Genetically derived toxoids for use as vaccines and adjuvants

Vaccine

Volumn 17, Issue SUPPL. 2, 1999, Pages

  Del Giudice, Giuseppe ^a   Rappuoli, Rino ^a  

^a CHIRON VACCINES   (Italy)

Author keywords

Adjuvants; Bacterial toxins; Mucosal immunity; Vaccines

Indexed keywords

BACTERIAL TOXIN; ESCHERICHIA COLI ENTEROTOXIN; IMMUNOLOGICAL ADJUVANT; PERTUSSIS VACCINE; TOXOID;

CONFERENCE PAPER; DRUG SAFETY; GENE TECHNOLOGY; IMMUNE RESPONSE; IMMUNOGENICITY; MUCOSAL IMMUNITY; PRIORITY JOURNAL; VACCINATION;

ADJUVANTS, IMMUNOLOGIC; CROSS REACTIONS; HUMANS; MUCOUS MEMBRANE; MUTATION; PERTUSSIS TOXIN; STRUCTURE-ACTIVITY RELATIONSHIP; TOXOIDS; VIRULENCE FACTORS, BORDETELLA;

EID: 0033215467     PISSN: 0264410X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0264-410X(99)00234-0     Document Type: Conference Paper

References (93)

1. Cossart P., Boquet P., Normark S., Rappuoli R. Cellular microbiology emerging. Science. 271:1996;315-316.
2. Hepler J.R., Gilman A.G. G proteins. Trends Biochem. Sci. 17:1992;383-387.
3. Gill D.M. Bacterial toxins: a table of lethal amounts. Microbiol. Rev. 46:1985;86-94.
4. Bennett M.J., Choe S., Eisenberg D. Refined structure of dimeric diphtheria toxin at 2.0 Angstrom resolution. Protein Sci. 3:1994;1444-1463.
5. Iwamoto R., Higashiyama S., Mitamura T., Taniguchi N., Klagsbrun M., Mekada E. Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9 which upregulates functional receptors and diphtheria toxin sensitivity. Embo. J. 13:1994;2322-2330.
6. Neville D.M. Jr, Hudson T.H. Transmembrane transport of diphtheria toxin, related toxins, and colicins. Ann. Rev. Biochem. 55:1986;195-224.
7. Van Ness B.G., Howard J.B., Bodley J.W. ADP-ribosylation of elongation factor 2 by diphtheria toxin. J. Biol. Chem. 255:1980;10710-10716.
8. Uchida T., Gill D.M., Pappenheimer A.M. Jr. Mutation in the structural gene for diphtheria toxin carried by temperate phage β Nature (New Biol.). 233:1971;8-11.
9. Uchida T., Pappenheimer A.M. Jr, Greany R. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 248:1973;3838-3844.
10. Giannini G., Rappuoli R., Ratti G. The amino acid sequence of two nontoxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 12:1984;4063-4069.
11. Pappenheimer A.M. Jr, Uchida T., Harper A.A. An immunological study of the diphtheria toxin molecule. Immunochemistry. 9:1972;891-906.
12. Porro M., Saletti M., Nencioni L., Tagliaferri L., Marsili I. Immunogenic correlation between cross-reacting material (CRM) 197 produced by a mutant of Corynebacterium diphtheriae and diphtheria toxoid. J. Infect. Dis. 142:1980;716-724.
13. Rappuoli R. Toxin inactivation and antigen stabilization: two different uses of formaldehyde. Vaccine. 12:1994;579-581.
14. Podda A., Vescia N., Donati D., Marsili I., Volpini G., Nencioni L., Mastroeni I., Rappuoli R., Fara G.M. Phase I clinical trial of a new vaccine against tetanus and diphtheria for adults. Ann. Ig. 3:1991;79-84.
15. Vadheim C., Greenberg D., Ericksen E., Hemenway L., Christenson P., Ward B., Mascola L., Ward J.I. Protection provided by Haemophilus influenzae type b conjugate vaccines in Los Angeles: a case-control study. Pediatr. Infect. Dis. J. 13:1994;274-280.
16. Costantino P., Viti S., Podda A., Velmonte M.A., Nencioni L., Rappuoli R. Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine. 10:1992;691-698.
17. Anderson E.L., Bowers T., Mink C.M., Kennedy D.J., Belshe R.B., Harakeh H., Pais L., Holder P., Carlone G.M. Safety and immunogenicity of meningococcal A and C polysaccharide conjugate vaccine in adults. Infect. Immun. 62:1994;3391-3395.
18. Twumasi P.A. Jr, Kumah S., Leach A., O'Dempsey T.J., Ceesay S.J., Todd J., Broome C.V., Carlone G.M., Pais L.B., Holder P.K., Plikaytis B.D., Greenwood B.M. A trial of a group A plus group C meningococcal polysaccharide-protein conjugate vaccine in African infants. J. Infect. Dis. 171:1995;632-638.
19. Lieberman J.M., Chiu S.S., Wong V.K., Partridge S., Chang S.J., Chiu C.Y., Gheesling L.L., Carlone G.M., Ward J.I. Safety and immunogenicity of a serogroups A/C Neisseria meningitidis oligosaccharide-protein conjugate vaccine in young children: a randomized controlled trial. JAMA. 275:1996;1499-1503.
20. Ahman H, Käyhty H, Tamminen P, Vuorela A, Malinoski F, Eskola J. Pentavalent pneumococcal oligosaccharide conjugate vaccine PncCRM is well tolerated and able to induce an antibody response in infants. Pediatr Infect Dis J 1996;15:134-9.
21. Pittman M. Pertussis toxin: the cause of harmful effects and prolonged immunity of whooping cough: a hypothesis. Rev. Infect. Dis. 1:1979;401-412.
22. Aricò B., Rappuoli R. Bordetella parapertussis and bronchiseptica contain transcriptionally silent pertussis toxin genes. J. Bacteriol. 169:1987;2847-2853.
23. Muñoz J.J. Biological activity of pertussigen (pertussis toxin). Sekura R.D., Moss J., Vaughan M. Pertussis toxin. 1985;1-18 Academic Press, Orlando.
24. Stein P.E., Boodhoo A., Armstrong G.D., Cockle S.A., Klein M.H., Read R.J. The crystal structure of pertussis toxin. Structure. 2:1994;45-57.
25. Domenighini M., Pizza M., Rappuoli R. Bacterial ADP-ribosyltransferases. Moss J., Iglewski B., Vaughan M., Tu A.T. Bacterial toxins and virulence factors in disease. 1995;59-80 Marcel Dekker, New York.
26. Lobet Y., Feron C., Desquesne G., Simoen E., Hauser P., Locht C. Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin. J. Exp. Med. 177:1993;79-87.
27. Sekura R.D., Zhang Y. Pertussis toxin: structural elements involved in the interaction with cells. Sekura R.D., Moss J., Vaughan M. Pertussis toxin. 1985;45-64 Academic Press, Orlando.
28. Nicosia A., Perugini M., Franzini C., Casagli M., Casagli M.C., Borri M.G., Antoni G., Almoni M., Neri P., Ratti G., Rappuoli R. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc. Natl. Acad. Sci USA. 83:1986;4631-4635.
29. Pizza M., Covacci A., Bugnoli M., Manetti R., Rappuoli R. The S1 subunit is important for pertussis toxin secretion. J. Biol. Chem. 265:1990;17759-17763.
30. Sato Y., Kimura M., Fukumi H. Development of a pertussis component vaccine in Japan. Lancet. 1:1984;122-126.
31. Edwards K.E., Meade B.D., Decker M.D., Reed G.F., Rennels M.B., Steinhoff M.C., Anderson E.L., Englund J.A., Pichichero M.E., Deloria M.A., Deforest A. Comparison of 13 acellular pertussis vaccines: overview and serologic response. Pediatrics. 96:1995;548-557.
32. National Bacteriological Laboratory, Sweden. A clinical trial of acellular pertussis vaccines in Sweden. Technical Report 1988, Stockholm.
33. Kimura A., Mountzouros K.T., Schad P.A., Cieplak W., Cowell J.L. Pertussis toxin analog with reduced enzymatic and biological activities is a protective immunogen. Infect. Immun. 58:1990;3337-3347.
34. Sato Y., Sato H., Chazono M., Ginnaga A., Tamura C. Characterization of mutant strains producing pertussis toxin cross reacting materials. Dev. Biol. Stand. 73:1991;93-107.
35. Nicosia A., Bartoloni A., Perugini M., Rappuoli R. Expression and immunological properties of the five subunits of pertussis toxin. Infect. Immun. 55:1987;963-967.
36. Bartoloni A., Pizza M., Bigio M., Nucci D., Ashworth L.A., Irons L.I., Robinson A., Burns D., Manclark C., Sato H., Rappuoli R. Mapping of a protective epitope of pertussis toxin by in vitro refolding of recombinant fragments. Biotechnology. 6:1989;709-712.
37. Pizza M., Covacci A., Bartoloni A., Perugini M., Nencioni L., de Magistris M.T., Villa L., Nucci D., Manetti R., Bugnoli M., Giovannoni F., Olivieri R., Barbieri J.T., Sato H., Rappuoli R. Mutants of pertussis toxin suitable for vaccine development. Science. 246:1989;497-500.
38. Nencioni N., Pizza M., Bugnoli M., de Magistris T., Di Timmaso A., Giovannoni F., Manetti R., Marsili I., Matteucci G., Nucci D., Olivieri R., Pileri P., Presentini R., Villa L., Kreeftenberg J.G., Silvestri S., Tagliabue A., Rappuoli R. Characterization of genetically inactivated pertussis toxin mutants: candidates for a new vaccine against whooping cough. Infect. Immun. 58:1990;1308-1315.
39. Nencioni L., Volpini G., Peppoloni S., Bugnoli M., de Magistris T., Marsili I., Rappuoli R. Properties of pertussis toxin mutant PT-9K/129G after formaldehyde treatment. Infect. Immun. 59:1991;625-630.
40. van der Pouw-Kraan T., Rensink I., Rappuoli R., Aarden L. Co-stimulation of T cells via CD28 inhibits human IgE production. Reversal by pertussis toxin. Clin. Exp. Immunol. 99:1995;473-478.
41. Kosecka U., Marshall J.S., Crowe S.E., Bienenstock J., Perdue M.H. Pertussis toxin stimulates hypersensitivity and enhances nerve-mediated antigen uptake in rat intestine. Am. J. Physiol. 30:1994;G745-53.
42. Roberts M., Bacon A., Rappuoli R., Pizza M., Cropley I., Douce G., Dougan G., Marinaro M., McGhee J., Chatfield S. A mutant pertussis toxin molecule that lacks ADP-rybosiltransferase activity, PT-9K/129G, is an effective mucosal adjuvant for intranasally delivered proteins. Infect. Immun. 63:1995;2100-2108.
43. Zumbihl R., Dornand J., Fischer T., Cabane S., Rappuoli R., Bouaboula M., Casellas P., Rouot B. IL-1 stimulates a diverging signaling pathway in EL4 6.1 thymoma cells. J. Immunol. 155:1995;181-189.
44. Brio G.A.C., Souza M.H.L.P., Melo-Filho A.A., Hewlett E.L., Lima A.A.M., Flores C.A., Ribeiro R.A. Role of pertussis toxin A subunit in neutrophil migration and vascular permeability. Infect. Immun. 65:1997;1114-1118.
45. Sindt K.A., Hewlett E.L., Redpath G.T., Rappuoli R., Gray L.S., Vandenberg S.R. Pertussis toxin activates platelets through an interaction with platelet glycoprotein Ib. Infect. Immun. 62:1994;3108-3114.
46. Ibsen P.H. The effect of formaldehyde, hydrogen peroxide and genetic detoxification of pertussis toxin on epitope recognition by murine monoclonal antibodies. Vaccine. 14:1996;359-368.
47. Podda A., Nencioni L., de Magistris M.T., Di Tommaso A., Bossù P., Nuti S., Pileri P., Peppoloni S., Bugnoli M., Ruggiero P., Marsili I., D'Errico A., Tagliabue A., Rappuoli R. Metabolic, humoral, and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT-9K/129G. J. Exp. Med. 172:1990;861-868.
48. Podda A., Carapella de Luca E., Titone L., Casadei A.M., Cascio A., Peppoloni S., Volpini G., Marsili I., Nencioni L., Rappuoli R. Acellular pertussis vaccine composed of genetically inactivated pertussis toxin: safety and immunogenicity in 12- to 24- and 2- to 4-month-old children. J. Pediatr. 120:1992;680-685.
49. Podda A., Nencioni L., Marsili I., Peppoloni S., Volpini G., Donati D., Di Tommaso A., de Magistris M.T., Rappuoli R. Phase I clinical trial of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin combined with FHA and 69 kDa. Vaccine. 9:1991;741-745.
50. Podda A., Carapella de Luca E., Titone L., Casadei A.M., Cascio A., Bartalini M., Volpini G., Peppoloni S., Marsili I., Nencioni L., Rappuoli R. Immunogenicity of an acellular pertussis vaccine composed of genetically inactivated pertussis toxin combined with filamentous hemagglutinin and pertactin in infants and children. J. Pediatr. 123:1993;81-84.
51. Podda A., Carapella de Luca E., Contu B., Furlan R., Maida A., Moiraghi A., Stramare D., Titone L., Uxa F., Di Pisa F., Peppoloni S., Nencioni L., Rappuoli R. Comparative study of a whole-cell pertussis vaccine and a recombinant acellular pertussis vaccine. J. Pediatr. 124:1994;921-926.
52. Di Tommaso A., Bartalini M., Peppoloni S., Podda A., Rappuoli R., de Magistris M.T. Acellular pertussis vaccines containing genetically detoxified pertussis toxin induce long-lasting humoral and cellular responses in adults. Vaccine. 15:1997;1218-1224.
53. Hewlett E.L. Acellular pertussis trial. Pediatrics. 98:1996;800.
54. Pichichero M.E., Deloria M.A., Rennels M.B., Anderson E.L., Edwards K.M., Decker M.D., Englund J.A., Steinhoff M.C., Deforest A., Meade B.D. A safety and immunogenicity comparison of 12 acellular pertussis vaccines and one whole-cell pertussis vaccine given as a fourth dose in 15- to 20-month-old children. Pediatrics. 100:1997;772-788.
55. Greco D., Salmaso S., Mastrantonio P., Giuliano M., Tozzi A.F., Anemona A., Ciofi degli Atti M.L., Giammanco A., Panei P., Blackwelder W.C., Klein D.L., Wassilak S.G.F. A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. N. Engl. J. Med. 334:1996;341-346.
56. Olin P, Gustafsson L, Rasmussen F, Hallander H, Heijbel H, Gottfarb P. Efficacy trial of acellular pertussis vaccines. Technical Report Trial II. Swedish Institute for Infectious Disease Control, Stockholm, 1997.
57. Olin P., Rasmussen F., Gustafsson L., Hallander H.O., Heijbel H. Randomised controlled trial of two-component, three-component, and five-component acellular pertussis vaccines compared with whole-cell pertussis vaccine. Lancet. 350:1997;1569-1577.
58. Salmaso S., Mastrantonio P., Wassilak S.G.F., Giuliano M., Anemona A., Giammanco A., Tozzi A.E., Ciofi degli Atti M.L., Greco D. Persistence of protection through 33 months of age provided by immunization in infancy with two three-component acellular pertussis vaccines. Vaccine. 13:1998;1270-1275.
59. Pierce N.S., Gowans J.L. Cellular kinetics of the intestinal immune response to cholera toxoid in rats. J. Exp. Med. 142:1975;1550-1563.
60. Clements J.D., Yancy R.J., Finkelstein R.A. Properties of homogenous heat-labile enterotoxin from E. coli. Infect. Immun. 29:1980;91-97.
61. Lycke N., Holmgren J. Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. Immunology. 59:1986;301-308.
62. Jackson R.J., Fujihashi K., Amano J.X., Kiyono H., Elson C.O., McGhee J.R. Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant. Infect. Immun. 61:1993;4272-4279.
63. Levine M.M., Kaper J.B., Black R.E., Clements M.L. New knowledge on pathogenesis of bacterial infections as applied to vaccine development. Microbiol. Rev. 47:1983;510-550.
64. Holmgren J., Lonnroth I., Svennerholm L. Tissue receptor for cholera exotoxin: postulated structure from studies with GM1 ganglioside and related gangliolipids. Inf. Imm. 8:1973;208-214.
65. Zhang R.G., Scott D.L., Westbrook M.L., Nance S., Spangler B.D., Shipley G.G.W. The three-dimensional crystal structure of cholera toxin. J. Mol. Biol. 251:1995;563-573.
66. 32P ]ADP-ribosylation of proteins. Methods in Enzymol. 195:1991;267-280.
67. Field M., Rao M.C., Chang E.B. Intestinal electrolyte transport and diarrheal disease: Part I. N. Eng. J. Med. 321:1989;800-806.
68. Dallas W.S., Falkow S. Amino acid homology between cholera toxin and Escherichia coli heat-labile toxin. Nature. 288:1980;499-501.
69. Domenighini M., Pizza M., Jobling M.G., Holmes R.K., Rappuoli R. Identification of errors among database sequence entries and comparison of correct amino acid sequences for the heat-labile enterotoxins of Escherichia coli and Vibrio cholerae. Mol. Microbiol. 15:1995;1165-1167.
70. Holmgren J., Svennerholm A.M., Jertborn M., Clements J., Sack D.A., Salenstedt R., Wigzell H. An oral B subunit-whole cell vaccine against cholera. Vaccine. 10:1992;911-914.
71. Holmgren J., Jertborn M., Svennerholm A.M. New and improved vaccines against cholera. II. Oral B subunit killed whole-cell cholera vaccine. Levine M.M., Woodrow G.C., Kaper J.B., Cobon G.S. New generation vaccines. 1997;459-468 Marcel Dekker, New York.
72. Wilson A.D., Robinson A., Irons L., Stokes C.R. Adjuvant action of cholera toxin and pertussis toxin in the induction of IgA antibody response to orally administered antigens. Vaccine. 11:1993;113-118.
73. Clements J.D., Hartzog N.M., Lyon F.L. Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens. Vaccine. 6:1988;269-277.
74. Lycke N., Tsuji T., Holmgren J. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. Eur. J. Immunol. 22:1992;2277-2281.
75. Douce G., Turcotte C., Cropley I., Roberts M., Pizza M., Domenighini M., Rappuoli R., Dougan G. Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants. Proc. Natl. Acad. Sci., USA. 92:1995;1644-1648.
76. Pizza M., Domenighini M., Hol W., Giannelli V., Fontana M.R., Giuliani M.M., Magagnoli C., Peppoloni S., Manetti R., Rappuoli R. Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis. Mol. Microbiol. 14:1994;51-60.
77. de Haan L., Verweij W.R., Feil I.K., Lijnema T.H., Hol W.G., Agsteribbe E., Wilschut J. Mutants of Escherichia coli heat-labile enterotoxin with reduced ADP-ribosylation activity or no activity retain the immunogenic properties of the native holotoxin. Infect. Immun. 64:1996;5413-5416.
78. Hase C.C., Thai L.S., Boesman-Finkelstein M., Mar V.L., Burnette W.N., Kaslow H.R., Stevens L.A., Moss J., Finkelstein R.A. Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs. Infect. Immun. 62:1994;3051-3057.
79. Fontana M.R., Manetti R., Giannelli V., Magagnoli C., Marchini A., Domenighini M., Rappuoli R., Pizza M. Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit. Infect. Immun. 63:1995;2356-2360.
80. Yamamoto S., Kiyono H., Yamamoto M., Imaoka K., Yamamoto M., Fujihashi K., Van Ginkel F.W., Noda M., Takeda Y., McGhee J.R. A non toxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity. Proc. Natl. Acad. Sci. USA. 94:1997;5267-5272.
81. Domenighini M., Magagnoli C., Pizza M., Rappuoli R. Common features of the NAD- binding and catalytic site of ADP-ribosylating toxins. Mol. Microbiol. 14:1994;41-50.
82. Grant C.C., Messer R.J., Cieplak W. Jr. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin. Infect. Immun. 62:1994;4270-4278.
83. Dickinson B.L., Clements J D. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect. Immun. 63:1995;1617-1623.
84. Di Tommaso A., Saletti G., Pizza M., Rappuoli R., Dougan G., Abrignani S., Douce G., de Magistris M.T. Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant. Infect. Immun. 64:1996;974-979.
85. Giuliani M.M., Del Giudice G., Giannelli V., Dougan G., Douce G., Rappuoli R., Pizza M. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J. Exp. Med. 187:1998;1123-1132.
86. Douce G., Fontana M.R., Pizza M., Rappuoli R., Dougan G. Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin. Infect. Immun. 65:1997;2821-2828.
87. Marchetti M., Rossi M., Giannelli V., Giuliani M.M., Pizza M., Censini S., Covacci A., Massari P., Pagliaccia C., Manetti R., Telford J.L., Douce G., Dougan G., Rappuoli R., Ghiara P. Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant. Vaccine. 16:1998;33-37.
88. Ghiara P., Rossi M., Marchetti M., Di Tommaso A., Vindigni C., Ciampolini F., Covacci A., Telford J.L., de Magistris M.T., Pizza M., Rappuoli R., Del Giudice G. Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection. Infect. Immun. 65:1997;4996-5002.
89. Giannelli V., Fontana M.R., Giuliani M.M., Guangcai D., Rappuoli R., Pizza M. Protease susceptibility and toxicity of heat-labile enterotoxins with a mutation in the active site or in the protease-sensitive loop. Infect. Immun. 65:1997;331-334.
90. Glenn G.M., Rao M., Matyas G.R., Alving C.R. Skin immunization made possible by cholera toxin. Nature. 391:1998;851.
91. Partidos C.D., Pizza M., Rappuoli R., Steward M.W. The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide. Immunology. 89:1996;483-487.
92. Nashar T.O., Webb H.M., Eaglestone S., Williams N.A., Hirst T.R. Potent immunogenicity of the B subunit of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets. Proc. Natl. Acad. Sci. USA. 93:1996;226-230.
93. Conference Coverage (ICAAC). New mucosal adjuvant safe in humans. Vaccine Weekly 1997; November 3.4.