Controlled release vaccine formulations

Abstract

The present disclosure provides, inter alia, a vaccine composition comprising human papilloma virus (VLP) of at least one type of human papilloma virus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82, where the vaccine composition provides an enhanced or comparable HPV vaccine response as compared to similar multi-dose vaccines.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 323,903, filed on March 25, 2022, the entire contents of which are incorporated herein by reference.

[0003] field

[0004] The present invention generally relates to preventing human papillomavirus (HPV) infection. More specifically, the present invention relates to pharmaceutical compositions and formulations comprising virus-like particles (VLPs) of HPV and PLGA, which can be administered as a single-dose vaccine. The present disclosure provides, among other things, a single-dose vaccine composition comprising an HPV vaccine, wherein a single administration of the vaccine composition provides a comparable or enhanced immune response compared to multiple administrations of the same HPV vaccine formulated. Methods of using the disclosed compositions and formulations are further provided.

[0005] background

[0006] 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 carcinogenic properties. HPV includes major (L1) and minor (L2) capsid proteins. More than 200 different HPV genotypes have been identified (Li et al., "Rational design of a triple-type humanpapillomavirus vaccine by compromising viral-type specificity," Nature, 9: 5360 (2018)), many of which are associated with pathology from benign proliferative warts to malignant cervical cancer (reviewed in McMurray et al., Int. J. Exp. Pathol. 82 (1): 15-33 (2001)). Those HPV types marked as "high risk" include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68 and 59 types. (Chan et al., “Human Papillomavirus Infection and Cervical Cancer: Epidemiology, Screening, and Vaccination—Review of CurrentPerspectives,” Journal of Oncology, Volume 2019, Article ID 3257939, 2019.)

[0007] HPV is the main pathogen of cervical cancer (one of the most common types of cancer in women) and squamous cell carcinomas of the anus, tonsils, tongue, vulva, vagina and penis. HPV16 and HPV18 are well known to be the most virulent of the high-risk HPV types, as they cause approximately 70% of all invasive cervical cancers in the world.

[0008] Papillomaviruses are small (50-60 runs), non-enveloped icosahedral DNA viruses encoding up to 8 early genes (E1-E7) and 2 late genes (L1-L2). The L1 protein is the major capsid protein and has 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 results in the self-assembly of virus-like particles (VLPs) (for review, see Schiller and Roden, Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed., Leeds, UK: Leeds Medical Information, pp. 101-12 (1996)).

[0009] VLP is similar to real virus particle in morphology, and can induce high titer of neutralizing antibody when applied to animals or humans. Because VLP does not contain potential oncogenic viral genome, they provide a safe alternative to using live virus in HPV vaccine development (reviewed in Schiller and Hidesheim, J Clin.Virol.19:67-74 (2000)). Therefore, L1 and L2 genes have been confirmed as immune targets for the development of preventive and therapeutic vaccines for HPV infection and disease.

[0010] VLP vaccines have been shown to effectively induce immune responses in human subjects vaccinated with bivalent HPV 16 and 18 VLP vaccines (Harper et al., Lancet 364(9447):1757-65(2004)), quadrivalent HPV 6, 11, 16 and 18 VLP vaccines (Villa et al., Vaccine 24:5571-5583(2006)) and multivalent HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 VLP vaccines. Three commercially available VLP vaccines against HPV are administered according to a 2 or 3 dose schedule. (GlaxoSmithKline Biologicals, Rixensart, Belgium) is a bivalent vaccine that protects against HPV 16 and 18. and 9 (Merck & Co., Inc., Rahway, NJ, USA) protects against two additional HPV types and seven additional HPV types, respectively, and prevents additional HPV-related anogenital disease, including wart formation. compared to, 9 increased the protection against anogenital malignancies from about 70% to about 90%. (Ibid., M. 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).

[0011] Overview

[0012] The present disclosure provides a pharmaceutical composition comprising: virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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; a polymer comprising poly(lactide-co-glycolide); and a pharmaceutically acceptable carrier.

[0013] In one aspect, the present disclosure provides a pharmaceutical composition suitable for parenteral administration, comprising: (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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; (b) a polymer comprising poly(lactide-co-glycolide); and (c) a pharmaceutically acceptable carrier, wherein the weight content of the HVP vaccine component in the composition is in the range of 0.01% to 20%; the weight content of the polymer in the pharmaceutical composition is in the range of 80% to 99.09%; and wherein the polymer has an intrinsic viscosity of 0.4-0.9 dl / g and a molar ratio of lactide to glycolide of 65:35 to 90:10.

[0014] In another aspect, the present disclosure provides a kit comprising: (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82; poly(lactide-co-glycolide); and a pharmaceutically acceptable carrier; and (b) a delivery device.

[0015] In another aspect, the present disclosure provides a pulsatile drug delivery system comprising: a microneedle assembly comprising a plurality of microneedles filled with a composition comprising: virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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; poly(lactide-co-glycolide); and a pharmaceutically acceptable carrier, wherein the microneedle assembly is configured to release the VLPs in a predetermined amount of VLPs at a predetermined time while the microneedle assembly remains embedded in a patient.

[0016] The foregoing summary of the technology is not intended to be limiting and other features and advantages of the technology will be apparent from the following detailed description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A A schematic representation of a formulation of the invention comprising a core-shell polymer is depicted. Figure 1B A graphical representation of the formulation when released as a liquid and burst over time is depicted.

[0019] Figure 2A Core-shell microspheres of the present invention are shown. Figure 2B A subcutaneous implant of the present invention is shown. Figure 2C Core-shell lyospheres of the present invention are shown. Figure 2D Microparticles of the present invention are shown.

[0020] Figure 3 is a SEM image of ProCept spray dried material. See, e.g., Example 1.

[0021] Figure 4A is a graphical comparison of Buchi vs ProCept HPV VLP potency. Figure 4B is a graphical comparison of Buchi vs ProCept AAHS particle size. See, e.g., Example 1.

[0022] Figure 5 is a diagram of the scheme of the present invention. See, for example, Example 2.

[0023] Figure 6 is a graphical representation of the HPV efficacy of different formulations after microparticle encapsulation. See, e.g., Example 2.

[0024] Fig. 7A is a graphical representation of the ELISA titers of various preparations. Figure 7B is a graphical representation of the ELISA titers of various formulations 120 days after vaccination. See, e.g., Example 2.

[0025] Figure 8 is a graphical depiction of a scheme for forming microparticles of the invention. See, e.g., Example 2.

[0026] Fig. 9A is a graphical depiction of one embodiment of the present invention showing antibody response to VLP HPV 16 over 80 days. Fig. 9B is a graphical depiction of one embodiment of the invention showing antibody response over 80 days to VLP HPV 18. See, e.g., Example 2.

[0027] Fig.10 is a graphical depiction of the effectiveness of various embodiments of the present invention. See, e.g., Example 3.

[0028] Fig.11A is a graphical depiction of the effectiveness of the HPV minitabs that do not include AAHS. Fig. 11B is a graphical depiction of the efficacy of HPV minitabs including AAHS. See, e.g., Example 3.

[0029] Figures 12A-12B is a graphical depiction of the immunogenicity evaluation of various formulations of the invention. See, e.g., Example 3.

[0030] Fig.13 A graphical depiction of the relative effectiveness of various embodiments of the invention is depicted. See, e.g., Example 4.

[0031] Various aspects described herein are described in further detail in the following subsections.

[0032] Details

[0033] Currently, there are multiple approved HPV vaccines that consist of engineered virus-like particles (VLPs) and are highly effective in protecting vaccinated patients against precancerous lesions and anogenital cancers and genital warts when administered as a multiple-dose regimen prior to natural infection in subjects over the age of 9. The compositions of the present invention are intended to produce immunity to HPV subtypes comparable to the 2-3 injection regimens of such HPV vaccines (including approved bivalent, quadrivalent or nonavalent HPV vaccines) through a single administration regimen.

[0034] Despite improvements, HPV vaccination rates worldwide remain suboptimal. Global coverage of HPV vaccination rates can be improved by reducing the number of health care practitioner visits required for vaccination, increasing education about HPV disease prevention, and alleviating the social stigma associated with vaccination. The proportion of adolescents who complete a two-dose vaccination series in the Americas and Europe is estimated to be less than 50%. Therefore, it is desirable to improve HPV vaccination rates by generating an improved vaccine that can generate immunity to HPV with a single administration to provide an immune response comparable to existing HPV vaccines that require 2 or more doses.

[0035] It is desirable to provide a vaccine that can be delivered alone or in combination with a solution / suspension in a single doctor's visit to greatly improve the number of individuals who will be protected from HPV infection. Accordingly, the present disclosure relates to improved methods for delivering HPV vaccines to individuals in need thereof. In some embodiments, the present disclosure provides a method for stably binding HPV to aluminum hydroxyphosphate sulfate adjuvant ("AAHS") or not bound to AAHS. In another embodiment, the present invention provides a method for preparing a spray-dried HPV-AAHS formulation.

[0036] definition

[0037] Listed below are definitions of various terms used herein. These definitions apply to the terms as used throughout the specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well known and commonly used in the art.

[0039] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element. In addition, the use of the term "including" and other forms, such as "include", "includes", and "included" is not limiting.

[0040] All ranges disclosed herein are inclusive of the recited endpoints and are independently combinable (e.g., the range "50 mg to 500 mg" includes the endpoints 50 mg and 500 mg and all intermediate values). The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values; they are imprecise enough to include values ​​approximating these ranges and / or values.

[0041] As used herein, the term "comprising" includes the embodiments of "consisting of" and "consisting essentially of." As used herein, the terms "comprising," "including," "having," "having," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words that require the presence of specified ingredients / steps and allow for the presence of other ingredients / steps. However, such descriptions should be construed as also describing compositions or methods as "consisting of" and "consisting essentially of" the listed components, which allows for the presence of only the specified components or compounds, and any acceptable carriers or fluids, and excludes other components or compounds.

[0042] AAHS: As used herein, the term "AAHS" refers to amorphous aluminum hydroxyphosphate sulfate adjuvant.

[0043] About: As used herein, the term "about" when used in this article about numerical value, refers to the same as the numerical value mentioned or the value similar to the numerical value mentioned in the context. Generally speaking, those skilled in the art who are familiar with this context will recognize that "about" is included in this context. The absolute amount and / or relative degree of difference. As used herein, the term "about" in quantitative terms refers to the value of its modification plus or minus 10% (if the value is not subdivided, such as the number of molecules or nucleotides, then rounded to the nearest integer). For example, in some embodiments, the term "about" can be included in 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the value range.

[0044] Adjuvant: As used herein, the term "adjuvant" refers to a composition or compound that can enhance the immune response to the relevant antigen. An adjuvant is a substance or combination of substances used in conjunction with a vaccine antigen to enhance (e.g., increase, accelerate, prolong and / or possibly target) a specific immune response to a vaccine antigen or to adjust to a different type (e.g., converting a Th1 immune response to a Th2 response, or converting a humoral response to a cytotoxic T cell response) to enhance the clinical effectiveness of a vaccine. In some embodiments, the adjuvant modifies the (Th1 / Th2) immune response. In some embodiments, the adjuvant enhances the intensity and lifespan of the immune response. In some embodiments, the adjuvant broadens the immune response to the antigen administered concomitantly. In some embodiments, the adjuvant can induce strong antibodies and T cell responses. In some embodiments, the adjuvant can increase the polyclonal capacity of the induced antibodies. In some embodiments, the adjuvant is used to reduce the amount of antigen necessary to induce the desired immune response and provide protection against the disease. In some embodiments, the adjuvant is used to reduce the number of injections required to induce a lasting immune response and provide protection against the disease in a clinical regimen. The adjuvanted formulations described herein can exhibit enhancement of humoral and / or cellular immunogenicity of vaccine antigens (eg, subunit vaccine antigens).The adjuvants of the present invention are not used to deliver antigens, antibodies, active pharmaceutical ingredients (APIs) or VLPs.

[0045] Administration: As used herein, the term "administering" refers to the act of providing an active agent, composition or formulation to a subject. Exemplary routes of administration to humans can be through the eye (ocular), mouth (oral), skin (transdermal), nose (nasal), lung (inhalation), rectum, vagina, oral mucosa (buccal), ear, by injection (e.g., intravenous (IV), subcutaneous, intratumoral, intraperitoneal, intramuscular (IM), intradermal (ID), etc.), etc.

[0046] Agent: As used herein, the term "agent" refers to particles, compounds, molecules or entities of any chemical class, 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 "agent" may refer to a compound, molecule or entity comprising a polymer or multiple polymers.

[0047] Antibody: As used herein, the term "antibody" (or "Ab") refers to any form of an antibody that exhibits the desired biological or binding activity. It is therefore used in the broadest sense and specifically encompasses, 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.

[0048] Antigen: As used herein, the term "antigen" refers to any antigen that can generate one or more immune responses. An antigen can be a protein (including a recombinant protein), a VLP, a polypeptide, or a peptide (including a synthetic peptide). An antigen can be an antigen that generates a humoral and / or CTL immune response.

[0049] API: As used herein, the term "API" refers to an active pharmaceutical ingredient, such as HPV VLP, which is a component of a composition or formulation disclosed herein that has biological activity (e.g., is capable of inducing an appropriate immune response) and confers a therapeutic or preventive benefit to a human or animal in need thereof. As used herein, API is a vaccine active ingredient.

[0050] Biocompatible: As used herein, the term "biocompatible" refers to materials that are non-toxic to the body, pharmaceutically acceptable, and non-carcinogenic.

[0051] Biodegradable: As used herein, the term "biodegradable" refers to materials that should degrade by bodily processes into products that the body can easily handle and should not accumulate in the body. The products of biodegradation should also be biocompatible with the body.

[0052] Body: As used herein, "body" preferably refers to the human body, but it should be understood that body may also refer to non-human animal bodies.

[0053] Controlled release: As used herein, "controlled release" refers to the rate at which an active agent or other type of substance is released into the body over time.

[0054] Dose: As used herein, the term "dose" refers to the amount of an agent, API, formulation, or pharmaceutical composition administered or recommended for administration at a specific time.

[0055] HPV and PV: As used herein, the terms "HPV" and "PV" refer to human papillomavirus and papillomavirus, respectively.

[0056] Microparticles: As used herein, the term "microparticles" refers to particles containing an active agent or other material dispersed or dissolved within a polymer that serves as a matrix or binder for the particle.

[0057] Microspheres: As used herein, the term "microspheres" refers to spherical or spheroidal particles composed of a drug uniformly dissolved and / or dispersed throughout a polymer material, with a particle size of 1-500 μm and usually prepared as an injectable suspension.

[0058] Multiple doses: As used herein, the term "multiple doses" refers to a vaccine composition or pharmaceutical composition in which more than one dose of a component is required in a clinical setting to induce a durable immune response and provide protection against a disease. One skilled in the art understands how to determine a durable immune response, such as by measuring antibody titers over a specified period of time.

[0059] Patient: As used herein, the term "patient" refers to any human being who will receive the HPV vaccine or pharmaceutical composition described herein. As defined herein, "patient" includes patients who have been infected with one or more types of HPV and patients who are to be prevented from infection with one or more types of HPV.

[0060] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to excipients (vehicles, additives) and compositions that can reasonably be administered to a subject to provide an effective dose of the active ingredient used and are "generally regarded as safe" when administered to humans, e.g., physiologically tolerable and generally do not produce allergic or similar adverse reactions, such as gastric upset, etc. In another embodiment, the term refers to molecular entities and compositions approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, more particularly humans.

[0061] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition containing an active drug or biological ingredient and one or more additional components, such as a composition in which the active agent is formulated with one or more pharmaceutically acceptable carriers. As used herein, the terms "pharmaceutical preparation" and "preparation" can be used interchangeably with "pharmaceutical composition". In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen, which shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. The pharmaceutical composition or preparation can be liquid or solid (e.g., lyophilized). Additional components that may be included as appropriate include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, fillers, stabilizers, lyoprotectants, solubilizers, emulsifiers, salts, adjuvants, tension enhancers, 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 can be specially formulated for administration in solid or liquid form, including those suitable for the following: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (such as those targeted for buccal, sublingual and systemic absorption), boluses, powders, granules, pastes applied to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection, as, for example, sterile solutions or suspensions or sustained release formulations; topical administration, such as creams, ointments or controlled release patches or sprays applied to the skin, lungs or oral cavity; intravaginal or intrarectal, such as as pessaries, creams or foams; sublingual; ophthalmic; transdermal; or nasal, lung and other mucosal surfaces. In some embodiments, the term formulation refers to a single dose of the vaccine, which can be included in any volume suitable for injection.

[0062] Single dose: As used herein, the term "single dose" refers to a vaccine composition that requires only one administration or injection in a clinical setting to induce a durable immune response and provide protection against a disease. Those skilled in the art understand how to determine a durable immune response, such as by measuring antibody titers over a specified period of time.

[0063] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments a prenatal human form). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. 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 does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics representing a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnosis and / or therapy is administered and / or has been administered.

[0064] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to 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, such as the amount necessary to elicit an immune response, treat, cure, prevent, or inhibit the development and progression of a disease or its symptoms and / or the amount necessary to improve symptoms or cause regression of the disease. The therapeutically effective amount can vary depending on the structure and potency of the active ingredient and the intended mode of administration. A person skilled in the art can readily determine the therapeutically effective amount of a given antibody or therapeutic protein or vaccine antigen.

[0065] Vaccine: As used herein, the term "vaccine" or "vaccine composition" refers to a substance used to stimulate antibody production and provide immunity to one or more diseases, prepared from the pathogen of the disease, its products or synthetic substitutes, treated to act as an antigen without inducing the disease. The vaccine composition may include at least one antigen or VLP in a pharmaceutically acceptable vehicle that can be used to induce an immune response in a subject. The vaccine composition is administered by dosages and techniques known to those skilled in the pharmaceutical or veterinary arts, taking into account factors such as the age, sex, weight, species and condition of the recipient animal and the route of administration.

[0066] Valence: As used herein, the term "valence" refers to the presence of a specified number of antigens in a molecule. For example, the terms divalent, divalent, 2valent, or 2-valent refer to two different antigens. Similarly, the terms tetravalent, 4valent, or 4-valent refer to four different antigens, and the terms ninevalent, ninevalent, or nine-valent refer to nine different antigens.

[0067] Viscosity: As used herein, viscosity refers to a measure of the resistance of a substance to deformation or flow at a given rate. Viscosity can be measured, for example, by using a viscometer at a given shear rate appropriately selected by one skilled in the art to accurately measure viscosity within the viscosity range of the relevant sample. The viscosity of the materials used herein is measured at 20° C. at a standard concentration (e.g., 1% in 1% acetic acid) using a viscometer (e.g., Brookfield DVII+pro).

[0068] Virus-like particles: As used herein, the term "virus-like particle" or "VLP" refers to an agent that is morphologically similar to true virus particles or provides an array display of antigens and is capable of inducing high antibody neutralization ratings after administration in animals. VLPs lack the viral genetic material of true virus particles and are therefore non-infectious.

[0069] Volume % or % by volume: As used herein, the term "volume %" or "% by volume" refers to the volume fraction of a polymer carrier (e.g., microparticles, microspheres, or minitabs) per 100 total volumes. Unless otherwise specified, percentages (%) reported herein are by volume.

[0070] Weight % or % by weight: As used herein, the term "weight %" or "% by weight" refers to the weight parts per 100 parts of the total weight of the polymer carrier (e.g., microparticles, microspheres, or minitabs). For example, 10 weight % active agent means 10 parts by weight of active agent and 90 parts by weight of polymer.

[0071] particle

[0072] The present disclosure also relates to a novel approach to delivering HPV bound to and not bound to AAHS by temperature programmed loading of HPV to prepare monolithic PLGA microparticles. Figure 1A-1B. The present disclosure also relates to a method for preparing a sterile sustained-release formulation of a 9-valent human papillomavirus (HPV) vaccine with or without an adjuvant by temperature-programmed loading of poly(lactic-co-glycolic acid) (PLGA) microparticles for HPV not bound to AAHS. Encapsulation of biomacromolecules in their native state within a polymer matrix remains challenging because harsh processing conditions are typically involved in traditional microencapsulation techniques (e.g., solvent evaporation, coacervation, spray drying, and microfluidics). The methods described herein significantly minimize the effects of common factors that cause protein instability during microencapsulation, such as organic solvent exposure, excessive heating, or mechanical stress conditions. In the case of HPV bound to AAHS, integrated PLGA microparticles are prepared by encapsulating HPV / AAHS via a double emulsion method.

[0073] In some embodiments, the HPV vaccine is delivered via microparticles. Figure 2C and 2D The microparticles of the invention may include a polymer. In some embodiments, the polymer is a lactide-co-glycolide copolymer, also known as poly(lactide-co-glycolide) and often abbreviated as PLGA ("PLGA"). See Figure 5 and Figure 6 .

[0074] In some embodiments, PLGA is "uncapped poly(lactide-co-glycolide)", which refers to poly(lactide-co-glycolide) with carboxyl end groups ("uncapped PLGA"). In some embodiments, PLGA is "capped poly(lactide-co-glycolide)", which refers to poly(lactide-co-glycolide) with ester groups ("capped PLGA").

[0075] Two copolymers, i.e. two PLGAs, are end-capped PLGAs with a high intrinsic viscosity of 0.4-0.9 dl / g, preferably 0.45-0.8 dl / g, more preferably 0.45-0.55 dl / g, and end-capped PLGAs with a low intrinsic viscosity of 0.1-0.45 dl / g, preferably 0.1-0.3 dl / g, more preferably 0.2-0.3 dl / g. In some embodiments, the weight ratio of the end-capped PLGA with a high intrinsic viscosity to the end-capped PLGA with a low intrinsic viscosity is (100:0):(50:50). In some embodiments, the weight ratio of the end-capped PLGA with a high intrinsic viscosity to the end-capped PLGA with a low intrinsic viscosity is (50-95):(5-50). In some embodiments, the weight ratio of the end-capped PLGA with a high intrinsic viscosity to the end-capped PLGA with a low intrinsic viscosity is (70-90):(10-30), more preferably 80:20. In some embodiments, the weight ratio of the end-capped PLGA with high intrinsic viscosity to the end-capped PLGA with low intrinsic viscosity is (90:10)-(70:30), more preferably 80:20. In some embodiments, the molar ratio of lactide to glycolide in the end-capped PLGA with high intrinsic viscosity is in the range of 65:35 to 90:10, preferably 75:25; the molar ratio of lactide to glycolide in the end-capped PLGA with low intrinsic viscosity is in the range of 50:50 to 75:25, preferably 50:50. In some embodiments, the molar ratio of lactide to glycolide in the end-capped PLGA with high intrinsic viscosity is in the range of 10:90 to 90:10, preferably 50:50; the molar ratio of lactide to glycolide in the end-capped PLGA with low intrinsic viscosity is in the range of 50:50 to 75:25, preferably 50:50.

[0076] In some embodiments, two copolymers, i.e. two PLGAs, are uncapped PLGAs with a high intrinsic viscosity of 0.4-0.9 dl / g, preferably 0.45-0.8 dl / g, more preferably 0.45-0.55 dl / g, and uncapped PLGAs with a low intrinsic viscosity of 0.1-0.35 dl / g, preferably 0.1-0.3 dl / g, more preferably 0.2-0.3 dl / g. The weight ratio of the uncapped PLGA with a high intrinsic viscosity to the uncapped PLGA with a low intrinsic viscosity is (50-95):(5-50), preferably (70-90):(10-30), more preferably 80:20. The molar ratio of lactide to glycolide in the uncapped PLGA with high intrinsic viscosity is in the range of 65:35 to 90:10, preferably 75:25; the molar ratio of lactide to glycolide in the uncapped PLGA with low intrinsic viscosity is in the range of 50:50 to 75:25, preferably 50:50.

[0077] The intrinsic viscosity of PLGA was determined by preparing an approximately 0.5% (w / v) solution of PLGA in chloroform and measuring the intrinsic viscosity of PLGA using a Cannon-Fenske glass capillary viscometer at 30°C.

[0078] PLGA can also be a high molecular weight PLGA with a molecular weight of 20,000-145,000, preferably 30,000-100,000, more preferably 35,000-60,000 and a low molecular weight PLGA with a molecular weight of 4,000 to 45,000, preferably 4,000-35,000, more preferably 15,000-35,000. The weight ratio of high molecular weight PLGA to low molecular weight PLGA is (50-100):(0-50), preferably (70-90):(10-30), more preferably 80:20. The molar ratio of lactide to glycolide in the high molecular weight PLGA is in the range of 40:60 to 90:10, preferably 50:50; the molar ratio of lactide to glycolide in the low molecular weight PLGA is in the range of 50:50 to 75:25, preferably 50:50. As used herein, the term "molecular weight" refers to the "weight average molecular weight", abbreviated as "molecular weight".

[0079] For ease of description, hereinafter, the molar ratio of lactide to glycolide in PLGA and the intrinsic viscosity of PLGA are indicated in brackets after PLGA. For example, "PLGA (50 / 50, 0.5E)" refers to a lactide-glycolide copolymer having an intrinsic viscosity of 0.5 dl / g and carboxyl end groups, wherein the molar ratio of lactide to glycolide is 50:50.

[0080] In some embodiments, the weight ratio of the capped PLGA (50 / 50, 0.5E) with high intrinsic viscosity to the uncapped PLGA (50 / 50, 0.25A) with low intrinsic viscosity in the present invention is 50:50.

[0081] Specifically, in some embodiments of the microsphere composition of the present invention, the weight content of VLP is between 0.01-20% and the weight content of PLGA is 80-99.09%, the weight ratio of the two PLGAs is 80:20, the molecular weights of the two PLGAs are 55,000-85,000 and 15,000-35,000, the intrinsic viscosities of the two PLGAs are 0.45-0.55 dL / g and 0.2-0.3 dL / g, and the molar ratios of lactide to glycolide in the two PLGAs are 75:25 and 50:50, respectively.

[0082] In some embodiments, the composition includes a capped PLGA that is ester capped, has a 50:50 ratio and an intrinsic viscosity of 0.45-0.6 dL / g.

[0083] In some embodiments, 9vHPV vaccine / AAHS / PLGA microparticles are first prepared by conventional water-in-oil-in-water (w / o / w) double emulsion method to prepare blank AAHS / PLGA microparticles, separated, and sterilized by gamma irradiation. Subsequently, sterile HPV VLPs are rehydrated and aseptically loaded into blank microparticles at elevated temperatures to accelerate mass transfer. Each HPV serotype is loaded separately in a separate container and can be combined in a desired ratio to prepare sterile 9V HPV / AAHS / PLGA microparticles.

[0084] To prepare sterile 9-valent HPV-loaded PLGA microparticles, sterile HPV VLPs were aseptically added to blank microparticles and incubated at ambient temperature for 2 days, followed by incubation at 37°C for another 2 days, which accelerates mass transfer. After loading, the particles were centrifuged and washed to remove unencapsulated HPV. Each HPV serotype was loaded separately in a separate container and could be combined in a desired ratio to prepare sterile 9V HPV / AAHS / PLGA microparticles.

[0085] Microspheres

[0086] The present disclosure also relates to a method for preparing core-shell particles, wherein the core of the microsphere can be dried in situ or started as a solid intermediate (see Figure 1A ).

[0087] In some embodiments, a burst release formulation is provided. In some embodiments, the initial burst release is provided by the active ingredient present in the liquid formulation, and the secondary burst release is provided by the microparticles (see Figure 1B ). In some embodiments, a composition comprising an HPV vaccine and PLGA is provided. In some embodiments, the composition further comprises AAHS. In some embodiments, the composition comprises a PLGA core containing an HPV vaccine and a shell associated with the core, wherein the shell comprises PLGA.

[0088] Microspheres having a core-shell configuration are particularly useful for controlled release applications. These types of microspheres can be produced using a variety of methods, including atomization techniques using multiphase nozzles, microfluidic devices for preparing double emulsions, coating methods in which the core is coated with a coating solution using atomized liquid or vapor deposition, or layered precipitation methods. Microspheres having a core-shell configuration can be designed so that each microsphere contains a single core or so that each microsphere contains more than one core. Core-shell microspheres can be particularly useful for encapsulating materials that are sensitive to solvents or the local environment associated with the material comprising the shell. See Figure 2A .

[0089] In some embodiments, a pulsatile drug delivery system is provided, comprising a microneedle assembly comprising a plurality of microneedles filled with a therapeutic agent, the microneedles comprising a biodegradable polymer, the microneedle assembly being configured to release the therapeutic agent in a predetermined amount at a predetermined time while the microneedle assembly remains embedded in a patient's body.

[0090] Spray-dried HPV VLPs

[0091] U.S. Patent No. 10,772,947 describes how HPVVLPs combined with hydroxyaluminum phosphate sulfate (AAHS) can be lyophilized to maintain the stability of HPVVLPs and the AAHS particle size. Although this method is acceptable for drug products in vials, lyophilized powders do not provide the physical properties required for powder filling or direct compaction. Lyophilization is a sublimation process that requires frozen formulations and heated at a temperature lower than the collapse temperature of the product, followed by secondary drying at a temperature higher than the collapse temperature to obtain low-density powders. This can be converted into a batch process, but there are additional process steps of batch drying, pulverizing and transferring before use to cause significant process losses. In addition, additional blending steps are required to prepare free-flowing particles for powder filling or compaction.

[0092] Spray drying is a bulk evaporative process that requires aspiration of a solution or suspension and exposure to high temperatures to dry. The resulting powder has a higher density than a lyophilized powder and can be designed as a free-flowing powder with a particle size suitable for powder filling or compaction processes. However, the temperatures used in the spray drying process require temperatures well above the HPV VLP melting temperature range of 59-61°C. In addition, the evaporative drying of the aspirated droplets results in the concentration of AAHS as the droplets dry, and this concentration has been well documented to cause aggregation. The method described in the present application comprises method parameters and formulation components for producing a free-flowing powder of HPV VLPs bound to AAHS that stabilizes the potency of the VLPs and prevents AAHS aggregation.

[0093] In some embodiments, stable spray-dried HPV VLPs with AAHS are provided as powders to be filled into vials / syringes for vaccine drug products, or used as intermediates for spray-dried and compacted vaccine tablets. Figure 3 , Figure 4A and Figure 4B The method involves spray drying HPV VLPs bound to AAHS in the presence of a disaccharide (trehalose or sucrose) in the presence or absence of a tripeptide at a moisture content <6% using a 2-3 fluid nozzle.

[0094] Vaccine Minitabs

[0095] The present disclosure also relates to a new way to deliver HPV bound and unbound to the AAHS described below as a controlled release minitab. The present invention describes a method for preparing a delayed release vaccine minitab that can be administered alone or in combination with an immediate release vaccine to provide protection against bacterial and viral pathogens. The liquid injection provides primary immunity, while the implant provides delayed release at the injection site and acts as a booster after weeks to months in the body. This solves the challenge of individuals needing to schedule follow-up visits with medical staff. Specifically, a method for preparing a delayed release vaccine minitab containing a prophylactic agent has been developed, comprising a.) providing a dry vaccine formulation containing ≤6% moisture, b.) compacting the dry formulation to obtain minitabs, c.) coating the minitabs with a polymer coating composition; and d.) isolating the delayed release vaccine minitab.

[0096] In some embodiments, a method for preparing a vaccine minitab is provided. In some embodiments, the vaccine minitab is administered to provide controlled release of the vaccine to the intended recipient. In some embodiments, the vaccine minitab provides a controlled release vaccine that can be administered alone or in combination with an immediate-release vaccine to provide protection against bacterial and viral pathogens. This challenge of obtaining wider coverage can be partially addressed by administering the vaccine minitab simultaneously with a vaccine liquid injection. The liquid injection provides primary immunization, while the vaccine minitab provides controlled release at the injection site after weeks to months in the body and acts as a booster. This solves the challenge that individuals need to arrange follow-up visits with medical staff.

[0097] In some embodiments, minitablets may contain vaccines selected from subunits, conjugates and / or live viruses (LVVs) in adjuvanted and unadjuvanted forms (i.e., virus-like particles (VLPs) and aluminum hydroxyphosphate sulfate (AAHS)), within a range of sizes and coated with various polymers for injection or surgical implantation to delay or prolong the release of the antigen. Compaction of the material into tablets requires pressures in the range of 50 MPa to 400 MPa. These stresses that occur during compaction can damage proteins, and due to the large size of many vaccines, they are more susceptible to these types of stresses than smaller proteins or small molecule drugs. The formulation and binder combination are important for achieving adequate compressibility and compactability. Once compacted, the minitablets are coated by plunging the minitabs into a compatible antisolvent containing the selected polymer.

[0098] The minitabs of the present invention include human papillomavirus VLPs. The core tablet can be a multi-granular core: containing one or more pellet types designed to release the drug in the post-gastric portion of the gastrointestinal tract, wherein these pellet types provide approximately 1 to 95% of the total core tablet weight; or a mini-tab core: containing one or more mini-tabs, also designed to release the drug in the post-gastric gastrointestinal tract, wherein these mini-tabs provide approximately 1 to 95% of the total core tablet weight. The enteric polymer layer can be prepared by spraying many different types of enteric polymers on the above-mentioned core tablet. Therefore, the enteric polymer coating delays the disintegration of the core tablet until the core tablet reaches the intestine. In addition, the multi-granules or mini-tabs that constitute the core tablet can be formulated with additional delayed and / or modified release materials, thereby further delaying and / or modifying the release of the active agent released therefrom.

[0099] In one embodiment, the immediate release portion is an immediate release drug layer, which can be sprayed onto the enteric coated core tablet, thereby serving as the immediate release portion of the drug delivery system. In one embodiment of the invention, the immediate release drug layer completely embeds the enteric polymer coated core tablet. In one embodiment of the invention, the immediate release drug layer does not completely embed the enteric polymer coating of the core tablet; in this embodiment, a portion of the enteric polymer coating is exposed.

[0100] Vaccine implants

[0101] The present disclosure also relates to a method for preparing a delayed-release vaccine implant containing a prophylactic agent, which comprises the following methods: a.) providing a dry vaccine formulation containing ≤5% moisture; b.) mixing the dry vaccine formulation with a molten polymer; c.) preparing an implant by extruding a polymer vaccine matrix; d.) coating the implant with a polymer coating composition; e.) isolating the delayed-release vaccine implant, wherein the extrudate temperature is above the HPV VLP Tm without degrading the HPV VLP.

[0102] In some embodiments, a method for preparing a vaccine implant suitable for subcutaneous implantation is provided. See, e.g. Figure 2B . In some embodiments, vaccine implants are administered to provide delayed release of the vaccine to the intended recipient. In some embodiments, vaccine implants provide controlled release vaccines that can be administered alone or in combination with immediate release vaccines to provide protection against bacterial and viral pathogens. This challenge of obtaining wider coverage can be partially addressed by such implants that can be administered simultaneously with liquid injections. The liquid injection provides the primary immunization, while the implant provides delayed release at the injection site after weeks to months in the body and acts as a booster. This addresses the challenge of individuals needing to schedule follow-up visits with medical personnel.

[0103] VLP

[0104] As described above, the pharmaceutical compositions and formulations of the present invention comprise at least one HPV VLP type, such as HPV 16 or 18. In specific embodiments of the compositions disclosed herein, the vaccine further comprises VLPs of at least one additional HPV type. In further embodiments, the at least one additional HPV type is selected from the group consisting of: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 69, 70, 73, and 82. In some embodiments, the at least one additional HPV type comprises HPV 16 and 18. In some embodiments, the at least one additional HPV type comprises HPV 6, 11, 16, and 18. In some embodiments, the at least one additional HPV type comprises HPV 6, 18, 52, and 58. In some embodiments, the at least one additional HPV type comprises HPV 6, 11, 16, 18, 31, 45, 52, and 58. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 59. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 53, and 58. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 53, and 59. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 45, 52, 58, and 59. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.In some embodiments, the at least one additional HPV type includes HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 69 and 70. In some embodiments, the at least one additional 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. The pharmaceutical composition of the present invention comprises HPV VLPs composed of recombinant L1 or recombinant L1+L2 proteins of HPV. HPV L1 or L1+L2 proteins can be recombinantly expressed by cloning L1 or L1+L2 DNA molecules into expression vectors containing suitable promoters and other appropriate transcriptional regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant proteins. Techniques for these manipulations are fully described by Sambrook et al. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989)), which is incorporated herein by reference. When the L1 protein is recombinantly expressed in a host cell, VLPs can self-assemble.

[0105] The recombinant HPV L1 protein of the present invention can be any full-length L1 protein sequence found in nature or any mutant or truncated L1 protein that can self-assemble into VLP. In a specific embodiment of the present invention, the pharmaceutical composition and vaccine described herein comprise HPV VLPs composed of recombinant HPV L1 protein and do not contain HPV L2 protein. In certain embodiments, the vaccine composition or pharmaceutical composition described herein comprises HPV VLPs composed of full-length recombinant HPV L1 protein. In other embodiments, HPV VLPs consist of truncated HPV L1 proteins, such as L1 proteins truncated at the C-terminus. The L1 protein sequence used in the present invention can be determined by isolating DNA from one or more clinical samples containing the selected HPV type, determining the sequence of the HPV L1 DNA sequence, and translating the DNA sequence into an amino acid sequence using the genetic code. Many exemplary L1 sequences suitable for the present invention can be found in the literature. See, e.g., U.S. Pat. Nos. 5,820,870; 7,250,170; 7,276,243; 7,482,428; 7,976,848; 7,498,036; 7,700,103; 7,744,892; and 5,437,951; Kirii et al. (Virology 185(1):424-427(1991)). Other L1 proteins useful in the compositions and formulations of the present invention include biologically active fragments and / or mutants of the HPV L1 sequence, including but not necessarily limited to amino acid substitutions, deletions, additions, amino-terminal truncations, and carboxyl-terminal truncations, such that these mutations provide L1 proteins or protein fragments capable of forming VLPs. See, e.g., International Publication No. WO2006 / 114312 and U.S. Pat. No. 6,599,508. Suitable host cells for expressing recombinant HPV L1 or recombinant L1+L2 and subsequently self-assembling VLPs include but are not limited to yeast cells, insect cells, mammalian cells or bacteria. In an exemplary embodiment of the present invention, VLPs are produced in yeast cells, such as selected from the following yeasts: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis and Schizosaccharomyces pombe. In a specific embodiment, HPV VLPs are produced in Saccharomyces cerevisiae cells. The expression of HPV VLPs in yeast cells provides cost-effectiveness and is easily adapted to the advantages of large-scale growth in fermentation tanks.

[0106] The present invention also includes pharmaceutical compositions comprising mutant forms of HPV VLPs, such as HPV VLPs comprising biologically active fragments and / or mutants of HPV L1 or L2 proteins, including but not necessarily limited to amino acid substitutions, deletions, additions, amino-terminal truncations, and carboxyl-terminal truncations, such that these mutations provide proteins or protein fragments with therapeutic or prophylactic uses and can be used for HPV VLP vaccine development. Any such mutant form of the HPV L1 protein should be able to form VLPs and stimulate an immune response to the desired HPV type when administered to humans.

[0107] In addition, those skilled in the art will recognize that the HPV L1 or L1+L2 protein for self-assembly VLP to be included in the compositions disclosed herein can be encoded by full-length wild-type HPV L1 or L2 polynucleotides, or can be encoded by fragments or mutants of known wild-type sequences. Wild-type polynucleotide sequences encoding mRNA expressing HPV L1 or L2 protein are available in the art. Any mutant polynucleotide will encode a protein or protein fragment that at least substantially simulates the pharmacological properties of HPV L1 or L2 protein (including the ability to form VLPs that can stimulate an immune response to the relevant HPV type when administered to humans). Any such polynucleotide includes but is not necessarily limited to: nucleotide substitutions, deletions, additions, amino-terminal truncations, and carboxyl-terminal truncations.

[0108] The amount of virus-like particles of each HPV type included in the preparations and compositions of the present invention depends on the immunogenicity of the expressed gene product. In general, the therapeutically effective dose of the VLP of any of the at least one HPV type is about 1 μg to about 300 μg. In some embodiments, the therapeutically effective dose of the VLP of any of the at least one HPV type is about 1 μg to 200 μg. In some embodiments, the therapeutically effective dose of the VLP of any of the at least one HPV type is about 1 μg to 100 μg. In some embodiments, the therapeutically effective dose of the VLP of any of the at least one HPV type is about 10 μg to 200 μg. In some embodiments, the therapeutically effective dose of the VLP of any of the at least one HPV type is about 10 μg to 100 μg. In some embodiments, the therapeutically effective dose of the VLP of any of the at least one HPV type is about 10 μg to 80 μg. In some embodiments, the therapeutically effective dose of the VLP of any of the at least one HPV type is preferably about 20 μg to 60 μg.

[0109] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0110] 15-160 μg of HPV type 6 L1 protein VLPs,

[0111] 20-200 μg of HPV type 11 L1 protein VLPs,

[0112] 30-280 μg of HPV 16 L1 protein VLPs,

[0113] 20-200 μg of HPV 18 L1 protein VLPs,

[0114] 10-120 μg of HPV type 31 L1 protein VLPs,

[0115] 10-120 μg of HPV 33 L1 protein VLPs,

[0116] 10-120 μg of HPV 45 L1 protein VLPs,

[0117] 10-120 μg of HPV type 52 L1 protein VLPs, and

[0118] 10-120 μg of VLPs of HPV type 58 L1 protein.

[0119] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0120] 15-120 μg of HPV type 6 L1 protein VLPs,

[0121] 20-150 μg of HPV type 11 L1 protein VLPs,

[0122] 30-210 μg of HPV 16 L1 protein VLPs,

[0123] 20-150 μg of HPV 18 L1 protein VLPs,

[0124] 10-90 μg of HPV type 31 L1 protein VLPs,

[0125] 10-90 μg of HPV 33 L1 protein VLPs,

[0126] 10-90 μg of HPV 45 L1 protein VLPs,

[0127] 10-90 μg of VLPs of HPV type 52 L1 protein, and

[0128] 10-90 μg of VLPs of HPV type 58 L1 protein.

[0129] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0130] 15-80 μg of HPV type 6 L1 protein VLPs,

[0131] 20-100 μg of HPV type 11 L1 protein VLPs,

[0132] 30-140 μg of HPV 16 L1 protein VLPs,

[0133] 20-100 μg of HPV 18 L1 protein VLPs,

[0134] 10-60 μg of HPV type 31 L1 protein VLPs,

[0135] 10-60 μg of HPV 33 L1 protein VLPs,

[0136] 10-60 μg of HPV 45 L1 protein VLPs,

[0137] 10-60 μg of VLPs of HPV type 52 L1 protein, and

[0138] 10-60 μg of VLPs of HPV type 58 L1 protein.

[0139] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0140] 15-40 μg of HPV type 6 L1 protein VLPs,

[0141] 20-50 μg of HPV type 11 L1 protein VLPs,

[0142] 30-70 μg of HPV 16 L1 protein VLPs,

[0143] 20-50 μg of HPV 18 L1 protein VLPs,

[0144] 10-30 μg of HPV type 31 L1 protein VLPs,

[0145] 10-30 μg of HPV 33 L1 protein VLPs,

[0146] 10-30 μg of HPV 45 L1 protein VLPs,

[0147] 10-30 μg of VLPs of HPV type 52 L1 protein, and

[0148] 10-30 μg of VLPs of HPV type 58 L1 protein.

[0149] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0150] 90 μg of HPV type 6 L1 protein VLPs,

[0151] 120 μg of HPV type 11 L1 protein VLPs,

[0152] 180 μg of HPV type 16 L1 protein VLPs,

[0153] 120 μg of HPV type 18 L1 protein VLPs,

[0154] 60 μg of HPV type 31 L1 protein VLPs,

[0155] 60 μg of HPV type 33 L1 protein VLPs,

[0156] 60 μg of HPV 45 L1 protein VLPs,

[0157] 60 μg of HPV type 52 L1 protein VLPs, and

[0158] 60 μg of VLPs of HPV type 58 L1 protein.

[0159] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0160] 60 μg of HPV type 6 L1 protein VLP,

[0161] 80 μg of HPV type 11 L1 protein VLPs,

[0162] 120 μg of HPV type 16 L1 protein VLPs,

[0163] 80 μg of HPV type 18 L1 protein VLPs,

[0164] 40 μg of HPV type 31 L1 protein VLPs,

[0165] 40 μg of HPV type 33 L1 protein VLPs,

[0166] 40 μg of HPV 45 L1 protein VLPs,

[0167] 40 μg of HPV type 52 L1 protein VLPs, and

[0168] · 40 μg of VLPs of HPV type 58 L1 protein.

[0169] In some embodiments, a 0.5 mL dose of a composition or vaccine comprising VLPs of at least one HPV type comprises:

[0170] · 30 μg of HPV type 6 L1 protein VLP,

[0171] 40 μg of HPV type 11 L1 protein VLPs,

[0172] 60 μg of HPV type 16 L1 protein VLPs,

[0173] 40 μg of HPV type 18 L1 protein VLPs,

[0174] 20 μg of HPV type 31 L1 protein VLPs,

[0175] 20 μg of HPV type 33 L1 protein VLP,

[0176] 20 μg of HPV 45 L1 protein VLPs,

[0177] 20 μg of HPV type 52 L1 protein VLPs, and

[0178] · 20 μg of VLPs of HPV type 58 L1 protein.

[0179] Aluminum adjuvant

[0180] The aluminum adjuvant of the present invention can 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) (see Klein et al., Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)A1 MAS NMR., JPharm. Sci. 89(3):311-21 (2000)). In exemplary embodiments of the present invention provided herein, the aluminum adjuvant is aluminum hydroxyphosphate or AAHS. The ratio of phosphate to aluminum in the aluminum adjuvant may be 0 to 1.3. In embodiments of this aspect of the present invention, the ratio of phosphate to aluminum is in the range of 0.1 to 0.70. In some embodiments, the ratio of phosphate to aluminum is in the range of 0.2 to 0.50.

[0181] Those skilled in the art can determine the optimal dose of aluminum adjuvant, which is both safe and effective in increasing the immune response to the targeted HPV type. For a discussion of the safety status of aluminum and the amount of aluminum contained in FDA-licensed vaccines, see Baylor et al., Vaccine 20: S18-S23 (2002). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 3600 μg / dose (200 to 7200 μg / mL concentration). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 2700 μg / dose (200 to 5400 μg / mL concentration). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 1800 μg / dose (200 to 3600 μg / mL concentration). In some embodiments, the aluminum adjuvant is present in an amount of about 100 to 900 μg / dose (200 to 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, there is less than 400 μg of aluminum adjuvant per dose of vaccine. In some embodiments of the formulations and compositions of the invention, less than 300 μg of aluminum adjuvant is present per dose of vaccine. In some embodiments of the formulations and compositions of the invention, less than 200 μg of aluminum adjuvant is present per dose of vaccine. In some embodiments of the formulations and compositions of the invention, less than 100 μg of aluminum adjuvant is present per dose of vaccine.

[0182] Chitosan adjuvant

[0183] Chitin, a polymer of N-acetylglucosamine (i.e., (1-4)-2-acetamido-2-deoxy-β-d-glucan), is an important component of the body of all crustaceans and is also present in the exoskeletons and cell walls of fungi, insects and yeasts. Chitosan, i.e., α-(1-4)-2-amino-2-deoxy-β-d-glucan, is a form of the naturally occurring polysaccharide chitin in which most of the acetylation groups have been removed. Chitosan is usually formed by deacetylation of chitin in the presence of alkali. The term "chitosan" refers to a class of molecules whose degree of deacetylation is different from that of chitin. For example, molecules with a degree of deacetylation of less than 75% are considered chitin. In contrast, molecules with a degree of deacetylation of more than 75% are considered chitosan. The degree of deacetylation of chitosan powder can be measured by NMR, UV (EP method) or IR. The degree of deacetylation of chitosan in solution can be measured by CZE (capillary zone electrophoresis), GC-MS, ion chromatography and SEC-UV.

[0184] In some embodiments, chitosan adjuvant includes chitosan. In some embodiments, chitosan adjuvant includes chitosan with 75% or greater deacetylation. In some embodiments, chitosan adjuvant includes chitosan with a deacetylation degree in the range of about 75-99%. In some embodiments, chitosan adjuvant includes chitosan with a deacetylation degree in the range of about 85-99%. In some embodiments, chitosan adjuvant includes chitosan with a deacetylation degree greater than 90%. In some embodiments, chitosan adjuvant includes chitosan with a deacetylation degree of about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% chitosan. The degree of deacetylation can be calculated according to any of the methods described herein.

[0185] In some embodiments, chitosan adjuvants include chitosan in the form of water-soluble chitosan with a degree of deacetylation in the range of about 75-99%. In some embodiments, chitosan adjuvants include water-soluble chitosan with a degree of deacetylation in the range of about 75-99%. In some embodiments, chitosan adjuvants include water-soluble chitosan with a degree of deacetylation in the range of about 85-99%. In some embodiments, chitosan adjuvants include water-soluble chitosan with a degree of deacetylation greater than 90%. In some embodiments, chitosan adjuvants include water-soluble chitosan with a degree of deacetylation of about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% water-soluble chitosan. In some embodiments, the water-soluble chitosan is chitosan hydrochloride.The degree of deacetylation can be calculated according to any of the methods described herein.

[0186] In some embodiments, chitosan adjuvant includes acid-soluble chitosan with a degree of deacetylation in the range of about 75-99%. In some embodiments, acid-soluble chitosan is chitosan hydrochloride. In some embodiments, chitosan adjuvant includes acid-soluble chitosan with a degree of deacetylation in the range of about 85-99%. In some embodiments, chitosan adjuvant includes acid-soluble chitosan with a degree of deacetylation greater than 90%. In some embodiments, chitosan adjuvant includes acid-soluble chitosan with a degree of deacetylation of about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. Deacetylation can be calculated according to any of the above methods.

[0187] In some embodiments, the chitosan adjuvant includes chitosan having a viscosity in the range of about 1cP to about 200cP when measured with a viscometer (e.g., Brookfield DVII+pro) at 20°C at a standard concentration (e.g., 1% in 1% acetic acid). In some embodiments, the viscosity of chitosan is in the range of about 1cP to about 100cP. In some embodiments, the viscosity of chitosan is in the range of about 5cP to about 100cP. In some embodiments, the viscosity of chitosan is in the range of about 1cP to about 50cP. In some embodiments, the viscosity of chitosan is in the range of about 5cP to about 50cP. In some embodiments, the viscosity of chitosan is in the range of about 1cP to about 25cP. In some embodiments, the viscosity of chitosan is in the range of about 5cP to about 25cP. In some embodiments, the viscosity of chitosan is in the range of about 1cP to about 20cP. In some embodiments, the viscosity of chitosan is in the range of about 5 cP to about 20 cP. In some embodiments, the viscosity of chitosan is in the range of about 1 cP to about 15 cP. In some embodiments, the viscosity of chitosan is in the range of about 5 cP to about 15 cP. In some embodiments, the viscosity of chitosan is in the range of about 1 cP to about 10 cP. In some embodiments, the viscosity of chitosan is in the range of about 5 cP to about 10 cP. In some embodiments, the viscosity of chitosan is less than 100. In some embodiments, the viscosity of chitosan is less than 75. In some embodiments, the viscosity of chitosan is less than 50. In some embodiments, the viscosity of chitosan is less than 40. In some embodiments, the viscosity of chitosan is less than 30. In some embodiments, the viscosity of chitosan is less than 20. In some embodiments, the viscosity of chitosan is less than 15. In some embodiments, the viscosity of chitosan is less than 10. In some embodiments, the viscosity of chitosan is less than 5. In some embodiments, the viscosity of chitosan is 1 cP, 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25 cP, 26 cP, 27 cP, 28 cP, 29 cP, 30 cP, 31 cP, 32 cP, 33 cP, 34 cP, 36 cP, 37 cP, 38 cP, 39 cP, 40 cP, 41 cP, 42 cP, 43 cP, 5cP, 26cP, 27cP, 28cP, 29cP, 30cP, 31cP, 32cP, 33cP, 34cP, 35cP, 36cP, 37cP, 38cP, 39cP, 40cP, 41cP, 42cP, 43cP, 44cP, 45cP, 46cP, 47cP, 48cP, 49cP or 50cP.

[0188] In some embodiments, the chitosan adjuvant includes a water-soluble chitosan having a viscosity in the range of about 1cP to about 200cP. In some embodiments, the water-soluble chitosan includes chitosan hydrochloride having a viscosity in the range of about 1cP to about 200cP when measured with a viscometer (e.g., Brookfield DVII+pro) at 20°C at a standard concentration (e.g., 1% in 1% acetic acid). In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 1cP to about 100cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 5cP to about 100cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 1cP to about 50cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 5cP to about 50cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 1cP to about 25cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 5cP to about 25cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 1cP to about 20cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 5cP to about 20cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 1cP to about 15cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 5cP to about 15cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 1cP to about 10cP. In some embodiments, the viscosity of chitosan hydrochloride is in the range of about 5cP to about 10cP. In some embodiments, the viscosity of chitosan hydrochloride is less than 100. In some embodiments, the viscosity of chitosan hydrochloride is less than 75. In some embodiments, the viscosity of chitosan hydrochloride is less than 50. In some embodiments, the viscosity of chitosan hydrochloride is less than 40. In some embodiments, the viscosity of chitosan hydrochloride is less than 30. In some embodiments, the viscosity of chitosan hydrochloride is less than 20. In some embodiments, the viscosity of chitosan hydrochloride is less than 15. In some embodiments, the viscosity of chitosan hydrochloride is less than 10. In some embodiments, the viscosity of chitosan hydrochloride is less than 5.In some embodiments, the viscosity of chitosan hydrochloride is 1 cP, 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25cP, 26cP, 27cP, 28cP, 29cP, 30cP, 31cP, 32cP, 33cP, 34cP, 35cP, 36cP, 37cP, 38cP, 39cP, 40cP, 41cP, 42cP, 43cP, 44cP, 45cP, 46cP, 47cP, 48cP, 49cP or 50cP.

[0189] In some embodiments, the chitosan adjuvant includes an acid-soluble chitosan having a viscosity in the range of about 1 cP to about 200 cP when measured with a viscometer (e.g., Brookfield DVII+pro) at a standard concentration (e.g., 1% in 1% acetic acid) at 20°C. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 1 cP to about 100 cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 5 cP to about 100 cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 1 cP to about 50 cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 5 cP to about 50 cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 1 cP to about 25 cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 5 cP to about 25 cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 1cP to about 20cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 5cP to about 20cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 1cP to about 15cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 5cP to about 15cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 1cP to about 10cP. In some embodiments, the viscosity of the acid-soluble chitosan is in the range of about 5cP to about 10cP. In some embodiments, the viscosity of the acid-soluble chitosan is less than 100. In some embodiments, the viscosity of the acid-soluble chitosan is less than 75. In some embodiments, the viscosity of the acid-soluble chitosan is less than 50. In some embodiments, the viscosity of the acid-soluble chitosan is less than 40. In some embodiments, the viscosity of the acid-soluble chitosan is less than 30. In some embodiments, the viscosity of the acid-soluble chitosan is less than 20. In some embodiments, the viscosity of the acid-soluble chitosan is less than 15. In some embodiments, the viscosity of the acid-soluble chitosan is less than 10. In some embodiments, the viscosity of the acid-soluble chitosan is less than 5.In some embodiments, the viscosity of the acid-soluble chitosan is 1 cP, 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25cP, 26cP, 27cP, 28cP, 29cP, 30cP, 31cP, 32cP, 33cP, 34cP, 35cP, 36cP, 37cP, 38cP, 39cP, 40cP, 41cP, 42cP, 43cP, 44cP, 45cP, 46cP, 47cP, 48cP, 49cP or 50cP.

[0190] In some embodiments, chitosan adjuvants include water-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity less than 50cP. In some embodiments, water-soluble chitosan includes chitosan hydrochloride with a degree of deacetylation greater than 75% and a viscosity less than 50cP. In some embodiments, chitosan adjuvants include chitosan hydrochloride with a degree of deacetylation greater than 75% and a viscosity less than 40cP. In some embodiments, chitosan hydrochloride has a degree of deacetylation greater than 75% and a viscosity less than 30cP. In some embodiments, chitosan hydrochloride has a degree of deacetylation greater than 75% and a viscosity less than 20cP. In some embodiments, chitosan hydrochloride has a degree of deacetylation greater than 75% and a viscosity less than 10cP. In some embodiments, chitosan hydrochloride has a degree of deacetylation greater than 75% and a viscosity less than 5cP. In some embodiments, chitosan hydrochloride has a degree of deacetylation greater than 85% and a viscosity less than 50cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 85% and a viscosity less than 40cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 85% and a viscosity less than 30cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 85% and a viscosity less than 20cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 85% and a viscosity less than 10cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 85% and a viscosity less than 5cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 90% and a viscosity less than 50cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 90% and a viscosity less than 40cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 90% and a viscosity less than 30cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 90% and a viscosity less than 20cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 90% and a viscosity less than 10cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 90% and a viscosity less than 5cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 95% and a viscosity less than 50cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 95% and a viscosity less than 40cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 95% and a viscosity less than 30cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 95% and a viscosity less than 20cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 95% and a viscosity less than 10cP. In some embodiments, chitosan hydrochloride has a deacetylation degree greater than 95% and a viscosity less than 5cP.

[0191] In some embodiments, the chitosan adjuvant includes an acid-soluble chitosan having a deacetylation degree greater than 75% and a viscosity less than 50 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 75% and a viscosity less than 40 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 75% and a viscosity less than 30 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 75% and a viscosity less than 20 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 75% and a viscosity less than 10 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 75% and a viscosity less than 5 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 85% and a viscosity less than 50 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 85% and a viscosity less than 40 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 85% and a viscosity less than 30 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 85% and a viscosity less than 20 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 85% and a viscosity less than 10 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 85% and a viscosity less than 5 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 90% and a viscosity less than 50 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 90% and a viscosity less than 40 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 90% and a viscosity less than 30 cP. In some embodiments, the acid-soluble chitosan has a degree of deacetylation greater than 90% and a viscosity less than 20 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 90% and a viscosity less than 10 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 90% and a viscosity less than 5 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 95% and a viscosity less than 50 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 95% and a viscosity less than 40 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 95% and a viscosity less than 30 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 95% and a viscosity less than 20 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 95% and a viscosity less than 10 cP. In some embodiments, the acid-soluble chitosan has a deacetylation degree greater than 95% and a viscosity less than 5 cP.

[0192] In some embodiments, the chitosan adjuvant includes chitosan in an amount of about 0.1 μg to about 200 mg. In some embodiments, the chitosan adjuvant includes chitosan in an amount of about 0.1 μg to about 100 mg. In some embodiments, the chitosan adjuvant includes chitosan in an amount of about 0.1 μg to about 50 mg. In some embodiments, the chitosan adjuvant includes chitosan in an amount of about 0.1 μg to about 25 mg. In some embodiments, the chitosan adjuvant includes chitosan in an amount of about 0.1 μg to about 20 mg. In some embodiments, the chitosan adjuvant includes chitosan in an amount of about 0.1 μg to about 100 mg. In some embodiments, the chitosan adjuvant includes less than 100 mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 90 mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 80 mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 70 mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 60mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 50mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 40mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 30mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 20mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 10mg of chitosan. In some embodiments, the chitosan adjuvant includes less than 5mg of chitosan. In some embodiments, the chitosan is water-soluble chitosan. In some embodiments, the chitosan is acid-soluble chitosan. In some embodiments, the chitosan is chitosan hydrochloride.

[0193] In some embodiments, the chitosan adjuvant includes a buffer. In some embodiments, the buffer is selected from any pharmaceutically acceptable buffer, including acetic acid, histidine, citrate, Bis-Tris, HEPES, phosphate, MES and combinations thereof. In some embodiments, the buffer is present in an amount of 1 mMol to about 100 mMol of the chitosan adjuvant.

[0194] In some embodiments, chitosan adjuvants include water-soluble chitosan and buffers. In some embodiments, chitosan adjuvants include chitosan hydrochloride and buffers. In some embodiments, chitosan adjuvants include buffers and water-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include buffers and chitosan hydrochloride with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include chitosan hydrochloride and histidine buffers. In some embodiments, chitosan adjuvants include buffers and chitosan hydrochloride with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include acetic acid buffers and chitosan hydrochloride with a degree of deacetylation greater than 85% and a viscosity of 1cp to 200cp. In some embodiments, the chitosan adjuvant comprises an acetic acid buffer and chitosan hydrochloride having a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, the chitosan adjuvant comprises an acetic acid buffer and chitosan hydrochloride having a degree of deacetylation greater than 85% and a viscosity of 5cp to 100cp. In some embodiments, the chitosan adjuvant comprises a sodium chloride buffer and chitosan hydrochloride having a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, the chitosan adjuvant comprises a sodium chloride buffer and chitosan hydrochloride having a degree of deacetylation greater than 85% and a viscosity of 5cp to 100cp.

[0195] In some embodiments, chitosan adjuvants include acid-soluble chitosan and buffers. In some embodiments, chitosan adjuvants include buffers and acid-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include acetic acid buffers and acid-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include acetic acid buffers and acid-soluble chitosan with a degree of deacetylation greater than 85% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include acetic acid buffers and acid-soluble chitosan with a degree of deacetylation greater than 85% and a viscosity of 5cp to 100cp. In some embodiments, chitosan adjuvants include acid-soluble chitosan and histidine buffers. In some embodiments, chitosan adjuvants include buffers and acid-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, the chitosan adjuvant comprises a sodium chloride buffer and an acid-soluble chitosan having a degree of deacetylation greater than 75% and a viscosity of 1 cp to 200 cp. In some embodiments, the chitosan adjuvant comprises a sodium chloride buffer and an acid-soluble chitosan having a degree of deacetylation greater than 85% and a viscosity of 5 cp to 100 cp.

[0196] In some embodiments, the chitosan adjuvant includes a tension regulator. In some embodiments, the tension regulator is selected from any pharmaceutically acceptable tension regulator, such as sodium chloride, potassium chloride, sucrose, trehalose and combinations thereof. In some embodiments, the tension regulator is present in an amount of 10mM to 500mM.

[0197] In some embodiments, chitosan adjuvants include water-soluble chitosan and tension regulators. In some embodiments, chitosan adjuvants include acid-soluble chitosan and tension regulators. In some embodiments, chitosan adjuvants include chitosan hydrochloride and tension regulators. In some embodiments, chitosan adjuvants include tension regulators and water-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include tension regulators and acid-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvants include sodium chloride and water-soluble chitosan with a degree of deacetylation greater than 85% and a viscosity of 5cp to 100cp. In some embodiments, chitosan adjuvants include sodium chloride and acid-soluble chitosan with a degree of deacetylation greater than 85% and a viscosity of 5cp to 100cp.

[0198] In some embodiments, the chitosan adjuvant includes a detergent. In some embodiments, the detergent is selected from any pharmaceutically acceptable detergent, such as polysorbate 80, polysorbate 20, poloxamer 188, and combinations thereof. In some embodiments, the detergent is present in an amount of 0.001 to 0.2% (w / v).

[0199] In some embodiments, chitosan adjuvant includes water-soluble chitosan and detergent. In some embodiments, chitosan adjuvant includes acid-soluble chitosan and detergent. In some embodiments, chitosan adjuvant includes chitosan hydrochloride and detergent. In some embodiments, chitosan adjuvant includes detergent and water-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp. In some embodiments, chitosan adjuvant includes detergent and acid-soluble chitosan with a degree of deacetylation greater than 75% and a viscosity of 1cp to 200cp.

[0200] HPV VLP vaccines

[0201] Any HPV VLP type vaccine is suitable for the pharmaceutical composition and method of the present invention. Known HPV VLP vaccines can be modified to include aluminum adjuvants and PLGA. New vaccines can be developed according to the invention described herein, which include at least one HPV type, optionally in the form of HPV VLPs adsorbed to aluminum adjuvants, and PLGA. In addition, new vaccines can be developed according to the invention described herein, which include at least one HPV type in the form of HPV VLPs adsorbed to aluminum adjuvants, and PLGA.

[0202] An exemplary HPV vaccine is a bivalent vaccine that protects against HPV 16 and 18, which is commercially known as (GlaxoSmithKline Biologicals, Rixensart, Belgium). Another exemplary HPV VLP vaccine is a non-infectious, recombinant tetravalent vaccine prepared from highly purified VLPs of the major capsid (L1) proteins of HPV types 6, 11, 16, and 18 and may be referred to herein by its proprietary name (Merck & Co., Inc., Rahway, NJ, USA) mentioned, see Bryan, JT Vaccine 25 (16): 3001-6 (2007); Shi et al., Clinical Pharmacology and Therapeutics 81 (2): 259-64 (2007). Another exemplary HPV VLP vaccine is the nine-valent vaccine commercially available for the prevention of HPV (which includes capsid (L1) proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58), which is referred to herein by its proprietary name 9 (Merck & Co., Inc., Rahway, NJ, USA) mentioned.

[0203] In some embodiments, the vaccine dose also includes aluminum adjuvant (as amorphous hydroxyaluminum sulfate phosphate), sodium chloride, L-histidine, polysorbate 80, sodium borate and water in addition to VLP. In some embodiments, the HPV vaccine includes 100-3500 μg aluminum adjuvant, 1-50 mg sodium chloride, 0.05-10 mg L-histidine, 1-100 μg polysorbate, 1-100 μg sodium borate and water. In some embodiments, the HPV vaccine includes about 500 μg aluminum adjuvant, about 9.56 mg sodium chloride, about 0.78 mg L-histidine, about 50 μg polysorbate 80, about 35 μg sodium borate and water for injection. Known HPV VLP vaccines can be modified according to the present invention to include aluminum adjuvant and PLGA.

[0204] In some embodiments of the present invention, pharmaceutical compositions and formulations comprise monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, hexavalent, octavalent or 9-valent HPV VLP-type vaccines or HPV VLPs as described herein. In specific embodiments, pharmaceutical compositions and formulations are 9-valent. In some embodiments, pharmaceutical compositions comprise HPV VLP-type vaccines or HPV VLPs as described herein having more than four different types of HPV VLPs. For example, pharmaceutical compositions and formulations of the present invention may include 8-valent, 9-valent, 10-valent, etc. HPV VLP-type vaccines or HPV VLPs as described herein. For example, pharmaceutical compositions comprising VLPs of HPV 16 and / or HPV 18 but not other HPV VLP types are included within the scope of the present invention. Pharmaceutical compositions comprising VLPs of HPV 16 and / or HPV 18 and / or HPV 18 are also contemplated herein. or 9. A multivalent vaccine comprising HPV VLPs of different HPV types.

[0205] In some embodiments, VLPs of HPV types 6 and 11 are included. In some embodiments, VLPs of HPV types 16, 31, and 35 are included. In some embodiments, VLPs of HPV types 18, 45, and 59 are included. In some embodiments, VLPs of HPV types 26, 51, and 69 are included. In some embodiments, VLPs of HPV types 33, 52, and 58 are included. In some embodiments, VLPs of HPV types 39, 68, and 70 are included. In some embodiments, VLPs of HPV types 53, 56, and 66 are included.

[0206] In some embodiments, VLPs of HPV types 16 and 18 are included. In some embodiments, VLPs of HPV types 6, 11, 16, and 18 are included. In some embodiments, VLPs of HPV types 6, 18, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 45, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 33, 45, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 59 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 53, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 53, and 59 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 35, 45, 52, and 58 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58, and 59 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 45, 52, 58, 59, and 68 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 45, 51, 52, 58, 59, and 69 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, 69, and 70 are included. In some embodiments, VLPs of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, and 70 are included.

[0207] In some embodiments, pharmaceutical compositions and formulations include HPV VLP-based vaccines and / or antigens as listed in Table 1 below:

[0208] Table 1:

[0209]

[0210]

[0211] In some embodiments, the compositions of the invention are administered as a single injection providing multiple doses of the composition.

[0212] In some embodiments, the composition comprises a buffer. In some embodiments, the composition comprises a buffer, wherein the buffer is selected from the group consisting of: acetic acid, histidine, citrate, Bis-Tris, HEPES, phosphate, MES, sodium chloride, and combinations thereof.

[0213] In some embodiments, the composition includes a salt.

[0214] In some embodiments, the composition includes a tonicity adjuster. In some embodiments, the tonicity adjuster is selected from the group consisting of sodium chloride, potassium chloride, sucrose, trehalose, and combinations thereof.

[0215] In some embodiments, the composition includes a detergent. In some embodiments, the detergent is selected from polysorbate 80, polysorbate 20, poloxamer 188, and combinations thereof.

[0216] Injectable composition

[0217] The preparations of the present invention are suitable for parenteral administration, for example, by intraarticular (in a joint), intravenous or intraperitoneal injection, preferably by intramuscular, intradermal or subcutaneous injection, including aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents and solutes that make the preparation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. The preparations can be presented in unit dose or multi-dose sealed containers such as ampoules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Cells transduced by RNA molecules can also be administered intravenously or parenterally.

[0218] The composition of the present invention is suitable for injection into a host body by needle. In one embodiment, the composition comprises microparticles suspended in an aqueous injection vehicle. The microparticles preferably have a mass median diameter in the range of at least about 10 μm to about 250 μm, preferably about 20 μm to about 150 μm. However, it should be understood that the present invention is not limited to microparticles within this size range, and smaller or larger microparticles may also be used.

[0219] The controlled release composition of the present invention can provide the following advantages: 1) it provides immediate release after entering the body without a lag period of drug release at high or low drug loading, 2) it is conducive to large-scale production (scale above 75L) during the production process, and 3) it is highly stable, so the in vivo release behavior remains unchanged after long-term storage.

[0220] Kit of the present invention

[0221] Also provided herein are kits comprising any of the pharmaceutical compositions of the vaccines described above and instructions for use.

[0222] Also provided herein are kits comprising a pharmaceutical composition comprising HPV VLPs of at least one type of HPV.

[0223] In some embodiments of the kit, the pharmaceutical composition of (a) comprises HPV VLPs of at least one type of human papillomavirus (HPV) selected from 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 of (a) is an HPV vaccine. In some embodiments, the HPV vaccine is a recombinant human papillomavirus bivalent (type 16 and type 18) vaccine. In some embodiments, the HPV vaccine is In some embodiments, the HPV vaccine is a recombinant human papillomavirus quadrivalent (types 6, 11, 16, 18) vaccine. In some embodiments, the HPV vaccine is In some embodiments, the HPV vaccine is a recombinant papillomavirus 9-valent vaccine. In some embodiments, the HPV vaccine is 9.

[0224] In some embodiments, the kit includes a buffer.In some embodiments, the kit includes a tonicity adjuster.In some embodiments, the kit includes a detergent.

[0225] In some embodiments of the kit, the kit includes a label or package insert including a description of the components and / or instructions for in vivo use of the components therein. In some embodiments, the kit includes instructions for administering (or inoculating) the pharmaceutical composition or HPV vaccine. In some embodiments, the kit includes instructions for mixing the pharmaceutical composition or HPV vaccine and then administering (or inoculating) the mixture to a patient.

[0226] Therapeutic methods of the present invention

[0227] 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 a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82.

[0228] Also provided herein is a method for inducing an immune response to human papillomavirus (HPV) in a human patient, comprising administering a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82.

[0229] 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 a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82.

[0230] Also provided herein is a method for preventing a human patient from being infected with a human papillomavirus (HPV), comprising administering to the patient a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82.

[0231] Also provided herein is a method for delivering a pharmaceutical composition to a host to induce neutralization titers to HPV antigens in the host, comprising administering to the host a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82, whereby administration of the pharmaceutical composition induces neutralization titers to HPV antigens in the host, and wherein a single dose of the pharmaceutical composition provides enhanced or comparable neutralization titers compared to multiple doses of the same pharmaceutical composition.

[0232] Also provided herein is a method for preventing cancer in a human patient caused by human papillomavirus (HPV) types 16, 18, 31, 33, 45, 52 and 58, comprising administering to the patient a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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.

[0233] Also provided herein is a method for preventing cancer in a human patient caused by HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58, comprising administering to the patient a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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 lesion or dysplastic lesion of the cervix, vulva, vagina and anus.

[0234] Also provided herein is a method for preventing cancer in a human patient caused by HPV types 6 and 11, comprising administering to the patient a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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 acuminatum.

[0235] Also provided herein is a method of preventing precancerous or dysplastic lesions in a human patient caused by HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73, and 82, comprising administering to the patient a polymer (e.g., poly(lactide-co-glycolide)) and an agent 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. , 55, 56, 58, 59, 66, 68, 73 and 82, wherein the lesions are selected from cervical intraepithelial neoplasia (CIN) grade 2 / 3, cervical adenocarcinoma in situ (AIS), cervical intraepithelial neoplasia (CIN) grade 1, vulvar intraepithelial neoplasia (VIN) grade 2 and grade 3, vaginal intraepithelial neoplasia (VaIN) grade 2 and grade 3, anal intraepithelial neoplasia (AIN) grade 1, 2 and grade 3 (1.1).

[0236] Also provided herein is a method for preventing HPV-related anogenital diseases in human patients caused by an HPV type selected from 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 the patient a pharmaceutical composition comprising a polymer (e.g., poly(lactide-co-glycolide)) and virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82.

[0237] Embodiments of the invention also include one or more pharmaceutical compositions described herein, which are (i) used for the following uses, (ii) used as a medicament or composition for the following uses, or (iii) used to prepare a medicament for the following uses: (a) treatment (e.g., treatment of humans); (b) pharmaceutical products; (c) inducing an immune response to the HPV types included in the vaccine; (d) reducing the likelihood of HPV infection in a patient; (e) preventing infection with the HPV types in the vaccine, (f) preventing or reducing the likelihood of cervical cancer, (g) preventing or reducing the likelihood of vulvar cancer, (h) preventing or reducing the likelihood of vaginal cancer, (i) preventing or reducing the likelihood of anal cancer, (j) preventing or reducing the likelihood of oropharyngeal cancer, (k) preventing or Reduce the likelihood of other head and neck cancers, (k) Prevent or reduce the likelihood of anal precancerous or dysplastic lesions, (l) Prevent or reduce the likelihood of genital warts or condyloma acuminatum, (m) Prevent or reduce the likelihood of cervical intraepithelial neoplasia (CIN) grade 2 / 3 lesions, (n) Prevent or reduce the likelihood of cervical adenocarcinoma in situ (AIS) lesions, (o) Prevent or reduce the likelihood of cervical intraepithelial neoplasia (CIN) grade 1 lesions, (p) Prevent or reduce the likelihood of vulvar intraepithelial neoplasia (VIN) grades 2 and 3 lesions, (q) Prevent or reduce the likelihood of vaginal intraepithelial neoplasia (VaIN) grades 2 and 3 lesions, (r) Prevent or reduce the likelihood of anal intraepithelial neoplasia (AIN) grades 1, 2, and 3 lesions.

[0238] In embodiment 1, a pharmaceutical composition is provided, comprising:

[0239] Virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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;

[0240] A polymer comprising poly(lactide-co-glycolide); and

[0241] A pharmaceutically acceptable carrier.

[0242] In embodiment 2, there is provided a pharmaceutical composition of embodiment 1, wherein the VLP is present in an amount of 0.01-20% by weight of the composition.

[0243] In embodiment 3, there is provided a pharmaceutical composition of embodiment 1, wherein the polymer is present in an amount of 80-99.09% by weight of the composition.

[0244] In embodiment 4, a pharmaceutical composition suitable for parenteral administration is provided, comprising:

[0245] (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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;

[0246] (b) a polymer comprising poly(lactide-co-glycolide); and

[0247] (c) a pharmaceutically acceptable carrier,

[0248] wherein the weight content of the HVP vaccine component in the composition is in the range of 0.01% to 20%; the weight content of the polymer in the pharmaceutical composition is in the range of 80% to 99.09%; and

[0249] The polymer has an intrinsic viscosity of 0.4-0.9 dl / g and a molar ratio of lactide to glycolide of 65:35 to 90:10.

[0250] In embodiment 5, there is provided a pharmaceutical composition according to any one of embodiments 1-4, wherein the poly(lactide-co-glycolide) is an ester-terminated poly(lactide-co-glycolide) having a molar ratio of lactide to glycolide of 50:50.

[0251] In embodiment 6, there is provided a pharmaceutical composition according to any one of embodiments 1-5, wherein the HPV VLPs of each of the at least one HPV type are present at a concentration of about 10 μg to about 300 μg per 0.5 mL of the pharmaceutical composition.

[0252] In embodiment 7, a pharmaceutical composition according to any one of embodiments 1-6 is provided, wherein the total VLP concentration is 10 μg to 2000 μg / 0.5 mL of the pharmaceutical composition.

[0253] In embodiment 8, there is provided a pharmaceutical composition according to any one of embodiments 1-7, wherein the total poly(lactide-co-glycolide) concentration is from 0.1 μg to about 200 mg per 0.5 mL of the pharmaceutical composition.

[0254] In embodiment 9, there is provided a pharmaceutical composition according to any one of embodiments 1-8, further comprising an adjuvant.

[0255] In embodiment 10, there is provided a pharmaceutical composition according to any one of embodiments 1-9, further comprising about 100 μg to about 3500 μg of an aluminum adjuvant.

[0256] In embodiment 11, there is provided a pharmaceutical composition of embodiment 10, wherein the HPV VLPs are adsorbed onto the aluminum adjuvant.

[0257] In embodiment 12, a composition of any one of embodiments 1-11 is provided, wherein each HPV VLP is present at a concentration of about 10 μg to about 300 μg / 0.5 mL of the pharmaceutical composition; wherein the total HPV VLP concentration is 10 μg to 2000 μg / 0.5 mL of the pharmaceutical composition; and wherein the polymer has an intrinsic viscosity of 0.4-0.9 dl / g, a molar ratio of lactide to glycolide of 65:35 to 90:10.

[0258] In embodiment 13, a pharmaceutical composition according to any one of embodiments 1-2 is provided, wherein the HPV VLP comprises HPV L1 protein and does not comprise HPV L2 protein.

[0259] In embodiment 14, a pharmaceutical composition according to any one of embodiments 1-13 is provided, wherein said VLP is associated with said poly(lactide-co-glycolide).

[0260] In embodiment 15, a pharmaceutical composition according to any one of embodiments 1-13 is provided, wherein said VLP is encapsulated in said poly(lactide-co-glycolide).

[0261] In embodiment 16, a pharmaceutical composition according to any one of embodiments 1-13 is provided, wherein said VLP is embedded in said poly(lactide-co-glycolide).

[0262] In embodiment 17, there is provided a pharmaceutical composition according to any one of embodiments 1-16, wherein the composition is in the form of microparticles.

[0263] In embodiment 18, a pharmaceutical composition according to any one of embodiments 1-16 is provided, wherein the composition is in the form of microspheres.

[0264] In embodiment 19, a pharmaceutical composition according to any one of embodiments 1-16 is provided, wherein the composition is in the form of a minitab.

[0265] In embodiment 20, a pharmaceutical composition according to any one of embodiments 1-19 is provided, wherein the composition is administered as a single injection providing multiple doses of the VLPs over a period of time.

[0266] In embodiment 21, a method of preventing a human patient from being infected by human papillomavirus (HPV) is provided, comprising administering to the patient the pharmaceutical composition of any one of embodiments 1-20.

[0267] In embodiment 22, a method of delivering a pharmaceutical composition to a host to induce neutralization titers to HPV antigens in the host is provided, comprising: administering the pharmaceutical composition of any one of embodiments 1-20 to the host.

[0268] In embodiment 23, a method of forming a biodegradable implant in situ in a patient in need thereof is provided, comprising the step of injecting the composition of any one of embodiments 1-19 into the patient.

[0269] In embodiment 24, a kit is provided, comprising:

[0270] (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82; poly(lactide-co-glycolide); and a pharmaceutically acceptable carrier; and

[0271] (b) Delivery device.

[0272] In embodiment 25, the kit of embodiment 24 is provided, further comprising instructions for administering the HPV vaccine and poly(lactide-co-glycolide) to a human patient.

[0273] In embodiment 26, a pulsatile drug delivery system is provided, comprising:

[0274] A microneedle assembly comprising a plurality of microneedles filled with a composition comprising:

[0275] (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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;

[0276] (b) poly(lactide-co-glycolide); and

[0277] (c) a pharmaceutically acceptable carrier,

[0278] Wherein the microneedle assembly is configured to release the VLPs in a predetermined amount of VLPs at a predetermined time while the microneedle assembly remains embedded in a patient's body.

[0279] In embodiment 27, the delivery system of embodiment 26 is provided, wherein the PLGA degrades over time to release the VLP into the patient. Example

[0280] The following examples are intended to be illustrative and should not be construed as further limiting.The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0281] Example 1: Spray Drying of HPV / AAHS

[0282] A formulation consisting of 0.54 mg / mL HPV VLPs, 8.7 mg / mL AAHS, 9.5% trehalose, 0.5% trileucine, 75 mM NaCl, 30 mM histidine, 0.01% PS-80 at pH 6.2 was spray dried using two spray dryers, ProCept and Buchi B-290. The parameters are listed in Table 2 below.

[0283] Table 2: Spray dryer conditions for ProCept and Buchi processes

[0284]

[0285]

[0286] The temperatures used in the process included an inlet temperature of 143C for the ProCept unit and 102C for the Buchi unit, well above the melting temperature of VLPs, 59-61C. The spray-dried material was transferred to glass vials, stoppered and stabilized at 37C for 0, 2 and 4 weeks. The samples were analyzed for potency using a Biacore 3000 and for AAHS particle size by static light scattering. The analysis showed that AAHS particle size and HPV potency were stable for up to 4 weeks at 37C. The moisture content was 4.6% and 5.7% for the Buchi and ProCept units, respectively. Figure 3 Depicted is an SEM image of the ProCept spray-dried material. Figure 4A and 4B Characterization of spray-dried material at 0, 2, and 4 weeks at 37°C in a sealed container is depicted. Specifically, Figure 4A Shown is the potency by Biacore analysis compared to a reference standard. Figure 4B The particle size of AAHS by static light scattering is depicted.

[0287] Example 2: HPV temperature programmed loading into PLGA microparticles

[0288] There are three FDA-approved preventive HPV vaccines: Cervarix TM , and 9. The latest HPV vaccination recommendation from the Advisory Committee on Immunization Practices (ACIP) remains a 2-dose or 3-dose regimen to obtain adequate protection against new HPV infections. However, immunization noncompliance occurs when people fail to complete the dose series, and this remains a public health problem. To improve vaccine coverage and adherence to the immunization schedule, a novel vaccine that can be administered during a single doctor's visit with currently marketed products has been developed. 9. A long-acting HPV preparation with controlled release of HPV administered in combination.

[0289] Successful encapsulation of HPV virus-like particles (VLPs, -20,000 kDa) was achieved with blank PLGA microparticles containing the adjuvant aluminum hydroxyphosphate sulfate (AAHS, also known as AAHS) by active loading of HPV VLPs.

[0290] like Figure 5 As shown in the schematic diagram of, microparticles are prepared as follows. First, AAHS concentrated with 4% trehalose is prepared. 10mL of AAHS stock solution in FAP buffer (500mMNaCl, 20mM histidine, 0.02 wt %PS80, pH 6.2) is transferred to a 15mL falcon tube, 0.87mL of 50% trehalose solution is added and mixed. The internal aqueous phase is prepared by centrifuging the AAHS solution containing trehalose at 1500rpm for 15 minutes at 15°C and removing all supernatants. 0.2mL of AAHS aqueous phase is added to 1mL of polymer oil phase, and then homogenized for 60 seconds at 8krpm with an IKA distributor (T25 Easy Clean Digital, equipped with S25N-8G dispersion tool). 2mL of 5% PVA (9-10kDa, 80% hydrolysis) is added to the obtained primary w / o emulsion and vortexed for 60 seconds at 2750rpm. Finally, the final w / o / w emulsion was added to 100 mL of 0.5% PVA solution and the microparticles were hardened for a minimum of 3 hours under rapid stirring. The microparticles were collected and graded by passing through 106um, 53um and 20um in sequence and rinsed repeatedly with DI water to remove residual PVA. The particles were collected and freeze-dried and then gamma irradiated at ~19-20 kGy for sterilization.

[0291] To prepare sterile blank AAHS / PLGA microparticles, 38-54 kDa PLGA ( RG 504, ester-capped, L:G ratio 50:50) was dissolved in 1 mL of dichloromethane to achieve a concentration of 300 mg / mL. Figure 6As shown in, all formulations were prepared with 100% 38-54 kDa PLGA and the following specifications as given in Table 3. Formulation A was homogenized at 8000 rpm for 60 seconds and vortexed at 3000 rpm for 60 seconds. Formulation B was homogenized at 5000 rpm for 45 seconds and vortexed at 1500 rpm for 30 seconds. Formulation C was homogenized at 6000 rpm for 45 seconds and vortexed at 1500 rpm for 35 seconds. Formulation D was homogenized at 15000 rpm for 60 seconds and vortexed at 3000 rpm for 60 seconds.

[0292] Table 3

[0293]

[0294] As shown in Figure 7, the potency of HPV 33 after encapsulation was largely retained (>80%). In addition, active loading achieved through the strong binding affinity of HPV to AAHS in blank PLGA microparticles resulted in high encapsulation efficiency (>85%). Compared to traditional encapsulation techniques, this provides advantages such as reducing the costs associated with using high-value, high-cost APIs in the loading process.

[0295] Sterile 9V HPV / AAHS / PLGA microparticles Figure 5 The formulation was prepared as shown in and described in detail above. The formulation was diluted with a buffer containing carboxymethylcellulose, sodium chloride, histidine, and polysorbate 80 to achieve different dosage levels. Fig. 7A The formulation was tested in a BALB / c mouse model with 8 mice per group using a single dose of microparticles ranging from 0.18 μg to 15 μg. To assess immunogenicity, serum samples from individual animals were evaluated for antibody levels to HPV types 16 and 18 using a multiplex assay. VLP-specific HPV antibody concentrations were determined on study days 14, 28, 42, 56, 70, 84, 120. Titers to HPV VLP-16 and HPV VLP-18 are shown in Figure 7B The HPV16 / 18 released from the formulation was immunogenic and successfully elicited HPV16 / 18 titers in a dose-dependent manner.

[0296] When used in combination with the adjuvant AAHS, the stability of HPV by emulsification is significantly enhanced. Figure 8 The w / o / w double emulsion method shown in Figure 1 was successfully encapsulated in PLGA microparticles. G9 / PLGA microparticles and 9V HPV / AAHS / PLGA microparticles prepared by the programmed temperature loading method showed non-inferior performance when used in combination with G9 compared to the baseline G9 prime and boost (day 28) in the rabbit model.

[0297] Example 3: Delayed Release Minitab

[0298] Minitabs according to the present invention are prepared by compaction. The tablet may contain a vaccine selected from subunits, conjugates and / or live viruses (LVV) in adjuvanted and non-adjuvanted forms (i.e., virus-like particles (VLPs) and aluminum hydroxyphosphate sulfate (AAHS)), which are within a certain size range and coated with various polymers for injection or surgical implantation to delay or prolong the release of antigens. The material is compacted into tablets requiring a pressure in the range of 50MPa to 400MPa. These stresses occurring during the compaction process can damage proteins, and due to the large size of many vaccines, they are more susceptible to these types of stresses than smaller proteins or small molecule drugs. The formulation and binder combination are important for achieving sufficient compressibility and compactability. Once compacted, the minitablet is coated by placing the minitabs into a compatible antisolvent containing the selected polymer.

[0299] In this example, a 9-valent HPV / aluminum adjuvanted vaccine (hereinafter referred to as "9vHPV vaccine") comprising capsid (L1) proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 was tested (the 9v vaccine further included sodium chloride, L-histidine, and polysorbate 80, and also contained aluminum (provided as AAHS) where indicated). The 9-valent HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 were diluted into a buffer having a final concentration of 5.0 wt% sucrose, 4.5 wt% trehalose, 3.0 wt% HPMC, 10 mM histidine, 75 mM NaCl, 0.01 v / v% PS80 with and without approximately 2 wt% AAHS. In addition, a lyophilized formulation without HPV or AAHS adjuvant was prepared as above as a placebo formulation. The three formulations (HPV-free AAHS, HPV+AAHS, and placebo) were dispensed into 2r vials, 0.5 mL per vial, half-seated with Igloo stoppers, and the vials were then placed on a lyophilizer shelf and lyophilized. HPV+AAHS was prepared as shown in Table 4 below.

[0300] Table 4

[0301] Liquid concentration (mg / mL) quality% 9vHPV 0.135 0.100 AAHS 2.18 1.63 sucrose 50.0 37.4 Trehalose 45.2 33.9 NaCl 4.38 3.28 Histidine 1.56 1.16 HPMC 30.0 22.5 PS80 0.100 0.070 total 133 100

[0302] At the end of the cycle, the vials were stoppered and removed from the lyophilizer. The lyophilized HPV from multiple vials were combined, ground through a sieve, blended with a binder (lactose, mannitol), and then lubricated with magnesium stearate to a final composition of 1% lyophilized formulation, 30% mannitol, 59% lactose, 1% magnesium stearate. The material was compacted to a compaction pressure of approximately 125 MPa using a 2 mm diameter round punch with a curvature. The minitabs were coated by immersing and stirring the minitabs in a 20% acetone solution of 85% / 15% PLGA. The coating thickness can be adjusted by repeating the coating process. The resulting minitabs are approximately 2 x 2 mm. Fig.10 As shown in , the efficacy of all 9 HPV types after coating was similar for all types in minitabs containing AAHS and minitabs without AAHS. In addition, it was found that in the formulation that did not include AAHS (see Fig.11A ) and preparations including AAHS (see Fig. 11B ), all 9 HPV types were stable at 4C over 6 months.

[0303] Mouse immunogenicity studies were designed to evaluate the efficacy of these formulations compared to a benchmark 9VHPV mouse dose. As summarized in Table 5 below, 12 different formulations were tested. Fig. 12A and 12B As shown in the immunogenicity assessment in , intramuscular ELISA titers of 9vHPV and 9vHPV+AAHS provided significant improvements over placebo. In contrast, subcutaneous 9vHPV+AAHS performed better than placebo and subcutaneous 9vHPV without AAHS.

[0304] Table 5

[0305]

[0306] IM = intramuscular; NA = not applicable; SC = subcutaneous

[0307] Example 4: Delayed Release Vaccine Implants Comprising VLPs in the Presence and Absence of Adjuvant

[0308] The method includes preparing a dry vaccine intermediate to <5% moisture using freeze drying, spray drying, spray freeze drying or microwave vacuum drying. The dry vaccine intermediate is then sieved or ground (if necessary) into a free-flowing powder. The powder is added to a polymer or polymer blend and extruded via a twin-screw extruder at a controlled rate and temperature.

[0309] In this example, HPV33 VLPs were lyophilized in the presence of 6.3% sucrose, 6.3% trehalose, 6.3% HPMC, 113mM NaCl, 12mM histidine and 0.012% PS80 and dried to moisture <5%. The lyophilized intermediate was crushed into a powder. The extrusion conditions are listed in Table 6 below. A total of 0.6 mg was used to start the extruder and confirm the appropriate parameters (1:1 HPV33 intermediate to PEG3350 ratio) and discarded. Another 2g HPV33 intermediate + 2g PEG3350 was extruded. See Figure 9.

[0310] Table 6: Extruder parameters

[0311] 7.5mm extruder: FDC-059w / 2mm die head

[0312] Motor rpm 125

[0313] Maximum torque 40%

[0314] Feed rate 30%

[0315] Zone 1 65℃

[0316] Zone 2 75°C

[0317] Zone 3 85℃

[0318] Zone 4 Outage

[0319] The extrudates were cut into segments for analysis. Evaluation of protein concentration, HPV33 particle size and potency. The extrudates were weighed and reconstituted in 1 mL 0.5 M NaCl, 20 mM histidine, 0.02% PS-80, pH 6.2.

[0320] For the two reconstituted extruded fragments, the protein concentrations were 495 and 330 ug / mL, respectively. The particle size of HPV33 was also measured after dilution with 0.5 M NaCl, 20 mM histidine, 0.02% PS-80, pH 6.2, and reconstituted to reduce the PEG concentration in the sample. The measurements were performed on a Malvern Nanosizer instrument. Controls of HPV33 alone and HPV33 spiked with the same PEG level as the sample were also measured.

[0321] Compared to the HPV 33 reference standard, the potency measured by SPR analysis was 110% and 89%, respectively. Fig.13 .

[0322] Table 7: Particle size of reconstituted extruded products measured by DLS

[0323] Sample Name Particle Size (d.nm)

[0324] HPV alone 60.97

[0325] HPV 72.55 doped with PEG

[0326] Extruded sample 1 71.51

[0327] Extruded sample 2 67.57

[0328] The disclosed subject matter is not limited in scope by the specific embodiments and examples described herein. In fact, various modifications of the present disclosure, other than those described, will be apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0329] All references (e.g., publications or patents or patent applications) cited herein are hereby incorporated by reference in their entirety and for all purposes, just as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

1. A pharmaceutical composition comprising: Virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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; A polymer comprising poly(lactide-co-glycolide); and A pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the VLP is present in an amount of 0.01-20% by weight of the composition.

3. The pharmaceutical composition of claim 1, wherein the polymer is present in an amount of 80-99.09% by weight of the composition.

4. A pharmaceutical composition suitable for parenteral administration, comprising: (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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; (b) a polymer comprising poly(lactide-co-glycolide); and (c) a pharmaceutically acceptable carrier, wherein the weight content of the VLP in the composition is in the range of 0.01% to 20%; the weight content of the polymer in the pharmaceutical composition is in the range of 80% to 99.09%; and The polymer has an intrinsic viscosity of 0.4-0.9 dl / g and a molar ratio of lactide to glycolide of 65:35 to 90:

10.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the poly(lactide-co-glycolide) is an ester-terminated poly(lactide-co-glycolide) having a molar ratio of lactide to glycolide of 50:

50.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the HPV VLPs of each of the at least one HPV type are present in a concentration of about 10 μg to about 300 μg / 0.5 mL of the pharmaceutical composition.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the total VLP concentration is 10 μg to 2000 μg / 0.5 mL of the pharmaceutical composition.

8. The pharmaceutical composition of any one of claims 1-7, wherein the total poly(lactide-co-glycolide) concentration is 0.1 μg to about 200 mg / 0.5 mL of the pharmaceutical composition.

9. The pharmaceutical composition according to any one of claims 1 to 8, further comprising an adjuvant.

10. The pharmaceutical composition according to any one of claims 1 to 9, further comprising about 100 μg to about 3500 μg of an aluminum adjuvant.

11. The pharmaceutical composition according to claim 10, wherein the HPV VLPs are adsorbed onto the aluminum adjuvant.

12. A composition according to any one of claims 1-11, wherein each HPV VLP is present at a concentration of about 10 μg to about 300 μg / 0.5 mL of the pharmaceutical composition; wherein the total HPV VLP concentration is 10 μg to 2000 μg / 0.5 mL of the pharmaceutical composition; and wherein the polymer has an intrinsic viscosity of 0.4-0.9 dl / g, a molar ratio of lactide to glycolide of 65:35 to 90:

10.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the HPV VLP comprises HPV L1 protein and does not comprise HPV L2 protein.

14. The pharmaceutical composition of any one of claims 1-13, wherein the VLP is associated with the poly(lactide-co-glycolide).

15. The pharmaceutical composition of any one of claims 1-13, wherein the VLP is encapsulated in the poly(lactide-co-glycolide).

16. The pharmaceutical composition of any one of claims 1-13, wherein the VLP is embedded in the poly(lactide-co-glycolide).

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the composition is in the form of microparticles.

18. The pharmaceutical composition according to any one of claims 1 to 16, wherein the composition is in the form of microspheres.

19. The pharmaceutical composition of any one of claims 1-18, wherein the composition is administered as a single injection providing multiple doses of VLPs over a period of time.

20. A method for preventing a human patient from being infected by human papillomavirus (HPV), comprising administering to the patient a pharmaceutical composition according to any one of claims 1 to 20.

21. A method of delivering a pharmaceutical composition to a host to induce neutralization titers to HPV antigens in the host, comprising: Administering the pharmaceutical composition according to any one of claims 1 to 19 to a host.

22. A method of forming a biodegradable implant in situ in a patient in need thereof, comprising the step of injecting a composition according to any one of claims 1 to 18 into the patient.

23. A kit comprising: (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 73 and 82; poly(lactide-co-glycolide); and a pharmaceutically acceptable carrier; and (b) Delivery device.

24. The kit of claim 23, further comprising instructions for administering the HPV vaccine and poly(lactide-co-glycolide) to a human patient.

25. A pulsatile drug delivery system comprising: A microneedle assembly comprising a plurality of microneedles filled with a composition comprising: (a) virus-like particles (VLPs) of at least one type of human papillomavirus (HPV) selected from 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; (b) poly(lactide-co-glycolide) ("PLGA"); and (c) a pharmaceutically acceptable carrier, Wherein the microneedle assembly is configured to release the VLPs in a predetermined amount of VLPs at a predetermined time while the microneedle assembly remains embedded in a patient's body.

26. The delivery system of claim 25, wherein the PLGA degrades over time to release the VLPs into the patient.

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