MXPA97001445A - Effective mutating enterotoxin as adjuvant oral no tox - Google Patents
Effective mutating enterotoxin as adjuvant oral no toxInfo
- Publication number
- MXPA97001445A MXPA97001445A MXPA/A/1997/001445A MX9701445A MXPA97001445A MX PA97001445 A MXPA97001445 A MX PA97001445A MX 9701445 A MX9701445 A MX 9701445A MX PA97001445 A MXPA97001445 A MX PA97001445A
- Authority
- MX
- Mexico
- Prior art keywords
- vaccine
- spp
- antigen
- adjuvant
- composition
- Prior art date
Links
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Abstract
The present invention relates to methods and compositions for the use of a novel mutant form of heat labile enterotoxin of E. coli, which has lost its toxicity but retained its immunological activity. This enterotoxin is used in combination with an unrelated antigen to achieve an increased immune response to said antigen when administered as part of a vaccine preparation or
Description
ENTERDTOXI TO EFFECTIVE MUTANT AS NON-TOXIC ORAL ADJUVANT The research described in this specification was supported in part by the United States Navy - Grant No. N00014-83- -0192. The government has certain rights over this invention. 1. FIELD OF THE INVENTION The present invention focuses directly on a mutant genetically distinct from the enterota :. thermal inactivation of E. coli (LT) and its. Use as an oral adjuvant to induce mucosal and serum antibodies. Specifically, the mutant LT is modified by an amino acid substitution that overrides its inherent tonicity but leaves the adjuvant properties of the molecule intact. 2. BACKGROUND OF THE INVENTION Microbial pathogens can infect a host by one of several mechanisms. They can penetrate an opening in the integument induced by trauma, they can be introduced by vectorial transmission, or they can interact with a mucosal surface. Most human pathogens initiate a disease by this same mechanism, that is, after the interaction with mucosal surfaces. Viral bacterial pathogens acting through this mechanism first come into contact with the mucosal surface where they can be fixed and then colonized or they can be absorbed by special absorption cells (M cells) in the epithelium lining the Peyer patches and other lymphoid follicles (Boc an and Cooper, 1973, Am. J. Ant. 136: 455-477; Ornen et al., 1986, J. Infect. Dis. 153: 1108-1118). Organisms that penetrate the lymphoid tissues can be killed easily within the lymphoid follicles, thus eliciting a potentially protective in unologic response when the antigens are delivered to immune cells within the follicles (eg, Vibrio cholerae). Alternatively, pathogenic organisms capable of surviving local defense mechanisms can spread from the follicles and subsequently cause a local or systemic disease.
(ie, Sal onella spp., pol i virus, rotavirus in immunocompetent omitted hosts). IgA secretory antibodies (slgA) directed against specific virulence determinants of infectious organisms play an important role in global mucosal immunity (Cebra et al., 1986, In: Vaccines 86, Brown et al. (Ed.), Cold Spring Harbor Laboratory, Ne < »York, pp. 129-133). In many cases, it is possible to avoid initial infection of the ucasales surfaces by stimulating the production of mucosal slgA levels focused towards relevant virulence determinants of the infectious organism. The secretion IgA can prevent the initial interaction of the pathogen with the mucosal surface by blocking the fixation and / or colonization, by neutralizing the toxins that act on the surface, or by preventing the invasion of the host cells. While significant research has been done to determine the role of cell-mediated immunity and serum antibodies in protecting against infectious agents, less is known about the regulation, induction, and secretion of slgA. Preparations of whole viruses or of deactivated whole cells administered parenterally are effective to cause delayed type hypersensitivity reactions of serum IgG against organisms that have a significant serum phase in their pathogenesis (eg, Salmonella typhy, hepatitis B). However, parenteral vaccines are not effective in eliciting mucosal slgA responses and are ineffective against bacteria that interact with mucosal surfaces and do not invade (eg, Vibrio cholerae). However, there is recent evidence according to which vaccines administered parenterally can be effective against at least one virus, rotavirus, which interacts primarily with mucosal surfaces (Conner et al., 1993, J. Virol. 67: 6633-6641). The protection probably results from the transudation of antigen-specific IgG on mucosal surfaces to neutralize the virus. Therefore, mechanisms that stimulate both serum and mucosal antibodies are important for effective vaccines. An oral immunization can be effective for the induction of specific slgA responses and the antigens are presented to the ucositos T and B and accessory cells contained within the Peyer patches where the preferential development of the B cells of IgA begins. The Peyer patches contain helper T cells (TH) that mediate the change from isotype to B cells directly from the IgM cells to IgA B cells. The patches also contain T cells that initiate the differentiation of terminal B cells. The primed B cells then migrate to the mesenteric lymph nodes and undergo differentiation, penetrate the thoracic duct, then the general circulation and subsequently disperse in all secretory tissues of the body including the lamina propria of the intestines and the respiratory tract. IgA is then produced by mature plasma cells, complexed with the secretory component bound to membranes, and transported on 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 Immu ty, Gallin and Fauci (ed.), Raven Press, New York, pp. 1-30; Tomasi and P_lut, 1985, In: Advances ín host defense echanisms. Vo] 4. Mucosal I munity, Gallip and Fauci (ed.), Raven Press, New York. p. 31-61). The existence of this unmucosal common mucous system explains in part the potential of live oral vaccines and oral immunization for protection against pathogenic organisms that initial infection first interacting with mucasales surfaces. Numerous strategies for oral immunization have been developed, including the use of attenuated bacterial mutants (eg, Sal onella spp.) As vehicles for heterologous antigens (Cñardenas and Clements, 1992, Clin.
Microb iol. P.e 5: 328-342? Elements et al., 1992, In:
Recombinant DNA Vaccines: Rationale and Strategy, Isaacson
(ed.), Marcel Decker, New York. p. 293-321; Clements and
Cárdenas, 1990, Res. Microbiol. 141: 981-993; Clements and El-Morshidy, 1984, Infect. Im a. 46: 564-569), encapsulation of antigens in microspheres composed of pal i-DL-lactide-glycolide (PGL), polymers, protein-like proteinoids (santiago et al., 1993, Pharmaceutical Research 10: 1243-1247), capsules of gelatine, different formulations of liposomes (Alving et al., 1986, Vaccine 4: 166-172, Garcon and Six, 1993, J. Immunol., 146: 3697-3702, Gould-Fogeri and Mannino, 1993, In: Liposo e Tehcnolsgy 2nd Edition, Vol. III »Gregoriadis (ed.)), Nanoparticle adsorption, use of lipophilic immunological stimulation complexes (ISC0MS) (Mowat and Donachie, 191, I unology Today 12: 383-385), and addition of bacterial products with known adjuvant properties (Clements et al., 1988, Vaccine 6: 269-277, Elson, 19B9, Immunology Today 146: 29-33, Lycfce and Holren, 1986, Immunolagy 59: 301-308; Lycfee et al., 1992, Eur. J. Iolol 22: 2277-2281). The two bacterial products with the most important potential to function as oral adjuvants are cholera toxin (CT), produced by several strains of V. cholerae, and the enterotoxin ter olábil (LT) produced by some enterotoxigenic strain of Eschepchia co) i . Although LT and CT have many characteristics in common, these characteristics are clearly different molecules with biochemical and immunological differences that make them unique. The extensive cholera area is the result of a potent exo-enterotoxin that causes the activation of adeny latcyclase and a subsequent increase in the intracellular levels of 3'-, 5 '-adenssxn cyclic onophosphate (cAMP). Cholera enterotoxin (CT) is a 84,000 dalton polytechnic protein composed of two immunologically dominant, non-covalently associated ("cholera-A" and "cholera-B") regions or domains (Finfcelstein and LoSpalluta , 1969, J. Exp. Med. 130: 185-202). Among them, the region of 56,000 daltons or coleagenoid is responsible for the binding of the toxin on the host cell membrane receptor, 6M1 (ceramide of g osi 1-N-ac ti lgalactosa ini 1- (sial il) -g lactosi 1-glucosyl), which is found on the surface of essentially all eukaryotic cells. Phenoid cholera is composed of 5 subunits not covalently associated, while region A (27,000 daltons) is responsible for several biological effects of the toxin. The ratio of the two subunits of CT in terms of the immunological properties of the molecule has been a considerable debate agent. On the one hand CT is an excellent immunogen that causes the development of serum and mucosal antitoxin antibody responses when administered orally. This finding is not new insofar as patients with cholera develop increases in titers of antitoxin antibodies during the convalescence of clinical cholera (Finkelstein, 1975, Curr Top, Microbial Immunol 69: 137-196). An essential finding of the people investigating the nature of this response was the observation that CT, unlike most other protein antigens, does not induce an oral tolerance against it (Elson and Ealding, 1984, J. Immunol. : 2892-2897; Elson and Ealding, 1984, J. Immunol., 132: 2736-2741). It was found that this was also true when only B subuphty was fed to mice, an observation was made. supported by trials in the field of cholera bacilli in Bangladesh where oral inunization with subunit B combined with dead whole cells elicited systemic and mucosal antitoxin antibody responses (Svennerholm et al., 1984, J. Infect. Dis. 149: 884-893). In addition to being a potent oral immunogenic, CT has numerous other immunological properties reported. As indicated above, Elson and Ealding (Elson and Ealding, 1984, J. Immunol, 133: 2892-2897) observed that orally administered CT does not induce tolerance against it. In addition, simultaneous oral administration of CT with a soluble protein antigen, lapple ocyanin (LH), resulted in the development of secretory IgA responses both against CT co or against KLH and also nullified the induction of oral tolerance against KLH. . These findings were subsequently confirmed and extended by Lycke and Holmgren (Lycke and Holmgren, 1986, Imology 59: 301-308). The confusion arises when one tries to define the function of subunits A and B of CT in relation to the adjuvant properties of the molecule. The following observations, in accordance with the summary by Elson (Elson, 1989, Immunology Today 146: 29-33), are the basis of this confusion: * CT does not induce oral tolerance against it (Elson and Ealding, 1984, J. Im unol 133: 2892-2897). • »CT does not induce tolerance or l against it (Elson and Ealding, 1984, J. I Unol 133: 2892-2897).
* CT can avoid the induction of tolerance against other antigens with which it is simultaneously supplied and also appears as an adjuvant for these antigens (Elson and Ealding, 1984, J. Im unol. 133: 2892-2897; Lycke and Holmgren, 1986, Immunology 59: 301-308). * CT can act as an adjuvant for CT-B (Elsan and Ealding, 1984, J. Immunol., 133: 2892-2897). * Thermally added CT shows little toxicity but is a potent oral immunogen (Pierce et al., 1983, Infect Immun 40: 1112-1118). * CT-B can serve as an immunological "vehicle" in a traditional hapten-vehicle configuration (Cebra et al., 1986, In: Vaccines 86, Broin et al. (Ed.), Cold Spring Harbor Laboratory, New York. pp. 129-133; MeKenzie and Halsey, 1984, J. Im unol. 133: 1818-1824). Numerous researchers came to the conclusion from these findings that the B subunit must possess some inherent adjuvant activity. The findings of Cebra et al. (Cebra et al., 1986, In: Vaccines 86, Brown et al. (Ed.), Cold Sprip Harbor Laboratory, New York, pp. 129-133), Lycke and Holmgren (Lycke and Holmgren, 1986, Immunology 59: 3 1-308), and Liang et al. (Liang et al., 1988, J. I munal.141: 1495-1501) argued against this conclusion. Cebra et al. (Cebra et al., 1986, In: Vaccines 86, Brown et al. (Ed.), Cold Spring Harbor Labora Ory, New York, pp. 129-133) demonstrated that purified CT-B was effective in raising the presence of anti-cholera toxin B cells specified in Peyer's patches when administered in antroduodenal fashion but, in contrast to CT, did not result in significant numbers of B cells assigned to IgA. Lycke and Holmgren (Lycke and Holmgren, 1986, I munology 59: 301-308) compared CT and CT-B to determine their ability to increase the 3 ucosal immunological response of the intestines to Kl H by measuring the cells that secrete immunobass. in the lamina propria of orally immunized mice. They found no increase in the cells that produce anti-LH in response to the doses of B subunit tested in their system. Finally Liang et al. (Liang et al., 1988, J. Immunol. 141: 3495-1501) na found no adjuvant effect when CT-B was administered orally in combination with the deactivated Senda i virus. When an adjuvant activity was observed for the isolated B subunit, it was typically for one of two reasons. First, a traditional method for the preparation of subunit B was to subject a holotoxin dissociation chromatography by gel filtration in the presence of a dissociating agent
(ie, guamdine HCl or formic acid). The isolated subunits were then pooled and the dissociating agent removed. Subunit B prepared by this technique is invariably contaminated with small amounts of subunit B in such a way that a small amount of holatoxin is reconstituted when renaturation is carried out. The second reason has to do with the definition of an immune vehicle. Like other soluble proteins, the B subunit can serve as an immunological vehicle for the presentation of antigens to the immune system. These antigens are sufficiently small to have limited immunogenic characteristics, they can be transformed to become immunogenic in a traditional hapten-vehicle configuration. In the same way, there is a "theoretical" immunological increase associated with subunit B, especially in the case of oral presentation, to the extent that subunit B binds to the surface of epithelial cells and can immobilize a fixed antigen for processing by the lymphoid tissues associated with the intestines. However, any potential advantage of this antigen stabilization mechanism can be compensated for by the distribution of the antigen in uniologically relevant tissues, i.e., the surface of the intestinal epithelial cells. In the context of the mucosal response, the immunologically relevant lesions are Peyer's patches, especially in the case of the activation of B cells that depend on antigen-specific T cells (Strober and Jacobs, 1985, En: Advances m host defense mech nisms, Vol 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York, pp. 1-30; To asi and Plaut, 1985, In: Advances ín host defense mechanis s. Val. 4. Mucosal Immumt-y, Gal n and Faucí (ed.), Raven Press, New York. p. 31-36; Brandtzaeg, 1989, Curr. Top. Microbiol. ImmunoJ. 146: 13-25). Therefore, events up to isotype change from IgM cells to IgA B cells occur in Peyer's patches. Antigens indicated on the surface of epithelial cells can contribute to the proliferation of B cells induced by antigen to the extent that positive class II hairy epithelial cells can act as antigen presenting cells for the activation of cells T in the secretory site, thereby reinforcing the production of cytokines, differentiation of terminal B cells, increased expression of the secretory component, as well as increased external transport of antigen-specific IgA (Toma i, TB, and AG Plaut, 1985, En: Advances in host defense echanis s. Vol. 4. Mucosal I munity, G 111 n and Faucí
(ed.), Raven Press, New York. p. 31-61). The relationship of these events may not have been clearly defined for sub-group B as a vehicle for other antigens and the use of the term "adjuvant" may seem inappropriate for this purpose.
It is clear that the adjuvant property of the molecule is found in the holotoxin in which the B subunit is required for receptor recognition and to facilitate the penetration of the A subunit into the cell. The A subunit also requires an adjuvant activity, probably on the basis of its enzymatic ADP-ribosylating activity and ability to increase the intracellular levels of cAMP (see below). The B subunit alone can act as a vehicle for other antigens insofar as when conjugated these antigens can be immobilized for processing by the lymphoid tissues associated with the intestines. Although LT and CT may have characteristics in common, these are clearly distinct molecules with biochemical and immunological differences that make them unique, including a difference of 20 * /. in nucleotide and amino acid sequence homology (Dallas and Falkow, 1980, Nature 288: 49-501). The two toxins have the same number and arrangement of subunits, the same biological mechanism of action, and the same specific activity in many in vitro trials (Clements and Finkelstein, 1979, Infect. Immun 24: 760-769, Clements et al. , 1980, Infect. Im a 24: 91-97). However, there are significant differences between these molecules that influence not only their enterotoxic properties, but also their ability to function as adjuvants. To begin with, unlike CT produced by V. cholerae, LT remains associated with the cell and is released only from E. coli during cell lysis (Clements and Fmkelstein, 1979, Infect. Immun. 24: 760- 769). CT is excised from the glass as soon as it is synthesized and can be easily identified in culture supernatants and purified from said supernatants. Therefore, in contrast to CT, LT is not fully biologically active when it is first isolated from the cell. Consistent with model A-B for bacterial toxins, LT requires proteolysis and disulfide reduction to be active. In the absence of the proteolytic process, the enzymatically active Al moiety can not dissociate from the A2 component and can not reach its target substrate (adenilate cyclase) on the basolateral surface of the intestinal epithelial cell. This is also true in the case of CT, but the proteases in the culture supernatant, to which the toxin is exposed during the purification, carry out the proteolysis. Since LT is not totally biologically active, it is difficult to identify them during purification using biological or vitro assays such as for example the adrenal cell test Y1 or the permeab1 factor assay. This difference in the activation of the Matter] isolated results in difference in response thresholds for LT and CT in biological systems. For example,
CT induces a detectable net fluid secretion in the mouse intestine at a dose of 5-10 μg. LT induces a detectable net secretion in the mouse intestine at levels greater than 100 μ. In the looped loyal rabbit i loop, the difference is dramatic and clear. In addition, in primates, LT has not been shown to induce a fluid secretion in any of the tested doses up to 1 milligram. That is 200 times the amount of CT reported that induces a positive fluid movement in humans. When LT is exposed to proteolytic enzymes with a similarity to trypsin, the molecule becomes indistinguishable from CT in a biological assay system. This was clearly demonstrated by Clements and Finkelstein
(Clements and Finkelstein, 1979, Infect. Immun 24: 760-769).
In addition to the differences indicated above, LT has an unusual affinity for matrices containing carbohydrates. Specifically LT, with a molecular weight of 90,000 is eluted from Sephadex (glucose) columns with an apparent molecular weight of 45,000 and from columns of Agarose (galactose) with an apparent molecular weight of 0. That is, it binds on matrices and contains galactose and it can be eluted from these matrices in pure form by the application of galactose. LT binds not only on the agarose in columns used for purification but, if it is more important, on other biological molecules that contain galactose, including glycoproteins and 1 ipopol isacids. This binding property, similar to the LT-dk reading, results in a broader distribution of receptors in mammalian cells for LT than for CT that binds solitarily with GM1. This may partly explain the reported differences in the ability of these two molecules to induce different responses to T helper cell lymphocytes (McGhee et al., 1994, Mucosa 1 Immunology Update, Spring 1994, Raven Press, New York page 21). In these studies reported by McGhee et al. (McGhee et al., 1994, Mucosal Im unolog Update, Spring 1994, Raven Press, New York, p.21), it was shown that oral immunization of mice with vaccines co or for example tetanus toxside (TT) with CT as Mucosal adjuvant selectively induces TH2-type cells in Peyer's patches and in spleens as shown by TH cells that produce IL-4 and IL-5, but not IL-2 or INF-gamma. (For a more complete review of the cytokine network, see Arai et al., 1990, Ann.Rev. Biochem. 59: 783-836). Importantly, when CT was used as a mucosal adjuvant it also increased antigen-specific IgE responses in addition to the IgA response. Such an increase in IgE responses severely compromises the safety of CT co or mucosal adjuvant due to the prospect of inducing immediate type hypersensitivity reactions. In contrast, LT induces both TH1 and TH2 cells and predominantly antigen-specific IgA responses without IgE responses when used as an orally administered mucosal adjuvant. The two molecules also have many immunological differences, as demonstrated by immune and fusion studies (Clements and Finkelstein, 1978, Infect. I am 21: 1036-1039; Clements and finkelstein, 1978, Infect. Im a. 22: 709-713), in vitro neutralization studies, and the partial protection against LT associated with E. coli caused by volunteers receiving vaccine against whole B subunit cholera (Cle ens et al., 1988, J. Infect. Dis. 158: 372-377). Taken together, these findings demonstrate that LT and CT are single molecules, despite their apparent simi larities, and that LT is a practical oral adjuvant while CT is not. The demonstration of the adjuvant properties of LT comes from an investigation of the influence of LT on the development of tolerance to antigens orally administered by one of the present inventors. It was not clarified yes. or not LT also influences the induction of oral tolerance or has the adjuvant effects demonstrated for CT, given the differences observed between the two molecules. Accordingly, the present inventors examined various parameters, including the effect of LT on oral tolerance to OVA and the function of the two subunits of LT in the observed response, the effect of varying the timing and route of administration of LT, the effect of a previous exposure to OVA on the ability of LT to influence tolerance _ OVA, the use of LT as an adjuvant with two non-related antigens, and the effect of the immunization route on anti-OVA responses. the results obtained from these studies (Clements et al., 1988, Vaccine 6: 26c? -277; Clements et al., 1988, Summary No. B91, 88th Ann. Meet. Am. Soc. Microbiol.) are summarized then: 1. The simultaneous administration of LT with OVA prevented the induction of tolerance to OVA and increased the serum anti-OVA response by 30 to 90 times compared to animals primed with OVA and primed with PBS, respectively. . This effect was determined as a function of the enzymatically active subunit A of the toxin since the B subunit alone could not influence the induction of tolerance. 2. Animals that received LT with OVA after being primed with OVA initially developed an anti-OVA response of serum and mucosal IgA significantly lower than the animals that had LT can OVA in the initial immunization, indicating that the exposure Pre-antigen reduces the effectiveness of LT to influence tolerance and its ability to act as an adjuvant. LT could not override the tolerance once established. This was also true for CT when animals were pre-immunized with OVA before oral ovalbumin plus CT and offers some explanation as to the beneficial observation that anti-body responses to non-white diet antigens do not increase when these adjuvants are employed. 3. The responses of serum and mucosal IgA in animals receiving LT in a single opportunity, being said opportunity of the first exposure to antigen, were equivalent to the responses after three OVA / LT priming processes, indicating that the Dedication to the response occurs early and at the first exposure to the antigen. It was also shown that the direction of response to predominantly either serum or mucosal IgA can be controlled by the administration or not of a parenteral booster dose. 4. The simultaneous administration of LT with two soluble protein antigens results in the development of serum and mucosal antibodies against both antigens if the animal has no previous immuno-logical experience with them. This was an important finding since a possible?
application of IT co or adjuvant would be for the development of mucosal antibodies against complex antigens, such as dead bacteria or viruses, where the ability to respond to multiple antigens would be important. Studies carried out by Tamura et al., (Ta ura et al., US Pat. No. 5,182,109) demonstrated that LT and / or CT administered i nally increased l a. titration of antibodies against a co-administered antigen. However, it is not presented anywhere in Tamura et al. that these toxins can induce a protective immunological response when administered orally. Clearly, LT has a significant immunoregulatory potential, both as a way to avoid the induction of tolerance to specific antigens and as an adjuvant to orally administered antigens and causes the production of both serum IgG and mucosal IgA against antigens with which it is supplied. This raises the possibility of an effective immunization program against several pathogens involving the oral administration of attenuated dead agents or relevant determinants of the virulence of specific agents. However, the fact that it is a "toxin" may stimulate a net secretory secretory response when they are proteol tically dissociated, such as proteases, or when administered orally at sufficiently high concentrations, may impede the investigation of their potential or avoid its use under appropriate conditions. This problem could be solved LT could "deto? Icate" without diminishing the adjuvant properties of the molecules. To observe how this can be achieved, it is necessary to further analyze the mechanism of action of LT and CT as well as the structural and functional relationships of these molecules. As previously indicated, both LT co or CT are synthesized as toxins from multiple subunits with components A and B. After the initial interaction of the toxin with the host cell membrane receptor, the B region facilitates the penetration of subunit A to through the cell membrane. When carrying out a thiol reduction, this component A is dissociated into two smaller polypeptide chains. One of these chains, the Al part, catalyzes the ADP-r ibosi lac ion of the GTP binding pratein (Gs) stimulatory in the adeni latcyclase enzyme complex on the lateral vessel surface of the epithelial cell and this results in a increase in intracellular levels of c MP. The resulting increase in cAMP causes the secretion of water and electrolytes in the small intestine by interacting with two ac ion transport mechanisms sensitive to MP that involve 1) the cotransport of NaCl ion through the brush border of hairy epithelial cells, and 2) Cl-dependent secretion of electrogenic Na + by crypt cells (Field, 1980, In: Secretary diarrhea, Field et al. (Ed.), Waverly Press, Baltimore, pp. 21-30). Subunit A is also the major portion associated with the immune increase by these toxins. This subunit then becomes a probable target for manipulation to dissociate the toxic and immune functions. logic of molecules. A recent report by Lycke et al, (Lycke et al., 1992, Eur. J. I mu.nol.22: 2277-2281) clarifies that alterations that affect the enzymatic activity of ADP ribosylation of the toxin and alter the The ability to increase the intracellular levels of cAMP also prevents the molecule from functioning as an adjuvant. Therefore, another approach to detoxification should be explored. 3. COMPENDIUM OF THE INVENTION The present invention is based on the surprising observation that a mutant form of LT, which has lost its. toxic effect and na has the activity ADP-ribosi 1 transferase, still retains its activity as an immunological adjuvant. The LT UTA form differs from the wild type by a single amino acid substitution, Argl92-Glyl92, which renders the trypsin-sensitive site insensitive. The loss of the protealitic site prevents the proteolytic processing of subunit A in its. toxic form. The native LT is non-toxic when it is first isolated from the bacteria but has the potential to be totally toxic when exposed to proteases or for example those found in the mammalian gut. The mutant form of LT no longer has the potential to become toxic due to proteolytic activation. This mutant LT (hereinafter referred to as mLT) retains the ability to increase the immune response of an animal (eg, IgG, IgA) to an antigen unrelated to LT or mLT without toxic side effects. Experimental data indicate that mLT is useful as an adjuvant for orally administered antigens; such administration results in the production of serum IgG and / or mucosal slgA against the antigen with which mLT is delivered. The present invention provides a method for the induction of a serum and / or mucosal immune response in a host to an orally administered antigen comprising administering to the host an effective amount of mLT in combination with administering orally an effective amount of the antigen. Preferably, the antigen and mLT are administered start 1 in a simultaneous dose. The present method and compositions provide an improved mode of oral immunization for the development of serum antibodies and mucasales against pathogenic microorganisms. The production of IgA antibody responses against pathogenic microorganisms that penetrate or invade through mucosal surfaces can be directed towards this surface, whereas a significant serum antibody response can develop to avoid infection by pathogenic microorganisms against which the serum antibody It provides protection. The present invention is useful for any specific antigen wherein a specific neutralizing antibody response would be useful to eliminate the physiological or diseased state related to this antigen. The present invention also provides a composition useful as a component of a vaccine against heterologous bacterial organisms expressing cholera-like enterotoxins, and methods for their use. The invention also provides a composition useful in these methods. The composition comprises an effective amount of mLT in combination with an effective amount of antigen. 4. BRIEF DESCRIPTION OF THE FIGURES The present invention will be understood more cavally with reference to the following detailed description of the invention, examples of specific embodiments of the invention as well as the relationship with the attached figures wherein: Figure 1 is a schematic diagram of plasmid pBD94, which encodes both subunits A and B under the control of the lac promoter. Plasmid pBD95 contains the single base substitution at residue 192 of amino acids of subunit A, which codes for Gly instead of Arg, which preserves the reading structure but removes the proteolytic site. The amino acid sequence corresponding to the region of trypsin sensitivity and the site of the amino acid substitution Argl92 ~ Glyl92 is shown. Figure 2 is a graphical demonstration of the dose-dependent increase in ADP-ribos and lagmat ina levels as a function of increasing amounts of CT.
Figure 3 is the accumulation of fluid after feeding 125 μg of native LT but not after feeding 125 μg of mLT to mice. The ratio between intestine and corpse is defined as the intestinal weight divided by the weight of the rest of the corpse. Figure 4 shows the ability of mLT to act as an immunogenic adjuvant. Figure 4A shows the ability of mLT to induce a serum IgG response to OVA. Figure 4B shows the ability of mLT to induce a slgA mucosl response to OVA. Figure 5 is an experimental demonstration that mLT retains the ability to avoid the induction of oral tolerance to LT. Figure 5A shows the ability of mLT to induce a serum IgG response to LT. Figure 5B shows the ability of mLT to induce a mucosal slgA response to IT. 5. DETAILED DESCRIPTION OF THE INVENTION The present invention encompasses a composition and methods for its use to promote the production of mucosal and serum antibodies against antigens simultaneously administered orally with a genetically modified bacterial toxin. The modified toxin is a form of the thermolabile enterotoxin (LT) of F .. coli that by means of genetic engineering has lost its sensitive site to trypsin turning the non-toxic molecule but nevertheless, unexpectedly, the molecule retains its capacity to act as adjuvant and immunologist. The mutant LT is referred to herein as mLT. The invention is based on the discovery that mLT is equally effective as LT as an immunological adjuvant, a surprising and unexpected result. mLT no longer has the enzymatic activity of ADP because the subunit A can no longer be processed proteo! technically. In contrast to the published studies of Lycke and colleagues, which clarify that alterations that affect the activity of LT ADP pbosi lation also prevent the molecule from functioning as an immunoallogenic adjuvant (Lycke et al., 1992, Fur. J. Immunal., 22: 2277-2281), the mLT described here retains its activity as an immunological adjuvant even when, as demonstrated in the effects, it does not have the ribosylation activity of ADP. The novel mutant form of the thermolabile enterotaxin of E. coli mLT, described herein, behaves as an adjuvant and causes the production of both serum IgG and slgA mucous 1 against antigens with which it is supplied. The utility of this surprising discovery is that an effective adjuvant amount of mLT can be used in an effective immunization program against several pathogens that involve the oral administration of an effective amount of adjuvant mLT mixed with killed or attenuated pathogens or relevant virulence determinants. of specific pathogens without fear of the actual or potential toxic side effects associated with the oral administration of CT or LT. The present invention overcomes the prior art insofar as the present invention can be used in several immunological applications where CT, LT, or CT or LT subunits may have been used, but now with mLT there are no real or potential side effects, such as for example, in relation to its use. In contrast to LT which, although not toxic when first isolated from bacteria, has the potential to be totally toxic when exposed to co proteases or those found in mammalian intestines, mLT has no potential to become toxic due to activation. proteol í tic. Another embodiment of the present invention is a component of the vaccine against whole, toxic organisms that express cholera-like toxins. The present inventors have shown that mLT is not subject to orally induced inological tolerances when administered (see below), therefore mLT may work and is highly desirable as a component of vaccines directed against enterotoxin organisms. Current technology provides vaccines against organisms that express cholera-like toxins that contain dead whole cells and B subunit of the toxin. By replacing subunit B with mLT in the vaccine, the vaccine is improved in two different ways. First mlT, which has both the A and B subunits will now induce an immune response not only for the B subunit but also for the A subunit. This provides a greater number of epitopes for effective neutralization. Second, the adjuvant activity inherent in mLT will increase the 3a immune response against the dead whole cell component of the vaccine.
In addition, other researchers (Hase et al., 1994, Infect. Immun. 62: 3051-3057) have shown that the A subunit, modified in such a way that it is no longer toxic by altering the active site of the enzym activity. ADP pbosylation, (as opposed to the proteological site which is the subject of the present invention) can induce an immunological response against the wild-type A subunit. However, the A subunit modified in this way no longer has immunoadjuvant adjuvant activity and is therefore less desirable as a vaccine component than mLT. Furthermore, since antibodies with mLT cross-react with LT and CT, mLT can be used in vaccines directed against many types of entertoxic bacterial organisms that express cholera-like toxins, such as Escherichia spp. and Vibrio spp- 5.1 mLT PRODUCTION LT wild type toxin is encoded in a naturally occurring plasmid found in strains of enterotoxigenic E. coli capable of producing this toxin. The present inventors have previously cloned the LT gene from a human isolate of E. coli designated H10407. This rises Ion consists of a 5.2 kb DNA fragment from the enterotoxin plasmid of H10407 inserted in the PstI site of plasmid pBR.322 (Clements et al, 1983, Infect.Immun.40: 653). This recombinant plasmid, designated pDF82, has been extensively characterized and expresses LT under the control of the native LT promoter. The next step in this process was the placement of the LT gene under the. control of a strong promoter, in this case the lac promoter in the pUC18 plasmid. This was achieved by isolating the genes for LT-A and LT-B separately and recnaining them in a cassette in the plasmid vector. This was an important step because it allowed the purification of reasonable amounts of LT and the derivation of mutants for subsequent analysis. This labeled plasmid, designated pBD94, is presented diagramatically in Figure 1. Both CT co or LT are synthesized with a trypsin-sensitive peptide bond linking the Al and A2 parts. This peptide bond must be cut so that the molecule is "toxic". This is also true in the case of diphtheria toxin, prototypic toxin A-B, and for several other bacterial toxins. If the A1-A2 linkage is not removed, either by bacterial protease or intestinal proteases in the lumen of the intestion, the Al part can not reach its target on the basolateral surface of the intestinal epithelial cell. In contrast to CT, LT is not totally biologically active when it is first isolated from the cell. LT also requires proteolysis to be fully active and proteolytic activation can not occur within the bacteria. Therefore, one way of altering the toxicity of the molecule without affecting the enzymatic activity of ADP is the removal by genetic manipulation of the trypsin-sensitive amino acids that bind the A3 and A2 components of the A subunit. it can not be proteolytically dissociated, it will not be toxic. An expert in the material could predict that the molecule, however, retains its. enzyme activity of ADP ribosylation and therefore its adjuvant function. Figure 1 shows the sequence of the region subtended by disulfide that will separate the Al and A2 parts. Within this region is a unique Arginine residue that is believed to be the cleavage site necessary to activate the toxic properties of the molecule. This region was changed by site-directed mutagenesis in such a way that the molecule becomes insensitive to proteolytic digestion and therefore becomes non-toxic. Site-directed mutagenesis is achieved by hybridization over a single-stranded DNA of a synthetic oligonucleotide, commentary of single-stranded annealing except for a region of non-correspondence near the center. It is this region that contains the change or the desired nucleotide changes. After hybridization with single strand white DNA, the oligonucleotide is extended with DNA polymerase to create a double-stranded structure. The cut is then sealed with DNA ligase and the duplex structure transformed into an E. coli host. The theoretical yield of the mutants using this procedure is 50 * 4 due to the semiconservative mode of DNA replication. In practice, the performance is much lower. However, there are numerous methods available to increase the yield and to select oligonucleotide-directed mutants, the E3 system used a second mutagen-linked oligonucleotide to create altered restriction sites in a double-mutation strategy. The next step was the replacement of another amino acid by Arg (ie, GGA = Gly replaces AGA = Arg), thus preserving the reading structure while removing the proteolytic site. MLT was then purified by affinity chromatography on agarase from a mutant (pBD95) which had been confirmed by sequencing. Alternative methods of purification will be apparent to those skilled in the art. This »mLT, called LTÍR192G) was then examined by gel electrophoresis of SDS-pol i cp lick for modification of the trypsin-sensitive bond. Samples with and without trypsin exposure were examined and said samples were compared with native LT (no modifications). mLT is not divided into Al and A2 when incubated with trypsin, indicating that protease sensitivity has been removed. 5.2 M0D0D OF ADMINISTRATION OF mLT AND NON-RELATED ANTIGENS According to the present invention, mLT can be administered in combination with any antigen and / or iologically relevant vaccine, in such a way that an immune response is achieved! increased to said antigen and / or vaccine. In a preferred embodiment, the mLT and the antigen are administered at the end in a pharmaceutical composition comprising an effective amount of mLT and an effective amount of antigen. The admission mode is oral. The respective amounts of mLT and antigen vary depending on the identity of the antigen used and the species of the animal to be immunized. In one embodiment, the initial administration of ml.T of antigen is followed by a boost of the relevant antigen. In another modality, no reinforcement is administered. The time of administration of reinforcement may vary, depending on the antigen and the species to be treated. Modifications in the oxidation range and the time of application of reinforcement for a specific antigen and spice are easily determined by routine experimentation. The reinforcement can be antigen alone or in combination with mLT. The method of administration of the reinforcement can be oral, nasal, or parenteral; however, if mLT is used in the boost, administration is preferably oral. The methods and compositions of the present invention are intended to be used in both immature and mature vertebrates, specifically birds, mammals, and humans.
Useful antigens, by way of example and not with a limiting purpose, could include antigens of pathogenic strains of bacteria (Streptococcus pyogenes, Strpstococcus pneumoruae, Neisseria gonorrheae, Neissepa meningi t idis, Corynebacter lum diphtepae, Clostpdium boti l. Num, Clostpdium perfpngens, Clostridium tetam, Hemophilus influenzae, Klebsiella pneu oniae, Klebsiella ozaenae, Lebsiella rh moscleromotis, Staphylococcus aureus, Vibro col eva.es, Eschepchia coli, Pseudomonas aerugmosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocol it ic, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysentepae, Shigella flexnep, Shigella sannei, Salmanella typhimupu, Treponema pallidum, Treponema pertenue, Treponema carateneu, Borre! la vincentii, Borrelia burgdarfep, Leptospira icteroemorrhag iae, Mycobactepum tuberculosis, To ^ oplasma gondii, Pneumocystis capnii, Francisella tularensis, Brucella abortus, Bruc she suis, Br? cell elitensis, Mycoplasma spp., Rickettsia prowazek i, Rickettsia tsutsugu ushi, Chlamydia app.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blasto yces dermat 11 ídis, Cryptococcus n aformans, Histoplas a capsulatum); protozoapos (Entomoeba histolytica, Tricho onas tenas, Tpchomonas hominis, Tpchomonas vaginalis, Trypanssoma gambiense, Trypanosoma rhoderi íepse, Trypanasoma cruzi, Leish anodonovapi, Leish ania tropica, Leish ania bra_ il lensi, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or helminths (Enterobius vermí cul รís ,s, Tpchuris tpchiura, Ascaps lumbr icoides, Tp chine lia sp ira lis, Strongy laides stercoralis, Schistosus japonicu, Schistosoma ansoni, Schistosoma haem tobium, as well as anto lostos a), presented either to unological system in the form of whole cells or in isolated parts of cultures designed to culture said organisms that are well known in the art, or protective antigens that said organisms obtained by genetic engineering techniques or by qu synthesis. ic. Other relavant antigens are pathogenic viruses (by way of example, but not with a limitation: Poxvipdae, Herpes i pdae, H rpses Si plex viurs 1, Herpes Simplex virus 2, Adenovipdae, Papovavir idae, Enteroví pdae, Picornavir idae, Parvov go idae, Reovipdae, Petrov i ridae, influenza virus, influenzae virus, mumps, measles, respiratory syncytial virus, rubella, Arbovipdae, Rhabdovir idae, Arenav i pdae, hepatitis A virus, hepatitis B virus , hepatitis C irus, hepatitis E virus, hepatitis A virus / non-B virus, Rhinovirus ri, Coronary virus, Potoviridae, as well as human immunodeficiency virus) either presented to the immune system ica in its entirety or in part isolated from cultures designed to cultivate such viruses that are well known in the art or protective antigens thereof obtained by genetic engineering techniques or by chemical synthesis. Additional examples of relevant antigens include, but are not limited to, vaccines. Examples of such vaccines include, but are not limited to, gppa vaccines, pertussis vaccine, diphtheria and tetanus toxoid vaccine combined with whooping cough, hepatitis A vaccine, hepatitis B vaccine, vaccine against hepatitis C, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, vaccine against measles, rub wave vaccine, mumps vaccine, mixed vaccine against measles, mumps and rubella, vaccine against papi 1 lo avi rus , vaccine against parvovirus, vaccine against respiratory syncytial virus, vaccine against Ly disease, polio vaccine, vaccine against malaria, vaccine against vapcella, vaccine against gonorrhea, vaccine against HIV, vaccine against schistosamiasis, vaccine against broken, icoplasma vaccine, pneu ococcal vaccine, meningococcal vaccine and others. These can be produced by common known processes. In general terms, such vaccines comprise either the entire organism or the cultured and isolated virus by techniques well known to those skilled in the art, or comprise relevant antigens of these organisms or else they are produced by techniques. of genetic engineering ob in chemical synthesis. S <; < Production is illustrated by the following, but not limited to: Influenza vaccine: a vaccine that comprises all or part of the agglutamine, neuraminidase, nucleoprotein, and matrix protein that are obtained by purification of a vaccine. virus, which is grown in embryonated eggs, with ether and detergents, or by means of genetic engineering techniques or chemical synthesis. Pertussis vaccine: a vaccine that comprises all or part of the whooping cough toxin, hemagglutinin, K-agglucin that are obtained from a toxinin avilurente with formalin that is extracted by precipitation. application of salt or ul racentpfugación from the culture broth or from bacterial cells of Bordetella pertussis, or by genetic engineering techniques to good chemical synthesis. Vaccine against diftep. and tetanus toxoid combined with pertussis: a mixed vaccine with pertussis, diphtheria and tetanus toxoid vaccine. Vaccine against Japanese encephalitis: a vaccine comprising the entirety or phase of an antigenic protein obtained by the racemic culture of a virus in mice, and by purification of the virus particles by centrifugation or alcohol. ethyl alcohol and its deactivation, either through genetic engineering techniques or chemical synthesis.
Vaccine against hepatitis B: a vaccine comprising all or part of an antigen protein obtained by the isolation and purification of the HBs antigen by precipitation by application of salt or ul tracentry, obtained from the blood that carries hepatitis, or by techniques of genetic engineering or by chemical synthesis. Measles vaccine: a vaccine comprising all or part of a virus grown in cultured chicken cells either roe and bponado, or a protective antigen obtained by genetic engineering or by chemical synthesis. Rubella vaccine: a vaccine comprising all or part of a virus grown in cultured chicken embryo cells or embryonated egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis. Vaccine against mumps: a vaccine that comprises all or part of a virus grown in cultured rabbit cells or egg-tight, or a protective antigen obtained by genetic engineering techniques or chemical synthesis. Mixed vaccine against measles, rub wave and mumps: a vaccine produced by mixing measles, ruberola and mumps vaccine. Rat vaccine: a vaccine comprising all or part of an i cultured in cultured MA 104 cells either isolated from the feces of a patient, or a protective antigen obtained by genetic engineering techniques or chemical synthesis. Vaccine against i cap 1 asthma: a vaccine comprising all or part of "neoplasm" cells cultured in a liquid culture medium for mycaplasma or a protective antigen obtained by genetic engineering techniques or chemical synthesis. The conditions for which effective prevention can be achieved by means of the present method will be apparent to those skilled in the art. The vaccine preparation compositions of the present invention can be prepared by mixing the antigens illustrated above and / or vaccines with mLT in a desired ratio. The preparation must be carried out in an aseptic manner, and c < The component must also be handled aseptically. Pyrogens or allergens still naturally removed m_ > It's totally possible. The preparation of antigens of the present invention can be used by preparation of the perse and L antigen in particular. In addition, the present invention encompasses a set of implements comprising an effective amount of antigen and an adjuvant effective amount of mLT. In use, the components of the implement set can either be mixed together and then adminisd orally or the components can be adminisd orally separately within a short period of time between administration of them. The vaccine preparation compositions of the present invention may be combined with either a liquid or solid pharmaceutical carrier, and the compositions may be in the form of tablets, capsules, powders, granules, suspensions or solutions. The compositions may also contain suitable preservatives, coloring and flavoring agents, or agents that produce a slow release. Potential vehicles which may be employed in the preparation of the pharmaceutical compositions of this invention include, without limitation, gelatin capsules, sugars, cellulose derivates, for example sodium carboxymethylcellulose, gelatin, talc, magnesium stearate, vegetable oil. such as peanut oil, etc., gl icen na, sorbí oi, agar and wa The vehicles can also serve as binders to facilitate the formation of tablets of the compositions for convenient oral administration. The vaccine preparation composition of this invention can be maintained in a stable storage form for easy use by means of other methods well known to those skilled in the art. For oral admiration, the vaccine preparation can be reconstituted in the form of a suspension in a buffered saline solution, milk, or any other physiologically compatible liquid medium. The medium can be made more pleasant by the addition of suitable coloring and flavoring agents as desired. The administration of the vaccine preparation compositions can be preceded by an oral dosage of an effective amount of a gastric acid neutralizing agent. While vain composites can be used for this purpose, sodium bicarbonate is preferred. Alternatively, the vaccine compositions can be administered in enteric coated capsules (i.e., capsules that dissolve only after they have passed through the stomach). 6. EXAMPLE The following examples are presented for the purpose of illustrating only the present invention but not to limit the scope of this invention in any way.
6. 1 CONSTRUCTION OF mLT The wild-type LT toxin is encoded in a naturally occurring plasmid which is found in strains of E. coli enterotogenicum capable of producing this toxin. The present inventors have previously cloned the LT gene from a human isolate of E. coli designated H10407. This subclone consists of a 5.2 kb DNA fragment from the enterotoxin plasmid of H10407 inserted into the PstI site of plasmid pB322 (Clements et al., 1983, Infect.Immun.40: 653). This recombinant plasmid, called pDF82, has been extensively characterized and expresses LT under the control of the native LT promoter. The next step in this process was the placement of the LT gene under the control of a strong promoter, in this case, the lac promoter in the pUC18 plasmid. This was achieved by isolating the LT-A and LT-B genes separately and by recombining them in a cassette in the plasmid vector. This was an important step because it allowed for the purification of reasonable amounts of LT and the derivation of mutants for subsequent analysis. This plasmid, designated pDF94, is presented diagrammatically in FIG. 1. Both CT and LT are synthesized with a peptide bond sensitive to trypsin that binds the Al and A2 parts. This peptide bond must be cut so that the molecule is not "toxic". This is also true in the case of the diphtheria toxin, the prototypical toxin A-B, and for several other bacterial toxins. If the A1-A2 linkage is not removed, either by bacterial proteases or by intestinal proteases in the lumen of the intestine, that is, by processing or by proteolytic activation, the Alna part can reach its target in the lateral basal surface of the intestinal epithelial cell. In contrast to CT, LT is not fully biologically active when it is first isolated from the cell. LT also requires proteolysis to be fully active and proteolytic activation does not occur within the bacteria. Accordingly, one means of altering the toxicity of the molecule without affecting the enzymatic activity of ADP ribosylation would be the removal by genetic manipulation of the trypsin-sensitive amino acids that link the Al and A2 components of the A subunit. If the na molecule can be Proteol, which is completely dissociated, will not be toxic. One skilled in the art can predict that the molecule will nevertheless retain its enzymatic ADP-ribosylating activity and therefore its adjuvant function. Figure 1 shows the sequence of the region subtended by disulfide separating the Al and A2 parts. Within this region is an arginine residue that is considered to be the cleavage site necessary to activate the toxic properties of the molecule. This region was changed by site-directed mutagenesis in such a way that the molecule becomes insensitive to prateolitic digestion and, therefore, non-toxic. Site-directed mutagenesis is achieved by hybridization to single-stranded DNA of a synthetic synthetic system complementary to single-strand annealing except for a region that does not correspond near the center. It is this region that contains the change or the desired nucleotide changes. After hybridization with the single-stranded blank DNA, the oligonucleotide is extended with DNA polymerase to create a double-stranded structure. The cut is then sealed with DNA 1 igase and the duplex structure is transformed into an E. coli host. The theoretical yield of mutants using this procedure is 50 !. due to the way a DNA replication is made. However, in 3 practice, the performance is much lower. However, there are several methods available to increase the yield and to select mutants directed towards oligonucleotides. The system used employs a second mutagenic oligonucleotide to create altered restriction sites in a double mutation strategy. The next step was to substitute another amino acid for Arg Ies, GGA = Gly replaces AGA = Arg), thus preserving the reading structure while eminating the proteolytic. mLT was then purified by affinity chromatography on agarose from a mutant (pBD95) which had been confirmed by sequencing. Alternative purification methods will be apparent to those skilled in the art. The LT mutant, called LT (R192G), was then examined by electrophoresis on a gel of SDS-pol iacp lick for modification of the trypsin-sensitive link. Samples with and without trypsin exposure were examined and compared with native LT (unmodified). mLT is not divided1 into A1 and A2 when incubated with trypsin, indicating in this way that protease sensitivity has been eliminated. 6.2 EFFECT OF mLT ON ADRENAL CELLS Y-1 It could be predicted by a person skilled in the art that mLT would not be active in the Y-1 adrenal cell test. This prediction would be based on previous findings (Clements and Finkelstein, 1979, Infect, Im, 24: 760-769) according to which LT not cut was more than 1000 times less active in this test system than CT and LT activated by trypsin treatment at the same level of biological activity of CT in this assay. The residual LT activity observed in this assay in the absence of trypsin activation was considered to be dependent on some residual protease activity that could not be explained. For example, trypsin is used in the adrenal cell subculture process Y-1. Therefore it was considered that an LT that could not be cut would be totally inactive in the adrenal cell assay Y-1. The results appear in Tab3 to I. TABLE I TOXIN TRIPSIN ACTIVATED SPECIFIC ACTIVITY (a) cholera toxin - 15 LT - 60 LT + 15 LT (R1 2G) - 48,800 LT (R192G) + 48,800 (a) Minimum dose (picograms per well) required to produce cell rounding ( , >; 50 * / í). Table I demonstrates the unexpected finding that mLT retained a basal level of activity in the Y-1 adrenal cell assay, even though it could not be proteolytically processed. Camo is shown in Table I, CT and native LT treated with trypsin present the same activity level
(15 pg) in adrenal cells Y-l. In contrast, mLT (48,000 pg) was more than l-OOO times less active than CT or native LT and could not be activated with trypsin. The residual basal activity undoubtedly reflects a difference and a way to the unknown fehca of activation of adrenal cells in the pathway that requires separation of the Al - A2 bond. 6.3. ENZYMATIC ACTIVITY OF RJB0SILACIO DE ADP DE mLT Due to the fact that the mutation replacing Argl92 with Glyl92 does not alter the enzymatic site of 3rd Al portion, one skilled in the art could predict that mLT retains its enzymatic activity of ADP pbosylation. To examine this property, the ADP-ribos i 11 transferase assay of NAD-Agmaa 11 na was used (Moss et al., 1993, J. Bial, Chem. 268: 6383-6387). As can be seen in Figure 2, CT produces a dose-dependent increase in ADP levels -pbssi lagmat ina, a function of the ADP-p bosi 1 -ransferase activity of this molecule. TABLE II Activity ADP-pbosi 1 transf rasa of CT, native LT, and LT (R192G) Expér mint 1 2 3 4 mean ± SEM
Not toxic ND 9.12 5.63 14.17 9.64 + 2.48 llμμgg CCTT N NDD 17.81 17.60 25.75 20.39 ± 2.68 lOμg CT ND 107.32 111.28 104.04 107.55 ± 2.09 lO μg CT 3 35511..5Í5 361.73 308.09 ND 340.46 ± 16.45
3 0 μg LT 1 177..3: 2 14.48 13.86 ND 15.22 ± 1.07 lOOμg LT + 164.10 189.89 152.96 ND 168.98 ± 10.94 Trypsin lOOμg 14.58 12.34 9.30 ND 12.07 ± 1.53
lOOμg LT 14.73 8.90 10.47 ND 11.37 ± 1.74 (P192G) + tr i psi na ND - No data 11 z 6 data expressed in fMols m? n-1 Table II demonstrates in tabular form the unexpected finding that my T did not present any detectable ADP-ribosilant enzymatic activity, with or without trypsin activation, even though the enzyme site na had been altered and there was neither a demonstrable baseline of activity in the Yl adrenal cell assay. 6.4 ENTERTAQUE ACTIVITY ICA DE mLT Due to the unexpected finding that mLT has no detectable ADP-pbosylative enzymatic activity, with or without activation by trypsin, even when the enzymatic site has not been altered and the additional finding that there is a basal level of activity in the Yl adrenal cell assay, it was uncertain whether mLT could retain some of its enterotoxic properties. An ideal adjuvant formulation of mLT would retain its ability to act as an immune adjuvant but would not have the actual or potential side effects, such as diarrhea, associated with the use of LT or CT. Figure 3 shows that mLT does not induce a net fluid secretion in the mouse model, even at a dose of 125μg. This dose is more than 5 times higher than the effective dose of adjuvant for LT in this model. It is important to note that a potential toxicity of native LT can be seen in _ > e level.
41?
6. 5 ADJUVANT ACTIVITY OF mLT A person skilled in the art could predict that since mLT does not possess any demonstrable ADP-pbasi 1 transferase activity and is not enteric, na would have an adjuvant activity. This prediction would be based on the report of Lycle et al., (Lyche et al., 1992, Eur. J. Im unol. 22: 2277-2281) where it is clarified that alterations that affect the enzymatic activity ADP-pbosi lante of the toxin and alter the ability to increase the intracellular levels of cAMP also prevent the molecule from functioning as an adjuvant. As demonstrated above, mLT na has no ADP pbosylation enzymatic activity only some undefined basal activity in Y-1 adrenal cells, and does not induce any net secretion of fluid in the patent mouse model. To examine the adjuvant activity of mLT, the following experiment was carried out. Three groups of BALB / c mice were immunized. The animals were inoculated mtragástpca with a flat-tip feeding needle (Popper Sons, Inc., New Hyde Park, New York). On day 0, the group was orally immunized as follows: group A received 0.5 ml of PBS containing 5 mg of OVA, group B received 0.5 ml of PBS containing 5 mg of OVA and 25 μg of LT native, and group C received 0.5 ml of PBS containing 5 mg of OVA and 25 μg of mLT. Each regime was or
administered again on days 7 and 14. On day 21, all animals received an i.p. with 1 μg of OVA in Maalox 20 * / .. One week after inoculation í.p. the animals were sacrificed and assayed for serum IgG and mucosal IgA antibodies directed against OVA and LT by means of ELISA. ELISA reagents and antisera were obtained from Sigma Chemical Ca. Samples for ELISA were diluted in a phosphate-buffered saline solution (pH 7.2) -0.05 * /, of Tween 20 (PBS-TWEEN). To determine anti-LT ions, microtiter plates were pre-coated with 1.5 μg per well of mixed gangliosides (type III), then with 1 μg per well of purified LT. Anti-OVA was determined in pre-coated microtiter plates with 10 μg per well of OVA. An i-LT and serum OVA were determined with rabbit antiserum against mouse IgG conjugated with alkaline phosphatase. Anti-OVA and anti-LT mucosal IgA were tested with goat antiserum against mouse IgA 'specific for alpha chain) followed by rabbit antiserum against goat IgG conjugated with alkaline phosphatase. The reactions were suspended with 3N NaOH. Values for IgG and IgA were determined from a standard curve with purified mouse ieloma proteins (M0PC 315, gA (IgA12> M0PC 21, gGl: Litton Bionetics, Inc., Charleston, SC).
6. 5.1 ANTI-OVA SERUM IgG As shown in Figure 4A, animals primed orally with OVA and LT developed an anti-OVA IgG s? Pc response. significantly higher after subsequent μarenteral immunization with OVA (4058 μg / ml) than animals primed with OVA alone and subsequently immunized) parenterally with OVA (no detectable anti-OVA response) (Student's t test p = .031). Significantly, animals primed orally with OVA and mLT also developed a significantly higher serum IgG anti-OVA response after subsequent parenteral immunization with OVA (1338 μg / ml) than animals primed with OVA alone and subsequently immunized parenterally with OVA (no detectable anti-OVA response) (Student's t test p = .0007). 6.5.2 slGA ANTI-OVA MUCOSAL As shown in Figure 4B, similar results were obtained when anti-OVA IgA responses were compared within the same groups of animals. Animals primed orally with OVA and LT developed a significantly higher mucosal IgA anti-OVA response after subsequent parenteral immunization with OVA (869 ng / ml) than animals primed with OVA alone and subsequently immunized parenterally with OVA (none detectable anti-OVA response) (Student's t-test p-.0131). Co, or above, animals primed orally with OVA and mLT also developed an anti-OVA IgA mucous response! significantly higher after a subsequent parenteral immunization with OVA (230 pg / ml) than animals primed with OVA alone and subsequently immunized parenterally with OVA (no detectable anti-OVA response) (Student's t test p = .0189). 6.5.3 ANTI-LT SERIAL IgG The ability of IT and mLT to elicit an anticorporal anti-LT response in these same animals was also examined. This was important insofar as it would provide an indication of whether the mutant LT could impede the induction of tolerance towards itself in addition to functioning as an adjuvant for other proteins. As shown in Figure 5A, animals primed orally with OVA and LT developed a significantly higher serum IgG anti-LT response after a subsequent parenteral immunization with OVA (342 μg / ml) than animals primed with OVA alone and subsequently immunized parenterally with OVA (no detectable anti-LT response) (Student's t test p = .0005). Animals primed orally with OVA and mLT also developed a significantly higher serum IgG anti-LT response after subsequent parenteral immunization with OVA (552 μg / ml) than animals primed with OVA alone and subsequently immunized with OVA (none detectable anti-LT response) (Student's t test p = .0026). 6.5.4 sl A MUCOSAL ANTI-LT As shown in Figure 5B, similar results were obtained when anti-LT IgA responses were compared with the responses of the same groups of animals. Animals primed orally with OVA and LT developed a mucosal IgG anti-LT response if significantly higher after subsequent parenteral immunization with OVA (4,328 ng / ml) than those primed with OVA alone and subsequently immunized p renterally with OVA
(no detectable anti-LT response) (Student's t test p = .0047). As above, animals primed orally with OVA and mLT also developed a significantly higher mucosal IgA anti-LT response after subsequent parenteral immunization with OVA (1463 ng / ml) than animals primed with room OVA and subsequently immunized parenterally with OVA (no detectable anti-LT response) (Student's t test p = .0323). 7. MICROSOFT DEPOSIT ISMOS The following plasmid was deposited with the American Type Culture Collection (ATCC), Rockville, MD, on August 18, 1994, and has received the access number indicated: Plasmid Accession number pBD95 in E. coli LTR192G ATCC 69683 The invention described and claimed herein is not limited in scope to the specific embodiments presented herein since these embodiments are only for the purpose of illustrating various aspects of the invention. Any equivalent mode is within the scope of this invention. Further modifications to the present invention in addition to the embodiments presented and described herein will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to be within the scope of the appended claims. Is it debt? also understand that all numbers and sizes of base pairs and amino acid residues presented for nucleotides and peptides are approximate numbers and quantities and are used for the purpose of describing the present invention. Numerous references are mentioned herein, which are incorporated herein in their entirety, by reference.
International Application Number: PCT / / MICROORGANISMS Optional leaf in relation to the microorganism indicated on pages 32-33 lines 35-37 and 1-3 of the description (in the original document in English.) In the translation page 53 regones 22-25 and page 54 line 1) A. IDENTIFICATION OF THE DEPOSIT Additional deposits are identified in an additional sheet
Name of depository institution American Type Culture Collection Address of depository institution (including postal code and country) 12301 Park lawn Drive Rock vi, ND 20852 US Deposit date: August 18, 1994 Accession number: 69683 B. ADDITIONAL INDICATIONS (Leave blank if they are not applicable). This information continues on a separate attached sheet: - C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE:
D. SUPPLY SEPAPADO DF INDICATIONS (leave blank if it is not applicable): -The indications that appear in the following list will be submitted to the International Bureau.
(Specify the general nature of the information, for example "deposit access number"): - E () This sheet was received with the International application l 5 when it was submitted (to be reviewed by the receiving office) Mrs. Rivera, Specialist For legal PCT Receiving Office (708) 305 3185 10 (Signed) (Authorized Official) () The date of reception ** (of the applicant) by the Office
International was: - (Ofici l autori ado) 15 Format PCT / PO / 134 (January 1981)
.OR
_-
Claims (25)
- CLAIMS 1. A composition comprising a mutant form of a bacterial enterotoxin, halotoxin which has a reduced toxicity in an effective adjuvant dose, but retains its adjuvant and ammunogenic activity.
- 2. The composition of the rei indication 1, which is rendered insensitive to proteolytic activation.
- 3. The composition of claim 1, which is a tantalum form of a thermolabile enterotoxin of E. eoli, holotoxin which is. distinct from the native LT to the extent that it has an adjuvant immunological activity but a reduced ADP riboscopy activity.
- 4. The composition of claim t, which is a mutant form of a heat-labile enterotole of E. coli, holotoxin that is distinct from native LT to the extent that the A subunit of holotoxin is rendered insensitive to activation proteolytic
- 5. The composition of the rei indication 1, which is encoded by the plasmid pE * D95 contained in E. coli LTP192G having the accession number of ATCC 69683, which expresses both the 3a subunit A and the subunit B of the enterotox ina olilabil of E. coli.
- 6. A vaccine preparation comprising an antigen in combination with the composition in accordance with 3 rd indication 1.
- 7. An oral vaccine preparation comprising an antigen in combination with the composition in accordance with claim 1.
- 8. The vaccine preparation of claim 6 or 7, wherein the antigen is a bacterial antigen of selected pathogenic bacteria within the group consisting of Streptococcus spp., Neissepa spμ., Corynebacter íu spp., Clostndium spp., Hemophilus spp., Lebsiella spp., Staphylococcus spp., Vibrio spp., Esche ichi spp., Pseudomonas spp., Campylabacter spp., Aeromonas spp., Bacillus spp., Edwardsiella spp., Yersinia spp., Shigella spp., Sal onella spp., Treponema spp., Borrelia spp., Leptospira spp., Mycobactenum spp., Toxoplasma spp., Pneu ocystis spp., Francisella spp., Brucella spp., Mycoplasma spp., Pickettsia spp., And Chlamydia spp.
- 9. The vaccine preparation of claim 6 or 7, wherein the antigen is selected from the group consisting of influenza vaccine, varicella vaccine, diphtheria toxoid, tetanus toxoid, pertussis vaccine, Japanese encephalitis vaccine, mixed pertussis vaccine, diphtheria toxaide and tetanus toxoid, vaccine against Ly disease, polio vaccine, malaria vaccine, herpes vaccine, HIV vaccine, vaccine against daddy 1 lomaví rus, hepatitis B vaccine, rotavirus vaccine, Ca p lobac ter vaccine, cholera vaccine, toxicogenic E. coli enterop vaccine, E. coli and enterotoxin vaccine, Salmonella vaccine, Shigella vaccine, vaccine against schistosomiasis, measles vaccine, rubella vaccine, mumps vaccine, combination vaccine against measles, rubella and mumps, and vaccine against mycoplasma.
- 10. A composition useful for the production of a protective immunogenic response against a pathogen in a host comprising a mixture of an effective amount of an antigen and an effective amount of an adjuvant of the composition in accordance with the indication.
- 11. An oral composition useful for the production of a protective immune response against a pathogen in a host, comprising a mixture of an effective amount of an antigen and an effective adjuvant amount of the composition according to claim 1.
- 12 A mutant enteroxyma, according to claim 1, for use in medicine.
- 13. A set of elements useful for producing a protective immune response in a host against a pathogen comprising two components: (a) an effective amount of antigen and (b) an effective adjuvant amount of a heat-inactivated enterotole of E. coli. i mutant, holotoxin that is distinct from LT and native CT to the extent that the A subunit of the holoto ina has been rendered insensitive to proteolytic activation, but holotoxin retains its immunological adjuvant activity, where both components are in an orally acceptable vehicle and said components can be administered either after they have been mixed together or separately within a short period of time between said administrations.
- 14. The composition of claim 1 or 10, wherein the amino acid Argl92 of the thermolabile enterotoxin of E. cali de tipa salvaje is replaced by Glyl92.
- 15. The vaccine preparation in accordance with rei indication 6 or 7, wherein the amino acid Argl92 of the thermolabile ertterotaxin of wild-type E. coli is replaced by Glyl92.
- 16. The set of elements of claim 13, wherein the amino acid Argl92 of the thermolabile enterotaxin of wild-type E. cali is replaced by Glyl92.
- 17. A method for creating or maintaining a protective immunological response or adaptation to an antigen in a host, comprising orally administering a mixture of an effective amount of the antigen and an effective adjuvant amount of a thermolabile enterotaxin of mutant E. coli, holoto i na which is distinct from LT and native CT to the extent that the A subunit of holotoxin has been rendered insensitive to proteolytic activation, but holotoxin retains its immunological adjuvant activity in an orally acceptable pharmaceutical carrier.
- 18. The method of claim 17, wherein a serum response is produced.
- 19. The method of claim 17, wherein a mucosal response occurs.
- The method of claim 17, wherein a subsequent reinforcement of antigen is provided.
- 21. The method of claim 17, wherein the antigen is derived from the group consisting of bacteria, viruses, protozanes, fungi, helminths, and other microrbic pathogens.
- 22. The method of claim 17, wherein the mixture is delivered in a single dose.
- 23. A method for the induction of a protective immune response against a enterotoxic bacterial organism comprising the use of mLT as a component of a vaccine targeted against the enterotoxic bacterial organism.
- The method of claim 23, wherein the enterotoxic bacterial organism is selected from the group consisting of bacterial enterotoxic organisms that express cholera-like toxins.
- 25. The method of claim 24, wherein the enterotoxic bacterial organism is selected from the group consisting of Eschepchia spp. and Vibrio spp.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08296848 | 1994-08-26 | ||
US08/296,848 US6019982A (en) | 1994-08-26 | 1994-08-26 | Mutant enterotoxin effective as a non-toxic oral adjuvant |
PCT/US1995/009005 WO1996006627A1 (en) | 1994-08-26 | 1995-07-18 | Mutant enterotoxin effective as a non-toxic oral adjuvant |
Publications (2)
Publication Number | Publication Date |
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MX9701445A MX9701445A (en) | 1998-05-31 |
MXPA97001445A true MXPA97001445A (en) | 1998-10-23 |
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