Transdermal delivery system for antigen

A transdermal liposome system delivers antigen to immune cells without perforation of the skin, and induces an immune response in an animal or human. The system uses liposomes to deliver a variety of antigens which can elicit an antigen-specific immune response (e.g., humoral and/or cellular effectors) after topical application of a formulation containing liposomes and antigen to intact skin of the animal or human.

BACKGROUND 
1. Field of the Invention 
The invention relates to transdermal delivery of antigen by a liposome 
formulation to induce an antigen-specific immune response. 
2. Description of the Related Art 
Liposomes are smectic mesophases, which have been defined in the following 
manner by D. M. Small (Handbook of Lipid Research, Vol. 4, Plenum, N.Y., 
pp. 49-50): "When a given molecule is heated instead of melting directly 
into anisotropic lipid it may instead pass through intermediate states 
called mesophases or liquid crystals, characterized by residual order in 
some directions but by lack of order in others . . . In general, the 
molecules of liquid crystals are somewhat longer than they are wide and 
have a polar or aromatic part somewhere along the length of the molecule. 
The molecular shape and the polar-polar or aromatic interaction permit the 
molecules to align in a partial ordered array . . . These structures 
characteristically occur in molecules that possess a polar group at one 
end. Liquid crystals with long range order in the direction of the long 
axis of the molecule are called smectic, layered, or lamellar liquid 
crystals . . . in the smectic states, the molecules may be in single or 
double layers, normal or tilted to the plane of the layer, and with frozen 
or melted aliphatic chains." 
As an example of work in the field of transdermal delivery of antigen, Paul 
et al. (1995) and Paul and Cevc (1995) (hereinafter "the Cevc lab") were 
not able to use liposomes for transdermal immunization. The Cevc lab used 
three different lipid formulations: mixed micelles, liposomes, and 
transfersomes in attempting to cause the transdermal delivery of antigen. 
Lipid was provided as an ethanol solution of soybean phosphatidylcholine 
(SPC); liposomes were formed by sonication, then freeze-thawed, and 
finally filtered for the purposes of sterilization and improved sample 
homogeneity. Mixed micelles contained SPC and bile salt (BS) in a mole 
ratio of 1:1, transferosomes contained SPC and BS in a mole ratio of 9:2, 
and liposomes contained SPC but no BS. 
Because they contain a significant proportion of bile salts, mixed micelles 
and transferosomes cannot be considered liposomes (i.e., smectic 
mesophases) as stated by D. M. Small (Handbook of Lipid Research, Vol. 4, 
Plenum, N.Y., p. 95): "Class IIIA lipids . . . exhibit lyotropic 
mesomorphism at low water concentrations and form liquid crystals . . . At 
higher water concentrations, however, these liquid crystals dissolve to 
form micelles. Aliphatic molecules such as soaps, lysolecithin, and 
aliphatic detergents, are representative of class IIA lipids. In class 
IIIB lipids . . . bulky aromatic ring systems often comprise the 
hydrophobic component of the molecule. These compounds form micelles, but 
do not form liquid crystals. Molecules typical of this class are bile 
salts (e.g., Na cholate, Na deoxycholate, and Na chenodeoxycholate), 
saponins, and rosin soaps." As noted above in Small's definition of 
smectic mesophases, liposomes are a type of liquid crystals. 
FIG. 1 of Paul et al. (1995) shows that only a formulation of antigen and 
transferosomes induced an immune response as measured by titer of 
antigen-specific antibody. Topically applied formulations of antigen in 
solution, antigen and mixed micelles, and antigen and liposomes (i.e., 
smectic mesophases) 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. 
Moreover, Paul and Cevc (1995) state 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 
conclude, "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." 
Despite the aforementioned contrary teaching, we have found that liposomes 
do provide a transdermal delivery system for antigen that can induce an 
antigen-specific immune response. 
SUMMARY OF THE INVENTION 
An objective of the invention is to provide a transdermal delivery system 
that induces an immune response (e.g., humoral and/or cellular effectors) 
in an animal or human. Such a system provides a simple means to present 
antigen to the immune system of the animal or human. 
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 objective of the invention is to positively 
and/or negatively regulate components of the immune system by using the 
transdermal delivery system to affect antigen-specific helper (Th1 and/or 
Th2) or delayed-type hypersensitivity (T.sub.DTH) T-cell subsets. 
In one embodiment of the invention, a formulation containing liposomes and 
antigen 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 topical 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. 
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. 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). 
In a third embodiment of the invention, a patch for use in the above 
methods is provided. The patch comprises a dressing, liposomes, and a 
therapeutically effective amount of antigen. 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. 
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. 
The antigen may be derived from a pathogen that can infect the organism 
(e.g., bacterium, virus, fungus, Rickettsia, 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, peptide, phospholipid, or protein. 
Protein may be obtained by recombinant means, chemical synthesis, or 
purification from a natural source. The molecular weight of the antigen 
may be greater than 500 daltons, preferably greater than 800 daltons, and 
more preferably greater than 1000 daltons. 
Liposomes may be multilamellar, paucilamellar, or unilamellar; the 
liposomes may be phospholipid liposomes containing phospholipid, sterol, 
or a mixture thereof. The phospholipid may be phosphatidylcholine, 
phosphatidylglycerol, diphosphatidylglycerol, phosphatidylserine, 
phosphatidylinositol, phosphatidic acid, lysophosphatide, sphingomyelin, 
or mixtures thereof. The sterol is a derivative based on the 
cyclopentanophenanthrene nucleus, and is preferably cholesterol, 
cholesterol esters, cholesterol sulphates, or mixtures thereof. A liposome 
may also contain a nonphospholipid such as, for example, ceramide, 
cerebroside, glycosphingolipid, sphingolipid, free fatty acids, 
eicosanoids, and lipid vitamins. Liposomes may contain a nonionic 
amphiphile such as, for example, polyoxyethylene fatty acid ester, 
polyoxyethylene fatty acid ether, diethanolamide, long chain acyl 
hexosamide, long chain acyl amino acid amide, long chain amino acid amine, 
polyoxyethylene sorbitan ester, polyoxyethylene glyceryl ester, 
polyoxyethylene glyceryl diester, glycerol stearate, glycerol distearate, 
glycerol oleate, glycerol dioleate, glycerol palmitate, glycerol 
dipalmitate, or mixtures thereof. Liposomes may contain an ionic 
amphiphile such as, for example, betaine, sarcosinate, monomeric alkyd, 
dimeric alkyd, dimethyl distearyl amine, or mixtures thereof. 
The formulation may further comprise an adjuvant. 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). 
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. 
The term "therapeutically 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, immunosuppression, 
modulation of autoimmune disease, potentiation of cancer 
immunosurveillance, or vaccination against an infectious disease caused by 
a pathogen. 
DETAILED DESCRIPTION OF THE INVENTION 
Liposomes of the invention 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. They may 
be composed of a variety of lipid components such as, for example, 
phospholipid, nonionic surfactant, synthetic or natural lipid, saturated 
or unsaturated lipid, and charged or neutral lipid, either with or without 
a sterol. Liposomes may be either multilamellar, paucilamellar, or 
unilamellar, and may be made in different sizes: small being less than 25 
nm, intermediate being 25 nm to 500 nm, and large being greater than 500 
nm. A typical liposome is composed of dimyristoyl phosphatidylcholine 
(DMPC), dimyristoyl phosphatidylglycerol (DMPG), and cholesterol, with or 
without lipid A, in a multilamellar configuration, and has a population of 
sizes from about 0.2 .mu.m to about 10 .mu.m. Antigen is delivered by the 
delivery system through intact skin to cells of the immune system, where 
an immune response is induced. 
Liposomes of the invention are used as a transdermal delivery system of 
agents that induce an immune response. These agents as a class can be 
called antigens. Antigen may be composed of chemicals such as, for 
example, carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, 
peptide, phospholipid, protein or any other material known to induce an 
immune response. Antigen may be provided as a whole organism such as, for 
example, a 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. 
Liposomes may be preformed and then mixed with antigen. Liposomes may also 
be formed so as to contain antigen inserted in the lipid bilayer, in the 
inner aqueous spaces, associated with the outer leaflet of the lipid 
bilayer, in the surrounding solution, or in any combination of these 
arrangements. The antigen may be dissolved or suspended, and then added to 
(a) the preformed liposomes in a lyophilized state, (b) dried lipids as a 
swelling solution or suspension, or (c) the solution of lipids used to 
form liposomes. The liposomes may either be used unwashed, or washed prior 
to use to remove antigen that is not associated with the liposome. 
The liposomes may contain a single antigen, more than one antigen, or the 
liposomes containing separate antigens may be mixed into a single liposome 
formulation. The multivalent antigen formulation may be used to induce an 
immune response to more than one antigen at the same time. 
The liposomes may be applied in the form of an emulsion, gel, solution, 
suspension, or other forms known in the art. 
In addition to the above ingredients, there may also be incorporated other 
pharmaceutically acceptable additives including, for example, diluents, 
binders, stabilizers, preservatives, and colorings. 
An object of the invention is to provide a novel means for vaccination 
through intact skin without the need for penetration of the skin. The 
transdermal delivery system provides a method whereby antigens can be 
delivered to the immune system, especially specialized immune cells 
underlying the skin. A mixture of antigen and liposomes; or antigen 
encapsulated in, attached to, or associated with the lipid bilayer of 
liposomes may be applied with or without adjuvants. 
Without being bound to any particular theory but only to provide an 
explanation for our observations, it is presumed that the transdermal 
liposome 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, B 
lymphocyte, or Kupffer cell) that presents processed antigen to a T 
lymphocyte. Passage of liposomes through the stratum corneum may not be 
necessary to deliver antigen to cells of the immune system. 
LIPOSOME LIPID 
Liposomes may be prepared using a variety of techniques and membrane lipids 
(reviewed in Gregoriadis, 1993). 
Natural sources may provide liposome lipid such as, for example, lecithin 
(i.e., phosphatidylcholine): egg yolk, soybean, and brain. Synthetic 
lipids are preferred for their chemical purity. Synthetic lecithins are 
available with fatty acyl chain lengths ranging from 4 to 19 carbons; 
preferred chain lengths are those in the biological range (12 to 24 
carbons). 
Liposomes of the invention may be formed from a phospholipid such as, for 
example, phosphatidylcholine, phosphatidylglycerol, 
diphosphatidylglycerol, phosphatidylserine, phosphatidic acid, 
phosphatidylinositol, lysophosphatide, and sphingomyelin. If a sterol is 
used to stabilize the lipid bilayer, it is preferably cholesterol, a 
cholesterol ester, or a cholesterol sulphate. Cholesterol may be 
recrystallized to avoid the possibility of immunosuppression or toxicity 
due to oxidation products. 
Liposomes of the invention may be formed from lipids extracted from the 
stratum corneum including, for example, ceramide and cholesterol 
derivatives (Wertz, 1992). 
Liposomes of the invention may contain a nonionic amphiphile as a major 
structural component (by weight) of the lipid bilayer. The nonionic 
amphiphile may be, for example, polyoxyethylene fatty acid ester, 
polyoxyethylene fatty acid ether, diethanolamide, long chain acyl 
hexosamide, long chain acyl amino acid amide, long chain amino acid amine, 
polyoxyethylene sorbitan ester, polyoxyethylene glyceryl ester, 
polyoxyethylene glyceryl diester, glycerol stearate, glycerol distearate, 
glycerol oleate, glycerol dioleate, glycerol palmitate, glycerol 
dipalmitate, or mixtures thereof. Nonionic amphiphiles that form liposomes 
in the presence of steroid are disclosed in U.S. Pat. No. 4,917,951, 
incorporated herein by reference. The lipid bilayer may contain an ionic 
amphiphile in lieu of some, or all, of the nonionic amphiphile. The ionic 
amphiphile may be, for example, betaine, sarcosinate, monomeric alkyd, 
dimeric alkyd, dimethyl distearyl amine, or mixtures thereof. Ionic 
amphiphiles are disclosed in U.S. Pat. No. 5,164,191, incorporated herein 
by reference. 
Stability, rigidity, and permeability of the liposome is altered by changes 
in lipid composition. Membrane fluidity is generally controlled by the 
fatty acyl chains of the lipid. The fatty acyl chain can exist in an 
ordered, rigid state or in a relatively disordered fluid state. Factors 
affecting rigidity include chain length and degree of saturation of the 
fatty acyl chains and temperature. Longer chains interact more strongly 
with each other so fluidity is greater with short chains; saturated fatty 
acyl chains are more flexible than unsaturated fatty acyl chains. 
Transition of the membrane from the rigid to the fluid state occurs as the 
temperature is raised above the melting temperature. The melting 
temperature is dependent on the length and degree of saturation of the 
fatty acyl chain. 
ANTIGEN 
Antigen of the invention may be expressed by recombinant means, preferably 
as a fusion with an affinity or epitope tag (Goeddel, 1990; Kriegler, 
1990; Ausubel et al., 1996); chemical synthesis of peptide, either free or 
conjugated to carrier proteins, may be used to obtain antigen of the 
invention (Bodanszky, 1993; Wisdom, 1994). 
Peptide lengths of 6 residues to 20 residues are preferred. Peptides may 
also by synthesized as branched structures such as those disclosed in U.S. 
Pat. Nos. 5,229,490 and 5,390,111, incorporated herein by reference. 
Antigenic peptides include, for example, synthetic or recombinant B-cell 
and T-cell epitopes, universal T-cell epitopes, and mixed T-cell epitopes 
from one organism or disease and B-cell epitopes from another. 
Antigen obtained through recombinant 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). 
Antigen includes, for example, toxins, toxoids, and/or subunits thereof 
(e.g., cholera toxin, tetanus toxoid). 
Antigen is solubilized prior to mixing with liposomes. Suitable buffers 
include, but are not limited to, phosphate buffered saline Ca.sup.++ 
/Mg.sup.++ free (PBS), normal saline, 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 one antigen soluble only at acid pH, acetate-PBS at acid pH was 
used as a diluent after solubilization in 10 mM acetic acid. 
Antigen can also be solubilized in a detergent (e.g., a cell membrane 
extract) along with the lipids themselves, and liposomes are then 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. 
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, 
Rickettsia, or parasite). 
Bacteria include, for example: anthrax, campylobacter, cholera, diptheria, 
enterotoxigenic E. coli, giardia, gonococcus, Hemophilus influenza B, 
Hemophilus influenza non-typable, meningococcus, pertussis, pneumococcus, 
salmonella, shigella, tetanus, and yersinia. 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, Norwalk, 
papilloma virus, parvovirus B19, rabies, rotavirus, rubella, rubeola, 
varicella, and yellow fever. Parasites include, for example: 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) and Leishmania 
(Frankenburg et al., 1996). 
ADJUVANT 
The formulation of liposomes and antigen may also contain an adjuvant. 
Adjuvants are substances that are used to specifically or nonspecifically 
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. Suitable adjuvants include, for example, an oil emulsion (e.g., 
complete or incomplete Freund's adjuvant), a chemokine (e.g., defensins 1 
or 2, RANTES, interleukin-8) or a cytokine (e.g., interleukin-1, -2, -6, 
or -12; .gamma.-interferon; tumor necrosis factor; 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, or a lipopolysaccharide (LPS) derivative (e.g., lipid 
A or monophosphoryl lipid A). 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). 
Lipid A is derived from the lipopolysaccharide (LPS) of gram-negative 
bacterial endotoxin. It is an outstanding adjuvant that can be 
incorporated into the liposome bilayer to induce an immune response to a 
liposome-associated antigen (Alving, 1993). Lipid A is actually a 
heterogeneous mixture of compounds having similar structures (Banerji and 
Alving, 1979). The methods ordinarily used to obtain lipid A can produce a 
crude fraction, which is then purified by ethylenediamine tetraacetic acid 
and chloroform extraction to give a purified lipid A that is chloroform 
soluble (Banerji and Alving, 1979). 
PREATION OF LIPOSOMES 
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. 
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. 
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 .mu.m Hg) for 1 hour in a dessicator. 
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 40.degree. C. The liposome-antigen formulation may be used 
immediately or stored for several years. 
It may be advantageous to employ the liposome-antigen mixture directly in 
the transdermal delivery system. But if removal of non-encapsulated 
antigen from the mixture is desired, approximately 20 volumes of buffer 
may be added to the mixture and the liposomes pelleted by centrifugation 
at 25,000 g to 30,000 g for 30 minutes at 20.degree. C. to 25.degree. C. 
After removal of the clear (or slightly turbid) supernatant fraction, the 
liposome pellet may be suspended to the desired final volume (10 mM to 200 
mM with respect to liposome lipids) with the appropriate buffer. 
Alternatively, liposomes may be formed as described above but without 
addition of antigen to the aqueous solution. Antigen may then be added to 
the preformed liposomes and, therefore, antigen would be in solution 
and/or associated with, but not encapsulated by, the liposomes. 
A method for forming liposomes containing at least two lipids or 
amphiphiles is disclosed in U.S. Pat. No. 5,260,065, incorporated herein 
by reference. 
A method and apparatus for forming liposomes without using a solvent are 
disclosed in U.S. Pat. Nos. 4,895,452 and 4,911,928, incorporated herein 
by reference. The lipid phase of the formulation is heated until flowing, 
and then blended with an excess of the aqueous phase under shear 
conditions until liposomes are formed. If an oil or a water immiscible 
component of the formulation (e.g., antigen or adjuvant) is to be 
encapsulated in the liposome or incorporated in the lipid bilayer, such a 
component can be blended first with the lipid phase before hydration by 
the aqueous phase. 
Other methods for forming liposomes are disclosed in U.S. Pat. Nos. 
4,089,801, 4,196,191, 4,235,871, 4,485,054, 4,508,703, 4,731,210, 
4,897,269, 4,963,297, 4,975,282, 5,008,050, 5,059,421, and 5,169,637, 
incorporated herein by reference. A method for obtaining an oil-in-water 
emulsion containing liposomes is disclosed in U.S. Pat. No. 3,957,971, 
incorporated herein by reference; a method for obtaining a water-in-oil 
emulsion containing liposomes is disclosed in U.S. Pat. No. 5,256,422, 
incorporated herein by reference. If the process for forming liposomes 
would denature the antigen in the formulation, the antigen will be mixed 
with the formed liposomes. Therefore, antigen would not be encapsulated by 
liposomes formed by such an antigen-denaturing process but, instead, the 
antigen would only be mixed in solution and/or associated with the 
liposomes. 
Lipid compositions and methods for forming unilamellar liposomes are 
disclosed in U.S. Pat. Nos. 4,853,228 and 5,008,050, incorporated herein 
by reference. 
Several types of liposomes, such as unilamellar, paucilamellar, or 
multilamellar vesicles, might be used as a transdermal delivery system for 
antigens. However, because they are easier to manufacture and require less 
handling, and consequently afford less chances for contamination, we have 
used multilamellar vesicles in the Examples below. 
TRANSDERMAL DELIVERY OF ANTIGEN 
Liposomes have been used as carriers in adjuvants to enhance the immune 
response to antigens mixed with, encapsulated in, attached to, or 
associated with liposomes. For previous vaccine applications using 
liposomes, the formulation was injected through the skin with a needles, 
as are the majority of licensed vaccines. 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 (transdermal immunization) represents a major 
advance for vaccine delivery avoiding the aforementioned drawbacks in 
needle use. 
The transdermal 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. 
Moreover, transdermal 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 the skin. A therapeutically 
effective amount of antigen sufficient to induce an immune response may be 
delivered transdermally 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. 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 into the 
blood stream will also result in delivery of antigen to the phagocytic 
cells of the liver, spleen, and bone marrow that are known to serve as the 
antigen presenting cells. 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. 
The transdermal liposome system may be applied directly to the skin and 
allowed to air dry, held in place with a dressing or patch or absorbent 
material, applied as an ointment, or otherwise held by a device such as a 
stocking or shirt or sprayed onto the skin to maximize contact of the 
liposomes 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, plastic film or COMFEEL (Coloplast), or a 
non-occlusive dressing such as, for example, DUODERM (3M) or OPSITE (Smith 
& Napheu). 
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. This could 
include application to large areas of skin including total immersion, or a 
skin cream. 
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. 
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. 
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 peptide based on 
a malaria protein, in association with self-major histocompatibility 
antigen. CTLs induced by immunization with the transdermal 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. 
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. 
The transdermal delivery 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. For 
example, 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 transdermal delivery system containing peptides or 
proteins 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). 
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 peptide 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 falcipacrum 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. 
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), all incorporated by 
reference herein. Vaccination with T-cell receptor peptide may induce an 
immune response that halts progression of autoimmune disease (Antel et 
al., 1996; Vandenbark et al., 1996). U.S. Pat. No. 5,552,300, incorporated 
by reference herein, also describes antigens suitable for treating 
autoimmune disease.

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. 
EXAMPLE 1 
As an example of the present invention applied to transdermal delivery of 
the antigen cholera toxin, multilamellar liposomes containing dimyristoyl 
phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol and 
lipid A (prepared according to Alving et al., 1993; Alving et al., 1995; 
Richards et al., 1995, incorporated herein by reference) were used to 
induce an immune response against cholera toxin. 
Dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol 
(DMPG), and cholesterol (Chol) were obtained from Avanti Polar Lipids 
(Alabaster, Ala.). Lipid A (primarily monophosphoryl) and cholera toxin 
(CT) were obtained from List Biological Laboratories (Campbell, Calif.). 
Stock solutions (180 mM DMPC, 20 mM DMPG, and 150 mM Chol) of the lipids 
in chloroform were stored at -20.degree. C. 
Five mls of each stock solution was mixed in a 100 ml pear shaped flask to 
give a solution of DMPC, DMPG, Chol and lipid A in a mole ratio of 
9:1:7.5:0.02, respectively. Using a rotary evaporator, the solvent was 
removed at 37.degree. C. under negative pressure (approximately 140 torr) 
for 10 minutes. The lipids were further dried under low vacuum (less than 
50 torr) for 1 hour in a dessicator. The liposomes were formed by removing 
the lipid film from the wall of the flask with 20 ml of swelling solution 
(sterile water), shaken by hand for 5-10 minutes and briefly vortexed, 
lyophilized, and stored at -20.degree. C. 
These liposomes were mixed in their lyophilized state with cholera toxin 
(CT) at 5 mg/ml in normal saline (0.154 M NaCl, pH 7.0) to achieve a final 
concentration of 100 mM with respect to the phospholipids. 
BALB/c mice were marked using a stencil (1.6 cm.times.2.5 cm) to make a 4 
cm.sup.2 square which was then gently shaved using an electric clipper. 
This was done without any indication of trauma to the skin of any of the 
mice. After shaving, the mice were allowed to rest for 24 hours prior to 
immunization. 
A total of 100 .mu.l of the liposome-antigen formulation was applied to 4 
cm.sup.2 of shaved skin on the back of each BALB/c mouse. Approximately 25 
.mu.l of the formulation was applied to the shaved skin, this volume would 
form a bead on the shaved skin. Using the smooth side of a bent 200 .mu.l 
pipette tip, the formulation was gently spread to hydrate the patch of 
shaved skin; the remaining volume was then applied to the patch of shaved 
skin and adhered to the hydrated skin without difficulty. The formulation 
was left on each mouse's back for two hours, this amount of time was 
sufficient for the formulation to turn into a very small layer, but not 
fully dry or powdery; the mouse did not groom its back during this time. 
After the one hour immunization, each mouse was gently held by the nape of 
its neck and the tail, and placed for 15 seconds in a stream of lukewarm 
tap water flowing over the patch of shaved skin and back towards the tail. 
The mouse was gently rubbed with gauze and washed a second time. 
Boosting immunization was repeated using the same liposome-antigen 
formulation and technique three weeks after primary immunization. 
Anti-cholera toxin antibodies elicited by transdermal immunization with the 
transdermal delivery system of the invention were measured using an 
enzyme-linked immunosorbent assay (ELISA) (Glenn et al., 1995, 
incorporated herein by reference). Solid-phase ELISA was performed in 
IMMULON-2 polystyrene plates (Dynatech Laboratories, Chantilly, Va.). 
Wells of the plate were incubated with antigen in PBS overnight (0.1 mg CT 
per 50 ml) at room temperature, and blocked with 0.5% (w/v) casein for 2 
hours at room temperature. Individual mouse sera diluted in a solution of 
0.5% (w/v) casein were added and incubated at room temperature. 
Horseradish peroxidase-conjugated goat anti-mouse IgG (heavy and light) 
(Bio-Rad Laboratories, Richmond, Calif.) was used as secondary antibody 
and 2,2'-azino-di(3-ethyl-benzthiazolone) sulphonic acid was used as 
substrate, reaction was performed for 30 minutes at room temperature. 
Absorbances were read at 405 nm on a KINETIC MICROPLATE READER (Molecular 
Devices, San Diego, Calif.) and subtracted for the reactivity of preimmune 
sera. The linear portion of the dilution series is determined and the 
result reported is the serum dilution at which the optical density would 
be equal to 1. 
Induction of an immune response to cholera toxin is demonstrated 
by.antigen-specific antibody titers in Table 1. Note that an initial 
antibody response was achieved after a single immunization and that a 
boosting response, as denoted by a rise in antibody optical density units, 
was seen at 4 weeks in all three animals. 
TABLE 1 
______________________________________ 
MOUSE #998 MOUSE #999 MOUSE #1000 
______________________________________ 
WEEK 1 233 283 27 
WEEK 4 4768 5702 8045 
______________________________________ 
Table 1. Antibody response to cholera toxin. BALB/c mice immunized with 
cholera toxin in liposomes containing lipid A, applied on the skin. 
Anti-cholera toxin antibodies measured using ELISA, reported in OD units 
on individual mice. Mice immunized at 0 and 3 weeks. 
EXAMPLE 2 
Lyophilized lipids were prepared, and the liposomes were formed in the 
presence of cholera toxin antigen as in Example 1. The formulation 
containing cholera toxin was used for transdermal immunization unwashed, 
or washed by adding approximately 20 volumes of buffer and pelleting the 
liposomes by centrifugation at 25,000 g to 30,000 g for 20 minutes at 
20.degree. C. to 25.degree. C. After removal of the clear (or slightly 
turbid) supernatant fraction, the liposome pellet was suspended to a final 
concentration of 500 .mu.g/ml cholera toxin as determined by a modified 
Lowry assay. 
Mice in the unwashed liposome group were immunized transdermally with 100 
.mu.l of liposome-antigen formulation (with or without lipid A) as in 
Example 1; mice in the washed liposome group were immunized transdermally 
with 500 .mu.l of liposome-antigen formulation (with or without lipid A) 
as in Example 1. The amount of cholera toxin used in the above cases was 
250 .mu.g total antigen. 
The saline groups had 100 .mu.l of 2.5 mg/ml (250 .mu.g) cholera toxin 
applied to the shaved areas for two hours. The oral immunization groups 
were fed the equivalent of 25 .mu.g of cholera toxin of the 
liposome-antigen formulation used for transdermal immunization. 
As described above, five BALB/c mice were immunized in each group, and then 
boosted three weeks later. 
Induction of an immune response to cholera toxin at week 4 was assayed 
(Table 2) by measuring antigen-specific antibody using the ELISA method 
described in Example 1. 
TABLE 2 
______________________________________ 
Immunization Group 
Antigen Dose (.mu.g) 
Antibody Response 
______________________________________ 
Unwashed L(LA + CT), 
250 9960 (834-22,476) 
transdermal 
Unwashed L(CT), 
250 49422 (7180-64306) 
transdermal 
Washed L(LA + CT), 
250 9 (1-29) 
transdermal 
Washed L(CT), transdermal 
250 5 (0-20) 
L(LA), transdermal 
250 8 (0-42) 
CT + Saline, transdermal 
250 28 (1-359) 
CT + Saline, oral 
25 116 (16-154) 
Unwashed L(LA + CT), oral 
25 53 (16-31) 
Unwashed L(CT), oral 
25 134 (10-335) 
______________________________________ 
Table 2. Antibody response to cholera toxin. BALB/c mice (n=5) immunized as 
described in Example 2 at week 0 and week 3 in groups as shown. 
Anti-cholera toxin IgG antibodies individually measured using ELISA on 
sera one week after boosting (week 4) and reported as the geometric mean 
and standard error of the mean of each group. OD units represent the serum 
dilution at which the optical density is equal to 1. Abbreviations: L is 
liposome, LA is lipid A, and CT is cholera toxin. 
The antigen-specific immune response associated with the groups of unwashed 
liposome-antigen formulation applied transdermally was measured to 
demonstrate the kinetics and maintenance of the immune response (Table 3) 
and the difference in IgG subclasses induced by liposome-antigen 
formulations with or without lipid A (Table 4). 
TABLE 3 
______________________________________ 
GROUP WEEK 1 WEEK 4 WEEK 6 
______________________________________ 
L(LA + CT) 
37 (11-27) 49,422 55,572 
(7,000-64,000) 
(36,000-87,000) 
L(CT) 28 (11-27) 9,960 19,533 
(800-22,000) 
(11,000-34,000) 
______________________________________ 
Table 3. Kinetics and maintenance of the antibody response to cholera 
toxin. BALB/c mice (n=5) immunized as described in Example 2 at week 0 and 
week 3 in groups as shown and antibody response determined at week 1, 4 
and 6. Anti-cholera toxin IgG antibodies individually measured using ELISA 
and reported as the geometric mean and standard error of the mean for each 
group. OD units represent the serum dilution at which the optical density 
is equal to 1. Abbreviations: L is liposome, LA is lipid A, and CT is 
cholera toxin. 
TABLE 4 
______________________________________ 
IgG1 IgG2a IgG2b IgG3 
Group (.mu.g/ml) (.mu.g/ml) 
(.mu.g/ml) 
(.mu.g/ml) 
______________________________________ 
L(LA + CT) 
45.3 (25-80) 
7.8 (4-16) 
3.1 (2-4) 
1.9 (1-5) 
L(CT) 19.2 (10-35) 
40.2 (20-83) 
8.9 (6-14) 
11.4 (6-22) 
______________________________________ 
Table 4. IgG subclass antibodies to cholera toxin at week 4. Anti-cholera 
toxin specific IgG subclass antibodies were measured using a quantitative 
ELISA as described in Glenn et al. (1995) and reported as the geometric 
mean and standard error of the mean. Abbreviations: L is liposome, LA is 
lipid A, and CT is cholera toxin. 
EXAMPLE 3 
Recombinant dengue-2 envelope protein (DEN-2 E, Smucny et al., 1995) may be 
used as an antigen in the transdermal delivery system described above. 
Transdermal application of the liposome-antigen formulation may be used to 
vaccinate against dengue-2 viral infection and assessed in either animal 
or human trials. 
Three-week old BALE/c mice will be immunized three times with a formulation 
containing about 1 .mu.g to 250 .mu.g of DEN-2E antigen, either with or 
without adjuvant using the transdermal delivery system. Boosting 
immunization will performed at three to four week intervals. Antibodies 
against DEN-2 E antigen may be measured by ELISA (Smucny et al., 1995) and 
assayed for cross-reactivity against envelope proteins of serotypes 1, 3 
and 4. The mice will be challenged subsequently with an intracerebral 
injection of 100 times the LD.sub.50 dose of mouse-adapted dengue-2 virus 
and observed for for one month for protection against lethal encephalitis. 
Pooled sera collected three weeks after the third immunization will be 
assayed for viral neutralizing activity using a plaque reduction 
neutralization assay (Russell and Nisalak, 1987). Generally, sera will be 
serially diluted in two-fold steps from 1:5 to 1:160, and distributed in 
24-well plates with 50 p.f.u. of the dengue-2 NGC strain. Suspensions of 
PS cells (porcine kidney cells as described in Summers and Smith, 1987) 
(approximately 8.times.10.sup.4 cells per well) in L15 medium supplemented 
with 3% fetal calf serum will be added to each well and the plates will be 
incubated at 37.degree. C. for 4 hours. An equal volume of 2% 
carboxymethylcellulose suspension in L15 supplemented with 3% fetal calf 
serum will then be added to each well. After six days of incubation at 
37.degree. C., the PS cells will be rinsed with PBS, fixed with formol 
solution in PBS, and then permeabilized with 0.5% Triton X-100 in PBS. 
Focus staining will be performed by successive incubations of cell layers 
at 37.degree. C. for 1 hour each with HMAF (1:100 in PBS) (hyperimmune 
ascites fluid to whole dengue virions) followed by peroxidase-labeled goat 
anti-mouse IgG (BIOSIS, Philadelphia, Pa.). The staining solution (300 
.mu.l of 0.05% 3,3'-diaminobenzidine tetrachloride, 0.3% H.sub.2 O.sub.2, 
0.1M Tris-HCl, pH 7.6) will then be added to each well. The reaction will 
be stopped by adding 200 .mu.l of 1M H2SO4. Stained foci will be observed 
under a microscope and quantitated. Neutralizing serum titers will be 
determined as the serum dilution yielding a 50% reduction in plaque number 
(Delenda et al., 1994). Variations of this technique are described by 
Smucny et al. (1995) and Fonseca et al. (1994). Other dengue envelope 
proteins (Delenda et al., 1994; Fonseca et al., 1994) may be used for 
transdermal immunization and the induction of antigen-specific immune 
response assessed in a similar fashion. 
Multiple dengue envelope proteins from serotypes 1-4 may be used in 
transdermal immunization: simultaneously, in succession, in the same 
liposome, in separate liposomes, with or without adjuvants as described 
previously. Induction of an antigen-specific immune response may be 
assayed as described above for each serotype, and cross-reactivity between 
serotypes may or may not occur. A multivalent vaccine against serotypes 
1-4 using recombinant dengue envelope proteins from serotypes 1-4 may be 
achieved though this means. 
The disclosures of all patents, as well as all other printed documents, 
cited in this specification are incorporated herein by reference in their 
entirety. 
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. 
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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