CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of, and relies on the filing date of, U.S. Provisional Application No. 62/340,924, filed 24 May 2016, the entire disclosure of which is incorporated herein by reference.

FIELD

[002] This application relates generally to immunogenic cochleate formulations and methods of administering the same to treat diseases, in particular, infectious diseases and cancers.

BACKGROUND

[003] Pandemic disease, such as pandemic influenza, presents unique challenges not seen with seasonal disease, particularly the lack of pre-existing immunity in much of the population, which translates to the need for an even higher number of vaccine doses in a short space of time. In a pandemic setting, rapid protection following vaccination is desirable. Due to the special circumstances surrounding pandemics, vaccine manufacturers, regulatory bodies and health authorities approach the development of pandemic vaccines in many ways. One solution is dose-sparing to maximize vaccine availability. However, dose sparing may not always be achievable due to insufficient efficacy of vaccine. Hence, for this reason alone there remains a need for more potent and efficacious vaccines or delivery mechanisms capable of enhancing efficacy, which are non-toxic and do not have adverse side-effects.

[004] Further, influenza, as well as other infectious disease, may be better prevented with vaccines that are administered via a mucosal rather than an intramuscular route. Mucosal administration of vaccines is a valuable approach for the induction of appropriate immune responses to microbial and other environmental antigens in systemic sites and peripheral blood as well as in most external mucosal surfaces. However, to date, only a few vaccines have become available for mucosal use due to the apparent need for vaccine replicating agents. Further, strategies suggested to enhance mucosal immunity without the need for such agents have proven disappointing. For example, Ogra et al, Clin. Microbiol. Rev., 2001, 14:430-445, has suggested the use of DNA vaccines to reduce virulence and enhance antigen load and, thus, enhance mucosal immunity. However, DNA-based vaccines were found to demonstrate low immunogenicity and accordingly are not in widespread use. Consequently, there is a clear and continued need to improve the efficacy of vaccines, including vaccines with reduced virulence and/or vaccines capable of enhancing mucosal immunity.

SUMMARY

[005] The present inventors have surprisingly discovered that antigen or nucleic acid encoding an antigen can be administered to a subject in much lower amounts than needed in conventional vaccines, even in the absence of adjuvant, provided that the antigen is encapsulated in a cochleate. Moreover, these vaccines can induce highly effective immune responses when mucosally administered. Thus, the present vaccine provides a highly effective and non-toxic method for preventing infectious disease without the need for large amounts of antigen or nucleic acid to encode the antigen. These and other benefits of the instant immunogenic cochleates are further described herein.

[006] More particularly, the present disclosure is directed to an immunogenic cochleate including: an antigen or a nucleic acid encoding an antigen in an amount effective to induce an immune response in a subj ect, wherein the antigen or the nucleic acid is encapsulated within the cochleate, and wherein the cochleate includes a lipid component and a multivalent cation component.

[007] In some implementations, a ratio of the lipid component to the antigen ranges from 1 : 1 to 20: 1 .

[008] In some implementations, a ratio of the lipid component to the nucleic acid encoding the antigen ranges from 5: 1 to 200: 1.

[009] The immunogenic cochleate of the present disclosure may also be formulated for mucosal administration, such as oral administration or more typically, intranasal administration. [0010] In some implementations, the nucleic acid encoding the antigen is a double stranded DNA plasmid or an mRNA.

[0011] In some implementations, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. The viral antigen may include or be obtained or derived from, for example, an influenza antigen, an HIV-1 antigen, a Herpes simplex virus 2 antigen or a human papilloma virus antigen. The fungal antigen may include or be obtained or derived from an Aspergillus antigen.

[0012] In some implementations, the antigen is a cancer antigen, such as one or more of RAGE-I, tyrosinase, MAGE-I, MAGE-2, NY-E80- 1 and Melan-A M ART- 1.

[0013] In some implementations, the immunogenic cochleate of the present disclosure further includes a nucleic acid encoding an adjuvant such as a chemokine or a cytokine.

[0014] In some implementations, the nucleic acid encoding the adjuvant comprises

RNA.

[0015] In some implementations, the nucleic acid encoding the adjuvant comprises

DNA.

[0016] In some implementations, the nucleic acid encoding the adjuvant comprises a double stranded DNA plasmid.

[0017] In some implementations, the adjuvant is a cytokine, such as IL-12.

[0018] In some implementations, the immunogenic cochleate is a vaccine, such as a prophylactic vaccine or a therapeutic vaccine.

[0019] The present disclosure is also directed to a method of preventing an infectious disease in a subject including: administering to the subject in need thereof a therapeutically effective amount of a formulation including a cochleate, wherein the cochleate includes an antigen or a nucleic acid encoding an antigen.

[0020] In some implementations, the method includes administering the formulation mucosally, such as orally or intranasally. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the compositions and methods disclosed herein.

[0022] FIG. 1 is a schematic representation of a cochleate. The insert depicts the lipid strata of a cochleate, which contains a phospholipid bilayer (circles and tails), multivalent cation (unshaded circles) and an exemplified cargo moiety (hatched circles) protected within the cochleate.

[0023] FIG. 2 depicts a schematic of a macrophage engulfing a cochleate and its cargo. The insert depicts the opening of the cochleate and release of the cargo inside the macrophage as described in the detailed description.

[0024] FIG. 3 depicts a schematic representation of an active pharmaceutical ingredient enchocleation process as described in the detailed description.

[0025] FIG. 4 depicts an exemplary preparation of geode cochleates as described in the detailed description.

[0026] FIG. 5A depicts a schematic representation of a membrane protein associated with the lipid bilayer of a cochleate. FIG. 5B depicts the expected location of a nucleic acid within a cochleate due to its negative charge.

[0027] FIGS. 6A and 6B depict scanning electron micrographs of cochleates containing an influenza protein and an empty cochleate, respectively.

[0028] FIG. 7 depicts the serum titers of different isotypes of influenza virus antibodies following oral or intramuscular administration of cochleate vaccines as described in Example 2.

[0029] FIG. 8 depicts protection of Balb/C mice from influenza virus challenge in lung and trachea after oral immunization as described in Example 3.

[0030] FIG. 9 depicts serum antibody titers from cochleate and commercial virus after administration in Balb/C mice as described in Example 4.

[0031] FIG. 10 depicts neutralizing antibody titers induced by commercial vaccines and vaccine cochleates of the present disclosure as described in Example 4. [0032] FIG. 11 describes enhancement of the immune response with adjuvant in vaccine cochleate formulations as described in Example 4.

[0033] FIG. 12 depicts mucosal antibody induction of influenza virus-specific IgA titer in saliva after oral administration as described in Example 5.

[0034] FIG. 13 depicts the induction of influenza virus-specific splenocyte proliferation after oral immunization as described in Example 6.

[0035] FIG. 14 depicts a schematic of the plasmid, pCMVHIV-1, described in Example 7.

[0036] FIG. 15 depicts the T cell proliferation following oral administration of encochleated Human Immunodeficiency Virus 1 (HIV-1) plasmids as described in Example 7.

[0037] FIG. 16 depicts the cytotoxic lymphocyte (CTL) response following oral administration of encochleated Human Immunodeficiency Virus 1 (HIV-1) plasmids as described in Example 7.

[0038] FIG. 17 depicts the cytotoxic lymphocyte (CTL) response following oral administration of encochleated Herpes Simplex Virus 2 (HSV-2) and interleukin-12 plasmids as described in Example 8.

[0039] FIG. 18 depicts an endonuclease digest of the pCMVHIV-lenv DNA plasmid DNA as described in Example 9.

DETAILED DESCRIPTION

[0040] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the invention and should not be interpreted as limiting the scope of the invention.

[0041] The present disclosure is directed to cochleates comprising immunogens, such as immunogenic cochleates, as described herein, which optionally contain adjuvants. As used herein, the term "immunogen" or "antigen" refers to any substrate that elicits an immune response in a subject, e.g., a humoral and/or a cellular immune response to the antigenic molecule of interest. Typically the antigen is a polypeptide or a glycoprotein. The immunogenic cochleate can also contain a nucleic acid encoding the antigen of interest.

[0042] In some implementations, the present cochleates enhance or induce a Thi type response. In this context, an immune response may occur in various ways. A substantial factor for a suitable (adaptive) immune response is the stimulation of different T-cell sub-populations. T-lymphocytes are typically divided into two sub-populations, the T-helper 1 (Thl) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Thl) and extracellular (Th2) pathogens (e.g. antigens). The two Th cell populations differ in the pattern of the effector proteins (cytokines), which are produced. Thus, Thl cells assist the cellular immune response by activation of macrophages and cytotoxic T-cells. Th2 cells, on the other hand, promote the humoral immune response by stimulating B-cells for conversion into plasma cells and by inducing formation of antibodies. The Thl/Th2 ratio therefore can play an important role in the induction and maintenance of an adaptive immune response. In connection with the present disclosure, the Thl/Th2 ratio of the (adaptive) immune response is preferably shifted in the direction towards a Thl response, to promote the induction of a cellular immune response.

[0043] In some implementations, the present cochleates activate macrophages and induce lFN-γ and IL-2, both known to be associated with the induction of Till type cell- mediated immune responses. In other implementations, the present cochleates facilitate circulating IgG2a or IgGl production, also indicating enhancement of a predominant Thl type response. Thus, an immunological response as used herein may be one that stimulates the production of cytotoxic T lymphocytes (CTLs), and/or the production or activation of helper T-cells. The antigen(s) as described herein may also elicit an antibody-mediated immune response. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of T-cells directed specifically to an antigen or antigens present in the immunogenic cochleate or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art. [0044] In some embodiments, the present cochleates may be formulated for mucosal (e.g., oral or intranasal administration) administration or may be administered mucosally. Such mucosal administration can induce an immune response, such as a secretory IgA antibody response at mucosal surfaces. Secretion of IgA antibodies is an indicator of the mucosal defense system. Secretory IgA can protect against pathogens which replicate on or enter via mucosal surfaces, by binding to infectious organisms and blocking their attachment to or invasion through mucosal surfaces.

[0045] The cochleates disclosed herein may or may not be immunoprotective (prophylactic) or therapeutic, however. Accordingly, the term "immunogenic" is not intended to be limited to vaccines.

[0046] In some implementations, the immunogenic composition of the present disclosure is a vaccine. As used herein, a vaccine encompasses an immunogenic composition that prevents, ameliorates, palliates, or eliminates disease from a host, such as the diseases described herein. In some implementation, the present vaccines are prophylactic vaccines. In other implementations, the present vaccines are therapeutic vaccines.

[0047] The immunogens of the present immunogenic compositions, as described herein below, are encapsulated into cochleates. Suitable cochleates and methods of making cochleates for use as immunogenic cochleates or vaccine cochleates of the present disclosure immediately follow.

Cochleates and Methods of Making the Same

[0048] Cochleates are anhydrous, stable, multi-layered lipid crystals which spontaneously form upon the interaction of negatively charged lipids, such as phosphatidylserine, and divalent cations, such as, calcium (see, for example, U.S. Pat. Nos. 4,078,052; 5,643,574; 5,840,707; 5,994,318; 6,153,217; 6,592,894, as well as PCT Publ. Nos. WO 2004/091572; WO 2004/091578; WO 2005/110361, WO 2012/151517, and WO2014/022414, and U.S. Pat. Publ. 2010/0178325; each of which is incorporated fully herein by this reference). Typically, these are referred to as crystal cochleates. [0049] Crystal cochleates have a unique multilayered structure consisting of a large, continuous, solid, phospholipid bilayer sheet or strata rolled up in a spiral or as stacked sheets, with no internal aqueous space (FIG. 1). This unique structure provides protection from degradation for associated "encochleated" molecules. Since the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact, even though the outer layers of the cochleate may be exposed to harsh environmental conditions or enzymes. Divalent cation concentrations in vivo in serum and mucosal secretions are such that the cochleate structure is maintained. Hence, the majority of cochleate-associated molecules are present in the inner layers of a solid, stable, impermeable structure. Once within the interior of a cell, however, the low calcium concentration results in the opening of the cochleate crystal and release of the molecule that had been formulated into cochleates (FIG. 2). Accordingly, cochleate formulations remain intact in physiological fluids, including mucosal secretions, plasma and gastrointestinal fluid, thereby mediating the delivery of biologically active compounds by many routes of administration, including intramuscular and mucosal, e.g., intranasal and oral.

[0050] Typical cochleate structures include a lipid strata comprising alternating divalent cations and phospholipid bilayers that include at least one negatively charged phospholipid. Typically, a cargo moiety, such as an immunogen and/or adjuvant as described herein, is sequestered within the lipid strata of the cochleate.

[0051] Cochleates can be made any known method. In one implementation the LC (Liposomes before Calcium) Dialysis Method is used, as described in U.S. Patent No. 5,994,318, which is herein incorporated by reference in its entirety. A summary of this process is shown in FIG. 3. In some implementations, this method may be used to formulate the cochleates in the immunogenic composition of the disclosure, such as a vaccine composition, when the immunogen of interest contains membrane proteins with intact transmembrane regions or peptides with hydrophobic tails. This method for encochleation involves the removal of detergent from a solution of lipid and material to be encochleated, followed by the addition of a multivalent cation. Briefly, material to be encochleated is added to (or purified in) a solution containing a detergent in a high-salt buffer. Typically, a non-ionic detergent, such as nOctyl β-D glucopyranoside (OCG), is used to promote reconstitution of membrane proteins into cochleates. The non-ionic detergent, in some implementations, may maintain the native conformation of the proteins and may be readily removed by dialysis. In some implementations, OCG concentrations of 2% (20 mg/ml), at a detergent to lipid weight ratio of 6: 1 , for example, may be employed. High salt concentrations, (e.g. 2M NaCl), may be also be used to help avoid protein aggregation and to promote high quality recovery into cochleates or to isolate or solubilize recombinant or purified viral or bacterial membrane proteins. In some implementations, physiologic salt concentrations may be used for soluble proteins and/or nucleic acids. In some implementations, the lipids, which are used to make the liposomes that form the cochleates are in a solvent, such as chloroform, and then dried to a thin film. In other implementations, the lipids are in powdered form. Typically, buffer containing the material to be encochleated is added and the lipid is suspended by agitation. The detergent may be removed by dialysis against buffer, resulting in the formation of small lipid vesicles. Other methods of detergent removal may be used (e.g. ultrafiltration). Multivalent cation is added by dialysis. Alternatively, liposomes may be removed from the bag or ultrafiltration unit, and multivalent cation may be added directly in small aliquots, or continuously by injection using a pump. The addition of multivalent cation results in the collapse of the liposomes, and the formation of the sheets of cation-chelated phospholipid bilayers, which roll up or stack to form cochleates containing the material of interest as described above.

[0052] In another embodiment, the cochleates of the present disclosure are formed using the DC (Direct Calcium) Dialysis method. In this method, detergent is removed from a solution of lipid and material to be encochleated by dialysis against a buffer containing multivalent cation. The removal of detergent and addition of multivalent cation therefore take place simultaneously, rather than sequentially as in the LC method. In some implementations, this method is used in the formulation of immunogenic compositions of the disclosure, such as vaccines, containing nucleic acids, such as DNA plasmids. The DC method is also described in U.S. Patent No. 5,994,318, which is herein incorporated in its entirety.

[0053] In other implementations, trapping methods are used for encochleation. Trapping involves the addition of multivalent cation to a suspension of material to be encochleated, and liposomes comprised mainly of negatively charged lipids. The addition of multivalent cation ions induces the collapse and fusion of the liposomes into large lipid bilayer sheets, which spontaneously roll up or stack into cochleates. Materials will be "trapped" in these structures between the sheets, or within or partially within, the lipid bilayers, depending on their characteristics of size, shape, charge, and hydrophobicity. In some implementations, trapping methods may be used to encochleate, DNA plasmids, oligonucleotides, RNA, such as siRNA and soluble proteins. Trapping methods are described in U.S. Publication No. 2014/186430, which is herein incorporated by reference in its entirety.

[0054] As recognized by an ordinary artisan, many parameters, including pH, salt concentration, agitation method and rate, cation type, concentration, and rate of addition, lipid composition, concentration, and ratio of lipid to other material, etc., affect the formulation, and can be varied in order to optimize the encochleation of a particular material.

[0055] In a typical implementation, the multivalent cation is a divalent metal cation, such as calcium, zinc, magnesium, and barium. In a more typical implementation, the divalent metal cation is calcium.

[0056] In some implementations, the ratio of protein, including membrane protein or peptide, to lipid ratios (wt/wt) ranges from 1:1 to 1:50, or any range in between, such as, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:15, 1:20 and 1:25, typically 1:4 to 1:12, such as 1:5 to 1:7. In some implementations, the ratio of nucleic acid, including DNA plasmids, oligonucleotides, RNA, including single stranded RNA, double stranded RNA and siRNA, ranges from 1:5 to 1:1000, or any range in between, such as, 1:10, 1:15, 1:20, 1:25, 1:50, 1:75, 1:100. 1:125, 1: 150, 1:200.1:300.1:400.1:500, 1:600, 1:700, 1:800 and 1:900, typically, l:100to 1:700, such as 1:200 to 1:500 or 1:250 to 1:300.

[0057] The liposome used during the formation of the cochleates may be multilamellar (MLV) or unilamellar (ULV), including small unilamellar vesicles (SUV). The concentration of lipid in these liposomal solutions can be from about 0.1 mg/ml to 500 mg/ml. Typically, the concentration of lipid is from about 0.5 mg/ml to about 50 mg/ml, more typically from about 1 mg/ml to about 25 mg/ml.

[0058] A size-regulating agent may be introduced during the method of making the cochleate. A size-regulating agent, as used herein, refers to an agent that reduces the particle size of a cochleate. As used herein, the term "particle size" refers to the particle diameter, or in case the particles are not spherical, to the largest extension in one direction of the particle. The particle size of cochleates can be measured using conventional methods, such as a submicron particle size analyzer. In certain embodiments, the size regulating agent is a lipid- anchored polynucleotide, a lipid-anchored sugar (glycolipid), or a lipid-anchored polypeptide. In other embodiments, the size regulating agent is a bile salt, such as oxycholate, cholate, chenodeoxycholate, taurocholate, glycocholate, taurochenodeoxycholate, glycochenodeoxy cholate, deoxycholate, or lithocholate. Bile salts are bile acids compounded with a cation, usually sodium. Bile acids are steroid acids found predominantly in the bile of mammals and are commercially available.

[0059] In certain embodiments, the size-regulating agent is added to the lipid or liposomes before formation of the precipitated cochleate. For example, in one embodiment, the size-regulating agent is introduced into a liposomal suspension from which cochleates will subsequently be formed (e.g., by addition of cation or dialysis). Alternatively, the size- regulating agent may be introduced to a lipid solution, before or after addition of a pharmacologically active agent.

[0060] Any suitable lipid can be used to make the cochleate. In one embodiment, the lipid includes one or more negatively charged lipids. As used herein, the term "negatively charged lipid" includes lipids having a head group bearing a formal negative charge in aqueous solution at an acidic, basic or physiological pH, and also includes lipids having a zwitterionic head group. In one embodiment, the negatively charged lipid is a phospholipid.

[0061] The cochleates can also include non-negatively charged lipids (e.g., positive and/or neutral lipids). Typically, the cochleates include a significant amount of negatively charged lipids. In certain embodiments, a majority of the lipid is negatively charged. In one embodiment, the lipid is a mixture of lipids, comprising at least 50% negatively charged lipid, such as a phospholipid. In another embodiment, the lipid includes at least 75% negatively charged lipid, such as a phospholipid. In other embodiments, the lipid includes at least 85%, 90%, 95% or 98% negatively charged lipid, such as a phospholipid. In yet other embodiments, the negatively charged lipid (e.g., phospholipid) comprises between 30%-70%, 35%-70%, 40%-70%, 45%-65%, 45%-70%, 40%-60%, 50%-60%, 45%-55%, 45%-65%, or 45%-50% of the total lipid in the cochleate. In certain embodiments, the negatively charged lipid (e.g., phospholipid) comprises between 40%-60% or 45%-55% of the total lipid in the cochleate. In some embodiments, the negatively charged lipid (e.g., phospholipid) comprises between 30%- 70%, 35%-70%, 40%-70%, 45%-65%, 45%-70%, 40%-60%, 50%-60%, 45%-55%, 45%-65%, or 45%-50% of the total lipid in the non-hydrophobic domain component of the cochleate. In certain embodiments, the negatively charged lipid (e.g., phospholipid) comprises between 40%-60% or 45%-55% of the total lipid in the non-hydrophobic domain component of the cochleate. In some embodiments, the negatively charged lipid is a phospholipid and comprises between 30%-70%, 35%-70%, 40%-70%, 45%-65%, 45%-70%, 40%-60%, 50%-60%, 45%- 55%, 45%-65%, or 45%-50% of the total phospholipid in the cochleate or in the non- hydrophobic domain component of the cochleate. In some embodiments, the negatively charged lipid is a phospholipid and comprises between 40%-60% or 45%-55% of the total phospholipid in the cochleate or in the non-hydrophobic domain component of the cochleate.

[0062] The negatively charged lipid can include egg-based lipids, bovine-based lipids, porcine-based lipids, plant-based lipids such as soy -based lipids, or similar lipids derived from other sources, including synthetically produced lipids. The negatively charged lipid can include phosphatidylserine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA), phosphatidylinositol (PI), and/or phosphatidyl glycerol (PG) and or a mixture of one or more of these lipids with other lipids. Additionally or alternatively, the lipid can include phosphatidylcholine (PC), phosphatidylethanolamine (PE), diphosphotidylglycerol (DPG), dioleoyl phosphatidic acid (DOPA), distearoyl phosphatidylserine (DSPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylglycerol (DPPG) and the like. In another embodiment, the phosphatidylserine is egg or bovine derived phosphatidylserine.

Cochleates containing soy lipids

[0063] In some typical embodiments, the cochleates, including the geode cochleates, described herein below, are prepared using legume-based phospholipids, more typically soy- based lipids. Such soy-based lipids can be natural or synthetic. Even more typically, the soy- based lipids are soy phospholipids, such as soy phosphatidylserine is in an amount of 40%- 74% by weight of the lipid component of the cochleates. Alternatively, the soy phosphatidylserine can be about 40%, 45%, 50%, 55%, 60%, 65% or 70% or any incremental value thereof, by weight of the lipid component of the cochleates. It is to be understood that all values, and ranges between these values and ranges are meant to be encompassed by the present disclosure. In a typical embodiment, the phospholipid comprises 45-70% soy phosphatidylserine. In a more typical embodiment, the phospholipid comprises 45-55% soy phosphatidylserine.

[0064] Soy phosphatidylserine is commercially available, e.g., from Avanti Polar Lipids, Inc. Alabaster, AL. Alternatively, soy phosphtidylserine can be purified from soy phospholipid compositions, which are mixtures of several soy phospholipids, according to well-known and standard purification techniques.

[0065] In some embodiments, neutral lipids are used in combination with the soy phosphatidylserine to make the instant cochleates. As used herein, the term "neutral lipids" include any of a number of lipid species, which exist either in an uncharged or neutral zwitterionic form at physiological pH and, thus, are included within the group of lipids lacking an anionic function. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the cochleate compositions described herein is generally guided by consideration of, e.g., cochleate size and stability. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In one group of embodiments, lipids containing saturated fatty acids with carbon chain lengths in the range of Ci4 to C22 can be used. In another group of embodiments, lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of Ci4 to C22 can be used. In yet another group of embodiments, lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of Cs to C12 can be used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used.

[0066] In some embodiments, the neutral lipids used in the present disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. The neutral lipids useful in the present disclosure may also be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.

[0067] In a typical implementation, 99.9% pure dioleoyl phosphatidylserine, 99.9% pure soy phosphatidylserine, 75% soy phosphatidylserine and 50% soy phosphatidylserine, are used to manufacture cochleates. The lipid composition of 99.9% pure phosphatidylserine is typically modified by the addition of neutral lipids, including, but not limited to sphingomyelin and/or phosphatidylcholine. When lower purity phosphatidylserine (e.g., 50% soy phosphatidylserine) is used as a starting material, the lower purity phosphatidylserine can be subjected to extraction steps to remove unwanted impurities, such as, nucleases.

Geode cochleates

[0068] In some implementations, the cochleate of the present disclosure is a geode cochleate, or a geodate, as described, for example, in U.S. Patent Publication 2013/0224284, the entire disclosure of which is incorporated herein by reference. Geode cochleates further comprise a lipid monolayer comprising a negatively charged phospholipid, where the lipid monolayer surrounds a hydrophobic domain, such as an oil, and a cargo moiety, such as an immunogen and/or adjuvant and/or bioactive agent as described herein, which is dispersed within the hydrophobic domain. Typically, the hydrophobic domain contains an immunogen, which is a protein or peptide antigen, or a polynucleotide with or without an association to stabilizing proteins, lipids, such as cholesterol, fatty acids, or a positively charged amphiphile such as tocopherol (vitamin E) or other positively charged amphiphiles as described herein. The lipid monolayer is sequestered within the lipid strata of the geode cochleate.

[0069] As used herein, a "hydrophobic domain" is a composition that is sufficiently hydrophobic in nature to allow formation of a lipid monolayer about its periphery. A hydrophobic domain typically includes a hydrophobic composition, such as oil or fat, associated with a cargo moiety, such as an immunogen and/or an adjuvant of the disclosure. In certain embodiments, the ratio between the hydrophobic domain (HD) and the phospholipid component of the geode cochleate (PPLGD) HD:PPLGD or the castor oil domain (COD) and phospholipid component of the geode cochleate (PPLGD) COD:PPLGD is 1 :20 or less, 1 : 15 or less, 1 : 10 or less, 1 :8 or less, 1 :6 or less, 1 :5 or less, 1 :4 or less, 1 :3.5 or less, 1 :3 or less, 1 :2.75 or less, 1 :2.5 or less, 1 :2.25 or less, 1 :2 or less, 1 : 1.75 or less, 1 : 1.5 or less, 1 : 1.25 or less 1 : 1 or less.

[0070] FIG. 4 shows an exemplary schematic of how geode cochleates can be made. In this exemplary method, a phospholipid (represented as an open ring) is combined with a hydrophobic domain (shaded circles), such as an oil, and mixed to form a stable emulsion comprising liposomes and lipid monolayers surrounding the hydrophobic domain. A cargo moiety, such as a protein or peptide, etc., may be dispersed within the hydrophobic domain. The hydrophobic domains have phospholipids imbedded in their surface. Without intending to be bound by any theory, it is believed that the hydrophobic acyl chains of the phospholipid are within the hydrophobic domains, resulting in the hydrophobic domains having a hydrophilic surface due to the coating of the phospholipid head groups and forming a stable emulsion. If the phospholipid is negatively charged, such as with phosphatidylserine, the addition of a divalent cation, such as calcium, induces the formation of a crystalline structure (or lipid strata) comprising alternating divalent cations and phospholipid bilayers. The lipid strata are represented with hatching. In a geode cochleate, the lipid monolayers surrounding the hydrophobic domain are "encrusted" or "entrapped" within the crystalline matrix, akin to a "geode."

Antigens

[0071] The antigen of the present disclosure may be a protein or peptide of bacterial, fungal, protozoan, or viral origin, or a fragment derived from these antigens, a carbohydrate, or a carbohydrate mimetic peptide. Exemplary antigens include viral lipids and glycoproteins, bacterial membrane proteins, and fungal proteins. The antigen may also be expressed by a tumor. Additionally, the antigenic molecule(s) may also include one or more nucleic acids including those in a plasmid encoding for at least one antigen. [0072] For example, in some implementations, the nucleic acid encoding an antigen of the disclosure is in a DNA plasmid, such as an expression vector. The expression vector typically includes a eukaryotic promoter operably linked to a gene encoding the antigen, a cloning site, a poiyadenyiation sequence, a selectable marker and a bacterial origin of replication, in other embodiments, the nucleic acid encoding the antigen is an mRNA. Generally, the antigen is from an infectious pathogen or a cancer antigen as described herein. In some implementations, as also described herein, an adjuvant may be encoded in the same or a different expression vector as that encoding an antigen of interest.

[0073] In some embodiments, the nucleic acid encoding the antigen has the functionality of DNA or mRNA but has synthetic modifications to the ribose- or deoxyribose- backbone of the nucleic acid in order to stabilize these molecules versus the effect of nucleases or to maximize their transcribing or translating properties. Such modifications are well-known to the ordinary artisan.

[0074] In some embodiments, the nucleic acid encoding the antigen is associated with a positively charged amphiphile. Without wishing to be bound by any particular theory, it is believed that enhanced binding of the nucleic acid and the cochleates may be achieved by first forming an association between the nucleic acid and a positively charged amphiphile. The transfection potential of DNA, for example, complexed with certain positively charged amphiphiles has been described, e.g., in U.S. Pat. No. 6,797,281, the contents of which are incorporated herein in its entirety by this reference. However, in general, increased transfection rates have been coupled with increased toxicity. Bogden et al, AACS PharmSci 4(2) (2002).

[0075] The addition of a positively charged amphiphile to a nucleic acid within a cochleate, however, may be advantageous, e.g., in delivery of the nucleic acid. For example, it is believed that encochleated nucleic acids, which are associated with positively charged amphiphiles may improve transfection into cells without the associated toxicity generally observed. Accordingly, in some embodiments, cochleates including nucleic acids associated with positively charged amphiphile have significantly no toxicity or undetectable toxicity in vivo and/or in vitro. Moreover, associating the nucleic acid to a positively charged amphiphile may also be advantageous for facilitating the transfer of the nucleic acid across membranes subsequent to administration. [0076] Examples of suitable positively charged amphiphiles include polycations, e.g., polyethylenimine (PEI), polyvinylamine, spermine, spermidine, histamine or cationic lipid. In some embodiments, the positively charged amphiphile enhances binding of the nucleic acid to the liposome prior to precipitation. Alternatively, the positively charged amphiphile is mixed with and binds to the liposome first and then the nucleic acid is added.

[0077] Further examples of positively charged amphiphiles according to the invention include, but are not limited to: N-[ -(2,3-dioleyloxy)propy[]-N,N,N-trimethylamrnonium chloride (DOTMA), ester of L-carnitine bromide with 2-hydroxyacetyl-I ,3-dipalmitoyl glycerol, ester of acetyl L-caraitine bromide with 2-hydroxyacetyl-l ,3-dipalmitoyl glycerol, ester of propionyl L-carnitine bromide with 2-hydroxyacetyl-l, 3-dipairnitoyl glycerol, ester of isobutyryl L-carnitine bromide with 2-hydroxyacetyl-l,3-dipalraitoyI glycerol, ester of isovaler l L-carnitine bromide with 2-h droxy acetyl- 1 ,3-dipalmitoyl glycerol, ester of L- caraitine bromide with 1 ,3-dihexanoyl-2-hy droxycetyl glycerol, ester of acetyl L-carnitine bromide with l,3-dihexanoyl-2-hydroxy acetyl glycerol, ester of propionyl L-carnitine bromide with l ,3-dihexanoyl-2-hydroxy acetyl glycerol, isovaleryl L-carnitine undecyl ester, isobutyryl L-carnitine undecyl ester, palniitoyl L-carnitine chloride undecyl ester, stearoyl L-caraitine chloride undecyl ester, stearoyl L-carnitine chloride tetradecyl ester, palniitoyl L-carnitine chloride tetradecyl ester, miristoyl L-carnitine chloride tetradecyl ester, palniitoyl L-carnitine bromide nexadecyl ester, and oleyl L-carnitine chloride oleyl ester. In some embodiments, the positively charged amphiphile is an L-carnitine derivative. In other embodiments, the positively charged amphiphile is a compound described in U.S. Pat. No. 6,797,281.

[0078] In some embodiments, the positively charged amphiphile does not include protamine, polyethylenimine (PEI), Lipofectamine, polyvinyl amine, spermine, spermidine, histamine, vitamin E or cationic lipid.

[0079] in some particular examples, the immunogen is an antigen derived from a pathogen involved in a sexually transmitted disease, such as Hepatitis (e.g., Hepatitis A, Hepatitis B, or Hepatitis E), Herpes simplex virus (1, 2), HIV/ AIDS, human papilloma virus, cytomegalovirus, Epstein-Barr virus, SARS, or Kaposi's sarcoma-associated herpes virus.

[0080] In some implementations, the antigen is from a pathogenic bacteria e.g., one or more of (or any combination of) Actinobacillus sp., Bacillus sp. (such as Bacillus anthracis, Bacillus cereus. Bacillus subtilis, Bacillus thuringiensis), Bordetetta sp. (such as Bordetella pertussis), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coif), Clostridium sp. (such as Clostridium perfringens, Clostridium, difficile, Clostridium botulinum and Clostridium tetarii), Escherichia cob, Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori) Mycoplasm sp. (such as Mycoplasma pneumoniae). Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnet). Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus). Streptococcus sp. (such as Streptococcus pneumoniae) among others.

[0081] In some embodiments, the antigen is a fungus that causes disease. Examples of fungal pathogens include Trichophyton rubrum, Epidermophyton floccosum, Microsporum cams, Pityrosporum orbicular e, Candida sp. (such as Candida albicans), Aspergillus sp. (such as Aspergillus fumigatus, Aspergillus flavus mil Aspergillus clavatus), Cryptococcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii and Cryptococcus albidus), Histoplasma sp. (such as Hisioplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirov ecu), and Stachybotrys (such as Stachybotrys chartarum).

[0082] In some implementations, the antigen is a retrovirus antigen. "Retroviruses" are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a D A intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term "lenti virus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the "immunodeficiency viruses" which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FiV). HIV-I is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as AIDS. "HIV disease" refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T cells.

[0083] Additional examples of viral pathogens that may be used as antigens or from which antigens may be derived and included in the instant immunogenic cochleates disclosed herein include Coronaviruses (such as SARS), Flaviruses (such as Yellow fever virus, West Nile vims, Hepatitis C virus, and Dengue fever virus), Filoviruses (such as Ebola vims and Marburg virus), Flexiviruses, Hepeviruses (such as Hepatitis E virus), human adenoviruses (such as human adenovirus A-F), human enteroviruses (such as human enterovirus A-D), human herpesviruses (such as human herpesvirus 1 (herpes simplex virus type 1), human herpesvirus 2 (herpes simplex virus type 2), human herpesvirus 3 (Varicella zoster virus), human herpesvirus 4 type 1 (Epstein-Barr virus type 1), human herpes vims 4 type 2 (Epstein- BaiT vims type 2), human herpesvirus 5 strain AD 169, human herpesvirus 5 strain Merlin Strain, human herpesvirus 6 A, human herpesvirus 6B, human herpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type P and Human Cyotomegalovims), human papillomaviruses human parvovirus 4, human respiratory syncytial viruses, human rhinoviruses, Picoraaviruses, Poxviruses (such as Variola and Cowpox), Sequiviruses, Reoviruses (such as Rotavirus), human parechoviruses, human parvoviruses (such as human parvovirus 4 and human parvovirus B 19), human respiratory syncytial viruses, human rhinoviruses (such as human rhinovirus A and human rhino virus B), human spumaretroviruses, human T-lymphotropic viruses (such as human T-lymphotropic virus 1 and human T- lymphotropic vims 2), Human polyoma viruses, Hypoviruses, Leviviruses, Luteovimses, Lymphocytic choriomeningitis viruses (LCM), Marnaviruses, Nama viruses, Nidovirales, Noda viruses, Parti ti viruses. Paramyxoviruses (such as Measles virus and Mumps virus), Picornaviruses (such as Poliovirus, the common cold virus).Rliabdoviruses (such as Rabies virus) influenza viruses (such as influenza A virus including seroty pes such as H1N1, H2N2, h3N2, H5NI , H7N7,HiN2, H9N2, FI7N2, H7N3, H10N7, H7N9, virus influenza B, C and D virus) and human parainfluenza viruses (such as human parainfluenza virus 1-3).

[0084] in some implementations, the antigen is a tumor antigen. A tumor antigen is an antigen produced by tumor cells that can stimulate tumor-specific T-cell immune responses. Exemplary tumor antigens include, but are not limited to, RAGE-I, tyrosinase, MAGE-I, MAGE-2, NY-ESO- 1 , Mel an -A/MART- 1 , glycoprotein (gp) 75, beta-catenin, preferentially expressed antigen of melanoma (PRAME), MUM-L Wilms tumor ( T)-i carcinoembryonic antigen (CEA), and PR-I.

[0085] in some examples, the immunogenic cochleates of the instant disclosure includes a single antigen directed to a particular disease or a single nucleic acid encoding for a single antigen. In other examples, the immunogenic cochleate includes antigen(s) directed to two or more diseases simultaneously ("mixed antigen"). The mixed antigen may be a mixture of two or more antigens, or an antigen that has antigenicities for two or more diseases simultaneously, e.g., a. recombinant protein. See for example FIGS. 5 A and 5B which depict multiple membrane proteins and multiple DNA plasmids, respectively in strata of cochleates.

Adjuvants

[0086] In some implementation, the immunogenic cochleates of the instant disclosure include an adjuvant. As used herein, an adjuvant is a vehicle used to enhance antigenicity; such as a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (MF- 59, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or to cause an influx of macrophages). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and for example, immunostimulatory DNA or RNA molecules.

[0087] In some examples, the present immunogenic cochleates may be combined into a formulation with one or more of the following adjuvants for administration into a host including chitosan, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4)); AVRIDINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylaminob- D- glucopyranosyl)-N-octadecyl-dodecanoyl- amide hydroacetate); CALCITRIOL™ (l-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-Al -protein- A-D-fragment fusion protein, subunit B of the cholera toxin; CRL 1005 (block copolymer PI 205); DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandroster- one); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i) N-acetylglucosaminyl- (PI -4)-N-acetylmuramyl-L-alanyl-D35 glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N- acetylglucosaminyl-(bl -4)-N-acetylmuramyl-L47 alanyl- D-isoglutamine); imiquimod (1 -(2- methypropyl)-l H-imidazo[4,5-c]quinoline-4-amine); IMMTHER™ (N-acetylglucosaminyl-N- acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes pre- pared from dehydration-rehydration vesicles); interferongamma; interleukin-1 beta; inter- leukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3. LOXORIBINE™ (7-allyl-8-oxoguanosine); LT 5 oral adjuvant (Eco/i labile enterotoxin- protoxin); MF59™; (squalenewater emulsion); MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720TM (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4'-monophosphoryl lipid A); RAMETIDE™ (Nac-Mur-L- Ala-D-Gln-OCH3); MURAPALMITINETM and DMURA-PALMITINE™ (Nac-Mur-L-Thr- D-isoGln-sn-glyceroldipalmitoyl); NAGO (neuraminidase- galactose oxidase); PLEURAN™ ( -glucan); PLURONIC L121™; PMMA (polymethylmethacrylate); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-l H- imidazo[4,5-c]quinoline-l -ethanol); SAF-1™ ("Syntex adjuvant formulation"); Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or ROBANE® (2,6,10,15, 19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl- 2,6,10,14,1 8,22-tetracosahexane); stearyltyrosine (octadecyltyrosine hydrochloride); THERAMID® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L- Aladipalmitoxypropylamide); Theronyl-MDP, plant derived adjuvants, including QS21, Quit A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including Tomatine, biopolymers, including PLG, PMM, Inulin, microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1 -10, ligands of murine TLR 1 -13, ISS-1018, 35 IC31 , Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat- labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC- 1 VI 50, and RSV fusion protein.

[0088] The immunogenic cochleates can additionally contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory capacity, if desired. In general, it is possible to use as auxiliary substance any agent that influences the immune system in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow an immune response to be enhanced and/or influenced in a targeted manner. Particularly desirable auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, such as one or more of IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-1 6, IL-1 7, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL.24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IFN-a, IFN-β, IFN-γ, GM-CSF, G-CSF, M-CSF, LT-β or TNF-a, growth factors, such as hGH.

[0089] In some implementations, the cytokines, interleukins or chemokines are encoded by a nucleic acid and incorporated into the expression vector of the antigen. In other implementations, the adjuvant is encoded by a nucleic acid in a separate expression vector.

[0090] In some embodiment, the cochleates do not contain antigen nor nucleic acids encoding antigen, but only contain one or more adjuvants, such as disclosed herein. In these embodiments, the antigen or nucleic acid encoding the antigen may be administered "naked", i.e. , without being encochleated. As shown in the examples, such cochleates may shift the Thl/Th2 ratio in the direction of the Th i response.

Pharmaceutical Compositions

[0091] The cochleates described herein can be prepared as a pharmaceutical composition. Suitable preparation forms for the pharmaceutical compositions disclosed herein include, for example, tablets, capsules, soft capsules, granules, powders, suspensions, emulsions, microemulsions, nanoemulsions, unit dosage forms, rings, films, suppositories, solutions, creams, syrups, transdermal patches, ointments and gels. [0092] The pharmaceutical compositions can include other pharmaceutically acceptable excipients, such as, a buffer (e.g., Tris-HCl, acetate, phosphate) of various pH and ionic strength; an additive such as albumin or gelatin to prevent absorption to surfaces; a protease inhibitor; a permeation enhancer; a solubilizing agent (e.g., glycerol, polyethylene glycerol); an anti-oxidant (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxy anisole); a stabilizer (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose); a viscosity increasing agent (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum); a sweetener (e.g. aspartame, citric acid); a preservative (e.g., Thimerosal, benzyl alcohol, parabens); a flow-aid (e.g., colloidal silicon dioxide), a plasticizer (e.g., diethyl phthalate, triethyl citrate); an emulsifier (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate); a polymer coating (e.g., poloxamers or poloxamines, hypromellose acetate succinate); a coating and film forming agent (e.g., ethyl cellulose, acrylates, polymethacrylates, hypromellose acetate succinate); a pharmaceutically acceptable carrier for liquid formulations, such as an aqueous (water, alcoholic/aqueous solution, emulsion or suspension, including saline and buffered media) or non-aqueous (e.g., propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate) solution, suspension, emulsion or oil; and a parenteral vehicle (for subcutaneous, intravenous, intraarterial, or intramuscular injection), including but not limited to, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.

[0093] In certain embodiments, the pharmaceutical composition comprises a salt, such as NaCl or a bile salt, such as oxycholate, cholate, chenodeoxycholate, taurocholate, glycocholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, or lithocholate. Bile salts are bile acids compounded with a cation, usually sodium. Bile acids are steroid acids found predominantly in the bile of mammals and are commercially available. In one embodiment, the bile salts comprise cholate. In another embodiment, the bile salts comprises deoxycholate. In yet another embodiment, the bile salts comprise cholate and deoxycholate. In another embodiment, the bile salts consist of cholate and deoxycholate.

[0094] In certain embodiments, the concentration of NaCl is 1 mM to 1M, ImM to 0.5M, ImM to O. lM, lmM to 50mM, l OmM to l OOmM, l OmM to 50 mM, O. lM to 1M, 0.1M to 0.5M, or 0.5M to 1M. In certain embodiments, the concentration of the bile salts is ImM to lOOmM, lmM to 50 mM, lmM to 25mM, 1 niM to lOmM, lmM to 5mM, O. lmM to 5mM, 0. lmM to lmM, or 0. lmM to 0.5mM bile salts.

[0095] These excipients are provided by way of example and it will be known to those of skill in the art that there will be other or different excipients that can provide the same chemical features as those listed herein.

Dosage and Administration

[0096] A pharmaceutical composition comprising a cochleate, as disclosed herein, is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. This includes, for example, injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical.

[0097] In certain embodiments, the immunogenic cochleate is administered mucosally. Typically, the mucosal administration is oral (including, for example, by administering a suspension, a tablet, a capsule, a softgel or other oral dosage form) or intranasal (e.g., inhalation). Mucosal administration can also include intra-vaginal or intra-rectal administration.

[0098] The immunogenic cochleates, including vaccine cochleates and other cochleate compositions described herein are typically administered to a mammal (e.g., dog, cat, pig, horse, rodent, non-human primate, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated mammal (e.g., induction of an immune response, protection against infectious disease(s), prevention or elimination of cancer in a mammal, etc. ). Such a therapeutically effective amount can be determined as described below.

[0099] Toxicity and therapeutic efficacy of the cochleate compositions described herein can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LDso (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Those compositions that exhibit large therapeutic indices are desired. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of desired compositions lies typically within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form emplo ed and the route of administration utilized,

[00100] Therapeutically effective amounts of the cochleate compositions described herein generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1 ug to about 25,000 ,g (e.g., i, 100, 500, 2000, 2500, 10,000, 15,000, 25,000 ug) of cochleate containing antigen or nucleic acid encoding the antigen, cochleate containing adjuvant only as disclosed herein for a 70 kg patient, followed by boosting dosages of from about 1 μg to about 2500 ug of the cochleate composition pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity and/or antibody responses in the patient's blood.

[00101] For treating a subject currently suffering from an infectious disease or cancer and/or who has just been diagnosed with cancer or an infectious disease, administration typically begins at the first sign of disease or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter, in chronic infection, loading doses followed by boosting doses may be required. For prophylactic use, administration may begin as soon as an individual becomes aware of prior to an expected exposure to an infectious disease or a predisposition to cancer.

[00102] As is well known in the medical and veterinary arts, dosage for any one subject, however, depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. Accordingly, an ordinary artisan may vary the suggested dosage depending upon the foregoing factors.

[00103] The dosage of the encochleated antigen is likely to be less than that of a non-encochleated version of the same antigen due, at least in part, to the enhanced pharmacokinetics of cochleates. Circulating protein antigen, without the protection afforded by cochleates, can be problematic due, in part, to inefficient loading by target cells or non- specific toxicity if antigen levels are increased. Free antigen in nucleic acid form can be further problematic. Nucleic acids must somehow avoid the high levels of nucleases in serum to enter the cells intact. Often, nucleic acids do not survive the harsh serum environment prior to cell entry. In addition, high doses of nucleic acids (in order to overcome the degradation by nucleic acids) typically induce significant local or systemic side-effects and toxicities.

[00104] In contrast, high calcium concentrations in gastrointestinal secretions, serum and interstitial fluid stabilize the antigen(or nucleic acid)-containing-cochleate crystal. Immunogenic cochleates enter the circulatory system, diffuse into tissues and/or are taken up by "activated" and/or infected cells. Thus, intracellular levels of antigen(or nucleic acid)- cochleates increase and reach high levels. The low intracellular calcium concentration causes the antigen(or nucleic acid)-cochleates to open, releasing their cargo from the cochleates. Accordingly, lower plasma levels are required to reach efficacious intracellular antigen (or nucleic acid) concentrations. These lower plasma levels may result in less systemic toxicity. Furthermore, the cochleates protect the protein antigens or nucleic acids from the harsh serum environment, thus allowing them to enter the cell intact.

[00105] Due to this increased efficacy, the encochleated antigen may be administered at a lower dosage, less frequently, or for a shorter duration than a non- encochleated version of the antigen. In certain implementations, the encochleated antigen is administered at a lower dosage than a non-encochleated version of the same antigen. In certain embodiments, the lower dosage is about 2X less, such as 3X less, such as 5X less, such as 10X less, such as 20X less than the recommended or indicated dosage of a non-encochleated version of the antigen, typically when the antigen is contained in the cochleate in the form of a peptide or protein. In certain embodiments, the lower dosage is about 15X less, such as about 20X less, such as about 100X less, such as about 200X less, such as about 500X less, such as about 1000X less than the recommended or indicated dosage of a non-encochleated version of the antigen, typically when the antigen is contained in the cochleate in the form of a nucleic acid encoding the antigen, which is expressed in vivo.

[00106] The cochleates as described herein can be used in a method of treating a subject with an infectious disease or cancer, including any infectious disease or cancer for which treatment with an antigen is indicated. In certain implementations, the infectious disease or condition includes a viral, fungal or bacterial condition as described herein.

[00107] In certain implementations, the cancer is selected from colon carcinomas, melanomas, renal carcinomas, lymphomas, acute myeloid leukaemia (AML), acute lym-phoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), gastrointestinal tumours, pulmonary carcinomas, gliomas, thyroid tumours, mammary carcinomas, prostate tumours, hepatomas, various virus-induced tumours such as, for example, papilloma virus-induced carcinomas (e.g. cervical carcinoma), adenocarcinomas, herpes virus-induced tumours (e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), heptatitis B-mduced tumours (hepatocell carcinoma), HTLV-1- and HTLV-2-induced lymphomas, acoustic neuromas/neuri nomas, cervical cancer, lung cancer, pharyngeal cancer, anal carcinomas, glioblastomas, lymphomas, rectal carcinomas, astrocytomas, brain tumours, stomach cancer, retinoblastomas, basaliomas, brain metastases, medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer, melanomas, thyroidal carcinomas, bladder cancer, Hodgkm's syndrome, meningiomas, Schneeberger disease, bronchial carcinomas, hypophysis tumour, Mycosis fungoides, oesophageal cancer, breast cancer, carcinoids, neurinomas, spinaliomas, Burkitt's lymphomas, laryngeal cancer, renal cancer, thymomas, corpus carcinomas, bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/neck tumours, oligodendrogliomas, vulval cancer, intestinal cancer, colon carcinomas, oesophageal carcinomas, wart involvement, tumours of the small intestine, craniopharyngiomas, ovarian carcinomas, soft tissue tumours/sarcomas, ovarian cancer, liver cancer, pancreatic carcinomas, cervical carcinomas, endometrial carcinomas, liver metastases, penile cancer, tongue cancer, gall bladder cancer, leukaemia, plasmacytomas, uterine cancer, lid tumor, prostate cancer, etc.

[00108] One embodiment is directed to a method of treating a subject with an infectious disease or cancer, the method comprising administering to the subject in need thereof a therapeutically effective amount of a formulation comprising a cochleate, wherein the cochleate comprises an antigen or a nucleic acid encoding an antigen. In certain embodiments, the method of treating a subject with an infectious disease or cancer comprises mucosally administering, e.g., orally or intransally, to the subject in need thereof a therapeutically effective amount of a formulation comprising a cochleate, wherein the cochleate comprises an antigen or a nucleic acid encoding an antigen.

[00109] One embodiment is directed to a method of treating a subject in need thereof with an encochleated antigen or a nucleic acid encoding an antigen, wherein the cochleate further contains an adjuvant as disclosed herein. In some embodiments, a commercial or other vaccine is administered to the subject and the subject is administered concurrently or in any order a cochleate comprising an adjuvant, such as an IL-12 adjuvant.

[00110] In other embodiments, the subject is administered an encochleated antigen and a separate formulation comprising an encochleated adjuvant in the same or different cochleate from that of the adjuvant.

[00111] Another embodiment is directed to a method of treating a subj ect in need thereof with an encochleated antigen and optionally an adjuvant, the method comprising administering to the subject a formulation comprising a cochleate, wherein the cochleate comprises a therapeutically effective amount of an adjuvant, and wherein the cochleate is a geode cochleate comprising: 1) a lipid monolayer comprising a negatively charged phospholipid, such as a soy phosphotidylserine, wherein the lipid monolayer surrounds a droplet of castor oil and the antigen is comprised within the droplet of castor oil; and 2) a lipid strata comprising alternating divalent cations and phospholipid bilayers comprising the negatively charged phospholipid, wherein the lipid strata is disposed about the lipid monolayer. In certain embodiments, the geode cochleate is administered mucosally (e.g., orally or intr anas ally).

EXAMPLES

[00112] The examples provided below are simply for illustrative purposes.

Those of skill in the art will be able to readily determine appropriate methods and equipment in order to produce suitable solid dispersion forms as described herein.

[00113] EXAMPLE 1. FORMATION OF COCHLEATES CONTAINING

ANTIGENS USING INFLUENZA VIRUSES

[00114] Viral Growth and Purification. Virus was grown and purified essentially as described by M.C. Hsu et al. Virology, 1979, 95: 476 (1979). Influenza virus was propagated in the allantoic sac of 10 or 11 day old embiyonated chicken eggs. Eggs were inoculated with 1-100 egg infectious doses (10 ³ to 10 ⁵ viral particles as determined by HA titer) in 0.1 ml of phosphate buffered saline (0.2 gm/L Cl, 0.2 gm/L KH2PO4, 8.0 gm/L NaCl, 1.14 gm/L Na ₂ H-P0 ₄ , 0.1 gm/L CaCl ₂ , 0.1 gm/L MgCl ₂ 6H ₂ 0 (pH 7.2)). Eggs were incubated at 37°C for 48 to 72 hours, followed by incubation at 4°C for 24 to 48 hours. Allantoic fluid was collected and clarified by centrifugation at 2,000 rotations per minute (rpm) for 20 minutes at 5°C. The supernatant was then centrifuged at 13,000 rpm for 60 minutes. The pellets were resuspended in phosphate buffered saline (pH 7.2) by vortexing and sonicating, followed by centrifugation at 5,000 rpm for 20 minutes. The pellet was resuspended by vortexing and sonicating, diluting, and centnfugiiig again at 5,000 rpm for 20 minutes. The two 5,000 rpm supernatants were combined and centrifuged at 13,000 rpm for 60 minutes. The resulting pellets were resuspended in phosphate-buffered saline by vortexing and sonicating, aJiquoted, and stored at ~70°C. Sterile technique and materials were used throughout viral inoculation, isolatio , and purification.

[00115] Extraction of Viral Glycoproteins and Lipids. Virus stored at -70°C was thawed, transferred to sterile thick-walled polycarbonate tubes, and diluted with buffer A (2 mM TES, 2 mM L-histidine, 100 mM NaCl (pH 7.4)). it was pelleted at 30,000 rpm for 1 hour at 5°C in a Beckman TY65 rotor. The supernatant was removed and the pellet resuspended to a concentration of 2 milligrams viral protein per milliliter of extraction buffer (2M NaCl, 0.02M sodium phosphate buffer (pH 7.4)) by vortexing and sonicating. The nonionic detergent β-D-octyl-glucopyranoside was then added to a co centration of 2% (w/v). The suspension was mixed, sonicated for 5 seconds, and placed in a 37°C water bath for 45 minutes. At .1 , 30, and 45 minute incubation times, the suspension was removed briefly for mixing and sonication. Nucleocapsids were pelleted by centrifugation at 30,000 rpm for 45 minutes in a TY65 rotor. The resulting clear supernatant was removed and used in the formation of viral gly coprotein-containing cochleates. Some modification of the above procedure may have to be employed with other membrane proteins. Such modifications are well known to those skilled in the art.

[00116] Formation of Cochleates. Large, unilamellar, non-protein containing, phospholipid vesicles were made by a modification of the calcium-EDTA-chelation technique described by D. Papahadjopoulos et al, Biochem. Biophys. Acta., 1975, 394: 483 (1975). Phosphatidylserine and cholesterol (9: 1 wt ratio) were dried down in a clean glass tube under a stream of nitrogen. The lipid was resuspended in buffer A (pH 7.4) to a concentration of 6 μΜοΙ/mi by vortexing for 7 minutes. The resulting suspension of multilamellar vesicles was converted to small unilamellar vesicles by sonication under nitrogen at 5°-10°C for approximately 20 minutes in a bath-type sonicator. (Model G1125P16, Laboratory Supplies Co., Hicksviile, New York). These vesicles were dialyzed at room temperature against two changes of 250 ml of buffer A (pH 7.4) with 3 mM CaCb. This results in the formation of an insoluble precipitate referred to as cochleate cylinders.

[00117] LC Cochleates. An amount of phosphatidylserine and cholesterol (9: 1 wt ratio) in extraction buffer and non-ionic detergent as described hereinabove was mixed with a pre-selected concentration of polynucleotide and the solution was vortexed for 5 minutes. The solution first was dialyzed overnight using a maximum ratio of 1 :200 (v/v) of dialysate to buffer A without divalent cations, followed by three additional changes of buffer leading to the formation of small protein lipid vesicles. The vesicles were converted to a cochleate precipitate, either by the direct addition of Ca ² + ions, or by dialysis against two changes of buffer A containing 3 mM Ca 2+ ions, followed by one containing buffer A with 6 mM Ca2+.

[00118] DC Cochleates. An amount of phosphatidylserine and cholesterol (9: 1 wt ratio) in extraction buffer and non-ionic detergent as described hereinabove was mixed with a pre-selected concentration of polynucleotide and the solution was vortexed for 5 minutes. The clear, colorless solution which resulted was dialyzed at room temperature against three changes (minimum 4 hours per change) of buffer A (2 mM TES N-Tris[hydroxymethyl]- methyl-2 aminoethane sulfonic acid, 2 mM L-histidine, 100 mM NaCl, pH 7.4, also identified as TES buffer) containing 3 mM CaCh. The final dialysis routinely used is 6 mM Ca2+, although 3 mM Ca2+ is sufficient and other concentrations may be compatible with cochleate formation. The ratio of dialyzate to buffer for each change was a minimum of 1 : 100. The resulting white calcium-phospholipid precipitates have been termed DC cochleates. When examined by light microscopy (x 1000, phase contrast, oil), the suspension contains numerous particulate structures up to several microns in diameter, as well as needle-like structures. Cocholeates containing influenza glyocoprotein is shown in FIG. 6A, empty cochleates are depicted in FIG. 6B.

[00119] EXAMPLE 2. CIRCULATING ANTIBODY RESPONSES TO

ORALLY DELIVERED PROTEIN-COCHLEATE VACCINES

[00120] Cochleate vaccines containing the glycoproteins and lipids from the envelope of influenza virus and phosphatidyiserine and cholesterol were given to mice by gradually dispensing 0.1 ml liquid into the mouth and allowing it to be comfortably swallowed. 5Qug dose of cochleate vaccine containing the glycoproteins from the influenza virus were administered to a first experimental group of mice (orally and intramuscularly) at week zero (full dose), week three (full dose) and weeks 13-15 (quarter dose). The data demonstrate that high circulating antibody titers can be achieved by simply drinking cochleate vaccines containing viral glycoproteins (FIG. 7). The response is boostable, increasing with repeated administration, and is directly related to the amount of glycoprotein in the vaccine,

[00121] FIG. 7 depicts the circulating antibody titers at 14 weeks after vaccine administration at week zero, three and 13 in the amounts described above. As shown in the figure, intramuscular immunization supported higher circulating antibody titers than oral. However, the oral route also gave strong circulating IgG titers (25,600 at 14 weeks). Significantly, and consistent with induction of immune responses at mucosal surfaces, (see below) the circulating IgA levels observed are extremely high following oral administration, and substantially higher than those generated by intramuscular immunization (a titer of 640 versus 10 at 14 weeks). Subtype analysis of serum from influenza cochleate immunized animals demonstrates the production of both IgGl and IgG2a. The partem seen in numerous experiments with flu and other protein cochleates is higher IgGl titers, but very substantial IgG2a titers, both increasing with subsequent immunizations. This antibody subtype distribution indicates the induction of both T helper cell type 1 and type 2 responses (supporting IgG2a and IgGl , respectively), and correlates with results of cytokine secretion assays. The ratios of IgGl to IgG2a are similar whether the cochleates are given orally or intramuscularly.

[00122] EXAMPLE 3. PROTECTION FROM INTRANASAL

CHALLENGE WITH LIVE INFLUENZA FOLLOWING ORAL IMMUNIZATION WITH GLYCOPROTEIN-COCHLEATES [00123] in order to determine whether oral administration of the cochleate influenza vaccine could lead to protective immunity in the respiratory tract, initial vaccine doses of 100, 50, 25, 12.5, 6, 3 and 0 _, ug of cochleates containing the influenza glycoproteins of Example 1 (groups 1 through 7 respectively) were administered to a second experimental group of mice at 0 and 3 weeks. A third immuni zation (at 15 weeks) was adminis tered at one fourth the dose used for the initial two immunizations. The immunized mice were subsequently challenged by intranasal application of 2.5 x 10 ⁹ particles of influenza virus at 16 weeks. Three days after viral challenge, the mice were sacrificed, and the lungs and trachea were obtained. The entire lung or trachea was triturated and sonicated, and aliquots were injected into embryonated chicken eggs to allow amplification of any virus present. After three days at 37°C, allantoic fluid was obtained from individual eggs, and hemagglutination (HA) titers were performed to quantitate the virus

[00124] A high degree of protection from viral replication in the trachea was achieved (FIG. 8). Of the 25 mice that received the five highest doses (100 μg to 6 μg), 22 had no virus in the trachea. In the 3 μ-g dose group, 4 out of 5 were infected. All five of the unvaccinated mice were infected.

[00125] The oral protein cochleate vaccine also provided excellent protection against viral replication in the lungs (FIG. 8). All mice that received 12.5 g or higher, (20 out of 20), were negative for virus. The 3ug and 6 sg dose groups had reduced viral burdens in the lungs when compared to the controls (data not shown).

[00126] EXAMPLE 4. DOSE EFFICACY OF INTRANASALLY

ADMINISTERED ANTIGEN CONTAINING COCHLEATES

[00127] A third experiment was conducted to assess the dose efficacy of intranasally administered influenza-containing cochleates in comparison to a commercially available influenza vaccine. In this experiment, mice were intranasally administered 1 ,2 μg or 12 μ-g influenza cochleates or a commercially available vaccine on day 0 and day 109. Neutralizing plasma antibody titers were assessed at week 20. As shown in FIG. 9, the TH1 antibody response may be increased by encochleation. Further, FIG. 10 demonstrates that the dose efficiency of the cochleate nano-particle formulation with neutralizing antibody titers is more than 10X higher than a commercial influenza vaccine at each dose level. Addition of adjuvant, such as 3-Q-desacyl-4'-monophosphoryl lipid A (MPL™ Adjuvant) can increase serum IgG antibody titer to 1000X times that observed using encochleation alone (FIG. 11).

[00128] EXAMPLE 5. IMMUNIZATION WITH PROTEIN-

COCHLEA TES LEADS TO PRODUCTION OF IgA

[00129] Balb C mice were orally administered influenza glycoprotein cochleates at zero, three and ten weeks (50 μ§ dose, 50 ^ig dose and 12.5 ,ug dose, respectively). At 12 weeks, all five mice had significant influenza glycoprotein-specific salivary IgA, with titers ranging from 10 to 160 (FIG. 12). Demonstration of high mucosal IgA antibody titers following oral immunization is of considerable significance and highly desired for protection against organisms invading through mucosal surfaces. Secretory IgA can protect against pathogens which replicate on or enter via mucosal surfaces by binding to infectious organisms and blocking their attachment to or invasion through mucosal surfaces.

[00130] EXAMPLE 6. PROLIFERATIVE RESPONSES ARE

GENERATED TO ANTIGENS CONTAINED IN COCHLEATES

[00131] Balb/C mice were immunized with cochleates containing 50 μg influenza glycoprotein at 0 and 3 weeks and with 12.5 ,ug at 13 weeks (oral or intramuscular). The mice were sacrificed at 14 weeks and their spleens removed. Figure 13 shows the proliferati ve response to ultraviolet light-irradiated influenza virus over several days in culture. Splenocytes were incubated in vitro with 16 μg/ml UV-inactivated influenza virus. Proliferation was determined by measuring the uptake of tritiated thymidine into DNA.

[00132] These data demonstrate that strong proliferative responses (up to 25 fold stimulation index) can be obtained with either oral or intramuscular administration of antigen cochleate formulations (FIG. 13). Thus, successful vaccination can be achieved since antigen specific cell-mediated memory can be stimulated.

[00133] EXAMPLE 7. DNA COCHLEATES FORMULATED WITH

PLASMIDS EXPRESSING HIV-1 PROTEINS (ORAL ADMINISTRATION)

[00134] DNA plasmids were formulated into cochleates using the DC dialysis method. Cochleates containing 3.5 μg or 17 μg of plasmid pCMVHIV-1 (FIG. 14), which expresses the HIV-1 proteins env (gpl60), rev and tat in mammalian cells, were given to Balb/c mice by oral administration (swallowing) or intramuscular injection at 0 and 4 weeks. Control animals were given cochleates without DNA. At week six, animals were sacrificed, splenocytes were isolated, and stimulated in vitro either with cell culture medium or peptide pi 8 from gpl 60. Stimulated cells were assayed for proliferation by measuring uptake of ¾ thymidine. Antigen specific cytotoxicity was assessed by chromium release induction from target cells expressing HIV gpl 60 (env).

[00135] Oral administration of the two 3.5 μg or 17 μg doses mediated the induction of antigen-specific splenocyte proliferation at a level six to eleven fold above background (FIG. 15). These dosages also yielded strong splenocyte cytolytic responses of 73 to 85% specific lysis at an effector cell to target cell ratio of one hundred to one (FIG. 16). These cellular responses were essentially the same as those obtained by analogous intramuscular injection of 17 μg of DNA cochleates. Very small quantities of encochleated DNA were required to induce these responses, whereas a higher dose, 50 μg of naked DNA given orally induced no cytoxic or proliferative responses. These data demonstrate that substantial levels of antigen-specific cytotoxic splenocyte and proliferative responses can be induced following oral administration of DNA cochleates.

[00136] EXAMPLE 8. DNA COCHLEATES FORMULATED WITH

PLASMIDS EXPRESSING HERPES SIMPLEX VIRUS-2 (HSV-2) PROTEINS AND PLASMIDS EXPRESSING IL-12 (INTRAMUSCULAR ADMINISTRATION)

[00137] Vaccine Formulation and Animal Inoculation: Encochleated plasmid preparations were formulated to allow for the comparison of encochleated or naked plasmids. HSV-2 glycoprotein D (gD2) DNA (made with the pc DNA 3.1 vector backbone) were mixed with encochleated or naked plasmid IL-12 DNA (made using the pED vector backbone). The IL-12 plasmid DNA consists of an equal mixture of two plasmid DNAs coding for the p35 and p40 subunits. The gD2 DNA or pc DNA 3.1 were administered at 25 mg/dose. IL-12 (each heterodimer plasmid) and pED plasmids were administered at 35 mg/dose. Mice were immunized via the intramuscular route with either the naked or encochleated vaccine mixtures at weeks 0, 4 and 8. Four weeks post the last immunization, the mice were sacrificed and spleens and serum samples collected. [00138] ELLSPOT analysis of cytokine secretion: Spleen cells were restimulated in vitro with inactivated HSV for four days, then the viable cells plated in wells of nitrocellulose plates which had been coated with capture antibody specific for either murine IFNy or IL4. After a 20 hour incubation, cells were washed from the wells and the appropriate biotinylated anti-cytokine antibody added. Wells were washed after an overnight incubation and the peroxidase conjugated goat anti-biotin antibody added. Following a 3 hour incubation at 37°C, the AEC substrate was added. The reaction was stopped by washing the wells with tap water. Spot forming cells (SFC) were counted using a dissecting microscope.

[00139] Table 1 shows that encochleation of gD plasmid increases the release of Thl-type cytokine (IFN-γ) about 3 times that of naked gD plasmid. See rows 1-3 in bold.

[00140] gD ELISA Wells of IMMULON® II were coated with gD, then washed and blocked prior to addition of serial two-fold dilutions of sera. After 1 hour at room temperature, plates were washed prior to the addition of biotinylated secondary antibody specific for either murine IgGl or IgG2a. Following a 1 hour incubation at room temperature, the plates were washed and Horseradish peroxidase-conjugated streptavidin was added to each well for a further 1 hour, then washed again prior to addition of 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS) substrate. Resulting color was quantitated at 405 nanometers using a Biotekplate reader. Endpoint titers were defined as being 2 standard deviations above the average OD of the sera obtained from naive mice.

[00141] The effect of gD, IL-12 encochleation on antibody isotype response is shown in Table 2, below. Encochleation of IL-12 without concomitant encochleation of gD can increase Thl antibody response (IgG2a). See column IgG2a, cells in bold.

[00142] CTL Assay: Spleen cell suspensions were restimulated with UV- irradiated, HSV infected autologous spleen cells. After 5 days of culture, the viable cell concentrations were adjusted to the cell concentration yielding the desired effector cell number and 100 ml aliquots added to wells of a round bottom 96-well plate. Each dilution was plated in triplicate. A20 targets cells, HSV-infected or uninfected, were labeled with 51 Cr, then added to the wells in 100 μΐ aliquots. After 4 hours incubation, 100 ml were harvested from wells and the gamma emissions counted. Specific release was determined by the following formula: Specific Release = (Experimental CPM-Spontaneous Release CPM) X 100 (Total CPM - Spontaneous Release).

[00143] The effect of encochleation on HSV specific CTL response is shown in

FIG. 17. Encochleation of gD and IL-12 plasmids induced 2X greater HSV-specific cytolytic T cell responses than Herpes infection. Naked DNA was inactive.

[00144] Virus Neutralization Assay: Sera were evaluated for HSV neutralizing titer using a microneutralization assay method. Test sera were heat inactivated for 30 minutes at 56°C, and then subjected to serial 2-fold dilution. An equal volume of HSV2 was added to each dilution. Guinea pig complement was also included. Mixtures were incubated for 1 hour at 37°C (5% CCh) with gentle rocking, then inoculated directly onto confluent Vero cell monolayers in 96-well plates. Virus, medium and complement controls were included in each assay. Following incubation, cells were overlaid with 1% methylcellulose/MEM. Plates were incubated at 37°C (5% CCh) until approximately 50 plaques could be counted in virus control wells. Plaques were enumerated and titers were defined as the reciprocal dilution of the last serum dilution yielding a greater than 50% plaque reduction.

[00145] Table 3 depicts the results of the neutralization assay. As shown in the

Table, encochleation of gD enhances antibody response in the mice.

TABLE 1. ELISpot analysis of cytokines

gD DNA IL-12 DNA 353 43 8.2 gD DNA pED DNA 235 40 5.9 pc DNA 3.1 pED DNA 168 53 3.2 Cochleate cochleate

pc DNA 3.1 pED DNA 148 58 2.6 Cochleate

pc DNA 3.1 IL-12 DNA 278 87 3.2 Cochleate cochleate

pc DNA 3.1 pED DNA 170 47 3.6 pc DNA 3.1 pED DNA 148 60 2.5 cochleate

pc DNA 3.1 IL-12 242 62 3.9

HSV2 900 58 15.5 naive 183 48 3.8

Table 2. Encochleation of IL-12 Adjuvant

Table 3 Antibody Neutralization Titer

EXAMPLE 9. ENCOCHLEATION OF DNA PLASMIDS PROTECTS AGAINST NUCLEASE DIGESTION

[00146] DNA plasmids were encapsulated into cochleates, which were prepared according to the DC method described above using 18: 1 PS (DOPS), Avanti Polar Lipids 840035, (l,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)). pCMVHIV-l env plasmid DNA was retrieved from the DC type-encapsulated DNA complexes, subjected to gel electrophoresis and endonuclease digestion. FIG. 18 shows that DNA-cochleate samples are at least partially protected from nuclease digestion. The apparent, only partial protection, is likely due to the fact that un-encochleated DNA (which is likely adsorbed onto the outside surfaces of the cochleate crystals) was not washed away or in any manner treated to remove such DNA. Further, the efficiency of encochleation of DNA into the cochleates is ~ 50% - 60%, indicating that a large amount of un-encochleated DNA was likely adsorbed onto the cochleate surface. Nevertheless, as indicated in the Figure, partial protection was still observed (Lanes 5,7). No protection was observed for naked DNA in a pure (empty) DC type cochleate (Lanes 4, 6).

[00147] Lanes 3, 5, and 7 (pCMVHIV-lenv plasmid DNA retrieved from cochleate); Lanes 2, 4, 6 (control, i.e., naked, unformulated DNA added to the pure DC (empty) type cochleate); Lane 1 (DNA marker (1/HindIII); Lanes 4, 5 : untreated plasmid DNA; Lane 4, 5 : EcoRI digested DNA at 37° C for 15 minutes; Lane 6, 7: EcoRI digested DNA at 37°C for 60 minutes.