Nanoemulsion Adjuvant Composition for Human Papillomavirus Vaccine

Abstract

This disclosure, along with other matters, provides a vaccine composition comprising a squalene nanoemulsion (SNE) adjuvant and HPV virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 507,269, filed on June 9, 2023, the disclosure of which is incorporated herein in its entirety.

[0002] The present invention generally relates to the prevention of human papillomavirus (HPV) diseases. More specifically, the present invention relates to a composition comprising human papillomavirus virus - like particles (VLPs) and a squalene nano - emulsion (SNE) adjuvant that can be administered as a vaccine. The SNE adjuvant comprises a surfactant and / or a terpene and / or a terpenoid - based oil and / or a cationic lipid or a mixture thereof. Further, methods of using the disclosed compositions and formulations are provided.

Background Art

[0003] Human papillomavirus (HPV) is a small, double-stranded DNA virus that infects the skin and internal squamous mucosal epithelium of men and women. HPV is classified based on its oncogenic properties. HPV contains a major (L1) capsid protein and a secondary (L2) capsid protein. More than 200 different 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 are associated with conditions ranging from benign proliferative warts to malignant cervical carcinomas (see McMurray et al., Int.J.Exp.Patel.82(1):15-33 (2001) for a review). HPV types classified as "high-risk" include types 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 causative agent of cervical cancer, one of the most common cancer types in women, as well as squamous cell carcinomas of the anus, tonsils, tongue, vulva, vagina, and penis. HPV16 and HPV18 are known to be the most toxic of the high-risk HPV types, as they cause approximately 70% of all invasive cervical cancers worldwide.

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

[0006] VLPs are morphologically similar to true virions and can induce high titer neutralizing antibodies when administered to animals or humans. Since VLPs do not contain potentially oncogenic viral genomes, they offer a safe alternative to the use of live viruses in HPV vaccine development (see Schiller and Hidesheim, J Clin. Virol. 19:67-74 (2000) for a review).

[0007] VLP-based vaccines have been shown to be effective in inducing immune responses in human subjects vaccinated against bivalent HPV 16 and 18 (Harper et al. Lancet 364(9447):1757-65(2004)), tetravalent HPV 6, 11, 16 and 18 (Villa et al. Vaccine 24:5571-5583(2006)), and polyvalent HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 VLP-based vaccines. Three commercially available VLP-based vaccines against HPV are administered according to two or three dose regimens. CERVARIX® (GlaxoSmithKline Biologicals, Rixensart, Belgium) is a bivalent vaccine that provides protection against HPV 16 and 18. GARDASIL® and GARDASIL® 9 (Merck & Co., Inc., Lethway, New Jersey, USA) protect against two and seven additional HPV strains, respectively, and prevent additional HPV-related anogenital diseases, including wart formation. Compared to GARDASIL®, GARDASIL® 9 protects against five additional high-risk strains, increasing protection against anogenital malignancies from approximately 70% to approximately 90%. (Ibid., M. Nygard, et al., "Evaluation of the long-term anti-human 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] Although improving, global HPV vaccination rates remain insufficient. Global HPV vaccination coverage can be improved by reducing the number of visits required for vaccination, enhancing education on HPV disease prevention, and mitigating the social stigma associated with vaccination. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Furthermore, currently approved HPV vaccines utilize aluminum-containing derivatives as adjuvants to enhance immunogenicity. While aluminum adjuvants increase the immunogenic response from baseline, it is unclear whether this response is sufficient for more multivalent HPV vaccines. Therefore, there is a need to identify other adjuvants that can enhance the immunogenicity of multivalent HPV vaccines more effectively than current aluminum adjuvants. [Means for solving the problem]

[0010] The present invention provides a composition comprising at least one human papillomavirus (HPV) type virus-like particle (VLP) and a squalene nanoemulsion (SNE) adjuvant, wherein the human papillomavirus (HPV) is 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 adsorbed onto an aluminum adjuvant, and the SNE adjuvant comprises sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20), or polysorbate 80 (PS-80), and squalene.

[0011] The present invention further provides a composition comprising at least one human papillomavirus (HPV) type virus-like particle (VLP) and a squalene nanoemulsion (SNE) adjuvant, wherein the human papillomavirus (HPV) is 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 the SNE adjuvant comprises sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20), or polysorbate 80 (PS-80), and squalene. In some embodiments, the composition also comprises an aluminum adjuvant. In some embodiments, the VLP is adsorbed to the aluminum adjuvant.

[0012] The present invention further provides a method for inducing an immune response to human papillomavirus (HPV) in a human patient, comprising administering to the patient a pharmaceutical composition comprising virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 squalene nanoemulsion (SNE) adjuvant, wherein the SNE adjuvant comprises sorbitan trioleate (SPAN-85); polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and squalene, and may further comprise an aluminum adjuvant.

[0013] The Disclosure further provides, in particular, a method for preventing or reducing the likelihood of infection of a human patient with human papillomavirus (HPV), comprising administering to a patient a pharmaceutical composition comprising virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 adsorbed to an aluminum adjuvant, and a squalene nanoemulsion (SNE) adjuvant, wherein the SNE adjuvant comprises sorbitan trioleate (SPAN-85); polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and squalene.

[0014] The present invention further provides a use for preventing or reducing the likelihood of infection of a human patient with human papillomavirus (HPV), comprising at least one human papillomavirus (HPV) type virus-like particle (VLP) and a squalene nanoemulsion (SNE) adjuvant, wherein the human papillomavirus (HPV) is 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 adsorbed to an aluminum adjuvant, and the SNE adjuvant comprises sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20), or polysorbate 80 (PS-80), and squalene.

[0015] The present invention also provides a kit comprising (1) a human papillomavirus (HPV) vaccine and (2) a squalene nanoemulsion (SNE) adjuvant, the SNE adjuvant comprising sorbitan trioleate (SPAN-85); polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and squalene, and which may further comprise an aluminum adjuvant.

[0016] Definition As used throughout this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0017] When used throughout this specification and the appended claims, the following abbreviations and definitions apply.

[0018] CLA ((13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine) HPV Human papillomavirus ID Intradermal IM Intramuscular ME Microemulsion MNS Microfluidic nanoemulsion self-assembly Mw Molecular weight NE Nanoemulsion NMWCO Nominal molecular weight cut-off PHE Prehomogenized emulsion PS-20 Polysorbate 20 PS-80 Polysorbate 80 SNE Squalen nanoemulsion SPAN-85 Sorbitan trioleate (One or more) VPL (One or more) virus-like particles (VLP) w / v Weight per volume When used throughout this specification and the appended claims, the following definitions and abbreviations apply.

[0019] AAHS: As used herein, the term "AAHS" refers to an amorphous aluminum hydroxyphosphate sulfate adjuvant.

[0020] About: As used herein, the term "about" when used in reference to a value refers to a value that is the same as or similar in context to the referenced value. Generally, one of ordinary skill in the art who is familiar with the context will understand the absolute amount and / or relative degree of difference subsumed by "about" in that context. For example, in some embodiments, the term "about" can encompass values within a range of, or less than, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the reference value.

[0021] Adjuvant: As used herein, the term “adjuvant” refers to a composition or compound capable of enhancing an immune response to an antigen of interest. An adjuvant is a substance or combination of substances used with a vaccine antigen to enhance a specific immune response to the vaccine antigen (e.g., increase, accelerate, prolong, and / or potentially target) or to modulate it to a different type (e.g., switch a Th1 immune response to a Th2 response, or a humoral response to a cytotoxic T-cell response) in order to increase the clinical efficacy of the vaccine. In some embodiments, the adjuvant modifies the immune response (Th1 / Th2). In some embodiments, the adjuvant enhances the intensity and duration of the immune response. In some embodiments, the adjuvant broadens the immune response to antigens administered simultaneously. In some embodiments, the adjuvant can induce a strong antibody and T-cell response. In some embodiments, the adjuvant can increase the polyclonal capacity of the induced antibody. In some embodiments, the adjuvant is used to reduce the amount of antigen required to induce a desired immune response and provide protection against disease. In some embodiments, adjuvants are used to induce a sustained immune response and reduce the number of injections required in a clinical regime to provide protection against disease. The adjuvant-containing formulations described herein may demonstrate enhancement of the humoral and / or cellular immunogenicity of vaccine antigens, such as subunit vaccine antigens. The adjuvants of the present invention are not used to deliver antigens, antibodies, active pharmaceutical ingredients (APIs), or VLPs.

[0022] Administration: As used herein, the term “administration” means the act of providing an active agent, composition, or preparation to the human body. Exemplary routes of administration to the human body may include the eye (ophthalmic), oral cavity (oral), skin (percutaneous), nasal cavity (transnasal), lung (inhalation), rectum, vagina, oral mucosa (buccal mucosa), ear, and injection (e.g., intravenous (IV), subcutaneous, intratumoral, intraperitoneal, intramuscular (IM), intradermal (ID), etc.).

[0023] Drugs: As used herein, the term “drug” refers to any chemical class of particles, compounds, molecules, or entities, including, for example, VLPs, small molecules, polypeptides (e.g., proteins), polynucleotides (e.g., DNA polynucleotides or RNA polynucleotides), sugars, lipids, or combinations or complexes thereof. In some embodiments, the term “drug” may refer to a compound, molecule, or entity comprising a polymer or a plurality thereof.

[0024] Alkenyl: As used herein, the term “alkenyl” refers to a linear, cyclic, or branched unsaturated aliphatic hydrocarbon having a specified number of carbon atoms. In one embodiment, the alkenyl group contains 8 to 24 carbon atoms (C8 to C24). 24 Alkenyl). In one embodiment, the alkenyl group is linear. In another embodiment, the alkenyl group is branched. In yet another embodiment, the alkenyl group is unsubstituted.

[0025] Alkyl: As used herein, the term "alkyl" refers to a linear, cyclic, or branched saturated aliphatic hydrocarbon having a specified number of carbon atoms. In one embodiment, an alkyl group contains 8 to 24 carbon atoms (C8 to C8). 24 (Alkyl). In one embodiment, the alkyl group is linear. In another embodiment, the alkyl group is branched. In yet another embodiment, the alkyl group is unsubstituted.

[0026] Antibody: As used herein, the term “antibody” (or “Ab”) refers to any form of antibody exhibiting a desired biological activity. Therefore, it is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, and chimeric antibodies.

[0027] Antigen: As used herein, the term “antigen” refers to any antigen capable of generating one or more immune responses. Antigens may be proteins (including recombinant proteins), VLPs, polypeptides, or peptides (including synthetic peptides). Antigens may be those that generate humoral and / or CTL immune responses.

[0028] API: As used herein, the term "API" means an active pharmaceutical ingredient, such as HPV VLP, that is a component of a composition or formulation disclosed herein, which is biologically active (e.g., capable of inducing an appropriate immune response) and provides a therapeutic or prophylactic benefit to a person or animal in need thereof. As used herein, API is a vaccine active ingredient.

[0029] Cationic lipids: As used herein, the term "cationic lipids" refers to lipid species that have a net positive charge at a selected pH, such as physiological pH. Cationic lipids may be used as components of multi-component SNE adjuvant formulations. Cationic lipids are described in U.S. Patent Publication Nos. 2008 / 0085870, 2008 / 0057080, 2009 / 0263407, 2009 / 0285881, 2010 / 0055168, 2010 / 0055169, 2010 / 0063135, 2010 / 0076055, 2010 / 0099738, 2010 / 0104629, 2013 / 0017239, and 2016 / 0361411, International Publication No. 2011 / 022460, 20 Those skilled in the art will understand that this may include, but is not limited to, what is disclosed in Patent Nos. 12 / 040184, 2011 / 076807, 2010 / 021865, 2009 / 132131, 2010 / 042877, 2010 / 146740, and 2010 / 105209, as well as U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 6,890,557, and 9,669,097.

[0030] Co-administration: As used herein, the terms “co-administration” or “to co-administer” in relation to SNE adjuvants and pharmaceutical formulations (e.g., HPV vaccines) refer to the co-administration of SNE adjuvants and pharmaceutical formulations (e.g., HPV vaccines), i.e., simultaneous or sequential administration, i.e., administration of the HPV vaccine followed by administration of the SNE adjuvant (or vice versa). That is, after administration of the HPV vaccine (or SNE adjuvant), the SNE adjuvant (or HPV vaccine) may be administered substantially immediately after the HPV vaccine (or SNE adjuvant), or the SNE adjuvant (or HPV vaccine) may be administered after the effective period of the HPV vaccine (or SNE adjuvant), where the effective period is generally within a period of 1, 2, 3, 5, 10, 15, 20, 25, 30, 45, or 60 minutes.

[0031] Composition: As used herein, the term “composition” refers to a formulation containing an active pharmaceutical or biological component (e.g., at least one human papillomavirus (HPV) type virus-like particle (VLP) and SNE) together with one or more additional components. The term “composition” is used interchangeably with “pharmaceutical composition” and “formulation.” A composition may be a liquid or a solid (e.g., lyophilized). Additional components that may be included as needed include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, cryoprotectants, solubilizers, emulsifiers, salts, adjuvants, isotonic agents, delivery vehicles and antimicrobial preservatives. A composition is nontoxic to the recipient at the dosage and concentration used.

[0032] Dosage: As used herein, the term “dose” means the amount of a drug, API, formulation, or pharmaceutical composition that is administered or recommended to be administered at a particular time.

[0033] HPV and PV: As used herein, the terms "HPV" and "PV" refer to human papillomavirus and papillomavirus, respectively.

[0034] Immunogenicity: As used herein, the terms “immunogenic” or “immunogenic” refer to the ability of an antigen to induce an immune response in a subject. The term “immunogenic composition” refers to the ability of a drug, API, formulation, or composition to induce an immune response in a subject. The pneumococcal conjugate composition of the present invention is an immunogenic composition.

[0035] Requiring treatment: "Requiring treatment" includes individuals who have been previously exposed to or infected with human papillomavirus or papillomavirus, individuals who have been previously vaccinated against human papillomavirus or papillomavirus, and individuals who are susceptible to infection or for whom a reduction in the likelihood of infection is desired, such as immunocompromised individuals, the elderly, children, adults, or healthy individuals.

[0036] Lipids: As used herein, the term “lipids” refers to any of the group of organic compounds characterized by being esters of fatty acids, which are insoluble in water or have low solubility in water but may be soluble in many organic solvents. Lipids can be divided into at least three classes: (1) “simple lipids,” including, for example, fats and oils, and waxes; (2) “complex lipids,” including, for example, phospholipids and glycolipids; and (3) “derived lipids,” including, for example, steroids.

[0037] Patient: As used herein, the term “patient” means any human being administered with the HPV vaccine or pharmaceutical composition described herein. As defined herein, “patient” includes persons who are already infected with one or more types of HPV, as well as persons who should be protected from infection with one or more types of HPV.

[0038] pharmaceutically acceptable: As used herein with respect to carriers, diluents, or excipients of a pharmaceutical composition, the term “pharmaceutically acceptable” means that the carrier, diluent, or excipient must be compatible with the other components of the composition and not with its recipient.

[0039] Pharmaceutical Composition: As used herein, the term “pharmaceutical composition” refers to a composition containing an active pharmaceutical or biological component together with one or more additional components, for example, a composition in which the active agent is formulated together with one or more pharmaceutically acceptable carriers. As used herein, the terms “pharmaceutical preparation” and “preparation” are used interchangeably with “pharmaceutical composition.” In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regime that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. The pharmaceutical composition or preparation may be liquid or solid (e.g., lyophilized). Additional components that may be included as needed include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, cryoprotectants, solubilizers, emulsifiers, salts, adjuvants, isotonic agents, delivery vehicles, and antimicrobial preservatives. The pharmaceutical composition or preparation is nontoxic to the recipient at the dose and concentration used. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including: oral administration, e.g., drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g., for subbuccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, e.g., as a sterile solution or suspension, or as a sustained-release formulation; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a suppository, cream, or foam; sublingual administration; ophthalmic administration; transdermal administration; or administration to the nasal cavity, lungs, and other mucosal surfaces.

[0040] Squalene Nanoemulsion: As used herein, the term “squalene nanoemulsion” or “SNE” refers to a formulation of emulsifiers and / or solubilizers and / or surfactants and / or lipids having adjuvant properties in an HPV vaccine. Specifically, SNE refers to an SNE adjuvant formulation comprising (1) sorbitan trioleate (SPAN-85); (2) polysorbate 20 (PS-20); (3) squalene; and any (4) cationic lipid.

[0041] Subject: As used herein, the term “Subject” means an organism, typically a mammal (e.g., a human, and in some embodiments, a prenatal human form). In some embodiments, the Subject is suffering from a disease, disorder, or condition related to the subject. In some embodiments, the Subject is susceptible to a disease, disorder, or condition. In some embodiments, the Subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the Subject exhibits no symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the Subject is a person having one or more characteristics characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the Subject is a patient. In some embodiments, the Subject is an individual who has been and / or has been diagnosed and / or treated.

[0042] Surfactants: As used herein, the term “surfactants” refers to stabilizing components in multi-component SNE adjuvant formulations, including polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens, particularly PS-20 and PS-80), copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trade name DOWFAX®, e.g., linear EO / PO block copolymers (poloxamers); octoxynols in which the number of repeating ethoxy(oxy-1,2-ethanediyl) groups can vary, particularly octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL Examples include nonylphenol ethoxylates such as CA-630 / NP-40; Tergitol® NP series; polyoxyethylene aliphatic ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span-85, Tween-85, or [2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec-9-enoyl]oxyethyl](Z)-octadec-9-enoate) and sorbitan monolaurate. In one embodiment, the surfactant is selected from sorbitan esters and poloxamers. In one embodiment, the surfactant is selected from polysorbate 20 (PS-20) and polysorbate 80 (PS-80).

[0043] Terpenes: As used herein, the term “terpene” refers to, but is not limited to, the stabilizing components in multicomponent SNE adjuvant formulations, including monoterpenes comprising geraniol, terpineol, limonene, myrcene, linalool, and pinene; sesquiterpenes comprising humulene, farnesene, and farnesol; diterpenes comprising cafestol, kaweol, sembren, and taxadiene; triterpenes comprising squalene and squalane; tetraterpenes comprising acyclic lycopene, monocyclic gamma-carotene, and bicyclic alpha-carotene and beta-carotene; and polyterpenes and norisoprenoids. In one embodiment, the terpene is an oxidative decomposition product of a terpene. In one embodiment, the terpene is squalene.

[0044] Therapeutic dose: As used herein, the term “therapeutic dose” (or “therapeutic effective dose”) means the amount of an active ingredient (e.g., a therapeutic protein, vaccine, or antibody) sufficient to produce a desired therapeutic effect in a human or animal, for example, the amount necessary to induce an immune response, treat, cure, prevent or inhibit the onset and progression of a disease or its symptoms, and / or the amount necessary to improve symptoms or cause regression of the disease. The therapeutic dose may vary depending on the structure and potency of the active ingredient and the intended mode of administration. Those skilled in the art can readily determine the therapeutic dose of a given antibody or therapeutic protein or vaccine antigen.

[0045] Vaccine: As used herein, the terms “vaccine” or “vaccine composition” refer to a substance or preparation prepared from a disease-causing substance, its product, or a synthetic substitute, which has been treated to act as an antigen without inducing disease, and which is used to stimulate antibody production and provide immunity against one or more diseases. A vaccine composition may contain 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 in doses and techniques known to those skilled in the art of medicine or veterinary medicine, taking into account factors such as the age, sex, weight, species and condition of the recipient animal and the route of administration.

[0046] Valency: As used herein, the term “valency” 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, and the terms nonavalent, 9-valent, or 9-valent refer to nine different antigens.

[0047] Virus-like particles: As used herein, the terms “virus-like particles” or “VLP” refer to agents that are morphologically similar to true virions or that provide an array display of antigens and can induce a high antibody-neutralizing evaluation after administration to animals. VLPs lack the viral genetic material of true virions and are therefore non-infectious. [Brief explanation of the drawing]

[0048] [Figure 1] Representative structures of cationic lipids: (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA); (6Z,9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,29-tetraen-18-amine (CLX); and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine (CLY). See Example 1. [Figure 2] CLA-SNE components: (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA), SPAN-85, PS-20, and squalene. See Example 1. [Figure 3] Characterization of CLA-SNE adjuvant bulk preparations using static light scattering (SLS). See Example 2. [Figure 4]The impact of the formulation process on the integration of CLA into SNE. See Example 2. [Figure 5A] Figures 5A-5D: Nanotracking analysis (NTA) of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 5A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 5B: SNE [40 mg / mL squalene]; Figure 5C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; and Figure 5D: SNE [8 mg / mL squalene]). See Example 3. [Figure 5B] Figures 5A-5D: Nanotracking analysis (NTA) of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 5A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 5B: SNE [40 mg / mL squalene]; Figure 5C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; and Figure 5D: SNE [8 mg / mL squalene]). See Example 3. [Figure 5C] Figures 5A-5D: Nanotracking analysis (NTA) of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 5A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 5B: SNE [40 mg / mL squalene]; Figure 5C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; and Figure 5D: SNE [8 mg / mL squalene]). See Example 3. [Figure 5D] Figures 5A-5D: Nanotracking analysis (NTA) of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 5A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 5B: SNE [40 mg / mL squalene]; Figure 5C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; and Figure 5D: SNE [8 mg / mL squalene]). See Example 3. [Figure 6A]Figures 6A-6D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for one month at 4°C, 25°C, and 37°C (Figure 6A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 6B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; Figure 6C: SNE [40 mg / mL squalene]; and Figure 6D: SNE [8 mg / mL squalene]). See Example 3. [Figure 6B] Figures 6A-6D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for one month at 4°C, 25°C, and 37°C (Figure 6A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 6B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; Figure 6C: SNE [40 mg / mL squalene]; and Figure 6D: SNE [8 mg / mL squalene]). See Example 3. [Figure 6C] Figures 6A-6D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for one month at 4°C, 25°C, and 37°C (Figure 6A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 6B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; Figure 6C: SNE [40 mg / mL squalene]; and Figure 6D: SNE [8 mg / mL squalene]). See Example 3. [Figure 6D] Figures 6A-6D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for one month at 4°C, 25°C, and 37°C (Figure 6A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene]; Figure 6B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene]; Figure 6C: SNE [40 mg / mL squalene]; and Figure 6D: SNE [8 mg / mL squalene]). See Example 3. [Figure 7A] This figure shows the CLA concentration (mg / mL) of CLA-SNE and SNE preparations, measured by UPLC-CAD, after being stored at 4°C, 25°C, and 37°C for one month. See Example 4. [Figure 7B]This figure shows the squalene concentrations (mg / mL) of CLA-SNE and SNE preparations, measured by UPLC-CAD, after being stored at 4°C, 25°C, and 37°C for one month. See Example 4. [Figure 8A] The CLA / squalene (w / w)% after dialysis is plotted against the "target" (w / w)% before self-assembly. The CLA / squalene w / w% ratio (X) was measured by reverse-phase ULC-CAD before and after self-assembly and nanoemulsion dialysis. See Example 5. [Figure 8B] The measured intensity-weighted Z-mean DLS diameter (X) of CLA-SNE nanoparticles after dialysis is plotted against the measured CLA / squalene (w / w)% after dialysis for each MNS formulation. See Example 5. [Figure 8C] The measured zeta potential of CLA-SNE squalene nanoparticles (X) after dialysis at pH 5.5 is plotted against the measured CLA / squalene (w / w)% after dialysis for each MNS preparation. See Example 6. [Figure 9] DLS Z-mean diameter of CLA-SNE samples formed and treated in an aqueous phase (20 mM L-histidine) with gradually increasing pH. See Example 6. [Figure 10] Final [CLA] (mg / mL) of the CLA-SNE sample formed and treated in an aqueous phase (20 mM L-histidine) with a gradually increasing pH. See Example 6. [Figure 11A] Graph showing HPV VLP 16 antibody levels in rhesus monkeys after two doses of 9vHPV vaccine combined with CLA-SNE or SNE adjuvant. See Example 7. [Figure 11B] Graph showing HPV VLP 18 antibody levels in rhesus monkeys after two doses of 9vHPV vaccine combined with CLA-SNE or SNE adjuvant. See Example 7. [Figure 12]Graph showing individual HPV VLP antibody levels in rhesus monkeys measured 54 weeks after two doses of 9vHPV vaccine combined with CLA-SNE or SNE adjuvant. See Example 7. [Figure 13A] Graph showing HPV VLP 16 antibody levels in rhesus monkeys after two doses of 9vHPV vaccine combined with CLA-SNE (3.96 mg) or SNE (12 mg) adjuvant. See Example 7. [Figure 13B] Graph showing HPV VLP 18 antibody levels in rhesus monkeys after two doses of 9vHPV vaccine combined with CLA-SNE (3.96 mg) or SNE (12 mg) adjuvant. See Example 7. [Figure 14] Graph showing individual HPV VLP antibody levels in rhesus monkeys measured at 32 weeks after two doses of 9vHPV vaccine combined with either 3.96 mg CLA-SNE or 12 mg SNE adjuvant. See Example 7. [Modes for carrying out the invention]

[0049] In one embodiment, the present invention provides a composition comprising at least one human papillomavirus (HPV) type virus-like particle (VLP) and a squalene nanoemulsion (SNE) adjuvant, wherein the human papillomavirus (HPV) is 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 the SNE adjuvant comprises sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20), or polysorbate 80 (PS-80), and squalene. In some embodiments, the composition also comprises an aluminum adjuvant. In some embodiments, the VLP is adsorbed to the aluminum adjuvant.

[0050] Squalene nanoemulsion The squalene nanoemulsion ("SNE") of the present invention refers to a formulation of an emulsifier and / or solubilizer and / or surfactant and / or lipid. In one embodiment, the present disclosure provides a composition comprising, in particular, four SNE components: (1) a cationic lipid; (2) sorbitan trioleate (SPAN-85); (3) polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and (4) squalene. In one embodiment, the SNE composition comprises a cationic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine ("CLA" or "CLA-SNE" if the cationic lipid is included in the SNE).

[0051] Cationic lipids and methods for producing cationic lipids are well known in the art.

[0052] In some embodiments, cationic lipids are used in U.S. Patent Applications Publications 2008 / 0085870, 2008 / 0057080, 2009 / 0263407, 2009 / 0285881, 2010 / 0055168, 2010 / 0055169, 2010 / 0063135, 2010 / 0076055, 2010 / 0099738, 2010 / 0104629, 2013 / 0017239, and 2016 / 0361411, International Publication 2011 / 022460, and the United Nations Patent Application Publication No. This includes any cationic lipid referred to in International Publication No. 2012 / 040184, International Publication No. 2011 / 076807, International Publication No. 2010 / 021865, International Publication No. 2009 / 132131, International Publication No. 2010 / 042877, International Publication No. 2010 / 146740, International Publication No. 2010 / 105209, and U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 6,890,557, and 9,669,097.

[0053] In some embodiments, cationic lipids useful in the compositions of the present invention are those represented by formula 1 below: [ka]

[0054] (In the formula, R l and R 2 These are methyl compounds, R 3 H is, n is either 1 or 2. L1 is C8~C 24 Alkyl and C8~C 24 Selected from Alkenil, L2 is selected from C4-C9 alkyl and C4-C9 alkenyl groups. It has the structure of, or any pharmaceutically acceptable salt or stereoisomer thereof.

[0055] In some embodiments, the cationic lipid is an aminoalkyl lipid. In some embodiments, the cationic lipid is an asymmetric aminoalkyl lipid. In one embodiment of the present invention, the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA), or (6Z,9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,29-tetraen-18-amine (CLX); or N,N-dimethyl-1-((1S,2R)-2-octylcyclopropyl)heptadecane-8-amine (CLY).

[0056] In another embodiment of the present invention, the cationic lipid is DLinDMA;DLinKC2DMA;DLin-MC3-DMA;CLinDMA;S-octylCLinDMA;(2S)-1-{7-[(3P)-cholest-5-en-3-yloxy]heptyloxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine;(2R)-1-{4-[(3P)-cholest-5-en-3-yloxy]butoxy}-3-[(4Z)-dec-4-en-1-yloxy]-N,N-dimethylpropan-2-amine;1- [(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)propane-2-yl]guanidine;1-[(2R)-1-{7-[(3β)-cholest-5-en-3-yloxy]heptyloxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propane-2-amine;1-[(2R)-1-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1- [Iloxy]propan-2-amine; (2S)-1-({6-[(3P)-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]propan-2-amine; (3β)-3-[6-{[(2S)-3-[(9Z)-octadec-9-en-1-yloxy]-2-(pyrrolidine-1-yl)propyl]oxy}hexyl}oxy]cholest-5-en; (2R)-1-{4-[(3P)-cholest-5-en-3-yloxy]butoxy}-3-(octyloxy)prop N-2-amine; (2R)-1-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-(pentyloxy)propane-2-amine; (2R)-1-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-3-(heptyloxy)-N,N-dimethylpropane-2-amine; (2R)-1-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(2Z)-pento-2-en-1-yloxy]propane-2-amine;(2S)-1-butoxy-3-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylpropane-2-amine; (2S)-1-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-3-[2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorononyloxy]-N,N-dimethylpropane-2-amine; 2-amino-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propane-1,3-diole 2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]nonyl}oxy)-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol;2-amino-3-({6-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest-5-en-3-yloxy]nonyl}oxy)-2-{[(9Z)-octadeca-9-en-1-yloxy]methyl}propan-1-ol;(20Z,23Z)-N,N-dimethylnonacosa-2 0,23-diene-10-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-9-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-8-amine; (13Z,16Z)-N,N-dimethyldocosa-13,16-diene-5-amine; (12Z,15Z)-N,N-dimethylhenicosa-12,15-diene-4-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-6-amine; (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene- 7-amine; (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-10-amine; (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-5-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-4-amine; (19Z,22Z)-N,N-dimethyloctacosa-19,22-diene-9-amine; (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-8-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-7-amine;(16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-6-amine; (22Z,25Z)-N,N-dimethylhentriaconta-22,25-diene-10-amine; (21Z,24Z)-N,N-dimethyltriaconta-21,24-diene-9-amine; (18Z)-N,N-dimethylheptacos-18-en-10-amine; (17Z)-N,N-dimethylhexacos-17-en-9-amine; (19Z,22Z)-N,N-dimethyloctacosa-19,22-diene-7-amine; N,N-dimethylheptacosa-10-amine N; (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine; 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine; (20Z)-N,N-dimethylheptacos-20-en-10-amine; (15Z)-N,N-dimethylheptacos-15-en-10-amine; (14Z)-N,N-dimethylnonacosa-14-en-10-amine; (17Z)-N,N-dimethylnonacosa-17-en-10-amine; (24Z)-N,N-dimethyltritriaconta-24-en-10- Amine; (20Z)-N,N-dimethylnonakos-20-en-10-amine; (22Z)-N,N-dimethylhenthriaconta-22-en-10-amine; (16Z)-N,N-dimethylpentacos-16-en-8-amine; (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine; 1-[(1S,2R) -2-Hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine; N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine; N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecane-10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine;Selected from N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine; N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine; 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine; 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine; and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-triene-10-amine; or pharmaceutically acceptable salts thereof, or any of the aforementioned stereoisomers.

[0057] In another embodiment of the present invention, the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0058] In another embodiment of the present invention, the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA).

[0059] In some embodiments, the present disclosure provides compositions comprising, in particular, three SNE components: (1) sorbitan trioleate (SPAN-85), (2) polysorbate 20 (PS-20) or polysorbate 80 (PS-80), and (3) squalene. In embodiments of this aspect of the present invention, the SNE does not contain cationic lipids.

[0060] In some embodiments, the SNE comprises 32-97 mol% squalene, 1-34 mol% SPAN-85, and 1-34 mol% PS-20 or PS-80.

[0061] In some embodiments, the SNE comprises 86-98 mol% squalene, 1-7 mol% SPAN-85, and 1-7 mol% PS-20 or PS-80.

[0062] In some embodiments, the SNE comprises 92-94 mol% squalene, 3-4 mol% SPAN-85, and 3-4 mol% PS-20 or PS-80.

[0063] In one embodiment of the present invention, the SNE comprises 92.91 mol% squalene, 3.98 mol% SPAN-85, and 3.11 mol% PS-20 or PS-80.

[0064] The disclosure also provides compositions comprising, in particular, four SNE components: (1) a cationic lipid; (2) sorbitan trioleate (SPAN-85); (3) polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and (4) squalene. A particular SNE composition comprises a cationic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine ("CLA" or "CLA-SNE" if the cationic lipid is included in the SNE).

[0065] In some embodiments, the SNE comprises 1 to 60 mol% cationic lipids, 32 to 97 mol% squalene, 1 to 4 mol% SPAN-85, and 1 to 4 mol% PS-20 or PS-80.

[0066] In some embodiments, the SNE comprises 10-14 mol% cationic lipids, 78-84 mol% squalene, 1-6 mol% SPAN-85, and 1-6 mol% PS-20 or PS-80.

[0067] In some embodiments, the SNE comprises 40-46 mol% cationic lipids, 44-52 mol% squalene, 1-8 mol% SPAN-85, and 1-8 mol% PS-20 or PS-80.

[0068] In one embodiment of the present invention, the SNE comprises 13.82 mol% cationic lipid, 80.07 mol% squalene, 3.43 mol% SPAN-85, and 2.68 mol% PS-20 or PS-80.

[0069] In one embodiment of the present invention, the SNE comprises 44.5 mol% cationic lipid, 51.56 mol% squalene, 2.21 mol% SPAN-85, and 1.72 mol% PS-20 or PS-80.

[0070] In some embodiments, the SNE includes a PS-20.

[0071] In some embodiments, the SNE includes the PS-80.

[0072] In some embodiments, the disclosure provides compositions comprising, among other things, surfactants, mixtures of surfactants, phospholipids, terpenes, terpenoids, triterpenes, or combinations thereof, one or more noncationic lipids.

[0073] In some embodiments, the surfactant is polyoxyethylene sorbitan ester surfactant (commonly referred to as Tweens), particularly PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the trade name DOWFAX®, e.g., linear EO / PO block copolymers; octoxynols with a varying number of repeating ethoxy(oxy-1,2-ethanediyl) groups, particularly octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); nonylphenol ethoxylates such as the Tergitol® NP series; polyoxyethylene aliphatic ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants), e.g., triethylene glycol monolauryl ether (Brij 30); and selected from the group consisting of sorbitan esters (commonly known as SPANs), such as sorbitan trioleates (SPAN-85, Tween-85, or [2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec-9-enoyl]oxyethyl](Z)-octadec-9-enoate) and sorbitan monolaurate.

[0074] In some embodiments, mixtures of surfactants, such as PS-20 / SPAN 85 or PS-80 / SPAN 85 mixtures, are used. Combinations of polyoxyethylene sorbitan esters, such as polyoxyethylene sorbitan monooleate (PS-80), and octoxynols, such as t-octylphenoxypolyethoxyethanol (Triton X-100), are also suitable. Another useful combination includes laureth-9 + polyoxyethylene sorbitan ester and / or octoxynol.

[0075] In some embodiments, the amount of surfactant or emulsifier is 0.01 to 10 mol%, particularly about 1 to 4 mol%, of polyoxyethylene sorbitan ester (PS-20 or PS-80, etc.), 0.001 to 10 mol%, particularly about 1 to 4 mol% w / v, particularly 0.01 to 0.1% w / v, of octylphenoxy or nonylphenoxy polyoxyethanol (Triton X-100, etc., or other detergents in the Triton series), and 0.1 to 20 mol%, preferably 0.5 to 10 mol%, particularly 1 to 4 mol%, or about 10% by mass ratio, of polyoxyethylene ether (Laureth 9, etc.).

[0076] In some embodiments, the phospholipids are selected from natural phospholipids including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidic acid salt) (PA), dipalmitoylphosphatidylcholine, monoacylphosphatidylcholine (lysoPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), N-acylPE, phosphoinositides, and phosphosphingolipids. Phospholipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE, DOPE), and phosphatidylserine (DOPS). Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0, and ethisin. In certain embodiments of the present invention, the phospholipid is phosphatidylserine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitreoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0077] In some embodiments, terpines are selected from monoterpenes comprising geraniol, terpineol, limonene, myrcene, linalool, or pinene; sesquiterpenes comprising humulene, farnesene, and farnesol; diterpenes comprising cafestol, kaweol, sembren, and taxadiene; triterpenes comprising squalene and squalane; acyclic lycopenes; monocyclic gamma-carotenes; and tetraterpenes comprising bicyclic alpha-carotenes and beta-carotenes; as well as polyterpenes and norisoprenoids. In some embodiments, terpine is squalene.

[0078] In one embodiment of this invention, SNE comprises 50 to 85 mol% squalene and 1 to 10 mol% nonionic surfactant. In one aspect of this embodiment, the nonionic surfactant comprises a mixture of PS-20 and SPAN-85 or a mixture of PS-80 and SPAN-85.

[0079] In one embodiment of this invention, SNE comprises 0 to 45 mol% cationic lipid, 50 to 85 mol% squalene, and 1 to 10 mol% nonionic surfactant. In one aspect of this embodiment, the nonionic surfactant comprises a mixture of PS-20 and SPAN-85 or a mixture of PS-80 and SPAN-85.

[0080] In one embodiment of the present invention, the SNE comprises one or more cationic lipids, one or more terpenes (e.g., squalene), and / or one or more sorbitan-based surfactants (e.g., PS-20 or PS-80; SPAN-85) in a specific molar ratio.

[0081] General methods for preparing SNEs (with and without cationic lipids) Generally, SNEs can be formed, for example, by first combining and mixing lipid components together, or by first utilizing a single lipid such as a cationic lipid. After mixing and blending (when combining and mixing lipid components together), an aqueous buffer is added and mixed with the initial lipids or lipid components to form a blended emulsion mixture. The blended emulsion components are first subjected to coarse homogenization, followed by fine homogenization. The resulting formulation is then subjected to a final filtration step and stored at 4°C. The lipid solution may contain, in a specific molar ratio, one or more cationic lipids, one or more terpenes (e.g., squalene), and one or more sorbitan-based surfactants (e.g., PS-20 or PS-80; SPAN-85).

[0082] VLP As described above, the pharmaceutical compositions and formulations of the present invention include at least one HPV VLP type, for example, HPV type 16 or 18. In certain embodiments of the compositions disclosed herein, the vaccine further includes a VLP 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 type 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, at least one HPV type includes HPV 6, 11, 16, 18, 31, 45, 52, and 58. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 33, 45, 52, and 58. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58. In some embodiments, at least one HPV type includes 6, 11, 16, 18, 31, 33, 45, 52, and 59. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 53, and 58. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 53, and 59. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 45, 52, 58, and 59. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68. In some embodiments, at least one HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.In some embodiments, 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, 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, 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, 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, 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, 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, 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.

[0083] The pharmaceutical composition of the present invention comprises an HPV VLP composed of recombinant HPV Ll or recombinant Ll+L2 protein. The HPV Ll or Ll+L2 protein can be recombinantly expressed by molecular cloning of Ll or Ll+L2 DNA into an expression vector containing a suitable promoter and other suitable transcriptional regulatory elements, and then transferred into a prokaryotic or eukaryotic host cell to produce the recombinant protein. Techniques for such operations are well described by Sambrook et al. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989)), which is incorporated herein by reference. The VLP can self-assemble once the Ll protein is recombinantly expressed in the host cell.

[0084] The recombinant HPV Ll protein of the present invention may be any full-length Ll protein sequence that can be found in nature, or any mutant or truncated Ll protein that can self-assemble into a VLP. In certain embodiments of the present invention, the pharmaceutical compositions and vaccines described herein include an HPV VLP composed of recombinant HPV L1 protein and do not contain HPV L2 protein. In certain embodiments, the vaccine composition or pharmaceutical composition described herein includes an HPV VLP composed of a full-length recombinant HPV L1 protein. In other embodiments, the HPV VLP is composed of a truncated HPV L1 protein, for example, a C-terminus truncated L1 protein. Ll protein sequences for use in the present invention can be determined by isolating DNA from one or more clinical samples containing an optimal HPV type, sequencing the HPV Ll DNA sequence, and translating the DNA sequence into an amino acid sequence using the genetic code. Many exemplary Ll sequences suitable for use in the present invention can be found in the literature. For example, see U.S. Patents 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, and Kirii et al. (Virology 185(1):424-427(1991)). Further Ll proteins useful for the compositions and formulations of the present invention include biologically active fragments and / or variants of the HPV Ll sequence, which include, but are not limited to, amino acid substitutions, deletions, additions, amino-terminal shortenings and carboxy-terminal shortenings, resulting in Ll proteins or protein fragments capable of forming VLPs. See, for example, International Publication No. 2006 / 114312 and U.S. Patent No. 6,599,508. Suitable host cells for recombinant HPV Ll or recombinant Ll+L2 expression and subsequent VLP self-assembly include, but are not limited to, yeast cells, insect cells, mammalian cells, or bacteria.In exemplary embodiments of the present invention, VLPs are produced in yeast cells selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluyveromyces lactis, and Schizosaccharomyces pombe. In specific embodiments, HPV VLPs are produced in Saccharomyces cerevisiae cells. Expression of HPV VLPs in yeast cells offers the advantages of being cost-effective and readily adaptable to large-scale growth in fermenters.

[0085] The present invention also includes pharmaceutical compositions comprising mutant HPV VLPs, such as HPV VLPs, which include fragments and / or variants of biologically active HPV L1 and / or L2 proteins, including, but not limited to, amino acid substitutions, deletions, additions, amino-terminal shortenings and carboxy-terminal shortenings, resulting in proteins or protein fragments that can be used for therapeutic or preventive purposes and may be useful in the development of HPV VLP vaccines. Any such mutant form of HPV L1 protein must be able to form VLPs and induce an immune response against a desired HPV type when administered to humans.

[0086] Furthermore, those skilled in the art will recognize that the HPV Ll or Ll+L2 proteins used to self-assemble VLPs for inclusion in the compositions disclosed herein may be encoded by full-length wild-type HPV Ll or L2 polynucleotides, or by fragments or variants of known wild-type sequences. Wild-type polynucleotide sequences encoding mRNA expressing HPV Ll or L2 proteins are available in the art. Any mutant polynucleotide encodes either a protein or protein fragment that at least substantially mimics the pharmacological properties of the HPV Ll or L2 protein, including the ability to form VLPs that can evoke an immune response against the HPV type of interest when administered to humans. Any such polynucleotides include, but are not limited to, nucleotide substitutions, deletions, additions, amino-terminus cleavages, and carboxyl-terminus cleavages.

[0087] The amount of virus-like particles of each HPV type contained in the formulations and compositions of the present invention depends on the immunogenicity of the expressed gene product. Generally, the therapeutic effective dose of any VLP of at least one HPV type is about 1 μg to about 300 μg. In some embodiments, the therapeutic effective dose of any VLP of at least one HPV type is about 1 μg to 200 μg. In some embodiments, the therapeutic effective dose of any VLP of at least one HPV type is about 1 μg to 100 μg. In some embodiments, the therapeutic effective dose of any VLP of at least one HPV type is about 5 μg to 200 μg. In some embodiments, the therapeutic effective dose of any VLP of at least one HPV type is about 5 μg to 100 μg. In some embodiments, the therapeutic effective dose of any VLP of at least one HPV type is about 10 μg to 200 μg. In some embodiments, the therapeutic effective dose of any VLP of at least one HPV type is about 10 μg to 100 μg. In some embodiments, the therapeutically effective dose of any VLP of at least one HPV type is about 10 μg to 80 μg. In some embodiments, it is preferable that the therapeutically effective dose of any VLP of at least one HPV type is about 20 μg to 60 μg.

[0088] In some embodiments, the dose of the composition is in the range of about 0.10 mL to about 1.5 mL. In some embodiments, the dose is in the range of about 0.25 mL to about 1.25 mL. In some embodiments, the dose is in the range of about 0.5 mL to about 1.0 mL. In some embodiments, the dose is 0.25 mL. In some embodiments, the dose is 0.5 mL. In some embodiments, the dose is 0.75 mL. In some embodiments, the dose is 1.0 mL. In some embodiments, the dose is 1.25 mL.

[0089] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • Contains 10-120 μg of HPV type 58 L1 protein VLP.

[0090] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • 10-120 μg of HPV type 59 L1 protein VLP, • 10-120 μg of HPV type 68 L1 protein VLP • 10-120 μg of HPV type 69 L1 protein VLP • 10-120 μg of HPV type 70 L1 protein VLP, • Contains 10-120 μg of HPV type 73 L1 protein VLP.

[0091] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 35 L1 protein VLP • 10-120 μg of HPV type 39 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 51 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 56 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • Contains 10-120 μg of HPV type 59 L1 protein VLP.

[0092] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 56 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • 10-120 μg of HPV type 59 L1 protein VLP, • 10-120 μg of HPV type 68 L1 protein VLP • 10-120 μg of HPV type 70 L1 protein VLP, • Contains 10-120 μg of HPV type 73 L1 protein VLP.

[0093] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 35 L1 protein VLP • 10-120 μg of HPV type 39 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 51 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 56 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • Contains 10-120 μg of HPV type 69 L1 protein VLP.

[0094] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 35 L1 protein VLP • 10-120 μg of HPV type 39 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 51 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • 10-120 μg of HPV type 59 L1 protein VLP, • Contains 10-120 μg of HPV type 69 L1 protein VLP.

[0095] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 35 L1 protein VLP • 10-120 μg of HPV type 39 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 51 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 56 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • 10-120 μg of HPV type 59 L1 protein VLP, • 10-120 μg of HPV type 68 L1 protein VLP • 10-120 μg of HPV type 69 L1 protein VLP • Contains 10-120 μg of HPV type 70 L1 protein VLP.

[0096] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 35 L1 protein VLP • 10-120 μg of HPV type 39 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 51 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 56 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • 10-120 μg of HPV type 59 L1 protein VLP, • 10-120 μg of HPV type 66 L1 protein VLP • 10-120 μg of HPV type 68 L1 protein VLP • 10-120 μg of HPV type 69 L1 protein VLP • Contains 10-120 μg of HPV type 70 L1 protein VLP.

[0097] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-160 μg of HPV type 6 L1 protein VLP • 20-200 μg of HPV type 11 L1 protein VLP • 30-280 μg of HPV type 16 L1 protein VLP • 20-200 μg of HPV type 18 L1 protein VLP • 10-120 μg of HPV type 31 L1 protein VLP • 10-120 μg of HPV type 33 L1 protein VLP • 10-120 μg of HPV type 35 L1 protein VLP • 10-120 μg of HPV type 39 L1 protein VLP • 10-120 μg of HPV type 45 L1 protein VLP • 10-120 μg of HPV type 51 L1 protein VLP • 10-120 μg of HPV type 52 L1 protein VLP, • 10-120 μg of HPV type 56 L1 protein VLP, • 10-120 μg of HPV type 58 L1 protein VLP, • 10-120 μg of HPV type 59 L1 protein VLP, • 10-120 μg of HPV type 66 L1 protein VLP • 10-120 μg of HPV type 68 L1 protein VLP • 10-120 μg of HPV type 69 L1 protein VLP • 10-120 μg of HPV type 70 L1 protein VLP, • Contains 10-120 μg of HPV type 73 L1 protein VLP.

[0098] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-120 μg of HPV type 6 L1 protein VLP • 20-150 μg of HPV type 11 L1 protein VLP • 30-210 μg of HPV type 16 L1 protein VLP • 20-150 μg of HPV type 18 L1 protein VLP • 10-90 μg of HPV type 31 L1 protein VLP • 10-90 μg of HPV type 33 L1 protein VLP • 10-90 μg of HPV type 45 L1 protein VLP • 10-90 μg of HPV type 52 L1 protein VLP • Contains 10-90 μg of HPV type 58 L1 protein VLP.

[0099] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-80 μg of HPV type 6 L1 protein VLP • 20-100 μg of HPV type 11 L1 protein VLP • 30-140 μg of HPV type 16 L1 protein VLP • 20-100 μg of HPV type 18 L1 protein VLP, • 10-60 μg of HPV type 31 L1 protein VLP • 10-60 μg of HPV type 33 L1 protein VLP • 10-60 μg of HPV type 45 L1 protein VLP • 10-60 μg of HPV type 52 L1 protein VLP • Contains 10-60 μg of HPV type 58 L1 protein VLP.

[0100] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 15-40 μg of HPV type 6 L1 protein VLP • 20-50 μg of HPV type 11 L1 protein VLP • 30-70 μg of HPV type 16 L1 protein VLP • 20-50 μg of HPV type 18 L1 protein VLP • 10-30 μg of HPV type 31 L1 protein VLP • 10-30 μg of HPV type 33 L1 protein VLP • 10-30 μg of HPV type 45 L1 protein VLP • 10-30 μg of HPV type 52 L1 protein VLP • Contains 10-30 μg of HPV type 58 L1 protein VLP.

[0101] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 90 μg of HPV type 6 L1 protein VLP, • 120 μg of HPV type 11 L1 protein VLP, • 180 μg of HPV type 16 L1 protein VLP, • 120 μg of HPV type 18 L1 protein VLP, • 60 μg of HPV type 31 L1 protein VLP, • 60 μg of HPV type 33 L1 protein VLP • 60 μg of HPV type 45 L1 protein VLP, • 60 μg of HPV type 52 L1 protein VLP, Contains 60 μg of HPV type 58 L1 protein VLP.

[0102] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 60 μg of HPV type 6 L1 protein VLP, • 80 μg of HPV type 11 L1 protein VLP, • 120 μg of HPV type 16 L1 protein VLP, • 80 μg of HPV type 18 L1 protein VLP, • 40 μg of HPV type 31 L1 protein VLP, • 40 μg of HPV type 33 L1 protein VLP, • 40 μg of HPV type 45 L1 protein VLP, • 40 μg of HPV type 52 L1 protein VLP, Contains 40 μg of HPV type 58 L1 protein VLP.

[0103] In some embodiments, the dose of a composition or vaccine comprising at least one HPV type VLP is • 30 μg of HPV type 6 L1 protein VLP, • 40 μg of HPV type 11 L1 protein VLP, • 60 μg of HPV type 16 L1 protein VLP, • 40 μg of HPV type 18 L1 protein VLP, • 20 μg of HPV type 31 L1 protein VLP, • 20 μg of HPV type 33 L1 protein VLP, • 20 μg of HPV type 45 L1 protein VLP, • 20 μg of HPV type 52 L1 protein VLP, Contains 20 μg of HPV type 58 L1 protein VLP.

[0104] Aluminum adjuvant The aluminum adjuvant of the present invention may be in the form of aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS), or so-called "alum" (KAl(SO4)-12H2O) (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 present invention provided herein, the aluminum adjuvant is aluminum hydroxyphosphate or AAHS. The phosphate-to-aluminum ratio in the aluminum adjuvant may be in the range of 0 to 1.3. In embodiments of this aspect of the present invention, the phosphate-to-aluminum ratio is in the range of 0.1 to 0.70. In some embodiments, the phosphate-to-aluminum ratio is in the range of 0.2 to 0.50.

[0105] Those skilled in the art will be able to determine the optimal dose of aluminum adjuvant that is safe and effective in increasing the immune response to one or more target HPV types. For a discussion of the safety profile of aluminum and the amount of aluminum included in FDA-approved vaccines, see Baylor et al., Vaccine 20:S18-S23 (2002). In some embodiments, the aluminum adjuvant is present in amounts of approximately 100–3600 μg / dose (200–7200 μg / mL concentration). In some embodiments, the aluminum adjuvant is present in amounts of approximately 100–2700 μg / dose (200–5400 μg / mL concentration). In some embodiments, the aluminum adjuvant is present in amounts of approximately 100–1800 μg / dose (200–3600 μg / mL concentration). In some embodiments, the aluminum adjuvant is present in amounts of approximately 100–900 μg / dose (200–1800 μg / mL concentration). In some embodiments of the formulations and compositions of the present invention, there is 200 to 300 μg of aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the present invention, there is 300 to 500 μg of aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the present invention, there is 400 to 1200 μg of aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the present invention, there is 1200 to 2000 μg of aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, there is less than 2000 μg of aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, there is less than 1500 μg of aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, there is less than 1000 μg of aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, there is less than 500 μg of aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, less than 400 μg of aluminum adjuvant is present per dose of vaccine.In some embodiments of the formulations and compositions of the present invention, less than 300 μg of aluminum adjuvant is present per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, less than 200 μg of aluminum adjuvant is present per dose of vaccine. In some embodiments of the formulations and compositions of the present invention, less than 100 μg of aluminum adjuvant is present per dose of vaccine.

[0106] HPV VLP-based vaccine Any HPVLP-based vaccine is suitable for use in the pharmaceutical compositions and methods of the present invention. Known HPV VLP vaccines can be modified to include both an aluminum adjuvant and an SNE adjuvant. Such HPV VLP vaccines can be in vitro mixed with the SNE adjuvant of the present invention (e.g., mixed by a clinician immediately before administration to a patient) or formulated together with the SNE adjuvant. Novel vaccines can be developed according to the present invention as described herein, comprising at least one HPV type, which may be in the form of HPV VLPs adsorbed to the aluminum adjuvant, in combination with the SNE adjuvant. Furthermore, novel vaccines can be developed according to the present invention as described herein, comprising at least one HPV type, which may be in the form of HPV VLPs adsorbed to the aluminum adjuvant, in combination with the SNE adjuvant.

[0107] One example of an exemplary HPV vaccine is a bivalent vaccine that protects against HPV 16 and 18, which is marketed as CERVARIX® (GlaxoSmithKline Biologicals, Lixensar, Belgium). Another exemplary HPV VLP vaccine is a non-infectious recombinant tetravalent vaccine prepared from highly purified VLPs of major capsid (L) proteins of HPV types 6, 11, 16, and 18, which may be referred to herein by its proprietary name GARDASIL® (Merck & Co., Inc., Lethway, New Jersey, USA) (see Bryan, JTVaccine 25(16):3001-6(2007); Shi et al. Clinical Pharmacology and Therapeutics 81(2):259-64(2007)). Another exemplary HPV VLP vaccine is a 9-valent vaccine marketed for the prevention of HPV (including HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 capsid (L1) proteins), which is referred herein by its proprietary name GARDASIL® 9 (Merck & Co., Inc., Lethway, New Jersey, USA).

[0108] In some embodiments, the vaccine dose includes, in addition to the VLP, an aluminum adjuvant (as amorphous aluminum hydroxyphosphate sulfate), sodium chloride, L-histidine, polysorbate 80, sodium borate, and water. In some embodiments, the HPV vaccine contains 100-3500 μg of aluminum adjuvant, 1-50 mg of sodium chloride, 0.05-10 mg of L-histidine, 1-100 μg of polysorbate, 1-100 μg of sodium borate, and water. In some embodiments, the HPV vaccine contains about 500 μg of aluminum adjuvant, about 9.56 mg of sodium chloride, about 0.78 mg of L-histidine, about 50 μg of polysorbate 80, about 35 μg of sodium borate, and water for injection. Known HPV VLP vaccines can be modified to include both the aluminum adjuvant and the SNE adjuvant according to the present invention.

[0109] In some embodiments of the present invention, the pharmaceutical compositions and formulations include an HPV VLP-based vaccine or HPV VLP as described herein, which is monovalent, divalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent, decavalent, elevenvalent, dodecavalent, thirteenvalent, or tetravalent. In certain embodiments, the pharmaceutical compositions and formulations are nonavalent. In other embodiments, the pharmaceutical compositions and formulations are decavalent. In other embodiments, the pharmaceutical compositions and formulations are dodecavalent. In certain embodiments, the pharmaceutical compositions and formulations are tetravalent. In some embodiments, the pharmaceutical compositions include an HPV VLP-based vaccine or HPV VLP as described herein, which includes more than four different types of HPV VLP. For example, the pharmaceutical compositions and formulations of the present invention may include an HPV VLP-based vaccine or HPV VLP as described herein, which is octavalent, nonavalent, decavalent, etc. For example, a pharmaceutical composition containing HPV 16 and / or HPV 18 VLPs without including other HPV VLP types falls within the scope of the present invention. This specification also envisions a multivalent vaccine containing HPV VLPs different from the HPV types included in GARDASIL® or GARDASIL® 9.

[0110] In some embodiments, VLPs for HPV types 6 and 11 are included. In some embodiments, VLPs for HPV types 16, 31, and 35 are included. In some embodiments, VLPs for HPV types 18, 45, and 59 are included. In some embodiments, VLPs for HPV types 26, 51, and 69 are included. In some embodiments, VLPs for HPV types 33, 52, and 58 are included. In some embodiments, VLPs for HPV types 39, 68, and 70 are included. In some embodiments, VLPs for HPV types 53, 56, and 66 are included.

[0111] In some embodiments, VLPs for HPV types 16 and 18 are included. In some embodiments, VLPs for HPV types 6, 11, 16, and 18 are included. In some embodiments, VLPs for HPV types 6, 18, 52, and 58 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 45, 52, and 58 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 33, 45, 52, and 58 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 59 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 45, 53, and 58 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 45, 53, and 59 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58, and 59 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 26, 31, 33, 35, 45, 51, 52, 58, 59, and 69 are included. In some embodiments, VLPs for HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are included.In some embodiments, VLPs include HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 73. In some embodiments, VLPs include HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 69, and 70. In some embodiments, VLPs include HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. In some embodiments, VLPs include HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70. In some embodiments, VLPs include HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 70, and 73.

[0112] In some embodiments, the pharmaceutical composition and formulation comprises an HPV VLP-based vaccine and / or antigen listed in Table I below. [Table 1]

[0113] TIFF2026522303000004.tif165152

[0114] The compositions of the present invention can be administered subcutaneously, topically, orally, on the mucous membrane, intravenously, or intramuscularly. The composition is administered in an amount sufficient to induce a defensive response. The composition can be administered via various routes, such as orally, parenterally, subcutaneously, on the mucous membrane, or intramuscularly. The dose administered may vary depending on the patient's general condition, sex, weight, and age, as well as the route of administration.

[0115] Compositions of the present invention, as highlighted in the various embodiments described above, are sometimes referred to as immunogenic compositions.

[0116] The compositions of the present invention, as highlighted in the various embodiments described above, may also be referred to as vaccines or vaccine compositions.

[0117] In one embodiment, SNE is provided as a composition comprising PS-20, sorbitan trioleate, squalene, and (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

[0118] In one embodiment, a composition is provided in which SNE comprises 5 to 15 mol% sorbitan trioleate, 25 to 35 mol% PS-20 or PS-80, 1 to 2.5 mol% squalene, and 55 to 65 mol% (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

[0119] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises up to 75 mol% cationic lipid, up to 30 mol% sorbitan trioleate, up to 30 mol% polysorbate 20 or polysorbate 80, and 25 to 85 mol% squalene.

[0120] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises up to 50 mol% cationic lipid, up to 10 mol% sorbitan trioleate, up to 10 mol% polysorbate 20 or polysorbate 80, and 50-80 mol% squalene.

[0121] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises up to 24 mol% cationic lipid, 1 to 8 mol% sorbitan trioleate, 1 to 8 mol% polysorbate 20 or polysorbate 80, and 60 to 75 mol% squalene.

[0122] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises about 10-14 mol% cationic lipid, 1-4 mol% sorbitan trioleate, 1-4 mol% polysorbate 20 or polysorbate 80, and 50-80 mol% squalene.

[0123] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 30-65 mol% cationic lipid, 5-30 mol% sorbitan trioleate, 10-40 mol% squalene, and 0.5-4 mol% PS-20 or PS-80.

[0124] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 55-65 mol% cationic lipid, 5-15 mol% sorbitan trioleate, 25-35 mol% squalene, and 1-2.5 mol% PS-20 or PS-80.

[0125] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 13-45 mol% cationic lipid, 2-4 mol% sorbitan trioleate, 50-82 mol% squalene, and 1.5-3 mol% PS-20 or PS-80.

[0126] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 13-14 mol% of cationic lipid, 1-2 mol% of sorbitan trioleate, 79-81 mol% of squalene, and 1-2 mol% of PS-20 or PS-80.

[0127] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 0 mol% cationic lipid, 8-10 mol% sorbitan trioleate, 80-84 mol% squalene, and 8-10 mol% PS-20 or PS-80.

[0128] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 20 mol% cationic lipid, 30 mol% sorbitan trioleate, 20 mol% squalene, and 30 mol% PS-20 or PS-80.

[0129] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises about 2 mol% cationic lipid, about 8 mol% sorbitan trioleate, about 82 mol% squalene, and about 8 mol% PS-20 or PS-80.

[0130] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 2 mol% cationic lipid, 8 mol% sorbitan trioleate, 82 mol% squalene, and 8 mol% PS-20 or PS-80.

[0131] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises about 13.82 mol% cationic lipid, about 3.43 mol% sorbitan trioleate, about 80.07 mol% squalene, and about 2.68 mol% PS-20 or PS-80.

[0132] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 13.82 mol% cationic lipid, 3.43 mol% sorbitan trioleate, 80.07 mol% squalene, and 2.68 mol% PS-20 or PS-80.

[0133] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises about 44.5 mol% cationic lipid, about 2.21 mol% sorbitan trioleate, about 51.56 mol% squalene, and about 1.72 mol% PS-20 or PS-80.

[0134] In one embodiment, a composition is provided in which a cationic lipid-containing SNE comprises 44.5 mol% cationic lipid, 2.21 mol% sorbitan trioleate, 51.56 mol% squalene, and 1.72 mol% PS-20 or PS-80.

[0135] In one embodiment, a composition is provided in which SNE comprises 32 mol% squalene, 34 mol% SPAN-85, and 34 mol% PS-20 or PS-80.

[0136] In one embodiment, SNE is provided as a composition comprising 98 mol% squalene, 1 mol% SPAN-85, and 1 mol% PS-20 or PS-80.

[0137] In one embodiment, a composition is provided in which SNE comprises 86 mol% squalene, 7 mol% SPAN-85, and 7 mol% PS-20 or PS-80.

[0138] In one embodiment, a composition is provided in which SNE comprises 92 mol% squalene, 4 mol% SPAN-85, and 4 mol% PS-20 or PS-80.

[0139] In one embodiment, a composition is provided in which SNE comprises 94 mol% squalene, 3 mol% SPAN-85, and 3 mol% PS-20 or PS-80.

[0140] In one embodiment, SNE is provided as a composition comprising 92.91 mol% squalene, 3.98 mol% SPAN-85, and 3.11 mol% PS-20 or PS-80.

[0141] In one embodiment, a composition is provided in which SNE comprises 62 mol% squalene, 17 mol% SPAN-85, and 17 mol% PS-20 or PS-80.

[0142] In each of the embodiments described above, the composition further comprises at least one HPV type HPV VLP.

[0143] In some embodiments, a vaccine composition is provided comprising (1) a cationic lipid in a concentration of about 2 μg / mL to about 400 mg / mL, and (2) an HPV VLP of at least one HPV type, wherein each HPV VLP, when present in the vaccine composition, is present at a concentration of about 1 μg to about 300 μg per 0.5 mL of the vaccine composition, and the total VLP concentration is about 10 μg to about 2000 μg per 0.5 mL of the vaccine composition. In some embodiments, a vaccine composition is provided comprising (1) a cationic lipid in a concentration of about 2 μg / mL to about 400 mg / mL, (2) an aluminum adjuvant in a concentration of about 100 μg to about 3500 μg, and (3) an HPV VLP of at least one HPV type, wherein each HPV VLP, when present in the vaccine composition, is present at a concentration of about 1 μg to about 180 μg per 0.5 mL of the vaccine composition, and the total VLP concentration is about 10 μg to about 2000 μg per 0.5 mL of the vaccine composition.

[0144] In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids, about 1 μg to about 2000 μg of HPV VLPs of at least two HPV types, and about 100 μg to about 2700 μg of aluminum adjuvant. In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids, about 4 HPV VLPs of at least four HPV types, and about 100 μg to about 3500 μg of aluminum adjuvant.

[0145] In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids and about 1 μg to about 100 μg of each HPV VLP present in the vaccine composition. In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids and 2 μg to about 600 μg of two HPV types (i.e., the vaccine is a bivalent HPV VLP vaccine). In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids and 4 μg to about 1200 μg of four HPV types (i.e., the vaccine is a quadrivalent HPV VLP vaccine). In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids and 9 μg to about 2700 μg of nine HPV types (i.e., the vaccine is a nonavalent HPV VLP vaccine). In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids and 20 μg to about 6000 μg of 20 HPV types (i.e., the vaccine is a 20-valent HPV VLP vaccine). In some embodiments, the vaccine composition also comprises about 100 μg to about 2700 μg of aluminum adjuvant.

[0146] In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids, 1 μg to about 300 μg of monovalent HPV VLPs, and 100 μg to about 2700 μg of aluminum adjuvant. In some embodiments, a vaccine composition is provided comprising about 2 μg / mL to about 400 mg / mL of cationic lipids, 1 μg to about 300 μg per VLP of bivalent HPV VLPs (i.e., HPV VLPs of two HPV types), and 100 μg to about 3500 μg of aluminum adjuvant. In some embodiments, a vaccine composition is provided comprising (1) about 2 μg / mL to about 400 mg / mL of cationic lipids, (2) 1 μg to about 300 μg per VLP of tetravalent HPV VLPs (i.e., HPV VLPs of four HPV types), and (3) 100 μg to about 3500 μg of aluminum adjuvant. In some embodiments, a vaccine composition is provided comprising (1) about 2 μg / mL to about 400 mg / mL of cationic lipids, (2) 1 μg to about 300 μg per VLP of 9-valent HPV VLPs (i.e., HPV VLPs for 9 HPV types), and (3) 100 μg to about 3500 μg of aluminum adjuvant. In some embodiments, a vaccine composition is provided comprising (1) about 2 μg / mL to about 400 mg / mL of cationic lipids, (2) 1 μg to about 300 μg per VLP of 20-valent HPV VLPs (i.e., HPV VLPs for 20 HPV types), and (3) 100 μg to about 3500 μg of aluminum adjuvant.

[0147] In some embodiments, the vaccine composition contains (1) 1 μg to about 300 μg of HPV VLP (HPV types 16 and 18) per VLP and (2) about 2 μg / mL to about 400 mg / mL of cationic lipids. In some embodiments, the vaccine composition contains (1) 1 μg to about 300 μg of HPV VLP (HPV types 6, 11, 16, and 18) per VLP and (2) about 2 μg / mL to about 400 mg / mL of cationic lipids. In some embodiments, the vaccine composition contains (1) 1 μg to about 300 μg of HPV VLP (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58) per VLP and (2) about 2 μg / mL to about 400 mg / mL of cationic lipids.

[0148] In some embodiments, the vaccine composition contains 1 μg to about 300 μg of HPV VLP (HPV types 16 and 18) per VLP, 100 μg to about 3500 μg of aluminum adjuvant, and about 2 μg / mL to about 400 mg / mL of cationic lipid. In some embodiments, the vaccine composition contains 1 μg to about 300 μg of HPV VLP (HPV types 6, 11, 16 and 18) per VLP, 100 μg to about 3500 μg of aluminum adjuvant, and about 2 μg / mL to about 400 mg / mL of cationic lipid. In some embodiments, the vaccine composition contains 1 μg to about 300 μg per VLP of HPV VLP (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58), 100 μg to about 3500 μg of aluminum adjuvant, and about 2 μg / mL to about 400 mg / mL of cationic lipids.

[0149] The vaccine of the present invention comprises a VLP containing an antigenic determinant necessary to induce the production of neutralizing antibodies in a target. The vaccine is expected to be sufficiently safe to administer without the risk of clinical infection, free from toxic side effects, stable, compatible with conventional carriers, and effectively administered. In some embodiments, the SNE adjuvant of the present invention is combined with a bivalent (types 16 and 18) recombinant human papillomavirus vaccine. In some embodiments, the SNE adjuvant of the present invention is combined with CERVARIX®. In some embodiments, the SNE adjuvant of the present invention is combined with a quadrivalent (types 6, 11, 16, and 18) recombinant human papillomavirus vaccine. In some embodiments, the SNE adjuvant of the present invention is combined with GARDASIL®. In some embodiments, the SNE adjuvant of the present invention is combined with a nonavalent recombinant human papillomavirus vaccine. In some embodiments, the SNE adjuvant of the present invention is combined with GARDASIL® 9.

[0150] The pharmaceutical compositions, formulations, and vaccines of the present invention may be administered subcutaneously, topically, orally, on the mucous membrane, intravenously, or intramuscularly. The pharmaceutical compositions, formulations, and vaccines are administered in amounts sufficient to induce a protective response. Vaccines, pharmaceutical compositions, and formulations can be administered via various routes, such as orally, parenterally, subcutaneously, on the mucous membrane, or intramuscularly. The dose administered may vary depending on the patient's general condition, sex, weight, and age, the route of administration, and the type of HPV VLP in the vaccine. Vaccines, pharmaceutical compositions, or formulations may be in the form of capsules, suspensions, elixirs, or solutions. Such vaccines, pharmaceutical compositions, or formulations may be formulated with immunologically acceptable carriers.

[0151] The present invention kit A kit comprising any of the above-mentioned pharmaceutical compositions and instructions for use is also provided herein.

[0152] (1) A kit comprising a pharmaceutical composition containing HPV VLP of at least one type of HPV, and (2) an SNE adjuvant is also provided herein.

[0153] In some embodiments of the kit, the pharmaceutical composition contains HPV VLP of at least one human papillomavirus (HPV) type 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 bivalent (types 16 and 18) recombinant human papillomavirus vaccine. In some embodiments, the HPV vaccine is CERVARIX®. In some embodiments, the HPV vaccine is a quadrivalent (types 6, 11, 16, and 18) recombinant human papillomavirus vaccine. In some embodiments, the HPV vaccine is GARDASIL®. In some embodiments, the HPV vaccine is a nonavalent recombinant papillomavirus vaccine. In some embodiments, the HPV vaccine is GARDASIL® 9.

[0154] In some embodiments of the kit, the SNE adjuvant is one of the SNE adjuvants described above herein. In some embodiments, the kit contains 0.1 μg to 100 mg of SNE. In some embodiments, the kit contains 2 μg / mL to about 400 mg / mL of cationic lipid. In some embodiments, the kit contains about 0.1 μg / mL to about 400 mg / mL of cationic lipid, and further contains SPAN-85, PS-20 or PS-80 and squalene. In another embodiment, the cationic lipid is CLA. In another embodiment, the cationic lipid is CLX. In another embodiment, the cationic lipid is CLY.

[0155] In some embodiments, the kit contains approximately 60 μg / mL to approximately 2.4 mg / mL of cationic lipids, further comprising 6 μg / mL to 240 μg / mL of SPAN-85, 6 μg / mL to 240 μg / mL of PS-20 or PS-80, and 60 μg / mL to 2.4 mg / mL of squalene. In another embodiment, the cationic lipid is CLA. In another embodiment, the cationic lipid is CLX. In another embodiment, the cationic lipid is CLY. In some embodiments, the kit contains approximately 6 μg / mL to 24 mg / mL of SPAN-85, 6 μg / mL to 24 mg / mL of PS-20 or PS-80, and 60 μg / mL to 240 mg / mL of squalene. In some embodiments, the kit contains SPAN-85 at approximately 2 μg / mL to 24 mg / mL, PS-20 or PS-80 at 2 μg / mL to 2.4 mg / mL, and squalene at 20 μg / mL to 24 mg / mL. In another embodiment, the kit contains SPAN-85 at 6 μg / mL to 2.4 mg / mL, PS-20 or PS-80 at 6 μg / mL to 2.4 mg / mL, and squalene at 60 μg / mL to 24 mg / mL.

[0156] In some embodiments, the kit contains 30 μg / mL to about 2.4 mg / mL of cationic lipids, and further contains 6 μg / mL to 14 mg / mL of SPAN-85, 6 μg / mL to 14 mg / mL of PS-20 or PS-80, and 60 μg / mL to 34 mg / mL of squalene. In another embodiment, the cationic lipid is CLA. In another embodiment, the cationic lipid is CLX. In another embodiment, the cationic lipid is CLY.

[0157] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising PS-20, sorbitan trioleate, squalene, and (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

[0158] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 5-15 mol% sorbitan trioleate, 25-35 mol% PS-20 or PS-80, 1-2.5 mol% squalene, and 55-65 mol% (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

[0159] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising up to 75 mol% cationic lipid, up to 30 mol% sorbitan trioleate, up to 30 mol% polysorbate 20 or polysorbate 80, and 25-85 mol% squalene.

[0160] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising up to 50 mol% cationic lipid, up to 10 mol% sorbitan trioleate, up to 10 mol% polysorbate 20 or polysorbate 80, and 50-80 mol% squalene.

[0161] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising up to 24 mol% cationic lipid, 1-8 mol% sorbitan trioleate, 1-8 mol% polysorbate 20 or polysorbate 80, and 60-75 mol% squalene.

[0162] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising up to approximately 10-14 mol% of cationic lipids, 1-4 mol% of sorbitan trioleate, 1-4 mol% of polysorbate 20 or polysorbate 80, and 50-80 mol% of squalene.

[0163] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 30-65 mol% cationic lipid, 5-30 mol% sorbitan trioleate, 10-40 mol% squalene, and 0.5-4 mol% PS-20 or PS-80.

[0164] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 55-65 mol% cationic lipid, 5-15 mol% sorbitan trioleate, 25-35 mol% squalene, and 1-2.5 mol% PS-20 or PS-80.

[0165] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 13-45 mol% cationic lipid, 2-4 mol% sorbitan trioleate, 50-82 mol% squalene, and 1.5-3 mol% PS-20 or PS-80.

[0166] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 13-14 mol% cationic lipid, 1-2 mol% sorbitan trioleate, 79-81 mol% squalene, and 1-2 mol% PS-20 or PS-80.

[0167] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 0 mol% cationic lipid, 8-10 mol% sorbitan trioleate, 80-84 mol% squalene, and 8-10 mol% PS-20 or PS-80.

[0168] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 20 mol% cationic lipid, 30 mol% sorbitan trioleate, 20 mol% squalene, and 30 mol% PS-20 or PS-80.

[0169] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising about 2 mol% cationic lipid, about 8 mol% sorbitan trioleate, about 82 mol% squalene, and about 8 mol% PS-20 or PS-80.

[0170] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 2 mol% cationic lipid, 8 mol% sorbitan trioleate, 82 mol% squalene, and 8 mol% PS-20 or PS-80.

[0171] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising approximately 13.82 mol% cationic lipid, approximately 3.43 mol% sorbitan trioleate, approximately 80.07 mol% squalene, and approximately 2.68 mol% PS-20 or PS-80.

[0172] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 13.82 mol% cationic lipid, 3.43 mol% sorbitan trioleate, 80.07 mol% squalene, and 2.68 mol% PS-20 or PS-80.

[0173] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising approximately 44.5 mol% cationic lipid, approximately 2.21 mol% sorbitan trioleate, approximately 51.56 mol% squalene, and approximately 1.72 mol% PS-20 or PS-80.

[0174] In one embodiment, the kit comprises an SNE adjuvant, the cationic lipid-containing SNE comprising 44.5 mol% cationic lipid, 2.21 mol% sorbitan trioleate, 51.56 mol% squalene, and 1.72 mol% PS-20 or PS-80.

[0175] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 32 mol% squalene, 34 mol% SPAN-85, and 34 mol% PS-20 or PS-80.

[0176] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 98 mol% squalene, 1 mol% SPAN-85, and 1 mol% PS-20 or PS-80.

[0177] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 86 mol% squalene, 7 mol% SPAN-85, and 7 mol% PS-20 or PS-80.

[0178] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 92 mol% squalene, 4 mol% SPAN-85, and 4 mol% PS-20 or PS-80.

[0179] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 94 mol% squalene, 3 mol% SPAN-85, and 3 mol% PS-20 or PS-80.

[0180] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 92.91 mol% squalene, 3.98 mol% SPAN-85, and 3.11 mol% PS-20 or PS-80.

[0181] In one embodiment, the kit comprises an SNE adjuvant, the SNE comprising 62 mol% squalene, 17 mol% SPAN-85, and 17 mol% PS-20 or PS-80.

[0182] In some embodiments, the kit includes a buffer. In some embodiments, the kit includes an isotonic modifier. In some embodiments, the kit includes a cleaning agent.

[0183] In some embodiments of the kit, the kit includes a label or packaging insert containing a description of its components and / or instructions for the in vivo use of some of its components. In some embodiments, the kit includes instructions for co-administering (or vaccinating) (1) a pharmaceutical composition or HPV vaccine and (2) an SNE adjuvant. In some embodiments, the kit includes instructions for mixing (1) a pharmaceutical composition or HPV vaccine and (2) an SNE adjuvant, and subsequently administering (or vaccinating) the mixture to a patient.

[0184] Therapeutic method of the present invention Also provided herein is a method for inducing an immune response to human papillomavirus (HPV) in a human patient, comprising administering to the patient a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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.

[0185] Also provided herein are methods for inducing an immune response to human papillomavirus (HPV) in a human patient, comprising administering an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 SNE adjuvant is formulated separately from the VLPs. In some embodiments, the SNE adjuvant is formulated together with the VLPs. In some embodiments, the SNE adjuvant and VLPs are mixed in a clinical setting before administration to the patient to form a pharmaceutical composition. In some embodiments, the SNE adjuvant and VLPs are administered sequentially to the patient.

[0186] A method for inducing an immune response to human papillomavirus (HPV) in a human patient is also provided herein, comprising co-administering to the patient (1) a pharmaceutical composition containing virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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) an SNE adjuvant.

[0187] Also provided herein is a method for preventing infection of a human patient with human papillomavirus (HPV) or reducing the likelihood of infection of a human patient, comprising administering to a patient a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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.

[0188] Furthermore, this specification also provides a method for delivering a pharmaceutical composition to a subject in which a neutralizing titer to an HPV antigen is induced, comprising administering the pharmaceutical composition to the subject a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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, thereby causing the administration of the pharmaceutical composition to induce a neutralizing titer to an HPV antigen in the subject, and providing the pharmaceutical composition with an enhanced or equivalent neutralizing titer compared to the same pharmaceutical composition if formulated without the SNE adjuvant.

[0189] A method for preventing cancer in a human patient caused by human papillomavirus (HPV) types 16, 18, 31, 33, 45, 52, and 58 is provided herein, comprising administering to the patient a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 cancer, vulvar cancer, vaginal cancer, anal cancer, oropharyngeal cancer, and other head and neck cancers.

[0190] The Specified also provides a method for preventing cancer in human patients caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, comprising administering to a patient a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 a precancerous or dysplastic lesion of the cervix, vulva, vagina, and anus.

[0191] Furthermore, this specification also provides a method for preventing cancer in human patients caused by HPV types 6 and 11, comprising administering to the patient a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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.

[0192] Furthermore, a method for preventing precancerous or dysplastic lesions in human patients 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, comprising an SNE adjuvant and HPV A method is provided herein that comprises administering to a patient a pharmaceutical composition comprising virus-like particles (VLPs) of at least one human papillomavirus (HPV) type selected from the group consisting of 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 lesion is selected from (1.1) 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 (VaIN) grade 2 and grade 3, and anal intraepithelial neoplasia (AIN) grades 1, 2, and 3.

[0193] Furthermore, the present invention provides a method for preventing HPV-related anogenital disease in human patients caused by an HPV type 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, comprising administering to a patient a pharmaceutical composition comprising an SNE adjuvant and virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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.

[0194] Embodiments of the present invention also include (a) treatment (e.g., of the human body); (b) medical treatment; (c) induction of an immune response to HPV types contained in the vaccine; (d) reduction of the likelihood of HPV infection in patients; (e) prevention of infection by HPV types contained 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; (l) prevention or reduction of the likelihood of genital warts or genital verruca; (m) intraepithelial cell carcinoma of the cervix The present invention provides one or more pharmaceutical compositions described herein for use in (1) use, (2) use as a pharmaceutical or composition, or (3) use in the manufacture of a pharmaceutical, in order to (1) use in

[0195] Embodiment 1 provides a composition comprising virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 squalene nanoemulsion (SNE) adjuvant, wherein the SNE adjuvant comprises sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20), or polysorbate 80 (PS-80), and squalene.

[0196] In Embodiment 2, there is provided the composition of Embodiment 1 prepared by mixing an HPV vaccine and a squalene nanoemulsion (SNE) adjuvant. The HPV vaccine contains HPV VLP and a pharmaceutically acceptable carrier, and the squalene nanoemulsion (SNE) adjuvant contains sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20) or polysorbate 80 (PS-80), and squalene.

[0197] In Embodiment 3, there is provided the composition of Embodiment 1 or 2, wherein the SNE adjuvant contains 6 μg / mL to 14 mg / mL of SPAN-85, 6 μg / mL to 14 mg / mL of PS-20 or PS-80, and 20 μg / mL to 240 mg / mL of squalene.

[0198] In Embodiment 4, there is provided the composition of Embodiments 1 to 3, wherein the SNE adjuvant further contains a cationic lipid.

[0199] In Embodiment 5, there is provided the composition of Embodiment 4, wherein the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine.

[0200] In Embodiment 6, there is provided the composition of Embodiment 4 or 5, which contains 30 μg / mL to 4.8 mg / mL of the cationic lipid.

[0201] In Embodiment 7, there is provided the composition of any one of Embodiments 1 to 6, wherein the SNE contains PS-20.

[0202] In Embodiment 8, there is provided the composition of any one of Embodiments 1 to 7, wherein the composition further contains a buffer solution.

[0203] In Embodiment 9, there is provided the composition according to Embodiment 8, wherein the buffer solution is selected from the group consisting of acetic acid, histidine, citrate, Bis-Tris, HEPES, phosphate, MES, sodium chloride, succinate, Tris, and combinations thereof.

[0204] Embodiment 10 provides the composition of Embodiment 8 or 9, wherein the buffer solution is present in an amount of about 1 mMol to about 100 mMol.

[0205] Embodiment 11 provides the compositions of Embodiments 1 to 10, wherein the composition further comprises a salt.

[0206] Embodiment 12 provides the composition of Embodiment 11, wherein the salt is NaCl.

[0207] Embodiment 13 provides the compositions of Embodiments 1 to 12, further comprising 5 mM to 40 mM histidine-7.0 and 25 mM to 300 mM NaCl at pH 5.1.

[0208] Embodiment 14 provides the compositions of Embodiments 1 to 12, further comprising about 20 mM histidine and about 75 mM NaCl at about pH 5.8.

[0209] Embodiment 15 provides the compositions of Embodiments 1 to 14, wherein the compositions include HPV type 16 and type 18 VLPs.

[0210] Embodiment 16 provides the compositions of Embodiments 1 to 15, wherein the composition includes VLPs of HPV types 6, 11, 16, and 18.

[0211] Embodiment 17 provides the compositions of Embodiments 1 to 16, which include VLPs of HPV types 31, 45, 52, and 58.

[0212] Embodiment 18 provides the compositions of Embodiments 1 to 17, which include VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58.

[0213] Embodiment 19 provides the compositions of Embodiments 1 to 18, further comprising aluminum.

[0214] Embodiment 20 provides the compositions of Embodiments 1 to 19, wherein the HPV VLP comprises recombinant HPV Ll or recombinant HPV Ll+L2 protein.

[0215] Embodiment 21 provides the compositions of Embodiments 1 to 19, wherein the HPV VLP contains HPV L1 protein but does not contain HPV L2 protein.

[0216] Embodiment 22 provides the compositions of Embodiments 1 to 19, wherein the HPV VLP consists of HPV L1 protein.

[0217] Embodiment 23 provides a method for inducing an immune response to human papillomavirus (HPV) in a human patient, the method comprising administering any of the pharmaceutical compositions of Embodiments 1 to 22 to the patient.

[0218] Embodiment 24 provides a method for preventing infection of a human patient with human papillomavirus (HPV) or reducing the likelihood of infection in a human patient, the method comprising administering a pharmaceutical composition of any of Embodiments 1 to 22 to the patient.

[0219] Embodiment 25 provides the use of any of the compositions from Embodiments 1 to 22 to prevent infection of human patients with human papillomavirus (HPV) or to reduce the likelihood of infection of human patients.

[0220] Embodiment 26 provides a kit comprising (a) a human papillomavirus (HPV) vaccine and (b) a squalene nanoemulsion (SNE) adjuvant, wherein the SNE comprises sorbitan trioleate (SPAN-85); polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and squalene.

[0221] Embodiment 27 provides a kit of Embodiment 26, further including instructions for administering the HPV vaccine and SNE adjuvant to a human patient.

[0222] All publications referred to in this specification are incorporated by reference for the purpose of describing and disclosing methodologies and materials that can be used in connection with the present invention. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0223] Although embodiments of the present invention have been described with reference to the accompanying drawings, it should be understood that the present invention is not limited to those exact embodiments, and various changes and modifications can be made by those skilled in the art without departing from the scope or spirit of the invention as defined by the appended claims.

[0224] The following examples illustrate the present invention but do not limit the present invention.

[0225] [Examples] [Example 1] Preparation of a squalen nanoemulsion (SNE) adjuvant system with or without the cationic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine SNE adjuvants can be prepared together with or without the cationic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (also known as CLA), or (6Z,9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,29-tetraen-18-amine (also known as CLX), or N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine (also known as CLY) (Figure 1). SNE is a multi-component emulsion formulation containing three stabilizing components, namely squalene, sorbitan trioleate (SPAN-85), and polysorbate 20 (PS-20), and a cationic lipid such as CLA (referred to as CLA-SNE, see Table II), and also includes formulations that do not contain cationic lipids (referred to as SNE, see Table III). This formulation was prepared by combining and mixing cationic lipids (if used), squalene, SPAN-85, and PS-20 or similar components (e.g., surfactants, oils, and solubilizers) together (Table II and Figure 2). After mixing and blending, histidine buffer was added and mixed with the initial emulsion components. The blended emulsion components were first subjected to coarse homogenization, followed by fine homogenization, as described below. The resulting formulation was subjected to a final 0.2 mm filtration step. By controlling several process parameters within each step, such as the order of addition, mixing time, pH, temperature, component concentration, homogenization, and microfluidization, an emulsion system with the desired attributes was obtained. [Table 2]

[0226] [Table 3]

[0227] Preparation of pharmaceutical products An emulsion of squalene and a solubilizer (referred to as the oil phase) was prepared by adding squalene, SPAN-85, PS-20, and CLA to a container. The oil phase was then mixed using magnetic stirring at 100-1000 RPM for 10-120 minutes. After mixing these components, an aqueous phase consisting of 20 mM histidine at pH 5.8 was slowly added to the oil phase while mixing with a magnetic stirrer. This preparation was then mixed again for 1 hour.

[0228] coarse homogenization Next, the oil-aqueous mixture (referred to as pre-homogenized emulsion or PHE) was homogenized and reduced in size to form a crude emulsion using a rotor stator homogenizer at ambient temperature. The homogenizer arm tip was immersed in the PHE and held in place near the bottom of the formulation container, and operated at 6–10 kRPM for 5–15 minutes. This process yielded a homogeneous microemulsion (ME) suspension of squalene emulsion particles ranging in diameter from 4 to 20 μm, suitable for further size reduction by microfluidization in a high-pressure homogenizer for the production of squalene nanoemulsions (SNEs).

[0229] Fine homogenization for generating squalene nanoemulsions (SNEs) After coarse homogenization, the emulsion was further processed using a high-pressure homogenizer / microfluidizer to produce nanometer-sized emulsion particles. ME was introduced into a high-pressure homogenizer (Microfluidics low volume Microfluidizer®, GEA Group PandaPlus 2000 or Bee International, NanoDeBEE, etc.) to establish a recirculation loop. A backflow heat exchanger, supplied by a controlled temperature unit with a 5°C setpoint, was included in the recirculation loop to neutralize the heat generated by high-pressure homogenization. 20 kPSI was selected as the operating setpoint for this process step to produce emulsion particles of the desired size and processability. The high-pressure homogenizer operates at a constant, unchangeable flow rate through the established recirculation loop. Using this measured flow rate and the volume of ME being processed, the theoretical time required for the entire formulation to pass through the recirculation loop once was calculated. Considering this calculated single-pass time, the high-pressure homogenizer was typically operated until at least 10 desired pass counts were reached, resulting in either SNE or CLA-SNE.

[0230] filtration After compounding, SNE or CLA-SNE was passed through a 0.8 / 0.2 μm PES filter. A flux of 42 LMH passing through the filter was selected considering the optimal mass yield and particle stability after filtration.

[0231] The volumetric size distribution of nanoemulsions during preparation was measured using laser diffraction or static light scattering (SLS) techniques with a Malvern Panalytical Ltd. MS3000 instrument. This data was then analyzed to calculate the size of particles forming the scattering patterns. Sample fractions of prehomogenized emulsion (PHE), microemulsion (ME), and squalene nanoemulsion (SNE) were obtained. These emulsion formulations were diluted in 5 mM histidine pH 5.8 and 2.5 mM NaCl buffers to achieve a target light shielding rate of 3%, and collected by SLS under 1200 RPM recirculation. Sample data sets were collected with 30-second scans per data set. Three data sets from each step of the CLA-SNE formulation process are summarized in Figure 3. The 20 mM histidine pH 5.8 buffer was a perfectly suitable formulation for bulk stability during the process and when stored in polymer containers (e.g., plastic). However, when stored in glass, nonspecific absorption of CLA-SNE or SNE onto the glass surface was observed. Screening evaluations of surfactants / solubilizers, buffers, and salts showed that several formulations successfully eliminated this stability issue by selecting a stabilizing formulation of 20 mM histidine, 0.05% PS-20, and 75 mM NaCl (data not shown).

[0232] [Example 2] Preparation of a squalene nanoemulsion (SNE) adjuvant system and addition of a cationic lipid, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine) or CLA as a free base immediately after microfluidization of the SNE. Two formulation processes were evaluated for incorporating CLA into nanoemulsion particles containing squalene formulated in PS-20, sorbitan trioleate (SPAN-85), and histidine pH 5.8 buffer. In the first process (referred to as Process 1), SNE was prepared using the process described in Example 1. In the second process (referred to as Process 2), only PS-20, sorbitan trioleate (SPAN-85), and squalene were combined and mixed together. After mixing and blending, histidine buffer was added and mixed with the initial emulsion components (PS-20, sorbitan trioleate, and squalene). The blended emulsion components were first subjected to coarse homogenization to produce a microemulsion ME, as described in Example 1, followed by microfluidization to produce a nanoemulsion (NE). In a separate glass container, 0.25 mg / mL of CLA was dissolved in 100% ethanol at room temperature. Next, a sufficient volume of this CLA ethanol solution to produce the desired final CLA concentration was added to an SNE containing PS-20, sorbitan trioleate, and squalene in histidine buffer pH 5.8, and then mixed at room temperature for 60 minutes. After incubation, the formulations were dialyzed overnight at 4°C with two buffer changes using 500 mL of buffer per 10 mL of sample under conditions of 5 mM histidine, 2.5 mM NaCl, pH 5.8. The two treated emulsions (process 1 and 2) were then examined for CLA incorporation into the SNE using UPLC-CAD. The results showed that no significant amount of CLA was incorporated compared to the individual formulations using process 2 (w / w)% targeted incorporation, indicating that process 1 is preferred for successful CLA incorporation and stability into the nanoemulsion (Figure 4).

[0233] [Example 3] Effects of time and temperature on the physical stability of nanoemulsion formulations using NTA and DLS As shown in Figures 5A-5D, nanoparticle tracking analysis (NTA) was used to evaluate the stability of the nanoemulsion systems (CLA-SNE or SNE) prepared as described in the Examples (previously mentioned). This technique collects video of directly tracked nanoparticle populations as they move by Brownian motion to extrapolate particle size and concentration. A Class 1, 635 nm laser focuses an 80 mm red laser beam through the liquid sample, irradiating the particles as rapid diffusion points of light. A CCD camera records video at 30 frames / second to track the movement of individual irradiated particles over time. System software identifies the center of each particle from the video and tracks the distance traveled independently to determine the mean square displacement. This tracking was performed simultaneously for all particles in the sample population in each frame until the raw data collected from the entire video was analyzed. By simultaneously measuring the mean square displacement of the individual tracked particles, their diffusion coefficient (Dt) and spherical equivalent hydrodynamic radius (rh) were determined by applying the Stokes-Einstein equations. Next, the software represents this accumulated data as particle size and concentration distributions. In addition to particle size and concentration, raw data information regarding the intensity or brightness of individual particles was collected. In summary, the data was fitted and plotted individually as particle intensity against particle size and particle concentration against particle size, and then plotted on a three-dimensional contour plot comparing the particle size, concentration, and intensity of the entire particle population.

[0234] When nanoemulsion formulations (CLA-SNE or SNE) were exposed to 37°C for up to one month, no significant changes in nanoemulsion particle concentration or size distribution were observed, as evaluated using NTA (Figure 5).

[0235] Nanoemulsions are prone to aggregation within the particle size range of 10–1000 nm; therefore, dynamic light scattering (DLS) is a suitable stability indicator technique for evaluating and quantifying aggregation phenomena. To evaluate the stability of the nanoemulsion systems prepared as described in the Examples (previously), the average particle size distribution was measured using dynamic light scattering (DLS). A DLS instrument uses a laser to irradiate particles in a solution, and then examines the change in the intensity of the scattered light over time as a result of Brownian motion. The correlation of the scattered light intensity over time with respect to the intensity at time 0 yields an exponential decay curve or correlation function. The decay rate of the correlation function with respect to time is much faster for smaller particles than for larger particles, which forms the basis for calculating particle size. When nanoemulsions were exposed to 4°C, 25°C, or 37°C for up to one month, no change in the size distribution of the nanoemulsions was observed by DLS (Figures 6A–6D). The Z-mean of CLA-SNE or SNE remained approximately 110 nm–180 nm.

[0236] [Example 4] Effects of time and temperature on the chemical stability of nanoemulsion formulations using UPLC-CAD To evaluate the chemical stability of the nanoemulsion systems prepared as described in the previously mentioned examples, ultrahigh-performance liquid chromatography (UPLC-CAD) combined with a charge aerosol detector was used to measure the stability of CLA (CLA-SNE only) and squalene concentration after storage at 4°C, 25°C, and 37°C for one month. Exposure of the nanoemulsions to 4°C, 25°C, and 37°C for up to one month affected the concentrations of CLA (Figure 7A) or squalene (Figure 7B) in the SNE, demonstrating excellent chemical stability in both the CLA-SNE adjuvant and the SNE adjuvant.

[0237] Furthermore, UPLC-CAD can quantify the generation of degradation products due to the chemical degradation of either squalene or CLA. When SNE or CLA-SNE adjuvant systems were exposed to high temperatures, no detectable degradation peaks were observed, indicating that the squalene and CLA components of CLA-SNE and SNE adjuvant systems have excellent thermal stability (data not shown).

[0238] [Example 5] Preparation of squalene nanoemulsion (SNE) adjuvant systems containing or not containing cationic lipids ((13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine) by microfluidic nanoemulsion self-assembly (MNS). Squalene nanoemulsion adjuvants are prepared with and without CLA (also known as CLA, Figure 1), an ionizable cationic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine). A microfluidic nanoemulsion self-assembly (MNS) process is used to prepare SNEs. SNEs are multi-component emulsion formulations consisting of three stabilizing components: squalene, sorbitan trioleate (SPAN-85), and polysorbate 20 (PS-20), and may contain CLA (referred to as CLA-SNE, Table IV) or not (referred to as SNE, Table V). The biophysical properties (e.g., particle size, chemical composition) and stability of MNS-prepared SNE / CLA-SNE formulations are very similar to those of the high-pressure micro-homogenization process used to prepare the SNE / CLA-SNE formulations described in Example 3. Essentially, the microfluidic nanoemulsion self-assembly (MNS) process described in this example is an alternative process for preparing squalene nanoemulsion (SNE) adjuvant systems. The nanoparticle self-assembly process described in this example was carried out using a “microfluidic” ethanol / aqueous mixing apparatus. However, the ethanol / aqueous flow nanoparticle self-assembly process described in the present invention is not limited to “microfluidic” mixing. Mixing a larger volume flow of a hydrophobic solvent with an aqueous solution can be achieved using the Tee mixing process outlined in Example 4.

[0239] SNE MNS formulations can generally be prepared by dissolving cationic lipids, squalene, SPAN-85, and PS-20 at target concentrations in a suitable non-aqueous solvent such as ethanol. The self-assembly procedure involves combining a flow of ethanol-dissolved hydrophobic emulsion components with a flow of aqueous emulsion solution. When the two solvent flows are combined, the hydrophobic molecules (i.e., cationic lipids, squalene, SPAN-85, and PS-20) interact with the aqueous solvent. The molecules then assemble into an emulsion of nano-sized particles, as described below. After the formation of the self-assembling emulsion of nanoparticles, residual ethanol can be removed from the squalene emulsion by several suitable means. In this example, ethanol was reduced to less than 0.1% (w / v) by overnight dialysis in an aqueous buffer. The resulting SNE formulation was sterilized by filtration through a sterile filter with a pore size of 0.2 μm. By controlling several process parameters within each step, such as the order or addition, mixing time, temperature, concentration of non-aqueous components, concentration of aqueous buffer components, aqueous pH, mixing ratio of non-aqueous solution to aqueous solution, total flow rate, and waste volume, an SNE adjuvant system with the desired attributes was obtained. [Table 4]

[0240] [Table 5]

[0241] Formulation preparation using microfluidic nanoemulsion self-assembly In this example, one SNE and four CLA-SNE formulations were prepared by a microfluidic nanoemulsion self-assembly procedure at 20 mM histidine pH 5.8 for biophysical characterization. The self-assembly nanoemulsion process was initiated with 15 mg / mL squalene, 1.5 mg / mL SPAN-85, and 1.5 mg / mL PS-20 completely dissolved in ethanol. Furthermore, each of the above ethanol solutions also contained either 0.75, 1.5, 5.0, or 15.0 mg CLA / mL of CLA. Thus, the initial “target” CLA / squalene (w / w)% for all five formulations were 0.0, 5.0, 10.0, 33.3, and 100 (w / w)% CLA / squalene. The aqueous buffer for all formulations was 20 mM histidine at pH 5.8. Using Precision NanoSystems, Inc.'s (Vancouver, British Columbia, Canada) benchtop NanoAssemblr® system, one SNE and four CLA-SNE-assisted nanoemulsions were self-assembled.

[0242] Self-assembled squalene nanoparticle formulations were prepared in the following manner: A 1 mL syringe was filled with slightly more than 0.7 mL of a hydrophobic compound mixture dissolved in ethanol, and a 3 mL syringe was filled with slightly more than 1.4 mL of aqueous 20 mM histidine pH 5.8 buffer. After filling both syringes with the appropriate amount of solution, the syringes were attached to a NanoAssemblr® device. The following microfluidic mixing parameters were programmed into NanoAssemblr®: a) total volume = 2 mL, b) flow ratio = 2:1 (aqueous vs. ethanol), c) total flow rate = 12 mL / min, d) starting waste volume = 0.25 mL, and e) ending waste volume = 0.05 mL. The device was activated, and the mixing process of ethanol and aqueous solution was started in just a few seconds. Approximately 2.0 mL of the post-mixed nanoparticle emulsion in approximately 30% ethanol was collected in 15 mL Falcon tubes for each of the five formulations. A new NanoAssemblr™ mixed cartridge was used for each of the five different SNE and CLA-SNE formulations described above. The ethanol concentration in each formulation was reduced by overnight dialysis. After dialysis, all samples were stored at 4°C before analytical characterization.

[0243] Analytical Characteristics Assessment Cationic lipids, CLA, and squalene were equally incorporated into the squalene-CLA-SNE nanoparticles prepared by MNS, as shown in Figure 8A. The CLA / squalene (w / w)% ratio after dialysis (i.e., y-axis) for removing process ethanol was compared to the CLA / squalene (w / w)% before self-assembly (i.e., x-axis) while in ethanol solution for all formulation samples described in this example. The "measured" CLA / squalene (w / w)% after MNS and dialysis was equal to the "target" (w / w)% up to at least 35 (w / w)%. Even with 100% "target" CLA / squalene (w / w)% before self-assembly, more than 70% of the available CLA was incorporated into the CLA-SNE nanoparticles relative to the squalene content in the MNS-prepared nanoparticle emulsion. The CLA / squalene (w / w)% ratio was measured by reverse-phase ULC-CAD. CLA was clearly incorporated into CLA-SNE by preparing it using a microfluidic nanoemulsion self-assembly (MNS) process.

[0244] The intensity-weighted Z-mean DLS diameter of CLA-SNE formulations prepared by the MNS process was measured using a Malvern ZetaSizer Ultra. Aliquots of the post-dialysis CLA-SNE samples from each formulation were diluted 50-fold or 100-fold with 2.0 mL of 20 mM histidine pH 5.8 buffer. The mean DLS diameter and standard deviation were plotted against the post-dialysis CLA / squalene (w / w)% measured for each formulation, and are shown in Figure 8B. Three DLS measurements were performed for each formulation at room temperature. Standard deviation bars are shown unless the standard deviation is smaller than the data point image. The intensity-weighted Z-mean DLS diameter of MNS-prepared CLA-SNE was in the range of approximately 150-280 nm, similar to that of CLA-SNE nanoparticles prepared by high-pressure homogenization. In this example, various MNS process parameters, such as those described above, were controlled to obtain CLA-SNE adjuvant systems with desired diameters.

[0245] Figure 8C shows the measured zeta potentials of CLA-SNE squalene nanoparticle formulations at pH 5.5, prepared using the MNS process. Zeta potentials were measured using a Malvern ZetaSizer Ultra. Aliquots of the post-dialysis CLA-SNE samples from each formulation were diluted 50-fold or 100-fold in 2.0 mL of 20 mM citrate BIS TRIS propane buffer at pH 5.5. Three zeta potential measurements were performed for each formulation at room temperature. Standard deviation bars are shown unless the standard deviation is smaller than the data point image. The zeta potential of the 0(w / w)%CLA CLA-SNE formulation, i.e., without CLA, was approximately -5 mV. As shown in Figure 8C, the addition of CLA significantly increased the zeta potential of the nanoparticles to approximately +10 mV.

[0246] [Example 6] Optimization of CLA-SNE preparation by changing the aqueous phase pH.

[0247] The CLA-SNE process involves the use of a reversible cationic CLA molecule with an observed pKa of 6.4. Addition of CLA to the SNE preparation process and the final matrix at pH 5.8 results in protonation of the CLA, which functions to impart an overall net positive charge to the CLA-SNE particles and any intermediates in the preparation process. CLA-SNE preparation was terminated at a 0.8 / 0.2 μm filtration event, which proved to be a difficult process step to perform, with significant filter contamination and consistently low product yields observed. However, filtration of SNE did not exhibit the same magnitude of filtration challenges and proved to be a more efficient nanoemulsion filtration process. Importantly, in the absence of CLA, SNE does not possess the strong positive charge observed in CLA-SNE. To prepare uncharged CLA-SNE for a more efficient CLA-SNE filtration process, a series of experiments were performed to adjust the pH of the aqueous phase (20 mM L-histidine) before use in CLA-SNE preparation. 20 mM L-histidine was prepared using pH targets of 5.0, 5.7, 5.8, 6.0, 6.2, 7.0, and 7.7. Each buffer was then used as the aqueous phase during the CLA-SNE preparation process, targeting 15 mg / mL of CLA as the target formulation, and the CLA-SNE preparation proceeded precisely as described in Example 3. After the homogenization process was completed, the particle size of the CLA-SNE intermediate was measured by DLS using a Malvern Panalytical Nano ZS. This was followed by filtration through a 0.8 / 0.2 μm PES filter. The particle size distribution was measured after filtration by DLS, and [CLA] was quantified by UPLC-CAD. In CLA-SNE samples prepared with 20 mM L-histidine at pH 7.0 or 7.7, complete filter blockage was observed immediately after the material was applied to the filter, making collection of the filtered material impossible. Quantification by DLS or ULC-CAD was also impossible, and a value of 0 was reported for illustrative purposes. In pre-filtered samples, a tendency for particle size to increase by DLS was observed as the pH of the aqueous buffer increased (Figure 9).This relationship was maintained in the filtered samples, with each CLA-SNE sample showing a moderate decrease in particle size after filtration, except for the samples at pH 7.0 and 7.7, which clearly exhibited complete filter blockage immediately, making material recovery impossible. In the filtered samples, it was observed that [CLA] decreased in the final CLA-SNE material as the pH of the aqueous phase increased (Figure 10). This relationship demonstrates that CLA-SNE preparation using the aqueous phase at lower pH results in increased process yield in final filtration and a more efficient SLA-CAN manufacturing process.

[0248] [Example 7] Immunogenicity and persistence of two doses of 9vHPV vaccine + CLA-SNE (0.33 mg, 1.32 mg, 3.96 mg) or SNE (2.16 mg) adjuvant in rhesus monkeys. The objective of the SD-HPV-062 study was to evaluate the immunogenicity of the 9vHPV vaccine (9vHPV+AAHS) in combination with escalating doses of CLA-SNE adjuvant (0.33 mg, 1.32 mg, or 3.96 mg (total lipids)) or SNE (2.16 mg (total lipids)) in a non-human primate nonclinical immunogenicity model. The group names are listed in Table VI. At week 0, groups of 4-5 rhesus monkeys were administered either the 9vHPV vaccine alone or the 9vHPV vaccine in combination with 0.33 mg, 1.32 mg, or 3.96 mg of CLA-SNE or 2.16 mg of SNE adjuvant. At week 24, all groups received a second dose of the 9vHPV vaccine, including the respective adjuvant and dose. The 1.0 mL dose was prepared by mixing 9vHPV vaccine with 0.5 mL of double-concentration CLA-SNE or SNE adjuvant and administering the mixture to rhesus monkey quadriceps muscles (0.5 mL x 2 quadriceps / NHP) within one hour of preparation. [Table 6]

[0249] Rhesus monkeys (n=4-5 / group) received two intramuscular injections of 9vHPV vaccine (weeks 0 and 24) in combination with either 3.96 mg, 1.32 mg, or 0.33 mg of CLA-SNE or 2.15 mg of SNE. Antibody levels against all nine HPV VLP types were monitored for 54 weeks. To assess immunogenicity, serum from individual animals was evaluated using a multiplex assay for antibody levels against all nine HPV types in the vaccine. VLP-specific HPV antibody concentrations were determined at weeks 0, 4, 6, 8, 12, 20, 24, 26, 28, 30, 32, 34, 36, 44, and 54 of the study.

[0250] Representative titers for HPV VLP-16 and HPV VLP-18 are shown in Figures 11A and 11B. Antibody concentrations (μg / mL) detected in serum against HPV VLP types 16 (Figure 1A) and 18 (Figure 1B) at weeks 0, 4, 6, 8, 12, 20, 24, 26, 28, 30, 32, 34, 36, 44, and 54. Data are presented as geometric mean concentrations and 95% confidence intervals.

[0251] As shown in the figure, the adjuvant effect of CLA-SNE on HPV antibody titers was clearly dose-dependent. For SNE, one dose was tested to match the highest dose of CLA-SNE water-in-oil emulsion concentration. Animals vaccinated with any CLA-SNE or 9vHPV vaccine in combination with SNE dose showed higher HPV16 antibody titers than the two-dose 9vHPV vaccine group at all time points except week 24 / 26, when HPV16 titers reached their maximum titer in all groups. Animals vaccinated with any CLA-SNE dose in combination with 9vHPV vaccine showed higher HPV18 antibody titers than the two-dose 9vHPV vaccine group at all time points. Animals vaccinated with 2.16 mg of SNE did not have a higher HPV18 titer post-administration, but showed higher geometric mean titers at each time point after the second dose. Importantly, these titers remained higher than those of the two-dose control group throughout the final week (54 weeks) of the study. As shown in Figure 12, rhesus monkeys (n=4-5 / group) received intramuscular injections of either two doses (weeks 0 and 24) of the 9vHPV vaccine or two doses (weeks 0 and 24) of the 9vHPV vaccine in combination with either 3.96 mg, 1.32 mg, or 0.33 mg of CLA-SNE or 2.16 mg of SNE. Antibody levels against all nine HPV VLP types were monitored for 54 weeks. Antibody concentrations (μg / mL) detected in serum against all nine VLP types at week 54 are shown. Data are presented as geometric mean concentrations and 95% confidence intervals.

[0252] To assess immunogenicity, serum from individual animals was evaluated using multiplex assays for antibody levels against all nine HPV types in the vaccine. VLP-specific HPV antibody concentrations were determined at weeks 0, 4, 6, 8, 12, 20, 24, 26, 28, 30, and 32 of the study. Data were collected at weeks 36, 44, and 54. Representative titers for HPV VLP-16 and HPV VLP-18 are shown in Figures 13A and 13B. Rhesus monkeys immunized with 9vHPV vaccine + 3.96 mg of VA-879 CLA-SNE achieved peak antibody titers for HPV16 and HPV18 four weeks after dose 1, remaining very stable until a boost at week 24, where titers were also very high and remained stable until week 32. Rhesus monkeys immunized with 9vHPV vaccine + 12 mg of VA-881SNE consistently showed higher geometric mean antibody titers after the second dose of the total vaccine at 24 weeks compared to a single dose of 9vHPV, as well as similar or higher titers for HPV16 and HPV18. These results indicate that increasing the amount of VA-881SNE to 12 mg of total lipids (10 mg squalene) results in higher geometric mean antibody titers against HPV VLPs.

[0253] Rhesus monkeys (n=5 / group) received two intramuscular injections of either 3.96 mg of CLA-SNE or 12 mg of SNE combined with the 9vHPV vaccine (at week 0 and week 24). Antibody levels against all nine HPV VLP types were monitored for 32 weeks. Figure 14 shows the serum antibody concentrations (μg / mL) for all nine VLP types at week 32. Data are presented as geometric mean concentrations and 95% confidence intervals. These findings apply to all nine VLP types included in the 9vHPV vaccine.

Claims

1. A composition comprising virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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 squalene nanoemulsion (SNE) adjuvant, wherein the SNE adjuvant comprises sorbitan trioleate (SPAN-85), polysorbate 20 (PS-20), or polysorbate 80 (PS-80), and squalene.

2. The composition according to claim 1, wherein the composition is prepared by mixing an HPV vaccine with an SNE adjuvant, the HPV vaccine comprising HPV VLP and a pharmaceutically acceptable carrier, and the SNE adjuvant comprising SPAN-85, PS-20 or PS-80 and squalene.

3. The composition according to claim 1 or claim 2, wherein the SNE adjuvant comprises SPAN-85 in a concentration of 6 μg / mL to 14 mg / mL, PS-20 or PS-80 in a concentration of 6 μg / mL to 14 mg / mL, and squalene in a concentration of 20 μg / mL to 240 mg / mL.

4. The composition according to any one of claims 1 to 3, wherein the SNE adjuvant further comprises a cationic lipid.

5. The composition according to claim 4, wherein the cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

6. The composition according to claim 4 or 5, comprising 30 μg / mL to 4.8 mg / mL of cationic lipids.

7. The composition according to any one of claims 1 to 6, wherein the SNE comprises PS-20.

8. The composition according to any one of claims 1 to 7, further comprising a buffer solution.

9. The composition according to claim 8, wherein the buffer solution is selected from the group consisting of acetic acid, histidine, citrate, Bis-Tris, HEPES, phosphate, MES, sodium chloride, succinate, Tris, and combinations thereof.

10. The composition according to either claim 8 or 9, wherein the buffer solution is present in an amount of about 1 mMol to about 100 mMol.

11. The composition according to any one of claims 1 to 10, wherein the composition further comprises a salt.

12. The composition according to claim 11, wherein the salt is NaCl.

13. The composition according to any one of claims 1 to 12, wherein the composition comprises 5 mM to 40 mM histidine and 25 mM to 300 mM NaCl at a pH of 5.1 to 7.

0.

14. The composition according to any one of claims 1 to 12, wherein the composition comprises about 20 mM histidine and 75 mM NaCl at pH 5.

8.

15. The composition according to any one of claims 1 to 14, wherein the composition comprises HPV type 16 and type 18 VLPs.

16. The composition according to any one of claims 1 to 15, wherein the composition comprises HPV type 6, type 11, type 16, and type 18 VLPs.

17. The composition according to any one of claims 1 to 16, wherein the composition comprises HPV types 31, 45, 52, and 58.

18. The composition according to any one of claims 1 to 17, wherein the composition comprises HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58.

19. The composition according to any one of claims 1 to 17, wherein the composition comprises HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.

20. The composition according to any one of claims 1 to 17, wherein the composition comprises HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 73 VLPs.

21. The composition according to any one of claims 1 to 17, wherein the composition comprises HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70 VLPs.

22. The composition according to any one of claims 1 to 21, wherein the composition further comprises aluminum.

23. The composition according to any one of claims 1 to 22, wherein the HPV VLP comprises recombinant HPV Ll or recombinant HPV Ll + L2 protein.

24. The composition according to any one of claims 1 to 22, wherein the HPV VLP comprises HPV L1 protein and does not contain HPV L2 protein.

25. The composition according to any one of claims 1 to 22, wherein the HPV VLP comprises HPV L1 protein.

26. A method for inducing an immune response to human papillomavirus (HPV) in a human patient, comprising administering to the patient a composition according to any one of claims 1 to 25.

27. A method for preventing infection of a human patient with human papillomavirus (HPV) or reducing the likelihood of infection, comprising administering a composition according to any one of claims 1 to 25 to the patient.

28. Use of the composition according to any one of claims 1 to 25 for preventing infection of a human patient by human papillomavirus (HPV) or for reducing the likelihood of infection of a human patient.

29. It's a kit, (a) Human papillomavirus (HPV) vaccine and (b) comprising a squalene nanoemulsion (SNE) adjuvant, wherein the SNE comprises sorbitan trioleate (SPAN-85); polysorbate 20 (PS-20) or polysorbate 80 (PS-80); and squalene. kit.

30. The kit according to claim 29, further comprising instructions for administering the HPV vaccine and SNE adjuvant to a human patient.

31. The kit according to claim 29 or 30, wherein the HPV vaccine comprises virus-like particles (VLPs) of at least one human papillomavirus (HPV) type 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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