synthesis of O-antigen of both serotypes have been cloned and expressed in E. coli K-1234. Using such strains as immunogens it has been possible to generate antisera which are highly protective in an infant mouse model. Whether this protection is due to a general steric hindrance effect, since much of the cell surface is covered in LPS, or whether it is due to a specific property of the O-antigen is not known. A specific effect could perhaps be inferred from the results of Finkelstein 33 who found that monoclonal antibodies directed against specific O-antigen determinants prevented adherence. In any case it is generally accepted that a vaccine capable of inducing antibodies to the V. cholerae O-antigen(s) is highly desirable.

Outer membrane proteins Outer membrane proteins have been implicated as important antigens by Sears et al.35 and by the use of hybrid strains expressing a non-01 (hence non-protective) LPS O-antigen 3~'. The outer membrane of V. cholerae contains numerous proteins including several major proteins 37-3'. These include 3-4 proteins in the range 44 000 to 47 000 daltons which probably represent the porins, a 35 000 dalton O m~A-like protein and the 26 000 dalton OmpV protein Under iron starvation conditions, thought to mimic the in vivo environment, several other proteins are derepressed and these are most likely associated with iron transport 44. No data is available on the importance of any of these proteins in pathogenesis, however, it seems likely that antibodies blocking iron uptake would effectively suppress in vivo proliferation of the vibrios. The OmpV protein appears to be strongly immunogenic and antibodies to it are readily detected in convalescent sera 46 (unpublished data). Immune sera can also be readily used to select clones encoding other surface proteins 47. However, the roles of these proteins in pathogenesis or whether they represent protective antigens have not been determined. Kabir has suggested the major outer membrane porin protein is immunolo~ically important and possibly a protective antigen 48 "4"~. However, what he refers to as a

Cell surface of Vibrio cholerae: P.A. Manning

single protein can be demonstrated under different electrophoretic conditions to contain at least three different proteins 3~. A variety of studies have implicated non-LPS components of the outer membrane as protective antigens. Antibodies, presumably to proteins, had both bactericidal and protective capacity which correlated with their ability to inhibit in vitro attachment of vibrios 32"5°'51. Recently, it has been demonstrated that these antigens are present on both E1 Tor and Classical strains, although the ability to express these determinants appears to have been lost from some old laboratory El Tor strains 52. The inability to detect significant differences in the outer membrane proteins between El Tor and Classical strains, both protective and non-protective, suggests that these non-LPS antigens are only minor proteins 39. However, the search for these proteins should continue since antibodies to the protective proteins are more protective on a weight basis than antibodies to LPS 5°.

The flagellum V. cholerae possesses a single polar sheathed flagellum responsible for its motility, a property neither necessary nor sufficient for virulence 51. However, motile strains have been reported to be more virulent for infant mice 5t'53. Together, this then suggests that the virulence is associated with a component of the flagellum itself• Antibodies to the flagellum are important 54"55, Yancey et al. and Eubanks et al. have described a nonLPS protective antigen associated with the flagellum, and this has been confirmed by Attridge and Rowley5~. This component is not one of the flagellar core proteins but may be one of a few proteins detected in the flagellar sheath 56. Further evidence for an adhesin on the flagellum comes from electron microscopic observations showing vibrios attaching to the epithelium terminally with flagella directed towards the mucus 57. Hranltsky et al. ~ have characterized a flagellar sheath protein and demonstrated that antibodies to this protein could react with a variety of strains. They suggested that this protein may represent the common vibrio H antigen. This protein is present on both the flagellum and the outer membrane consistent with the conclusions of Das and Chatterjee 59 that the sheath is identical to and continuous with the outer membrane. This may be the flagella associated protective antigen. •

58

•

Construction of vaccine strains Recombinant DNA technology has provided us with tremendous possibilities for generating bacteria with new combinations of antigens or in which specific toxic factors have been deleted. Attenuated V. cholerae lacking the genes for cholera toxin have been constructed s'6°. However, they have the disadvantage of still producing diarrhoea in healthy volunteers 6~. One could imagine that deletion of all the factors responsible for these side effects could make the strains too feeble to effectively colonize and immunize. It is also possible to produce a strain which is more effectively able to stimulate an antibody response than the original organism. For example, it is thought that IgA antibody in the gut is required for protection 6263, necessitating the induction of a local immune response

in the intestine. This response is achieved by cells entering the Peyer's patch lymphoid tissue, which is accomplished probably via the dome cells sampling the gut contents during the course of a V. cholerae infection. If the important antigens were present on the surface of an organism which actively invaded the Peyer's patches (e.g. Salmonella typhi in man) then this would be expected to improve the stimulation. Attenuated salmonellae unable to proliferate in vivo but still capable of Peyer's patch invasion would appear to be well suited to this task °4-°°. Thus, one strategy for constructing vaccine strains is to introduce the genes for the relevant antigens from V. cholerae into attenuated S. typhi which could then be administered orally. This approach has the advantage over attenuated V. cholerae in that all toxic agents of the V. cholerae are eliminated and only the protective antigens are included. It does, however, have the disadvantage that only a limited number of antigens can easily be engineered into such strains, but this does not appear to be a problem with the limited experience to date. For example, the live oral typhoid vaccine strain. S. typhi Ty21a 67, has been used as a carrier for the V. cholerae LPS O-antigen. Oral immunization of volunteers with such a strain has led to good levels of specific IgA in the gut (see report this issue). The combination of such a strain with a similar one expressing the appropriate protein determinant(s) could be expected to provide excellent protection against cholera (as well as typhoid as a consequence of the carrier organism).

Acknowledgements The author is grateful for the continued support of the National Health and Medical Research Council of Australia.

References 1 Carpenter, C.C.J., Greenough, W.B. and Gordon, R.S. Pathogenesis and pathophysiology of cholera. In: Cholera. (Eds Barua, ,D. and Burrows, W)W.B. Saunders, Philadelphia 1974, pp. 129-140 2 Newland, J.W., Green, B.A., Foulds, J. and Holmes, R.K. Cloning of extracellular DNase and construction of a DNase negative strain of Vibrio cholerae. Infect. Immun. 1985, 47, 691 3 Focareta, T. and Manning, P.A. Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization 0t the deoxyribonuclease and its periplasmie localization in Escherichia coil K-12. Gene 1987, in press 4 Schneider, D.R. and Parker, C.D. Isolation and characterization of protease-deficient mutants of Vibrio cholerae. J. Infect. Dis. 1978, 138, 143 5 Manning, P.A., Brown, M.H. and Heuzenroeder, M.W. Cloning of the structural gene (hly) for the haemolysin of Vibrio cholerae El Tor strain 017. Gene 1984, 31,235 6 Goldberg, S.L. and Murphy, J.R. Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor• J. Bacteriol. 1984, 160, 239 7 Kaper, J.B. and Levine, M.M. Cloned cholera enterotoxin genes in study and prevention of cholera. 1981, Lancet ii, 1162 8 Mekalanos, J.J., Swartz, D.J., Pearson, G.D.N, Harford, N., Groyne, F. and de Wilde, M., Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature 1983, 806, 551 9 Gaastra, W. and De Graaf, F.K. Host specific fimbrial adhesins of non-invasive enterotoxigenic Escherichia coil strains. Microbiol. Rev. 1982, 46, 129 10 Tramont, E.C. and Boslego, J.W. Pilus Vaccines. Vaccine 1985, 3, 3 11 Tweedy, J.M., Park, R.R.W.A. and Hodgkins, W. Evidence for the presence of fimbriae (pill) on vibrio species. J. Gen. MicrobioL 1986, 51,235 12 Faris, A., Lindahl, M. and Wadstrom, T. High surface hydrophobicity of haemagglutinating Vibrio cholerae and other vibrios. Aust. MicrobioL 1982, 7, 357

Vaccine, Vol. 5, June 1987 85

Cell s u r f a c e o f Vibrio cholerae: P.A. M a n n i n g

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29 30

31

32

33

34

35

86

Ehara, M., Ishibashi, M., Watanabe, S., Iwanaga, M., Shimotori, S. and Naito, T. Fimbriae of Vibrio cholerae 01 : observation of fimbriae on the organisms adherent to the intestinal epithelium and development of a new medium to enhance fimbriae production. Trop. Med. 1986, 28, 21 Ehara, M., Ishibashi, M., Ichinose, Y., Iwanaga, M., Shimotori, S. and Naito, T. Purification and partial characterization of fimbriae of Vibrio cholerae 01. Vaccine, in press Hanne, L.F. and Finkelstein, R.A. Characterization and distribution of the hemagglutinins produced by Vibrio cholerae. Infect. Immun. 1982, 36, 209 Franzon, V.L. and Manning, P.A. Molecular cloning and expression in r--scnerichia coil K-12 of the gene for a hemagglutinin from Vibrio cnolerae. Infect. Immun. 1986, 52, 279 Finkelstein, R.A. and Hanne, L.F. Purification and characterization of the soluble hemagglutinin (cholera lectin) produced by Vibrio cholerae. Infect. Immun. 1982, 36, 1199 Svennerholm, A.M., Str6mberg, G.J. and Holmgren, J. Purification of Vibrio cholerae soluble hemagglutinin and development of enzyme linked immunosorbent assays for antigen and antibody quantitations. Infect. Immun. t983, 41,237 Van Dongen, W.M.A.M., De Graaf, F.K. Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin. J. Gen. Microbiol. 1986, 132, 2225 Booth, B.A., Boesman-Finkelstein, M., Finkelstein, RA. Vibrio cholerae soluble hemagglutinin/protease is a metalloenzyme. Infect. Immun. 1983, 42, 639 Booth, B.A., Boesman-Finkelstein, M, Finkelstein, R.A. Vibrio cholerae hemagglutinin/protease nicks cholera enterotoxin. Infect. Immun. 1984, 45,559 Finkelstein, R.A., Boesman-Finkelstein, M. and Holt, P. Vibrio cholerae hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: FM. Burnet revisted. Proc. Natl Acad. Sci. USA 1983, 80, 1092 Lankford, C.E. and Legsomburana, U. Virulence factors of Choleragenic Vibrios. In: Proceedings of the Cholera Research Symposium, Honolulu, Hawaii. (Eds Bushnell, O.H. and Brookslyser, C.S.) US Government Printing Office, Washington, DC 1965, pp. 109-120 Attridge, S.R. and Rowley, D. The specificity of Vibrio cholerae adherence and the significance of the slime agglutinin as a second mediator of in vitro attachment. J. Infect. Dis. 1983, 147, 873 Jones, G.E, Abrams, G.D. and Freter, R. Adhesive properties of Vibrio cholerae: adhesion to isolated rabbit brush border membranes and hemagglutinating activity. Infect. Immun. 1976, 14, 232 Jones, G.W. and Freter, R. Adhesive properties of Vibrio cholerae: nature of interaction with isolated rabbit brush border membranes and human erythrocytes. Infect. Immun. 1976, 14, 240 Booth, B.A. and Finkelstein, R.A. Presence of hemagglutinin/ protease and other potential virulence factors in 01 and non-01 Vibrio cholerae. J. Infect. Dis. 1986, 154, 183 Finkeistein, R.A., Arita, M., Clements, J.D. and Nelson, E.T. isolation and purification of an adhesive factor ('cholera-lectin') from Vibrio cholerae. In: Proceedings of the 13th Joint Conference on Cholera. (Eds Craig, J.P. and Fukumi, H.) U.S. Japan Cooperative Medical Science Program. NIH Publication no 78-1590. US Government Printing Office, Washington DC, 1978, pp. 137-151 Sakazaki, R. and Tamura, K. Somatic antigen variation in Vibrie cholerae. Jap. J. Med. Sci. Biol. 1971, 24, 93 Redmond, J.W., Korsch, MJ. and Jackson, G.D.F. Immunochemical studies on the O-antigens of Vibrio cholerae. Partial characterization of an acid-labile antigenic determinant. Aust. J. Exp. Biol. Med. Sci. 1973, 51,229 Chitnis, D.S., Sharma, K.D. and Kamat, R.S. Role of somatic antigen of Vibrio cholerae in adhesion to intestinal mucosa. J. Med. MicrobioL 1982, 5, 53 Neoh, S.H and Rowley, D. Protection of infant mice against cholera by antibodies to three antigens of Vibrio cholerae. J. Infect. Dis. 1972, 126, 41 Booth, B.A., Sciortino, C.V., Finkelstein, C.A. Adhesins of Vibrio cholerae. In: Microbial. Lectins and Agglutinins, (Ed. D. Mirelman) John Wiley and Sons, New York, 1985, pp. 169-182 Manmng, P.A., Heuzenroeder, MW., Yeadon, J., Leavesley, D.[., Reeves, P.R. and Rowiey, D. Molecular cloning and expression in Escherichia coil K-12 of the O-antigens of the Inaba and Ogawa serotypes of the Vibrio cholerae 01 lipopolysaccharides and their potential for vaccine development. Infect. Immun. 1986, 53, 272 Sears, S.D., Richardson, K., Young, C., Parker, C.D. and Levine, M.M. Evaluation of the human immune response to outer membrane proteins of Vibrio cholerae. Infect. Immun. 1984, 44, 439

Vaccine, Vol. 5, June 1987

36

37 38

39 40

41

42

43

44

46 47

48 49

50

51

52

53 54

55

56

57

58

59

60

61

62

63

Bhaskaran, K. Isolation of hybrid vibrio strains for immunological research in cholera. In: Proceedings of a Symposium on Bacterial Vaccines. Zagreb, Yugoslavia, Yugoslav Academy of Arts and Science, 1972, pp. 247-261 Kabir, S. Composition, and immunochemical properties of outer membrane proteins of Vibrio cholerae. J. Bacteriol. 1980, 144, 382 Kelley, J.T. and Parker, C.D. Identification and preliminary characterization in Vibrio cholerae outer membrane proteins. J. Bacteriol. 1981, 145, 1018 Manning, P.A., Imbesi, F. and Haynes, D.R. Cell envelope proteins in Vibrio cholerae. FEMS Microbiol. Lett. 1982, 14, 159 Aim, R.A., Braun, G., Morona, R. and Manning, P.A. Detection of an OmpA-like protein in Vibrio cholerae. FEMS Microbiol. Lett. 1986, 37, 99 Stevenson, G., Leavesley, D.I., Lagnado, C.A., Heuzenroeder, M.W. and Manning, P.A. Purification of the 25kDal Vibrio cholerae major outer membrane protein and the molecular cloning of its gene: ompV. Eur. J. Biochem. 1985, 148, 385 Pohlner, J., Meyer, T.F., Jalajakumari, M.B. and Manning, P.A. Nucleotide sequence of ompV, structural gene for a major Vibrio cholerae outer membrane protein. Molec. Gen. Genet. 1986, 205, 494 Pohlner, J., Meyer, T.F. and Manning, P.A. Serological properties and processing in Escherichia coil K-12 of OmpV fusion proteins of Vibrio cholerae. Molec. Gen. Genet. 1986, 205,501 Sciortino, C.V. and Finkelstein, R.A. Vibrio cholerae expresses ironregulated outer membrane proteins in vivo. Infect. Immun. 1983, 42, 990 Manning, P.A. and Haynes, D.R. A common immunogenic Vibrio outer membrane protein. FEMS Microbiol. Lett. 1984, 24, 297 Manning, P.A., Bartowsky, E.J., Leavesley, D.I., Hackett, J.A. and Heuzenroeder, M.W. Molecular cloning using immune sera of a 22 kDal minor outer membrane protein of Vibrio cholerae. Gene 1985, 34, 950 Kabir, S. Immunochemical properties of the major outer membrane protein of Vibrio cholerae. Infect. Immun. 1983, 39, 452 Kabir, S. Composition and immunochemical properties of the cell surface proteins of Vibrio cholerae. J. Gen. Microbiol. 1986, 132. 2235 Neoh, S.H. and Rowley, D. The antigens of Vibrio cholerae involved in the vibriocidal action of antibody and complement. J. Infect. Dis. 1970, 121, 505 Attridge, S.R. and Rowley, D. Prophylactic significance of the nonlipopolysaccharide antigens of Vibrio cholerae. J. Infect. Dis. 1983. 148, 931 Sharma, DP., Attridge, S., Hackett, J. and Rowley, D. Non-lipopolysaccharide protective antigens shared by Classical and El Tor biotypes of Vibrio cholerae. J. Infect. Dis. 1987, in press Guentzel, M.N. and Berry, L.J. Motility as a virulence factor for Vibrio cholerae. Infect. Immun. 1975, 11,890 Yancey, R.I., Willis, D.L. and Berry, J.J. Flagella-induced immunity against experimental cholera in adult rabbits. Infect. Immun. 1979, 25, 220 Eubanks, E.R., Guentzel, MN. and Berry, L.J. Evaluation of surface components of Vibrio cholerae as protective immunogens. Infect. Immun. 1977, 15, 533 Richardson, K. and Parker, C.D. identification and occurrence of Vibrio cholerae flagellar core proteins in isolated outer membrane. Infect. Immun. 1985, 47, 674 Nelson, E.T., Clements, J.D. and Finkelstein, R.A. Vibrio cholerae adherence and colonization in experimental cholera: Electron microscopic studies. Infect. Immun. 1976, 14, 527 Hranitsky, K.W., Mulholland, A., Larson, A.D., Eubanks, E.R. and Hart, L.T. Characterization of a flagellar sheath protein of Vibrio cholerae. Infect. Immun. 1980, 27, 597 Das, J. and Chatterjee, SN. Electron microscopic studies on some ultrastructural aspects of Vibrio cholerae. Indian J. Med. Res. 1966, 54, 330 Kaper, J.B., Lockman, H., Baldini, M.M. and Levine, M.M. Recombinant nontoxigenic Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature 1984, 308, 655 Levine, M.M., Kaper, J.B., Black, R.E., Clements, M.L. and Morris, J.G. In: Proceedings 19th Joint Conference on Cholera, US-Japan Cooperative Medical Science Program, 1983, p. 56 Waldman, RH., Bencic, Z., Sinha, R., Deb, B.C., Sakazaki, R.. Tamura, K. et aL Cholera immunology: II Serum and intestinal secretion antibody responses after naturally occurring cholera. J. Infect. Dis. 1972, 126, 401 Finkelstein, R.A. Immunology of Cholera. Curr. Top. Microbiol. Immunol. 1975, 69, 167

Cell surface of Vibrio cholerae: P.A. Manning 64

Hohmann, A., Schmidt, G. and Rowley, D. Intestinal and serum antibody responses in mice after oral immunization with Salmonella, E. coil and Salmonella/E. coil hybrid strains. Infect. Immun. 1977, 25, 27 65 Srisart, P., Reynolds, B.L. and Rowley, D. The correlation between serum IgA antibody levels and resistance to infection with Salmonella typhimurium after oral immunization with various Salmonellae. Aust. J. Exp. BioL Med. Sci. 1985, 63, 177

66 Stevenson, G. and Manning, P.A. Galactose-epimeraseless (GalE) of Salmonella typhimurium is a good potential live oral vaccine carrier for fimbrial antigens. FEMS Microbiol. Lett. 1985, 28, 317 67 Germanier, R. and FL~rer, E. Isolation and characterization of GalE mutant Ty21a of Salmonella typhi: a candidate strain for live oral typhoid vaccine. J. Infect. Dis. 1975, 131,553

Vaccine, Vol. 5, June 1987

87