U.S. patent application number 11/334349 was filed with the patent office on 2006-11-30 for adjuvant for transcutaneous immunization.
This patent application is currently assigned to The Government of the United States. Invention is credited to Carl R. Alving, Gregory M. Glenn.
Application Number | 20060269593 11/334349 |
Document ID | / |
Family ID | 27669393 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269593 |
Kind Code |
A1 |
Glenn; Gregory M. ; et
al. |
November 30, 2006 |
Adjuvant for transcutaneous immunization
Abstract
A transcutaneous immunization system delivers antigen to immune
cells without perforation of the skin, and induces an immune
response in an animal or human. The system uses an adjuvant,
preferably an ADP-ribosylating exotoxin, to induce an
antigen-specific immune response (e.g., humoral and/or cellular
effectors) after transcutaneous application of a formulation
containing antigen and adjuvant to intact skin of the animal or
human. The efficiency of immunization may be enhanced by adding
hydrating agents (e.g., liposomes), penetration enhancers, or
occlusive dressings to the transcutaneous delivery system. This
system may allow activation of Langerhans cells in the skin,
migration of the Langerhans cells to lymph nodes, and antigen
presentation.
Inventors: |
Glenn; Gregory M.;
(Gaithersburg, MD) ; Alving; Carl R.; (Bethesda,
MD) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
The Government of the United
States
|
Family ID: |
27669393 |
Appl. No.: |
11/334349 |
Filed: |
January 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09266803 |
Mar 12, 1999 |
7037499 |
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11334349 |
Jan 19, 2006 |
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08896085 |
Jul 17, 1997 |
5980898 |
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09266803 |
Mar 12, 1999 |
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08749164 |
Nov 14, 1996 |
5910306 |
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08896085 |
Jul 17, 1997 |
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Current U.S.
Class: |
424/450 ;
424/144.1; 424/184.1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61K 2039/55527 20130101; A61K 9/06 20130101; A61K
2039/53 20130101; A61K 39/39 20130101; A61K 9/08 20130101; A61K
9/7023 20130101; A61P 37/04 20180101; B82Y 5/00 20130101; A61K
2039/55511 20130101; A61P 37/00 20180101; A61K 9/107 20130101; A61K
2039/55555 20130101; A61K 2039/54 20130101; A61K 2039/55544
20130101; A61K 9/127 20130101 |
Class at
Publication: |
424/450 ;
424/144.1; 424/184.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61K 9/127 20060101
A61K009/127 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] The U.S. government may retain certain rights in this
invention.
Claims
1-49. (canceled)
50: A method of inducing an immune response to at least one antigen
comprising applying a formulation to hydrated skin of an organism,
wherein the formulation comprises an antigen which is derived from
a pathogen, and wherein an effective amount of the antigen induces
the immune response to the at least one antigen in the
organism.
51: The method of claim 50, wherein the pathogen is selected from
the group consisting of bacterium, virus, fungus and parasite.
52: The method of claim 50, wherein the antigen is selected from
the group consisting of carbohydrate, glycolipid, glycoprotein,
lipid, protein, lipoprotein, phospholipid, and polypeptide.
53: The method of claim 50, wherein the pathogen is a live or an
attenuated live virus and the antigen is expressed by the live or
attenuated live virus.
54: The method of claim 51, wherein the bacterium is anthrax.
55: The method of claim 51, wherein the virus is rabies.
56: The method of claim 50, wherein the formulation is a cream or
gel or emulsion or ointment or lotion or paste or solution or
suspension.
57: The method of claim 50, wherein the formulation is applied with
a patch.
58: The method of claim 50, wherein the formulation further
comprises a dressing.
59: The method of claim 58, wherein the dressing is occlusive or
non-occlusive.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation in-part of U.S. application Ser. No.
08/749,164 filed Nov. 14, 1996.
BACKGROUND
[0003] The invention relates to transcutaneous immunization, and
adjuvants useful therein, to induce an antigen-specific immune
response.
[0004] Transcutaneous immunization requires both passage of an
antigen through the outer barriers of the skin, which are normally
impervious to such passage, and an immune response to the antigen.
We showed in U.S. application Ser. No. 08/749,164 that using
cholera toxin as an antigen elicits a strong antibody response that
is highly reproducible; the antigen could be applied in a saline
solution to the skin, with or without liposomes. In the present
application, we show transcutaneous immunization using adjuvants
such as, for example, bacterial exotoxins, their subunits, and
related toxins.
[0005] There is a report of transdermal immunization with
transferosomes by Paul et al. (1995). In this publication, the
transferosomes are used as a carrier for proteins (bovine serum
albumin and gap junction proteins) against which the
complement-mediated lysis of antigen-sensitized liposomes is
directed. An immune response was not induced when solution
containing the protein was placed on the skin; only transferosomes
were able to transport antigen across the skin and achieve
immunization. As discussed in U.S. application Ser. No. 08/749,164,
transferosomes are not liposomes.
[0006] FIG. 1 of Paul et al. (1995) showed that only a formulation
of antigen and transferosomes induced an immune response, assayed
by lysis of antigen-sensitized liposomes. Formulations of antigen
in solution, antigen and mixed micelles, and antigen and liposomes
(i.e., smectic mesophases) applied to the skin did not induce an
immune response equivalent to that induced by subcutaneous
injection. Therefore, there was a positive control (i.e., antigen
and transfersomes) to validate their negative conclusion that a
formulation of antigen and liposomes did not cause transdermal
immunization.
[0007] Paul et al. (1995) stated on page 3521 that the skin is an
effective protective barrier that is "impenetrable to substances
with a molecular mass at most 750 DA", precluding non-invasive
immunization with large immunogen through intact skin. Therefore,
the reference would teach away from using a molecule like cholera
toxin (which is 85,000 daltons) because such molecules would not be
expected to penetrate the skin and, therefore, would not be
expected to achieve immunization. Thus, skin represents a barrier
that would make penetration by an adjuvant or antigen like cholera
toxin unexpected without the disclosure of the present
invention.
[0008] Paul and Cevc (1995) stated on page 145, "Large molecules
normally do not get across the intact mammalian skin. It is thus
impossible to immunize epicutaneously with simple peptide or
protein solutions." They concluded, "The dermally applied liposomal
or mixed micellar immunogens are biologically as inactive as simple
protein solutions, whether or not they are combined with the
immunoadjuvant lipid A."
[0009] Wang et al. (1996) placed a solution of ovalbumin (OVA) in
water on the skin of shaved mice to induce an allergic type
response as a model for atopic dermatitis. Mice were anesthetized
and covered with an occlusive patch containing up to 10 mg of OVA,
which was placed on the skin continuously for four days. This
procedure was repeated after two weeks.
[0010] In FIG. 2 of Wang et al. (1996), an ELISA assay done to
determine the IgG2a antibody response showed no IgG2a antibody
response to OVA. However, IgE antibodies that are associated with
allergic responses could be detected. In a further experiment, the
mice were more extensively patched with OVA in solution for four
days every two weeks. This was repeated five times, i.e., the mice
wore patches for a total of 20 days. Again, the high dose of OVA
did not produce significant IgG2a antibodies. Significant levels of
IgE antibodies were produced.
[0011] The authors stated on page 4079 that "we established an
animal model to show that epicutaneous exposure to protein Ag, in
the absence of adjuvant, can sensitize animals and induce a
dominant Th2-like response with high levels of IgE". Extensive
epicutaneous exposure to high doses of protein antigen could not
produce significant IgG antibodies but could induce IgE antibodies,
the hallmark of an allergic type reaction. Thus, Wang et al. (1996)
teaches that OVA exposure as described is a model for atopic
dermatitis and not a mode of immunization. Therefore, following the
teaching of the reference, one would have expected that
transcutaneous immunization with antigen would induce high levels
of IgE antibodies if it were to pass through the skin and induce an
immune response. Instead, we have unexpectedly found that antigen
placed on the skin in a saline solution with adjuvant induces high
levels of IgG and some IgA, but not IgE.
[0012] In contrast to the cited references, the inventors have
found that application to the skin of antigen and adjuvant provides
a transcutaneous delivery system for antigen that can induce an
antigen-specific immune response of IgG or IgA. The adjuvant is
preferably an ADP-ribosylating exotoxin. Optionally, hydration,
penetration enhancers, or occlusive dressings may be used in the
transcutaneous delivery system.
SUMMARY OF THE INVENTION
[0013] An object of the invention is to provide a system for
transcutaneous immunization that induces an immune response (e.g.,
humoral and/or cellular effectors) in an animal or human. The
system provides simple application to intact skin of an organism of
a formulation comprised of antigen and adjuvant to induce a
specific immune response against the antigen. In particular, the
adjuvant may activate antigen presenting cells of the immune system
(e.g., Langerhans cells in the epidermis, dermal dendritic cells,
dendritic cells, macrophages, B lymphocytes) and/or induce the
antigen presenting cells to phagocytose the antigen. The antigen
presenting cells then present the antigen to T and B cells. In the
instance of Langerhans cells, the antigen presenting cells then may
migrate from the skin to the lymph nodes and present antigen to
lymphocytes (e.g., B and/or T cells), thereby inducing an
antigen-specific immune response.
[0014] In addition to eliciting immune reactions leading to
generation of an antigen-specific B lymphocyte and/or T lymphocyte,
including a cytotoxic T lymphocyte (CTL), another object of the
invention is to positively and/or negatively regulate components of
the immune system by using the transcutaneous immunization system
to affect antigen-specific helper (Th1 and/or Th2) or delayed-type
hypersensitivity (T.sub.DTH) T-cell subsets.
[0015] In a first embodiment of the invention, a formulation
containing antigen and adjuvant is applied to intact skin of an
organism, the antigen is presented to immune cells, and an
antigen-specific immune response is induced without perforating the
skin. The formulation may include additional antigens such that
transcutaneous application of the formulation 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 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.
[0016] In a second embodiment of the invention, the above method is
used to treat an organism. If the antigen is derived from a
pathogen, the treatment vaccinates the organism against infection
by the pathogen or against its pathogenic effects such as those
caused by toxin secretion. A formulation that includes a tumor
antigen may provide a cancer treatment; a formulation that includes
an autoantigen may provide a treatment for a disease caused by the
organism's own immune system (e.g., autoimmune disease).
[0017] In a third embodiment of the invention, a patch for use in
the above methods is provided. The patch comprises a dressing, and
effective amounts of antigen and adjuvant. The dressing may be
occlusive or non-occlusive. The patch may include additional
antigens such that application of the patch 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. For effective treatment,
multiple patches may be applied at frequent intervals or constantly
over a period of time.
[0018] Moreover, in a fourth embodiment of the invention, the
formulation is applied to intact skin overlying more than one
draining lymph node field using either single or multiple
applications. The formulation may include additional antigens such
that application to intact skin 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.
[0019] The products and methods may be used therapeutically to
treat existing disease, protectively to prevent disease, or to
reduce the severity and/or duration of disease.
[0020] In addition to antigen and adjuvant, the formulation may
comprise a hydrating agent (e.g., liposomes), a penetration
enhancer, or both. For example, the formulation may comprise
AQUAPHOR (an emulsion of petrolatum, mineral oil, mineral wax, wool
wax, panthenol, bisabol, and glycerin), emulsions (e.g., aqueous
creams), oil-in-water emulsions (e.g., oily creams), anhydrous
lipids and oil-in-water emulsions, anhydrous lipids and
water-in-oil emulsions, fats, waxes, oil, silicones, and humectants
(e.g., glycerol).
[0021] The antigen may be derived from a pathogen that-can infect
the organism (e.g., bacterium, virus, fungus, or parasite), or a
cell (e.g., tumor cell or normal cell). The antigen may be a tumor
antigen or an autoantigen. Chemically, the antigen may be a
carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein,
phospholipid, polypeptide, or chemical or recombinant conjugate of
the above. The molecular weight of the antigen may be greater than
500 daltons, preferably greater than 800 daltons, and more
preferably greater than 1000 daltons.
[0022] Antigen may be obtained by recombinant means, chemical
synthesis, or purification from a natural source. Preferred are
proteinaceous antigen or conjugates with polysaccharide. Antigen
may be at least partially purified in cell-free form.
Alternatively, antigen may be provided in the form of a live virus,
an attenuated live virus, or an inactivated virus.
[0023] Inclusion of an adjuvant may allow potentiation or
modulation of the immune response. Moreover, selection of a
suitable antigen or adjuvant may allow 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.sub.DTH).
[0024] Preferably, the adjuvant is an ADP-ribosylating exotoxin or
a subunit thereof. Optionally, an activator of Langerhans cells may
be used.
[0025] Optionally, antigen, adjuvant, or both may be provided in
the formulation by means of a nucleic acid (e.g., DNA, RNA, cDNA,
cRNA) encoding the antigen or adjuvant as appropriate. This
technique is called genetic immunization.
[0026] The term "antigen" as used in the invention, is meant to
describe a substance that induces a specific immune response when
presented to immune cells of an organism. An antigen may comprise a
single immunogenic epitope, or a multiplicity of immunogenic
epitopes recognized by a B-cell receptor (i.e., antibody on the
membrane of the B cell) or a T-cell receptor. A molecule may be
both an antigen and an adjuvant (e.g., cholera toxin) and, thus,
the formulation may contain only one component.
[0027] The term "adjuvant" as used in the invention, is meant to
describe a substance added to the formulation to assist in inducing
an immune response to the antigen.
[0028] The term "effective amount" as used in the invention, is
meant to describe that amount of antigen which induces an
antigen-specific immune response. Such induction of an immune
response may provide a treatment such as, for example,
immunoprotection, desensitization, immunosuppression, modulation of
autoimmune disease, potentiation of cancer immunosurveillance, or
therapeutic vaccination against an established infectious
disease.
[0029] 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 set of lymph nodes (e.g.,
cervical, axillary, inguinal, epitrochelear, popliteal, those of
the abdomen and thorax).
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows cholera toxin (CT) induces enhanced major
histocompatibility complex (MHC) class II expression on Langerhans
cells (LC), changes in LC morphology, and loss of LCs (presumably
through migration). BALB/c mice (H-2.sup.d) were transcutaneously
immunized with cholera CT or its B subunit (CTB) in saline solution
on the ear (7,000 anti-CT ELISA units after a single immunization).
Previous experiments had established that mice were readily
immunized when using the skin of the ear. After 16 hours, epidermal
sheets were prepared and stained for MHC class II molecules (scale
bar is 50 .mu.m). Panels indicate (A) saline alone as a negative
control, (B) transcutaneous immunization with CT in saline, (C)
transcutaneous immunization with CTB in saline, and (D) intradermal
injection with tumor necrosis factor-.alpha. (10 .mu.g) as a
positive control.
DETAILED DESCRIPTION OF THE INVENTION
[0031] A transcutaneous immunization system delivers agents to
specialized cells (e.g., antigen presentation cell, lymphocyte)
that produce an immune response (Bos, 1997). These agents as a
class are called antigens. Antigen may be composed of chemicals
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 means.
[0032] Processes for preparing a pharmaceutical formulation are
well-known in the art, whereby the antigen and adjuvant is combined
with a pharmaceutically acceptable carrier vehicle. Suitable
vehicles and their preparation are described, for example, in
Remington's Pharmaceutical Sciences by E. W. Martin. Such
formulations will contain an effective amount of the antigen and
adjuvant together with a suitable amount of vehicle in order to
prepare pharmaceutically acceptable compositions suitable for
administration to a human or animal. The formulation may be applied
in the form of an cream, emulsion, gel, lotion, ointment, paste,
solution, suspension, or other forms known in the art. In
particular, formulations that enhance skin hydration, penetration,
or both are preferred. There may also be incorporated other
pharmaceutically acceptable additives including, for example,
diluents, binders, stabilizers, preservatives, and colorings.
[0033] Increasing hydration of the stratum corneum will increase
the rate of percutaneous absorbtion of a given solute (Roberts and
Walker, 1993). As used in the present invention, "penetration
enhancer" does not include substances such as, for example: water,
physiological buffers, saline solutions, and alcohols which would
not perforate the skin.
[0034] An object of the present invention is to provide a novel
means for immunization through intact skin without the need for
perforating the skin. The transcutaneous immunization system
provides a method whereby antigens and adjuvant can be delivered to
the immune system, especially specialized antigen presentation
cells underlying the skin such as, for example, Langerhans
cells.
[0035] Without being bound to any particular theory but only to
provide an explanation for our observations, it is presumed that
the transcutaneous immunization delivery system carries antigen to
cells of the immune system where an immune response is induced. The
antigen may pass through the normal protective outer layers of the
skin (i.e., stratum corneum) and induce the immune response
directly, or through an antigen presenting cell (e.g., macrophage,
tissue macrophage, Langerhans cell, dendritic cell, dermal
dendritic cell, B lymphocyte, or Kupffer cell) that presents
processed antigen to a T lymphocyte. Optionally, the antigen may
pass through the stratum corneum via a hair follicle or a skin
organelle (e.g., sweat gland, oil gland).
[0036] Transcutaneous immunization with bacterial ADP-ribosylating
exotoxins (bAREs) may target the epidermal Langerhans cell, known
to be among the most efficient of the antigen presenting cells
(APCs) (Udey, 1997). We have found that bAREs activate Langerhans
cells when applied epicutaneously to the skin in saline solution.
The Langerhans cells direct specific immune responses through
phagocytosis of the antigens, and migration to the lymph nodes
where they act as APCs to present the antigen to lymphocytes (Udey,
1997), and thereby induce a potent antibody response. Although the
skin is generally considered a barrier to invading organisms, 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 via the skin (Udey, 1997).
[0037] According to Udey (1997): [0038] "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 uninflammed epidermis, an 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 . . . [0039] "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 local 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."
[0040] We envision that the potent antigen presenting capability of
the epidermal Langerhans cells can be exploited for
transcutaneously delivered vaccines. A transcutaneous immune
response using the skin immune system would require delivery of
vaccine antigen only to Langerhans cells in the stratum corneum
(the outermost layer of the skin consisting of cornified cells and
lipids) via passive diffusion and subsequent activation of 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 cells (Stingl et al., 1989). If antigens other that bAREs
(for example BSA) were to be phagocytosed by the Langerhans cells,
then these antigens could also be taken to the lymph node for
presentation to T-cells and subsequently induce an immune response
specific for that antigen (e.g., BSA). Thus, a feature of
transcutaneous immunization is the activation of the Langerhans
cell, presumably by a bacterial ADP-ribosylating exotoxin,
ADP-ribosylating exotoxin binding subunits (e.g., cholera toxin B
subunit), or other Langerhans cell activating substance.
[0041] The mechanism of transcutaneous immunization via Langerhans
cells activation, migration and antigen presentation is clearly
shown by the upregulation of MHC class II expression in the
epidermal Langerhans cells from epidermal sheets transcutaneously
immunized with CT or CTB. In addition, the magnitude of the
antibody response induced by transcutaneous immunization and
isotype switching to predominantly IgG is generally achieved with
T-cell help (Janeway and Travers, 1996), and activation of both Th1
and Th2 pathways is suggested by the production of IgG1 and IgG2a
(Paul and Seder, 1994; Seder and Paul, 1994). Alternatively, a
large antibody response may be induced by a thymus-independent
antigen type 1 (TI-1) which directly activates the B cell (Janeway
and Travers, 1996).
[0042] The spectrum of more commonly known skin immune responses is
represented by contact dermatitis and atopy. Contact dermatitis, a
pathogenic manifestation of LC activation, is directed by
Langerhans cells which phagocytose antigen, migrate to lymph nodes,
present antigen, and sensitize T cells for the intense destructive
cellular response that occurs at the affected skin site (Dahl,
1996; Leung, 1997). 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 (Dahl, 1996;
Leung, 1997).
[0043] Transcutaneous immunization with cholera toxin-and related
bAREs on the other hand is a novel immune response with an absence
of superficial and microscopic post-immunization skin findings
(i.e., non-inflamed skin) shown by the absence of lymphocyte
infiltration 24, 48 and 120 hours after immunization. This
indicates that Langerhans cells "comprise all of the accessory cell
activity that is present in uninflammed 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 the absence of
anti-CT IgE antibody.
[0044] Thus, we have found that bacterial-derived toxins applied to
the surface of the skin can activate Langerhans cells and induce a
potent immune response manifested as high levels of
antigen-specific circulating IgG antibodies. Such adjuvants may be
used in transcutaneous immunization to enhance the IgG antibody
response to proteins not otherwise immunogenic by themselves when
placed on the skin.
[0045] Transcutaneous targeting of Langerhans cells may also be
used to deactivate their antigen presenting function, thereby
preventing immunization or sensitization. Techniques to deactivate
Langerhans cells include, for example, the use of interleukin-10
(Peguet-Navarro et al., 1995), monoclonal antibody to
interleukin-1.beta. (Enk et al., 1993), or depletion via
superantigens such as through staphylococcal enterotoxin-A (SEA)
induced epidermal Langerhans cell depletion (Shankar et al.,
1996).
[0046] Transcutaneous immunization may be induced via the
ganglioside GM1 binding activity of CT, LT or subunits such as CTB
(Craig and Cuatrecasas, 1975). Ganglioside GM1 is a ubiquitous cell
membrane glycolipid found in all mammalian cells (Plotkin and
Mortimer, 1994). When the pentameric CT B subunit binds to the cell
surface a hydrophilic pore is formed which allows the A subunit to
penetrate across the lipid bilayer (Ribi et al., 1988).
[0047] We have shown that transcutaneous immunization by CT or CTB
may require ganglioside GM1 binding activity. When mice were
transcutaneously immunized with CT, CTA and CTB, only CT and CTB
resulted in an immune response. CTA contains the ADP-ribosylating
exotoxin activity but only CT and CTB containing the binding
activity were 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 cell may be activated by CTB
binding to its cell surface.
Antigen
[0048] Antigen of the invention may be expressed by recombinant
means, preferably as a fusion with an affinity or epitope tag
(Summers and Smith, 1987; Goeddel, 1990; Ausubel et al., 1996);
chemical synthesis of an oligopeptide, either free or conjugated to
carrier proteins, may be used to obtain antigen of the invention
(Bodanszky, 1993; Wisdom, 1994). Oligopeptides are considered a
type of polypeptide.
[0049] Oligopeptide lengths of 6 residues to 20 residues are
preferred. Polypeptides may also by synthesized as branched
structures such as those disclosed in 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.
[0050] Antigen obtained through recombinant means or peptide
synthesis, as well as antigen of the invention obtained from
natural sources or extracts, may be purified by means of the
antigen's physical and chemical characteristics, preferably by
fractionation or chromatography (Janson and Ryden, 1989; Deutscher,
1990; Scopes, 1993).
[0051] 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 injection, or the oral or intranasal routes.
[0052] Antigen includes, for example, toxins, toxoids, subunits
thereof, or combinations thereof (e.g., cholera toxin, tetanus
toxoid).
[0053] Antigen may be solubilized in a buffer. Suitable buffers
include, but are not limited to, phosphate buffered saline
Ca.sup.++/Mg.sup.++ free (PBS), normal saline (150 mM NaCl in
water), and 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 present invention.
[0054] Hydrophobic antigen can be solubilized in a detergent, for
example a polypeptide containing a membrane-spanning domain.
Furthermore, for formulations containing liposomes, an antigen in a
detergent solution (e.g., a 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 such as, for example, those from a virus (e.g., hepatitis
A) need not be soluble per se, but can be incorporated directly
into a liposome in the form of a virosome (Morein and Simons,
1985).
[0055] Plotkin and Mortimer (1994) provide antigens which can be
used to vaccinate animals or humans to 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 parasite).
[0056] Bacteria include, for example: anthrax, campylobacter,
cholera, diphtheria, enterotoxigenic E. coli, giardia, gonococcus,
Helicobacter pylori (Lee and Chen, 1994), Hemophilus influenza B,
Hemophilus influenza non-typable, meningococcus, pertussis,
pneumococcus, salmonella, shigella, Streptococcus B, tetanus,
Vibrio cholerae, and yersinia.
[0057] Viruses include, for example: adenovirus, dengue serotypes 1
to 4 (Delenda et al., 1994; Fonseca et al., 1994; Smucny et al.,
1995), ebola (Jahrling et al., 1996), enterovirus, hepatitis
serotypes A to E (Blum, 1995; Katkov, 1996; Lieberman and
Greenberg, 1996; Mast, 1996; Shafara et al., 1995; Smedila et al.,
1994; U.S. Pat. Nos. 5,314-,808 and 5,436,126), herpes simplex
virus 1 or 2, human immunodeficiency virus (Deprez et al., 1996),
influenza, measles, Norwalk, papilloma virus, parvovirus B19,
polio, rabies, rotavirus, rubella, rubeola, vaccinia, vaccinia
constructs containing genes coding for other antigens such as
malaria antigens, varicella, and yellow fever.
[0058] Parasites include, for example: Entamoeba histolytica (Zhang
et al., 1995); Plasmodium (Bathurst et al., 1993; Chang et al.,
1989, 1992, 1994; Fries et al., 1992a, 1992b; Herrington et al.,
1991; Khusmith et al., 1991; Malik et al., 1991; Migliorini et al.,
1993; Pessi et al., 1991; Tam, 1988; Vreden et al., 1991; White et
al., 1993; Wiesmueller et al., 1991), Leishmania (Frankenburg et
al., 1996), and the Helminthes.
Adjuvant
[0059] The formulation also contains an adjuvant, although a single
molecule may contain both adjuvant and antigen properties (e.g.,
cholera toxin) (Elson and Dertzbaugh, 1994). Adjuvants are
substances that are used to specifically or non-specifically
potentiate an antigen-specific immune response. Usually, the
adjuvant and the formulation are mixed prior to presentation of the
antigen but, alternatively, they may be separately presented within
a short interval of time.
[0060] Adjuvants include, for example, an oil emulsion (e.g.,
complete or incomplete Freund's adjuvant), a chemokine (e.g.,
defensins 1 or 2, RANTES, MIP1-.alpha., MIP-2, interleukin-8) or a
cytokine (e.g., interleukin-1.beta., -2, -6, -10 or -12;
.gamma.-interferon; tumor necrosis factor-.alpha.; or
granulocyte-monocyte-colony stimulating factor) (reviewed in Nohria
and Rubin, 1994), a muramyl dipeptide derivative (e.g., murabutide,
threonyl-MDP or muramyl tripeptide), a heat shock protein or a
derivative, a derivative of Leishmania major LeIF (Skeiky et al.,
1995), cholera toxin or cholera toxin B, a lipopolysaccharide (LPS)
derivative (e.g., lipid A or monophosphoryl lipid A), or
superantigen (Saloga et al., 1996). Also, see Richards et al.
(1995) for adjuvants useful in immunization.
[0061] An 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.sub.DTH)
(Glenn et al., 1995).
[0062] Cholera toxin is a bacterial exotoxin from the family of
ADP-ribsoylating exotoxins (referred to as bAREs). Most bAREs are
organized as A:B dimer with a binding B subunit and an A subunit
containing the ADP-ribosyltransferase. Such toxins include
diphtheria, Pseudomonas exotoxin A, cholera toxin (CT), E. coli
heat-labile enterotoxin (LT), pertussis toxin, C. botulinum toxin
C2, C. botulinum toxin C3, C. limosum exoenzyme, B. cereus
exoenzyme, Pseudomonas exotoxin S, Staphylococcus aureus EDIN, and
B. sphaericus toxin.
[0063] Cholera toxin is an example of a bARE that is organized with
A and B subunits. The B subunit is the binding subunit and consists
of a B-subunit pentamer which is non-covalently bound to the A
subunit. The B-subunit pentamer is arranged in a symmetrical
doughnut-shaped structure that binds to GM.sub.1-ganglioside on the
target cell. The A subunit serves to ADP ribosylate the alpha
subunit of a subset of the hetero trimeric GTP proteins (G
proteins) including the Gs protein which results in the elevated
intracellular levels of cyclic AMP. This stimulates release of ions
and fluid from intestinal cells in the case of cholera.
[0064] Cholera toxin (CT) and its B subunit (CTB) have adjuvant
properties when used as either an intramuscular or oral immunogen
(Elson and Dertzbaugh, 1994; Trach et al., 1997). Another antigen,
heat-labile enterotoxin from E. coli (LT) is 80% homologous at the
amino acid level with CT and possesses similar binding properties;
it also appears to bind the GM.sub.1-ganglioside receptor in the
gut and has similar ADP-ribosylating exotoxin activities. Another
bARE, Pseudomonas exotoxin A (ETA), binds to the
.alpha..sub.2-macroglobulin receptor-low density lipoprotein
receptor-related protein (Kounnas et al., 1992). bAREs are reviewed
by Krueger and Barbieri (1995).
[0065] The examples below show that cholera toxin (CT),-its B
subunit (CTB), E. coli heat-labile enterotoxin (LT), and pertussis
toxin are potent adjuvants for transcutaneous immunization,
inducing high levels of IgG antibodies but not IgE antibodies. Also
shown is that CTB without CT can also induce high levels of IgG
antibodies. Thus, both bAREs and a derivative thereof can
effectively immunize when epicutaneouly applied to the skin in a
simple solution.
[0066] When an adjuvant such as CT is mixed with BSA, a protein not
usually immunogenic when applied to the skin, anti-BSA antibodies
are induced. An immune response to diphtheria toxoid was induced
using pertussis toxin as adjuvant, but not with diphtheria toxoid
alone. Thus, bAREs can act as adjuvants for non-immunogenic
proteins in an transcutaneous immunization system.
[0067] Protection against the life-threatening infections
diphtheria, pertussis, and tetanus (DPT) can be achieved by
inducing high levels of circulating anti-toxin antibodies.
Pertussis may be an exception in that some investigators feel that
antibodies directed to other portions of the invading organism are
necessary for protection, although this is controversial (see
Schneerson et al., 1996) and most new generation acellular
pertussis vaccines have PT as a component of the vaccine (Krueger
and Barbieri, 1995). The pathologies in the diseases caused by DPT
are directly related to the effects of their toxins and anti-toxin
antibodies most certainly play a role in protection (Schneerson et
al., 1996).
[0068] 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 such as CT. Genetically toxoided toxins which have
altered ADP-ribosylating exotoxin activity, but not binding
activity, are envisioned to be especially useful as non-toxic
activators of antigen presenting cells used in transcutaneous
immunization.
[0069] We envision that CT can also act as an adjuvant to induce
antigen-specific CTLs through transcutaneous immunization (see
Bowen et al., 1994; Porgador et al., 1997 for the use of CT as an
adjuvant in oral immunization).
[0070] 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.
[0071] 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 difficulty 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. This approach is described by Burnette et al. (1994),
Rappuoli et al. (1995), and Rappuoli et al. (1996). Such
genetically toxoided exotoxins could be useful for transcutaneous
immunization system in that they would not create a safety concern
as the toxoids would not be considered toxic. Additionally, several
techniques exist to chemically toxoid toxins which can address the
same problem (Schneerson et al., 1996). These techniques could be
important for certain applications, especially pediatric
applications, in which ingested toxins (e.g., diphtheria toxin)
might possibly create adverse reactions. optionally, an activator
of Langerhans cells may 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); lipopolysaccharides, lipid A, or derivatives
thereof; bacterial DNA (Stacey et al., 1996); cytokines (e.g.,
tumor necrosis factor-.alpha., interleukin-1.beta., -10, -12); and
chemokines (e.g., defensins 1 or 2, RANTES, MIP-1.alpha., MIP-2,
interleukin-8).
[0072] If an immunizing antigen has sufficient Langerhans cell
activating capabilities then a separate adjuvant may not be
required, as in the case of CT which is both antigen and adjuvant.
It is envisioned that whole cell preparations, live viruses,
attenuated viruses, DNA plasmids, and bacterial DNA could be
sufficient to immunize transcutaneously. It may be possible to use
low concentrations of contact sensitizers or other activators of
Langerhans cells to induce an immune response without inducing skin
lesions.
Liposomes and Their Preparation
[0073] Liposomes are closed vesicles surrounding an internal
aqueous space. The internal compartment is separated from the
external medium by a lipid bilayer composed of discrete lipid
molecules. In the present invention, antigen may be delivered
through intact skin to specialized cells of the immune system,
whereby an antigen-specific immune response is induced.
Transcutaneous immunization may be achieved by using liposomes;
however, as shown in the examples, liposomes are not required to
elicit an antigen-specific immune response.
[0074] Liposomes may be prepared using a variety of techniques and
membrane lipids (reviewed in Gregoriadis, 1993). Liposomes may be
pre-formed and then mixed with antigen. The antigen may be
dissolved or suspended, and then added to (a) the pre-formed
liposomes in a lyophilized state, (b) dried lipids as a swelling
solution or suspension, or (c) the solution of lipids used to form
liposomes. They may also be formed from lipids extracted from the
stratum corneum including, for example, ceramide and cholesterol
derivatives (Wertz, 1992).
[0075] Chloroform is a preferred solvent for lipids, but it may
deteriorate upon storage. Therefore, at one- to three-month
intervals, chloroform is redistilled prior to its use as the
solvent in forming liposomes. After distillation, 0.7% ethanol can
be added as a preservative. Ethanol and methanol are other suitable
solvents.
[0076] The lipid solution used to form liposomes is placed in a
round-bottomed flask. Pear-shaped boiling flasks are preferred,
particularly those flasks sold by Lurex Scientific (Vineland, N.J.,
cat. no. JM-5490). The volume of the flask should be more than ten
times greater than the volume of the anticipated aqueous suspension
of liposomes to allow for proper agitation during liposome
formation.
[0077] Using a rotary evaporator, solvent is removed at 37.degree.
C. under negative pressure for 10 minutes with a filter aspirator
attached to a water faucet. The flask is further dried under low
vacuum (i.e., less than 50 mm Hg) for 1 hour in a dessicator.
[0078] To encapsulate antigen into liposomes, an aqueous solution
containing antigen may be added to lyophilized liposome lipids in a
volume that results in a concentration of approximately 200 mM with
respect to liposome lipid, and shaken or vortexed until all the
dried liposome lipids are wet. The liposome-antigen mixture may
then be incubated for 18 hours to 72 hours at 4.degree. C. The
liposome-antigen formulation may be used immediately or stored for
several years. It is preferred to employ such a formulation
directly in the transcutaneous immunization system without removing
unencapsulated antigen. Techniques such as bath sonication may be
employed to decrease the size of liposomes, which may augment
transcutaneous immunization.
[0079] Liposomes may be formed as described above but without
addition of antigen to the aqueous solution. Antigen may then be
added to the pre-formed liposomes and, therefore, antigen would be
in solution and/or associated with, but not encapsulated by, the
liposomes. This process of making a liposome-containing formulation
is preferred because of its simplicity. Techniques such as bath
sonication may be employed to alter the size and/or lamellarity of
the liposomes to enhance immunization.
[0080] Although not required to practice the present invention,
hydration and/or penetration of the stratum corneum may be enhanced
by adding liposomes to the formulation. Liposomes have been used as
carriers with adjuvants to enhance the immune response to antigens
mixed with, encapsulated in, attached to, or associated with
liposomes.
Transcutaneous Delivery of Antigen
[0081] Efficient immunization can be achieved with the present
invention because transcutaneous delivery of antigen may target the
Langerhans cell. These cells are found in abundance in the skin and
are efficient antigen presenting cells leading to T-cell memory and
potent immune responses (Udey, 1997). Because of the presence of
large numbers of Langerhans cells in the skin, the efficiency of
transcutaneous delivery may be related to the surface area exposed
to antigen and adjuvant. In fact, the reason that transcutaneous
immunization is so efficient may be that it targets a larger number
of these efficient antigen presenting cells than intramuscular
immunization.
[0082] We envision the present invention will enhance access to
immunization, while inducing a potent immune response. Because
transcutaneous immunization does not involve penetration of the
skin and the complications and difficulties thereof, the
requirements of trained personnel, sterile technique, and sterile
equipment are reduced. Furthermore, the barriers to immunization at
multiple sites or to multiple immunizations are diminished.
Immunization by a single application of the formulation is also
envisioned.
[0083] Immunization may be achieved using epicutaneous application
of a simple solution of antigen and adjuvant impregnated in gauze
under an occlusive patch, or by using other patch technologies;
creams, immersion, ointments and sprays are other possible methods
of application. The immunization could be given by untrained
personnel, and is amenable to self-application. Large-scale field
immunization could occur given the easy accessibility to
immunization. Additionally, a simple immunization procedure would
improve access to immunization by pediatric patients and the
elderly, and populations in Third World countries.
[0084] For previous vaccines, their formulations were injected
through the skin with needles. Injection of vaccines using needles
carries certain drawbacks including the need for sterile needles
and syringes, trained medical personnel to administer the vaccine,
discomfort from the injection, and potential complications brought
about by puncturing the skin with the needle. Immunization through
the skin without the use of needles (i.e., transcutaneous
immunization) represents a major advance for vaccine delivery by
avoiding the aforementioned drawbacks.
[0085] The transcutaneous delivery system of the invention is also
not concerned with penetration of intact skin by sound or
electrical energy. Such a system that uses an electrical field to
induce dielectric breakdown of the stratum corneum is disclosed in
U.S. Pat. No. 5,464,386.
[0086] Moreover, transcutaneous immunization may be superior to
immunization using needles as more immune cells would be targeted
by the use of several locations targeting large surface areas of
skin. A therapeutically effective amount of antigen sufficient to
induce an immune response may be delivered transcutaneously either
at a single cutaneous location, or over an area of intact skin
covering multiple draining lymph node fields (e.g., cervical,
axillary, inguinal, epitrochelear, popliteal, those of the abdomen
and thorax). Such locations close to numerous different lymphatic
nodes at locations all over the body will provide a more widespread
stimulus to the immune system than when a small amount of antigen
is injected at a single location by intradermal subcutaneous or
intramuscular injection.
[0087] Antigen passing through or into the skin may encounter
antigen presenting cells which process the antigen in a way that
induces an immune response. Multiple immunization sites may recruit
a greater number of antigen presenting cells and the larger
population of antigen presenting cells that were recruited would
result in greater induction of the immune response. It is
conceivable that absorption through the skin may deliver antigen to
phagocytic cells of the skin such as, for example, dermal dendritic
cells, macrophages, and other skin antigen presenting cells;
antigen may also be delivered to phagocytic cells of the liver,
spleen, and bone marrow that are known to serve as the antigen
presenting cells through the blood stream or lymphatic system. The
result would be widespread distribution of antigen to antigen
presenting cells to a degree that is rarely, if ever achieved, by
current immunization practices.
[0088] The transcutaneous immunization system may be applied
directly to the skin and allowed to air dry; rubbed into the skin
or scalp; held in place with a dressing, patch, or absorbent
material; 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 absorbant 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, Inc.), plastic film, COMFEEL (Coloplast) or vaseline;
or a non-occlusive dressing such as, for example, DUODERM (3M) or
OPSITE (Smith & Napheu). An occlusive dressing completely
excludes the passage of water.
[0089] The 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.
[0090] Genetic immunization has been described in U.S. Pat. Nos.
5,589,466 and 5,593,972. The nucleic acid(s) contained in the
formulation may encode the antigen, the adjuvant, or both. The
nucleic acid may or may not be capable of replication; it may be
non-integrating and non-infectious. The nucleic acid may further
comprise a regulatory region (e.g., promoter, enhancer, silencer,
transcription initiation and termination sites, RNA splice acceptor
and donor sites, polyadenylation signal, internal ribosome binding
site, translation initiation and termination sites) operably linked
to the sequence encoding the antigen or adjuvant. The nucleic acid
may be complexed with an agent that promotes transfection such as
cationic lipid, calcium phosphate, DEAE-dextran, polybrene-DMSO, or
a combination thereof. The nucleic acid may comprise regions
derived from viral genomes. Such materials and techniques are
described by Kriegler (1990) and Murray (1991).
[0091] An immune response may comprise humoral (i.e.,
antigen-specific antibody) and/or cellular (i.e., antigen-specific
lymphocytes such as B cells, CD4.sup.+ T cells, CD8.sup.+ T cells,
CTL, Th1 cells, Th2 cells, and/or T.sub.DTH cells) effector arms.
Moreover, the immune response may comprise NK cells that mediate
antibody-dependent cell-mediated cytotoxicity (ADCC) The immune
response induced by the formulation of the invention may include
the elicitation of antigen-specific antibodies and/or cytotoxic
lymphocytes (CTL, reviewed in Alving and Wassef, 1994). Antibody
can be detected by immunoassay techniques, and the detection of
various isotypes (e.g., IgM, IgD, IgA1, IgA2, secretory IgA, IgE,
IgG1, IgG2, IgG3, or IgG4) may be expected. An immune response can
also be detected by a neutralizing assay.
[0092] Antibodies are protective proteins produced by B
lymphocytes. They are highly specific, generally targeting one
epitope of an antigen. Often, antibodies play a role in protection
against disease by specifically reacting with antigens derived from
the pathogens causing the disease. Immunization may induce
antibodies specific for the immunizing antigen, such as cholera
toxin. These antigen-specific antibodies are induced when antigen
is delivered through the skin by liposomes.
[0093] CTLs are particular protective immune cells produced to
protect against infection by a pathogen. They are also highly
specific. Immunization may induce CTLs specific for the antigen,
such as a synthetic oligopeptide based on a malaria protein, in
association with self-major histocompatibility antigen. CTLs
induced by immunization with the transcutaneous delivery system may
kill pathogen infected cells. Immunization may also produce a
memory response as indicated by boosting responses in antibodies
and CTLs, lymphocyte proliferation by culture of lymphocytes
stimulated with the antigen, and delayed type hypersensitivity
responses to intradermal skin challenge of the antigen alone.
[0094] In a viral neutralization assay, serial dilutions of sera
are added to host cells which are then observed for infection after
challenge with infectious virus. Alternatively, serial dilutions of
sera may be incubated with infectious titers of virus prior to
innoculation of an animal, and the innoculated animals are then
observed for signs of infection.
[0095] The transcutaneous immunization system of the invention may
be evaluated using challenge models in either animals or humans,
which evaluate the ability of immunization with the antigen to
protect the subject from disease. Such protection would demonstrate
an antigen-specific immune response. In lieu of challenge,
achieving anti-diphtheria antibody titers of 5 IU/ml or greater is
generally assumed to indicate optimum protection and serves as a
surrogate marker for protection (Plotkin and Mortimer, 1994).
[0096] Furthermore, the Plasmodium faciparum challenge model may be
used as to induce an antigen-specific immune response in humans.
Human volunteers may be immunized using the transcutaneous
immunization system containing oligopeptides or proteins
(polypeptides) derived from the malaria parasite, and then exposed
to malaria experimentally or in the natural setting. The Plasmodium
yoelii mouse malaria challenge model may be used to evaluate
protection in the mouse against malaria (Wang et al., 1995).
[0097] Alving et al. (1986) injected liposomes comprising lipid A
as an adjuvant for inducing an immune response to cholera toxin
(CT) in rabbits and to a synthetic protein consisting of a malaria
oligopeptide containing four tetra-peptides (Asn-Ala-Asn-Pro)
conjugated to BSA. The authors found that the immune response to
cholera toxin or to the synthetic malaria protein was markedly
enhanced by encapsulating the antigen within the liposomes
containing lipid A, compared to similar liposomes lacking lipid A.
Several antigens derived either from the circumsporozoite protein
(CSP) or from merozoite surface proteins of Plasmodium falciparum
have been encapsulated in liposomes containing lipid A. All of the
malaria antigens that have been encapsulated in liposomes
containing lipid A have been shown to induce humoral effectors
(i.e., antigen-specific antibodies), and some have been shown to
induce cell-mediated responses as well. Generation of an immune
response and immunoprotection in an animal vaccinated with a
malaria antigen may be assayed by immunofluorescence to whole,
fixed malaria sporozoites or CTLs killing of target cells
transfected with CSP.
[0098] Mice may be transcutaneously immunized with cholera toxin,
and then challenged intranasally with an LD.sub.70 (40 .mu.g) dose
of cholera toxin and observed for protection. Mallet et al.
(personal communication) have found that C57BL/6 mice develop a
fatal hemorrhagic pneumonia in response to intranasal challenge
with CT. Alternatively, the mice may be challenged with an
intraperitoneal dose of CT (Dragunsky et al., 1992). Cholera
toxin-specific IgG or IgA antibody may provide protection against
cholera toxin challenge (Pierce, 1978; Pierce and Reynolds,
1974).
[0099] Vaccination has also been used as a treatment for cancer and
autoimmune disease. For example, vaccination with a tumor antigen
(e.g., prostate specific antigen) may induce an immune response in
the form of antibodies, CTLs and lymphocyte proliferation which
allows the body's immune system to recognize and kill tumor cells.
Tumor antigens useful for vaccination have been described for
melanoma (U.S. Pat. Nos. 5,102,663, 5,141,742, and 5,262,177),
prostate carcinoma (U.S. Pat. No. 5,538,866), and lymphoma (U.S.
Pat. Nos. 4,816,249, 5,068,177, and 5,227,159). Vaccination with
T-cell receptor oligopeptide may induce an immune response that
halts progression of autoimmune disease (U.S. Pat. Nos. 5,612,035
and 5,614,192; Antel et al., 1996; Vandenbark et al., 1996). U.S.
Pat. No. 5,552,300 also describes antigens suitable for treating
autoimmune disease.
[0100] The following is meant to be illustrative of the present
invention; however, the practice of the invention is not limited or
restricted in any way by the examples.
EXAMPLES
Immunization Procedure
[0101] BALB/c mice of 6 to 8 weeks were shaved with a #40 clipper.
This shaving could be done without any signs of trauma to the skin.
The shaving was done from the mid-thorax to just below the nape of
the neck. The mice were then allowed to rest for 24 hours. Prior to
this the mice had been ear-tagged for identification, and pre-bled
to obtain a sample of pre-immune serum. Mice were also
transcutaneously immunized without shaving by applying 50 .mu.l of
immunizing solution to each ear.
[0102] The mice were then immunized in the following way. Mice were
anesthetized with 0.03-0.06 ml of a 20 mg/ml solution of xylazine
and 0.5 ml of 100 mg/ml ketamine; mice were immobilized by this
dose of anesthesia for approximately one hour. The mice were placed
ventral side down on a warming blanket.
[0103] The immunizing solution was then placed on the dorsal shaved
skin of a mouse in the following manner: a 1.2 cm.times.1.6 cm
stencil made of polystyrene was laid gently on the back and a
saline-wetted sterile gauze was used to partially wet the skin
(this allowed even application of the immunizing solution), the
immunizing solution was then applied with a pipet to the area
circumscribed by the stencil to yield a 2 cm.sup.2 patch of
immunizing solution. Care was used not to scrape or rub the skin
with the pipet tip. The immunizing solution was spread around the
area to be covered with the smooth side of the pipet tip.
[0104] The immunizing solution (between about 100 .mu.l to about
200 .mu.l) was left on the back of the mouse for 60 minutes. At the
end of 60 minutes, the mouse was held gently by the nape of the
neck and the tail under a copious stream of lukewarm tap water, and
washed for 10 seconds. The mouse was then gently patted dry with a
piece of sterile gauze and a second washing was performed for 10
seconds; the mouse was then patted dry a second time and left in
the cage. The mice appeared to exhibit no adverse effects from the
anesthesia, immunization, washing procedure, or toxicity from the
exotoxins. No skin irritation, swelling or redness was seen after
the immunization and the mice appeared to thrive. Immunization
using the ear was performed as described above except that fur was
not removed prior to immunization.
Antigen
[0105] The following antigens were used for immunization and ELISA,
and were mixed using sterile PBS or normal saline. Cholera toxin or
CT (List Biologicals, Cat #101B, lot #10149CB), CT B subunit (List
Biologicals, Cat #BT01, lot #CVXG-14E), CT A subunit (List
Biologicals, Cat #102A, lot #CVXA-17B), CT A subunit (Calbiochem,
Cat #608562); pertussis toxin, salt-free (List Biologicals, lot
#181120a); tetanus toxoid (List Biologicals, lots #1913a and
#1915a); Pseudomonas exotoxin A (List Biologicals, lot #ETA25a);
diphtheria toxoid (List Biologicals, lot #15151); heat-labile
enterotoxin from E. coli (Sigma, lot #9640625); bovine serum
albumin or BSA (Sigma, Cat #3A-4503, lot #31F-0116); and Hemophilus
influenza B conjugate (Connaught, lot#6J81401).
ELISA--IgG(H+L)
[0106] Antibodies specific for CT, LT, ETA, pertussis toxin,
diphtheria toxoid, tetanus toxoid, Hemophilus influenza B
conjugate, and BSA were determined using ELISA in a technique
similar to Glenn et al. (1995). All antigens were dissolved in
sterile saline at a concentration of 2 .mu.g/ml. Fifty microlilters
of this solution (0.1 .mu.g) per well was put on IMMULON-2
polystyrene plates (Dynatech Laboratories, Chantilly, Va.) and
incubated at room temperature overnight. The plates were then
blocked with a 0.5% casein/0.05% Tween 20 blocking buffer solution
for one hour. Sera was diluted with 0.5% casein/0.05% Tween 20
diluent; dilution series were done in columns on the plate.
Incubation was for 2 hours at room temperature.
[0107] The plates were then washed in a PBS-0.05% Tween 20 wash
solution four times, and goat anti-mouse IgG(H+L) horseradish
peroxidase (HRP)-linked (Bio-Rad Laboratories, Richmond, Calif.,
Cat #170-6516) secondary antibody was diluted in casein diluent at
a dilution of 1/500 and left on the plates for one hour at room
temperature. The plates were then washed four times in the
PBS-Tween wash solution. One hundred microliters of
2,2'-azino-di(3-ethyl-benzthiazolone)sulphonic acid substrate
(Kirkegaard and Perry) were added to each well and the plates were
read at 405 nm after 20-40 minutes of development. Results are
reported as the geometric mean of individual sera and standard
error of the mean of ELISA units (the serum dilution at which the
absorbance in equal to 1.0) or as individual antibody responses in
ELISA units.
ELISA--IgG(.gamma.), IgM(.mu.) and IgA(.alpha.)
[0108] IgG(.gamma.), IgM(.mu.) and IgA(.alpha.) anti-CT antibody
levels were determined using ELISA with a technique similar to
Glenn et al. (1995). CT was dissolved in sterile saline at a
concentration of 2 .mu.g/ml. Fifty microliters of this solution
(0.1 .mu.g) per well were put on IMMULON-2 polystyrene plates
(Dynatech Laboratories, Chantilly, Va.) and incubated at room
temperature overnight. The plates were then blocked with a 0.5%
casein-Tween 20 blocking buffer solution for one hour. Sera was
diluted and casein diluent and serial dilutions were done on the
plate. This was incubated for two hours at room temperature.
[0109] The plates were then washed in a PBS-Tween wash solution
four times and goat anti-mouse IgG(.gamma.) HRP-linked (Bio-Rad
Laboratories, Richmond, Calif., Cat #172-1038), goat anti-mouse
IgM(.mu.) HRP-linked (BioRad Laboratories, Richmond, Calif., Cat
#172-1030), or goat anti-mouse IgA HRP-linked (Sigma, St. Louis,
Mo., Cat #1158985) secondary antibody was diluted in casein diluent
in a dilution of 1/1000 and left on the plates for one hour at room
temperature. The plates were then washed four times in a PBS-Tween
wash solution. One hundred microliters of 2,2'-azino-di(3-ethyl
benzthiazolone) sulphonic acid substrate from (Kirkegaard and
Perry, Gaithersburg, Md.) were added to the wells and the plates
were read at 405 nm. Results are reported as the geometric mean of
individual sera and standard error of the mean of ELISA units (the
serum dilution at which the absorbance in equal to 1.0).
ELISA--IgG Subclass
[0110] Antigen-specific IgG (IgG1, IgG2a, IgG2b, and IgG3) subclass
antibody against CT, LT, ETA, and BSA was performed as described by
Glenn et al. (1995). The solid phase ELISA was performed in
IMMULON-2 polystyrene plates (Dynatech Laboratories, Chantilly,
Va.). Wells were incubated with the respective antigens in saline
overnight (0.1 .mu.g/50 .mu.l) and blocked with 0.5% casein-Tween
20. Individual mouse sera diluted in 0.5% casein were serially
diluted, and incubated at room temperature for four hours.
Secondary antibody consisted of horseradish peroxidase-conjugated
goat anti-mouse isotype-specific antibody (IgG1, IgG2a, IgG2b,
IgG3, The Binding Site, San Diego, Calif.). A standard curve for
each subclass was determined using mouse myeloma IgG1, IgG2a,
IgG2b, and IgG3 (The Binding Site, San Diego, Calif.). Standard
wells were coated with goat anti-mouse IgG(H+L) (Bio-Rad
Laboratories, Richmond, CA, Cat #172-1054) to capture the myeloma
IgG subclass standards which were added in serial dilutions. The
myeloma IgG subclass was also detected using the
peroxidase-conjugated goat anti-mouse subclass-specific antibody.
Both the test sera and myeloma standards were detected using
2,2'-azino-di(3-ethyl-benzthiazolone) sulphonic acid (Kirkegaard
and Perry, Gaithersburg, Md.) as substrate. Absorbances were read
at 405 nm. Individual antigen specific subclasses were quantitated
using the values from the linear titration curve computed against
the myeloma standard curve and reported as .mu.g/ml.
ELISA--IgE
[0111] Antigen-specific IgE antibody quantitation was performed
using a protocol from Pharmingen Technical Protocols, page 541 of
the Research Products Catalog, 1996-1997 (Pharmingen, San Diego,
Calif.). Fifty microliters of 2 .mu.g/ml purified anti-mouse IgE
capture mAb (Pharmingen, Cat# 02111D) in 0.1 M NaHCO.sub.3 (pH 8.2)
were added to IMMUNO plates(Nunc, Cat #12-565-136). Plates were
incubated overnight at room temperature, washed three times with
PBS-Tween 20, blocked with 3% BSA in PBS for two hours, and washed
three times with PBS-Tween. Sera were diluted in 1% BSA in PBS,
added at dilutions of 1/100, and diluted serially down the columns
(e.g., 1/100, 1/200, et cetera). Purified mouse IgE standards
(Pharmingen, Cat # 0312D) were added with a starting dilution of
0.25 .mu.g/ml and serially diluted down the columns. Plates were
incubated for two hours and washed five times with PBS-Tween.
[0112] Biotinylated anti-mouse IgE mAB (Pharmingen, Cat #02122D) to
2 .mu.g/ml in 1% BSA in PBS, incubated for 45 minutes and washed
five times with PBS-Tween. Avidin-peroxidase (Sigma A3151, 1:400 of
1 mg/ml solution) was added for 30 min and plates were washed six
times with PBS-Tween. Both the test sera and IgE standards were
detected using 2,2'-azino-di(3-ethyl-benzthiazolone)sulphonic acid
(Kirkegaard and Perry, Gaithersburg, Md.) as substrate. Absorbances
were read at 405 nm. Individual antigen specific subclasses were
quantitated using the values from the linear titration curve
computed against the IgE standard curve and reported as
.mu.g/ml.
Liposome Preparation
[0113] Where liposomes were included in the formulation for
transcutaneous immunization, multilamellar liposomes composed of
dimyristoyl phosphatidyl choline, dimyristoyl phosphatidyl
glycerol, cholesterol were prepared according to Alving et al.
(1993). Dimyristoyl phosphatidylcholine, dimyristoyl
phosphatidylglycerol, and cholesterol were purchased from Avanti
Polar Lipids Inc. (Alabaster, Ala.). Stock solutions of the lipids
in chloroform were removed from -20.degree. C. freezer where they
were stored.
[0114] The lipids were mixed in a molar ratio of 0.9:0.1:0.75
dimyristoyl phosphatidyl choline, dimyristoyl phosphatidyl
glycerol, and cholesterol in a pear shaped flask. Using a rotary
evaporator, the solvent was removed at 37.degree. C. under negative
pressure for 10 minutes. The flask was further dried under low
vacuum for two hours in a dessicator to remove residual solvent.
The liposomes were swollen at 37 mM phospholipid using sterile
water, lyophilized and stored at -20.degree. C. These liposomes
were mixed in their lyophilized state with normal saline (pH 7.0)
to achieve a designated phospholipid concentration in the saline.
Alternatively, the dried lipids were swollen to make liposomes with
normal saline (pH 7.0) and were not lyophilized.
Example 1
[0115] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized using 100 .mu.l of
immunization solution which was prepared as follows: liposomes
prepared as described above for "Liposome Preparation" were mixed
with saline to form the liposomes. The pre-formed liposomes were
then diluted in either saline (liposome alone group) or with CT in
saline to yield an immunizing solution containing liposomes at
10-150 mM phospholipid with 100 .mu.g of CT per 100 .mu.l of
immunizing solution. CT was mixed in saline to make an immunizing
solution containing 100 .mu.g of CT per 100 .mu.g of solution for
the group receiving CT alone. Solutions were vortexed for 10
seconds prior to immunization.
[0116] The mice were immunized transcutaneously at 0 and 3 weeks.
Antibody levels were determined using ELISA as described above for
"ELISA IgG(H+L)" 3 weeks after the boosting immunization, and
compared against pre-immune sera. As shown in Table 1, the level of
anti-CT antibodies induced by CT without liposomes was not
different from the level of anti-CT antibodies generated using
liposomes except in the mice where 150 mM liposomes were used. CT
in saline alone was able to immunize mice against CT to produce
high antibody titers. TABLE-US-00001 TABLE 1 Anti-CT antibodies
Group ELISA Units SEM CT alone 27,482 (16,635-48,051) CT + 150 mM
Liposomes 4,064 *(2,845-5,072) CT + 100 mM Liposomes 35,055
(25,932-44,269) CT + 50 mM Liposomes 9,168 (4,283-12,395) CT + 25
mM Liposomes 18,855 (12,294-40,374) CT + 10 mM Liposomes 28,660
(18,208-31,498) 50 mM Liposomes 0 *Significantly different from the
Group CT alone (P < 0.05)
Example 2
[0117] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized at 0 and 3 weeks
using 100 .mu.l of immunization solution prepared as follows: BSA
was mixed in saline to make an immunizing solution containing 200
.mu.g of BSA per 100 .mu.l of saline for the group receiving BSA
alone; BSA and CT were mixed in saline to make an immunizing
solution containing 200 .mu.g of BSA and 100 .mu.g of CT per 100
.mu.l of saline for the group receiving BSA and CT. Where liposomes
were used, the liposomes were prepared as described above for
"Liposome Preparation", and were first mixed with saline to form
the liposomes. They were then diluted in BSA or BSA and CT in
saline to yield an immunizing solution containing liposomes at 50
mM phospholipid with 200 .mu.g of BSA per 100 .mu.l of immunizing
solution, or 200 .mu.g BSA+100 .mu.g CT per 100 .mu.l of immunizing
solution. Solutions were vortexed for 10 seconds prior to
immunization.
[0118] The antibodies were determined using ELISA as described
above for "ELISA IgG(H+L)" on sera 3 weeks after the second
immunization. The results are shown in Table 2. BSA alone, with or
without liposomes, was not able to elicit an antibody response.
However, the addition of CT stimulated an immune response to BSA.
CT acted as a adjuvant for the immune response to BSA, and anti-BSA
antibodies of high titer were produced. TABLE-US-00002 TABLE 2
Anti-BSA antibodies Group ELISA Units SEM BSA in saline 0 BSA + 50
mM Liposomes 0 CT + BSA in saline 8,198 (5,533-11,932) CT + BSA +
50 mM 3,244 (128-3,242)
Example 3
[0119] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized at 0 and 3 weeks
using 100 .mu.l of immunization solution prepared as follows: LT
was mixed in saline to make an immunizing solution containing 100
.mu.g of LT per 100 .mu.l of saline for the group receiving LT
alone. Where liposomes were used the liposomes prepared as
described above for "Liposome Preparation", and were first mixed
with saline to form the liposomes. The pre-formed liposomes were
then diluted in LT in saline to yield an immunizing solution
containing liposomes at 50 mM phospholipid with 100 .mu.g of LT per
100 .mu.l of immunizing solution. Solutions were vortexed for 10
seconds prior to immunization.
[0120] The anti-LT antibodies were determined using ELISA as
described above for "ELISA IgG(H+L)" 3 weeks after the second
immunization. The results are shown in Table 3. LT was clearly
immunogenic both with and without liposomes, and no significant
difference between the groups could be detected. LT and CT are
members of the family of bacterial ADP-ribosylating exotoxins
(bAREs). They are organized as A:B proenzymes with the
ADP-ribosyltransferase activity contained in the A subunit and the
target cell binding a function of the B subunit. LT is 80%
homologous with CT at the amino acid level and has a similar
non-covalently bound subunit organization, stoichiometry (A:B5),
the same binding target, ganglioside GM1, and is similar in size
(MW .about.80,000). The similarities of LT and CT appear to
influence their immunogenicity by the transcutaneous route as
reflected by the similar magnitude of the antibody response to both
CT and LT (Tables 1 and 3). TABLE-US-00003 TABLE 3 Anti-LT
antibodies Group ELISA Units SEM LT in saline 23,461
(20,262-27,167) LT + 50 mM Liposomes 27,247 (19,430-38,211)
Example 4
[0121] C57BL/6 mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized once using 100
.mu.l of immunization solution prepared as follows: LT was mixed in
saline to make an immunizing solution containing 100 .mu.g of LT
per 100 .mu.l of saline. The solution was vortexed for 10 seconds
prior to immunization.
[0122] The anti-LT antibodies were determined using ELISA as
described above for "ELISA IgG (H+L)" 3 weeks after the single
immunization. The results are shown in Table 4. LT was clearly
immunogenic with a single immunization and antibodies were produced
by 3 weeks. Rapid enhancement of antibody titers and responses to
single immunization would be a useful aspect of the transcutaneous
immunization method. It is conceivable that a rapid single
immunization would be useful in epidemics, for travelers, and where
access to medical care is poor. TABLE-US-00004 TABLE 4 Anti-LT
antibodies Mouse Number ELISA Units 5141 6,582 5142 198 5143 229
5144 6,115 5145 17,542 Geo Mean 2,000
Example 5
[0123] C57BL6 mice at 8 to 12 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized once using 100
.mu.l of immunization solution prepared as follows: CT was mixed in
saline to make an immunizing solution containing 100 .mu.g of CT
per 100 .mu.l of saline. The solution was vortexed for 10 seconds
prior to immunization.
[0124] The anti-CT antibodies were determined using ELISA as
described above for "ELISA IgG (H+L)" 3 weeks after the single
immunization. The results are shown in Table 5. CT was highly
immunogenic with a single immunization. Rapid enhancement of
antibody titers and responses to single immunication may be a
useful aspect of the transcutaeous immunization method. It is
conceivable that a rapid single immunization would be useful in
epidemics, for travelers, and where access to medical care is poor.
TABLE-US-00005 TABLE 5 Anti-CT antibodies Mouse Number ELISA Units
2932 18,310 2933 30,878 2934 48,691 2935 7,824 Geo Mean 21,543
Example 6
[0125] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized at 0 and 3 weeks
using 100 .mu.l of immunization solution prepared as follows: ETA
was mixed in saline to make an immunizing solution containing 100
.mu.g of ETA per 100 .mu.l of saline for the group receiving ETA
alone. Where liposomes were used, the liposomes were prepared as
described above for "Liposome Preparation", and were first mixed
with saline to form the liposomes. The pre-formed liposomes were
then diluted with ETA in saline to yield an immunizing solution
containing liposomes at 50 mM phospholipid with 100 .mu.g of ETA
per 100 .mu.l of immunizing solution. Solutions were vortexed for
10 seconds prior to immunization.
[0126] The antibodies were determined using ELISA as described
above for "ELISA IgG(H+L)" on sera 3 weeks after the second
immunization. The results are shown in Table 6. ETA was clearly
immunogenic both with and without liposomes, and no significant
difference between the groups could be detected. ETA differs from
CT and LT in that ETA is a single 613 amino acid peptide with A and
B domains on the same peptide and binds to an entirely different
receptor, the .alpha.2-macroglobulin receptor/low density
lipoprotein receptor-related protein (Kounnas et al., 1992).
Despite the dissimilarities between ETA and CT in size, structure,
and binding target, ETA also induced a transcutaneous antibody
response. TABLE-US-00006 TABLE 6 Anti-ETA antibodies Group ELISA
Units SEM ETA in saline 3,756 (1,926-7,326) ETA + 50 mM Liposomes
857 (588-1,251)
Example 7
[0127] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized using 100 .mu.l of
immunization solution which was prepared as follows: CT was mixed
in saline to make 100 .mu.g of CT per 100 .mu.l of immunizing
solution, LT was mixed in saline to make 100 .mu.g of LT per 100
.mu.l of immunizing solution, ETA was mixed in saline to make 100
.mu.g of ETA per 100 .mu.l of immunizing solution, and CT and BSA
were mixed in saline to make 100 .mu.g of CT per 100 .mu.l of
immunizing solution and 200 .mu.g of BSA per 100 .mu.l of
immunizing solution. Solutions were vortexed for 10 seconds prior
to immunization.
[0128] The mice were immunized transcutaneously at 0 and 3 weeks
and the antibody levels were determined using ELISA as described
above for "ELISA IgG Subclass", three weeks after the boosting
immunization and compared against the pre-immune sera. The IgG
subclass response to CT, BSA and LT had similar levels of IgG1 and
IgG2a reflecting activation of T help from both Th1 and Th2
lymphocytes (Seder and Paul, 1994), whereas the IgG subclass
response to ETA consisted of almost exclusively IgG1 and IgG3,
consistent with a Th2-like response (Table 7). Thus, it appears
that all IgG subclasses can be produced using transcutaneous
immunization. TABLE-US-00007 TABLE 7 IgG subclasses of induced
antibodies Imm. Antibody IgG1 IgG2a IgG2b IgG3 Antigen Specificity
(.mu.g/.mu.l) (.mu.g/.mu.l) (.mu.g/.mu.l) (.mu.g/.mu.l) CT CT 134
25 27 0 CT + BSA BSA 108 17 12 5 LT LT 155 28 10 8 ETA ETA 50 0 1
10
Example 8
[0129] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized using 100 .mu.l of
immunization solution which was prepared as follows: LT was mixed
in saline to make an immunizing solution containing 100 .mu.g of LT
per 100 .mu.l of saline for the group receiving LT alone, CT was
mixed in saline to make an immunizing solution containing 100 .mu.g
of CT per 100 .mu.l of saline for the group receiving CT alone, ETA
was mixed in saline to make an immunizing solution containing 100
.mu.g of ETA per 100 .mu.l of saline for the group receiving ETA
alone, and BSA and CT were mixed in saline to make an immunizing
solution containing 100 .mu.g of BSA and 100 .mu.g of CT per 100
.mu.l of saline for the group receiving BSA and CT.
[0130] The mice were immunized transcutaneously at 0 and 3 weeks
and the antibody levels were determined using ELISA as described
above for "ELISA IgE", one week after the boosting immunization and
compared against the pre-immune sera. As shown in Table 8, no IgE
antibodies were found although the sensitivity of detection was
0.003 .mu.g/ml. IgG antibodies were determined in the same mice
using "ELISA IgG(H+L)" on sera 3 weeks after the second
immunization. The IgG antibody response to LT, ETA, CT and BSA are
shown to indicate that the animals were successfully immunized and
responded with high titers of antibodies to the respective
antigens. TABLE-US-00008 TABLE 8 IgE antibodies to LT, ETA, CT and
BSA Antibody Group Specificity IgE (.mu.g/ml) IgG (ELISA Units) LT
Anti-LT 0 23,461 ETA Anti-ETA 0 3,756 CT Anti-CT 0 39,828 CT + BSA
Anti-BSA 0 8,198
Example 9
[0131] BALB/c mice at 6 to 8 weeks of age immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized at 0 and 3 weeks
using 100 ml of immunization solution which was prepared as
follows: CT was mixed in saline to make an immunizing solution
containing 100 mg of CT per 100 ml of immunizing solution. The
immunization solution was vortexed for 10 seconds prior to
immunization.
[0132] The mice were immunized transcutaneously at 0 and 3 weeks
and the antibody levels were determined using ELISA as described
above for "ELISA IgG(H+L)" and "ELISA IgG(.gamma.)". Determinations
were done at 1 and 4 weeks after the initial immunization, and
compared against the pre-immune sera. As shown in Table 9, high
levels of anti-CT IgG(.gamma.) antibodies were induced by CT in
saline. Small amounts of IgM could be detected by using IgM(.mu.)
specific secondary antibody. By 4 weeks, the antibody response was
primarily IgG. Data are reported in ELISA units. TABLE-US-00009
TABLE 9 IgG(.gamma.) and IgM(.mu.) Imm. Group Week IgG(.gamma.)
IgM(.mu.) CT 1 72 168 CT 4 21,336 38 L( ) + CT 1 33 38 L( ) + CT 4
22,239 70
Example 10
[0133] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized once using 100
.mu.l of immunization solution prepared as follows: CT was mixed in
saline to make an immunizing solution containing 100 .mu.g of CT
per 100 .mu.l of saline. The solution was vortexed for 10 seconds
prior to immunization. The mice were immunized transcutaneously at
0 and 3 weeks. Antibody levels were determined using ELISA as
described above for "ELISA IgG (H+L)" 5 weeks after the boosting
immunization, and compared against pre-immune sera. As shown in
Table 10, serum anti-CT IgA antibodies were detected.
TABLE-US-00010 TABLE 10 Anti-CT IgA antibodies Mouse Number IgA
(ng/ml) 1501 232 1502 22 1503 41 1504 16 1505 17
Example 11
[0134] BALB/c mice at 6 to 8 weeks of age were immunized
transcutaneously as described above for "Immunization Procedure",
in groups of five mice. The mice were immunized using 100 .mu.l of
immunization solution which was prepared as follows: CT was mixed
in saline to make an immunizing solution containing 100 .mu.g of CT
per 100 .mu.l of immunizing solution. The immunization solution was
vortexed for 10 seconds prior to immunization.
[0135] The mice were immunized with 100 .mu.l of immunizing
solution transcutaneously at 0 and 3 weeks and the antibody levels
were determined using ELISA as described above for "ELISA IgG(H+L)"
and "ELISA IgG(.gamma.)". Antibody determinations were done at 8
weeks after the initial immunization and compared against the
pre-immune sera. As shown in Table 11, high levels of serun anti-CT
antibodies were induced by CT in saline. Lung wash IgG could be
detected by ELISA using IgG(H+L) or IgG(.gamma.) specific antibody.
The antibody found on the lung mucosal surface is diluted by the
lavage method used to collect mucosal antibody and, thus, the exact
amounts of antibody detected are not as significant as the mere
presence of detectable antibody.
[0136] Lung washes were obtained after sacrificing the mouse. The
trachea and lungs were exposed by gentle dissection and trachea was
transected above the bifurcation. A 22 gauge polypropylene tube was
inserted and tied off on the trachea to form a tight seal at the
edges. Half a milliliter of PBS was infused using a 1 ml syringe
attached to the tubing and the lungs were gently inflated with the
fluid. The fluid was withdrawn and reinfused for a total of 3
rounds of lavage. The lung wash was then frozen at -20.degree.
C.
[0137] Table 11 shows the IgG(H+L) and IgG(.gamma.) antibody
response to cholera toxin in the sera and lung washes at 8 weeks.
Data are expressed in ELISA units. Antibodies were clearly
detectable for all mice in the lung washes. The presence of
antibodies in the mucosa may be important for protection against
mucosally active diseases. TABLE-US-00011 TABLE 11 Mucosal Antibody
to CT Animal# Imm. Group IgG(H + L) IgG(.gamma.) Source 1501 CT 133
34 Lungs 1502 CT 75 12 Lungs 1503 CT 162 28 Lungs 1504 CT 144 18
Lungs 1505 CT 392 56 Lungs Geo Mean 156 26 1501 CT 34,131 13,760
Sera 1502 CT 11,131 2,928 Sera 1503 CT 21,898 10,301 Sera 1504 CT
22,025 8,876 Sera 1505 CT 34,284 10,966 Sera Geo Mean 23,128
8,270
Example 12
[0138] BALB/c mice were immunized transcutaneously at 0 and 3 weeks
as described above for "Immunization Procedure", in groups of four
mice. Liposomes were prepared as described above for "Liposome
Preparation", and were first mixed with saline to form the
liposomes. The pre-formed liposomes were then diluted with either
CT, CTA or CTB in saline to yield an immunizing solution containing
liposomes at 50 mM phospholipid with 50 .mu.g of antigen (CT, CTA
or CTB) per 100 .mu.l of immunizing solution. Solutions were
vortexed for 10 seconds prior to immunization.
[0139] The antibodies were determined using ELISA as described
above for "ELISA IgG(H+L)", one week after the boosting
immunization and compared against the pre-immune sera. The results
are shown in Table 12. CT and CTB were clearly immunogenic whereas
CTA was not. Thus, the B subunit of CT is necessary and sufficient
to induce a strong antibody response. TABLE-US-00012 TABLE 12
Antibodies to CT, CTA and CTB Group Anti-CT Anti-CTA Anti-CTB CT +
50 mM Liposomes 12,636 136 7,480 CTB + 50 mM Liposomes 757 20 1,986
CTA + 50 mM Liposomes 0 0 0
Example 13
[0140] BALB/c mice were immunized transcutaneously as described
above for "Immunization Procedure", in groups of five mice. Mice
were immunized at 0 and 3 weeks with 100 .mu.g of diphtheria toxoid
and 10 .mu.g of pertussis toxin per 100 .mu.l of saline solution.
Solutions were vortexed for 10 seconds prior to immunization.
[0141] The antibodies were quantitated using ELISA as described for
"ELISA IgG(H+L)". Anti-diphtheria toxoid antibodies were detected
only in animals immunized with both pertussis toxin and diphtheria
toxoid. The highest responder had anti-diphtheria toxoid antibody
ELISA units of 1,038. Thus, a small amount of pertussis toxin acts
as an adjuvant for diphtheria toxoid antigen. The toxoid alone did
not induce an immune response suggesting that the toxoiding process
has affected the portion of the molecule responsible for the
adjuvant effects found in the ADP-ribosylating exotexin.
TABLE-US-00013 TABLE 13 Antibody to Diphtheria Mouse Number
Immunizing Antigen IgG ELISA Units 4731 DT + PT 1,039 4732 DT + PT
1 4733 DT + PT 28 4734 DT + PT 15 4735 DT + PT 20 4621 DT 0 4622 DT
0 4623 DT 0 4624 DT 0 4625 DT 0
Example 14
[0142] BALB/c mice were immunized transcutaneously as described
above for "Immunization Procedure", in groups of five mice. Mice
were immunized once at 0 weeks with 50 .mu.g of pertussis toxin per
100 .mu.l of saline solution. The solution was vortexed for 10
seconds prior to immunization.
[0143] The antibodies were quantitated using ELISA as described for
"ELISA IgG(H+L)". Anti-pertussis toxin antibodies were detected at
8 weeks in animals immunized with pertussis. The highest responder
had anti-petussis toxin antibody ELISA units of 73. Thus, pertussis
toxin acts as an adjuvant for itself and immunizes after a single
immunization. TABLE-US-00014 TABLE 14 Antibody to Pertussis Mouse
Number Immunizing Antigen IgG ELISA Units 4731 PT 56 4732 PT 60
4733 PT 3 4734 PT 13 4735 PT 73
Example 15
[0144] BALB/c mice were immunized transcutaneously as described
above for "Immunization Procedure", in groups of five mice. Mice
were immunized once at 0 weeks with 50 .mu.g of tetanus toxoid and
100 .mu.g of cholera toxin per 100 .mu.l of saline solution. The
solution was vortexed for 10 seconds prior to immunization.
[0145] The antibodies were quantitated using ELISA as described for
"ELISA IgG(H+L)". Anti-tetanus toxoid antibodies were detected at 8
weeks in animal 5173 at 443 ELISA units.
Example 16
[0146] The possibility that oral immunization occurred through
grooming after epicutaneous application and subsequent washing of
the site of application was evaluated using .sup.125I-labeled CT to
track the fate of the antigen/adjuvant. Mice were anesthetized,
transcutaneously immunized as described above for "Immunization
Procedure" with 100 .mu.g of .sup.125I-labeled CT (150,000
cpm/.mu.g CT). Control mice remained anesthetized for 6 hours to
exclude grooming, and experimental mice were anesthetized for one
hour and then allowed to groom after washing. Mice were sacrificed
at 6 hours and organs weighed and counted for 125I on a Packard
gamma counter. A total of 2-3 .mu.g of CT was detected on the
shaved skin at the site of immunization (14,600 cpm/.mu.g tissue)
while a maximum of 0.5 .mu.g of CT was detected in the stomach (661
cpm/.mu.g tissue) and intestine (9 cpm/.mu.g tissue).
[0147] Oral immunization (n=5) with 10 .mu.g if CT in saline at 0
and 3 weeks (without NaHCO.sub.3) induced a 6 week mean IgG
antibody response of <1,000 ELISA units whereas transcutaneous
immunization with 100 .mu.g of CT, shown above to result in less
than 5 .mu.g of CT retained in the skin after washing, resulted in
an anti-CT response of 42,178 ELISA units at 6 weeks. Induction of
an immune response to orally fed CT requires the addition of
NaHCO.sub.3 to the immunizing solution (Piece, 1978; Lycke and
Holmgren, 1986). Thus, oral immunization does not significantly
contribute to the antibodies detected when CT is applied
epicutaneously to the skin.
Example 17
[0148] In vivo evidence of Langerhans cell activation was obtained
using cholera toxin (CT) in saline applied epicutaneously to the
skin, specifically the ears of the mouse, where large populations
of Langerhans cells can be readily visualized (Enk et al., 1993;
Bacci et al., 1997), and staining for major histocompatibility
complex (MHC) class II molecules which is upregulated in activated
Langerhans cells (Shimada et al., 1987).
[0149] BALB/c mouse ears were coated on the dorsal side with either
100 .mu.g of CT in saline, 100 .mu.g of CTB in saline, saline
alone, or an intradermal injection of the positive controls 100
.mu.g LPS or 10 .mu.g TNF.alpha., for one hour while the mouse was
anesthetized. The ears were then throughly washed and, after 24
hours, the ears were removed and epidermal sheets were harvested
and stained for MHC class II expression as described by Caughman et
al. (1986). Epidermal sheets were stained with MKD6
(anti-I-A.sup.d) or negative control Y3P (anti-I-A.sup.k), and goat
anti-mouse FITC F(ab).sub.2 was used as a second step reagent. Mice
transcutaneously immunized on the ear (as described above without
shaving) had previously been found to have anti-CT antibodies of
7,000 ELISA units three weeks after a single immunization.
[0150] Enhanced expression of MHC class II molecules as detected by
staining intensity, the reduced number of Langerhans cells
(especially with cholera toxin), and changes in Langerhans cell
morphology were found in the epidermal sheets of the mice immunized
with CT and CTB comparable to controls (FIG. 1), suggesting that
the Langerhans cells were activated by the epicutaneously applied
cholera toxin (Aiba and Katz, 1990; Enk et al., 1993).
Example 18
[0151] Langerhans cells represent the epidermal contingent of a
family of potent accessory cells termed `dendritic cells`.
Langerhans cells (and perhaps related cells in the dermis) are
thought to be required for immune responses directed against
foreign antigens that are encountered in skin. The `life cycle` of
the Langerhans cell is characterized by at least two distinct
stages. Langerhans cells in epidermis (the `sentinels`) can ingest
particulates and process antigens efficiently, but are weak
stimulators of unprimed T cells. In contrast, Langerhans cells that
have been induced to migrate to lymph nodes after contact with
antigen in epidermis (the `messengers`) are poorly phagocytic and
have limited antigen-processing capabilities, but are potent
stimulators of naive T cells. If Langerhans cells are to fulfill
both their `sentinel` and `messenger` roles, they must be able to
persist in epidermis, and also be able to exit epidermis in a
controlled fashion after exposure to antigen. Thus, regulation of
Langerhans cell-keratinocyte adhesion represents a key control
point in Langerhans cell trafficking and function. Langerhans cells
express E-cadherin (Blauvelt et al., 1995), a homophilic adhesion
molecule that is prominently represented in epithelia.
Keratinocytes also express this adhesion molecule, and E-cadherin
clearly mediates adhesion of murine Langerhans cells to
keratinocytes in vitro. It is known that E-cadherin is involved in
the localization of Langerhans cells in epidermis. See Stingl et
al. (1989) for a review of the characterization and properties of
Langerhans cells and keratinocytes.
[0152] The migration of epidermal Langerhans cells (LC) and their
transport of antigen from the skin to draining lymph nodes are
known to be important in the induction of cutaneous immune
responses, such as contact sensitization. While in transit to the
lymph nodes, Langerhans cells are subject to a number of phenotypic
changes required for their movement from the skin and acquisition
of the capacity for antigen presentation. In addition to the
upregulation of MHC class II molecules, are alterations in the
expression of adhesion molecules that regulate interactions with
the surrounding tissue matrix and with T lymphocytes. The migration
of the Langerhan cell is known to be associated with a marked
reduction in the expression of E-cadherin (Schwarzenberger and
Udey, 1996, and a parallel upregulation of ICAM-1 (Udey, 1997).
[0153] Transcutaneous immunization with bacterial ADP ribosylating
exotoxins (bARE's) target the Langerhans cells in the epidermis.
The bAREs activate the Langerhans cell, transforming it from its
sentinel role to its messenger role. Ingested antigen is then taken
to the lymph node where it is presented to B and T cells (Streilein
and Grammer, 1989; Kripke et al., 1990; Tew et al., 1997). In the
process, the epidermal Langerhans cell matures into an
antigen-presenting dendritic cell in the lymph node (Schuler and
Steinman, 1985); lymphocytes entering a lymph node segregate into
B-cell follicles and T-cell regions. The activation of the
Langerhans cell to become a migratory Langerhans cell is known to
be associated with not only a marked increase in MHC class II
molecules, but also marked reduction in the expression of
E-cadherin, and upregulation of ICAM-1.
[0154] We envision that cholera toxin (CT) and its B subunit (CTB)
upregulate the expression of ICAM-1 and downregulate the expression
of E-cadherin on Langerhans cells as well as upregulate the
expression of MHC class II molecules on the Langerhans cell. CT or
CTB acts as an adjuvant by freeing the sentinel Langerhans cell to
present antigens such as BSA or diphtheria toxoid phagocytosed by
the Langerhans cell at the same location and time as the encounter
with the CT or CTB when they are acting as adjuvant. The activation
of a Langerhans cells to upregulate the expression of ICAM-1 and
dowregulate the expression of E-cadherin may be mediated by
cytokine release including TNF.alpha. and IL-1.beta. from the
epidermal cells or the Langerhans cells themselves.
[0155] This method of adjuvancy for transcutaneous immunization is
envisioned to work for any compound that activates the Langerhans
cell. Activation could occur in such manner as to downregulate the
E-cadherin and upregulate ICAM-1. Langerhans cells would then carry
antigens made of mixtures of such Langerhans cell-activating
compounds and antigens (such as diphtheria toxoid or BSA) to the
lymph nodes where the antigens are presented to T cells and evoke
an immune response. Thus, the activating substance such as a bARE
can be used as an adjuvant for an other wise transcutaneously
non-immunogenic antigen such as Diphtheria toxoid by activating the
Langerhans cell to phagocytose the antigen such as diphtheria
toxoid, migrate to the lymph node, mature into a dendritic cell,
and present the antigen to T cells.
[0156] The T-cell helper response to antigens used in
transcutaneous immunization may be influenced by the application of
cytokines and/or chemokines. For example, interleukin-10 (IL-10)
may skew the antibody response towards a Th2 IgG1/IgE response
whereas anti-IL-10 may enhance the production of IgG2a
(Bellinghausen et al., 1996).
[0157] The disclosures of all patents, as well as all other printed
documents, cited in this specification are incorporated herein by
reference in their entirety.
[0158] While the present invention has been described in connection
with what is presently considered to be practical and preferred
embodiments, it is understood that the present invention is not to
be limited or restricted to the disclosed embodiments but, on the
contrary, is intended to cover various modifications and equivalent
arrangements included within the spirit and scope of the appended
claims.
[0159] Thus, it is to be understood that variations in the
described invention will be obvious to those skilled in the art
without departing from the novel aspects of the present invention
and such variations are intended to come within the scope of the
claims below.
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* * * * *