Chapter 33 : Genetics as a Route toward Mucosal Vaccine Development

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A goal in vaccinology is to obtain fully antigenically defined vaccines that can be delivered via mucosal routes, thus avoiding the requirement for injections with needles. Mucosal presentation of some antigens induces a tolerance effect on the immune system when the same antigen is subsequently administered systemically. This phenomenon may be controlled by mechanisms which have evolved to limit potentially damaging immune responses to dietary antigens. Thus, the immune system may be able to partially discriminate between dietary antigens and antigenic components of pathogens. The immune system is also organized into compartments associated with different tissues of the body. The majority of antigens are poor mucosal immunogens. In practical terms, potentially the quickest route toward the development of mucosal vaccines is the use of attenuated variants of mucosal pathogens. Genetic analysis of the genes required for virulence has uncovered an array of different genes covering a wide range of functions. The construction of candidate human vaccine strains requires the use of precise gene replacement systems in order to ensure that any introduced mutation is fully characterized. A number of different bacteria have been considered for use as antigen delivery systems, including members of the genera and . Genetic instability has been a major problem encountered with the use of live vaccines for the expression of heterologous antigens. Cholera enterotoxin (CT) and CT-B are potent mucosal antigens. Studies are under way to determine the characteristics of the protein that contributes to this property.

Citation: Dougan G. 1994. Genetics as a Route toward Mucosal Vaccine Development, p 491-506. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch33

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Figure 1

Persistence and immunogenicity of SH and SL1344 derivatives following intravenous (IV) inoculation into BALB/c mice. The horizontal arrow on the left axis indicates the dose of vaccine administered. Levels of bacteria in the livers (●) and spleens (▲) were measured as described previously ( ). L1 to L4 and S1 to S4 refer to the number of organs (liver [L] or spleen [S]) cleared of bacteria at a particular sampling point. Protection was monitored at 4 or 8 weeks as indicated. Each plus sign represents a log unit of protection against virulent challenge with parenterally administered virulent (A) Results for SH SH and SH as indicated. (B) Results for SH

Citation: Dougan G. 1994. Genetics as a Route toward Mucosal Vaccine Development, p 491-506. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch33
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1. Benjamin, W. H.,, P. Hall,, and D. E. Briles. 1991. A hemA mutation renders Salmonella typhimurium avirulent in mice, yet capable of eliciting protection against intra-venous infection with 5. typhimurium. Microb. Pathog. 11:289296.
2. Bienenstock, J.,, M. McDermott,, D. Befus,, and L. M. O'Neil. 1978. A common mucosal immunological system involving the bronchus, breast and bowel. Adv. Exp. Med. Biol. 107:5369.
3. Bowe, F.,, P. O'Goara,, D. Masked,, M. Cafferkey,, and G. Dougan. 1989. In vivo properties of Yersinia enterocolitica aroA mutants. Infect, lmmun. 57:32343236.
4. Brown, F.,, G. Dougan,, and D. Snary. 1984. Genetically engineered vaccines: problems and promises. Microbiol. Sci. 1:123126.
5. Carrier, M. J.,, S. N. Chatfield,, G. Dougan,, V. T. A. Nowicka,, D. O'Callaghan,, J. E. Beesley,, S. Milano,, E. Cillais,, and F. Y. Liew. 1992. Expression of human interleukin-1 B in Salmonella typhimurium: a model system for the delivery of recombinant therapeutic proteins in vivo. J. Immunol. 148:11761181.
6. Chamberlain, L.,, R. Strugnell,, G. Dougan,, C. E. Hormaeche,, and R. De Marco De Hormaeche. 1993. Neisseria gonorrhoeae strain MS11 harbouring a mutation in gene aroA is attenuated and immunogenic. Microb. Pathog. 15:5163.
7. Charles, I.,, and G. Dougan. 1990. Gene expression and the development of live enteric vaccines. TIBtech. 8:117121.
8. Charles, I.,, G. Dougan,, D. Pickard,, S. N. Chatfield,, M. Smith,, P. Novotny,, P. M. Morrissey,, and N. F. Falrweather. 1988. Molecular cloning and characterisation of P. 69, a protective outer membrane protein from Bordetella pertussis. Proc. Natl. Acad. Sci. USA 89:35543558.
9. Chatfield, S. Personal communication.
10. Chatfield, S. N.,, I. G. Charles,, A. J. Makoff,, M. D. Oxer,, G. Dougan,, D. Pickard,, D. Slater,, and N. F. Falrweather. 1992. Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine. Bio/Technology 10:888892.
11. Chatfield, S. N.,, N. F. Fairwether,, I. Charles,, D. Pickard,, M. M. Levine,, D. Hone,, M. Posada,, R. A. Strugnell,, and G. Dougan. 1992. Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine. Vaccine 10: 5360.
12. Clements, J. D.,, F. L. Lyon,, K. L. Lowe,, A. L. Farrand,, and S. El-Morshidy. 1986. Oral immunization of mice with attenuated Salmonella enteritidis containing a recombinant plasmid which codes for production of the B subunit of heat-labile enterotoxin of Escherichia coli. Infect, lmmun. 53: 685693.
13. Curtiss, R., III,, S. M. Kelly,, P. A. Gulig,, and K. Nakayama. 1989. Selective delivery of antigens by recombinant bacteria. Curr. Top. Microbiol. Immunol. 146:3554.
14. Curtiss, R. R.,, and S. M. Kelly. 1987. Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclase receptor protein are avirulent and immunogenic. Infect. lmmun. 55:30353043.
15. Dallas, W. S.,, D. M. Gill,, and S. Falkow. 1979. Cistrons encoding Escherichia coli heat-labile toxin. J. Bacteriol. 139:850858.
16. Dorman, C. J.,, S. N. Chatfield,, C. H. Higgins,, C. Hayward,, and G. Dougan. 1989. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: omp mutants are attenuated in vivo. Infect, lmmun. 57:21362140.
17. Douce, G.,, M. Pizza,, I. Cropley,, M. Roberts,, R. Rappuoli,, and G. Dougan. Submitted for publication.
18. Douce, G.,, M. Pizza,, R. Rappuoli,, and G. Dougan. Submitted for publication.
19. Dougan, G.,, S. N. Chatfield,, D. Pickard,, D. O'Callaghan,, and D. Maskell. 1988. Construction and characterisation of Salmonella vaccine strains harbouring mutations in two different aro genes. J. Infect. Dis. 158:13291335.
20. Elson, C. J.,, and W. Ealding. 1984. Generalised systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J. Immunol. 132:27362743.
21. Fairweather, N. F.,, S. N. Chatfield,, A. J. Makoff,, R. A. Strugnell,, J. Bester,, D. J. Maskell,, and G. Dougan. 1990. Oral vaccination against tetanus using a live, rationally attenuated Salmonella carrier. Infect, lmmun. 58:13231329.
22. Fairweather, N. F.,, V. Lyness,, and D. Maskell. 1987. Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli. Infect, lmmun. 55:25412545.
23. Fields, P. I.,, R. V. Swanson,, C. G. Heidaris,, and F. Heffron. 1986. Mutants of Salmonella typhimurium that cannot survive inside macrophages are avirulent. Proc. Natl. Acad. Sci. USA 83: 51895193.
24. Germanier, R.,, and E. Furer. 1975. Isolation and characterisation of a galE mutant Ty21a of Salmonella typhi: a candidate strain for a live oral typhoid vaccine. J. Infect. Dis. 131:553558.
25. Hoiseth, S. K.,, and B. A. D. Stacker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (London) 291:238239.
26. Holmgren, J.,, and C. Czerkinsky. 1992. Cholera as a model for research on mucosal immunity and development of oral vaccines. Curr. Opin. Immunol. 4:387392.
27. Hone, D.,, S. Attridge,, L. Van den Bosch,, and J. Hackett. 1988. A chromosomal integration system for stabilisation of heterologous genes in Salmonella based vaccine strains. Microb. Pathog. 5: 407418.
28. Johnson, K. J.,, I. G. Charles,, I. A. Miller,, D. Pickard,, P. O'Goara,, G. Costa,, T. AH,, and C. E. Hormaeche. 1991. The role of a stress-response protein in Salmonella typhimurium virulence. Mol. Microbiol. 5:401407.
29. Jones, P. W.,, G. Dougan,, C. Hayward,, N. Mackenzie,, P. Collins,, and S. N. Chatfield. 1990. Oral vaccination of calves against experimental salmonellosis using a double aro mutant of Salmonella typhimurium. Vaccine 9:2934.
30. Karnell, A.,, P. D. Cam,, N. Verma,, and A. A. Lindberg. 1993. aroD deletion attenuates Shigella flexneri strain 2457T and makes it a safe and efficatious oral vaccine in monkeys. Vaccine 11: 830836.
31. Levine, M. M.,, D. Herrington,, J. R. Murphy,, J. G. Morriss,, G. Losonsky,, B. Tall,, A. Lindberg,, S. Svenson,, S. Baqar,, M. F. Edwards,, and B. A. D. Stocker. 1987. Safety, infectivity, immunogenic-ity and in vivo stability of two attenuated auxotrophic mutant strains of Salmonella typhi, 541Ty and 543Ty as live oral vaccines in humans. J. Clin. Invest. 79:888902.
32. Lewis, D. J. M.,, L. R. R. Castello-Branco,, P. Novotny,, G. Dougan,, and G. E. Griffin. 1993. Circulating cellular immune response to oral immunisation of humans with cholera toxin B-subunit. Vaccine 11:119121.
33. Lewis, D. J. M.,, P. Novotny,, G. Dougan,, and G. E. Griffin. 1991. The early cellular and humoral immune response to primary and booster oral immunisation with cholera toxin B-subunit in humans. Eur. J. Immunol. 21:20872094.
34. Lipscombe, M.,, I. G. Charles,, M. Roberts,, G. Dougan,, J. Tite,, and N. F. Fairweather. 1991. Intranasal immunisation using B subunit of the Escherichia coli heat labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen. Mol. Microbiol. 5:13851392.
35. Lycke, N.,, and J. Holmgren. 1986. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology 59:301308.
36. Lycke, N.,, T. Tsuji,, and J. Holmgren. 1992. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins are linked to their ADP-ribosyl-transferase activity. Eur. J. Immunol. 22:22772281.
37. Maskell, D. J.,, K. J. Sweeney,, D. O'Callaghan,, C. E. Hormaeche,, F. Y. Liew,, and G. Dougan. 1987. Salmonella typhimurium aroA mutants as carriers of the Escherichia coli heat-labile entero-toxin B subunit to the murine secretory and systemic immune systems. Microb. Pathog. 2:211221.
38. McGhee, J. R.,, J. Mestecky,, M. T. Dertzbaugh,, J. H. Eldridge,, M. Hirasawa,, and H. Kiyono. 1992. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10: 7581.
39. Miller, I. A.,, S. N. Chatfield,, G. Dougan,, L. DeSUva,, H. Joysey,, and C. H. Hormaeche. 1989. Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains. Mol. Gen. Genet. 215:312316.
40. Miller, S. I.,, A. M. Kukral,, and J. J. Mekalanos. 1989. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence in mice. Proc. Natl. Acad. Sci. USA 86:50545058.
41. Minor, P. 1992. The molecular biology of polio vaccines. J. Gen. Virol. 73:30653077.
42. Morrissey, P.,, and G. Dougan. 1986. Cloning and characterisation of the E. coli 987P adhesion fimbriae determinant. Gene 43:7984.
43. Nakayama, K.,, S. M. Kelley,, and R. Curtiss III. 1988. Construction of an asd expression-cloning vector: stable maintenance and high level expression of cloned genes in salmonella based vaccine strains. Bio/Technology 6:693697.
44. O'Callaghan, D.,, D. Maskell,, F. Y. Liew,, C. S. F. Easmon,, and G. Dougan. 1988. Characterization of aromatic-dependent and purine-dependent Salmonella typhimurium: studies on attenuation, persistence, and ability to induce protective immunity in BALB/c mice. Infect. lmmun. 56: 419423.
45. O'Callaghan, D.,, D. Maskell,, J. Tite,, and G. Dougan. 1990. Immune responses in BALB/c mice following immunisation with aromatic compound or purine dependent Salmonella typhimurium strains. Immunology 69:184189.
46. O'Gaora, P.,, M., Roberts,, F. Bowe,, C. Hormaeche,, R. De Marco de Hormaeche,, M. Cafferkey,, J. Tite,, and G. Dougan. 1990. Yersinia enterocolitica aroA mutants as carriers of the B subunit of the Escherichia coli heat-labile enterotoxin to the murine immune system. Microb. Pathog. 9: 105116.
47. O'Hagan, D. T. 1990. Novel non-replicating antigen delivery systems. Curr. Opin. Infect. Dis. 3: 393401.
48. Oxer, M. D.,, C. M. Bentley,, J. G. Doyle,, T. C. Peakman,, I. G. Charles,, and A. J. Makoff. 1991. High level heterologous expression in E. coli using the anaerobically-activated nirB promoter. Nucleic Acids Res. 19:18891897.
49. Peakman, T.,, J. Crouzet,, J. F. Jayaux,, S. Busby,, S. Mohan,, N. Harborne,, J. Wooton,, R. Nicholson,, and J. Cole. 1990. Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysB region of the Escherichia coli chromosome. Eur. J. Biochem. 191:315324.
50. Poirier, T. P.,, M. Kehoe,, and E. H. Beachey. 1988. Protective immunity evoked by oral administration of attenuated aroA Salmonella typhimurium expressing streptococcal M protein. J. Exp. Med. 168:2536.
51. Rappuoli, R.,, A. Podda,, M. Pizza,, A. Covacci,, A. Bartolini,, M. Magistris,, and L. Nencioni. 1992. Progress towards the development of new vaccines against whooping cough. Vaccine 10: 10271032.
52. Roberts, M.,, I. Cropley,, S. N. Chatfield,, and G. Dougan. 1993. Protection of mice against respiratory Bordetella pertussis infection by intranasal immunisation with P.69 and FHA. Vaccine 11: 866872.
53. Sadoff, J. C.,, R. W. Ballou,, L. S. Baron,, W. R. Marjaran,, R. N. Brey,, W. T. Hockmeyer,, J. Young,, J. J. Cryz,, J. Ou,, G. H. Lowell,, and J. D. Chulay. 1988. Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria. Science 240:236240.
54. Shahin, R. D.,, D. F. Amsbaugh,, and M. F. Leef. 1992. Mucosal immunization with filamentous hemagglutinin protects against Bordetella pertussis respiratory tract infection. Infect. lmmun. 60: 14821488.
55. Shipley, P.,, G. Dougan,, and S. Falkow. 1981. Identification and cloning of the genetic determinant that encodes the K88ac adherence antigen. J. Bacteriol. 145:920925.
56. So, M.,, W. S. Dallas,, and S. Falkow. 1978. Characterization of an Escherichia coli plasmid encoding for the synthesis of heat-labile toxin: molecular cloning of the toxin determinant. J. Bacteriol. 139:850858.
57. Strugnell, R.,, G. Dougan,, S. N. Chatfield,, I. C. Charles,, N. F. Falrweather,, J. Tite,, L. Jing Li,, J. Beesely,, and M. Roberts. 1992. Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis. Infect. lmmun. 60:39944002.
58. Strugnell, R.,, D. Maskell,, N. F. Fairweather,, D. Pickard,, A. Cockayne,, C. Penn,, and G. Dougan. 1990. Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains. Gene 88:5763.
59. Svennerholm, A.-M.,, and J. Holmgren,. 1986. Oral combined B subunit-whole cell cholera vaccine, p. 3343. In J. Holmgren,, A. Lindberg,, and R. Mollby (ed.), Development of Vaccines and Drugs against Diarrhoea. 11th Nobel Conference, Stockholm, 1985. Student Lituratur, Lund, Sweden.
60. Tacket, C. O.,, D. M. Hone,, R. Curtiss HI,, S. M. Kelly,, G. Losonsky,, L. Guers,, A. M. Harris,, R. Edelman,, and M. M. Levine. 1992. Comparison of the safety and immunogenicity of δaroC, δaroA and δcya δcrp Salmonella typhi strains in adult volunteers. Infect. lmmun. 60:536544.
61. Tacket, C. O.,, D. M. Hone,, G. A. Losonsky,, L. Guers,, R. Adelman,, and M. M. Levine. 1992. Clinical acceptability and immunogenicity of CVD908 Salmonella typhi vaccine strain. Vaccine 10:443446.
62. Tite, J. P.,, S. M. Russell,, G. Dougan,, D. O'Callaghan,, I. Jones,, G. Browniee,, and F. Y. Liew. 1988. Antiviral immunity induced by recombinant nucleoprotein of influenzae A virus. I. Characteristics and cross-reactivity of T-cell responses. J. Immunol. 141:39803987.
63.World Health Organization. Ten Years of Progress 1984-1993. The Programme for Vaccine Development. World Health Organization, Geneva.
64. Wu, H.-Y.,, and M. W. Russell. 1993. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholerae toxin B subunit. Infect. Immun. 61: 314322.


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Table 1

Effects of level of attenuation on immunogenicity of SLI344 (wild type) defined mutants in BALB/c mice

The mutations were all in the S. typhimurium SL1344 background. Wild type refers to the parent strain. All mutations were introduced by using precise genetic methods. Antibody production was monitored by an enzyme-linked immunosorbent assay against whole, inactivated SL1344 bacteria. Data are based on work published previously ( ).

The cellular response was measured by using a number of parameters but, in particular, refers to the ability of immunized animals to control an intravenous challenge with L. monocytogenes 2 weeks after vaccination with a single intravenous dose of the attenuated SL1344 derivative.

—, abscesses and scattered deaths.

Animals were protected against salmonellosis by using a single oral dose of 109 bacteria.

Citation: Dougan G. 1994. Genetics as a Route toward Mucosal Vaccine Development, p 491-506. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch33
Generic image for table
Table 2

Influence of route of vaccination and formaldehyde treatment on secretory immune response to pertussis toxoid Ptx-crm in BALB/c mice

Levels of antibody were measured by an enzyme-linked immunosorbent assay against Ptx-crm. Ptx-crm + formaldehyde is material treated with formaldehyde to remove the cell-binding activity but retain the holotoxin formation ability. Letters indicate titers in individual mice, i.n., intranasal; s.c, subcutaneous.

Specific anti-Ptx IgA titers in nasal lavage fluid following intranasal or subcutaneous immunization of mice. Each anti-Ptx IgA value has been corrected for total IgA in each individual lavage.

Citation: Dougan G. 1994. Genetics as a Route toward Mucosal Vaccine Development, p 491-506. In Miller V, Kaper J, Portnoy D, Isberg R (ed), Molecular Genetics of Bacterial Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555818340.ch33

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