WO2014130938A1

WO2014130938A1 - Particle formulations for delivery of tlr agonists and antigens

Info

Publication number: WO2014130938A1
Application number: PCT/US2014/017994
Authority: WO
Inventors: Lynda TUSSEY; Alan Shaw; Robert Becker; Scott Umlauf; Uma KAVITA; Andrew Loxley; Garry Thomas Gwozdz
Original assignee: Vaxinnate Corporation; Particle Sciences, Inc.
Priority date: 2013-02-25
Filing date: 2014-02-24
Publication date: 2014-08-28

Abstract

The present invention relates to formulations comprising one or more TLR5 agonists and one or more antigens adsorbed or attached to the same particles or to different particles. In particular, the present invention relates to vaccine compositions in which antigens and flagellin have been attached to a particle through non-covalent bonding. The present invention relates to improved vaccines which may enhance immunogenicity of the vaccine and/or reduce reactogenicity of the vaccine when administered.

Description

PARTICLE FORMULATIONS FOR DELIVERY OF TLR AGONISTS AND ANTIGENS RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 61/850,871 , filed on February 25, 2013.

[0002] The entire teachings of the above application are incorporated herein by reference. BACKGROUND OF THE INVENTION

[0003] The present invention relates to formulations comprising one or more TLR5 agonists and one or more antigens adsorbed or attached to particles. In particular, the present invention relates to vaccine compositions in which antigens and flagellin have been attached to a particle through non-covalent bonding. The present invention relates to improved vaccines which may enhance immunogenicity of the vaccine and/or reduce reactogenicity of the vaccine when administered.

SUMMARY OF THE INVENTION

[0004] A number of vaccine formats which fuse protein antigens to Toll-like Receptors (TLR) agonists such as flagellin have been developed. Toll-like Receptors refer to a family of receptor proteins that are homologous to the Drosophila melanogaster Toll protein. Toll-like Receptors are Type I transmembrane signaling receptor proteins characterized by an extracellular leucine-rich repeat domain and an intracellular domain homologous to an interieukin 1 receptor. HA is the major protective antigen for influenza and a subunit of HA referred to as HAl-2 appears to be the minimally protective subunit as demonstrated in preclinical lethal challenge models. A longer subunit referred to as HAl-1 genetically fused to flagellin has also been shown to be protective in the preclinical models. In addition to using different lengths of the HA antigen, vaccine formats which differ in the attachment point of the vaccine antigen to flagellin have also been developed. Such fusions typically have a one-to-one or two-to-one correspondence of antigen to TLR agonist. Furthermore, some of the genetic fusion forms of antigens coupled to TLR 5 agonists as described above tend to be somewhat reactogenic.

Therefore, there exists a need to provide an alternative vehicle for delivering TLR 5 agonist and antigen combinations such that the ratio of TLR 5 agonist and antigen can be altered.

[0005] WO 2008/121926 describes pharmaceutical formulations containing particles comprised of hydrophobic organic material co-dissolved or co-dispersed with an active pharmaceutical ingredient.

[0006] US Patent No. 5,716,637 describes nanoelmusion of particles comprising a liquid core composed of a lipid which is solid or liquid at room temperature, which is stabilized by a phospholipid envelope.

[0007] WO 2004/069227 describes a process for the preparation of a stable dispersion of solid particles, in an aqueous medium comprising by combining a pyrrole carboxamide compound, a water-miscible organic solvent and an inhibitor with an aqueous phase comprising water and a stabilizer.

[0008] Bodmeier, et al. J. Microencapsulation 9(1): 89-98 (1992) describes forming microparticles by a melt dispersion technique, in which the drug-wax melt was emulsified into a heated aqueous phase followed by cooling to form the microparticles.

[0009] The present invention relates to improved TLR 5/antigen formulations which alter the ratio of TLR 5 agonist to antigen such that tolerability of the vaccine is improved while retaining the TLR 5 activation activity. The use of particles as a means of presenting antigens and TLR 5 agonists permits the ratio of antigen to TLR 5 agonist to be altered such that optimization of immunogenicity to the antigen and reduced reactogenicity to the TLR 5 agonist might be attained. The use of particles also permits the use of non-proteinaceous antigens in combination with TLR 5 agonists as well as the use of existing purified vaccine antigen preparations in combination with TLR 5 agonists. SUMMARY OF THE INVENTION

[0010] The invention relates to particle compositions comprising one or more TLR 5 agonists and one or more antigens on the same particle and methods of making these particles. The invention further relates to methods of manufacturing these particle compositions.

[0011] The invention also relates to particle compositions where the TLR 5 agonist(s) and antigen(s) are located on the same particle and the ratio of TLR 5 agonist to antigen different than 1 : 1 as in the case of fusions of TLR 5 agonist with antigen. The particle compositions of the present invention include particles where the ratio of the TLR agonist to antigen is less than 1 : 1 or more than 1 : 1 on either a weight to weight basis or molecule to molecules basis.

[0012] The invention also relates to particle compositions which include surfactant(s) as well as TLR 5 agonist(s) and antigen(s). Surfactants include but are not limited to cetyl

trimethylammonium bromide and/or cetylpyridinium bromide.

[0013] The invention relates to particle compositions in which the antigen(s) and/or TLR 5 agonist(s) are absorbed or attached to the particles by non-covalent interactions such as hydrophilic or electrostatic interactions.

[0014] The invention also relate to methods of making immunologic formulations comprising preparing a hot aqueous surfactant solution, adding the hot surfactant to a molten lipid, adding this mixture to an aqueous phase, cooling the mixture and adding TLR 5 agonist(s) and/or antigen(s).

[0015] The particle compositions of the invention may also provide immunologic compositions that are more potent than the antigen alone. The particle compositions of the invention may also provide immunologic compositions in which the TLR 5 agonist provokes a TLR 5 mediate response, but in which the immune response to the TLR 5 agonist is reduced compared to the TLR 5 agonist in solution when administered.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a chart of the tunability of the surface charge of the particles of the invention by selection of particular surfactants in the manufacture of the particles.

[0017] Figure 2 is a graph of the IL-8 response (TLR5 bioassay) of flagellin and T1BT adsorbed to CTAB and CPB formulations.

[0018] Figure 3 is a graph of the IgG antibody response to T1BT.* Sera collected from individual mice following the second immunization with the various formulations shown in the figure above were diluted and the amount of anti-TlBT* IgG was measured by ELISA and quantified using a 4-parameter logistic standard curve generated using known amounts of mouse IgG. Results shown are the geometric mean titers with 95% confidence interval of the 10 individual mouse sera within each group. The CTAB particles containing Flagellin and T1BT* at a ratio of 0.1 : 1 generate the highest amount of anti-TlBT IgG relative the control formulation without Flagellin.

[0019] Figure 4A and 4B are charts of IgG antibody responses to Flagellin at day 20 and day 35 respectively. Sera collected from individual mice following the first and second

immunization with the various formulations shown in the figure above were diluted and the amount of anti-Flagellin IgG was measured by ELISA and quantified using a 4-parameter logistic standard curve generated using known amounts of mouse IgG as described in the methods section. Results shown are the geometric mean titers with 95% confidence interval of the 10 individual mouse sera samples within each group.

[0020] Figure 5 is a graph of IgG antibody response to T1BT* and Flagellin. Sera collected from individual mice following the second immunization with the various formulations shown in the figure above were diluted and the amount of anti-TlBT* IgG or anti-Flagellin IgG was measured by ELISA and quantified using a 4-parameter logistic standard curve generated using known amounts of mouse IgG. Results shown are the geometric mean titers for each group of 10 mice. Ratios of Flagellin and T1BT* that generate the highest anti-TlBT* antibodies with the CTAB particles do not generate anti-Flagellin antibodies.

[0021] Figure 6 is a graph of IgG antibody response to T1BT.* Sera collected from individual mice following the second immunization with the various formulations shown in the figure above were diluted and the amount of anti-TlBT* IgG was measured by ELISA and quantified using a 4-parameter logistic standard curve generated using known amounts of mouse IgG. Results shown are the geometric mean titers with 95% confidence interval of the 10 individual mouse sera within each group. The CPB particles containing Flagellin and T1BT* at a ratio of 0.02: 1 generate the highest amount of anti-TlBT IgG relative the control formulation without Flagellin.

[0022] Figure 7A and 7B are graphs of IgG antibody response to Flagellin at day 20 and day 35 respectively. Sera collected from individual mice following the first and second

[0023] Figure 8 is a graph of IgG antibody response to HA. Sera collected from individual mice following the second immunization with the various CTAB formulations shown in the figure above were diluted and the amount of anti-HA IgG was measured by ELISA and quantified using a 4-parameter logistic standard curve generated using known amounts of mouse IgG. Results shown are the geometric mean titers with 95% confidence interval of the 10 individual mouse sera within each group.

[0024] Figures 9A and 9B are graphs of IgG antibody response to Flagellin at day 20 and 35 respectively. Sera collected from individual mice following the first and second immunization with the various formulations shown in the figure above were diluted and the amount of anti- flagellin IgG was measured by ELISA and quantified using a 4-parameter logistic standard curve generated using known amounts of mouse IgG as described in the methods section. Results shown are the geometric mean titers with 95% confidence interval of the 10 individual mouse sera samples within each group.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention relates to particle compositions comprising one or more TLR 5 agonists and one or more antigens and methods of making these particles. The invention further relates to methods of manufacturing these particle compositions.

[0026] Preferred method of making the particles of the invention is via a melt-emulsification- chill process in which a hot aqueous surfactant solution comprising a 1% cationic surfactant solution is added to melted Yellow Carnauba wax (YC) comprised of 40% aliphatic esters, 21% diesters of 4-hydroxycinnamic acid, 13% ω-hydroxycarboxylic acids and 12% fatty acid alcohols after which the mixture is then sonicated and cooled. This process produces a solidified lipid particle having surfactant molecules oriented such that their heads are oriented on the outer surface of the particle while the hydrophilic tails are oriented into the interior of the particle.

[0027] Lipids that can be used to make particles include carnauba wax, bees wax, behenyl alcohol (docosanol), cetyl alcohol, microcrystalline triglycerides such as dynasan 118 (glyceryl tristearate) and polyethylene wax. A preferred lipid is carnauba wax including a carnauba comprising aliphatic esters, diesters of 4-hydroxycinnamic acid, ω-hydroxycarboxylic acids and fatty acid alcohols.

[0028] Surfactants that may be used in making the particles include cationic, anionic and non-ionic surfactants. Surfactants include, but are not limited to cetyl triammonium bromide (CTAB), N-[l -(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate DOTAP, cetylpyridinium bromide (CPB), polysorbate surfactants such as Tween 20, Tween 80

(polyoxyethylene sorbitan monoloaurate) ,polyethylene stearyl ether such as Brij 70, sodium stearate, sodium myristate, sodium dodecyl sulfate, Dioctyl sodium sulfosuccinate such as AOT and combinations thereof. The surfactant may present in a level from about 0.01% to about 10%, or from about 0.05% to about 5% or from about 0.1% to about 2% or from about 0.5% to about 2% or from about 1.0% to about 2.0% .

[0029] The surface charge on the particle can be altered to optimize attachment of the TLR 5 agonist and/or antigen by adjusting the type of surfactant used in the production of the particles. Figure 1 demonstrates how the surface charge of the particles can be altered based on the selection of the surfactant.

[0030] Particle size is preferably less than Ι τη with the particles ranging in size between about lOnm to l OOOnm or between about 20nm to about 900 nm or from about 30nm to abut 800nm or from about 40nm to about 700nm or from about 50nm to about 650nm or from about lOOnm to about 750nm or from about 200nm to about 750nm or from about 300nm to about 750 nm or from about 300nm to about 650nm or from about 400nm to about 750nm or from about 400nm to about 660nm or from about 500nm to about 750nm or from about 500nm to about 650nm. Particle shape may be, but is not limited to spheres, prolate and oblate spheroids, cylindrical, and irregular shapes.

[0031] Antigens that can be used in combination with flagellin in the compositions and methods of the present invention are any antigen that will provoke an immune response in a human. Antigens may be, but are not limited to protein, peptide, carbohydrate, glycoprotein, lipopetide, and subunit antigens. Antigens used in the compositions of the present invention include viral antigens such as influenza viral antigens (e.g. hemagglutinin (HA) protein from influenza A, B and/or C where the influenza viral hemagglutinin protein may be at least one member selected from the group consisting of HI, H2, H3, H5, H7 and H9, matrix 2 (M2) protein, neuraminidase), respiratory synctial virus (RSV) antigens (e.g. fusion protein, attachment glycoprotein), papillomaviral (e.g. human papilloma virus (HPV), such as an E6 protein, E7 protein, LI protein and L2 protein), Herpes Simplex, rabies virus and flavivirus viral antigens (e.g. Dengue viral antigens, West Nile viral antigens), hepatitis viral antigens including antigens from HBV and HC. Antigens used in the compositions of the present invention include bacterial antigens including those from Streptococcus pneumonia, Haemophilus influenza, Staphylococcus aureus, Clostridium difficile and enteric gram-negative pathogens including Escherichia, Salmonella, Shigella, Yersinia, Klebsiella, Pseudomonas, Enter obacter, Serratia, Proteus. Antigens used in the compositions of the present invention include fungal antigens including those from Candida spp., Aspergillus spp., Crytococcus neoformans, Coccidiodes spp., Histoplasma capsulatum, Pneumocystis carinii, Paracoccidiodes brasiliensis, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Antigens for HPV vaccines would include El, E2, E3, E4, E5, E6, E7, and the N terminus of HPVL2 alone or in combination. Antigens for RSV vaccines include but are not limited to RSV F or G proteins, alone or in combination. Antigens for malaria vaccines include, but are not limited to the CSP1 protein, other pre-erythrocytic stage antigens and transmission-blocking antigens (such as, Pfs25, Pfs48 and homologues) alone or in combination. Antigens used in the present invention also include tumor antigens and/or tumor associate antigens such as, but not limited to PSA, CEA, Mart-1, gplOO, TRP-1, MAGE, NY-ESO-1, PAP, Mucin-1 and PSMA.

[0032] Embodiments of the present invention include particles with flagellin and dengue antigen(s) adsorbed thereto. The Dengue disease is caused by four mosquito-borne, serologically related flaviviruses know as DEN 1 (also referred herein as Den 1), DEN 2 (Den-2), DEN 3 (Den 3) or DEN 4 (Den 4). The compositions of the invention include antigens sequences such as those sequences disclosed in WO 2009/128949 (PCT/US2009/002427), Izquierdo et al., 2008 - 814669, Mota et al., 2005, Khanam et al, 2006, Pattnaik et al., 2007, Tripathi et al., 2008; A. Zulueta, et al., Virus Research 121 (2006) 65-73 herein incorporated by reference. In a preferred embodiment the antigen is T1BT, a 45 amino acid peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein.

[0033] The compositions and methods of the present invention employ a TLR-5 agonist in combination with an antigen. In preferred embodiments of the present invention the TLR-5 agonist is flagellin and in particular the type 2 flagellin of Salmonella typhimurium, however any of a variety of fiagellins capable of binding and triggering TLR5 may be used for this invention including engineered fiagellins as described in WO 2009/128950 herein incorporated by reference. [0034] Unlike the prior art methods of combining a TLR agonist with an antigen which required a one-to-one correspondence of agonist to antigen, the present invention relates to compositions that do not require a one-to-one correspondence of the agonist to antigen. In the present compositions the TLR agonist may be present in lesser amount than the antigen when both the agonist and antigen are coupled to the particle. For example the ratio of TLR agonist to antigen may be less than about 1 :2 or 1 :3 or 1 :4 or 1 :5 or 1 :6 or 1 :7 or 1 :8 or 1 :9 or 1 : 10 or from about 1 :20 or 1 :30 or 1 :50 or 1 : 100 or 1 :250 or 1 :500 or 1 :1000 or 1 : 10,000 as measured by the number of molecules of TLR 5 agonist and the number of molecules of antigen. Also the ratio of TLR agonist to antigen may be less than about 1 :2 or 1 :3 or 1 :4 or 1 :5 or 1 :6 or 1 :7 or 1 :8 or 1 :9 or 1 : 10 or from about 1 :20 or 1 :30 or 1 :50 or 1 : 100 or 1 :250 or 1 :500 or 1 : 1000 or 1 : 10,000 as measured by a weight to weight comparison of TLR 5 agonist to the antigen.

[0035] The compositions of the present invention may comprise more than one type of antigen whether the antigen is from the same organism or a different organism. The

compositions of the present invention may comprise more than one type of TLR 5 agonist such as more than one type of flagellin from the same organism or from different organisms.

[0036] The dose of TLR 5 agonist and antigen may be selected to optimize the immunogenic response while attempting to keep reactogenicity low. At least one dose selected from the group consisting of a Λ μg, 0.5μg, l μg dose, 2μg dose, 3μg dose, 4μg dose, 5μg dose, 6μg dose, 7μg dose, 8μg dose, 9μg dose, 10μg dose, 15μg dose, 20μg dose, 25μg dose and a 30μg dose may be sufficient to induce an immune response in humans. The dose of the TLR 5 agonist and antigen may be administered to the human within a range of doses including from about Λ μg to about 500μg, ^g to about 100μg, ^g to about 50μg, from about ^g to about 30μg, from about ^g to about 25μg, from about ^g to about 20μg, from about ^g to about \ 5μξ, from about ^ig to about 10μg, from about 2μg to about 50μg, 2μg to about 30μg, from about 2μg to about 20μg, from about 2μg to about l μg, from about 2μg to about 8μg, from about 3μg to about 50μg, 3μg to about 30μg, from about 3μg to about 20μg, from about 3μg to about 10μ from about 3μg to about 8μ¾ from about 3μg to about 5μg, from about 4μg to about 50μg, 4μg to about 30μ¾ from about 4μg to about 20μg, from about 4 g to about 10μg, from about 4μg to about 8μg, from about 5μg to about 50μg, 5μg to about 30μg, from about 5μg to about 20μg, from about 5μg to about l0μg, from about 5μg to about 9μg, and from about 5μg to about 8μg. With respect to compositions comprising flagellin and antigen the dosage refers to the amount of protein present in the vaccine given to the human. Some of the protein quantity relates to the antigen and some of the protein quantity relates to the flagellin.

[0037] The immunogenic compositions for use according to the present invention may be delivered as a standard 0.01 - 2.0 ml injectable dose and contain from about O. ^g to about 50μg of antigen. In a preferred embodiment of the immunogenic compositions for use according to the present invention is a 0.1 ml injectable dose and contains about ^g of antigen, l ^g of carnauba wax and 0.14μg of surfactant. The vaccine volume may be between 0.05 and 1.0 ml or between about 0.05 and 0.5ml or about 0.10 to about 0.25 ml. A vaccine dose according to the present invention may be provided in a smaller volume than conventional dosing. Low volume doses according to the present invention are suitably below 0.5ml, typically below 0.3ml and usually not less than 0.01 ml.

[0038] Thus, the present invention provides methods for optimizing the antigenicity (ie. increasing the antigenicity) and/or the reactogenicity (ie decreasing the reacotgenicity) of TLR 5/antigen combinations either as composed on single particles or as composed on separate particles, including antigen/flagellin combinations that have been poorly immunogenic and/or highly reactogenic. The immunogenic particle compositions comprising TLR 5 agonist(s) and antigen(s) may be more immunogenic than the combination of TLR 5 agonist and antigen as a fusion protein. For example the immunogenicity of the antigen as measured by the antibody response to the antigen may be greater for the particle compositions of the present invention than the antigen alone in solution or as a flagellin-antigen fusion by greater than about 10% or greater than about 20% or greater than about 30% or greater than about 40% or greater than about 50% or greater than about 60% or greater than about 70% or greater than about 80% or greater than about 90% or greater than about 100%) or greater than about 200% or greater than about 500% or greater than about 1000%. The immunogenic particle compositions comprising TLR 5 agonist(s) and antigen(s) may be less reactogenic than the combination of TLR 5 agonist and antigen as a fusion protein. For example the immune response to the TLR 5 agonist is undesired and may be less for the particle compositions of the present invention than the antigen alone in solution or as a flagellin-antigen fusion by less than about 10% or less than about 20% or less than about 30%> or less than about 40% or less than about 50% or less than about 60% or less than about 70% or less than about 80% or less than about 90% or less than about 100% or less than about 200% or less than about 500% or less than about 1000%. While the TLR 5 agonist is less reactogenic in the compositions of the present invention the particle compositions of the present invention still maintain the ability to trigger a TL 5 response.

[0039] Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0040] Within this disclosure, any indication that a feature is optional is intended to provide adequate support (e.g., under 35 U.S.C. 1 12 or Art. 83 and 84 of EPC) for claims that include closed or exclusive or negative language with reference to the optional feature. Exclusive language specifically excludes the particular recited feature from including any additional subject matter. For example, if it is indicated that A can be drug X, such language is intended to provide support for a claim that explicitly specifies that A consists of X alone, or that A does not include any other drugs besides X. "Negative" language explicitly excludes the optional feature itself from the scope of the claims. For example, if it is indicated that element A can include X, such language is intended to provide support for a claim that explicitly specifies that A does not include X. Non-limiting examples of exclusive or negative terms include "only," "solely," "consisting of," "consisting essentially of," "alone," "without", "in the absence of (e.g., other items of the same type, structure and/or function)" "excluding," "not including", "not", "cannot," or any combination and/or variation of such language.

[0041] Similarly, referents such as "a," "an," "said," or "the," are intended to support both single and/or plural occurrences unless the context indicates otherwise. For example "a dog" is intended to include support for one dog, no more than one dog, at least one dog, a plurality of dogs, etc. Non-limiting examples of qualifying terms that indicate singularity include "a single", "one," "alone", "only one," "not more than one", etc. Non-limiting examples of qualifying terms that indicate (potential or actual) plurality include "at least one," "one or more," "more than one," "two or more," "a multiplicity," "a plurality," "any combination of," "any permutation of," "any one or more of," etc. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. [0042] Where ranges are given herein, the endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0043] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0044] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that the various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXAMPLES

EXAMPLE 1

A: Manufacture of particles

[0045] Particles to which VAX102 and TlBT*peptides were adsorbed were prepared by adding the premixed peptide pairs to preformed cationic wax particle dispersions, followed by simple mixing as described below. VAX 102 is a recombinant protein comprising Salmonella typhimurium flagellin type 2 (STF2; TLR5 ligand) fused to Human M2e.

[0046] Stock cationic particles were prepared by a melt-emulsification-chill process as follows. 10 g Yellow Carnauba wax (YC) comprised of 40% aliphatic esters, 21% diesters of 4- hydroxycinnamic acid, 13% ω-hydroxycarboxylic acids and 12% fatty acid alcohols was melted in a 250 mL glass beaker at 90°C. In a second 250 mL glass beaker, 90 mL of 1% cationic surfactant solution (either cetyl triammonium bromide (CTAB) or cetylpyridinium bromide (CPB)) was heated to 90°C. The hot aqueous surfactant solution was added to the molten YC wax with tip sonication for 3 minutes to form an emulsion. The emulsion was then rapidly cooled using an ice-bath with an overhead stirrer. The 1% w/w dispersion was prepared by diluting this emulsion stock with water. [0047] VAX 102 was diluted with water from 1 1 mg/mL stock to either 1 mg/mL or 10 mg/mL working solutions. HA was used as-received at 1 mg/mL. T1BT* solutions were prepared in a 10% DMSO / water mixture and used immediately to avoid antigen degradation.

[0048] The required volumes of each peptide solution, according to the formulation table below (Table 1), were pipetted into a sterile 2 mL plastic vial and mixed by inversion. The required volume of 1% w/w cationic particle dispersion and the required volume of water, according to the tables below, was pipetted into a separate sterile 2 mL plastic vial and mixed by inversion. Each final formulation was prepared by transferring all of the appropriate peptide solution from the first vial to the appropriate particle dispersion in the second vial by Eppendorf pipette, with drop wise addition. The final mixture was then inverted three times to yield the final formulation ready for use.

Table 1

B: TLR5 Responses to Adsorbed Particles e.g. 1

[0049] TLR5 activity of the CTAB and CPB particles incorporating flagellin and T1BT* was measured by the ability of the various formulations to trigger IL-8 cytokine production from HEK293 cells expressing the TLR5 receptor. Binding of flagellin to the TLR5 receptor triggers the release of IL-8 into the cell media in a concentration-dependent manner, and is measured via a sandwich ELISA as described below. Positive controls for IL-8 production include flagellin conjugated to 4XM2e as well as flagellin alone. IL-8 sandwich ELISA Method:

[0050] HEK293 cells placed into 96-well tissue culture plates were stimulated with each control and particle test article for 16-18 hours at 37°C. Dose-response curves of fiagellin (STF2.His) and VAX102 (STF2.4xM2e-vaccine incorporating fiagellin) starting at 40 μg/mL with a 1 : 12 dilution scheme and/or at single point 278 ng/mL dilutions were prepared and used as controls in order to compare with test articles prepared in the same manner. Wells containing cells in media only were also included to determine background IL-8 production. A

commercially available sandwich ELISA kit (BD Biosciences, Franklin Lakes, NJ) containing all essential reagents was used to measure the amount of IL-8 secreted into the culture media during the incubation period. Using the anti IL-8 antibody, an ELISA plate was coated for each tissue culture plate and incubated overnight at 2-8°C. The following day the ELISA plate was blocked to prevent non-specific binding. After the blocking step, samples (tissue culture plate supernatants) were added to the ELISA plate followed by the subsequent incubations with primary/detection antibody, secondary antibody, TMB for the colorimetric reaction and 1M H2S04 to stop this reaction. The plates were read on a plate reader (SpectraMax 190, Molecular Devices, Sunnydale, CA) and the data analyzed with relevant software (SoftMax Pro 5.2, Molecular Devices). IL-8 concentrations in medium were calculated from an IL-8 standard curve fit with a 4-parameter logistic curve.

[0051] IL-8 production in response to T1BT* and fiagellin adsorbed particles can be seen in Figure 2. Particles with CPB and CTAB formulations to which varying ratios of VAX 102 to a constant concentration of T1BT* epitopes were adsorbed were diluted and treated as points on one dose-response curve. The results in Figure 2 show that the lipid nanoparticles with Fiagellin and T1BT* electrostatically adsorbed retain TLR5 triggering activity.

C: Responses to T1BT and Fiagellin

[0052] Formulations listed in Table 2 which contained CTAB and CPB were made as described in Example 1A and Table 1. Table 2

[0053] 140 mice (BALB/c) were divided into 14 groups of 10 mice and injected subcutaneously with 0, 1 ml of the formulations described in Table 2. The first dose was administered on day 0 and a second dose was administered on day 21. Blood samples were collected from each mouse on day 20 and day 35. A standard ELISA measurement was done on individual blood samples as described below to detect the presence of anti-TlBT antibodies and anti-Flagellin antibodies. The results of the ELISA assay are described below and shown in Figures 3 and 4.

T1BT and Flagellin ELISA Method:

[0054] Costar flat-bottom ELISA plates (Corning, NY) were coated overnight with 2 μg/ml T1 BT peptide or 1 μg/ml Flagellin protein and a standard curve of mouse polyclonal IgG (AbD Serotec, Raleigh, NC, USA) at 5-fold dilutions starting at 5 μg/ml . After overnight incubation at 2-80C, plates were washed and blocked (BD Diluent with Tween 20, BD Biosciences, San Diego, CA, USA). For the T1 BT ELISAs, sera from individual mice were diluted 5.0 fold several times starting at a dilution of 1 : 16 in a separate plate. For the Flagellin ELISAs, sera from individual mice were diluted 5.0 fold starting at a dilution of 1 :50. The diluted sera samples were transferred to the blocked and washed T1BT or Flagellin coated ELISA plates. After incubation for 1.5-2 hours at 250C and washing, plates were incubated with a 1 :5000- 10,000 dilution of goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Jackson Immunochemicals West Grove, PA USA), for 30-45 minutes at 250C, washed and developed with TMB substrate (ThermoScientific) and H2S04 stop solution. Plates were read at 450 nM. The concentration of the anti-TlBT* or anti-Flagellin antibodies in the sera were calculated from the standard curve fitted using a 4-parameter logistic equation in SoftMax Pro 5.2 (Molecular Devices, Sunnyvale, CA, USA) and adjusted for the dilution factor.

Antibody Response to TIBT*

[0055] Anti-TlBT specific IgG were measured in the individual blood samples of mice injected with various formulations of CTAB and CPB particles incorporating Flagellin and TIBT*. Results for each group were expressed as the geometric mean anti-TlBT* titers with a 95% confidence interval. For the CTAB formulations, a strong anti-TlBT* IgG response (geo mean of 12.6 μg/ml) was seen in group 12 at a molar ratio of 11 1 of TIBT* to Flagellin (Fig 2, STF2:T1BT of 0.1 : 1). The control CTAB formulation in group 14 lacking the Flagellin adjuvant (STF2:T1BT of 0: 1) showed very low amounts of anti-TlBT* IgG antibodies (geo mean of 0.83 μg/ml). There was a difference of 15.2 fold between groups 12 and 14. An increase in anti- T1BT* IgG antibodies were also noted in two other groups of the CTAB formulations relative to control group 14; 3-fold higher levels at a molar ratio of 557 (group 13 STF2:T1BT of 0.02: 1) and 4-fold higher levels at a molar ratio of 37 of TIBT* to Flagellin (group 1 1 STF2:T1BT of 0.3 : 1). The levels of anti-TlBT IgG in groups 11, 12, and 13 were higher than those observed in the control group containing covalently conjugated Flagellin and TIBT* (group 2, STF2.T1BT 25 μg) The adjuvant effect of Flagellin on generating anti-TlBT* antibodies decreased at a molar ratio of 1 1 (group 10 STF2:T1BT of 1 : 1). Incorporation of Flagellin in the CPB particles showed a 1.2-1.3 fold increase (groups 7 and 8 STF: TIBT of 0.1 : 1 and 0.02: 1) respectively over control group 9. Taken together, these results may indicate that a strong antibody response to TIBT* can be generated at certain optimal ratios, below 1 : 1 of the Flagellin adjuvant to the TIBT* antigen, in the presence of particles such as CTAB. Secondly, these results demonstrate that the Flagellin adjuvant and TIBT* need not be conjugated directly to the particles.

Antibody Response to Flagellin

[0056] Anti-Flagellin antibodies were measured as described above in the individual blood samples of mice within each group at both day 20, following the first dose and at day 35 after the second dose. Results were plotted as geometric mean anti-Flagellin IgG titers for each group with a 95 % confidence interval. Figure 3 shows that anti-Flagellin antibodies are detected only after the second dose of vaccines and only in the formulations containing higher amounts of Flagellin (groups 5, 6 and 10). More significantly, none of the CTAB formulations that elicit high anti-TlBT* IgG titers (groups 11, 12 and 13) generate an anti-Flagellin response.

Similarly, the CPB formulation groups 7 and 8 that show small increases in anti-TlBT* titers also did not generate anti-Flagellin antibodies. All three positive control formulations containing Flagellin directly conjugated to TIBT* (groups 2, 3, and 4) generate high levels of anti-Flagellin antibodies.

[0057] The observation that anti-TlBT* antibodies can be made in high amounts in the absence of a Flagellin IgG response when both are presented on the CTAB or CPB particles at certain ratios is significant since it minimizes theoretical adverse reaction concerns stemming from high levels of anti-Flagellin antibodies.

Example 2

A: Manufacture of particles

[0058] Particles to which VAX102 and TIBT* or VAX102 and HA peptides were adsorbed were prepared as described in example 1 using the required volumes of each peptide solution, according to the formulation table below (Table 3).

Table 3

B: Responses to TIBT, Flagellin and HA

[0059] Formulations listed in Table 4 which contained CTAB and CPB were made as described in section A of example 1, section A and Table 3 of example 2.

Table 4:

[0060] 210 mice (BALB/c) were divided into 21 groups of 10 mice and injected

subcutaneously with 0.1 ml of the formulations described in Table 4. The first dose was administered on day 0 and a second dose was administered on day 21. Blood samples were collected from each mouse on day 20 and day 35. A standard ELISA measurement was done on individual blood samples as described below to detect the presence of anti TIBT antibodies, anti- Flagellin antibodies and anti-HA antibodies. The results of the ELISA assay are described below and shown in Figures 6, 7 and 8.

TIBT Flagellin and ELISA Method:

[0061] TIBT* and Flagellin ELISAs were done as described in section C of example 1. For the TIBT ELISAs, sera from individual mice were diluted 5.0 fold several times starting at a dilution of 1 : 10 in a separate plate. For the HA ELISAs Costar flat-bottom ELISA plates (Corning, NY) were coated overnight with 4 μg/ml HA protein and a standard curve of mouse polyclonal IgG (AbD Serotec, Raleigh, NC, USA) at 5-fold dilutions starting at 5 μg/ml . Sera for HA ELISAs were diluted 5-fold starting from a dilution of 1 : 10. All other steps of the ELISA were as described in section C of example 1. The concentration of the anti-TlBT*, anti- HA or anti-Flagellin antibodies in the sera were calculated from the standard curve fitted using a

4- parameter logistic equation in SoftMax Pro 5.2 (Molecular Devices, Sunnyvale, CA, USA) and adjusted for the dilution factor.

Antibody Response to T1BT*

[0062] Anti-TlBT specific IgG were measured in the individual blood samples of mice injected with various formulations of CTAB and CPB particles incorporating Flagellin and T1BT*. Results for each group were expressed as the geometric mean anti-TlBT* titers with a 95% confidence interval. For the CTAB formulations, the highest anti-TlBT* IgG responses are observed at a Flagellin to T1BT* ratio of 0.02: 1 and are at 1.9 fold higher levels (Fig 6) relative to the formulation without the Flagellin control (group 13 compared with group 14, Table 4). These levels of anti-TlBT IgG are 2.5 fold higher than those observed in the control group containing covalently conjugated Flagellin and T1BT* (group 2, STF2.T1BT 25 μg).

[0063] For the CPB formulations, the highest anti-TlBT* IgG titers are also seen at a Flagellin to T1BT ratio of 0.02: 1 (group 7 and Fig 6) and are 2.7 fold higher compared with the control group 8 lacking the Flagellin adjuvant.

Antibody Response to Flagellin

[0064] Anti-Flagellin antibodies were measured as described above in the individual blood samples of mice within each group at both day 20, following the first dose and at day 35 after the second dose. Results were plotted as geometric mean anti-Flagellin IgG titers for each group with a 95 % confidence interval (Fig 7). As observed in example 1, there are no anti-Flagellin antibodies being generated even at day 35 against the CTAB formulation that shows a small increase in anti-TlBT* IgG titers (Flagellin to T1BT ratio of 0.02: 1). There is approximately a

5- fold increase in the level of anti-Flagellin antibodies at day 35 in the CPB formulation (0.02: 1 of Flagellin to T1BT) that shows the highest increase in anti-TlBT* titers relative to the non- Flagellin control.

Antibody response to a complex HA antigen:

[0065] The hemagglutinin (HA) antigen from influenza B strain (B Brisbane) conjugated to Flagellin was incorporated into the CTAB nanoparticles at various concentrations as shown in Table 4 to determine the immune response level to the more complex HA antigen compared to the much smaller T1BT* peptide. 69-fold higher levels of anti-HA antibodies were detected in the formulation containing a 0.1 : 1 ratio of Flagellin to HA relative to the formulation without any HA (0.3:0 Figure 8). The highest IgG titer was observed in the 0.1 : 1 STF2: HA formulation. These responses tended to decrease with a higher amount of Flagellin in the formulation. In comparison, the control HA protein without the nanoparticles showed a highly variable response between the individual mice within the group.

Antibody response to the flagellin portion of a complex HA antigen

[0066] Anti-flagellin antibodies were measured in both the day 20 and day 35 sera samples. About 24 μg/ml of anti-flagellin antibodies are detected in the formulation (0.1 : 1) that shows the highest anti-HA response levels (Figs 9A and B).

Claims

CLAIMS What is claimed is:

1. A composition comprising a particle to which one or more TLR5 agonists and one or more antigens is attached.

2. The composition of claim 1 wherein the particle is comprised of carnauba wax.

3. The composition of claim 2 wherein the ratio of the TLR5 agonist to antigen is less than about 1 :5 on a weight to weight basis

4. The composition of claim 2 wherein the ratio of the TLR5 agonist to antigen is less than about 1 :20 on a molecule to molecule basis

5. The composition of claim 3 wherein the TLR5 agonist is flagellin.

6. The composition of claim 5 wherein the antigen is selected from the group consisting of T1BT and HA.

7. The composition of claim 2 wherein the carnauba wax is comprised of aliphatic esters, diesters of 4-hydroxycinnamic acid ω-hydroxycarboxylic acids and fatty acid alcohols.

8. The composition of claim 1 further comprising a surfactant.

9. The composition of claim 8 wherein the surfactant is selected from the group consisting of cetyl triammonium bromide (CTAB), N-[l-(2,3-Dioleoyloxy)]-N,N,N- trimethylammonium propane methylsulfate DOTAP, cetylpyridinium bromide (CPB), Tween 20, Tween 80 (polyoxyethylene sorbitan monoloaurate), Brij 70, sodium stearate, sodium myristate, sodium dodecyl sulfate, dioctyl sodium sulfosuccinate and

combinations thereof.

10. The composition of claim 9 wherein the surfactant is cetyl trimethylammonium bromide.

1 1. The composition of claim 9 wherein the surfactant is cetylpyridinium bromide.

12. The composition of claim 9 wherein the TLR5 agonist and antigens are attached to the particle through electrostatic interactions.

13. The composition of claim 9 wherein the TLR5 agonist and antigens are attached to the particle through covalent attachment

14. The composition of claim 9 wherein the TLR5 agonist and antigens are attached to the particle through hydrophilic interactions.

15. The composition of claim 9 wherein the TLR5 agonist and antigens are attached to the particle through hydrophobic interactions.

16. The composition of claim 9 wherein the TLR5 agonist and antigens are attached to the particle through non-covalent interactions.

17. A method of making a vaccine formulation comprising:

(a) preparing a hot aqueous surfactant solution;

(b) adding the hot surfactant to a molten lipid to form a hot lipid/surfactant mixture;

(c) adding the hot lipid/surfactant mixture to an aqueous phase to form a second mixture;

(d) sonicate the second mixture;

(e) cool the second mixture;

(f) add TLR5 agonist and antigens.

18. The method of claim 17 wherein the antigen is selected from the group consisting of T1BT and HA.

19. The method of claim 17 wherein the lipid is carnauba wax.

20. The method of claim 19 wherein the carnuba wax is comprised of aliphatic esters,

diesters of 4-hydroxycinnamic acid co-hydroxycarboxylic acids and fatty acid alcohols.

21. The method of claim 17 wherein the surfactant is selected from the group consisting of cetyl triammonium bromide (CTAB), N-[l-(2,3-Dioleoyloxy)]-N,N,N- trimethylammonium propane methylsulfate DOTAP, cetylpyridinium bromide (CPB), Tween 20, Tween 80 (polyoxyethylene sorbitan monoloaurate), Brij 70, sodium stearate, sodium myristate, sodium dodecyl sulfate, dioctyl sodium sulfosuccinate and combinations thereof.

22. The method of claim 21 wherein the surfactant is cetyl triammonium bromide.

23. The method of claim 21 wherein the surfactant is cetylpyridinium bromide.

24. The method of claim 17 wherein the TLR5 agonist is flagellin.