Mutant enterotoxin effective as a non-toxic adjuvant

Details Mutant enterotoxin effective as a non-toxic adjuvant Mutant enterotoxin effective as a non-toxic adjuvant

1999-08-04

2002-08-20

Devi, S. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S236100, C424S234100, C424S235100, C424S200100, C424S184100, C424S278100, C424S282100, C530S350000, C530S825000

Reexamination Certificate

active

06436407

ABSTRACT:

1. FIELD OF THE INVENTION
The present invention is directed towards a hgenetically distinct mutant of
E. coli
heat-labile enterotoxin (LT) and its use as an adjuvant to induce mucosal and serum antibodies and cell mediated immune responses. Specifically, the mutant LT is modified by a single amino acid substitution that reduces its inherent toxicity but leaves intact the adjuvant properties of the molecule.
2. BACKGROUND OF THE INVENTION
Microbial pathogens can infect a host by one of several mechanisms. They may enter through a break in the integument induced by trauma, they may be introduced by vector transmission, or they may interact with a mucosal surface. The majority of human pathogens initiate disease by the last mechanism, i.e., following interaction with mucosal surfaces. Bacterial and viral pathogens that act through this mechanism first make contact with the mucosal surface where they may attach and then colonize, or be taken up by specialized absorptive cells (M cells) in the epithelium that overlay Peyer's patches and other lymphoid follicles [Bockman and Cooper, 1973, Am. J. Anat. 136:455-477; Owen et al., 1986, J. Infect. Dis. 153:1108-1118]. Organisms that enter the lymphoid tissues may be readily killed within the lymphoid follicles, thereby provoking a potentially protective immunological response as antigens are delivered to immune cells within the follicles (e.g.,
Vibrio cholerae
). Alternatively, pathogenic organisms capable of surviving local defense mechanisms may spread from the follicles and subsequently cause local or systemic disease (i.e., Salmonella spp., poliovirus, rotavirus in immunocompromised hosts).
Secretory IgA (sIgA) antibodies directed against specific virulence determinants of infecting organisms play an important role in overall mucosal immunity [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133]. In many cases, it is possible to prevent the initial infection of mucosal surfaces by stimulating production of mucosal sIgA levels directed against relevant virulence determinants of an infecting organism. Secretory IgA may prevent the initial interaction of the pathogen with the mucosal surface by blocking attachment and/or colonization, neutralizing surface acting toxins, or preventing invasion of the host cells. While extensive research has been conducted to determine the role of cell mediated immunity and serum antibody in protection against infectious agents, less is known about the regulation, induction, and secretion of sIgA. Parenterally administered inactivated whole-cell and whole-virus preparations are effective at eliciting protective serum IgG and delayed type hypersensitivity reactions against organisms that have a significant serum phase in their pathogenesis (i.e.,
Salmonella typhi,
Hepatitis B). However, parenteral vaccines are not effective at eliciting mucosal sIgA responses and are ineffective against bacteria that interact with mucosal surfaces and do not invade (e.g.,
Vibrio cholerae
). There is, however, recent evidence that parenterally administered vaccines may be effective against at least one virus, rotavirus, that interacts primarily with mucosal surfaces [Conner et al., 1993, J. Virol. 67:6633-6641]. Protection is presumed to result from transudation of antigen specific IgG onto mucosal surfaces for virus neutralization. Therefore, mechanisms that stimulate both serum and mucosal antibodies and cell mediated immunity are important for effective vaccines.
Mucosal immunization can be effective for induction of specific sIgA responses if the antigens are presented to the T and B lymphocytes and accessory cells contained within the Peyer's patches where preferential IgA B-cell development is initiated. The Peyer's patches contain helper T (TH)-cells that mediate B-cell isotype switching directly from IgM cells to IgA B-cells. The patches also contain T-cells that initiate terminal B-cell differentiation. The primed B-cells then migrate to the mesenteric lymph nodes and undergo differentiation, enter the thoracic duct, then the general circulation, and subsequently seed all of the secretory tissues of the body, including the lamina propria of the gut and respiratory tract. IgA is then produced by the mature plasma cells, complexed with membrane-bound Secretory Component, and transported onto the mucosal surface where it is available to interact with invading pathogens [Strober and Jacobs, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 1-30; Tomasi and Plaut, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61]. The existence of this common mucosal immune system explains in part the potential of live attenuated vaccines and mucosal immunization for protection against pathogenic organisms that initiate infection by first interacting with mucosal surfaces.
A number of strategies have been developed for mucosal immunization, including the use of attenuated mutants of bacteria (i.e., Salmonella spp.) as carriers of heterologous antigens [Cárdenas and Clements, 1992, Clin. Microbiol. Rev. 5:328-342; Clements et al., 1992, In: Recombinant DNA Vaccines: Rationale and Strategy, Isaacson (ed.), Marcel Decker, New York. p.p. 293-321; Clements and Cárdenas, 1990, Res. Microbiol. 141:981-993; Clements and El-Morshidy, 1984, Infect. Immun. 46:564-569], encapsulation of antigens into microspheres composed of poly-DL-lactide-glycolide (PGL), protein-like polymers—proteinoids [Santiago et al., 1993, Pharmaceutical Research 10:1243-1247], gelatin capsules, different formulations of liposomes [Alving et al., 1986, Vaccine 4:166-172; Garcon and Six, 1993, J. Immunol. 146:3697-3702; Gould-Fogerite and Mannino, 1993, In: Liposome Technology 2nd Edition. Vol. III, Gregoriadis (ed.)], adsorption onto nanoparticles, use of lipophilic immune stimulating complexes (ISCOMS) [Mowat and Donachie, 1991, Immunology Today 12:383-385], and addition of bacterial products with known adjuvant properties [Clements et al., 1988, Vaccine 6:269-277; Elson, 1989, Immunology Today 146:29-33; Lycke and Holmgren, 1986, Immunology 59:301-308; Lycke et al., 1992, Eur. J. Immunol. 22:2277-2281]. The two bacterial products with the greatest potential to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of
V. cholerae,
and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of
Escherichia coli.
Although LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique.
The extensive diarrhea of cholera is the result of a potent exo-enterotoxin which causes the activation of adenylate cyclase and a subsequent increase in intracellular levels of cyclic 3′-,5′-adenosine monophosphate (cAMP). The cholera enterotoxin (CT) is an 84,000 dalton polymeric protein composed of two major, non-covalently associated, immunologically distinct regions or domains (“cholera-A” and “cholera-B”) [Finkelstein and LoSpalluto, 1969, J. Exp. Med. 130: 185-202]. Of these, the 56,000 dalton region, or choleragenoid, is responsible for binding of the toxin to the host cell membrane receptor, G
M1
(galactosyl-N-acetylgalactosaminyl-(sialyl)-galactosyl-glucosyl ceramide), which is found on the surface of essentially all eukaryotic cells. Choleragenoid is composed of five non-covalently associated monomers, while the A region (27,000 daltons) is responsible for the diverse biological effects of the toxin.
The relationship of the two subunits of CT with respect to the immunologic properties of the molecule has been a source of considerable debate. On the one hand, CT is an excellent immunogen that provokes the development of both serum and mucosal antitoxin antibody responses when delivered mucosally. This finding is

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