Mutant enterotoxin effective as a non-toxic oral adjuvant

Methods and compositions are provided herein for the use of a novel mutant form of E. coli heat-labile enterotoxin which has lost its toxicity but has retained its immunologic 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 an oral vaccine preparation.

1. FIELD OF THE INVENTION 
The present invention is directed towards a genetically distinct mutant of 
E. coli heat-labile enterotoxin (LT) and its use as an oral adjuvant to 
induce mucosal and serum antibodies. Specifically, the mutant LT is 
modified by a single amino acid substitution that abolishes 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, N.Y. 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 are important for effective 
vaccines. 
Oral 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 oral vaccines and oral immunization for protection 
against pathogenic organisms that initiate infection by first interacting 
with mucosal surfaces. 
A number of strategies have been developed for oral immunization, including 
the use of attenuated mutants of bacteria (i.e., Salmonella spp.) as 
carriers of heterologous antigens [Cardenas 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 Cardenas, 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 [Sanitago 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 oral 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.sub.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 subunits, 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 
orally. This finding is not new in that cholera patients are known to 
develop rises in titers of antitoxin antibodies during convalescence from 
clinical cholera [Finkelstein, 1975, Curr. Top. Microbiol. Immunol. 
69:137-196]. One key finding of those investigating the nature of this 
response was the observation that CT, unlike most other protein antigens, 
does not induce oral tolerance against itself [Elson and Ealding, 1984, J. 
Immunol. 133:2892-2897; Elson and Ealding, 1984, J. Immunol. 
132:2736-2741]. This was also found to be true when just the B-subunit was 
fed to mice, an observation substantiated by the cholera vaccine field 
trials in Bangladesh in which oral immunization with B-subunit combined 
with killed whole cells gave rise to mucosal as well as systemic antitoxin 
antibody responses [Svennerholm et al., 1984 J. Infect. Dis. 149:884-893]. 
In addition to being a potent oral immunogen, CT has a number of other 
reported immunologic properties. 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 itself. Moreover, 
simultaneous oral administration of CT with a soluble protein antigen, 
keyhole limpet hemocyanin (KLH), resulted in the development of secretory 
IgA responses against both CT and KLH and also abrogated the induction of 
oral tolerance against KLH. These findings were subsequently confirmed and 
extended by Lycke and Holmgren [Lycke and Holmgren, 1986, Immunology 
59:301-308]. The confusion arises when one attempts to define the role of 
the A and B subunits of CT with respect to the adjuvant properties of the 
molecule. The following observations, as summarized by Elson [Elson, 1989, 
Immunology Today 146:29-33], are the basis for that confusion: 
CT does not induce oral tolerance against itself [Elson and Ealding, 1984, 
J. Immunol. 133:2892-2897]. 
CT-B does not induce oral tolerance against itself [Elson and Ealding, 
1984, J. Immunol. 133:2892-2897]. 
CT can prevent the induction of tolerance against other antigens with which 
it is simultaneously delivered and also serve as an adjuvant for those 
antigens [Elson and Ealding, 1984, J. Immunol. 133:2892-2897; Lycke and 
Holmgren, 1986, Immunology 59:301-308]. 
CT can act as an adjuvant for CT-B [Elson and Ealding, 1984, J. Immunol. 
133:2892-2897]. 
Heat aggregated CT has little toxicity but is a potent oral immunogen 
[Pierce et al., 1983, Infect. Immun. 40:1112-1118]. 
CT-B can serve as an immunologic "carrier" in a traditional hapten-carrier 
configuration [Cebra at al., 1986, In: Vaccines 86, Brown et al. (ed.), 
Cold Spring Harbor Laboratory, New York. p.p. 129-133; McKenzie and 
Halsey, 1984, J. Immunol. 133:1818-1824]. 
A number of researchers have concluded 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 Spring Harbor Laboratory, New York. p.p. 129-133], Lycke and Holmgren 
[Lycke and Holmgren, 1986, Immunology 59:301-308], and Liang et al. [Liang 
et al., 1988, J. Immunol. 141:1495-1501] would argue against that 
conclusion. Cebra et al. [Cebra et al., 1986, In: Vaccines 86, Brown et 
al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133] 
demonstrated that purified CT-B was effective at raising the frequency of 
specific anti-cholera toxin B-cells in Peyer's patches when given 
intraduodenally but, in contrast to CT, did not result in significant 
numbers of IgA committed B-cells. Lycke and Holmgren [Lycke and Holmgren, 
1986, Immunology 59:301-308] compared CT and CT-B for the ability to 
enhance the gut mucosal immune response to KLH by measuring immunoglobulin 
secreting cells in the lamina propria of orally immunized mice. They found 
no increase in anti-KLH producing cells in response to any dose of 
B-subunit tested in their system. Finally, Liang et al. [Liang et al., 
1988, J. Immunol. 141:1495-1501] found no adjuvant effect when CT-B was 
administered orally in conjunction with inactivated Sendai virus. 
Where adjuvant activity has been observed for isolated B-subunit, it has 
typically been for one of two reasons. First, a traditional method of 
preparing B-subunit has been to subject holotoxin to dissociation 
chromatography by gel filtration in the presence of a dissociating agent 
(i.e., guanidine HCl or formic acid). The isolated subunits are then 
pooled and the dissociating agent removed. B-subunit prepared by this 
technique is invariably contaminated with trace amounts of A-subunit such 
that upon renaturation a small amount of holotoxin is reconstituted. The 
second reason has to do with the definition of an immunologic carrier. 
Like many other soluble proteins, B-subunit can serve as an immunologic 
vehicle for presentation of antigens to the immune system. If those 
antigens are sufficiently small as to be poorly immunogenic, they can be 
made immunogenic in a traditional hapten-carrier configuration. Likewise, 
there is a "theoretical" immune enhancement associated with B-subunit, 
especially for oral presentation, in that B-subunit binds to the surface 
of epithelial cells and may immobilize an attached antigen for processing 
by the gut associated lymphoid tissues. However, any potential advantage 
to this mechanism of antigen stabilization may be offset by the 
distribution of the antigen across non-immunologically relevant tissues, 
i.e., the surface of intestinal epithelial cells. In context of the 
mucosal responsiveness, the immunologically relevant sites are the Peyer's 
patches, especially for antigen-specific T cell-dependent B cell 
activation [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; Brandtzaeg, 1989, Curr. Top. Microbiol. Immunol. 
146:13-25]. Thus, the events up to isotype switching from IgM cells to IgA 
B-cells occurs in the Peyer's patches. Antigens localized on the 
epithelial cell surface may contribute to antigen induced B cell 
proliferation in that the class II positive villous epithelial cells may 
act as antigen presenting cells for T cell activation at the secretory 
site, thereby increasing cytokine production, terminal B cell 
differentiation, increased expression of secretory component, and 
increased external transport of antigen specific IgA [Tomasi, T. B., and 
A. G. 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 relationships of these events have not been clearly defined 
for B-subunit as a carrier of other antigens and use of the term 
"adjuvant" would seem inappropriate for such an effect. 
It is clear that the adjuvant property of the molecule resides in the 
holotoxin in which B-subunit is required for receptor recognition and to 
facilitate penetration of the A-subunit into the cell. The A-subunit is 
also required for adjuvant activity, presumably as a function of its 
ADP-ribosylating enzymatic activity and ability to increase intracellular 
levels of cAMP (see below). The B-subunit alone may act as a carrier of 
other antigens in that when conjugated to those antigens they can be 
immobilized for processing by the gut associated lymphoid tissues. 
Although LT and CT have many features in common, these are clearly distinct 
molecules with biochemical and immunologic differences which make them 
unique, including a 20% difference in nucleotide and amino acid sequence 
homology [Dallas and Falkow, 1980, Nature 288:499-501]. The two toxins 
have the same subunit number and arrangement, same biological mechanism of 
action, and the same specific activity in many in vitro assays [Clements 
and Finkelstein, 1979, Infect. Immun. 24:760-769; Clements et al., 1980, 
Infect. Immun. 24:91-97]. 
There are, however, 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 cell associated and is only released from E. coli during cell 
lysis [Clements and Finkelstein, 1979, Infect. Immun. 24:760-769]. CT is 
secreted from the vibrio as soon as it is synthesized and can be readily 
identified in, and purified from, culture supernatants. Consequently, in 
contrast to CT, LT is not fully biologically active when first isolated 
from the cell. Consistent with the A-B model for bacterial toxins, LT 
requires proteolysis and disulfide reduction to be fully active. In the 
absence of proteolytic processing, the enzymatically active A.sub.1 moiety 
is unable to dissociate from the A.sub.2 component and cannot reach its 
target substrate (adenylate cyclase) on the basolateral surface of the 
intestinal epithelial cell. This is also true for CT, but proteases in the 
culture supernatant, to which the toxin is exposed during purification, 
perform the proteolysis. Since LT is not fully biologically active, it is 
difficult to identify during purification using in vitro biological assays 
such as the Y-1 adrenal cell assay or permeability factor assay. 
This difference in activation of the isolated material results in 
differences in response thresholds for LT and CT in biologic systems. For 
instance, CT induces detectable net fluid secretion in the mouse intestine 
at a dose of 5-10 .mu.g. LT induces detectable net secretion in the mouse 
intestine at levels above 100 .mu.g. In the rabbit ligated ileal loop, the 
difference is dramatic and clear cut. Moreover, in primates LT has been 
shown not to induce fluid secretion at any dose tested up to 1 milligram. 
This is 200 times the amount of CT reported to induce positive fluid 
movement in humans. When LT is exposed to proteolytic enzymes with 
trypsin-like specificity, the molecule becomes indistinguishable from CT 
in any biologic assay system. This was demonstrated clearly by Clements 
and Finkelstein [Clements and Finkelstein, 1979, Infect. Immun. 
24:760-769]. 
In addition to the above reported differences, LT has an unusual affinity 
for carbohydrate containing matrices. Specifically, LT, with a molecular 
weight of 90,000, elutes from Sephadex columns (glucose) with an apparent 
molecular weight of 45,000 and from Agarose columns (galactose) with an 
apparent molecular weight of 0. That is, it binds to galactose containing 
matrices and can be eluted from those matrices in pure form by application 
of galactose. LT binds not only to agarose in columns used for 
purification, but more importantly, to other biological molecules 
containing galactose, including glycoproteins and lipopolysaccharides. 
This lectin-like binding property of LT results in a broader receptor 
distribution on mammalian cells for LT than for CT which binds only to 
G.sub.M1. This may account in part for the reported differences in the 
abilities of these two molecules to induce different helper T lymphocyte 
responses [McGhee et al., 1994, Mucosal Immunology Update, Spring 1994, 
Raven Press, New York. p. 21]. 
In these studies reported by McGhee et al. [McGhee et al., 1994, Mucosal 
Immunology Update, Spring 1994, Raven Press, New York. p. 21], it was 
shown that oral immunization of mice with vaccines such as tetanus toxoid 
(TT) with CT as a mucosal adjuvant selectively induces T.sub.H 2 type 
cells in Peyer's patches and spleens as manifested by TH cells which 
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 
enhanced antigen-specific IgE responses in addition to the IgA response. 
Such enhancement of IgE responses seriously compromises the safety of CT 
as a mucosal adjuvant due to the prospect of inducing immediate-type 
hypersensitivity reactions. In contrast, LT induces both T.sub.H 1 and 
T.sub.H 2 cells and predominantly antigen-specific IgA responses without 
IgE responses when used as an orally administered mucosal adjuvant. 
The two molecules also have many immunologic differences, as demonstrated 
by immunodiffusion studies [Clements and Finkelstein, 1978, Infect. Immun. 
21:1036-1039; Clements and Finkelstein, 1978, Infect. Immun. 22:709-713], 
in vitro neutralization studies, and the partial protection against LT 
associated E. coli diarrhea in volunteers receiving B-subunit whole cell 
cholera vaccine [Clemens et al., 1988, J. Infect. Dis. 158:372-377]. 
Taken together, these findings demonstrate that LT and CT are unique 
molecules, despite their apparent similarities, and that LT is a practical 
oral adjuvant while CT is not. 
The demonstration of the adjuvant properties of LT grew out of an 
investigation of the influence of LT on the development of tolerance to 
orally administered antigens by one of the present inventors. It was not 
clear whether or not LT would also influence the induction of oral 
tolerance or exhibit the adjuvant effects demonstrated for CT, given the 
observed differences between the two molecules. Consequently, the present 
inventors examined a number of parameters, including the effect of LT on 
oral tolerance to OVA and the role of the two subunits of LT in the 
observed response, the effect of varying the timing and route of delivery 
of LT, the effect of prior exposure to OVA on the ability of LT to 
influence tolerance to OVA, the use of LT as an adjuvant with two 
unrelated antigens, and the effect of route of immunization on anti-OVA 
responses. The results obtained from these studies [Clements et al., 1988, 
Vaccine 6:269-277; Clements et al., 1988, Abstract No. B91, 88th Ann. 
Meet. Am. Soc. Microbiol.] are summarized below: 
1. Simultaneous administration of LT with OVA was shown to prevent the 
induction of tolerance to OVA and to increase the serum anti-OVA IgG 
response 30 to 90 fold over OVA primed and PBS primed animals, 
respectively. This effect was determined to be a function of the 
enzymatically active A-subunit of the toxin since the B-subunit alone was 
unable to influence tolerance induction. 
2. Animals fed LT with OVA after an initial OVA prime developed a 
significantly lower serum IgG and mucosal IgA anti-OVA response than those 
fed LT with OVA in the initial immunization, indicating that prior 
exposure to the antigen reduces the effectiveness of LT to influence 
tolerance and its ability to act as an adjuvant. LT was not able to 
abrogate tolerance once it had been established. This was also found to be 
true for CT when animals were pre-immunized with OVA prior to oral 
ovalbumin plus CT and offers some insight into the beneficial observation 
that antibody responses to nontarget dietary antigens are not increased 
when these adjuvants are used. 
3. Serum IgG and mucosal IgA responses in animals receiving LT on only a 
single occasion, that being upon first exposure to antigen, were 
equivalent to responses after three OVA/LT primes, indicating that 
commitment to responsiveness occurs early and upon first exposure to 
antigen. It was also demonstrated that the direction of the response to 
either predominantly serum IgG or mucosal IgA can be controlled by whether 
or not a parenteral booster dose is administered. 
4. Simultaneous administration of LT with two soluble protein antigens 
results in development of serum and mucosal antibodies against both 
antigens if the animal has no prior immunologic experience with either. 
This was an important finding since one possible application of LT as an 
adjuvant would be for the development of mucosal antibodies against 
complex antigens, such as killed bacteria or viruses, where the ability to 
respond to multiple antigens would be important. 
Studies by Tamura et al., [Tamura et al., U.S. Pat. No. 5,182,109] 
demonstrated that LT and/or CT administered intranasally enhanced the 
antibody titer against a co-administered antigen. However, nowhere in 
Tamura et al. is it taught that these toxins can induce a protective 
immune response when administered orally. 
Clearly, LT has significant immunoregulatory potential, both as a means of 
preventing the induction of tolerance to specific antigens and as an 
adjuvant for orally administered antigens and it elicits the production of 
both serum IgG and mucosal IgA against antigens with which it is 
delivered. This raises the possibility of an effective immunization 
program against a variety of pathogens involving the oral administration 
of killed or attenuated agents or relevant virulence determinants of 
specific agents. However, the fact that this "toxin" can stimulate a net 
lumenal secretory response when proteolytically cleaved, as by gut 
proteases, or when administered in high enough concentrations orally, may 
hinder investigation into its potential or prevent its use under 
appropriate conditions. This problem could be resolved if LT could be 
"detoxified" without diminishing the adjuvant properties of the molecules. 
In order to appreciate how this might be accomplished, it is necessary to 
further analyze the mechanism of action of the LT and CT and the 
structural and functional relationships of these molecules. As indicated 
previously, both LT and CT are synthesized as multisubunit toxins with A 
and B components. After the initial interaction of the toxin with the host 
cell membrane receptor, the B region facilitates the penetration of the 
A-subunit through the cell membrane. On thiol reduction, this A component 
dissociates into two smaller polypeptide chains. One of these, the A.sub.1 
piece, catalyzes the ADP-ribosylation of the stimulatory GTP-binding 
protein (G.sub.S) in the adenylate cyclase enzyme complex on the 
basolateral surface of the epithelial cell and this results in increasing 
intracellular levels of cAMP. The resulting increase in cAMP causes 
secretion of water and electrolytes into the small intestine through 
interaction with two cAMP-sensitive ion transport mechanisms involving 1) 
NaCl co-transport across the brush border of villous epithelial cells, and 
2) electrogenic Na.sup.+ dependent Cl.sup.- secretion by crypt cells 
[Field, 1980, In: Secretory diarrhea, Field et al. (ed.), Waverly Press, 
Baltimore. p.21-30]. The A subunit is also the principal moiety associated 
with immune enhancement by these toxins. This subunit then becomes a 
likely target for manipulation in order to dissociate the toxic and 
immunologic functions of the molecules. A recent report by Lycke et al. 
[Lycke et al., 1992, Eur. J. Immunol. 22:2277-2281] makes it clear that 
alterations that affect the ADP-ribosylating enzymatic activity of the 
toxin and alter the ability to increase intracellular levels of cAMP also 
prevent the molecule from functioning as an adjuvant. Consequently, 
another approach to detoxification must be explored. 
3. SUMMARY 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 is devoid of 
ADP-ribosyltransferase activity, still retains its activity as an 
immunological adjuvant. The mutant form of LT differs from the wild-type 
by a single amino acid substitution, Arg.sub.192 -Gly.sub.192, rendering a 
trypsin sensitive site insensitive. The loss of the proteolytic site 
prevents the proteolytic processing of the A subunit into its toxic form. 
Native LT is not toxic when first isolated from the bacterium but has the 
potential to be fully toxic when exposed to proteases such as those found 
in the mammalian intestine. The mutant form of LT no longer has the 
potential to become toxic due to proteolytic activation. This mutant LT 
(hereinafter mLT) retains the capability of enhancing an animal's immune 
response (e.g., IgG, IgA) to an antigen unrelated to LT or mLT with no 
toxic side effects. Experimental evidence shows that mLT has utility as an 
adjuvant for orally administered antigens; such administration results in 
the production of serum IgG and/or mucosal sIgA against the antigen with 
which the mLT is delivered. The present invention provides a method for 
induction of a serum and/or mucosal immune response in a host to any 
orally administered antigen which comprises administering to the host an 
effective amount of mLT in conjunction with oral administration of an 
effective amount of the antigen. Preferably, the antigen and the mLT are 
administered initially in a simultaneous dose. 
The present method and compositions provide an improved mode of oral 
immunization for development of serum and mucosal antibodies against 
pathogenic microorganisms. Production of IgA antibody responses against 
pathogenic microorganisms which penetrate or invade across mucosal 
surfaces can be directed to that surface, while a significant serum 
antibody response can be developed to prevent infection by pathogenic 
microorganisms against which serum antibody is protective. The present 
invention is useful for any specific antigen where a specific neutralizing 
antibody response would be useful in ablating the physiological or disease 
state associated with that antigen. 
The present invention also provides a composition useful as a component of 
a vaccine against enterotoxic bacterial organisms expressing cholera-like 
enterotoxins and methods for its 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.

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 
that are simultaneously orally administered with a genetically modified 
bacterial toxin. The modified toxin is a form of the heat-labile 
anterotoxin (LT) of E. coli which through genetic engineering has lost its 
trypsin sensitive site rendering the molecule non-toxic but yet, 
unexpectedly, retains its ability to act as an immunological adjuvant. The 
mutant LT is herein termed "mLT". The invention is based on the discovery 
that mLT is as effective as LT as an immunological adjuvant, an unexpected 
and surprising result. mLT no longer has the enzymatic activity of 
ADP-ribosylation because the A subunit can no longer be proteolytically 
processed. In contrast to published studies of Lycke and colleagues, which 
made it clear that alterations that effect the ADP-ribosylating activity 
of LT also prevent the molecule from functioning as an immunologic 
adjuvant [Lycke et al., 1992, Eur. J. Immunol. 22:2277-2281], the 
presently described mLT retains activity as an immunological adjuvant 
although, as demonstrated in the examples, it does not have 
ADP-ribosylating activity. 
The novel mutant form of the heat-labile enterotoxin of E. coli, mLT, 
described herein, behaves as an adjuvant and elicits the production of 
both serum IgG and mucosal sIgA against antigens with which it is 
delivered. The utility of this surprising discovery is that an adjuvant 
effective amount of mLT may be utilized in an effective immunization 
program against a variety of pathogens involving the oral administration 
of an effective amount of mLT adjuvant in admixture with killed or 
attenuated pathogens or relevant virulence determinants of specific 
pathogens with no fear of the real or potential toxic side-effects 
associated with oral administration of CT or LT. 
The present invention supersedes the prior art, in that the present 
invention may be used in a variety of immunological applications where CT, 
LT, or subunits of CT or LT may have been used, but now with mLT there are 
no real or potential side-effects, such as diarrhea, associated with its 
use. In contrast to LT, which although not toxic when first isolated from 
the bacterium, has the potential to be fully toxic when exposed to 
proteases such as those found in the mammalian intestine, mLT does not 
have the potential to become toxic due to proteolytic activation. 
Another embodiment of the present invention is as a component of a vaccine 
against enterotoxic organisms which express cholera-like toxins. The 
present inventors have shown that mLT is not subject to orally induced 
immune tolerance when administered (see below), therefore mLT can function 
and is highly desired as a component of vaccines directed against 
enterotoxic organisms. Current technology provides for vaccines against 
cholera-like toxin expressing organisms containing killed whole cells and 
the B subunit of the toxin. By replacing the B subunit 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 
to the B subunit but to the A subunit as well. This provides for more 
epitopes for effective neutralization. Second, the adjuvant activity 
inherent in mLT will enhance the immune response against the killed whole 
cell component of the vaccine. 
Further, other investigators [Hase et. al., 1994, Infect. Immun. 
62:3051-3057] have shown that the A subunit, modified so that it is no 
longer toxic by altering the active site of the ADP-ribosylating enzymatic 
activity, (as opposed to the proteolytic site which is the subject of the 
current invention) can induce an immune response against the wild type A 
subunit. However, the A subunit so modified now lacks immunologic adjuvant 
activity and is therefore less desirable as a vaccine component than mLT. 
Moreover, since antibodies against mLT cross-react with LT and CT, mLT can 
be used in vaccines directed against many types of enterotoxic bacterial 
organisms that express cholera-like toxins, such as Escherichia spp. and 
Vibrio spp. 
5.1 PRODUCTION OF mLT 
The wild-type LT toxin is encoded on a naturally occurring plasmid found in 
strains of enterotoxigenic E. coli capable of producing this toxin. The 
present inventors had 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 pBR322 [Clements et al, 1983, Infect. Immun. 40:653]. This 
recombinant plasmid, designated pDF82, has been extensively characterized 
and expresses LT under control of the native LT promoter. The next step in 
this process was to place the LT gene under the control of a strong 
promoter, in this case the lac promoter on plasmid pUC18. This was 
accomplished by isolating the genes for LT-A and LT-B separately and 
recombining them in a cassette in the vector plasmid. This was an 
important step because it permitted purification of reasonable quantities 
of LT and derived mutants for subsequent analysis. This plasmid, 
designated pBD94, is shown diagrammatically in FIG. 1. 
Both CT and LT are synthesized with a trypsin sensitive peptide bond that 
joins the A.sub.1 and A.sub.2 pieces. This peptide bond must be nicked for 
the molecule to be "toxic". This is also true for diphtheria toxin, the 
prototypic A-B toxin, and for a variety of other bacterial toxins. If the 
A.sub.1 -A.sub.2 bond is not removed, either by bacterial proteases or 
intestinal proteases in the lumen of the bowel, the A.sub.1 piece cannot 
reach its target on the basolateral surface of the intestinal epithelial 
cell. In contrast to CT, LT is not fully biologically active when first 
isolated from the cell. LT also requires proteolysis to be fully active 
and the proteolytic activation does not occur inside of the bacterium. 
Therefore, one means of altering the toxicity of the molecule without 
affecting the ADP-ribosylating enzymatic activity would be to remove by 
genetic manipulation the trypsin sensitive amino acids that join the 
A.sub.1 and A.sub.2 components of the A subunit. If the molecule cannot be 
proteolytically cleaved, it will not be toxic. One skilled in the art 
would predict that the molecule should, however, retain its 
ADP-ribosylating enzymatic activity and consequently, its adjuvant 
function. 
FIG. 1 shows the sequence of the disulfide subtended region that separates 
the A.sub.1 and A.sub.2 pieces. Within this region is a single Arginine 
residue which is believed to be the site of cleavage necessary to activate 
the toxic properties of the molecule. This region was changed by 
site-directed mutagenesis in such a way as to render the molecule 
insensitive to proteolytic digestion and, consequently, nontoxic. 
Site-directed mutagenesis is accomplished by hybridizing to single stranded 
DNA a synthetic oligonucleotide which is complementary to the single 
stranded template except for a region of mismatch near then center. It is 
this region that contains the desired nucleotide change or changes 
Following hybridization with the single stranded target DNA, the 
oligonucleotide is extended with DNA polymerase to create a double 
stranded structure. The nick is then sealed with DNA ligase and the duplex 
structure is transformed into an E. coli host. The theoretical yield of 
mutants using this procedure is 50% due to the semi-conservative mode of 
DNA replication. In practice, the yield is much lower. There are, however, 
a number of methods available to improve yield and to select for 
oligonucleotide directed mutants. The system employed utilized 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 (i.e., GGA=Gly 
replaces AGA=Arg), thus preserving the reading frame while eliminating the 
proteolytic site. mLT was then purified by agarose affinity chromatography 
from one mutant (pBD95) which had been confirmed by sequencing. Alternate 
methods of purification will be apparent to those skilled in the art. This 
mutant LT, designated LT.sub.(R192G) was then examined by 
SDS-polyacrylamide gel electrophoresis for modification of the trypsin 
sensitive bond. Samples were examined with and without exposure to trypsin 
and compared with native (unmodified) LT. mLT does not dissociate into 
A.sub.1 and A.sub.2 when incubated with trypsin, thereby indicating that 
sensitivity to protease has been removed. 
5.2 MODE OF ADMINISTRATION OF mLT AND UNRELATED ANTIGENS 
In accordance with the present invention, mLT can be administered in 
conjunction with any biologically relevant antigen and/or vaccine, such 
that an increased immune response to said antigen and/or vaccine is 
achieved. In a preferred embodiment, the mLT and antigen are administered 
simultaneously in a pharmaceutical composition comprising an effective 
amount of mLT and an effective amount of antigen. The mode of 
administration is oral. The respective amounts of mLT and antigen will 
vary depending upon the identity of the antigen employed and the species 
of animal to be immunized. In one embodiment, the initial administration 
of mLT and antigen is followed by a boost of the relevant antigen. In 
another embodiment no boost is given. The timing of boosting may vary, 
depending on the antigen and the species being treated. The modifications 
in dosage range and timing of boosting for any given species and antigen 
are readily determinable by routine experimentation. The boost may be of 
antigen alone or in combination with mLT. The mode of administration of 
the boost may either be oral, nasal, or parenteral; however, if mLT is 
used in the boost, the administration is preferably oral. 
The methods and compositions of the present invention are intended for use 
both in immature and mature vertebrates, in particular birds, mammals, and 
humans. Useful antigens, as examples and not by way of limitation, would 
include antigens from pathogenic strains of bacteria (Streptococcus 
pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria 
meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, 
Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, 
Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, 
Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas 
aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus 
cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, 
Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, 
Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema 
pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, 
Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma 
gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, 
Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, 
Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides 
immitis, Aspergillus fumigatus, Candida albicans, Blastomyces 
dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa 
(Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, 
Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, 
Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania 
braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium 
falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, 
Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, 
Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, 
Schistosoma haematobium, and hookworms) either presented to the immune 
system in whole cell form or in part isolated from media cultures designed 
to grow said organisms which are well know in the art, or protective 
antigens from said organisms obtained by genetic engineering techniques or 
by chemical synthesis. 
Other relevant antigens would be pathogenic viruses (as examples and not by 
limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes 
Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, 
Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, 
parainfluenza viruses, mumps, measles, respiratory syncytial virus, 
rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, 
Hepatitis 3 virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B 
Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human 
Immunodeficiency Virus) either presented to the immune system in whole or 
in part isolated from media cultures designed to grow such viruses which 
are well known in the art or protective antigens therefrom obtained by 
genetic engineering techniques or by chemical synthesis. 
Further examples of relevant antigens include, but are not limited to, 
vaccines. Examples of such vaccines include, but are not limited to, 
influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoid 
combined with pertussis vaccine, hepatitis A vaccines hepatitis B vaccine, 
hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, 
herpes vaccine, measles vaccines rubella vaccine, mumps vaccine, mixed 
vaccine of measles, mumps and rubella, papillomavirus vaccine, parvovirus 
vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio 
vaccine, malaria vaccine, varicella vaccine, gonorrhea vaccine, HIV 
vaccines schistosomiasis vaccine, rota vaccine, mycoplasma vaccine 
pneumococcal vaccine, meningococcal vaccine and others. These can be 
produced by known common processes. In general, such vaccines comprise 
either the entire organism or virus grown and isolated by techniques well 
known to the skilled artisan or comprise relevant antigens of these 
organisms or viruses which are produced by genetic engineering techniques 
or chemical synthesis. Their production is illustrated by, but not limited 
to, as follows: 
Influenza vaccine: a vaccine comprising the whole or part of hemagglutinin, 
neuraminidase, nucleoprotein and matrix protein which are obtainable by 
purifying a virus, which is grown in embryonated eggs, with ether and 
detergent, or by genetic engineering techniques or chemical synthesis. 
Pertussis vaccine: a vaccine comprising the whole or a part of pertussis 
toxin, hemagglutinin and K-agglutin which are obtained from avirulent 
toxin with formalin which is extracted by salting-out or 
ultracentrifugation from the culture broth or bacterial cells of 
Bordetella pertussis, or by genetic engineering techniques or chemical 
synthesis. 
Diphtheria and tetanus toxoid combined with pertussis vaccine: a vaccine 
mixed with pertussis vaccine, diphtheria and tetanus toxoid. 
Japanese encephalitis vaccine: a vaccine comprising the whole or part of an 
antigenic protein which is obtained by culturing a virus intracerebrally 
in mice and purifying the virus particles by centrifugation or ethyl 
alcohol and inactivating the same, or by genetic engineering techniques or 
chemical synthesis. 
Hepatitis B vaccine: a vaccine comprising the whole or part of an antigen 
protein which is obtained by isolating and purifying the HBs antigen by 
salting-out or ultracentrifugation, obtained from hepatitis carrying 
blood, or by genetic engineering techniques or by chemical synthesis. 
Measles vaccine: a vaccine comprising the whole or part of a virus grown in 
a cultured chick embryo cells or embryonated egg, or a protective antigen 
obtained by genetic engineering or chemical synthesis. 
Rubella vaccine: a vaccine comprising the whole or part of a virus grown in 
cultured chick embryo cells or embryonated egg, or a protective antigen 
obtained by genetic engineering techniques or chemical synthesis. 
Mumps vaccine: a vaccine comprising the whole or part of a virus grown in 
cultured rabbit cells or embryonated egg, or a protective antigen obtained 
by genetic engineering techniques or chemical synthesis. 
Mixed vaccine of measles, rubella and mumps: a vaccine produced by mixing 
measles, rubella and mumps vaccines. 
Rota vaccine: a vaccine comprising the whole or part of a virus grown in 
cultured MA 104 cells or isolated from the patient's feces, or a 
protective antigen obtained by genetic engineering techniques or chemical 
synthesis. 
Mycoplasma vaccine: a vaccine comprising the whole or part of mycoplasma 
cells grown in a liquid culture medium for mycoplasma or a protective 
antigen obtained by genetic engineering techniques or chemical synthesis. 
Those conditions for which effective prevention may be achieved by the 
present method will be obvious to the skilled artisan. 
The vaccine preparation compositions of the present invention can be 
prepared by mixing the above illustrated antigens and/or vaccines with mLT 
at a desired ratio. The preparation should be conducted strictly 
aseptically, and each component should also be aseptic. Pyrogens or 
allergens should naturally be removed as completely as possible. The 
antigen preparation of the present invention can be used by preparing the 
antigen per se and the mLT separately 
Further, the present invention encompasses a kit comprising an effective 
amount of antigen and an adjuvant effective amount of mLT. In use, the 
components of the kit can either first be mixed together and then 
administered orally or the components can be administered orally 
separately within a short time of each other. 
The vaccine preparation compositions of the present invention can be 
combined with either a liquid or solid pharmaceutical carrier, and the 
compositions can be in the form of tablets, capsules, powders, granules, 
suspensions or solutions. The compositions can also contain suitable 
preservatives, coloring and flavoring agents, or agents that produce slow 
release. Potential carriers that can be used in the preparation of the 
pharmaceutical compositions of this invention include, but are not limited 
to, gelatin capsules, sugars, cellulose derivations such as sodium 
carboxymethyl cellulose, gelatin, talc, magnesium stearate, vegetable oil 
such as peanut oil, etc, glycerin, sorbitol, agar and water. Carriers may 
also serve as a binder to facilitate tabletting of the compositions for 
convenient oral administration. 
The vaccine preparation composition of this invention may be maintained in 
a stable storage form for ready use by lyophilization or by other means 
well known to those skilled in the art. For oral administration, the 
vaccine preparation may be reconstituted as a suspension in buffered 
saline, milk, or any other physiologically compatible liquid medium. The 
medium may be made more palatable by the addition of suitable coloring and 
flavoring agents as desired. 
Administration of the vaccine preparation compositions may be preceded by 
an oral dosage of an effective amount of a gastric acid neutralizing 
agent. While many compounds could be used for this purpose, sodium 
bicarbonate is preferred. Alternatively, the vaccine compositions may be 
delivered in enteric coated capsules (i.e., capsules that dissolve only 
after passing through the stomach). 
6. EXAMPLES 
The following examples are presented for purposes of illustration only and 
are not intended to limit the scope of the invention in any way. 
6.1 CONSTRUCTION OF mLT 
The wild-type LT toxin is encoded on a naturally occurring plasmid found in 
strains of enterotoxigenic E. coli capable of producing this toxin. The 
present inventors had 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 pBR322 [Clements et al., 1983, Infect. Immun. 40:653]. 
This recombinant plasmid, designated pDF82, has been extensively 
characterized and expresses LT under control of the native LT promoter. 
The next step in this process was to place the LT gene under the control 
of a strong promoter, in this case the lac promoter on plasmid pUC18. This 
was accomplished by isolating the genes for LT-A and LT-B separately and 
recombining them in a cassette in the vector plasmid. This was an 
important step because it permitted purification of reasonable quantities 
of LT and derived mutants for subsequent analysis. This plasmid, 
designated pDF94, is shown diagrammatically in FIG. 1. 
Both CT and LT are synthesized with a trypsin sensitive peptide bond that 
joins the A.sub.1 and A.sub.2 pieces. This peptide bond must be nicked for 
the molecule to be "toxic". This is also true for diphtheria toxin, the 
prototypic A-B toxin, and for a variety of other bacterial toxins. If the 
A.sub.1 -A.sub.2 bond is not removed, either by bacterial proteases or 
intestinal proteases in the lumen of the bowel, i.e., proteolytic 
processing or activation, the A.sub.1 piece cannot reach its target on the 
basolateral surface of the intestinal epithelial cell. In contrast to CT, 
LT is not fully biologically active when first isolated from the cell. LT 
also requires proteolysis to be fully active and the proteolytic 
activation does not occur inside of the bacterium. Therefore, one means of 
altering the toxicity of the molecule without affecting the 
ADP-ribosylating enzymatic activity would be to remove by genetic 
manipulation the trypsin sensitive amino acids that join the A.sub.1 and 
A.sub.2 components of the A subunit. If the molecule cannot be 
proteolytically cleaved, it will not be toxic. One skilled in the art 
would predict that the molecule should, however, retain its 
ADP-ribosylating enzymatic activity and consequently, its adjuvant 
function. 
FIG. 1 shows the seqguence of the disulfide subtended region that separates 
the A.sub.1 and A.sub.2 pieces. Within this region is a single Arginine 
residue which is believed to be the site of cleavage necessary to activate 
the toxic properties of the molecule. This region was changed by 
site-directed mutagenesis is such a way as to render the molecule 
insensitive to proteolytic digestion and, consequently, nontoxic. 
Site-directed mutagenesis is accomplished by hybridizing to single stranded 
DNA a synthetic oligonucleotide which is complementary to the single 
stranded template except for a region of mismatch near then center. It is 
this region that contains the desired nucleotide change or changes. 
Following hybridization with the single stranded target DNA, the 
oligonucleotide is extended with DNA polymerase to create a double 
stranded structure. The nick is then sealed with DNA ligase and the duplex 
structure is transformed into an E. coli host. The theoretical yield of 
mutants using this procedure is 50% due to the semi-conservative mode of 
DNA replication. In practice, the yield is much lower. There are, however, 
a number of methods available to improve yield and to select for 
oligonucleotide directed mutants. The system employed utilized 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 (i.e., GGA=Gly 
replaces AGA=Arg), thus preserving the reading frame while eliminating the 
proteolytic site. mLT was then purified by agarose affinity chromatography 
from one mutant (pBD95) which had been confirmed by sequencing. Alternate 
methods of purification will be apparent to those skilled in the art. This 
mutant LT, designated LT.sub.(R192G) was then examined by 
SDS-polyacrylamide gel electrophoresis for modification of the trypsin 
sensitive bond. Samples were examined with and without exposure to trypsin 
and compared with native (unmodified) LT. mLT does not dissociate into 
A.sub.1 and A.sub.2 when incubated with trypsin, thereby indicating that 
sensitivity to protease has been removed. 
6.2 EFFECT OF mLT ON Y-1 ADRENAL CELLS 
It would be predicted by one skilled in the art that mLT would not be 
active in the Y-1 adrenal cell assays. This prediction would be based upon 
previous findings [Clements and Finkelstein, 1979, Infect. Immun. 
24:760-769] that un-nicked LT was more than 1,000 fold less active in this 
assay system than was CT and that trypsin treatment activated LT to the 
same level of biological activity as CT in this assay. It was presumed 
that the residual activity of LT observed in this assay in the absence of 
trypsin activation was a function of some residual protease activity which 
could not be accounted for. For instance, trypsin is used in the process 
of subculturing Y-1 adrenal cells. It was therefore assumed that LT that 
could not be nicked would be completely inactive in the Y-1 adrenal cell 
assay. Results are shown in Table I. 
TABLE I 
______________________________________ 
Toxin Trypsin Activated 
Specific Activity.sup.a 
______________________________________ 
Cholera Toxin 
- 15 
LT - 60 
LT + 15 
LT.sub.(R192G) - 48,800 
LT.sub.(R192G) + 48,800 
______________________________________ 
.sup.a Minimum dose (picograms per well) required to produce (&gt;50%) cell 
rounding. 
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. As shown in Table I, CT and native LT treated 
with trypsin have the same level of activity (15 pg) on Y-1 adrenal cells. 
By contrast, mLT (48,000 pg) was &gt;1,000 fold less active than CT or native 
LT and could not be activated by trypsin. The residual basal activity 
undoubtedly reflects a different and here-to-fore unknown pathway of 
adrenal cell activation than that requiring separation of the A.sub.1 
-A.sub.2 linkages 
6.3 ADP-RIBOSYLATING ENZYMATIC ACTIVITY OF mLT 
Because the mutation replacing Arg.sub.192 with Gly.sub.192 does not alter 
the enzymatic site of the A.sub.1 moiety, one skilled in the art would 
predict that mLT would retain its ADP-ribosylating enzymatic activity. To 
examine this property, the NAD-Agmatine ADP-ribosyltransferase Assay was 
employed [Moss et al., 1993, J. Biol. Chem. 268:6383-6387]. As shown in 
FIG. 2, CT produces a dose-dependent increase in the levels of 
ADP-ribosylagmatine, a function of the ADP-ribosyltransferase activity of 
this molecule. 
TABLE II 
______________________________________ 
ADP-Ribosyltransferase Activity of CT, native LT, and LT.sub.(R192G) 
Experiment 
1 2 3 4 Mean .+-. SEM 
______________________________________ 
No Toxin 
ND 9.12 5.63 14.17 9.64 .+-. 2.48 
1 .mu.g CT ND 17.81 17.66 25.75 20.39 .+-. 2.68 
10 .mu.g CT ND 107.32 111.28 104.04 107.55 .+-. 2.09 
100 .mu.g CT 351.55 361.73 308.09 ND 340.46 .+-. 16.45 
100 .mu.g LT 17.32 14.48 13.86 ND 15.22 .+-. 1.07 
100 .mu.g LT + 164.10 189.89 152.96 ND 168.98 .+-. 10.94 
Trypsin 
100 .mu.g 14.58 12.34 9.30 ND 12.07 .+-. 1.53 
LT.sub.(R192G) 
100 .mu.g 14.73 8.90 10.47 ND 11.37 .+-. 1.74 
LT.sub.(R192G) + 
Trypsin 
______________________________________ 
ND = Not Done data expressed in fMoles min.sup.-1 
Table II demonstrates in tabular form the unexpected finding that mLT 
lacked any detectable ADP-ribosylating enzymatic activity, with or without 
trypsin activation, even though the enzymatic site had not been altered 
and there was a demonstratable basal level of activity in the Y-1 adrenal 
cell assay. 
6.4 ENTEROTOXIC ACTIVITY OF mLT 
Because of the unexpected finding that mLT lacks any detectable 
ADP-ribosylating enzymatic activity, with or without trypsin activations 
even though the enzymatic site has not been altered and the additional 
finding that there is a basal level of activity in the Y-1 adrenal cell 
assay, it was unclear whether mLT would retain any of its enterotoxic 
properties. An ideal adjuvant formulation of mLT would retain its ability 
to act as an immunological adjuvant but would lack the real or potential 
side-effects, such as diarrhea, associated with the use of LT or CT. FIG. 
3 demonstrates that mLT does not induce net fluid secretion in the patent 
mouse model, even at a dose of 125 .mu.g. This dose is more than five 
times the adjuvant effective dose for LT in this model. Importantly, the 
potential toxicity of native LT can be seen at this level. 
6.5 ADJUVANT ACTIVITY OF mLT 
One skilled in the art would predict that since mLT possessed no 
demonstrable ADP-ribosyltransferase activity and is not enterotoxic, it 
would lack adjuvant activity. This prediction would be based upon the 
report by Lycke et al. [Lycke et al., 1992, Eur. J. Immunol. 22:2277-2281] 
where it is made clear that alterations that affect the ADP-ribosylating 
enzymatic activity of the toxin and alter the ability to increase 
intracellular levels of cAMP also prevent the molecule from functioning as 
an adjuvant. As demonstrated above, mLT has no ADP-ribosylating enzymatic 
activity and only some undefined basal activity in Y-1 adrenal cells, and 
induces no net fluid secretion in the patent mouse model. 
In order to examine the adjuvant activity of mLT the following experiment 
was performed. Three groups of BALB/c mice were immunized Animals were 
inoculated intragastrically with a blunt tipped feeding needle (Popper & 
Sons, Inc., New Hyde Park, N.Y.). On day 0 each group was immunized orally 
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 .mu.g of native LT, 
and Group C received 0.5 ml of PBS containing 5 mg of OVA and 25 .mu.g of 
mLT. Each regimen was administered again on days 7 and 14. On day 21, all 
animals were boosted i.p. with 1 .mu.g of OVA in 20% Maalox. One week 
after the i.p. inoculation animals were sacrificed and assayed for serum 
IgG and mucosal IgA antibodies directed against OVA and LT by ELISA. 
Reagents and antisera for the ELISA were obtained from Sigma Chemical Co. 
Samples for ELISA were serially diluted in phosphate buffered saline (pH 
7.2)-0.05% Tween 20 (PBS-TWEEN). For anti-LT determinations, microtiter 
plates were precoated with 1.5 .mu.g per well of mixed gangliosides (Type 
III), then with 1 .mu.g per well of purified LT. Anti-OVA was determined 
on microtiter plates precoated with 10 .mu.g per well of OVA. Serum 
anti-LT and anti-OVA were determined with rabbit antiserum against mouse 
IgG conjugated to alkaline phosphatase. Mucosal anti-LT and anti-OVA IgA 
were assayed with goat antiserum against mouse IgA [alpha-chain specific] 
followed by rabbit antiserum against goat IgG conjugated to alkaline 
phosphatase. Reactions were stopped with 3N NaOH. Values for IgG and IgA 
were determined from a standard curve with purified mouse myeloma proteins 
(MOPC 315, gA(IgA12); MOPC 21, gG1: Litton Bionetics, Inc., Charleston, 
S.C.). 
6.5.1 SERUM IgG ANTI-OVA 
As shown in the FIG. 4A, animals primed orally with OVA and LT developed a 
significantly higher serum IgG anti-OVA response following subsequent 
parenteral immunization with OVA (4,058 .mu.g/ml) than those primed with 
OVA alone and subsequently immunized parenterally with OVA (No detectable 
anti-OVA response) (Student t-test p=0.031). Significantly, animals primed 
orally with OVA and mLT also developed a significantly higher serum IgG 
anti-OVA response following subsequent parenteral immunization with OVA 
(1,338 .mu.g/ml) than those primed with OVA alone and subsequently 
immunized parenterally with OVA (No detectable anti-OVA response) (Student 
t-test p=0.0007). 
6.5.2 MUCOSAL sIgA ANTI-OVA 
As shown in the FIG. 4B, similar results were obtained when anti-OVA IgA 
responses were compared within these same groups of animals. Animals 
primed orally with OVA and LT developed a significantly higher mucosal IgA 
anti-OVA response following subsequent parenteral immunization with OVA 
(869 ng/ml) than those primed with OVA alone and subsequently immunized 
parenterally with OVA (No detectable anti-OVA response) (Student t-test 
p=0.0131). As above, animals primed orally with OVA and mLT also developed 
a significantly higher mucosal IgA anti-OVA response following subsequent 
parenteral immunization with OVA (230 ng/ml) than those primed with OVA 
alone and subsequently immunized parenterally with OVA (No detectable 
anti-OVA response) (Student t-test p=0.0189). 
6.5.3 SERUM IgG ANTI-LT 
The ability of LT and mLT to elicit an anti-LT antibody response in these 
same animals was also examined. This was important in that it would 
provide an indication of whether the mutant LT was able to prevent 
induction of tolerance to itself in addition to functioning as an adjuvant 
for other proteins. As shown in FIG. 5A, animals primed orally with OVA 
and LT developed a significantly higher serum IgG anti-LT response 
following subsequent parenteral immunization with OVA (342 g/ml) than 
those primed with OVA alone and subsequently immunized parenterally with 
OVA (No detectable anti-LT response) (Student t-test p=0.0005). Animals 
primed orally with OVA and mLT also developed a significantly higher serum 
IgG anti-LT response following subsequent parenteral immunization with OVA 
(552 .mu.g/ml) than those primed with OVA alone and subsequently immunized 
parenterally with OVA (No detectable anti-LT response) (Student t-test 
p=0.0026). 
6.5.4 MUCOSAL sIgA ANTI-LT 
As shown in the FIG. 5B, similar results were obtained when anti-LT IgA 
responses were compared within these same groups of animals. Animals 
primed orally with OVA and LT developed a significantly higher mucosal IgA 
anti-LT response following subsequent parenteral immunization with OVA 
(4,328 ng/ml) than those primed with OVA alone and subsequently immunized 
parenterally with OVA (No detectable anti-LT response) (Student t-test 
p=0.0047). As above, animals primed orally with OVA and mLT also developed 
a significantly higher mucosal IgA anti-LT response following subsequent 
parenteral immunization with OVA (1,463 ng/ml) than those primed with OVA 
alone and subsequently immunized parenterally with OVA (No detectable 
anti-LT response) (Student t-test p=0.0323). 
7. DEPOSIT OF MICROORGANISMS 
The following plasmid was deposited with the American Type Culture 
Collection (ATCC), Rockville, Md. on Aug. 18, 1994, and has been assigned 
the indicated accession number: 
______________________________________ 
Plasmid Accession Number 
______________________________________ 
pBD95 ATCC 69683 
______________________________________ 
The invention described and claimed herein is not to be limited in scope by 
the specific embodiments herein disclosed since these embodiments are 
intended as illustration of several aspects of the invention. Any 
equivalent embodiments are intended to be within the scope of this 
invention. Indeed, various modifications of the invention in addition to 
those shown and described herein will become apparent to those skilled in 
the art from the foregoing description. Such modifications are also 
intended to fall within the scope of the appended claims. 
It is also to be understood that all base pair and amino acid residue 
numbers and sizes given for nucleotides and peptides are approximate and 
are used for purposes of description. 
A number of references are cited herein, the entire disclosures of which 
are incorporated herein, in their entirety, by reference. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 5 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - GGTTGTGGAA ATTCATCAAG AACAATTACA GGTGATACTT GTAAT - # 
- #45 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Gly Cys Gly Asn Ser Ser Arg Thr Ile Thr Gl - #y Asp Thr Cys Asn 
1 5 - # 10 - # 15 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - TCATCAGGAA CAATTACA - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - GGTTGTGGAA ATTCATCAGG AACAATTACA GGTGATACTT GTAAT - # 
- #45 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - Gly Cys Gly Asn Ser Ser Gly Thr Ile Thr Gl - #y Asp Thr Cys Asn 
1 5 - # 10 - # 15 
__________________________________________________________________________