Liposomal adjuvant compositions for human papillomavirus vaccines

Abstract

The present disclosure provides a vaccine composition that includes, among other things, HPV virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82 and a liposomal adjuvant, and methods of inducing an immune response to HPV or methods of preventing infection of or reducing the likelihood of infection by HPV using the compositions.

Description

LIPOSOMAL ADJUVANT COMPOSITIONS FOR HUMAN PAPILLOMAVIRUS VACCINESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application 63 / 730,534, filed December 11, 2024, which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The invention relates generally to the prevention of human papillomavirus (HPV) disease. More specifically, the invention relates to a composition comprising HPV virus-like particles (VLPs) and a liposomal adjuvant, which can be administered as a vaccine. The liposomal adjuvant comprises a glycolipid, such as a glucopyranosyl lipid A (“GLA”) and a saponin, such as QS-21. Further provided are methods of using the disclosed compositions and formulations.BACKGROUND

[0003] Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect the skin and internal squamous mucosal epithelia of men and women. HPVs are classified based on their carcinogenic properties. HPVs include major (LI) and minor (L2) capsid proteins. Over 200 distinct HPV genotypes have been identified (Li et al., "‘Rational design of a triple-type human papillomavirus vaccine by compromising viral-type specificity,” Nature, 9:5360 (2018)), many of which have been associated with pathologies ranging from benign proliferative warts to malignant carcinomas of the cervix (for review, see McMurray et al., Int. J. Exp. Pathol. 82(1): 15-33 (2001)). Those HPV types labeled as “high-risk” include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68, and 59. (Chan et al., “Human Papillomavirus Infection and Cervical Cancer:Epidemiology, Screening, and Vaccination — Review of Current Perspectives,” Journal of Oncology, vol. 2019, Article ID 3257939, 2019.)

[0004] HPV is the primary etiological agent in cervical cancer, one of the most common cancer types in women, as well as squamous cell carcinomas of the anus, tonsil, tongue, vulva, vagina, and penis. HPV16 and HPV18 are well known as the most virulent of the high-risk HPV types as they cause approximately 70% of all invasive cervical cancer in the world.

[0005] Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode up to eight early (El- E7) and two late (L1-L2) genes. The LI protein is the major capsid protein and has a Mw of 55-60 kDa. Expression of the LI protein or a combination of the LI andL2 proteins in yeast, insect cells, mammalian cells or bacteria leads to self-assembly of virus-like particles (VLPs) (for review, see Schiller and Roden, in Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information, pp 101-12 (1996)).

[0006] VLPs are morphologically similar to authentic virions and are capable of inducing high titers of neutralizing antibodies upon administration into animals or humans. Because VLPs do not contain the potentially oncogenic viral genome, they present a safe alternative to the use of live virus in HPV vaccine development (for review, see Schiller and Hidesheim, J Clin. Virol. 19: 67-74 (2000)).

[0007] VLP-based vaccines have proven to be effective at inducing immune responses in human subjects vaccinated with bivalent HPV 16 and 18 (Harper et al. Lancet 364 (9447): 1757-65 (2004)), quadrivalent HPV 6, 11, 16, and 18 (Villa et al. Vaccine 24: 5571-5583 (2006)) and multi-valent HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 VLP-based vaccines. Three approved VLP-based vaccines against HPV are administered according to 2 or 3 dose regimens. CERVARIX® (GlaxoSmithKline Biologicals, Rixensart, Belgium), is a bivalent vaccine protective against HPV 16 and 18. GARDASIL® and GARDASIL®9 (Merck & Co., Inc., Rahway, NJ, USA) protect against two and seven additional HPV types, respectively, and prevent additional HPV-related anogenital diseases, including wart formation. The additional five strains in GARDASIL®9 compared to GARDASIL® are “high-risk” strains, which increase protection from about 70% of anogenital malignancies to about 90%. (Id., M. Nygard. et al., " Evaluation of the long-term antihuman papillomavirus 6 (HPV6), 11, 16, and 18 immune responses generated by the quadrivalent HPV vaccine,” Clinical and Vaccine Immunology, vol. 22, no. 8, pp. 943-948, 2015.)

[0008] Though improving, worldwide HPV vaccination rates remain suboptimal. The worldwide coverage of HPV vaccination can be improved by reducing the number of healthcare practitioner visits required for vaccination, increasing education on HPV disease prophylaxis, and alleviating the social stigma associated with vaccination.

[0009] Moreover, licensed HPV vaccines currently utilize aluminum-containing derivatives as adjuvants to increase immunogenicity. Even though aluminum adjuvants increase immunogenic responses from baseline, it is unknown whether the immunogenic response is sufficient for higher valency HPV vaccines. Therefore, there is a need to identify other adjuvants that can provide increased immunogenicity for multivalent HPV vaccines over the current aluminum adjuvant.SUMMARY OF THE INVENTION

[0010] The invention provides a composition comprising virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and a liposomal adjuvant, wherein the liposomal adjuvant comprises a glycolipid and a saponin. In an embodiment, the VLPs of at least one type of HPV are adsorbed on an aluminum adjuvant.

[0011] The invention further provides a composition wherein the composition is made by mixing an HPV vaccine and the liposomal adjuvant; wherein the HPV vaccine comprises HPV VLPs of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and a pharmaceutically acceptable earner and the liposomal adjuvant comprises a glucopyranosyl lipid A (“GLA”) and QS-21. In some embodiments, the composition also includes an aluminum adjuvant. In some embodiments, the VLPs are adsorbed on an aluminum adjuvant.

[0012] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula I:OHFormula I,wherein:Ri is -P(O)(OH)2;R2is selected from H, -C(O)CH2CH(OH)C11alkyl, and -C(O)CH2CH(OC(O)C11-C13alkyl)C11alkyl;R3is -C(O)CH2CH(OC(O)C11-C13alkyl)C11alkyl;R4is selected from H, -C(O)CH2CH(OH)C11alkyl and -C(O)CH2CH(OC(O)C11-C13alkyl)C11alkyl;R5is selected from -C(O)CH2CH(OH)C11alkyl and -C(O)CH2CH(OC(O)C13-C15alkyl)C11alkyl; andRe is H.

[0013] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula II:Formula II,wherein:R1is selected from H and -C(O)C11-C13alkyl;R2is C11alkyl;R3is C11-C13alkyl;R4is C11alkyl;R5is selected from H and -C(O)CH2CH(OH)C11alkyl;R6is selected from H and C(O)C13-C15alkyl; andR7is C11alkyl,or a pharmaceutically accepted salt thereof.

[0014] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula II A:Formula IIA,wherein:R1is selected from H and -C(O)C11-C13alkyl;R2is C11alkyl;R3is C11-C13alkyl;R4is C11alkyl;R5is selected from H and -C(O)CH2CH(OH)C11alkyl;R6is selected from H and C(O)C13-C15alkyl; andR7is C11alkyl,or a pharmaceutically accepted salt thereof.

[0015] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula IIIA:MeFormula IIIA.

[0016] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula IV:Me MeFormula IV.

[0017] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula V:MeFormula V.

[0018] The invention further provides compositions, wherein the glycolipid has the structure set forth in Formula VI:MeFormula VI.

[0019] In an embodiment, the invention further provides compositions, wherein the liposomal adjuvant further comprises l,2-dioleoyl-sn-glycero-3-phosphocholine (“DOPC”). In another embodiment, the invention provides compositions, wherein the liposomal adjuvant further comprises cholesterol. In another embodiment, the invention provides compositions, wherein the composition further comprises a buffer. In another embodiment, the invention provides compositions, wherein the composition comprises a salt.

[0020] The invention further provides a method of preventing infection of or reducing the likelihood of infection of a human patient by a human papillomavirus (HPV) comprising administration to the patient the composition comprising virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and a liposomal adjuvant, whereinthe liposomal adjuvant comprises a glycolipid and a saponin. In an embodiment, the VLPs of at least one type of HPV are adsorbed on an aluminum adjuvant.

[0021] The invention further provides a kit comprising: (a) a human papillomavirus (HPV) vaccine comprising VLPs of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82; and (b) a liposomal adjuvant, wherein the liposomal adjuvant comprises a glycolipid and a saponin.DEFINITIONS

[0022] As used throughout the specification and in the appended claims, the singular forms "a." “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0023] As used throughout the specification and appended claims, the following abbreviations and definitions apply:HPV human papillomavirusID intradermalIM intramuscularMw molecular weightVLP(s) virus-like particle(s) (VLPs)v / v volume per volume

[0024] As used throughout the specification and appended claims, the following definitions and abbreviations apply:

[0025] AAHS: As used herein, the term “AAHS” refers to an amorphous aluminum hydroxyphosphate sulfate adjuvant.

[0026] About: As used herein, the term “about,” when used herein in reference to a value, refers to a value that is the same as or, in context, is similar to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the absolute amount and / or relative degree of difference encompassed by “about” in that context. For example, in some embodiments, the term “about” encompasses a range of values that within 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%, 1%, or less of the referenced value.

[0027] Adjuvant. As used herein, the term “adjuvant” refers to a composition or compound that is capable of enhancing the immune response against an antigen of interest. Adjuvants are substances or combinations of substances that are used in conjunction with a vaccine antigen to enhance (e.g., increase, accelerate, prolong and / or possibly target) the specific immune response to the vaccine antigen or modulate to a different type (e.g., switch a Thl immune response to aTh2 response, or a humoral response to a cytotoxic T cell response) in order to enhance the clinical effectiveness of the vaccine. In some embodiments, the adjuvant modifies (Thl / Th2) the immune response. In some embodiments, the adjuvant boosts the strength and longevity of the immune response. In some embodiments, the adjuvant broadens the immune response to a concomitantly administered antigen. In some embodiments, the adjuvant is capable of inducing strong antibody and T cell responses. In some embodiments, the adjuvant is capable of increasing the polyclonal ability of the induced antibodies. In some embodiments, the adjuvant is used to decrease the amount of antigen necessary to provoke the desired immune response and provide protection against the disease. In some embodiments, the adjuvant is used to decrease the number of injections needed in a clinical regimen to induce a durable immune response and provide protection against the disease. Adjuvant containing formulations described herein may demonstrate enhancements in humoral and / or cellular immunogenicity of vaccine antigens, for example, subunit vaccine antigens. Adjuvants of the invention are not used as vehicles to deliver antigens, antibodies, active pharmaceutical ingredients (APIs), or VLPs.

[0028] Administration. As used herein, the term ’administration" refers to the act of providing an active agent, composition, or formulation to a subject. Exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), rectal, vaginal, oral mucosa (buccal), ear, by injection (e.g., intravenously (IV). subcutaneously, intratumorally, intraperitoneally, intramuscularly (IM), intradermally (ID) and the like.

[0029] Agent. As used herein, the term “agent” refers to a particle, compound, molecule, or entity of any chemical class including, for example, a VLP, a small molecule, polypeptide (e.g., a protein), polynucleotide (e.g., a DNA polynucleotide or an RNA polynucleotide), saccharide, lipid, or a combination or complex thereof. In some embodiments, the term “agent” can refer to a compound, molecule, or entity that includes a polymer, or a plurality thereof.

[0030] Alkyl. As used herein, the term “alkyd” refers to a straight chain, cyclic or branched saturated aliphatic hydrocarbon having the specified number of carbon atoms. In one embodiment, an alkyl group contains from 8 to 24 carbon atoms (C8-C24 alkyl). In one embodiment, an alkyl group is linear. In another embodiment, an alkyl group is branched. In another embodiment the alkyl group is unsubstituted.

[0031] Antibody. As used herein, the term “antibody” (or “Ab”) refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, and chimeric antibodies.

[0032] Antigen: As used herein, the term ‘'antigen” refers to any antigen that can generate one or more immune responses. The antigen may be a protein (including recombinant proteins), VLP, polypeptide, or peptide (including synthetic peptides). The antigen may be one that generates a humoral and / or CTL immune response.

[0033] API. As used herein, the term ‘‘API” refers to an active pharmaceutical ingredient, e.g.. HPV VLPs, which is a component of the compositions or formulations disclosed herein that is biologically active (e.g., capable of inducing an appropriate immune response) and confers a therapeutic or prophylactic benefit to a person or animal in need thereof. As used herein, an API is a vaccine active ingredient.

[0034] Co-administration: As used herein, the term “co-administration” or “co-administering” in relation to the liposomal adjuvant and a pharmaceutical formulation (e.g., an HPV vaccine) refers to administration of a liposomal adjuvant and a pharmaceutical formulation (e.g., an HPV vaccine) concurrently, i.e., simultaneously in time, or sequentially, i.e., administration of an HPV vaccine followed by administration of a liposomal adjuvant (or vice versa). That is, after administration of the HPV vaccine (or liposomal adjuvant), the liposomal adjuvant (or HPV vaccine) can be administered substantially immediately after the HPV vaccine (or liposomal adjuvant) or the liposomal adjuvant (or the HPV vaccine) can be administered after an effective time period after the HPV vaccine (or liposomal adjuvant); the effective time period is the amount of time period is generally within 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, or 60 minutes.

[0035] Composition. As used herein, the term “composition” refers to a formulation containing an active pharmaceutical or biological ingredient (for example, a virus-like particle (VLP) of at least one type of human papillomavirus (HPV) and a liposomal adjuvant), along with one or more additional components. The term “composition” is used interchangeably with “pharmaceutical composition” and “formulation.” The compositions can be liquid or solid (e.g., lyophilized). Additional components that may be included as appropriate include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, lyo-protectants. solubilizers, emulsifiers, salts, adjuvants, tonicity enhancing agents, delivery vehicles, and anti -microbial preservatives. Compositions are nontoxic to recipients at the dosages and concentrations employed.26095

[0036] Dose: As used herein, the term “dose” means a quantity of an agent, API, formulation, or pharmaceutical composition administered or recommended to be administered at a particular time.

[0037] Effective amount. As used herein, the term “effective amount” refers to an amount of active agent, API, or composition that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, an effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “effective amount” does not in fact require successful treatment be achieved in a particular subject. Rather, an effective amount can be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to subjects in need of such treatment. In some embodiments, reference to an effective amount can be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, an effective amount of a particular agent or therapy can be formulated and / or administered in a single dose. In some embodiments, an effective agent can be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0038] HPV and PV As used herein, the terms “HPV” and “PV” refer to human papillomavirus and papillomavirus, respectively.

[0039] Immunogenic. As used herein, the term “immunogenic” or “immunogenicity” refers to the ability of an antigen to provoke an immune response in a subject. The term “immunogenic composition” refers to the ability of an agent, API, formulation, or composition to provoke an immune response in a subject. The pneumococcal conjugate compositions of the invention are immunogenic compositions.

[0040] In need of treatment. Those “in need of treatment” include those previously exposed to or infected with human papillomavirus or papillomavirus, those who were previously vaccinated against human papillomavirus or papillomavirus, as well as those prone to have an infection or any person in which a reduction in the likelihood of infection is desired, e.g., the immunocompromised, the elderly, children, adults, or healthy individuals.

[0041] Lipid. As used herein, the term “lipid” refers to any of a group of organic compounds that are esters of fatty acids and are characterized by being insoluble in water or having low26095solubility in water but may be soluble in many organic solvents. Lipids can be divided in at least three classes: (1) “simple lipids,” which include, e.g., fats and oils as well as waxes; (2) “compound lipids,” which include, e.g., phospholipids and glycolipids; and (3) “derived lipids,” which include, e.g., steroids.

[0042] Patient: As used herein, the term “patient” refers to any human being that is to receive the HPV vaccines, or pharmaceutical compositions, described herein. As defined herein, “patient” includes those already infected with one or more types of HPV as well as those in which infection with one or more types of HPV is to be prevented.

[0043] Pharmaceutically acceptable: As used herein with respect to a carrier, diluent, or excipient of a pharmaceutical composition, the term “pharmaceutically acceptable” indicates that a earner, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0044] Pharmaceutical composition. As used herein, the term “pharmaceutical composition,” refers to a composition containing an active pharmaceutical or biological ingredient, along with one or more additional components, e.g., a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. As used herein, the terms “pharmaceutical formulation” and “formulation” are used interchangeably with “pharmaceutical composition.” In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. The pharmaceutical compositions or formulations can be liquid or solid (e.g., lyophilized). Additional components that may be included as appropriate include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, lyo-protectants. solubilizers, emulsifiers, salts, adjuvants, tonicity enhancing agents, deliver}’ vehicles, and anti-microbial preservatives. The pharmaceutical compositions or formulations are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, a pharmaceutical composition can be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity;26095intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary. and to other mucosal surfaces.

[0045] Subject: As used herein, the term “subject’’ refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0046] Surfactants’. As used herein, the term “surfactants” refers to stabilizing ingredients in a multi-component liposomal adjuvant formulation and include the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens, especially PS-20 and PS-80), copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers (poloxamers); octoxynols, which can vary in the number of repeating ethoxy (oxy-1, 2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)poly ethoxyethanol (IGEPAL CA-630 / NP-40); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span-85, Tween-85 or [2-[(2A,35'.4A)-4-hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec-9-enoyl] oxy ethyl] (Z)-octadec-9-enoate) and sorbitan monolaurate. In an embodiment, surfactants are selected from sorbitan esters and poloxamers. In an embodiment, surfactants are selected from polysorbate-20 (PS-20) and polysorbate-80 (PS-80).

[0047] Therapeutically Effective Amount: As used herein, the term “therapeutically effective amount” (or “therapeutically effective dose”) refers to an amount of the active ingredient (e.g., therapeutic protein, vaccine, or antibody) sufficient to produce the desired therapeutic effect in a human or animal, e.g., the amount necessary to elicit an immune response, treat, cure, prevent, or inhibit development and progression of a disease or the symptoms thereof and / or the amount necessary’ to ameliorate symptoms or cause regression of a disease. Therapeutically effective amount may vary depending on the structure and potency of the active ingredient and the26095contemplated mode of administration. One of skill in the art can readily determine a therapeutically effective amount of a given antibody or therapeutic protein or vaccine antigen.

[0048] Vaccine. As used herein, the term ‘"vaccine” or “vaccine composition” refers to a substance or preparation used to stimulate the production of antibodies and provide immunity against one or several diseases, prepared from the causative agent of a disease, its products, or a synthetic substitute, treated to act as an antigen without inducing the disease. A vaccine composition may include at least one antigen or VLP in a pharmaceutically acceptable vehicle useful for inducing an immune response in a subject. The vaccine composition is administered by doses and techniques known to those skilled in the pharmaceutical or veterinary fields, taking into account factors such as the age, sex, weight, species, and condition of the recipient animal and the route of administration.

[0049] Valent: As used herein, the term "‘valent” refers to the presence of a specified number of antigens in a vaccine. For example, the terms bi-valent, bivalent, 2 valent, or 2-valent refer to two different antigens. Similarly, the terms quadrivalent, 4 valent, or 4-valent refer to four different antigens, the terms nonaval ent, 9 valent or 9-valent refer to nine different antigens, the terms 14-valent or 14 valent refer to fourteen different antigens, and the terms 20 valent or 20-valent refer to twenty different antigens.

[0050] Virus-Like Particles. As used herein, the term “virus-like particles” or “VLPs” refers to agents that are morphologically similar to authentic virions or provide an arrayed display of an antigen and are capable of inducing high antibody neutralization ratings after administration in an animal. VLPs lack the viral genetic material of the authentic virions and are thus non-infectious.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG 1 A is a graphical representation of HPV VLP 6 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0052] FIG IB is a graphical representation of HPV VLP 11 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0053] FIG 1 C is a graphical representation of HPV VLP 16 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.26095

[0054] FIG ID is a graphical representation of HPV VLP 18 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0055] FIG IE is a graphical representation of HPV VLP 31 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0056] FIG IF is a graphical representation of HPV VLP 33 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0057] FIG 1G is a graphical representation of HPV VLP 35 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0058] FIG 1H is a graphical representation of HPV VLP 39 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9

[0059] FIG II is a graphical representation of HPV VLP 45 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0060] FIG 1J is a graphical representation of HPV VLP 51 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0061] FIG IK is a graphical representation of HPV VLP 52 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0062] FIG IL is a graphical representation of HPV VLP 56 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0063] FIG IM is a graphical representation of HPV VLP 58 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.

[0064] FIG IN is a graphical representation of HPV VLP 59 antibody levels in Rhesus Macaques after two-doses of a 14vHPV vaccine alone or in combination with a liposomal adjuvant. See Example 9.26095DETAILED DESCRIPTION

[0065] The invention generally relates to compositions that include an HPV vaccine antigen and an adjuvant, wherein the adjuvant comprises a glycolipid, such as a glucopyranosyl lipid A (‘ GLA”) and a saponin, such as QS-21. In one aspect, the invention provides a composition comprising virus-like particles (VLPs) of at least one ty pe of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55. 56, 58, 59, 66, 68, 69, 70, 73, and 82 and liposomal adjuvant, wherein the adjuvant comprises a glycolipid, such as a glucopyranosyl lipid A (‘'GLA’’) and a saponin, such as QS-21. In some embodiments, the composition also includes an aluminum adjuvant. In some embodiments, the VLPs are adsorbed on an aluminum adjuvant.The Liposomal Adjuvant

[0066] The liposomal adjuvant of the invention is a multi-component composition composed of structural lipids (such as l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol) in combination with a glycolipid (e.g., Glucopyranosyl Lipid A) and saponin molecules (such as, Quillaja Saponaria 21 (QS-21).Structural Lipids

[0067] In an embodiment, a combination of lipids are included to provide structure for the liposomal adjuvant. In an embodiment, a phospholipid provides structure for the liposomal adjuvant. In an embodiment, a sterol provides structure for the liposomal adjuvant. In an embodiment, a phospholipid and a sterol provide structure for the liposomal adjuvant.

[0068] In an embodiment, the liposomal adjuvant includes a phospholipid. In an embodiment, the phospholipid is DOPC. In an embodiment, the liposomal adjuvant may include a phospholipid selected from: phospholipids, aminolipids and sphingolipids. In some embodiments, the structural lipid includes a phospholipid selected from: phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleryl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. In some embodiments, the liposomal adjuvant includes a neutral lipid selected from: sphingolipid, glycosphingolipid families, diacylglycerols and S-acyloxyacids. In some embodiments, the liposomal adjuvant includes a neutral lipid selected from: phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine. monoacyl-phosphatidylcholine (lyso PC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), N-acyl-PE, phosphoinositides, and phosphosphingolipids.26095In some embodiments, the structural lipid includes a neutral lipid selected from: phosphatidic acid (DMPA, DPP A, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC. DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidyl ethanolamine (DMPE, DPPE, DSPE DOPE), and phosphatidylserine (DOPS). In some embodiments, the liposomal adjuvant includes a neutral lipid selected from: fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (Cl 8: 1), linoleic acid (C18:2), linolenic acid (C 18:3), arachidonic acid (C20:4), C20:0. C22:0 and lecithin. In some embodiments, the phospholipid includes l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0069] In some embodiments, the liposomal adjuvant includes 40-90 mole% phospholipid. In some embodiments, the liposomal adjuvant includes 45-85 mole% phospholipid. In some embodiments, the liposomal adjuvant includes 50-85 mole% phospholipid. In some embodiments, the liposomal adjuvant includes 55-75 mole% phospholipid. In some embodiments, the liposomal adjuvant includes 60-70 mole% phospholipid. In some embodiments, the liposomal adjuvant includes 60-65 mole% phospholipid. In some embodiments, the liposomal adjuvant includes about 65 mole% phospholipid. In an embodiment, the phospholipid is DOPC.

[0070] In some embodiments, the liposomal adjuvant includes about 60 pg / mL to about 300 mg / mL phospholipid. In some embodiments, the liposomal adjuvant includes about 80 pg / mL to about 50 mg / mL phospholipid. In some embodiments, the liposomal adjuvant includes about 1 mg / mL to about 10 mg / mL phospholipid. In some embodiments, the liposomal adjuvant includes about 2.5 mg / mL to about 3.5 mg / mL phospholipid. In some embodiments, the liposomal adjuvant includes about 3.0 mg / mL phospholipid. In an embodiment, the phospholipid is DOPC.

[0071] In some embodiments, the liposomal adjuvant includes a sterol, such as cholesterol. In some embodiments, the structural lipid includes cholesterol. In some embodiments, the liposomal adjuvant includes 10-40 mole% cholesterol. In some embodiments, the liposomal adjuvant includes 15-25 mole% cholesterol. In some embodiments, the liposomal adjuvant includes 10-20 mole% cholesterol. In some embodiments, the liposomal adjuvant includes 20-30 mole% cholesterol. In some embodiments, the liposomal adjuvant includes 10-15 mole% cholesterol. In some embodiments, the liposomal adjuvant includes 25-35 mole% cholesterol. In some embodiments, the liposomal adjuvant includes 30-35 mole% cholesterol. In some embodiments, the liposomal adjuvant includes about 30 mole % cholesterol.

[0072] In some embodiments, the liposomal adjuvant includes about 0.05 pg / mL to about 10 mg / mL sterol. In some embodiments, the liposomal adjuvant includes about 0.2 pg / mL to about 3.0 mg / mL sterol. In some embodiments, the liposomal adjuvant includes about 0.25 mg / mL to26095about 2.0 mg / mL sterol. In some embodiments, the liposomal adjuvant includes about 0.5 mg / mL to about 1.5 mg / mL sterol. In some embodiments, the liposomal adjuvant includes about 0.75 mg / mL sterol. In an embodiment, the sterol is cholesterol.Glycolipid

[0073] In an embodiment, liposomal adjuvant composition includes a glycolipid having the structure set forth in Formula I;OHFormula I,wherein: Ri is -P(O)(OH)2;R2is selected fromH, -C(O)CH2CH(OH)Cn alkyl, and -C(O)CH2CH(OC(O)Cn-Ci3 alkyljCn alkyl;R3 is -C(O)CH2CH(OC(O)Cu-Ci3 alkyljCn alkyl;R4is selected from H, -C(O)CH2CH(OH)Cn alkyl, and - C(O)CH2CH(OC(O)Cn-Ci3 alkyljCn alkyl;Rs IS selected from -C(O)CH2CH(OH)Cn alkyl, and -C(O)CH2CH(OC(O)Ci3-Ci5 alkyl)C11 alkyl; andwherein Re is H,or a pharmaceutically acceptable salt thereof.

[0074] In an embodiment, the glycolipid has the structure set forth in Formula II;Formula II26095wherein:Ri is H and -C(0)Cn-Ci3 alkyl;R2is C11alkyl;R3 is C11-C13 alkyl;R4is C11alkyl;Rs is H or -C(O)CH2CH(OH)Cn alkyl;Rs is H or -C(O)Ci3-Ci5 alkyl; andR7is C11alkyl,or a pharmaceutically acceptable salt thereof.

[0075] In an embodiment, the glycolipid has the structure set forth in Formula IIA;Formula IIAwherein:Ri is H, -C(O)Cn-Ci3 alkyl;R2is C11alkyl;R3is C11-C13alkyl;R4is C11alkyl;R5IS H or -C(O)CH2CH(OH)CII alkyl;Rs is H or -C(O)Ci3-Ci5 alky l; andR7is C11alkyl,or a pharmaceutically acceptable salt thereof.

[0076] In an embodiment, the glycolipid has the structure set forth in Formula III:26095MeFormula III, or a pharmaceutically acceptable salt thereof.

[0077] In an embodiment, the glycolipid has the structure set forth in Formula IIIA:26095MeFormula IIIA, or a pharmaceutically acceptable salt thereof.

[0078] In an embodiment, the glycolipid has the structure set forth in Formula IV:26095Me MeFormula IV, or a pharmaceutically acceptable salt thereof.

[0079] In an embodiment, the glycolipid has the structure set forth in Formula IVA:26095Formula IVA, or a pharmaceutically acceptable salt thereof.

[0080] In an embodiment, the glycolipid has the structure set forth in Formula V:26095MeFormula V, or a pharmaceutically acceptable salt thereof.

[0081] In an embodiment, the glycolipid has the structure set forth in Formula VI:MeFormula VI or a pharmaceutically acceptable salt thereof.

[0082] In one aspect under any one structures set forth in Formulas I-VI, the pharmaceutically acceptable salt is an ammonium, sodium, potassium, calcium, or organic amine salt such as an ethylamine, di ethylamine, or tri ethylamine salt. In one aspect under any one of Formulas I-VI, the pharmaceutically acceptable salt is an ammonium salt.

[0083] In some embodiments, the liposomal adjuvant includes 0.25-20 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.5-10 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.25-5 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.25-2.5 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.5-2.25 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.5-2.0 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.75- 1.90 mole% glycolipid. In some embodiments, the liposomal adjuvant includes 0.95-1.85 mole%glycolipid. In some embodiments, the liposomal adjuvant includes 1.0-1.75 mole% glycolipid. In some embodiments, the liposomal adjuvant includes about 1.44 mole % glycolipid. In an embodiment, the glycolipid is GLA.

[0084] In an embodiment, the glycolipid is selected from the structure set forth in Formula III, Formula IIIA, Formula IV, Formula IVA, Formula V, and Formula VI, or combinations thereof.

[0085] In some embodiments, the liposomal adjuvant includes about 0.01 pg / mL to about 10 mg / mL glycolipid. In some embodiments, the liposomal adjuvant includes about 0.02 pg / mL to about 3.0 mg / mL glycolipid. In some embodiments, the liposomal adjuvant includes about 0.03 mg / mL to about 2.0 mg / mL glycolipid. In some embodiments, the liposomal adjuvant includes about 0.05 mg / mL to about 1.0 mg / mL glycolipid. In some embodiments, the liposomal adjuvant includes about 0.15 mg / mL glycolipid. In an embodiment, the glycolipid is GLA.

[0086] In an embodiment, the glycolipid is selected from the structure set forth in Formula III, Formula IIIA, Formula IV, Formula IVA, Formula V, and Formula VI, or combinations thereof. Saponins

[0087] In an embodiment, the liposomal adjuvant includes a saponin. In an embodiment, the saponin is a derivative of Quillaja saponaria Molina quil A, suitably an immunologically active fraction of Quil A, such as QS7, QS17, QS18 or QS-21, in particular QS-21. In an embodiment, the Quil A saponin is selected from QS7, QS17, QS18, QS-21, and combinations thereof. In an embodiment, the saponin is selected from QS7, QS-21, and combinations thereof. In one embodiment, the saponin is selected from QS-17, QS-18, and QS-21. or combinations thereof. In an embodiment, the saponin is QS-21.

[0088] In some embodiments QS-21 comprises commercially available QS-21 purified from Quillaja saponaria Molina tree bark. Commercially available QS-21 purified from Quillaja saponaria Molina tree bark can comprise one or more of QS-21-H, QS-21-Apiose and QS-21-xylose as depicted below:

[0089] In one embodiment, the QS-21 comprises QS-21-H, QS-21-Apiose and QS-21-xylose. In another embodiment, the QS-21 comprises QS-21-Apiose and QS-21-xylose. See N. E. Jacobsen et aL. Carbohydr. Res.. 280 (1996) 1-14.26095

[0090] In an embodiment, the saponin, such as Quil A and in particular QS-21, is at least 90% pure, such as at least 95% pure, especially at least 98% pure, or in particular 99% pure.

[0091] In some embodiments, the liposomal adjuvant includes 0.25-20 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.5-10 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.25-5 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.25-2.5 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.5-2.25 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.5-2.0 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.75-1.75 mole% saponin. In some embodiments, the liposomal adjuvant includes 0.95-1.5 mole% saponin. In some embodiments, the liposomal adjuvant includes 1.0- 1.5 mole% saponin. In some embodiments, the liposomal adjuvant includes about 1.27 mole % saponin. In an embodiment, the saponin is QS-21.

[0092] In some embodiments, the liposomal adjuvant includes about 0.01 pg / mL to about 10 mg / mL saponin. In some embodiments, the liposomal adjuvant includes about 0.02 pg / mL to about 3.0 mg / mL saponin. In some embodiments, the liposomal adjuvant includes about 0.03 mg / mL to about 2.0 mg / mL saponin. In some embodiments, the liposomal adjuvant includes about 0.05 mg / mL to about 1.0 mg / mL saponin. In some embodiments, the liposomal adjuvant includes about 0.15 mg / mL saponin. In an embodiment, the saponin is QS-21.Buffer

[0093] In some embodiments, the liposomal adjuvant includes a buffer. In some embodiments, the buffer is selected from any pharmaceutically acceptable buffer, including acetic acid, histidine, citrate, Bis-Tris, HEPES, phosphate, MES, disodium hydrogen phosphate dihydrate, potassium dihydrogen phosphate, salt, and combinations thereof. In some embodiments, the buffer is present in an amount of ImMol to about 100 mMol.

[0094] In some embodiments, the liposomal adjuvant includes about 60 pg / mL to about 300 mg / mL buffer. In some embodiments, the liposomal adjuvant includes about 80 pg / mL to about 50 mg / mL buffer. In some embodiments, the liposomal adjuvant includes about 1 mg / mL to about 25 mg / mL buffer. In some embodiments, the liposomal adjuvant includes about 2.5 mg / mL to about 20 mg / mL buffer. In some embodiments, the liposomal adjuvant includes about 13.0 mg / mL buffer. In an embodiment, the buffer includes about 1.0 mg / mL to about 20 mg / mL disodium hydrogen phosphate dihydrate, about 1.0 mg / mL to about 20 mg / mL potassium dihydrogen phosphate, and about 1.0 mg / mL to about 20 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.25 mg / mL to about 10 mg / mL disodium hydrogen phosphate dihydrate, about 2.5 mg / mL to about 15 mg / mL potassium dihydrogen phosphate, and26095about 2.5 mg / mL to about 15 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.5 mg / mL to about 5 mg / mL disodium hydrogen phosphate dihydrate, about 3.5 mg / mL to about 10 mg / mL potassium dihydrogen phosphate, and about 3.5 mg / mL to about 10 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.6 mg / mL disodium hydrogen phosphate dihydrate, about 5.6 mg / mL potassium dihydrogen phosphate, and about 5.8 mg / mL sodium chloride.General Methods of Making the Liposomal Adjuvant

[0095] The liposomal adjuvant of the invention is a multi-component composition composed of structural lipids (such as l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol) in combination with a glycolipid (e.g., Glucopyranosyl Lipid A) and one or more saponin molecules (such as, e.g., Quillaja Saponaria 21 (QS-21). In an embodiment, the liposomal adjuvant is prepared by first solubilizing the structural lipids (e.g., DOPC, cholesterol) and glycolipid (e.g., GLA) at desired mass ratios (for example, see Table I) in an organic solvent (such as ethanol, chloroform, isopropyl alcohol) to form a lipidic mixture. The organic solvent is then removed from the lipidic mixture by heat and / or a vacuum driven drying procedure (e.g., a rotatory evaporation) to form a dry thin lipidic film. In an embodiment, the thin lipidic film is then reconstituted with an aqueous buffer (e.g., histidine, sodium and potassium phosphate buffer, and combinations thereof) to trigger the formation of a polydisperse mixture of liposomes. In an embodiment, the poly dispersity of liposome mixture is then reduced by passage of liposomes under high pressure (e.g., using an extrusion device through a defined pore size membrane) or high shear (such as, e.g., microfluidizer through a narrow chamber) to achieve a defined liposome particle size distribution, lamellarity. In an embodiment, the formed liposomes are then mixed with one or more saponin molecules (such as QS-21) dissolved in an aqueous matrix (such as, e.g., histidine buffers, phosphate buffers, and combinations thereof), which leads to binding of the saponin molecules to the cholesterol component of the liposomes. In an embodiment, the resulting structural lipid / glycolipid / saponin liposome formulation is then sterile filtered to form the liposomal adjuvant. Several process parameters within each step, including but not limited to lipid dissolution (mixing time, pH, temperature, concentration of components), solvent removal (temperature, pressure, time), lipid film hydration (time, temperature), and liposome sizing (pressure, membrane pore size, flow rate, # of passes) may be controlled to yield a liposomal adjuvant with desired attributes. Additionally, alternate formulation platforms like bulk-mixing or Tee- mixing may be used to make liposomal adjuvants containing glycolipids and saponins.26095Table I: Composition of a Representative Liposomal AdjuvantTarget Content of Each Molecular Content of Mass ratioComponent Concentration and Lipid and range Weight Each Lipid and range (mg / mL) and ranges(Mass %) (g / mol) range (Mole %) Phospholipid 2.25-3.75 10-30 55.56-92.59 786.11 48.38-80.63Sterol 0.56-0.94 2-15 13.89-23.15 386.65 24.59-40.98Glycolipid 0.12-0.18 0-3 2.78-4.63 1763.46 1.08-1.80Saponin 0.12-0.18 0-5 2.78-4.63 1990.13 0.96-1.595-300mM buffer,Buffer Matrix N / A at pH 5.0-8.0Lipid Stock Solution

[0096] In an embodiment, the structural lipids (for example, DOPC and cholesterol) (with or without GLA) are dissolved in an organic solvent e.g., methanol, ethanol chloroform, isopropyl alcohol (IP A)) or a mixture of solvents (e.g., 80:20 ethanol: IP A; 90:10 chloroform: methanol) to desired concentrations, ranging from 10-30 mg / mL DOPC, 2.5-10 mg / mL cholesterol and 0.5-5 mg / mL GLA. The stock solutions are then prepared separately for all 3 components or prepared as a combined stock solutions containing 1-3 of the components based on ease of manufacturing. Lipid dissolution parameters such as starting mass and ratios of lipids (micrograms- grams), solvent volume mixing speeds (100-600rpm), temperature (40-60°C) and time (30 min- 3hrs) are ranged appropriately to achieve complete lipid dissolution towards targeted scale.

[0097] In an embodiment, 100-200 mg DOPC and 20-100 mg cholesterol are together dissolved in 1-50 mL ethanol by heating at 30-60°C for 1-25 minutes to complete dissolution; 1-50 mg GLA is then dissolved in l-50mL ethanol at 30-100°C for 1-60 minutes with sonication to complete dissolution. In an embodiment, each lipid is dissolved individually, e.g., 1-50 mg / mL DOPC is dissolved in chloroform. 1-50 mg / mL cholesterol is dissolved in chloroform, and 1-50 mg / mL GLA is dissolved in a 9: 1 mixture of chloroform: methanol under ambient temperatures with swirl mixing. In an embodiment, all lipid components (e.g., 1-50 g DOPC, 1-25 g cholesterol, 0.25-25 g GLA), were dissolved together in 1-10 L ethanol by mixing at 100-500 RPM at temperatures (30-70°C) for 60-120 mins.

[0098] In one embodiment, 150mg DOPC and 37.7mg cholesterol were together dissolved in 5mLethanol by heating at 40°C for 5 min to complete dissolution. 11mg GLA was then dissolved26095in 11mL ethanol at 60°C for 25 min with sonication to complete dissolution. In one embodiment, each lipid was dissolved individually under ambient temperatures with swirl mixing. 25mg / mL DOPC was dissolved in chloroform, lOmg / mL cholesterol was dissolved in chloroform, and 5mg / mL GLA was dissolved in a 9:1 mixture of chloroform: methanol. In one embodiment, the lipids components (36g DOPC, 9 g Cholesterol, 1.8 g GLA), were dissolved together in 3.6L ethanol by mixing at 350 RPM at temperatures (50°C) for 120 mins.Preparation Thin-lipidic Film

[0099] In an embodiment, thin lipidic films are prepared via dehydration of the lipid stock solution described above. In an embodiment, the individual lipid stock solutions are combined at a target mass ratio of DOPC: cholesterol: GLA (such as a 20:5: 1, ratio) or prepared at the target ratios. In an embodiment, the lipid stock solution is then dehydrated using a rotatory evaporator to make a dry thin lipid film. In an embodiment, the dehydration parameters such as starting volumes of lipid mix volumes (10 mL-3.6L), rotatory speed (40-280rpm) temperature (ambient -50°C) and time (40min-8 hrs) are ranged appropriately to achieve complete dehydration.

[0100] In an embodiment, the rotary evaporation of 1 ImL lipid mix is performed at ambient temperature (e.g., 23°C), pressure 22mbar and rotary speed 150 rpm for 40 mins to form a thin lipid film on the inner wall of the round bottom flask. In an embodiment, the drying time range from (40 minutes - 16 hours). In an embodiment, the rotatory evaporation is performed at room temperature at 280rpm and pressure is reduced from 600mbar to 15mbar at a rate of 100mbar / 15-30mins and a total drying time of approximately 1-5 hours. In an embodiment, the rotatory evaporation is performed at room temperature (e.g., about 23°C) to about 50°C at 40-110 rpm speed with -0.07 to 0.1 MPa vacuum for a total of about 4-10 hours. In an embodiment, the dehydrated thin film is stored under vacuum or purged with nitrogen gas at ambient temperature for approximately 5-25 hours to ensure the complete removal of residual ethanol.Preparation of Liposomes

[0101] In an embodiment, the dried lipidic film described above is then reconstituted in an aqueous buffer (e.g., histidine buffer, sodium buffer, or potassium phosphate buffer) to form liposomes. Rehydration parameters such as temperature, volume, mixing speeds and buffers are ranged.

[0102] In an embodiment, the lipid film is rehydrated with a buffer at ambient temperature of about 23°C to about 50°C with constant mixing to form liposomes with l-15mg / mL phospholipid, 0.25-2.25mg / mL sterol and 0.05-1.25mg / mL glycolipid concentration. In an embodiment, the lipid film is rehydrated with buffer at ambient temperature of about 23 °C to about 50°C with manual swirling to form liposomes with l-15mg / mL phospholipid, 0.25-260952.25mg / mL sterol and 0.05-1.25mg / mL glycolipid concentration. In an embodiment, the lipid thin fdm is resuspended with a buffer at ambient temperature of about 23 °C to about 50°C over 30 minutes with 80 rpm mixing to form liposomes having the target of l-15mg / mL phospholipid, 0.25-2.25mg / mL sterol and 0.05-1.25mg / mL glycolipid.

[0103] In an embodiment, the lipid fdm is rehydrated with 30mL of lOmM (Na / K) phosphate, pH 6.2. lOOmM NaCl buffer at (ambient-50°C) with constant mixing to form liposomes with 5mg / mL DOPC, 1.25mg / mL cholesterol and 0.25mg / mL GLA concentration. In an embodiment, the lipid fdm is rehydrated with 20mL of lOmM His pH 6.2, 325mM NaCl, 0.01% PS80 buffer at 50°C with manual swirling to form liposomes with 6 mg / mL DOPC, 1.5mg / mL cholesterol and 0.3mg / mL GLA concentration. In an embodiment, the lipid thin fdm is resuspended with a 5.4 L of 50 mM (Na / K) phosphate, pH 6.2, lOOmM NaCl buffer at 50°C over 30 minutes with 80 rpm mixing to form liposomes having the target of 6.7 mg / mL DOPC, 1.7 mg / mL cholesterol and 0.3 mg / mL GLA concentration.

[0104] In an embodiment, the poly disperse (in terms of particle size, lamellarity) mixture of liposomes are subjected to pressure or flow driven extrusion through a defined pore sized membrane or high shear (such as microfluidizer through a narrow chamber) to achieve a defined liposome particle size distribution, lamellarity. A step-wise liposome sizing procedure where extrusion starts by passing liposomes through a large (400nm) pore size membrane and step- wise decreases to smaller pore size membrane 200nm,100nm and 80nm to achieve target liposome size is also used. Process parameters such as pressure, flow rate, membrane pore size, temperature and number of passes are ranged to achieve the optimal size range and lamellarity.

[0105] In an embodiment, the rehydrated liposomes are extruded in batch mode, through an extruder device with a lOOnm pore size membrane under a pressure from 200-400 psi at ambient-50°C and passed 6 times to achieve a low poly dispersity (poly dispersity index, PDI —0.1), lamellarity(~2) and ~120nm mean particle size. In an embodiment, the extrusion is performed under constant flow rate (3L / min) through an extruder device with a lOOnm pore size membrane at 40°C temperature and passed 35 times through the membrane to achieve low poly dispersity (PDI —0.1), lamellarity (~ 2), mean particle size-lOOnm. In an embodiment, the liposomes are extruded via a continuous mode of extrusion under constant flow rate at ~3.5 L / min through an extruder device with a 100 nm pore size membrane at 40°C for a total of 30 minutes to achieve low polydispersity(PDI —0.1), lamellarity -2 and particle size -100 nm.

[0106] In an embodiment, a buffer including 30mL of 50mM (Na / K) phosphate, pH 6.2, lOOmM NaCl buffer is used to rehydrate lipid film at (ambient-50°C) with constant mixing to get 5mg / mL DOPC, 1.25mg / mL cholesterol and 0.25mg / mL GLA concentration. The rehydrated26095liposomes are extruded in batch mode, through an extruder device with a 1 OOnm pore size membrane under a pressure from 200-400 psi at ambient-50°C and passed 6 times to achieve a low poly dispersity (poly dispersity index, PDI —0.1), lamellarity(~2) and ~120nm mean particle size.

[0107] In an embodiment, a buffer including 20mL of lOmM His pH 6.2, 325mM NaCl, 0.01% PS80 buffer is used to rehydrate lipid film at 50°C with manual swirling to get 6 mg / mL DOPC, 1.5mg / mL Cholesterol and 0.3mg / mL GLA concentration. Extrusion is performed under constant flow rate (3L / min) through an extruder device with a lOOnm pore size membrane at 40°C temperature and passed 35 times through the membrane to achieve low poly dispersity (PDI —0.1), lamellarity (-2), and a mean particle size~100nm.

[0108] In an embodiment, the lipid thin film is resuspended with 5.4 L of 50 mM (Na / K) phosphate, pH 6.2, lOOmM NaCl buffer at 50°C over 30 minutes with 80 rpm mixing to the target of 6.7 mg / mL DOPC, 1.7 mg / mL cholesterol and 0.3 mg / mL GLA concentration.Continuous mode of extrusion of the mixture is performed under constant flow rate at ~3.5 L / min through an extruder device with a 100 nm pore size membrane at 40°C for a total of 30 minutes to achieve low polydispersity(PDI —0.1), lamellarity -2 and particle size -100 nm.Liposomal Adjuvant Formation

[0109] In an embodiment, the final liposomal adjuvant is formed by first dissolving saponin in an aqueous buffer and mixing the resulting solution with the liposomes described above. The liposomal adjuvant is formed by saponin binding to the cholesterol component in the liposomes. The dissolution conditions such as mass of saponin, buffers, volume and mixing time and temperature are optimized to ensure complete dissolution and stability of saponin.

[0110] In an embodiment, approximately 1-100 mL of saponin stock was prepared at ambient condition with a range of 1 to 10 mg / mL saponin concentration in a buffer solution with gentle manual swirl. In an embodiment, the buffer solution includes, e.g., approximately 50 mM (Na / K.) phosphate, at pH 6.2 and approximately lOOmM NaCl buffer or approximately 20 mM histidine, pH 6.2 buffer. In an embodiment, up to IL of saponin solution are prepared with stir plate at 200-500 rpm mixing speed to assist complete dissolution.

[0111] In an embodiment, 2g QS-21 is dissolved in 800 mL of 50 mM (Na / K) phosphate, pH 6.2, 100 mM NaCl formulation buffer at room temperature with stir mixing at 500 rpm for 50 minutes to prepare the QS-21 stock solution. In an embodiment, a 5 mg QS-21 is dissolved in 5 mL of 20 mM histidine, pH 6.2 formulation buffer at room temperature with gentle swirl. In an embodiment. 5 mg QS-21 was dissolved in 0.5 mL of 50 mM (Na / K) phosphate, pH 6.2, 100 mM NaCl buffer at room temperature with gentle swirl.26095

[0112] In an embodiment, saponin and the extruded liposomes are mixed with a targeted ratio of saponin: cholesterol at 1:5 to form a liposomal solution, the mixing parameters such as rate of addition and mixing speed, and time are ranged. In an embodiment, the mixing volume is adjusted to achieve the targeted adjuvant concentration. In an embodiment, a low volume (example < 10 mL) of saponin stock solution is added dropwise with a volumetric pipette with gentle mix, while high volume (10 mL to IL) is added with a peristaltic pump (10-20 ml / min) to mix with the extruded liposome solution at 100-500 rpm stir mixing depending on the total solution volume. In an embodiment, 0.8mL of 10 mg / mL QS-21 stock solution was added with pipette and mixed thoroughly with 30 mL of extruded liposomes and 20 mL buffer (50 mM (Na / K) phosphate, pH 6.2, 100 mM NaCl). In an embodiment, the calculated volume 630g of QS-21 stock solution and 4000g formulation buffer is combined with the 5000g extruded liposome at 20 mL / min with 150 rpm mixing, and then stir-mixed thoroughly at room temperature at 120 rpm for at least 30 minutes as the final adjuvant formulation (composition as described in Table I above) before filtration.

[0113] In an embodiment, the liposomal solution is sterile filtered through a 0.22pm filter. In an embodiment, 0.22 pm syringe filters were used to filter ~ 60 mL of liposomal solution. In another instance, large capacity capsule filters units were connected to peristaltic pump to sterile filter approximately 10L of liposomal solution with less than 25 psi pressure throughout the filtration step.The VLPs

[0114] As stated above, the pharmaceutical compositions and formulations of the invention comprise at least one HPV VLP type, such as HPV 16 or 18. In particular embodiments of the compositions disclosed herein, the vaccine further comprises VLPs of at least one additional HPV type. In further embodiments, the at least one additional HPV type is selected from the group consisting of: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82. In some embodiments, the at least one HPV type includes HPV 16 and 18. In some embodiments, the at least one HPV ty pe includes HPV 6, 11, 16, and 18. In some embodiments, the at least one HPV type includes HPV 6, 18, 52. and 58. In some embodiments, the at least one HPV type includes HPV 6, 11, 16. 18. 31. 45. 52, and 58. In some embodiments, the at least one HPV ty pe includes HPV 6, 11, 16, 18, 33, 45, 52, and 58. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58. In some embodiments, the at least one HPV type includes 6, 11, 16, 18, 31, 33. 45, 52, and 59. In some embodiments, the at least one HPV ty pe includes HPV 6, 11, 16, 18, 31. 33. 45. 53. and 58. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 53, and 59. In some embodiments, the26095at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 45, 52, 58, and 59. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68. In some embodiments, the at least one HPV ty pe includes HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. In some embodiments, the at least one HPV type includes HPV 6. 11. 16, 18, 26, 31, 33, 35, 45, 51, 52, 58, 59, and 69. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 31. 33. 35. 39. 45. 51. 52, 56, 58, 59, and 68. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 73. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 69, and 70. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18. 31. 33. 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70. In some embodiments, the at least one HPV type includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, and 73.

[0115] The pharmaceutical compositions of the invention comprise HPV VLPs comprised of recombinant LI or recombinant LI + L2 proteins of HPV. HPV LI or LI + L2 protein can be expressed recombinantly by molecular cloning of LI or LI + L2 DNA into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokary otic or eukaryotic host cells to produce recombinant protein. Techniques for such manipulations are fully described by Sambrook et al. (Molecular Cloning: A Laboratory’ Manual; Cold Spring Harbor Laboratory’, Cold Spring Harbor, New York, (1989)), which is hereby incorporated by reference. VLPs can self-assemble when LI protein is recombinantly expressed in a host cell.

[0116] The recombinant HPV LI proteins of the invention may be any full-length LI protein sequence that can be found in nature or any mutated or truncated LI protein that is capable of self-assembling into VLPs. In particular embodiments of the invention, the pharmaceutical compositions and vaccines described herein comprise HPV VLPs comprised of recombinant HPV LI protein and do not contain HPV L2 protein. In certain embodiments, the vaccine compositions or pharmaceutical compositions described herein comprise HPV VLPs comprised of a full-length recombinant HPV LI protein. In other embodiments, the HPV VLPs are comprised of truncated HPV LI protein, e.g., LI protein that are truncated at the C-terminal end. LI protein sequences for use in the invention can be determined by isolating DNA from one or more clinical samples containing an HPV type of choice, determining the sequence of the HPV LI DNA sequence, and translating the DNA sequence into an amino acid sequence using the26095genetic code. Many exemplary LI sequences suitable for use in the invention can be found in the literature. See, e.g., U. S. Patent Nos. 5,820,870; 7,250,170; 7,276,243; 7,482,428; 7,976.848; 7,498,036; 7,700,103; 7,744,892; and 5,437,951; Kirii et al. (Virology 185(1): 424-427 (1991)). Further LI proteins that are useful in the compositions and formulations of the invention include biologically active fragments and / or mutants of an HPV LI sequence, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy -terminal truncations, such that these mutations provide LI proteins or protein fragments that are capable of forming a VLP. See, e.g., International Publication WO 2006 / 114312 and US Patent No. 6,599,508. Appropriate host cells for the expression of recombinant HPV LI or recombinant LI + L2 and subsequent self-assembly of VLPs include, but are not limited to yeast cells, insect cells, mammalian cells or bacteria. In exemplary embodiments of the invention, the VLPs are produced in yeast cells such as a yeast selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe. In particular embodiments, the HPV VLPs are produced in Saccharomyces cerevisiae cells. Expression of HPV VLPs in yeast cells offers the advantages of being cost-effective and easily adapted to large-scale growth in fermenters.

[0117] The invention also includes pharmaceutical compositions comprising mutant forms of HPV VLPs, such as HPV VLPs that comprise biologically active fragments and / or mutants of an HPV LI and / or L2 protein, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide proteins or protein fragments of therapeutic or prophylactic use and would be useful for HPV VLP vaccine development. Thus, mutant forms of an HPV LI protein that are capable of forming VLPs and of provoking an immune response against the desired HPV type when administered to a human are included in embodiments of the compositions of the invention.

[0118] Additionally, one of skill in the art will recognize that the HPV LI or LI + L2 proteins, which are used to self-assemble VLPs for inclusion in the compositions disclosed herein, may be encoded by a full-length wild-type HPV LI or LI + L2 polynucleotide, or may be encoded by a fragment or mutant of the known wild-type sequence. Wild-type polynucleotide sequences that encode mRNA expressing HPV LI or L2 protein are available in the art. Any mutant polynucleotide will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of an HPV LI or L2 protein, including the ability to form VLPs that are able to provoke an immune response against the HPV type of interest when26095administered to a human. Such mutant polynucleotides include both nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations.

[0119] The amount of virus-like particles of each HPV type to be included in the formulations and compositions of the invention will depend on the immunogenicity of the expressed gene product. In general, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 1 pg to about 300 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 1 pg to 200 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 1 pg to 100 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 5 pg to 200 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 5 pg to 100 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 10 pg to 200 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 10 pg to 100 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 10 pg to 80 pg. In some embodiments, a therapeutically effective dose of VLPs of any of the at least one HPV type is about 20 pg to 60 pg.

[0120] In some embodiments, a dose of a composition is in the range of about 0.10 mL to about 1.5 mL. In some embodiments, a dose is the range of about 0.25 mL to about 1.25 mL. In some embodiments, a dose is in the range of about 0.5 mL to about 1.0 mL. In some embodiments, the dose is a 0.25 mL dose. In some embodiments, the dose is a 0.5 mL dose. In some embodiments, the dose is a 0.75 mL dose. In some embodiments, the dose is a 1.0 mL dose. In some embodiments, the dose is a 1.25 mL dose.

[0121] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 L 1 protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10- 120 pg of VLPs of HPV Type 31 L 1 protein,• 10-120 pg of VLPs of HPV Type 33 LI protein.• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein, and• 10-120 pg of VLPs of HPV Type 58 LI protein.

[0122] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein.26095• 20-200 pg of VLPs of HPV Type 11 LI protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10-120 pg of VLPs of HPV Type 31 LI protein,• 10-120 pg of VLPs of HPV Type 33 LI protein,• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein.• 10-120 pg of VLPs of HPV Type 58 LI protein,• 10-120 pg of VLPs of HPV Type 59 LI protein,• 10-120 pg of VLPs of HPV Type 68 LI protein,• 10-120 pg of VLPs of HPV Type 69 LI protein,• 10-120 pg of VLPs of HPV Type 70 LI protein, and• 10-120 pg of VLPs of HPV Type 73 LI protein.

[0123] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 LI protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10-120 pg of VLPs of HPV Type 31 LI protein,• 10-120 pg of VLPs of HPV Type 33 LI protein.• 10-120 pg of VLPs of HPV Type 35 LI protein,• 10-120 pg of VLPs of HPV Type 39 LI protein,• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 51 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein,• 10-120 pg of VLPs of HPV Type 56 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein, and• 10-120 pg of VLPs of HPV Type 59 LI protein.

[0124] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg ofVLPs ofHPV Type 6 LI protein,• 20-200 pg ofVLPs ofHPV Type 11 LI protein,• 30-280 pg ofVLPs ofHPV Type 16 LI protein,• 20-200 pg ofVLPs ofHPV Type 18 LI protein,• 10-120 pg ofVLPs ofHPV Type 31 LI protein,26095• 10-120 pg of VLPs of HPV Type 33 LI protein,• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein,• 10-120 pg of VLPs of HPV Type 56 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein,• 10-120 pg of VLPs of HPV Type 59 LI protein,• 10-120 pg of VLPs of HPV Type 68 LI protein.• 10-120 pg of VLPs of HPV Type 70 LI protein, and• 10-120 pg of VLPs of HPV Type 73 LI protein.

[0125] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 LI protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10-120 pg of VLPs of HPV Type 31 LI protein,• 10-120 pg of VLPs of HPV Type 33 LI protein,• 10-120 pg of VLPs of HPV Type 35 LI protein,• 10-120 pg of VLPs of HPV Type 39 LI protein,• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 51 LI protein.• 10-120 pg of VLPs of HPV Type 52 LI protein,• 10-120 pg of VLPs of HPV Type 56 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein, and• 10-120 pg of VLPs of HPV Type 69 LI protein.

[0126] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 LI protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein.• 10-120 pg ofVLPs ofHPV Type 31 LI protein,• 10-120 μg of VLPs of HPV Type 33 L1 protein,• 10-120 μg of VLPs of HPV Type 35 L1 protein,• 10-120 μg of VLPs of HPV Type 39 L1 protein,• 10-120 μg of VLPs of HPV Type 45 L1 protein,26095• 10-120 pg of VLPs of HPV Type 51 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein,• 10-120 pg of VLPs of HPV Type 59 LI protein, and• 10-120 pg of VLPs of HPV Type 69 LI protein.

[0127] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 LI protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10-120 pg of VLPs of HPV Type 31 LI protein.• 10-120 pg of VLPs of HPV Type 33 LI protein,• 10-120 pg of VLPs of HPV Type 35 LI protein,• 10-120 pg of VLPs of HPV Type 39 LI protein,• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 51 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein,• 10-120 pg of VLPs of HPV Type 56 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein,• 10-120 pg of VLPs of HPV Type 59 LI protein.• 10-120 pg of VLPs of HPV Type 68 LI protein,• 10-120 pg of VLPs of HPV Type 69 LI protein, and• 10-120 pg of VLPs of HPV Type 70 LI protein.

[0128] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 LI protein,• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10-120 pg of VLPs of HPV Type 31 LI protein.• 10-120 μg of VLPs of HPV Type 33 L1 protein,• 10-120 μg of VLPs of HPV Type 35 L1 protein,• 10-120 μg of VLPs of HPV Type 39 L1 protein,• 10-120 μg of VLPs of HPV Type 45 L1 protein,• 10-120 μg of VLPs of HPV Type 51 L1 protein,26095• 10-120 pg of VLPs of HPV Type 52 LI protein,• 10-120 pg of VLPs of HPV Type 56 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein,• 10-120 pg of VLPs of HPV Type 59 LI protein,• 10-120 pg of VLPs of HPV Type 66 LI protein,• 10-120 pg of VLPs of HPV Type 68 LI protein,• 10-120 pg of VLPs of HPV Type 69 LI protein.• 10-120 pg of VLPs of HPV Type 70 LI protein.

[0129] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-160 pg of VLPs of HPV Type 6 LI protein,• 20-200 pg of VLPs of HPV Type 11 LI protein.• 30-280 pg of VLPs of HPV Type 16 LI protein,• 20-200 pg of VLPs of HPV Type 18 LI protein,• 10-120 pg of VLPs of HPV Type 31 LI protein,• 10-120 pg of VLPs of HPV Type 33 LI protein,• 10-120 pg of VLPs of HPV Type 35 LI protein,• 10-120 pg of VLPs of HPV Type 39 LI protein,• 10-120 pg of VLPs of HPV Type 45 LI protein,• 10-120 pg of VLPs of HPV Type 51 LI protein,• 10-120 pg of VLPs of HPV Type 52 LI protein.• 10-120 pg of VLPs of HPV Type 56 LI protein,• 10-120 pg of VLPs of HPV Type 58 LI protein,• 10-120 pg of VLPs of HPV Type 59 LI protein,• 10-120 pg of VLPs of HPV Type 66 LI protein,• 10-120 pg of VLPs of HPV Type 68 LI protein,• 10-120 pg of VLPs of HPV Type 69 LI protein,• 10-120 pg of VLPs of HPV Type 70 LI protein, and• 10-120 pg of VLPs of HPV Type 73 LI protein.

[0130] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-120 μg of VLPs of HPV Type 6 L1 protein,• 20-150 μg of VLPs of HPV Type 11 L1 protein,• 30-210 μg of VLPs of HPV Type 16 L1 protein,• 20-150 μg of VLPs of HPV Type 18 L1 protein,• 10-90 μg of VLPs of HPV Type 31 L1 protein.26095• 10-90 pg of VLPs of HPV Type 33 LI protein,• 10-90 μg of VLPs of HPV Type 45 L1 protein,• 10-90 pg of VLPs of HPV Type 52 LI protein, and• 10-90 pg of VLPs of HPV Type 58 LI protein.

[0131] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-80 pg of VLPs of HPV Type 6 LI protein,• 20-100 pg of VLPs of HPV Type 11 LI protein.• 30-140 pg of VLPs of HPV Type 16 LI protein,• 20-100 pg of VLPs of HPV Type 18 LI protein,• 10-60 pg of VLPs of HPV Type 31 LI protein,• 10-60 pg of VLPs of HPV Type 33 LI protein,• 10-60 pg of VLPs of HPV Type 45 LI protein,• 10-60 pg of VLPs of HPV Type 52 LI protein, and• 10-60 pg of VLPs of HPV Type 58 LI protein.

[0132] In some embodiments, a dose of a composition or vaccine of the invention includes:• 15-40 pg of VLPs of HPV Type 6 LI protein,• 20-50 pg of VLPs of HPV Type 11 LI protein,• 30-70 pg of VLPs of HPV Type 16 LI protein,• 20-50 pg of VLPs of HPV Type 18 LI protein,• 10-30 pg of VLPs of HPV Type 31 LI protein,• 10-30 pg of VLPs of HPV Type 33 LI protein,• 10-30 pg of VLPs of HPV Type 45 LI protein,• 10-30 pg of VLPs of HPV Type 52 LI protein, and• 10-30 pg of VLPs of HPV Type 58 LI protein.

[0133] In some embodiments, a dose of a composition or vaccine of the invention includes:• 90 pg of VLPs of HPV Type 6 LI protein,• 120 pg of VLPs of HPV Type 11 LI protein,• 180 pg of VLPs of HPV Type 16 LI protein,• 120 pg of VLPs of HPV Type 18 LI protein,• 60 pg of VLPs of HPV Type 31 LI protein,• 60 pg of VLPs of HPV Type 33 LI protein,• 60 pg of VLPs of HPV Type 45 LI protein,• 60 pg of VLPs of HPV Type 52 LI protein, and• 60 pg of VLPs of HPV Type 58 LI protein.26095

[0134] In some embodiments, a dose of a composition or vaccine of the invention includes:• 60 pg of VLPs of HPV Type 6 LI protein,• 80 pg of VLPs of HPV Type 11 LI protein,• 120 pg of VLPs of HPV Type 16 LI protein,• 80 pg of VLPs of HPV Type 18 LI protein,• 40 pg of VLPs of HPV Type 31 LI protein,• 40 pg of VLPs of HPV Type 33 LI protein,• 40 pg of VLPs of HPV Type 45 LI protein,• 40 pg of VLPs of HPV Type 52 LI protein, and• 40 pg of VLPs of HPV Type 58 LI protein.

[0135] In some embodiments, a dose of a composition or vaccine of the invention includes:• 30 pg of VLPs of HPV Type 6 LI protein,• 40 pg of VLPs of HPV Type 11 LI protein,• 60 pg of VLPs of HPV Type 16 LI protein,• 40 pg of VLPs of HPV Type 18 LI protein,• 20 pg of VLPs of HPV Type 31 LI protein,• 20 pg of VLPs of HPV Type 33 LI protein,• 20 pg of VLPs of HPV Type 45 LI protein,• 20 pg of VLPs of HPV Type 52 LI protein, and• 20 pg of VLPs of HPV Type 58 LI protein.The Aluminum Adjuvant

[0136] The aluminum adjuvant of the invention may be in the form of aluminum hydroxide (Al(0H)3), aluminum phosphate (AIPO4), aluminum hydroxy phosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS) or so-called “alum” (KAl(SO₄)·12H₂O) (see Klein et al., Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)A1 MAS NMR., J Pharm. Sci. 89(3): 311-21 (2000)). In exemplary embodiments of the invention provided herein, the aluminum adjuvant is aluminum hydroxyphosphate or AAHS. The ratio of phosphate to aluminum in the aluminum adjuvant can range from 0 to 1.3. In embodiments of this aspect of the invention, the phosphate to aluminum ratio is within the range of 0.1 to 0.70. In some embodiments, the phosphate to aluminum ratio is within the range of 0.2 to 0.50.

[0137] One of skill in the art will be able to determine an optimal dosage of aluminum adjuvant that is both safe and effective at increasing the immune response to the targeted HPV type(s). For a discussion of the safety’ profile of aluminum, as well as amounts of aluminum included in FDA-licensed vaccines, see Baylor et al., Vaccine 20: S18-S23 (2002). In some embodiments, the26095aluminum adjuvant is present in the amount of about 100 to 3600 pg / dose (200 to 7200 pg / mL concentration). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 2700 pg / dose (200 to 5400 pg / mL concentration). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 1800 pg / dose (200 to 3600 pg / mL concentration). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 900 pg / dose (200 to 1800 pg / mL concentration). In some embodiments of the formulations and compositions of the invention, there is between 200 and 300 pg aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the invention, there is between 300 and 500 pg aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the invention, there is between 400 and 1200 pg aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the invention, there is between 1200 and 2000 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 2000 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 1500 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 1000 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 500 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 400 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 300 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 200 pg aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, there is less than 100 pg aluminum adjuvant per dose of vaccine.The HPV VLP-based Vaccine

[0138] Any HPV VLP-based vaccine is suitable for use in the pharmaceutical compositions and methods of the invention. Known HPV VLP vaccines can be modified to include both an aluminum adjuvant and a liposomal adjuvant. Such HPV VLP vaccines can be field mixed with a liposomal adjuvant of the invention (e.g., mixed by the clinician just prior to administration to a patient) or can be formulated together with the liposomal adjuvant. New vaccines can be developed according to the invention described herein that comprise at least one HPV type, optionally in the form of an HPV VLP adsorbed to an aluminum adjuvant, in combination with a liposomal adjuvant. Additionally, new vaccines can be developed according to the invention26095described herein that comprise at least one HPV type in the form of an HPV VLP adsorbed to an aluminum adjuvant in combination with a liposomal adjuvant.

[0139] One exemplary HPV vaccine is a bivalent vaccine protective against HPV 16 and 18, which is known commercially as CERVARIX® (GlaxoSmithKline Biologicals, Rixensart, Belgium). Another exemplary HPV VLP vaccine is a non-infectious recombinant, quadrivalent vaccine prepared from highly purified VLPs of the major capsid (LI) protein of HPV types 6, 11, 16. and 18, and may be referred to herein by its proprietary name GARDASIL* (Merck & Co., Inc., Rahway, NJ, USA), see Bryan, J. T. Vaccine 25(16): 3001-6 (2007); Shi et al. Clinical Pharmacology and Therapeutics 81(2): 259-64 (2007). Another exemplary HPV VLP vaccine is the nine-valent vaccine marketed for prevention of HPV (that includes the capsid (LI) protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58), which is referred to herein by its proprietary name GARDASIL®9 (Merck & Co., Inc., Rahway, NJ, USA).

[0140] In some embodiments, the vaccine dose includes, in addition to VLPs, an aluminum adjuvant (as amorphous aluminum hydroxyphosphate sulfate), sodium chloride, L-histidine, polysorbate 80, sodium borate, and water. In some embodiments, the HPV vaccine includes 100-3500 pg aluminum adjuvant. 1-50 mg sodium chloride, 0.05-10 mg L-histidine, 1-100 pg polysorbate, 1-100 pg sodium borate, and water. In some embodiments, the HPV vaccine includes about 500 pg aluminum adjuvant, about 9.56 mg sodium chloride, about 0.78 mg L-histidine, about 50 pg polysorbate 80, about 35 pg sodium borate, and water for injection. Known HPV VLP vaccines can be modified to include both an aluminum adjuvant and a liposomal adjuvant in accordance with the invention.

[0141] In some embodiments of the invention, the pharmaceutical compositions and formulations comprise HPV VLPs described herein, or may be made by field-mixing a formulated HPV VLP-based vaccine with a liposomal adjuvant, wherein the compositions and formulations are monovalent, bivalent, trivalent, quadrivalent, 5-valent, 6-valent, 7-valent, 8-valent, 9-valent, 10-valent, 11-valent, 12-valent, 13-valent, 14-valent, 15-valent, 16-valent, 17-valent, or 18-valent. In particular embodiments, the pharmaceutical compositions and formulations are 9-valent. In other embodiments, the pharmaceutical compositions and formulations are 10-valent. In other embodiments, the pharmaceutical compositions and formulations are 12-valent. In particular embodiments, the pharmaceutical compositions and formulations are 14-valent. In particular embodiments, the pharmaceutical compositions and formulations are 15-valent. In particular embodiments, the pharmaceutical compositions and formulations are 16-valent. In particular embodiments, the pharmaceutical compositions and formulations are 17-valent. In particular embodiments, the pharmaceutical compositions and26095formulations are 18-valent. In some embodiments, the pharmaceutical compositions comprise more than four different types of HPV VLPs. For example, the pharmaceutical compositions and formulations of the invention may be 8-valent, 9-valent, 10-valent, and so forth. For example, pharmaceutical compositions comprising VLPs of HPV 16 and / or HPV 18, without the inclusion of other HPV VLP types, are included within the scope of the invention. Multi-valent vaccines comprising HPV VLPs that are different than the HPV types included in GARDASIL® or GARDASIL®9 are also contemplated herein.

[0142] In some embodiments, VLPs of HPV types 6 and 11 are included. In some embodiments, VLPs of HPV types 16, 31, and 35 are included. In some embodiments, VLPs of HPV types 18, 45, and 59 are included. In some embodiments, VLPs of HPV types 26, 51, and 69 are included. In some embodiments, VLPs of HPV types 33. 52. and 58 are included. In some embodiments, VLPs of HPV types 39, 68, and 70 are included. In some embodiments, VLPs of HPV types 53, 56, and 66 are included.

[0143] In some embodiments, VLPs of HPV types 16 and 18 are included. In some embodiments, VLPs of HPV types 6, 11, 16, and 18 are included. In some embodiments. VLPs of HPV types 6, 18, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11.16, 18, 31, 45, 52, and 58 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 33, 45, 52, and 58 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 45, 52, and 59 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 45, 53, and 58 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 45, 53, and 59 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58, and 59 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 26, 31, 33, 35, 45, 51, 52, 58, 59, and 69 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33. 35. 39. 45. 51. 52, 56, 58, 59, and 73 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 69, and 70 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73 are included. In some embodiments, VLPs ofHPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70 are included.26095In some embodiments, VLPs of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, and 73 are included.

[0144] In some embodiments, the pharmaceutical compositions and formulations comprise antigens as listed in Table II below or are made by combining an HPV VLP vaccine disclosed below with a liposomal adjuvant, as described herein:Table II:Name Antigen Adjuvant Party CERVARIX® LI VLP of HPV- 16 and HPV- Aluminum GlaxoSmithKline (2vHPV vaccine) 18 hydroxide and Biologies (Rixensart,MPL Belgium) GARDASIL® LI VLP ofHPV-6, HPV-11, AHSS Merck & Co., Inc., (4vHPV vaccine) HPV- 16 and HPV- 18 Rahway NJ USA GARDASIL® 9 LI VLP ofHPV-6, HPV-11, AHSS Merck& Co., Inc., (9vHPV vaccine) HPV- 16, HPV- 18, HPV-31, Rahway NJ USA HPV-33, HPV-45, HPV-52and HPV-58CECOLIN® LI VLP of HPV- 16 and HPV- Aluminum Xiamen Innovax 18 hydroxideGEOCOLIN® LI VLP ofHPV-6 and HPV- Aluminum Xiamen Innovax 11 hydroxideLI capsomers LI capsomers of HPV- 16 unknown R. Garcea, University of Colorado- Boulder RG1-VLP HPV- 16 L1-L2 (17-36) VLP Aluminum R. Kimbauer, NCI, hydroxide Pathovax LLC L2-AAV L2 peptides of HPV-16 and unknown 2A Pharma HPV-31 displayed on AAVVLPL2 multimer Fusion protein of L2 ~11-88 Alum Sanofi, BravoVax ofHPV-6, HPV-16, HPV- 18,HPV-31 andHPV-39L2-thioredoxin L2 peptide displayed on unknown M. Muller, DKFZ thioredoxinAX03 L2 peptide displayed on unknown Agilvax, NIAID bacteriophageL1-E7 VLP HPV-16 LI -E7 VLP None Medigene AG TA-CIN HPV-16 L2E7E6 fusion None Cantabprotein Pharmaceuticals,Xenova26095Name Antigen Adjuvant PartyTA-GW HPV-6 L2E7 fusion protein Aluminum Cantabhydroxide or Pharmaceuticals, GSKAS03

[0145] Compositions of the invention may be administered subcutaneously, topically, orally, on the mucosa, intravenously, or intramuscularly. The compositions are administered in an amount sufficient to elicit a protective response. Compositions can be administered by various routes, for example, orally, parenterally, subcutaneously, on the mucosa, or intramuscularly. The dose administered may vary depending on the general condition, sex, weight and age of the patient, and the route of administration.

[0146] Compositions of the invention, as highlighted in the various embodiments above, may be referred to as immunogenic compositions.

[0147] Compositions of the invention, as highlighted in the various embodiments above may also be referred to as vaccines or vaccine compositions.

[0148] In an embodiment, a liposomal adjuvant 14vHPV vaccine is prepared by combining the liposomal adjuvant described above with an HPV antigen. The HPV antigen component of the vaccine, may include multiple purified virus-like particles (VLPs) of the major capsid (LI) protein of HPV types (such as 6. 11. 16. 18, 31, 33, 45, 52, 58, 35, 39, 51,56. 59. 68) or chimeric sequence of one or more HPV types or additional types any other combinations. In an embodiment, the HPV antigen is adsorbed on a preformed aluminum-containing adjuvant (e.g., amorphous aluminum hydroxyphosphate sulfate). In an embodiment, the liposomal adjuvant and HPV antigen component(s) are prepared separately and mixed to prepare the final vaccine at the time of dosing. In an embodiment, the liposomal adjuvant and HPV antigen component(s) are formulated together as a single composition.

[0149] In an embodiment, a 9-valent HPV vaccine formulation comprising HPV VLP antigens adsorbed on aluminum is prepared from the purified virus-like particles (VLPs) of the major capsid (LI) protein of HPV Types 6, 11, 16. 18. 31. 33. 45, 52, and 58) adsorbed on preformed aluminum-containing adjuvant (amorphous aluminum hydroxyphosphate sulfate) (hereinafter “9vHPV Vaccine”). In an embodiment, a 5 -valent HPV vaccine formulation comprising HPV VLP antigens adsorbed on aluminum is prepared from the purified virus-like particles (VLPs) of the major capsid (LI) protein of HPV Types 35, 39, 51, 56 and 59) adsorbed on preformed aluminum-containing adjuvant (amorphous aluminum hydroxyphosphate sulfate) (hereinafter “5vHPV Vaccine”). In an embodiment, the 9vHPV vaccine is mixed with the liposomal adjuvant at the time of dosing. In an embodiment, the 5vHPV vaccine is mixed with the liposomal26095adjuvant at the time of dosing. In an embodiment, the 9vHPV Vaccine and the 5 -valent HPV and liposomal adjuvant are combined to form a liposomal adjuvant 14- valent HPV vaccine.

[0150] In each of the embodiments described above the composition further comprises HPV VLPs of at least one HPV type.

[0151] In some embodiments, a vaccine composition is provided that includes (1) 0.1 pg to 600 mg / mL liposomal adjuvant, and (2) HPV VLPs of at least one HPV type, wherein each of the HPV VLPs, when present in the vaccine composition, are present in a concentration of about 1 pg to about 300 pg per 0.5 mL of the vaccine composition and wherein the total VLP concentration is between about 10 pg to about 2000 pg per 0.5 mL of the vaccine composition. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) about 100 pg to about 3500 pg aluminum adjuvant, and (3) HPV VLPs of at least one HPV type, wherein each of the HPV VLPs, when present in the vaccine composition, are present in a concentration of about 1 pg to about 180 pg per 0.5 mL of the vaccine composition and wherein the total VLP concentration is between about 10 pg to about 2000 pg per 0.5 mL of the vaccine composition.

[0152] In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant, about 1 pg to about 2000 pg HPV VLPs of at least two HPV types, and about 100 pg to about 2700 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant. HPV VLPs of at least four HPV types, and about 100 pg to about 3500 pg aluminum adjuvant.

[0153] In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant, and 1 pg to about 100 pg of each HPV VLP present in the vaccine composition. In some embodiments, a vaccine composition is provided that about 2 pg / mL to about 400 mg / mL liposomal adjuvant and 2 pg to about 600 pg of HPV VLPs of two HPV types (i.e., the vaccine is a bivalent HPV VLP vaccine). In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant and 4 pg to about 1200 pg of HPV VLPs of four HPV types (i.e., the vaccine is a quadrivalent HPV VLP vaccine). In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant and 9 pg to about 2700 pg of HPV VLPs of nine HPV types (i.e., the vaccine is 9-valent HPV VLP vaccine). In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant and 20 pg to about 6000 pg of HPV VLPs of twenty HPV types (i.e., the vaccine is a2609520-valent HPV VLP vaccine). In some embodiments, the vaccine composition also includes about 100 pg to about 2700 pg aluminum adjuvant.

[0154] In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant, 1 pg to about 300 pg of a monovalent HPV VLP, and 100 pg to about 2700 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes about 2 pg / mL to about 400 mg / mL liposomal adjuvant, 1 pg to about 300 pg. per VLP. of HPV VLPs of two HPV types, and 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of a HPV VLPs of four HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 9 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 14 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 15 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant. (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 16 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 17 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 18 HPV ty pes, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 19 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant. In some embodiments, a vaccine composition is provided that includes (1) about 2 pg / mL to about 400 mg / mL liposomal adjuvant, (2) 1 pg to about 300 pg, per VLP, of HPV VLPs of 20 HPV types, and (3) 100 pg to about 3500 pg aluminum adjuvant.

[0155] In some embodiments, the vaccine composition includes (1) 1 pg to about 300 pg, per VLP. of HPV VLPs (HPV types 16 and 18) and (2) about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes (1) 1 pg to about26095300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, and 18,) and (2) about 2 pg / rnL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes (1) 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58) and (2) about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes (1) 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) and (2) about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes (I) 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 68) and (2) about 2 pg / rnL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes (1) 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and (2) about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes (1) 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73) and (2) about 2 pg / mL to about 400 mg / mL liposomal adjuvant.

[0156] In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 16 and 18), 100 pg to about 3500 pg of an aluminum adjuvant, and about 2 pg / rnL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, and 18,), 100 pg to about 3500 pg of an aluminum adjuvant, and about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58), 100 pg to about 3500 pg of an aluminum adjuvant, and about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58. and 59), 100 pg to about 3500 pg of an aluminum adjuvant, and about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 68), 100 pg to about 3500 pg of an aluminum adjuvant, and about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), 100 µg to about 3500 µg of an aluminum adjuvant, and about 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes 1 pg to about 300 pg, per VLP, of HPV VLPs (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58. 59. 68. and 73), 100 pg to26095about 3500 µg of an aluminum adjuvant, and about 2 µg / mL to about 400 mg / mL liposomal adjuvant.

[0157] In some embodiments of the vaccine composition, the liposomal adjuvant is any of the liposomal adjuvants described herein. In some embodiments, the vaccine composition includes 0.1 pg to 600 mg / mL of a liposomal adjuvant. In some embodiments, the vaccine composition includes 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the vaccine composition includes about 0.1 pg / mL to about 400 mg / mL liposomal adjuvant.

[0158] In some embodiments, the vaccine composition includes about 60 pg / mL to about 300 mg / mL phospholipid. In some embodiments, the vaccine composition includes about 80 pg / mL to about 50 mg / mL phospholipid. In some embodiments, the vaccine composition includes about 1 mg / mL to about 10 mg / mL phospholipid. In some embodiments, the vaccine composition includes about 2.5 mg / mL to about 3.5 mg / mL phospholipid. In some embodiments, the vaccine composition includes about 3.0 mg / mL phospholipid. In an embodiment, the phospholipid is DOPC.

[0159] In some embodiments, the vaccine composition includes about 0.05 pg / mL to about 10 mg / mL sterol. In some embodiments, the vaccine composition includes about 0.2 pg / mL to about 3.0 mg / mL sterol. In some embodiments, the vaccine composition includes about 0.25 mg / mL to about 2.0 mg / mL sterol. In some embodiments, the vaccine composition includes about 0.5 mg / mL to about 1.5 mg / mL sterol. In some embodiments, the vaccine composition includes about 0.75 mg / mL sterol. In an embodiment, the sterol is cholesterol.

[0160] In some embodiments, the vaccine composition includes about 0.01 pg / mL to about 10 mg / mL glycolipid. In some embodiments, the vaccine composition includes about 0.02 pg / mL to about 3.0 mg / mL glycolipid. In some embodiments, the vaccine composition includes about 0.03 mg / mL to about 2.0 mg / mL glycolipid. In some embodiments, the vaccine composition includes about 0.05 mg / mL to about 1.0 mg / mL glycolipid. In some embodiments, the vaccine composition includes about 0.15 mg / mL glycolipid. In an embodiment, the glycolipid is GLA.

[0161] In some embodiments, the vaccine composition includes about 0.01 pg / mL to about 10 mg / mL saponin. In some embodiments, the vaccine composition includes about 0.02 pg / mL to about 3.0 mg / mL saponin. In some embodiments, the vaccine composition includes about 0.03 mg / mL to about 2.0 mg / mL saponin. In some embodiments, the vaccine composition includes about 0.05 mg / mL to about 1.0 mg / mL saponin. In some embodiments, the vaccine composition includes about 0.15 mg / mL saponin. In an embodiment, the saponin is QS-21.

[0162] In some embodiments, the vaccine composition includes about 60 pg / mL to about 300 mg / mL buffer. In some embodiments, the vaccine composition includes about 80 pg / mL to about2609550 mg / mL buffer. In some embodiments, the vaccine composition includes about 1 mg / mL to about 25 mg / mL buffer. In some embodiments, the vaccine composition includes about 2.5 mg / mL to about 20 mg / mL buffer. In some embodiments, the vaccine composition includes about 13.0 mg / mL buffer. In an embodiment, the buffer includes about 1.0 mg / mL to about 20 mg / mL disodium hydrogen phosphate dihydrate, about 1.0 mg / mL to about 20 mg / mL potassium dihydrogen phosphate, and about 1.0 mg / mL to about 20 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.25 mg / mL to about 10 mg / mL disodium hydrogen phosphate dihydrate, about 2.5 mg / mL to about 15 mg / mL potassium dihydrogen phosphate, and about 2.5 mg / mL to about 15 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.5 mg / mL to about 5 mg / mL disodium hydrogen phosphate dihydrate, about 3.5 mg / mL to about 10 mg / mL potassium dihydrogen phosphate, and about 3.5 mg / mL to about 10 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.6 mg / mL disodium hydrogen phosphate dihydrate, about 5.6 mg / mL potassium dihydrogen phosphate, and about 5.8 mg / mL sodium chloride.

[0163] In an embodiment, the vaccine composition includes about 2.5 mg / mL to about 3.5 mg / mL phospholipid, about 0.5 mg / mL to about 1.5 mg / mL sterol, about 0.05 mg / mL to about 1.0 mg / mL glycolipid, about 0.05 mg / mL to about 1.0 mg / mL saponin, and about 2.5 mg / mL to about 20 mg / mL buffer.

[0164] In an embodiment, the vaccine composition includes about 2.5 mg / mL to about 3.5 mg / mL DOPC. about 0.5 mg / mL to about 1.5 mg / mL cholesterol, about 0.05 mg / mL to about 1.0 mg / mL GLA, about 0.05 mg / mL to about 1.0 mg / mL QS-21, and about 2.5 mg / mL to about 20 mg / mL buffer. In an embodiment, the vaccine composition includes about 2.5 mg / mL to about 3.5 mg / mL DOPC, about 0.5 mg / mL to about 1.5 mg / mL cholesterol, about 0.05 mg / mL to about 1.0 mg / mL GLA, about 0.05 mg / mL to about 1.0 mg / mL QS-21, 1.0 mg / mL to about 20 mg / mL disodium hydrogen phosphate dihydrate, about 1.0 mg / mL to about 20 mg / mL potassium dihydrogen phosphate, and about 1.0 mg / mL to about 20 mg / mL sodium chloride.

[0165] The vaccines of the invention comprise VLPs containing the antigenic determinants required to induce the generation of neutralizing antibodies in the subject. The vaccines are expected to be sufficiently safe to be administered without the risk of clinical infection, have no toxic side effects, are stable, compatible with conventional carriers and can be administered effectively. In some embodiments, a liposomal adjuvant of the invention is combined with a Human Papillomavirus Bivalent (Types 16 and 18) Vaccine, Recombinant. In some embodiments, the liposomal adjuvant of the invention is combined with CERVARIX®. In some embodiments, a liposomal adjuvant of the invention is combined with a Human Papillomavirus26095Quadrivalent (Types 6, 11, 16, 18) Vaccine, Recombinant. In some embodiments, a liposomal adjuvant of the invention is combined with GARDASIL®. In some embodiments, a liposomal adjuvant of the invention is combined with a Human Papillomavirus 9-valent Vaccine, Recombinant. In some embodiments, a liposomal adjuvant of the invention is combined with GARDASIL® 9.

[0166] Pharmaceutical compositions, formulations, and vaccines of the invention may be administered subcutaneously, topically, orally, on the mucosa, intravenously, or intramuscularly. The pharmaceutical compositions, formulations, and vaccines are administered in an amount sufficient to elicit a protective response. Vaccines, pharmaceutical compositions and formulations can be administered by various routes, for example, orally, parenterally, subcutaneously, on the mucosa, or intramuscularly. The dose administered may vary depending on the general condition, sex, weight and age of the patient, the route of administration and the type of HPV VLP in the vaccine. The vaccine, pharmaceutical composition, or formulation may be in the form of a capsule, suspension, elixir or solution. Such vaccine, pharmaceutical compositions or formulations may be formulated with an immunologically acceptable carrier. Kits of the Invention

[0167] Also provided herein are kits including any of the pharmaceutical compositions as described above and instructions for use.

[0168] Also provided herein are kits including (1) a pharmaceutical composition comprising HPV VLPs of at least one type of HPV and (2) a liposomal adjuvant.

[0169] In some embodiments of the kits, the pharmaceutical composition comprises HPV VLPs of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82. In some embodiments, the pharmaceutical composition is an HPV vaccine. In some embodiments, the HPV vaccine is a Human Papillomavirus Bivalent (Types 16 and 18) Vaccine, Recombinant. In some embodiments, the HPV vaccine is CERVARIX®. In some embodiments, the HPV vaccine is a Human Papillomavirus Quadrivalent (Types 6, 11, 16, 18) Vaccine, Recombinant. In some embodiments, the HPV vaccine is GARDASIL®. In some embodiments, the HPV vaccine is a Papillomavirus 9-valent Vaccine, Recombinant. In some embodiments, the HPV vaccine is GARDASIL® 9.

[0170] In an embodiment, the kit includes about 1 pg to about 300 pg of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 1 pg to 200 pg of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 1 pg to 100 pg of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 5 pg to26095200 pg of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 5 pg to 100 pg of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 10 pg to 200 pg a therapeutically effective dose of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 10 pg to 100 pg of VLPs of any of the at least one HPV type. In some embodiments, the kit includes about 10 pg to 80 pg of any of the at least one HPV type. In some embodiments, the kit includes about 20 pg to 60 pg of VLPs of any of the at least one HPV type.

[0171] In some embodiments of the kits, the liposomal adjuvant is any of the liposomal adjuvants described herein. In some embodiments, the kit includes 0.1 pg to 100 mg of a liposomal adjuvant. In some embodiments, the kit includes 2 pg / mL to about 400 mg / mL liposomal adjuvant. In some embodiments, the kit includes about 0.1 pg / mL to about 400 mg / mL liposomal adjuvant.

[0172] In some embodiments, the kit includes about 60 pg / mL to about 300 mg / mL phospholipid. In some embodiments, the kit includes about 80 pg / mL to about 50 mg / mL phospholipid. In some embodiments, the kit includes about 1 mg / mL to about 10 mg / mL phospholipid. In some embodiments, the kit includes about 2.5 mg / mL to about 3.5 mg / mL phospholipid. In some embodiments, the kit includes about 3.0 mg / mL phospholipid. In an embodiment, the phospholipid is DOPC.

[0173] In some embodiments, the kit includes about 0.05 pg / mL to about 10 mg / mL sterol. In some embodiments, the kit includes about 0.2 pg / mL to about 3.0 mg / mL sterol. In some embodiments, the kit includes about 0.25 mg / mL to about 2.0 mg / mL sterol. In some embodiments, the kit includes about 0.5 mg / mL to about 1.5 mg / mL sterol. In some embodiments, the kit includes about 0.75 mg / mL sterol. In an embodiment, the sterol is cholesterol.

[0174] In some embodiments, the kit includes about 0.01 pg / mL to about 10 mg / mL glycolipid. In some embodiments, the kit includes about 0.02 pg / mL to about 3.0 mg / mL glycolipid. In some embodiments, the kit includes about 0.03 mg / mL to about 2.0 mg / mL glycolipid. In some embodiments, the kit includes about 0.05 mg / mL to about 1.0 mg / mL glycolipid. In some embodiments, the kit includes about 0.15 mg / mL glycolipid. In an embodiment, the glycolipid is GLA.

[0175] In some embodiments, the kit includes about 0.01 pg / mL to about 10 mg / mL saponin. In some embodiments, the kit includes about 0.02 pg / mL to about 3.0 mg / mL saponin. In some embodiments, the kit includes about 0.03 mg / mL to about 2.0 mg / mL saponin. In some embodiments, the kit includes about 0.05 mg / mL to about 1.0 mg / mL saponin. In some26095embodiments, the kit includes about 0.15 mg / mL saponin. In an embodiment, the saponin is QS-21.

[0176] In some embodiments, the kit includes about 60 pg / mL to about 300 mg / mL buffer. In some embodiments, the kit includes about 80 pg / mL to about 50 mg / mL buffer. In some embodiments, the kit includes about 1 mg / mL to about 25 mg / mL buffer. In some embodiments, the kit includes about 2.5 mg / mL to about 20 mg / mL buffer. In some embodiments, the kit includes about 13.0 mg / mL buffer. In an embodiment, the buffer includes about 1.0 mg / mL to about 20 mg / mL disodium hydrogen phosphate dihydrate, about 1.0 mg / mL to about 20 mg / mL potassium dihydrogen phosphate, and about 1.0 mg / mL to about 20 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.25 mg / mL to about 10 mg / mL disodium hydrogen phosphate dihydrate, about 2.5 mg / mL to about 15 mg / mL potassium dihydrogen phosphate, and about 2.5 mg / mL to about 15 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.5 mg / mL to about 5 mg / mL disodium hydrogen phosphate dihydrate, about 3.5 mg / mL to about 10 mg / mL potassium dihydrogen phosphate, and about 3.5 mg / mL to about 10 mg / mL sodium chloride. In an embodiment, the buffer includes about 1.6 mg / mL disodium hydrogen phosphate dihydrate, about 5.6 mg / mL potassium dihydrogen phosphate, and about 5.8 mg / mL sodium chloride.

[0177] In an embodiment, the kit includes about 2.5 mg / mL to about 3.5 mg / mL phospholipid, about 0.5 mg / mL to about 1.5 mg / mL sterol, about 0.05 mg / mL to about 1.0 mg / mL glycolipid, about 0.05 mg / mL to about 1.0 mg / mL saponin, and about 2.5 mg / mL to about 20 mg / mL buffer.

[0178] In an embodiment, the kit includes about 2.5 mg / mL to about 3.5 mg / mL DOPC, about 0.5 mg / mL to about 1.5 mg / mL cholesterol, about 0.05 mg / mL to about 1.0 mg / mL GLA, about 0.05 mg / mL to about 1.0 mg / mL QS-21, and about 2.5 mg / mL to about 20 mg / mL buffer. In an embodiment, the kit includes about 2.5 mg / mL to about 3.5 mg / mL DOPC, about 0.5 mg / mL to about 1.5 mg / mL cholesterol, about 0.05 mg / mL to about 1.0 mg / mL GLA, about 0.05 mg / mL to about 1.0 mg / mL QS-21, 1.0 mg / mL to about 20 mg / mL disodium hydrogen phosphate dihydrate, about 1.0 mg / mL to about 20 mg / mL potassium dihydrogen phosphate, and about 1.0 mg / mL to about 20 mg / mL sodium chloride.

[0179] In an embodiment, the kits include instructions for admixing (1) the pharmaceutical composition or HPV vaccine and (2) the liposomal adjuvant and subsequentially administering (or vaccinating) the admixture to a patient.Methods of Treatment of the Invention

[0180] Also provided herein is a method of inducing an immune response to a human papillomavirus (HPV) in a human patient comprising administering to the patient a26095pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82.

[0181] Also provided herein is a method of inducing an immune response to a human papillomavirus (HPV) in a human patient including administering a liposomal adjuvant and virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82. In some embodiments, the liposomal adjuvant is formulated separately from the VLPs. In some embodiments, the liposomal adjuvant is formulated with the VLPs. In some embodiments, the liposomal adjuvant and VLPs are field-mixed to form a pharmaceutical composition prior to administration to the patient. In some embodiments, the liposomal adjuvant and VLPs are administered sequentially to a patient.

[0182] Also provided herein is a method of inducing an immune response to a human papillomavirus (HPV) in a human patient including co-administering to the patient (1) a pharmaceutical composition comprising virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82 and (2) a liposomal adjuvant.

[0183] Also provided herein is a method of preventing infection of or reducing the likelihood of infection of a human patient by a human papillomavirus (HPV) including administration to the patient a pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82.

[0184] Also provided herein is a method of delivering a pharmaceutical composition to a subject that induces a neutralizing titer against an HPV antigen in the subject that includes administering to the subject a pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, whereby the administration of the pharmaceutical composition induces a neutralizing titer against the HPV antigen in the subject, and wherein the pharmaceutical composition provides enhanced or comparable neutralizing titers when compared to the same pharmaceutical composition when the same composition is formulated without a liposomal adjuvant.

[0185] Also provided herein is a method for preventing cancer of a human patient cancers caused by human papillomavirus (HPV) Types 16, 18, 31, 33, 45, 52, and 58, including administration to the patient a pharmaceutical composition including a liposomal adjuvant and26095virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, wherein the cancer is cervical, vulvar, vaginal, anal, oropharyngeal, and other head and neck cancers.

[0186] Also provided herein is a method for preventing cancer of a human patient caused by HPV Types 6, 11, 16, 18, 31, 33, 45, 52, and 58 including administration to the patient a pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one ty pe of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, wherein the cancer is cervical, vulvar, vaginal, and anal precancerous or dysplastic lesions.

[0187] Also provided herein is a method for preventing cancer of a human patient caused by HPV Types 6 and 11, including administration to the patient a pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, wherein the cancer is genital warts or condyloma acuminata.

[0188] Also provided herein is a method for preventing precancerous or dysplastic lesions of a human patient caused by HPV Types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, including administration to the patient a pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, wherein the lesions are selected from cervical intraepithelial neoplasia (CIN) grade 2 / 3, cervical adenocarcinoma in situ (AIS), cervical intraepithelial neoplasia (CIN) grade 1, vulvar intraepithelial neoplasia (VIN) grade 2 and grade 3, vaginal intraepithelial neoplasia (ValN) grade 2 and grade 3, anal intraepithelial neoplasia (AIN) grades I, 2, and 3. (1.1).

[0189] Also provided herein is a method for preventing HPV -related anogenital disease of a human patient caused by HPV Types selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58. 59, 66, 68, 73, and 82 including administration to the patient a pharmaceutical composition including a liposomal adjuvant and virus-like particles (VLPs) of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82.

[0190] Embodiments of the invention also include one or more of the pharmaceutical compositions described herein (1) for use in, (2) for use as a medicament or composition for, or (3) for use in the preparation of a medicament for: (a) therapy (e.g., of the human body); (b)medicine; (c) induction of an immune response against HPV types included in the vaccine (d) decreasing the likelihood of HPV infection in a patient; (e) prevention of infection with HPV ty pes in the vaccine, (f) prevention or reduction of the likelihood of cervical cancer, (g) prevention or reduction of the likelihood of vulvar cancer, (h) prevention or reduction of the likelihood of vaginal cancer, (i) prevention or reduction of the likelihood of anal cancer, (j) prevention or reduction of the likelihood of oropharyngeal cancer, (k) prevention or reduction of the likelihood of other head and neck cancers, (k) prevention or reduction of the likelihood of precancerous or dysplastic anal lesions, (1) prevention or reduction of the likelihood of genital warts or condyloma acuminata, (m) prevention or reduction of the likelihood of Cervical intraepithelial neoplasia (CIN) grade 2 / 3 lesions, (n) prevention or reduction of the likelihood of cervical adenocarcinoma in situ (AIS) lesions, (o) prevention or reduction of the likelihood of Cervical intraepithelial neoplasia (CIN) grade 1 lesions, (p) prevention or reduction of the likelihood of Vulvar intraepithelial neoplasia (VIN) grade 2 and grade 3 lesions, (q) prevention or reduction of the likelihood of Vaginal intraepithelial neoplasia (ValN) grade 2 and grade 3 lesions, (r) prevention or reduction of the likelihood of Anal intraepithelial neoplasia (AIN) grades 1, 2, and 3 lesions.

[0191] In embodiment 1, a composition is provided that includes virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39. 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and liposomal adjuvant, wherein the liposomal adjuvant comprises a glycolipid and a saponin.

[0192] In embodiment 2, the composition of embodiment 1 is provided, wherein the composition is made by mixing an HPV vaccine and the liposomal adjuvant; wherein the HPV vaccine comprises HPV VLPs of at least one type of HPV selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33. 35. 39. 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and a pharmaceutically acceptable carrier and the liposomal adjuvant comprises a glucopyranosyl lipid A (“GLA”) and QS-21.

[0193] In embodiment 3, the composition of embodiment 1 is provided, wherein the liposomal adjuvant comprises a glucopyranosyl lipid A (“GLA’") and QS-21.

[0194] In embodiment 4. the composition of embodiment 1-3 is provided, wherein the glycolipid has the structure set forth in Formula I:OHFormula I,wherein R1 is -P(O)(OH)2; R2 is selected from H, -C(O)CH2CH(OH)C11 alkyl and -C(O)CH2CH(OC(O)C11-C13 alkyl)C11 alkyl; R3 is selected from -C(O)CH2CH(OC(O)C11-C13 alkyl)C11 alkyl; R4 is selected from H, -C(O)CH2CH(OH)C11 alkyl, and -C(O)CH2CH(OC(O)C11-C13 alkyl)C11 alkyl; R5 is selected from -C(O)CH2CH(OH)C11 alkyl and -C(O)CH2CH(OC(O)C13-C15 alkyl)C11 alkyl; and R6 is H.

[0195] In embodiment 5, the composition of any one of embodiments 1-4 is provided, wherein the glycolipid has the structure set forth in Formula II:Formula II.wherein R1 is H, -C(O)C11-C13 alkyl; R2 is C11 alkyl; R3 is C11-C13 alkyl; wherein R4 is C11 alkyl; R5 is H, -C(O)CH2CH(OH)C11 alkyl; R6 is H, -C(O)C13-C15 alkyl; and R7 is C11 alkyl.

[0196] In embodiment 6. the composition of any one of embodiments 1-5 is provided, wherein the glycolipid has the structure set forth in Formula IIIA:MeFormula IIIA.

[0197] In embodiment 7, the composition of any one of embodiments 1-5 is provided, wherein the glycolipid has the structure set forth in Formula IV:26095Me MeFormula IV.

[0198] In embodiment 8, the composition of any one of embodiments 1-5 is provided, wherein the glycolipid has the structure set forth in Formula V:26095MeFormula V.

[0199] In embodiment 9, the composition of any one of embodiments 1-5 is provided, wherein the glycolipid has the structure set forth in Formula VI:26095MeFormula VI.

[0200] In embodiment 10, the composition of any one of embodiments 1-9 are provided, wherein the liposomal adjuvant further comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0201] In embodiment 11, the composition of any one of embodiments 1-10 are provided, wherein the liposomal adjuvant further comprises cholesterol.

[0202] In embodiment 12, the composition of any one of embodiments 1-11 is provided, wherein the composition further comprises a buffer.

[0203] In embodiment 13, the composition of any one of embodiment 12 is provided, wherein the buffer is selected from the group consisting of: acetic acid, histidine, citrate, Bis-Tris,26095HEPES, potassium dihydrogen phosphate, disodium phosphate, MES, sodium chloride, succinate, and Tris, or combinations thereof.

[0204] In embodiment 14, the composition of any one of embodiments 12-13 is provided, wherein the buffer is present in the amount of about ImMol to about lOOmMol, at a pH of 5.1-7.0.

[0205] In embodiment 15, the composition of any one of embodiments 1-14 is provided, wherein the composition further comprises a salt.

[0206] In embodiment 16, the composition of any one of embodiment 15 is provided, wherein the salt is NaCl.

[0207] In embodiment 17, the composition of any one of embodiments 1-16 is provided, wherein the composition comprises 5 mM - 100 mM potassium hydrogen phosphate, 1 mM to 50 mM disodium phosphate, and 25 mM - 300 mM NaCl.

[0208] In embodiment 18, the composition of any one of embodiments 1-16 is provided, wherein the composition comprises 40 mM - 50 mM potassium hydrogen phosphate, 5 mM to 15 mM disodium phosphate, and 50 mM - 150 mM NaCl.

[0209] In embodiment 19, the composition of any one of embodiments 1-18 is provided, wherein the composition comprises VLPs of HPV types 16 and 18.

[0210] In embodiment 20, the composition of any one of embodiments 1-18 is provided, wherein the composition comprises VLPs of HPV types 6, 11, 16, and 18.

[0211] In embodiment 21, the composition of any one of embodiments 1-18 is provided, wherein the composition comprises VLPs of HPV types 31, 45, 52, and 58.

[0212] In embodiment 22, the composition of any one of embodiments 1-18 is provided, wherein the composition comprises VLPs of HPV ty pes 6, 11, 16, 18, 31, 33, 45, 52, and 58.

[0213] In embodiment 23, the composition of any one of embodiments 1-18 is provided, wherein the composition comprises VLPs of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.

[0214] In embodiment 24, the composition of any one of embodiments 1-18 is provided, wherein the composition comprises VLPs of HPV types 6, 11, 16, 18, 31, 33, 35. 39, 45, 51, 52, 56. 58, 59, and 73.

[0215] In embodiment 25, the composition of any one of embodiments 1 -18 is provided, wherein the composition comprises VLPs of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70.

[0216] In embodiment 26, the composition of any one of embodiments 1-25 is provided, wherein the composition further comprises aluminum.26095

[0217] In embodiment 27, the composition of any one of embodiments 1-26 is provided, wherein the HPV VLPs comprise recombinant HPV LI or recombinant HPV LI + L2 protein.

[0218] In embodiment 28, the composition of any one of embodiments 1-26 is provided, wherein the HPV VLPs comprise HPV LI protein and do not comprise HPV L2 protein.

[0219] In embodiment 29, the composition of any one of embodiments 1-26 is provided, wherein the HPV VLPs consist of HPV LI protein.

[0220] In embodiment 30, a method of preventing infection of or reducing the likelihood of infection of a human patient by a human papillomavirus (HPV) is provided comprising administration to the patient the composition of any one of embodiments 1-29.

[0221] In embodiment 31, a kit is provided comprising: (a) a human papillomavirus (HPV) vaccine comprising HPV VLPs of at least one HPV type; and (b) a liposomal adjuvant, wherein the liposomal adjuvant comprises a glycolipid and a saponin.

[0222] In embodiment 32, the kit of embodiment 30 is provided further comprising instructions for administering to a human patient the HPV vaccine and the liposomal adjuvant.

[0223] In embodiment 33, the kit of any one of embodiments 31-32 is provided wherein the HPV vaccine comprises virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82.

[0224] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methodologies and materials that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0225] Having described embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be used by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0226] The following examples illustrate, but do not limit the invention.EXAMPLESExample 1: Preparation of Formulation Buffer

[0227] A buffer formulation was prepared by dispensing water for injection (WFI) into a buffer preparation vessel. 15.54 grams of powdered di-sodium hydrogen phosphate dibasic, 54.31 grams of potassium dihydrogen phosphate, and 56.75 grams sodium chloride were then added to the vessel. The resulting formulation was stirred using a stir bar until dissolution of ingredients26095was adjusted to pH 6.2±0.1. WFI was added to bring the buffer to the final target volume of 10L. The buffer was then filtered through a 0.22 pg filter to form the Formulation Buffer.Example 2: Preparation of HPV Buffer

[0228] An additional buffer was prepared that combined 10mM histidine, 325 mM NaCl, and 0.01% PS-80, pH 6.2 to form the HPV Buffer.Example 3: Preparation of a Liposomal Adjuvant

[0229] The liposomal adjuvant formulation was prepared by mixing 2mL of 10.0 mg / mL of cholesterol / chloroform stock solution (Avanti’s Cat# 700000 dissolved in chloroform), 3.2 mL of 25mg / mL DOPC / chloroform stock solution (Avanti’s Cat# 850375C), and 4mL of chloroform, and 2mL of a glycolipid that is the ammonium salt of Formula IIIA above (2mg / mL) in chloroform: methanol (9: 1 v / v ratio) solution. The individual lipid solutions were then pooled together to make a lipid stock solution in a round bottom flask.

[0230] The round bottom flask containing the lipid stock solution was attached to a rotatory evaporator. The rotation rate was set at 280 rpm and a water bath at 20°C, the pressure set point was incrementally reduced to prevent boiling from 1000mbar down to 15mbar gradually and the rotatory evaporation process was continued until the lipid stock solution formed a thin lipidic film. The round bottom flask was purged with nitrogen gas to complete the drying process.

[0231] The thin lipidic film in the round bottom flask was then reconstituted with 14.0 mL of the Formulation Buffer described in Example 1 (50 mM Na / K Phosphate, 100 mM NaCl pH 6.2). The combination was then heated at 50°C until a polydisperse mixture of liposomes was formed.

[0232] The resulting polydisperse liposome mixture was then passed through a high-pressure extruder device fitted with a 100nm pore size polycarbonate membrane. The pressure was adjusted manually between 200psi - 380psi to drive the liposomes through the polycarbonate membrane. The extruder temperature was controlled at 50°C during the extrusion. The liposomes were extruded 6 times (6 passes) through the membrane to get the particle size ~100nm. The resulting liposomes were placed in a glass bottle.Example 4: Preparation of Liposomal Adjuvant Formulation

[0233] QS-21 (Desert King International Adjuvant QS-21, Fisher Scientific cat # NC0949192) was dissolved in 10mg / mL of the Formulation Buffer described in Example 1 (50 mM Na / K Phosphate, 100 mM NaCl pH 6.2). 0.4mL of 10mg / mL QS-21 solution was added to the glass bottle containing the extruded liposomes and mixed well. An additional 7.2 mL of the Formulation Buffer described in Example 1 (50 mM Na / K Phosphate, 100 mM NaCl pH 6.2) was added to the glass bottle to arrive at the final liposomal formulation having a Formula IIIA concentration of 0.2mg / mL and QS-21 concentration of 0.2mg / mL. The mass ratio of 1,2-26095dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, Quillaja Saponaria 21 (QS-21) and the ammonium salt of Formula IIIA was 20:5:1:1 by weight.Example 5: Preparation of 14vHPV Vaccine Formulation

[0234] To prepare a 14 valent HPV vaccine formulation, 9-valent HPV antigen on aluminum was first prepared combining purified virus-like particles (VLPs) of the major capsid (LI) protein of HPV Types 6, 11, 16, 18, 31, 33, 45, 52, and 58) adsorbed on Amorphous Aluminum Hydroxyphosphate Sulfate or AAHS in the HPV Buffer described in Example 2 (hereinafter “9vHPV Vaccine”) and a 5-valent HPV antigen was then prepared by combining purified VLPs of the major capsid (LI) protein of HPV Types 35, 39, 51, 56 and 59) adsorbed on AAHS in the HPV Buffer described in Example 2 (hereinafter “5vHPV Vaccine”).

[0235] The 9vHPV Vaccine and 5vHPV Vaccine were mixed well by inversion. One mL of 9vHPV Vaccine was added to 2.9 mL HPV Buffer of Example 2, followed by the addition of 0.4 mL of 5vHPV Vaccine. The components were mixed again by inversion to form the 14vHPV Vaccine Formulation.Example 6: Preparation of Liposomal Adjuvant 14vHPV Vaccine Formulation (Group 1)

[0236] 0.3 mL of the 14vHPV Vaccine Formulation described in Example 5 was added to 5.7 mL HPV Buffer of Example 2. Each 1.0 mL dose of 14vHPV Vaccine Formulation consisted of 18 pg 9vHPV VLPs (total amount of types 6, 11, 16, 18, 31, 33, 45, 52, and 58) adsorbed to 33 pg total AAHS, and 5 pg 5vHPV VLPs (1 pg of each type 35, 39, 51, 56, and 59) adsorbed to 1.8 pg total AAHS.Example 7: Preparation of Liposomal Adjuvant 14vHPV Vaccine Formulation (Group 4)

[0237] 0.3 mL of the 14vHPV Vaccine Formulation described in Example 5 was added to 2.7 mL HPV Buffer of Example 2, followed by the addition of 3 mL of the Liposomal Adjuvant described in Example 4 (“Liposomal Adjuvant 14vHPV Vaccine Formulation”). Each 1.0 mL dose of the Liposomal Adjuvant 14vHPV Vaccine Formulation consisted of 18 pg 9vHPV VLPs (total amount of types 6, 11, 16, 18, 31, 33, 45, 52, and 58) adsorbed to 33 pg total AAHS, and 5 pg 5vHPV VLPs (1 pg of each type 35, 39, 51, 56, and 59) adsorbed to 1.8 pg total AAHS. Example 8: Immunogenicity and Durability of a Two Dose 14vHPV Vaccine + Liposomal Adjuvant in Rhesus Macaques

[0238] Groups of 4 rhesus monkeys were inoculated intramuscularly with two doses of 9vHPV Vaccine plus 5vHPV Vaccine (1.3x / 20) (“14vHPV Vaccine”) or 9vHPV Vaccine plus 5vHPV Vaccine (1.3x / 20) plus the Liposomal Adjuvant (2.03 mg) described in Example 4 (“Liposomal Adjuvant 14vHPV Vaccine”). The group designations are described below in Table III. The 9vHPV and 5vHPV components were administered at approximately 1 / 20ththe targeted human26095dose, at Weeks 0 and 8. The 14vHPV Vaccine Formulation was a field mix of the 9vHPV Vaccine and the 5vHPV Vaccine in an HPV Buffer of Example 2. The Liposomal Adjuvant 14vHPV Vaccine Formulation was a field mix of the 9vHPV Vaccine, the 5vHPV Vaccine, and the Liposomal Adjuvant of Example 4, in an HPV buffer. The formulations were prepared to the dose concentration described in Table III. Each l. OmL dose was administered intramuscularly to the right quadricep within 3 hours of preparation. Serum samples were collected at Weeks 1 (prestudy), 2, 4, 6, 8 (prior to 2ndvaccination), 10, 12, 14, 16, 20, and 28. Sera were evaluated for antibody levels against the 14 VLPs of the HPV types in the 14vHPV Vaccine by ELISA to assess immunogenicity.Table IIIDose LevelNo. of Liposomal Vol.dDosing Group’ Animals Treatment 9vHPVb+ 5vHPVcAdjuvant ROA (mL) Schedule ~1 / 20thhuman dose618 pg total VLP / 33 pg1 14vHPV Vaccine Weeks 0, 4 AAHS 0 IM 1.0 Formulation 8 + 1 pg per type / 1.8 pgAAHS~ 1 / 20thhuman dose Liposomal618 pg total VLP / 33 pg4 Adjuvant Weeks 0, 4 AAHS 2.03 mg6IM 1.0 14vHPV Vaccine 8 + 1 pg per type / 1.8 pgFormulationAAHS5vHPV=5-valent human papillomavirus antigen component: 9vHPV=9- valent human papillomavirus antigen component; 14vHPV Vaccine Formulation = 5vHPV+9vHPV; Liposomal Adjuvant 14vHPV Vaccine Formulation = 5vHPV+9vHPV + Liposomal Adjuvant; AAHS=amorphous aluminum hydroxyphosphate sulfate; HPV=human papillomavirus; IM=intramuscular(ly ); No.=number; ROA=route of administration; Liposomal Adjuvant =MSD proprietary liposomal vaccine adjuvant; VLP=virus-like particle; Vol.=volume.aThe study protocol included additional groups not relevant to this program.bThe dose level of 9vHPV refers to the total amount of VLPs and total 33 pg AAHS at ~1 / 20thof 1,3x / 20.6The dose level of 5vHPV refers to 1 pg of each of the 5 VLPs and total 1.8 pg AAHS at ~1 / 20thof 1,3x / 20.dThe 1.0-mL dose was administered to the right quadriceps.6One rhesus monkey dose of 9vHPV is equivalent to ~1 / 20thof one human dose of GARDASIL®9.Example 9: ELISA to Evaluate Serum Antibody Levels Against HPV VLPs

[0239] An ELISA was used to determine the titers of rhesus monkey serum antibodies that bind to the VLPs of the 14 HPV types in the vaccine. Maxisorp black 384-well plates were coated with 0.8 pg / mL of one of the 14 HPV VLPs (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52. 56.58, and 59) in 20 mM histidine, 0.5 M NaCl, pH 6.2, for overnight incubation at 4 °C. After coating, plates were blocked with PBST with 3% nonfat dry milk for 30 minutes. Serum samples starting at 1:50 were diluted 4-fold across 10 concentrations in blocking buffer and incubated for 2 hours. Pooled NHP sera from animals immunized with 9vHPV Vaccine / 5vHPV Vaccine was26095included as a positive control on each assay plate. VLP-bound antibodies were detected by a 1:10000 dilution of goat anti-human IgG (Fcγ fragment specific)-HRP antibody that was incubated for 1 hour. Plates were washed 6 times with PBST after each step. Plates were developed with West Pico PLUS Chemiluminescent HRP Substrate that was prepared according to the manufacturer’s instructions. The luminescent assay signal generated by HRP cleavage of the chemiluminescent substrate was read on an EnVision plate reader (PerkinElmer, Waltham. Massachusetts. USA) after a 15-minute incubation.

[0240] A study was conducted to evaluate the immunogenicity of a 2-dose administration of the Liposomal Adjuvant 14vHPV Vaccine Formulation in an NHP nonclinical immunogenicity model. As set forth in Example 9, Groups of 4 rhesus monkeys were inoculated IM with 2 doses of the 14vHPV Vaccine Formulation or 2 doses of the Liposomal Adjuvant 14vHPV Vaccine Formulation, where the 14vHPV Vaccine Formulation was administered at approximately l / 20th of the targeted human dose. Doses were administered at Weeks 0 and 8.

[0241] To assess immunogenicity in both the 14vHPV Vaccine Formulation and Liposomal Adjuvant 14vHPV Vaccine Formulation, serum samples collected at Weeks -1 (prestudy), 2, 4, 6, 8 (prior to 2nd vaccination), 10, 12, 14, 16, 20, and 28, were evaluated for antibody levels against the 14 VLPs of the HPV types by ELISA. The antibody titers against the 14 HPV VLPs in both the 14vHPV Vaccine Formulation and Liposomal Adjuvant 14vfHPV Vaccine Formulation are presented in [FIGs. 1A-1N]. Rhesus monkeys (n=4 / group) were injected IM with 2 doses (Weeks 0 and 8, indicated by arrows) of 14vHPV Vaccine Formulation or Liposomal Adjuvant 14vHPV Vaccine Formulation, where the 14vHPV Vaccine Formulation was administered at approximately l / 20th of the targeted human dose. Serum samples were collected at Weeks 1 (prestudy), 2, 4, 6, 8 (prior to second vaccination), 10, 12, 14, 16, 20, and 28.

[0242] FIGs. 1 A - IN show longitudinal antibody levels against 14 HPV VLPs after a 2-dose IM administration of 14vHPV vaccine formulation or liposomal adjuvant 14xHPV vaccine formulation at approximately 1 / 20 of target human dose in rhesus monkeys. Specifically, the data shows interpolated serum antibody titers against the 14 VLPs of HPV types 6 (FIG. 1A), 11 (FIG. IB), 16 (FIG. 1C), 18 (FIG. ID), 31 (FIG. IE), 33 (FIG. IF), 35 (FIG. 1G), 39 (FIG. 1H), 45 (FIG. 11). 51 (FIG. 1 J), 52 (FIG. IK), 56 (FIG. IL), 58 (FIG. IM), and 59 (FIG. IN), in the 14-valent HPV formulations through Week 28. The data are presented as geometric mean titers. Error bars represent the 95% CI of the GMTs.

[0243] Both the 14vHPV Vaccine Formulation and Liposomal Adjuvant 14vHPV Vaccine Formulation elicited antibody titers against the legacy 9 HPV VLPs (types 6. 11, 16, 18, 31, 33, 45, 52, and 58) and the additional 5 HPV VLPs (types 35, 39, 51, 56, and 59) at 2 weeks PD126095(Week 2). A second dose of each formulation administered at Week 8 boosted the antibody titers against all 14 VLPs at Week 10. Titers following Week 10 decreased relative to peak levels and were detected for all 14 HPV VLPs through Week 28 in both treatment groups.Liposomal Adjuvant 14vHPV Vaccine Formulation elicited generally higher antibody GMTs than those of the 14vHPV Vaccine Formulation against many HPV VLPs at 4 weeks PD1, and against all 14 HPV VLPs at 2 weeks PD2 (Week 10) through Week 28.

[0244] The Liposomal Adjuvant, as described above in Example 4, enhanced immunogenicity of the 114vHPV Vaccine Formulation. Liposomal Adjuvant 14vHPV Vaccine Formulation was immunogenic in rhesus monkeys at a dose supportive of those planned for clinical studies.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and a liposomal adjuvant, wherein the liposomal adjuvant comprises a glycolipid and a saponin.

2. The composition of claim 1, wherein the liposomal adjuvant comprises a glucopyranosyl lipid A (“GLA”) and QS-21.

3. The composition of any one of claims 1-2. wherein the composition is made by mixing an HPV vaccine and the liposomal adjuvant; wherein the HPV vaccine comprises HPV VLPs of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82 and a pharmaceutically acceptable carrier and the liposomal adjuvant comprises a glucopyranosyl lipid A (“GLA”) and QS-21.

4. The composition any one of claims 1-3, wherein the glycolipid has the structure set forth in Formula I:OHFormula I,wherein:Ri is -P(O)(OH)2;R2 is selected fromH, -C(O)CH2CH(OH)Cn alkyl, and -C(O)CH2CH(OC(O)Cn-Ci3alkyl)Ci i alkyl:R? IS -C(O)CH2CH(OC(O)CII-CI? alkyl)Cn alkyl;R4IS selected fromH, -C(O)CH2CH(OH)Cn alkyl, and -C(O)CH2CH(OC(O)Cn-Ci3 alkyl)Ci i alkyl;Rs is selected from -C(O)CH2CH(OH)Cn alkyl and -C(O)CH2CH(OC(O)Ci3-Ci5alkyl)Cn alkyl: andRs is H, ora pharmaceutically acceptable salt thereof.

5. The composition any one of claims 1-4, wherein the glycolipid has the structure set forth in Formula II:Formula II,wherein: Ri is H, C(O)Cn-Ci3 alkyl:R2is CH alkyl:R3 is C11-C13 alkyl;R4is C11alkyl;Rs is H, C(O)CH2CH(OH)CII alkyl;Rs is H, C(O)Ci3-Ci5 alkyl; andR7 is C11 alkyl;or a pharmaceutically acceptable salt thereof.

6. The composition any one of claims 1-5, wherein the glycolipid has the structure set forth in Formula IIIA:MeFormula IIIA, or a pharmaceutically acceptable salt thereof.

7. The composition any one of claims 1-5, wherein the glycolipid has the structure set forth in Formula IVA:26095Me MeFormula IVA, or a pharmaceutically acceptable salt thereof.

8. The composition any one of claims 1-5, wherein the glycolipid has the structure set forth in Formula V:MeFormula V, or a pharmaceutically acceptable salt thereof.

9. The composition of claims 1-5, wherein the glycolipid has the structure set forth in Formula VI:MeFormula VI, or a pharmaceutically acceptable salt thereof.

10. The composition any one of claims 1-9, wherein the liposomal adjuvant further comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

11. The composition any one of claims 1-10, wherein the liposomal adjuvant further comprises cholesterol.

12. The composition any one of claims 1-11, wherein the composition further comprises a buffer.

13. The composition any one of claim 12, wherein the buffer is selected from the group consisting of acetic acid, histidine, citrate, Bis-Tris, HEPES. potassium dihydrogen phosphate, disodium phosphate, MES, sodium chloride, succinate, Tris, and combinations thereof.

14. The composition any one of claims 12-13, wherein the buffer is present in the amount of about ImMol to about lOOmMol.

15. The composition any one of claims 1-14, wherein the composition further comprises a salt.

16. The composition any one of claim 15, wherein the salt is NaCl.

17. The composition any one of claims 1-16, wherein the composition comprises 5 mM - 100 mM potassium hydrogen phosphate at pH 5.1 - 7.0, 1 mM to 50 mM disodium phosphate at pH 5.1 - 7.0, and 25 mM - 300 mM NaCl at pH 5.1 - 7.0.

18. The composition any one of claims 1-16, wherein the composition comprises 40 mM - 50 mM potassium hydrogen phosphate at pH 5.1 - 7.0, 5 mM to 15 mM disodium phosphate at pH 5.1 - 7.0, and 50 mM - 150 mM NaCl at pH 5.1 - 7.0.

19. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV types 16 and 18.

20. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV types 6, 11, 16, and 18.

21. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV types 31, 45, 52, and 58.

22. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58.2609523. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56.

58. and 59.

24. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV ty pes 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 73.

25. The composition any one of claims 1-18, wherein the composition comprises VLPs of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70.

26. The composition any one of claims 1-25, wherein the composition further comprises aluminum.

27. The composition any one of claims 1-26, wherein the HPV VLPs comprise recombinant HPV LI or recombinant HPV LI + L2 protein.

28. The composition any one of claims 1-26, wherein the HPV VLPs comprise HPV LI protein and do not comprise HPV L2 protein.

29. The composition any one of claims 1-26, wherein the HPV VLPs consist of HPV LI protein.

30. A method of preventing infection of or reducing the likelihood of infection of a human patient by a human papillomavirus (HPV) comprising administration to the patient the composition of claims 1-29.

31. A kit comprising:(a) a human papillomavirus (HPV) vaccine comprising HPV VLPs of at least one HPV ty pe; and(b) a liposomal adjuvant, wherein the liposomal adjuvant comprises a glycolipid and a saponin.

32. The kit of claim 31, further comprising instructions for administering to a human patient the HPV vaccine and the liposomal adjuvant.

33. The kit any one of claims 31-32, wherein the HPV vaccine comprises virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from the group consisting of HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82.

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