Vaccine for transcutaneous immunization (05-May-2009)

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Phase Application of International Application No. PCT/USO2/04254, filed Feb. 13, 2002, which claims the benefit of U.S. Provisional Application No. 60/268,016, filed Feb. 13, 2001; U.S. Provisional Application No. 60/304,110, filed Jul. 11, 2001; U.S. Provisional Application No. 60/310,447, filed Aug. 8, 2001; and U.S. Provisional Application No. 60/310,483, filed Aug. 8, 2001, all of which are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERAL SPONSORSHIP

The U.S. federal government has certain rights in this invention as provided for under contracts MRMC/DAMD17-01-0085 and NIH/AI 45227-01.

FIELD OF THE INVENTION

The invention relates to vaccines and transcutaneous immunization to treat infections by pathogens such as, for example, enterotoxigenic Escherichia coli (ETEC) and/or other symptoms of diarrheal disease caused thereby.

BACKGROUND OF THE INVENTION

Skin, the largest human organ, plays an important part in the body's defense against invasion by infectious agents and contact with noxious substances. But this barrier function of the skin appears to have prevented the art from appreciating that transcutaneous immunization provided an effective alternative to enteral, mucosal, and parenteral administration of vaccines.

Anatomically, skin is composed of three layers: the epidermis, the dermis, and subcutaneous fat. Epidermis is composed of the basal, the spinous, the granular, and the cornified layers; the stratum corneum comprises the cornified layer and lipid. The principal antigen presenting cells of the skin, Langerhans cells, are reported to be in the mid- to upper-spinous layers of the epidermis in humans. Dermis contains primarily connective tissue. Blood and lymphatic vessels are confined to the dermis and subcutaneous fat.

The stratum corneum, a layer of dead skin cells and lipids, has traditionally been viewed as a barrier to the hostile world, excluding organisms and noxious substances from the viable cells below the stratum corneum. Stratum corneum also serves as a barrier to the loss of moisture from the skin: the relatively dry stratum corneum is reported to have 5% to 15% water content while deeper epidermal and dermal layers are relatively well hydrated with 85% to 90% water content. Only recently has the secondary protection provided by antigen presenting cells (e.g., Langerhans cells) been recognized. Moreover, the ability to immunize through the skin with or without penetration enhancement (i.e., transcutaneous immunization) using a skin-active adjuvant has only been recently described. Although undesirable skin reactions such as atopy and dermatitis were known in the art, recognition of the therapeutic advantages of transcutaneous immunization (TCl) might not have been appreciated in the past because the skin was believed to provide a barrier to the passage of molecules larger than about 500 daltons.

We have shown that a variety of adjuvants are effectively administered by TCl to elicit systemic and regional antigen-specific immune responses to a separate, co-administered antigen. See WO 98/20734, WO 99/43350, and WO 00/61184; U.S. Pat. Nos. 5,910,306 and 5,980,898; and U.S. patent application Ser. Nos. 09/257,188; 09/309,881; 09/311,720; 09/316,069; 09/337,746; and 09/545,417. For example, adjuvants like ADP-ribosylating exotoxins are safe and effective when applied epicutaneously, in contrast to the disadvantages associated with their use when administered by an enteral, mucosal, or parenteral route.

U.S. Pat. Nos. 4,220,584 and 4,285,931 use E. coli heat-labile enterotoxin to immunize against E. coli-induced diarrhea. Rabbits were intramuscularly injected with the immunogen and Freund's adjuvant. Protection against challenge with toxin and neutralization of toxic effects on ileal loop activity was shown. U.S. Pat. No. 5,182,109 describes combining vaccine and toxin (e.g., E. coli heat-labile toxin) and administration in injectable, spray, or oral form. Neutralization was demonstrated with colostrum of immunized cows. Mutant versions of enterotoxin have also been described to retain immunogenicity and eliminate toxicity (e.g., U.S. Pat. Nos. 4,761,372 and 5,308,835).

Novel and inventive vaccine formulations, as well as processes for making and using them, are disclosed herein. In particular, TCl and the advantages derived therefrom in human vaccination to treat diarrheal disease are demonstrated. An important showing is that competition among different antigens in a multivalent vaccine was not an obstacle when administered by transcutaneous immunization. Other advantages of the invention are discussed below or would be apparent from the disclosure herein.

SUMMARY OF THE INVENTION

Immunogens comprised of at least one adjuvant and/or one or more antigens capable of inducing an immune response against pathogens like enterotoxigenic E. coli (ETEC) are provided for immunization. The adjuvant may be an ADP-ribosylating exotoxin (e.g., E. coli heat-labile enterotoxin, cholera toxin, diphtheria toxin, pertussis toxin) or derivatives thereof having adjuvant activity; the antigen may be derived from a bacterial toxin (e.g., heat-labile or heat-stable enterotoxin) or a colonization or a virulence factor (e.g., CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS17, PCF 0166) or peptide fragments or conjugates thereof having immunogenic activity. Subunit or whole-cell vaccines comprised of an immunogen and a patch are also provided, along with methods of making the aforementioned products and of using them for immunization. An immune response which is specific for molecules associated with pathogens (e.g., toxins, membrane proteins) may be induced by various routes (e.g., enteral, mucosal, parenteral, transcutaneous). Other traveler's diseases of interest that can be treated include campylobacteriosis (Campylobacter jejuni), giardiasis (Giardia intestinalis), hepatitis (hepatitis virus A or B), malaria (Plasmodium falciparum, P. vivax, P. ovale, and P. malariae), shigellosis (Shigella boydii, S. dysenteriae, S. flexneri, and S. sonnei), viral gastroenteritis (rotavirus), and combinations thereof. Effectiveness may be assessed by clinical or laboratory criteria. Protection may be assessed using surrogate markers or directly in controlled trials. Further aspects of the invention will be apparent to a person skilled in the art from the following detailed description and claims, and generalizations thereto.

DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D. Individual IgG and IgA peak fold rise in antibody titer to LT (A and B) and CS6 (C and D) among human volunteers immunized with adjuvant combined with antigen (LT+CS6), or with antigen alone (CS6). The transverse bar represents the median peak fold rise in antibody titer.

FIG. 2A-2D. Kinetics of the anti-LT (A and B) and anti-CS6 (C and D) IgA and IgG antibody responses among volunteers immunized and boosted (arrows) using the transcutaneous route. The circles indicate the geometric mean titer by the day after the first immunization, the bars denote the corresponding 95% confidence intervals.

*p<0.05,**p<0.01,***p<0.001, NS non-significant, Wilcoxon signed rank test, comparing antibody titer responses between boosting immunizations.

FIG. 3A-3D. Individual peak number of anti-LT (A and B) and anti-CS6 (C and D) ASC per 106 PBMC among responders to the immunization with adjuvant combined with antigen (LT+CS6), by the immunization after which the peak value was attained.

FIG. 4A-4C. Serum IgG response to TCl with CS3 and CS6 with and without LTR192G adjuvant. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol and tape stripped 10 times to disrupt the stratum corneum. Gauze patches were affixed to an adhesive backing and loaded with a 25 μl volume of 25 μg CS6 or 25 μg CS6 with 10 μg LTR192G. The patches were applied to the prepared skin and allowed to remain in place for ˜18 hr. A group of mice was intradermally injected with a 25 μl of CS6(25 μg) at the base of the tail. All mice received a vaccination on day 0, 14 and 28. Serum samples were collected 14 days after the third vaccination (day 42). Panels show serum IgG titer to CS3 (A), serum IgG titer to CS6 (B), and serum IgG titer to LTR192G (C).

FIG. 5A-5B. Serum IgG response to TCl with divalent and trivalent ETEC subunit vaccines. The vaccination site at the base of the tail was prepared using the procedure described in FIG. 4. Gauze patches, affixed to an adhesive backing were loaded with 25 μl volume consisting of the following mixtures: 25 μg CS3 and 10 μg LTR192G; 25 μg CS3, 25 μg CS6 and 10 μg LTR192G. The patches were applied to the prepared skin and allowed to remain in place for ˜18 hr. All mice received a transcutaneous vaccine on day 0 and 14. Serum was collected 10 days after the second immunization (day 24). Panels show serum IgG titer to CS3 (A) and serum IgG titer to CS6 (B).

FIG. 6A-6B. Serum IgG response to TCl using a CS3, CS6 and LTR192G multivalent vaccines. The vaccination site at the base of the tail was prepared using the procedure described in FIG. 4. Gauze patches affixed to an adhesive backing were loaded with 25 μl volume consisting of the following mixtures: 25 μg CS3; 25 μg CS6; 25 μg each CS3 and CS6; 25 μg each CS3 and CS6 and 10 μg LTR192G. The patches were applied to the pretreated skin and allowed to remain in place for ˜18 hr. All mice received two transcutaneous vaccinations on day 0 and 14. Serum was collected 10 days after the second immunization (day 24). Panels show serum IgG titer to CS3 (A) and serum IgG titer to CS6 (B).

FIG. 7A-7B. Lack of antibody cross-reactivity between CS3 and CS6. The site at the base of the tail was prepared using the procedure described in FIG. 4. Gauze patches affixed to an adhesive backing were loaded with 25 μl volume consisting of the following mixtures: 25 μg CS3 with 10 μg LTR192G (panels A and B) and 25 μg CS6 with 10 μg LTR192G (panels C and D). The patches were applied overnight (˜18 hr). All mice received two transcutaneous vaccinations on day 0 and 14. Serum was collected 10 days after the second immunization (day 24). Serum IgG titers for CS3 (panels A and C) and CS6 (panels B and D) were determined.

FIG. 8A-8C. Serum IgG subclasses elicited by transcutaneous vaccination with CS3 with and without LTR192G. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then mildly abraded with emery paper 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: 25 [Image Omitted] μg CS3 or 25 μg CS3 with or without 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice received three transcutaneous vaccinations on day 0, 14 and 28. Serum samples were collected 30 days after the third vaccination (day 58). Panels show total serum IgG titers to CS3 (A), serum IgG1 subclass to CS3 (B), and serum IgG2a subclass to CS3 (C).

FIG. 9A-9C. Serum IgG subclasses elicited by TCl with CS6 with and without and LTR192G. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then mildly abraded with emery paper 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: 25 μg CS6 or 25 μg CS3 with or without 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice received three transcutaneous vaccinations on day 0, 14, and 28. Serum samples were collected 30 days after the third vaccination (day 58). Panels show total serum IgG titers to CS6 (A), serum IgG1 subclass to CS6 (B), and serum IgG2a subclass to CS6 (C).

FIG. 10A-10C. Serum IgG subclasses elicited by TCl with LTR192G. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then mildly abraded with emery paper 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice received three transcutaneous vaccinations on day 0, 14 and 28. Serum samples were collected 30 days after the third vaccination (day 58). Panels shown total serum IgG titers to LTR192G (A), serum IgG1 subclass to LTR192G (B), and serum IgG2a subclass to LTR192G (C).

FIG. 11A-11C. Serum IgG subclasses elicited by LTR192G co-administered with CS3 or CS6. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then mildly abraded with emery paper 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following: 25 μg CS3 with 10 μg LTR192G; and 25 μg CS6 with 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice received three transcutaneous vaccinations on day 0, 14 and 28. Serum samples were collected 30 days after the third vaccination (day 58). Panels show total serum IgG titers to LTR192G (A), serum IgG1 subclass to LTR192G (B), and serum IgG2a subclass to LTR192G (C).

FIG. 12A-12H. Detection of CS3 specific fecal IgA (upper panels) and IgG (lower panels) following TCl. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: phosphate buffered saline (panels A and E); 25 μg CS3 (panels B and F); and 25 μg CS3 with 10 μg LTR192G (panels C and G). The patches were applied overnight (˜18 hr). A group of mice was vaccinated by intradermal (ID) injection of 25 μg CS3 (panels D and H). All mice received three vaccinations on day 0, 14 and 28. Fecal samples were collected one week after the third immunization (day 35). The samples were processed and evaluated for fecal IgA (panels A-D) and IgG (panels E-H) against CS3.

FIG. 13A-13H. Detection of CS6 specific fecal IgA (upper panels) and IgG (lower panels) following TCl. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: phosphate buffered saline (panels A and E); 25 μg CS6 (panels B and F); and 25 μg CS6 with 10 μg LTR192G (panels C and G). The patches were applied overnight (˜18 hr). A group of mice was vaccinated by intradermal (ID) injection of 25 μg CS6 (panels D and H). All mice received three vaccinations on day 0, 14 and 28. Fecal samples were collected one week after the third immunization (day 35). The samples were processed and evaluated for fecal IgA (panels A-D) and IgG (panels E-H) against CS6.

FIG. 14A-14H. Detection of LTR192G specific fecal IgA (upper panels) and IgG (lower panels) following TCl. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: phosphate buffered saline (panels A and E); 10 μg LTR192G (panels B and F); and 25 μg CS3 with 10 μg LTR192G (panels C and G); and 25 μg CS6 and 10 μg LTR192G (panel D and H). The patches were applied overnight (˜18 hr). All mice received three vaccinations on day 0, 14 and 28. Fecal samples were collected one week after the third immunization (day 35). The samples were processed and evaluated for fecal IgA (panels A-D) and IgG (panels E-H) against LTR192G.

FIG. 15A-15D. Detection of CS3, CS6 and LTR192G specific antibody secreting cells (ASC) in the spleen of mice transcutaneously vaccinated with monovalent and divalent ETEC subunit vaccines. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: phosphate buffered saline (vehicle); 25 μg CS3; 25 μg CS6; 25 μg CS3 with 10 μg LTR192G; and 25 μg CS6 with 10 μg LTR192G. The patches were applied overnight (˜18 hr). In addition, groups of mice were vaccinated by intradermal (ID) injection at the base of the tail with 25 μg of CS3 or CS6. All mice were vaccinated three times on day 0, 14 and 28. The spleen was harvested 30 days after the third immunization (day 58). Panels show CS3-specific IgA-ASC (A) and IgG-ASC (B); CS6-specific IgA-ASC (C) and IgG-ASC (D), and LTR192G-specific IgA-ASC (A and C) and IgG-ASC (B and D).

FIG. 16A-16B. Detection of CS3, CS6 and LTR192G specific antibody secreting cells (ASC) in the spleen of mice transcutaneously vaccinated with trivalent ETEC subunit vaccine. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of a mixture of the following formulation: phosphate buffered saline (vehicle); 25 μg CS3; 25 μg CS6; 25 μg CS3 with 10 μg LTR192G; 25 μg CS3/25 g CS6/10 μg LTR192G. The patches were applied overnight (˜18 hr). Mice were vaccinated three times on day 0, 14 and 28. The spleen was harvested 30 days after the third immunization (day 58). Panels show IgA-ASC specific for CS3, CS6 and LTR192G (A) and IgG-ASC specific for CS3, CS6 and LTR192G (B).

FIG. 17A-17B. Detection of CS3, CS6 and LTR192G specific antibody secreting cells (ASC) in the inguinal lymph nodes of mice transcutaneously vaccinated with monovalent and divalent ETEC subunit vaccines. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of the following mixtures: phosphate buffered saline (vehicle); 25 μg CS3; 25 μg CS6; 25 μg CS3 with 10 μg LTR192G; and 25 μg CS6 with 10 μg LTR192G. The patches were applied overnight (˜18 hr). In addition, separate groups of mice were vaccinated by intradermal (ID) injection at the base of the tail with 25 μg of CS3 or CS6. All mice were vaccinated three times on day 0, 14 and 28. Inguinal lymph nodes were collected 30 days after the third immunization (day 58). Panels show CS3-specific IgG-ASC (A), CS6-specific IgG-ASC (B), and LTR192G-specific IgG-ASC (A and B).

FIG. 18. Detection of CS3, CS6 and LTR192G specific antibody secreting cells (IgG-ASC) in the inguinal lymph nodes of mice transcutaneously vaccinated with trivalent ETEC subunit vaccine. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then tape stripped 10 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl of a mixture consisting of 25 μg CS3, 25 μg CS6 and 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice were vaccinated three times on day 0, 14 and 28. Inguinal lymph nodes were collected 30 days after the third immunization (day 58).

FIG. 19A-19B. Serum IgG response to TCl with CFA/I with and without LTR192G adjuvant. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol and mildly abraded with emery paper 5 times to disrupt the stratum corneum. Gauze patches were affixed to an adhesive backing and loaded with a 25 μl volume of 25 μg CFA/I and 25 μg CFA/I with 10 μg LTR192G. The patches were applied to the prepared skin and allowed to remain in place for ˜18 hr. Separate groups of mice was intradermally injected with a 25 μl of CFA/I (25 μg) at the base of the tail. All mice received a vaccination on day 0 and 14. Serum samples were collected 10 days after the second vaccination (day 24). Panels show serum IgG titer to CFA/I (A) and serum IgG titer to LTR192G (B).

FIG. 20A-20H. Detection of CFA/I specific fecal IgA (upper panels) and IgG (lower panels) following TCl. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then mildly abraded with emery paper 5 times. Gauze patches were affixed to an adhesive backing and loaded with 25 μl volume of 25 μg of the following mixtures: phosphate buffered saline (panels A and E); 25 of the following mixtures: phosphate buffered saline (Panels A and E); 25 μg CFA/I (panels B and F); and 25 μg CFA/I with 10 μg LTR192G (panels C and G). The patches were applied overnight (˜18 hr). A group of mice was vaccinated by intradermal (D) injection of 25 μg CFA/I (panels D and H). All mice received three vaccinations on day 0, 14 and 28. Fecal samples were collected two weeks after the third immunization (day 42). The samples were processed and evaluated for fecal IgA (panels A-D) and IgG (panels E-H) against CFA/I.

FIG. 21. Serum IgG response to TCl with a tetravalent ETEC subunit vaccine. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was mildly abraded with emery paper 5 times. Gauze patches, affixed to an adhesive backing were loaded with a mixture consisting of 25 μg CFA/I, 25 μg CS3, 25 μg CS6 and 10 μg LTR192G. The patches were applied to the prepared skin and allowed to remain in place for ˜18 hr. All mice received a transcutaneous vaccination on day 0 and 14. Serum was collected 10 days after the second immunization (day 24).

FIG. 22A-22F. Detection of fecal IgA (upper panels) and IgG (lower panels) antibodies to colonization factor antigens following TCl with the tetravalent ETEC vaccine. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol. The hydrated skin was then mildly abraded with emery paper. Gauze patches were affixed to an adhesive backing and loaded with the tetravalent vaccine: 25 μg CFA/I, 25 μg CS, 25 μg CS6 and 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice were vaccinated by intradermal (D) injections of 25 μg CFA/I (panels D and H). All mice received three vaccinations on day 0, 14 and 28. Fecal samples were collected two weeks after the third immunization (day 42). The samples were processed and evaluated for fecal IgA to CFA/I(A), CS3 (B), and CS6 (C). Processed samples were also evaluated for fecal IgG to CFA/I (D), CS3 (E), and CS6 (F).

FIG. 23A-23F. Detection of fecal IgA (upper panels) and IgG (lower panels) antibodies to LTR192G following TCl with the tetravalent ETEC vaccine. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration and abrasion as described in FIG. 18. Gauze patches were affixed to an adhesive backing and loaded with the following: 10 g LTR192G (mLT); 25 μg CFA/I and LTR192G; or 25 μg CFA/I, 25 μg CS, 25 μg CS6 and 10 μg LTR192G. The patches were applied overnight (˜18 hr). All mice received three vaccinations on day 0,14 and 28. Fecal samples were collected one week after the third immunization (day 35). Samples were processed and evaluated for fecal IgA to LTR192G (panels A-c) and for fecal IgG to LTR192G (panels D-F).

FIG. 24. Transcutaneous vaccination with CS3 and LTR192G subunit vaccines elicit serum antibodies that recognize CS3 expressing ETEC whole cells. Mice were shaved at the base of the tail by standard procedures. The shaved skin was tape stripped 10 times immediately prior to application of the patch. A gauze patch affixed to an adhesive backing was loaded with 25 μg CS3 and 10 μg LTR192G immediately prior to application. The patch was applied for ˜18 hr. A group of 10 mice received two patches on day 0 and day 14. Serum was collected 10 days after the second immunization (day 24). The serum was evaluated for antibodies to CS3, LTR192G and ETEC whole cells (E243778).

FIG. 25. Transcutaneous vaccination with killed enterotoxigenic E. coli whole cells (EWC). EWC were prepared by culturing ETEC (strain E243778) in bacterial broth. The cells were harvested by centrifugation and inactivated by overnight (room temperature) fixation with 2.5% formalin. The inactivated, killed whole cells were washed with phosphate buffered saline to remove the formalin. Prior to immunization, the mice were shaved at the base of the tail. The shaved skin was tape stripped 10 times immediately prior to application of the patch. The gauze patch on an adhesive backing was loaded with 10⁹ EWC's and 10 μg LTR192G. A group of 10 mice received were transcutaneously vaccinated on day 0 and 14. Serum was collected 10 days after the second immunization. Sera were evaluated for antibodies to EWC and LTR192G using the ELISA method as described in Materials and Methods. The results in FIG. 22 show that transcutaneous vaccination with killed bacterial whole cells did elicit antibodies that recognized whole cells and LTR192G adjuvant. These results demonstrate that killed ETEC bacteria can be applied to skin with the adjuavant and elicit specific immunity. These results are significant in that this is the first demonstration that TCl is applicable for subunit vaccines and for delivery of killed whole cell vaccines.

FIG. 26A-26B. Transcutaneous vaccination with a multivalent ETEC vaccine consisting of multiple colonization factors and two enterotoxins, LT and ST. Mice were shaved on the dorsal caudal surface at the base of the tail 48 hr prior to vaccination. The shaved skin was pretreated by hydration with 10% glycerol and 70% isopropyl alcohol and tape stripped 10 times to disrupt the stratum comeum. Gauze patches were affixed to an adhesive backing and loaded with a 25 μl volume of 25 μg CS3/25 μg CS6; 26 μg CS3/25 μg CS6/10 μg LTR192G and 25 μg CS3/25 μg CS6/10 μg LTR192G/8 μg STa. The patches were applied to the prepared skin and allowed to remain in place for ˜m18 hr. All mice received a vaccination on day 0, 14 and 28. Serum samples were collected 14 days after the second vaccination (day 42). Panels show serum IgG titer to CS3 (A) and serum IgG titer to CS6 (B).

FIG. 27. Wet and dry patch formulations are suitable for manufacturing articles for TCl. In these studies LT was used as an example for preparing different liquid and patch formulations. Briefly, LT was formulated in phosphate buffered saline and 5% lactose; LT was blended with an adhesive (KLUCEL) and spread as a thin film over an occlusive backing and allowed to air-dry at room temperature; LT solution was directly applied to a gauze patch surface and air-dried prior to use; and LT solution was applied to a gauze patch and administered as a fully hydrated patch. For mice receiving the liquid LT formulation, 10 μg LT was applied directly to the skin for 1 hr (with or without covering with gauze) and rinsed off. For mice receiving patches, the different patch formulations were applied for ˜24 hr before removal. The skin was hydrated with 10% glycerol and 70% isopropyl alcohol followed by mildly disrupting the stratum comeum with a pumice-containing swab (PDI/NicePak). All mice received two vaccinations on day 0 and day 14 with an equivalent of 10 μg (˜1 cm² area). Serum was collected two weeks later (day 28) and evaluated for serum antibodies to LT. Aqueous solutions, protein-in-adhesive, air-dried and fully hydrated patch formulations are suitable for transcutaneous delivery of ETEC antigens.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

A system for transcutaneous immunization (TCl) is provided which induces an immune response (e.g., humoral and/or cellular effector specific for an antigen) in an animal or human. The delivery system provides simple, epicutaneous application of a formulation comprised of one or more adjuvants, antigens, and/or polynucleotides (encoding adjuvant and/or antigen) to the skin of an animal or human subject. An antigen-specific immune response is thereby elicited against one or more pathogens like enterotoxigenic E. coli (ETEC) with or without the aid of chemical and/or physical penetration enhancement. At least one ingredient or component of the formulation (i.e., antigen or adjuvant) may be provided in dry form prior to administration of the formulation and/or as part of a patch. This system may also be used in conjunction with conventional enteral, mucosal, or parenteral immunization techniques.

Activation of one or more of adjuvant, antigen, and antigen presenting cell (APC) may assist in the promoting the immune response. The APC processes the antigen and then presents one or more epitopes to a lymphocyte. Activation may promote contact between the formulation and the APC (e.g., Langerhans cells, other dendritic cells, macrophages, B lymphocytes), uptake of the formulation by the APC, processing of antigen and/or presentation of epitopes by the APC, migration and/or differentiation of the APC, interaction between the APC and the lymphocyte, or combinations thereof. The adjuvant by itself may activate the APC. For example, a chemokine may recruit and/or activate antigen presenting cells to a site. In particular, the antigen presenting cell may migrate from the skin to the lymph nodes, and then present antigen to a lymphocyte, thereby inducing an antigen-specific immune response. Furthermore, the formulation may directly contact a lymphocyte which recognizes antigen, thereby inducing an antigen-specific immune response.

In addition to eliciting immune reactions leading to activation and/or expansion of antigen-specific B-cell and/or T-cell populations, including antibodies and cytotoxic T lymphocytes (CTL), the invention may positively and/or negatively regulate one or more components of the immune system by using transcutaneous immunization to affect antigen-specific helper (Th1 and/or Th2) or delayed-type hypersensitivity T-cell subsets (T_DTH). This can be exemplified by the differential behavior of cholera toxin and E. coli heat-labile enterotoxin which can result in different T-helper responses. The desired immune response induced by the invention is preferably systemic or regional (e.g., mucosal) but is usually not undesirable immune responses (e.g., atopy, dermatitis, eczema, psoriasis, or other allergic or hypersensitivity reactions). As seen herein, the immune responses elicited are of the quantity and quality that provide therapeutic or prophylactic immune responses useful treatment of infectious disease.

TCl may be practiced with or without skin penetration. For example, chemical or physical penetration enhancement techniques may be used as long as the skin is not perforated through the dermal layer. Hydration of the intact or skin before, during, or immediately after application of the formulation is preferred and may be required in some or many instances. For example, hydration may increase the water content of the topmost layer of skin (e.g., stratum corneum or superficial epidermis layer exposed by penetration enhancement techniques) above 25%, 50% or 75%.

Skin may be swabbed with an applicator (e.g., adsorbent material on a pad or stick) containing hydration or chemical penetration agents or they may be applied directly to skin. For example, aqueous solutions (e.g., water, saline, other buffers), acetone, alcohols (e.g., isopropyl alcohol), detergents (e.g., sodium dodecyl sulfate), depilatory or keratinolytic agents (e.g., calcium hydroxide, salicylic acid, ureas), humectants (e.g., glycerol, other glycols), polymers (e.g., polyethylene or propylene glycol, polyvinyl pyrrolidone), or combinations thereof may be used or incorporated in the formulation. Similarly, abrading the skin (e.g., abrasives like an emery board or paper, sand paper, fibrous pad, pumice), removing a superficial layer of skin (e.g., peeling or stripping with an adhesive tape), microporating the skin using an energy source (e.g., heat, light, sound, electrical, magnetic) or a barrier disruption device (e.g., gun, microneedle), or combinations thereof may act as a physical penetration enhancer. See WO98/29134 for microporation of skin and U.S. Pat. No. 6,090,790 for microneedles and U.S. Pat. No. 6,168,587 for transdermal guns which might be adapted for use in transcutaneous vaccination. The objective of chemical or physical penetration enhancement in conjunction with TCl is to remove at least the stratum corneum or deeper epidermal layer without perforating the skin through to the dermal layer. This is preferably accomplished with minor discomfort at most to the human or animal subject and without bleeding at the site. For example, applying the formulation to intact skin may not involve thermal, optical, sonic, or electromagnetic energy to perforate layers of the skin below the stratum corneum or epidermis.

Formulations which are useful for vaccination are also provided as well as processes for their manufacture. The formulation may be in dry or liquid form. A dry formulation is more easily stored and transported than conventional vaccines, it breaks the cold chain required from the vaccine's place of manufacture to the locale where vaccination occurs. Without being limited to any particular mode of action, another way in which a dry formulation may be an improvement over liquid formulations is that high concentrations of a dry active component of the formulation (e.g., one or more adjuvants and/or antigens) may be achieved by solubilization directly at the site of immunization over a short time span. Moisture from the skin (e.g., perspiration) and an occlusive dressing may hasten this process. In this way, it is possible that a concentration approaching the solubility limit of the active ingredient may be achieved in situ. Alternatively, the dry, active ingredient of the formulation per se may be an improvement by providing a solid particulate form that is taken up and processed by antigen presenting cells. These possible mechanisms are discussed not to limit the scope of the invention or its equivalents, but to provide insight into the operation of the invention and to guide the use of this formulation in immunization and vaccination.

The formulation may be provided as a liquid: cream, emulsion, gel, lotion, ointment, paste, solution, suspension, or other liquid forms. Dry formulations may be provided in various forms: for example, fine or granulated powders, uniform films, pellets, and tablets. The formulation may be dissolved and then dried in a container or on a flat surface (e.g., skin), or it may simply be dusted on the flat surface. It may be air dried, dried with elevated temperature, freeze or spray dried, coated or sprayed on a solid substrate and then dried, dusted on a solid substrate, quickly frozen and then slowly dried under vacuum, or combinations thereof. If different molecules are active ingredients of the formulation, they may be mixed in solution and then dried, or mixed in dry form only. Compartments or chambers of the patch may be used to separate active ingredients so that only one of the antigens or adjuvants is kept in dry form prior to administration; separating liquid and solid in this manner allows control over the time and rate of the dissolving of at least one dry, active ingredient.

A “patch” refers to a product which includes a solid substrate (e.g., occlusive or non-occlusive surgical dressing) as well as at least one active ingredient. Liquid may be incorporated in a patch (i.e., a wet patch). One or more active components of the formulation may be applied on the substrate, incorporated in the substrate or adhesive of the patch, or combinations thereof. A dry patch may or may not use a liquid reservoir to solubilize the formulation.

Formulation in liquid or solid form may be applied with one or more adjuvants and/or antigens both at the same or separate sites or simultaneously or in frequent, repeated applications. The patch may include a controlled-release reservoir or a rate-controlling matrix or membrane may be used which allows stepped release of adjuvant and/or antigen. It may contain a single reservoir with adjuvant and/or antigen, or multiple reservoirs to separate individual antigens and adjuvants. The patch may include additional antigens such that application of the patch induces an immune response to multiple antigens. In such a case, antigens may or may not be derived from the same source, but they will have different chemical structures so as to induce an immune response specific for different antigens. Multiple patches may be applied simultaneously; a single patch may contain multiple reservoirs. For effective treatment, multiple patches may be applied at intervals or constantly over a period of time; they may be applied at different times, for overlapping periods, or simultaneously. At least one adjuvant and/or adjuvant may be maintained in dry form prior to administration. Subsequent release of liquid from a reservoir or entry of liquid into a reservoir containing the dry ingredient of the formulation will at least partially dissolve that ingredient.

Solids (e.g., particles of nanometer or micrometer dimensions) may also be incorporated in the formulation. Solid forms (e.g., nanoparticles or microparticles) may aid in dispersion or solubilization of active ingredients; assist in carrying the formulation through superficial layers of the skin; provide a point of attachment for adjuvant, antigen, or both to a substrate that can be opsonized by antigen presenting cells, or combinations thereof. Prolonged release of the formulation from a porous solid formed as a sheet, rod, or bead acts as a depot.

The formulation may be manufactured under aseptic conditions acceptable to appropriate regulatory agencies (e.g., Food and Drug Administration) for biologicals and vaccines. Optionally, components such as dessicants, excipients, stabilizers, humectants, preservatives, adhesives, patch materials, or combinations thereof may be included in the formulation even though they are immunologically inactive. They may, however, have other desirable properties or characteristics.

A single or unit dose of formulation suitable for administration is provided. The amount of adjuvant or antigen in the unit dose may be anywhere in a broad range from about 0.001 μg to about 10 mg. This range may be from about 0.1 μg to about 1 mg; a narrower range is from about 5 μg to about 500 μg. Other suitable ranges are between about 1 μg and about 10 μg, between about 10 μg and about 50 μg, between about 50 μg and about 200 μg, and between about 1 mg and about 5 mg. A preferred dose for a toxin is about 50 μg or 100 μg or less (e.g., from about 1 μg to about 50 μg or 100 μg). The ratio between antigen and adjuvant may be about 1:1 (e.g., E. coli heat-labile enterotoxin when it is both antigen and adjuvant) but higher ratios may be suitable for poor antigens (e.g., about 1:10 or less), or lower ratios of antigen to adjuvant may also be used (e.g., about 10:1 or more). The native ratios between LT and ETEC antigens may be used for whole-cell or lysate formulations.

A formulation comprising adjuvant and antigen or polynucleotide may be applied to skin of a human or animal subject, antigen is presented to immune cells, and an antigen-specific immune response is induced. This may occur before, during, or after infection by pathogen. Only antigen or polynucleotide encoding antigen may be required, but no additional adjuvant, if the immunogenicity of the formulation is sufficient to not require adjuvant activity. The formulation may include an additional antigen such that application of the formulation induces an immune response against multiple antigens (i.e., multivalent). In such a case, antigens may or may not be derived from the same source, but the antigens will have different chemical structures so as to induce immune responses specific for the different antigens. Antigen-specific lymphocytes may participate in the immune response and, in the case of participation by B lymphocytes, antigen-specific antibodies may be part of the immune response. The formulations described above may include dessicants, excipients, humectants, stabilizers, preservatives, adhesives, and patch materials known in the art.

The invention is used to treat a subject (e.g., a human or animal in need of treatment such as prevention of disease, protection from effects of infection, therapy of existing disease or symptoms, or combinations thereof). When the antigen is derived from a pathogen, the treatment may vaccinate the subject against infection by the pathogen or against its pathogenic effects such as those caused by toxin secretion. The invention may be used therapeutically to treat existing disease, protectively to prevent disease, to reduce the severity and/or duration of disease, to ameliorate symptoms of disease, or combinations thereof.

The application site may be protected with anti-inflammatory corticosteroids such as hydrocortisone, triamcinolone and mometazone or non-steroidal anti-inflammatory drugs (NSAID) to reduce possible local skin reaction or modulate the type of immune response. Similarly, anti-inflammatory steroids or NSAID may be included in the patch material, or liquid or solid formulations; and corticosteroids or NSAID may be applied after immunization. IL-10, TNF-α, other immunomodulators may be used instead of the anti-inflammatory agents. Moreover, the formulation may be applied to skin overlying more than one draining lymph node field using either single or multiple applications. The formulation may include additional antigens such that application induces an immune response to multiple antigens. In such a case, the antigens may or may not be derived from the same source, but the antigens will have different chemical structures so as to induce an immune response specific for the different antigens. Multi-chambered patches could allow more effective delivery of multivalent vaccines as each chamber covers different antigen presenting cells. Thus, antigen presenting cells would encounter only one antigen (with or without adjuvant) and thus would eliminate antigenic competition and thereby enhancing the response to each individual antigen in the multivalent vaccine.

The formulation may be epicutaneously applied to skin to prime or boost the immune response in conjunction with penetration techniques or other routes of immunization. Priming by transcutaneous immunization (TCl) with either single or multiple applications may be followed with enteral, mucosal, parenteral, and/or transdermal techniques for boosting immunization with the same or altered antigens. Priming by enteral, mucosal, parenteral, and/or transdermal immunization with either single or multiple applications may be followed with transcutaneous techniques for boosting immunization with the same or altered antigens. It should be noted that TCl is distinguished from conventional topical techniques like mucosal or transdermal immunization because the former requires a mucous membrane (e.g., lung, mouth, nose, rectum) not found in the skin and the latter requires perforation of the skin through the dermis. The formulation may include additional antigens such that application to skin induces an immune response to multiple antigens.

In addition to antigen and adjuvant, the formulation may comprise a vehicle. For example, the formulation may comprise an AQUAPHOR, FREUND, RIBI or SYNTEX emulsion; water-in-oil emulsions (e.g., aqueous creams, ISA-720), oil-in-water emulsions (e.g., oily creams, ISA-51, MF59), microemulsions, anhydrous lipids and oil-in-water emulsions, other types of emulsions; gels, fats, waxes, oil, silicones, and humectants (e.g., glycerol).

Antigen may be derived from any pathogen that infects a human or animal subject (e.g., bacterium, virus, fungus, or protozoan). The chemical structure of the antigen may be described as one or more of carbohydrate, fatty acid, and protein (e.g., glycolipid, glycoprotein, lipoprotein). Proteinaceous antigen is preferred. The molecular weight of the antigen may be greater than 500 daltons, 800 daltons, 1000 daltons, 10 kilodaltons, 100 kilodaltons, or 1000 kilodaltons. Chemical or physical penetration enhancement may be preferred for macromolecular structures like cells, viral particles, and molecules of greater than one megadalton (e.g., CS6 antigen), but techniques like hydration and swabbing with a solvent may be sufficient to induce immunization across the skin. Antigen may be obtained by recombinant techniques, chemical synthesis, or at least partial purification from a natural source. It may be a chemical or recombinant conjugates: for example, linkage between chemically reactive groups or protein fusion. Antigen may be provided as a live cell or virus, an attenuated live cell or virus, a killed cell, or an inactivated virus. Alternatively, antigen may be at least partially purified in cell-free form (e.g., cell or viral lysate, membrane or other subcellular fraction). Because most adjuvants would also have immunogenic activity and would be considered antigens, adjuvants would also be expected to have the aforementioned properties and characteristics of antigens.

The choice of adjuvant may allow potentiation or modulation of the immune response. Moreover, selection of a suitable adjuvant may result in the preferential induction of a humoral or cellular immune response, specific antibody isotypes (e.g., IgM, IgD, IgA1, IgA2, IgE, IgG1, IgG2, IgG3, and/or IgG4), and/or specific T-cell subsets (e.g., CTL, Th1, Th2 and/or T_DTH). The adjuvant is preferably a chemically activated (e.g., proteolytically digested) or genetically activated (e.g., fusions, deletion or point mutants) ADP-ribosylating exotoxin or B subunit thereof. Adjuvant, antigen, or both may optionally be provided in the formulation with a polynucleotide (e.g., DNA, RNA, cDNA, cRNA) encoding the adjuvant or antigen as appropriate. Covalently closed, circular DNA such as plasmids are preferred forms of the polynucleotide; however, linear forms may also be used. The polynucleotide may include a region such as an origin of replication, centromere, telomere, promoter, enhancer, silencer, transcriptional initiation or termination signal, splice acceptor or donor site, ribosome binding site, translational initiation or termination signal, polyadenylation signal, cellular localization signal, protease cleavage site, polylinker site, or combinations thereof as are found in expression vectors.

An “antigen” is an active component of the formulation which is specifically recognized by the immune system of a human or animal subject after immunization or vaccination. The antigen may comprise a single or multiple immunogenic epitopes recognized by a B-cell receptor (i.e., secreted or membrane-bound antibody) or a T-cell receptor. Proteinaceous epitopes recognized by T-cell receptors have typical lengths and conserved amino acid residues depending on whether they are bound by major histocompatibility complex (MHC) Class I or Class II molecules on the antigen presenting cell. In contrast, proteinaceous epitopes recognized antibody may be of variable length including short, extended oligopeptides and longer, folded polypeptides. Single amino acid differences between epitopes may be distinguished. The antigen is capable of inducing an immune response against a molecule of a pathogen (e.g., a CS6 antigen is capable of inducing a specific immune response against the CS6 molecule of ETEC). Thus, antigen is usually identical or at least derived from the chemical structure of a specific molecule of the pathogen, but mimetics which are only distantly related to such chemical structures may also be successfully used.

An “adjuvant” is an active component of the formulation to assist in inducing an immune response to the antigen. Adjuvant activity is the ability to increase the immune response to a heterologous antigen (i.e., antigen which is a separate chemical structure from the adjuvant) by inclusion of the adjuvant itself in a formulation or in combination with other components of the formulation or particular immunization techniques. As noted above, a molecule may contain both antigen and adjuvant activities by chemically conjugating antigen and adjuvant or genetically fusing coding regions of antigen and adjuvant; thus, the formulation may contain only one ingredient or component.

The term “effective amount” is meant to describe that amount of adjuvant or antigen which induces an antigen-specific immune response. A “subunit” immunogen or vaccine is a formulation comprised of active components (e.g., adjuvant, antigen) which have been isolated from other cellular or viral components of the pathogen (e.g., membrane or polysaccharide components like endotoxin) by recombinant techniques, chemical synthesis, or at least partial purification from a natural source.

Induction of an immune response may provide a treatment such as, for example, prophylactic or therapeutic vaccination for an infectious disease. A product or method “induces” when its presence or absence causes a statistically significant change in the immune response's magnitude and/or kinetics; change in the induced elements of the immune system (e.g., humoral vs. cellular, Th1 vs. Th2); effect on the health and well-being of the subject; or combinations thereof.

The term “draining lymph node field” as used in the invention means an anatomic area over which the lymph collected is filtered through a set of defined lymph nodes (e.g., cervical, axillary, inguinal, epitrochelear, popliteal, those of the abdomen and thorax). Thus, the same draining lymph node field may be targeted by immunization (e.g., enteral, mucosal, parenteral, transcutaneous, transdermal) within the few days required for antigen presenting cells to migrate to the lymph nodes if the sites and times of immunization are spaced to bring different components of the formulation together (e.g., two closely spaced patches with either adjuvant or antigen may be effective when neither alone could successfully used). For example, a patch delivering adjuvant by the transcutaneous technique may be placed on the same arm as is injected with a conventional vaccine to boost its effectiveness in elderly, pediatric, or other immunologically compromised populations. In contrast, applying patches to different limbs may prevent an adjuvant-containing patch from boosting the effectiveness of a patch containing only antigen.

Without being bound to any particular theory for the operation of the invention but only to provide an explanation for our observations, we hypothesize that this transcutaneous delivery system carries antigen to cells of the immune system where an immune response is induced. The antigen may pass through the normally present protective outer layers of the skin (i.e., stratum corneum) and induce the immune response directly, or through an antigen presenting cell population in the epidermis (e.g., macrophage, tissue macrophage, Langerhans cell, other dendritic cells, B lymphocyte, or Kupffer cell) that presents processed antigen to lymphocytes. Thus, with or without penetration enhancement techniques, the dermis is not penetrated as in subcutaneous injection or transdermal techniques. Optionally, the antigen may pass through the stratum corneum via a hair follicle or a skin organelle (e.g., sweat gland, oil gland).

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins (bARE) as an example, may target the epidermal Langerhans cell, known to be among the most efficient of the antigen presenting cells (APC). Maturation of APC may be assessed by morphology and phenotype (e.g., expression of MHC Class II molecules, CD83, or co-stimulatory molecules). We have found that bARE appear to activate Langerhans cells when applied epicutaneously to intact skin. Adjuvants such as trypsin-cleaved bARE may enhance Langerhans cell activation. Langerhans cells direct specific immune responses through phagocytosis of the antigens, and migration to the lymph nodes where they act as APC to present the antigen to lymphocytes, and thereby induce a potent antibody response. Although the skin is generally considered a barrier to pathogens, the imperfection of this barrier is attested to by the numerous Langerhans cells distributed throughout the epidermis that are designed to orchestrate the immune response against organisms invading through the skin. According to Udey (Clin Exp Immunol, 107:s6-s8, 1997):

• • Langerhans cells are bone-marrow derived cells that are present in all mammalian stratified squamous epithelia. They comprise all of the accessory cell activity that is present in uninflamed epidermis, and in the current paradigm are essential for the initiation and propagation of immune responses directed against epicutaneously applied antigens. Langerhans cells are members of a family of potent accessory cells (‘dendritic cells’) that are widely distributed, but infrequently represented, in epithelia and solid organs as well as in lymphoid tissue.
  • It is now recognized that Langerhans cells (and presumably other dendritic cells) have a life cycle with at least two distinct stages. Langerhans cells that are located in epidermis constitute a regular network of antigen-trapping ‘sentinel’ cells. Epidermal Langerhans cells can ingest particulates, including microorganisms, and are efficient processors of complex antigens. However, they express only low levels of MHC class I and II antigens and costimulatory molecules (ICAM-1, B7-1 and B7-2) and are poor stimulators of unprimed T cells. After contact with antigen, some Langerhans cells become activated, exit the epidermis and migrate to T-cell-dependent regions of regional lymph nodes where they localize as mature dendritic cells. In the course of exiting the epidermis and migrating to lymph nodes, antigen-bearing epidermal Langerhans cells (now the ‘messengers’) exhibit dramatic changes in morphology, surface phenotype and function. In contrast to epidermal Langerhans cells, lymphoid dendritic cells are essentially non-phagocytic and process protein antigens inefficiently, but express high levels of MHC class I and class II antigens and various costimulatory molecules and are the most potent stimulators of naive T cells that have been identified.”

The potent antigen presenting capability of Langerhans cells can be exploited for transcutaneously-delivered immunogens and vaccines. An immune response using the skin's immune system may be achieved by delivering the formulation only to Langerhans cells in the stratum corneum (i.e., the outermost layer of the skin consisting of cornified cells and lipids) and subsequently activating the Langerhans cells to take up antigen, migrate to B-cell follicles and/or T-cell dependent regions, and present the antigen to B and/or T lymphocytes. If antigens other that bARE (e.g., toxin, colonization or virulence factor) are to be phagocytosed by Langerhans cells, then these antigens could also be transported to the lymph node for presentation to T lymphocytes and subsequently induce an immune response specific for that antigen. Thus, a feature of TCl is the activation of the Langerhans cell, presumably by bARE or derivatives thereof, chemokines, cytokines, PAMP, or other Langerhans cell activating substance including contact sensitizers and adjuvants. Increasing the size of the skin population of Langerhans cells or their state of activation would also be expected to enhance the immune response (e.g., acetone pretreatment). In aged subjects or Langerhans cell-depleted skin (i.e., from UV damage), it may be possible to replenish the population of Langerhans cells (e.g., tretinoin pretreatment).

Adjuvants such as bARE are known to be highly toxic when injected or given systemically. But if placed on the surface of intact skin (i.e., epicutaneous), they are unlikely to induce systemic toxicity. Thus, the transcutaneous route may allow the advantage of adjuvant effects without systemic toxicity. A similar absence of toxicity could be expected if the skin were penetrated only below the stratum corneum (e.g., near or at the epidermis), but not through the dermis. Thus, the ability to induce activation of the immune system through the skin confers the unexpected advantage of potent immune responses without systemic toxicity.

The magnitude of the antibody response induced by affinity maturation and isotype switching to predominantly IgG antibodies is generally achieved with T-cell help, and activation of both Th1 and Th2 pathways is suggested by the production of IgG1 and IgG2a. Alternatively, a large antibody response may be induced by a thymus-independent antigen type 1 (TI-1) which directly activates the B lymphocyte or could have similar activating effects on B lymphocytes such as up-regulation of MHC Class II, B7, CD40, CD25, and ICAM-1 molecules.

The spectrum of commonly known skin immune responses is represented by atopy and contact dermatitis. Contact dermatitis, a pathogenic manifestation of Langerhans cell activation, is directed by Langerhans cells which phagocytose antigen, migrate to lymph nodes, present antigen, and sensitize T lymphocytes that migrate to the skin and cause the intense destructive cellular response that occurs at affected skin sites. Such responses are not generally known to be associated with antigen-specific IgG antibodies. Atopic dermatitis may utilize the Langerhans cell in a similar fashion, but is identified with Th2 cells and is generally associated with high levels of IgE antibody.

On the other hand, transcutaneous immunization with bARE provides a useful and desirable immune response. There are usually no findings typical of atopy or contact dermatitis given the high levels of IgG that are induced. Cholera toxin or E. coli heat-labile enterotoxin epicutaneously applied to skin can achieve immunization in the absence of lymphocyte infiltration 24, 48 and 120 hours after immunization. The minor skin reactivity seen in preclinical trials were easily treated. This indicates that Langerhans cells engaged by transcutaneous immunization as they “comprise all of the accessory cell activity that is present in uninflamed epidermis, and in the current paradigm are essential for the initiation and propagation of immune responses directed against epicutaneously applied antigens” (Udey, 1997). The uniqueness of the transcutaneous immune response here is also indicated by the both high levels of antigen-specific IgG antibody, and the type of antibody produced (e.g., IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA) and generally the absence of antigen specific IgE antibody. Transcutaneous immunization could conceivably occur in tandem with skin inflammation if sufficient activation of antigen presenting cells and T lymphocytes were to occur in a transcutaneous response coexisting with atopy or contact dermatitis.

Transcutaneous targeting of Langerhans cells may also be used in tandem with agents to deactivate all or part of their antigen presenting function, thereby modifying immunization or preventing sensitization. Techniques to modulate Langerhans activation or other skin immune cells include, for example, the use of anti-inflammatory steroidal or non-steroidal agents (NSAID); cyclosporin, FK506, rapamycin, cyclophosphamide, glucocorticoids, or other immunosuppressants; interleukin-10; interleukin-1 monoclonal antibodies (mAB) or soluble receptor antagonists (RA); interleukin-1 converting enzyme (ICE) inhibitors; or depletion via superantigens such as through Staphylococcal enterotoxin A (SEA) induced epidermal Langerhans cell depletion. Similar compounds may be used to modify the innate response of Langerhans cells and induce different T-helper responses (Th1 or Th2) or may modulate skin inflammatory responses to decrease potential side effects of the immunization. Similarly, lymphocytes may be immunosuppressed before, during or after immunization by administering immunosuppressant separately or by coadministration of immunosuppressant with the formulation. For example, it may be possible to induce a potent systemic protective immune responses with agents that would normally result in allergic or irritant contact hypersensitivity but adding inhibitors of ICE may alleviate adverse skin reactions.

TCl may be accompished through the ganglioside GM1 binding activity of CT, LT, or subunits thereof (e.g., CTB or LTB). Ganglioside GM1 is a ubiquitous cell membrane glycolipid found in all mammalian cells. When the pentameric CT B subunit binds to the cell surface, a hydrophilic pore is formed which allows the A subunit to insert across the lipid bilayer. Other binding targets on the APC may be utilized. The LT B subunit binds to ganglioside GM1 in addition to other gangliosides and its binding activities may account for its the fact that LT is highly immunogenic on the skin.

TCl with bARE or B subunit-containing fragments or conjugates thereof may require their GM1 ganglioside binding activity. When mice were transcutaneously immunized with CT, CTA and CTB, CT and CTB were required for induction of an immune response. CTA contains the ADP-ribosylating exotoxin activity but only CT and CTB containing the binding activity are able to induce an immune response indicating that the B subunit was necessary and sufficient to immunize through the skin. We conclude that the Langerhans cells or other APC may be activated by CTB binding to its cell surface resulting in a transcutaneous immune response.

Antigen

A transcutaneous immunization system delivers agents to specialized cells (e.g., antigen presentation cell, lymphocyte) that produce an immune response. These agents as a class are called antigens. Antigen may be composed of chemical structures such as, for example, carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, phospholipid, polypeptide, conjugates thereof, or any other material known to induce an immune response. Antigen may be provided as a whole organism such as, for example, a bacterium or virion; antigen may be obtained from an extract or lysate, either from whole cells or membrane alone; or antigen may be chemically synthesized or produced by recombinant technology.

Antigen of the invention may be expressed by recombinant technology, preferably as a fusion with an affinity or epitope tag; chemical synthesis of an oligopeptide, either free or conjugated to carrier proteins, may be used to obtain antigen of the invention. Oligopeptides are considered a type of polypeptide. Oligopeptide lengths of 6 residues to 20 residues are preferred. Polypeptides may also by synthesized as branched structures (e.g., U.S. Pat. Nos. 5,229,490 and 5,390,111). Antigenic polypeptides include, for example, synthetic or recombinant B-cell and T-cell epitopes, universal T-cell epitopes, and mixed T-cell epitopes from one organism or disease and B-cell epitopes from another. Antigen obtained through recombinant technology or peptide synthesis, as well as antigen obtained from natural sources or extracts, may be purified by the antigen's physical and chemical characteristics, preferably by fractionation or chromatography. Recombinants may combine B subunits or chimeras of bARE. A multivalent antigen formulation may be used to induce an immune response to more than one antigen at the same time. Conjugates may be used to induce an immune response to multiple antigens, to boost the immune response, or both. Additionally, toxins may be boosted by the use of toxoids, or toxoids boosted by the use of toxins. Transcutaneous immunization may be used to boost responses induced initially by other routes of immunization such as by oral, nasal or parenteral routes. Antigen includes, for example, toxins, toxoids, subunits thereof, or combinations thereof (e.g., cholera toxin, tetanus toxoid); additionally, toxins, toxoids, subunits thereof, or combinations thereof may act as both antigen and adjuvant. Such oral/transcutaneous or transcutaneous/oral immunization may be especially important to enhance mucosal immunity in diseases where mucosal immunity correlates with protection.

Antigen may be solubilized in a buffer or water or organic solvents such as alcohol or DMSO, or incorporated in gels, emulsion, microemulsions, and creams. Suitable buffers include, but are not limited to, phosphate buffered saline Ca⁺⁺/Mg⁺⁺ free, phosphate buffered saline, normal saline (150 mM NaCl in water), and Hepes or Tris buffer. Antigen not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS. In the case of antigen soluble only at acid pH, acetate-PBS at acid pH may be used as a diluent after solubilization in dilute acetic acid. Glycerol may be a suitable non-aqueous buffer for use in the invention.

A hydrophobic antigen can be solubilized in a detergent or surfactant, for example a polypeptide containing a membrane-spanning domain. Furthermore, for formulations containing liposomes, an antigen in a detergent solution (e.g., cell membrane extract) may be mixed with lipids, and liposomes then may be formed by removal of the detergent by dilution, dialysis, or column chromatography. Certain antigens (e.g., membrane proteins) need not be soluble per se, but can be inserted directly into a lipid membrane (e.g., a virosome), in a suspension of virion alone, or suspensions of microspheres or heat-inactivated bacteria which may be taken up by activate antigen presenting cells (e.g., opsonization). Antigens may also be mixed with a penetration enhancer as described in WO 99/43350.

Many antigens are known in the art which can be used to vaccinate human or animal subjects and induce an immune response specific for particular pathogens, as well as methods of preparing antigen, determining a suitable dose of antigen, assaying for induction of an immune response, and treating infection by a pathogen (e.g., bacterium, virus, fungus, or protozoan).

The effect of Escherichia coli infection of mammals is dependent on the particular strain of organism. Many beneficial E. coli are present in the intestines. Since the initial association with diarrheal illness, five categories of diarrheagenic E. coli have been identified: enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroaggregative (EAggEC), and enteroinvasive (EIEC). They are grouped according to characteristic virulence properties, such as elaboration of toxins and colonization factors and/or by specific types of interactions with intestinal epithelial cells. ETEC are the most common of the diarrheagenic E. coli and pose the greatest risk to travelers. Strains which have been cultured from humans include B7A (CS6, LT, STa), H10407 (CFA/I, LT, STa) and E24377A (CS3, CS1, LT, STa). They may be used singly or in combination as whole-cell sources of antigen providing a variety of different toxins and colonization factors.

There is a need for vaccines which are specific against enterotoxigenic E. coli that give rise to antibodies that cross-react with and cross-protect against the more common colonization and virulence factors. The CS4-CFA/I family of fimbrial proteins are found on some of the more prevalent enterotoxigenic E. coli strains: there are six members of this family of ETEC antigens, CFA/I, CS1, CS2, CS4, CS17, and PCF 0166.

Colonization factor antigens (CFA) of ETEC are important in the initial step of colonization and adherence of the bacterium to intestinal epithelia. In epidemiological studies of adults and children with diarrhea, CFA/I is found in a large percentage of morbidity attributed to ETEC. The CFA/I is present on the surfaces of bacteria in the form of pili (fimbriae), which are rigid, 7 nm diameter protein fibers composed of repeating pilin subunits. The CFA/I antigens promote mannose-resistant attachment to human brush borders with an apparent sialic acid sensitivity. Hence, it has been postulated that a vaccine that establishes immunity against these proteins may prevent attachment to host tissues and subsequent disease.

Other antigens including CS3, CS5, and CS6. CFA/I, CS3 and CS6 may occur alone, but with rare exception CS1 is only found with CS3, CS2 with CS3, CS4 with CS6 and CS5 with CS6. Serological studies show these antigens occur in strains accounting for up to about 75% or as little as about 25% of ETEC cases, depending on the location of the study.

Consensus peptides have been described in U.S. Pat. No. 5,914,114 which raise antibodies against the antigens of all members of the E. coli family CS4-CFA/I. While the N-terminus of members of this family shows a high degree of identity, the remainder of the sequence of the proteins shows less relatedness across the strains. Consensus peptides encompass known linear B- and T-cell epitopes, and bears a high degree of evolutionary relatedness across the six different colonization factors. For example, consensus peptides have the amino acid sequence (an amino acid residue may be added to either termini or modified internally to provide a reactive linkage): VEKNITVTASVDPTIDLLQADGSALPSAVALTYSPA (SEQ ID NO: 1) and VEKNITVTASVDPTIDLLQADGSALPASVALTYSPA (SEQ ID NO: 2).

These consensus peptides were constructed based on the homologous regions of the CFA/I, CS1, CS2, CS4, CS17, and PCF 0166 antigens.

TABLE 1
Alignment of antigens of the CS4-CFA/I family
(SEQ ID NOS: 3-8)

Antigen	Amino Acid Sequence
CFA/I	VEKNITVTASVDPVIDLLQADGSALPSAVALTYSPAS
CS1	VEKTISVTASVDPTVDLLQSDGSALPNSVALTYSPAV
CS2	AEINITVTASVDPVIDLLQA
CS4	VEKNITVTASVDPTIDILQADGSYLPTAVELTYSPAA
CS17	VEKNITVRASVDKLIDLLQADGTSLPDSIALTYSVA
PCF0166	VEKNITVTASVDPTIDILQANGSAL

CS6, a component of colonization factor IV (CFA/IV), can also be found in more than about 25% of ETEC strains in serological surveys (e.g., soldiers in the Middle East). The nucleotide sequences of CS3 and CS6 antigens, along with a process for producing them, are described in U.S. Pat. No. 5,698,416.

Other antigens which may be used are toxins that cause enteric disease such as, for example, shiga toxin and E. coli enterotoxins. Heat-labile enterotoxin (LT) is described below, but heat-stable enterotoxins (e.g., STa, STb) which cause disease symptoms may also be neutralized by antibody. LT is a periplasmic toxin and ST is an extracellular toxin. STa is methanol soluble and STb is methanol insoluble. Two different precursors are used: STa is a 18-19 amino acid peptide and STb is a 48 amino acid peptide with no sequence similarity between them. Conjugates between LT and ST or ST multimers may also be used (see U.S. Pat. No. 4,886,663).

It would be advantageous for a vaccine to be developed for a broad range of common traveler's diseases, especially enteric infectious diseases. For example, campylobacteriosis (Campylobacter jejuni), giardiasis (Giardia intestinalis), hepatitis (hepatitis virus A or B), malaria (Plasmodium falciparum, P. vivax, P. ovale, and P. malarae), shigellosis (Shigella boydii, S. dysenterae, S. flexneri, and S. sonnet), viral gastroenteritis (rotavirus), and combinations thereof may be treated by including antigens derived from the responsible pathogen. Systemic or mucosal antibodies that neutralize toxicity or block attachment and entry into the cell are desirable. An immune response which is specific for molecules associated with pathogens (e.g., toxins, membrane proteins) may be induced by various routes of administration (e.g., enteral, mucosal, parenteral, transcutaneous).

Adjuvant

The formulation contains an adjuvant, although a single molecule may contain both adjuvant and antigen properties (e.g., E. coli heat-labile enterotoxin). Adjuvants are substances that are used to specifically or non-specifically potentiate an antigen-specific immune response, perhaps through activation of antigen presenting cells (e.g., dendritic cells in various layers of the skin, especially Langerhans cells). See also Elson et al. (in Handbook of Mucosal Immunology, Academic Press, 1994). Although activation may initially occur in the epidermis or dermis, the effects may persist as the dendritic cells migrate through the lymph system and the circulation. Adjuvant may be formulated and applied with or without antigen, but generally, activation of antigen presenting cells by adjuvant occurs prior to presentation of antigen. Alternatively, they may be separately presented within a short interval of time but targeting the same anatomical region (e.g., the same draining lymph node field).

Adjuvants include, for example, chemokines (e.g., defensins, HCC-1, HCC4, MCP-1, MCP-3, MCP4, MIP-1α, MIP-1β, MIP-1δ, MIP-3α, MIP-2, RANTES); other ligands of chemokine receptors (e.g., CCR1, CCR-2, CCR-5, CCR-6, CXCR-1); cytokines (e.g., IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12; IFN-γ; TNF-α; GM-CSF); other ligands of receptors for those cytokines, immunostimulatory CpG motifs in bacterial DNA or oligonucleotides; muramyl dipeptide (MDP) and derivatives thereof (e.g., murabutide, threonyl-MDP, muramyl tripeptide); heat shock proteins and derivatives thereof; Leishmania homologs of elF4a and derivatives thereof; bacterial ADP-ribosylating exotoxins and derivatives thereof (e.g., genetic mutants, A and/or B subunit-containing fragments, chemically toxoided versions); chemical conjugates or genetic recombinants containing bacterial ADP-ribosylating exotoxins or derivatives thereof; C3d tandem array; lipid A and derivatives thereof (e.g., monophosphoryl or diphosphoryl lipid A, lipid A analogs, AGP, AS02, AS04, DC-Chol, Detox, OM-174); ISCOMS and saponins (e.g., QUIL A, QS-21); squalene; superantigens; or salts (e.g., aluminum hydroxide or phosphate, calcium phosphate). See also Nohria et al. (Biotherapy, 7:261-269, 1994) and Richards et al. (in Vaccine Design, Eds. Powell et al., Plenum Press, 1995) for other useful adjuvants.

Adjuvant may be chosen to preferentially induce antibody or cellular effectors, specific antibody isotypes (e.g., IgM, IgD, IgA1, IgA2, secretory IgA, IgE, IgG1, IgG2, IgG3, and/or IgG4), or specific T-cell subsets (e.g., CTL, Th1, Th2 and/or T_DTH). For example, antigen presenting cells may present Class II-restricted antigen to precursor CD4+ T cells, and the Th1 or Th2 pathway may be entered. T helper cells actively secreting cytokine are primary effector cells; they are memory cells if they are resting. Reactivation of memory cells produces memory effector cells. Th1 characteristically secrete IFN-γ (TNF-β and IL-2 may also be secreted) and are associated with “help” for cellular immunity, while Th2 characteristically secrete IL-4 (IL-5 and IL-13 may also be secreted) and are associated with “help” for humoral immunity. Depending on disease pathology, adjuvants may be chosen to prefer a Th1 response (e.g., antigen-specific cytolytic cells) vs. a Th2 response (e.g., antigen-specific antibodies).

Unmethylated CpG dinucleotides or similar motifs are known to activate B lymphocytes and macrophages (see U.S. Pat. No. 6,218,371). Other forms of bacterial DNA can be used as adjuvants. Bacterial DNA is among a class of structures which have patterns allowing the immune system to recognize their pathogenic origins to stimulate the innate immune response leading to adaptive immune responses. These structures are called pathogen-associated molecular patterns (PAMP) and include lipopolysaccharides, teichoic acids, unmethylated CpG motifs, double-stranded RNA, and mannins. PAMP induce endogenous signals that can mediate the inflammatory response, act as costimulators of T-cell function and control the effector function. The ability of PAMP to induce these responses play a role in their potential as adjuvants and their targets are antigen presenting cells such as dendritic cells and macrophages. The antigen presenting cells of the skin could likewise be stimulated by PAMP transmitted through the skin. For example, Langerhans cells, a type of dendritic cell, could be activated by PAMP in solution on the skin with a transcutaneously poorly immunogenic molecule and be induced to migrate and present this poorly immunogenic molecule to T-cells in the lymph node, inducing an antibody response to the poorly immunogenic molecule. PAMP could also be used in conjunction with other skin adjuvants such as cholera toxin to induce different costimulatory molecules and control different effector functions to guide the immune response, for example from a Th2 to a Th1 response.

Most ADP-ribosylating exotoxins (bARE) are organized as A:B heterodimers with a B subunit containing the receptor binding activity and an A subunit containing the ADP-ribosyltransferase activity. Exemplary bARE include cholera toxin (CT) E. coli heat-labile enterotoxin (LT), diphtheria toxin, Pseudomonas exotoxin A (ETA), pertussis toxin (PT), C. botulinum toxin C2, C. botulinum toxin C3, C. limosum exoenzyme, B. cereus exoenzyme, Pseudomonas exotoxin S, S. aureus EDIN, and B. sphaeticus toxin. Mutant bARE, for example containing mutations of the trypsin cleavage site (e.g., Dickenson et al., Infect Immun, 63:1617-1623, 1995) or mutations affecting ADP-ribosylation (e.g., Douce et al., Infect Immun, 65:28221-282218, 1997) may be used.

CT, LT, ETA and PT, despite having different cellular binding sites, are potent adjuvants for transcutaneous immunization, inducing IgG antibodies but not IgE antibodies. CTB without CT can also induce IgG antibodies. Thus, both bARE and a derivative thereof can effectively immunize when epicutaneously applied to the skin. Native LT as an adjuvant and antigen, however, is clearly not as potent as native CT. But activated bARE can act as adjuvants for weakly immunogenic antigens in a transcutaneous immunization system. Thus, therapeutic immunization with one or more antigens could be used separately or in conjunction with immunostimulation of the antigen presenting cell to induce a prophylactic or therapeutic immune response.

In general, toxins can be chemically inactivated to form toxoids which are less toxic but remain immunogenic. We envision that the transcutaneous immunization system using toxin-based immunogens and adjuvants can achieve anti-toxin levels adequate for protection against these diseases. The anti-toxin antibodies may be induced through immunization with the toxins, or genetically-detoxified toxoids themselves, or with toxoids and adjuvants. Genetically toxoided toxins which have altered ADP-ribosylating exotoxin activity or trypsin cleavage site, but not binding activity, are envisioned to be especially useful as non-toxic activators of antigen presenting cells used in transcutaneous immunization and may reduce concerns over toxin use.

bARE can also act as an adjuvant to induce antigen-specific CTL through transcutaneous immunization. The bARE adjuvant may be chemically conjugated to other antigens including, for example, carbohydrates, polypeptides, glycolipids, and glycoprotein antigens. Chemical conjugation with toxins, their subunits, or toxoids with these antigens would be expected to enhance the immune response to these antigens when applied epicutaneously. To overcome the problem of the toxicity of the toxins (e.g., diphtheria toxin is known to be so toxic that one molecule can kill a cell) and to overcome the problems of working with such potent toxins as tetanus, several workers have taken a recombinant approach to producing genetically-produced toxoids. This is based on inactivating the catalytic activity of the ADP-ribosyl transferase by genetic deletion. These toxins retain the binding capabilities, but lack the toxicity, of the natural toxins. Such genetically toxoided exotoxins would be expected to induce a transcutaneous immune response and to act as adjuvants. They may provide an advantage in a transcutaneous immunization system in that they would not create a safety concern as the toxoids would not be considered toxic. Activation through a technique such as trypsin cleavage, however, would be expected to enhance the adjuvant qualities of LT through the skin which lacks trypsin-like enzymes. Additionally, several techniques exist to chemically modify toxins and can address the same problem. These techniques could be important for certain applications, especially pediatric applications, in which ingested toxins might possibly create adverse reactions.

Adjuvant may be biochemically purified from a natural source (e.g., pCT or pLT) or recombinantly produced (e.g., rCT or rLT). ADP-ribosylating exotoxin may be purified either before or after proteolysis (i.e., activation). B subunit of the ADP-ribosylating exotoxin may also be used: purified from the native enzyme after proteolysis or produced from a fragment of the entire coding region of the enzyme. The subunit of the ADP-ribosylating exotoxin may be used separately (e.g., CTB or LTB) or together (e.g., CTA-LTB, LTA-CTB) by chemical conjugation or genetic fusion.

Point mutations (e.g., single, double, or triple amino acid substitutions), deletions (e.g., protease recognition site), and isolated functional domains of ADP-ribosylating exotoxin may also be used as adjuvant. Derivatives which are less toxic or have lost their ADP-ribosylation activity, but retain their adjuvant activity have been described. Specific mutants of E. coli heat-labile enterotoxin include LT-K63, LT-R72, LT (H44A), LT (R192G), LT (R192G/L211A), and LT (Δ192-194). Toxicity may be assayed with the Y-1 adrenal cell assay (Clements and Finkelstein, Infect Immun, 24:760-769, 1979). ADP-ribosylation may be assayed with the NAD-agmatine ADP-ribosyltransferase assay (Moss et al., J Biol Chem, 268:6383-6387, 1993). Particular ADP-ribosylating exotoxins, derivatives thereof, and processes for their production and characterization are described in U.S. Pat. Nos. 4,666,837; 4,935,364; 5,308,835; 5,785,971; 6,019,982; 6,033,673; and 6,149,919.

An activator of Langerhans cells may also be used as an adjuvant. Examples of such activators include: inducers of heat shock protein; contact sensitizers (e.g., trinitrochlorobenzene, dinitrofluorobenzene, nitrogen mustard, pentadecylcatechol); toxins (e.g., Shiga toxin, Staph enterotoxin B); lipopolysaccharide (LPS), lipid A, or derivatives thereof; bacterial DNA; cytokines (e.g., TNF-α, IL-1β, IL-10, IL-12); members of the TGFβ superfamily, calcium ions in solution, calcium ionophores, and chemokines (e.g., defensins 1 or 2, RANTES, MIP-1α, MIP-2, IL-8).

If an immunizing antigen has sufficient Langerhans cell activating capabilities then a separate adjuvant may not be required, as in the case of LT which is both antigen and adjuvant. Alternatively, such antigens can be considered not to require an adjuvant because they are sufficiently immunogenic. It is envisioned that live cell or virus preparations, attenuated live cells or viruses, killed cells, inactivated viruses, and DNA plasmids could be effectively used for transcutaneous immunization. It may also be possible to use low concentrations of contact sensitizers or other activators of Langerhans cells to induce an immune response without inducing skin lesions.

Other techniques for enhancing activity of adjuvants may be effective, such as adding surfactants and/or phospholipids to the formulation to enhance adjuvant activity of ADP-ribosylating exotoxin by ADP-ribosylation factor. One or more ADP-ribosylation factors (ARF) may be used to enhance the adjuvanticity of bARE (e.g., ARF1, ARF2, ARF3, ARF4, ARF5, ARF6, ARD1). Similarly, one or more ARF could be used with an ADP-ribosylating exotoxin to enhance its adjuvant activity.

Undesirable properties or harmful side effects (e.g., allergic or hypersensitive reaction; atopy, contact dermatitis, or eczema; systemic toxicity) may be reduced by modification without destroying its effectiveness in transcutaneous immunization. Modification may involve, for example, removal of a reversible chemical modification (e.g., proteolysis) or encapsulation in a coating which reversibly isolates one or more components of the formulation from the immune system. For example, one or more components of the formulation may be encapsulated in a particle for delivery (e.g., microspheres, nanoparticles) although we have shown that encapsulation in lipid vesicles is not required for transcutaneous immunization and appears to have a negative effect. Phagocytosis of a particle may, by itself, enhance activation of an antigen presenting cell by upregulating expression of MHC Class I and/or Class II molecules and/or costimulatory molecules (e.g., CD40, B7 family members like CD80 and CD86).

Formulation

Processes for manufacturing a pharmaceutical formulation are well known. The components of the formulation may be combined with a pharmaceutically-acceptable carrier or vehicle, as well as any combination of optional additives (e.g., at least one diluent, binder, excipient, stabilizer, dessicant, preservative, coloring, or combinations thereof. See, generally, Ullmann's Encyclopedia of Industrial Chemistry, 6^th Ed. (electronic edition, 1998); Remington's Pharmaceutical Sciences, 22^nd (Gennaro, 1990, Mack Publishing); Pharmaceutical Dosage Forms, 2^nd Ed. (various editors, 1989-1998, Marcel Dekker); and Pharmaceutical Dosage Forms and Drug Delivery Systems (Ansel et al., 1994, Williams & Wilkins).

Good manufacturing practices are known in the pharmaceutical industry and regulated by government agencies (e.g., Food and Drug Administration). Sterile liquid formulations may be prepared by dissolving an intended component of the formulation in a sufficient amount of an appropriate solvent, followed by sterilization by filtration to remove contaminating microbes. Generally, dispersions are prepared by incorporating the various sterilized components of the formulation into a sterile vehicle which contains the basic dispersion medium. For production of solid forms that are required to be sterile, vacuum drying or freeze drying can be used. Solid dosage forms (e.g., powders, granules, pellets, tablets) or liquid dosage forms (e.g., liquid in ampules, capsules, vials) can be made from at least one active ingredient or component of the formulation.

Suitable procedures for making the various dosage forms and production of patches are known. The formulation may also be produced by encapsulating solid or liquid forms of at least one active ingredient or component, or keeping them separate in compartments or chambers. The patch may include a compartment containing a vehicle (e.g., a saline solution) which is disrupted by pressure and subsequently solubilizes the dry formulation of the patch. The size of each dose and the interval of dosing to the subject may be used to determine a suitable size and shape of the container, compartment, or chamber.

Formulations will contain an effective amount of the active ingredients (e.g., antigen and adjuvant) together with carrier or suitable amounts of vehicle in order to provide pharmaceutically-acceptable compositions suitable for administration to a human or animal. Formulation that include a vehicle may be in the form of an cream, emulsion, gel, lotion, ointment, paste, solution, suspension, or other liquid forms known in the art; especially those that enhance skin hydration.

The relative amounts of active ingredients within a dose and the dosing schedule may be adjusted appropriately for efficacious administration to a subject (e.g., animal or human). This adjustment may depend on the subject's particular disease or condition, and whether therapy or prophylaxis is intended. To simplify administration of the formulation to the subject, each unit dose would contain the active ingredients in predetermined amounts for a single round of immunization.

There are numerous causes of protein instability or degradation, including hydrolysis and denaturation. In the case of denaturation, the protein's conformation is disturbed and the protein may unfold from its usual globular structure. Rather than refolding to its natural conformation, hydrophobic interaction may cause clumping of molecules together (i.e., aggregation) or refolding to an unnatural conformation. Either of these results may entail diminution or loss of antigenic or adjuvant activity. Stabilizers may be added to lessen or prevent such problems.

The formulation, or any intermediate in its production, may be pretreated with protective agents (i.e., cryoprotectants and dry stabilizers) and then subjected to cooling rates and final temperatures that minimize ice crystal formation. By proper selection of cryoprotective agents and the use of preselected drying parameters, almost any formulation might be cryoprepared for a suitable desired end use.

It should be understood in the following discussion of optional additives like excipients, stabilizers, dessicants, and preservatives are described by their function. Thus, a particular chemical may act as some combination of excipient, stabilizer, dessicant, and/or preservative. Such chemicals would be considered immunologically-inactive because it does not directly induce an immune response, but it increases the response by enhancing immunological activity of the antigen or adjuvant: for example, by reducing modification of the antigen or adjuvant, or denaturation during drying and dissolving cycles.

Stabilizers include cyclodextrin and derivatives thereof (see U.S. Pat. No. 5,730,969). Suitable preservatives such as sucrose, mannitol, sorbitol, trehalose, dextran, and glycerin can also be added to stabilize the final formulation. A stabilizer selected from nonionic surfactants, D-glucose, D-galactose, D-xylose, D-glucuronic acid, salts of D-glucuronic acid, trehalose, dextrans, hydroxyethyl starches, and mixtures thereof may be added to the formulation. Addition of an alkali metal salt or magnesium chloride may stabilize a polypeptide, optionally including serum albumin and freeze-drying to further enhance stability. A polypeptide may also be stabilized by contacting it with a saccharide selected from the group consisting of dextran, chondroitin sulfuric acid, starch, glycogen, insulin, dextrin, and alginic acid salt. Other sugars that can be added include monosaccharides, disaccharides, sugar alcohols, and mixtures thereof (e.g., glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol). Polyols may stabilize a polypeptide, and are water-miscible or water-soluble. Suitable polyols may be polyhydroxy alcohols, monosaccharides and disaccharides including mannitol, glycerol, ethylene glycol, propylene glycol, trimethyl glycol, vinyl pyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose, and polymers thereof. Various excipients may also stabilize polpeptides, including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinyl pyrrolidone, hydrolyzed gelatin, and ammonium sulfate.

Single-dose formulations can be stabilized in poly(lactic acid) (PLA) and poly (lactide-co-glycolide) (PLGA) microspheres by suitable choice of excipient or stabilizer. Trehalose may be advantageously used as an additive because it is a non-reducing saccharide, and therefore does not cause aminocarbonyl reactions with substances bearing amino groups such as proteins.

It is conceivable that a formulation that can be administered to the subject in a dry, non-liquid form, may allow storage in conditions that do not require a cold chain. An antigen (e.g., CS6) in solution may be mixed in solution with an adjuvant such as LT and is placed on a gauze pad with an occlusive backing such as plastic wrap and allowed to dry. This patch can then be placed on skin with the gauze side in direct contact with the skin for a period of time and can be held in place covered with a simple occlusive such as plastic wrap and adhesive tape. The patch may have many compositions. The substrate may be cotton gauze, combinations of rayon-nylon or other synthetic materials and may have occlusive solid backings including polyvinyl chloride, rayons, other plastics, gels, creams, emulsions, waxes, oils, parafilm, rubbers (synthetic or natural), cloths, or membranes. The patch can be held onto the skin and components of the patch can be held together using various adhesives. One or more of the adjuvant and/or antigen may be incorporated into the substrate or adhesive parts of the patch.

A liquid or quasi-liquid formulation may be applied directly to the skin and allowed to air dry; rubbed into the skin or scalp; placed on the ear, inguinal, or intertriginous regions, especially in animals; placed on the anal/rectal tissues; held in place with a dressing, patch, or absorbent material; immersion; otherwise held by a device such as a stocking, slipper, glove, or shirt; or sprayed onto the skin to maximize contact with the skin. The formulation may be applied in an absorbent dressing or gauze. The formulation may be covered with an occlusive dressing such as, for example, AQUAPHOR (an emulsion of petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin from Beiersdorf), plastic film, COMFEEL (Coloplast) or VASELINE petroleum jelly; or a non-occlusive dressing such as, for example, TEGADERM (3M), DUODERM (3M) or OPSITE (Smith & Napheu). An occlusive dressing excludes the passage of water. Such a formulation may be applied to single or multiple sites, to single or multiple limbs, or to large surface areas of the skin by complete immersion. The formulation may be applied directly to the skin. Other substrates that may be used are pressure-sensitive adhesives such as acrylics, polyisobutylenes, and silicones. The formulation may be incorporated directly into such substrates, perhaps with the adhesive per se instead of adsorption to a porous pad (e.g., gauze) or bilious strip (e.g., filter paper).

1. A method of preventing traveler's diarrhea in a human that is to be subjected to exposure to pathogens causing traveler's diarrhea comprising applying a vaccine transcutaneously to the skin of the human, prior to exposure to pathogens causing traveler's diarrhea, wherein the vaccine comprises an effective amount of heat-labile enterotoxin of E. coli (LT) to prevent traveler's diarrhea.

2. A method of preventing traveler's diarrhea in a human that is to be subjected to exposure to pathogens causing traveler's diarrhea comprising applying a vaccine transcutaneously to the skin of the human, prior to exposure to pathogens causing traveler's diarrhea, wherein the vaccine comprises an effective amount of heat-labile enterotoxin of E. coli (LT) to treat traveler's diarrhea.

3. The method of claim 1 or 2, wherein the vaccine comprises LT and an adjuvant.

4. The method of claim 3, wherein the adjuvant is an ADP-ribosylating exotoxin or a derivative thereof having adjuvant activity.

5. The method of claim 1 or 2, wherein the vaccine comprises LT and an E. coli colonization factor antigen (CFA).

6. The method of claim 5, wherein the E. coli colonization factor antigen is selected from the group consisting of CFA/I, CS1, CS2, CS 4, CS5, CS6, CS17 and PCF 0166.

7. The method of claim 1, wherein the vaccine comprises LT and heat stable enterotoxin of E. coli (ST).

8. The method of claim 7, wherein the vaccine comprises a carrier or an excipient.

9. The method of claim 7, wherein LT is conjugated to ST.

10. The method of claim 1, wherein the method comprises pretreating the skin prior to administering the vaccine.

11. The method of claim 10, wherein pretreating comprises chemical penetration enhancement, physical penetration enhancement, or both.

12. The method of claim 1 or 2, wherein the vaccine is applied using a patch.

13. The method of claim 12 wherein the vaccine comprises a carrier or an excipient.

14. The method of claim 1, wherein LT is genetically detoxified.

15. The method of claim 1 or 2, wherein LT is a mutant form of the enterotoxin.

16. The method of claim 1 or 2, wherein LT is both an antigen and an adjuvant.

17. The method of claim 16, wherein LT is a mutant form of the enterotoxin.