WO2024102417A2 - Bivalent virus-like particle compositions and methods of use - Google Patents

Bivalent virus-like particle compositions and methods of use Download PDF

Info

Publication number
WO2024102417A2
WO2024102417A2 PCT/US2023/037041 US2023037041W WO2024102417A2 WO 2024102417 A2 WO2024102417 A2 WO 2024102417A2 US 2023037041 W US2023037041 W US 2023037041W WO 2024102417 A2 WO2024102417 A2 WO 2024102417A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
hpv
vlp
amino acid
vaccine composition
Prior art date
Application number
PCT/US2023/037041
Other languages
French (fr)
Inventor
Joshua Weiyuan WANG
Original Assignee
Pathovax Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pathovax Llc filed Critical Pathovax Llc
Publication of WO2024102417A2 publication Critical patent/WO2024102417A2/en

Links

Definitions

  • This invention relates to virus-like particle (VLP)-based bivalent vaccine compositions for therapeutic and prophylactic uses.
  • VLP virus-like particle
  • HPV human papillomaviruses
  • VLP viruslike particles
  • Gardasil®9 human papillomavirus 9-valent vaccine, recombinant; GlaxoSmithKline
  • Gardasil®9 human papillomavirus 9-valent vaccine, recombinant; GlaxoSmithKline
  • the extent of protection against these specific HPV types from clinical trials has shown between 98-100% efficacy.
  • important questions remain such as extent of vaccine cross protection against all HPV oncogenic types especially those not covered by Garasil-9, the extent of HPV type replacement and the potential role caused by HPV latent infections.
  • Recent evidence has suggested that HPV type replacement is a possibility although the subject still remains highly controversial. In two studies by Gray et al ( Int. J.
  • Solid organ and bone marrow transplants are life-saving modalities. With improvements in immune-suppression and surgical techniques, post-transplant survival has improved and the focus has shifted to improving quality of life after transplantation(Moloney et al Br J Dermatol. 2005).
  • OTRs organ transplant recipients
  • the subdominance of the L2 immunogen could be improved upon displaying regions of the L2 immunogen on the surface of virus-like particles or other carrier proteins (See, Roden et al., Papillomavirus-like particles (VLP) as broad spectrum human papillomavirus (HPV) vaccines, US9149503B2, US10046026B2; Embers et al. (2002), J. Virol. 76, 9798-805; Gaukroger et al. (1996), J. Gen. Virol. 77 (Pt 7), 1577-83, Kawana et al. (1999), J. Virol. 73, 6188-90; Pastrana et al. (2005a) Virology 337, 365-72; Roden et al. (2000) Virology 270, 254-257; (Gambhira et al. (2007a) J. Virol. 81, 11585-92).
  • the present invention provides cost-effective, simple vaccine formulations that address the inadequacies of the currently available HPV vaccines, and which can be administered in fewer doses than the current HPV vaccine solutions.
  • the present invention relates to a virus-like particle (VLP)-based bivalent vaccine composition, which includes:
  • a human papilloma virus 16 (HPV 16) LI protein into which is inserted, into a DE loop of the HPV 16 LI protein, a surface-displayed HPV 16 L2 peptide, wherein the surface-displayed HPV 16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 12); and
  • HPV18 human papilloma virus 18 LI protein into which is inserted, into a DE loop of the HPV 18 LI protein, a synthetic surface-displayed HPV L2 peptide
  • VLP-based bivalent vaccine compositions of the invention can be provided in a kit, e.g., for convenient clinical use.
  • Another embodiment of the invention is a method for immunizing or vaccinating a subject against a HPV, which involves administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
  • Still another embodiment of the invention is a method for inducing an immune response against HPV in a subject, involving administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
  • an inventive method for treating a HPV infection in a subject having a HPV infection, or at risk of being exposed to HPV comprises administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
  • the invention includes a method for preventing HPV-associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer, in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
  • Figure 1 demonstrates comparable HPV18 neutralization titers among all tested Candidates 4, 6, 7, 9 and 10 (see, Table 2 and Example 2, herein), as well as Gardasil® or Cervarix®. These results show that the insertion of additional epitopes into the DE loop region of HPV18 LI protein did not compromise the other HPV18 type-specific epitopes.
  • Figure 2 shows an ELISA assay using a concatenated protein called “RGlx22” to evaluate the cross-antibody reactivity generated by the HPV18 chimeric VLP candidates (Candidate 4, 6, 7, 9 and 10; see, Table 2 and Example 2, herein) following vaccination.
  • HPV16 RG1VLP A positive control known to react with the RG1X22 peptide was included: HPV16 RG1VLP. Negative controls were wild type HPV 18 VLP with no peptide inserted into the DE-loop, and Cervarix®, both of which contain no L2 peptide inserts in the DE-loops of their VLPs.
  • FIG 3 shows results from ELISA assays using a concatenated protein called “RGlx22” to evaluate the cross-antibody reactivity generated by HPV16RG1-VLP (“RGVax prototype”) co-formulated with either HPV18 chimeric VLP Candidate 4, 9 or 10, following vaccination.
  • Vaccinated mouse sera from Cervarix® which contains no L2 peptides in the DE-loops of their VLPs was used as a negative control.
  • Figures 4A-C show neutralization assay results comparing sera from mice vaccinated with either 2 or 3 doses of Cervarix® or HPV16RG1-VLP, co-formulated with HPV18 chimeric VLP Candidate 10 (“RGVax prototype + #10”). No detectable difference was observed in HPV16 LI titers ( Figure 4A), 18 LI titers ( Figure 4B), and L2 reactive antibody response (Figure 4C) between a 2-dose and 3-dose vaccination schedule, offering an advantage of fewer doses with the inventive vaccine composition, compared to previously known vaccine formulations.
  • Figure 5 show the results of neutralization assays against multiple HPV types using sera from either a 2-dose or 3-dose vaccination of HPV16 RG1 VLP, co-formulated with either HPV18-Beta 1-VLP, HPV18-Beta 2 VLP or Cervarix®. Pooled sera from each group of 5 mice were tested (triplicate studies).
  • FIG. 6 shows a schematic alignment illustrating how Beta 1 and Beta 2 epitopes were designed.
  • Both Beta 1 and Beta 2 epitopes contain a bio-informatically designed oligopeptide insert corresponding, respectively, to the amino acid residue positions 14-34 or positions 17-36 region of minor capsid protein L2 from the major seven HPV types 1, 4, 5, 8, 23, 38 and 76, well known to be associated with cutaneous HPV disease as well as cutaneous cancers; such pathogenic HPV types are commonly called “HPV beta types.”
  • the Beta 3 epitope is designed by overlapping Beta 1 and Beta 2.
  • Figure 6 shows, in the hash- bordered box, the alignment at amino acid residue positions 14-38 (in descending order): “HPV1-L2 SEQ1” (DIYPSCKISNTCPPDIQNKKIEHTTF/SEQ ID NO:28); “HPV4-L2 SEQ2” (NLYAKCQLSGNCLPDVKNKVEADTL//SEQ ID NO:29); “HPV5-L2 SEQ3” (HIYQTCKQAGTCPPDVINKVEQTTV//SEQ ID NO:30); “HPV8-L2 SEQ4” (HIYQTCKQAGTCPPDVINKVEQTTV//SEQ ID NO:31); “HPV23-L2 SEQ5” (DIYKGCKASGTCPPDVLNKVEQNTL//SEQ ID NO:32); “HPV38-L2 SEQ6” (DIYRGCKASNTCPPDVINKVEQSTE/SEQ ID NO:33); and “HPV76-L2 SEQ
  • Figures 7A-C shows transmission electron microscopy (TEM) imaging of HPV 18 Beta 1 ( Figure 7A), Beta 2 ( Figure 7B), or Beta 3 Figure 7C), following expression and purification.
  • Figure 8 summarizes the results from ELISA assays testing mouse vaccinated sera from HPV18 Beta 1, HPV18 Beta2, or HPV18 Beta3, as well as HPV16 RG1-VLP and Gardasil®9 against HPV types 1, 2/27 (HPV2 and HPV27 having the same amino acid sequence), 4, 5/8 (HPV5 and HPV8 having the same amino acid sequence), 38, 76, 92.
  • Reactivity is determined by “+” as the minimal whereby O.D600 is between 0.1 to 0.3, “++” as OD600 is between 0.3-0.99 and “+++” as O.D600 more than 1.0.
  • O.D600 values that were less than 0.1 absorbance were considered non- reactive and denoted as
  • Figures 9A-E show results from passive transfer of rabbit vaccinated sera with
  • FIGs 10A-C illustrates the structure and results of a vaccine study evaluating HPV16 RG-1VLP (“16 RG1 monovalent”) or HPV16 RG1-VLP+ HPV18 Beta 2 (“RG1 Bi-valent”) or Gardasil®9 against in vivo co-infection and disease caused by 18 different HPV types.
  • Figure 10A shows the experimental scheme of this vaccine study. Ten rabbits were vaccinated per group at months 0, 1 and 2. As a negative control, ten rabbits were also vaccinated with just aluminum hydroxide (Alhydrogel® alone; “Alum control”).
  • the term “antigen” is a molecule capable of being bound by an antibody, B-cell or T-cell receptor.
  • An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the stimulation of B- and/or T- lymphocytes.
  • the structural aspect of an antigen that gives rise to a biological response is referred to herein as an “antigenic determinant” or “epitope” and are synonymous.
  • B- lymphocytes respond to foreign antigenic determinants via antibody production, whereas T- lymphocytes are the mediator of cellular immunity.
  • antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies, B-cell receptors, or in the context of an MHC (both canonical and non-canonical), by T-cell receptors.
  • An antigenic determinant or epitope need not be a contiguous/consecutive sequence or segment of protein and may include various sequences that are not immediately adjacent to one another.
  • an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, B-cell receptor or in the context of T-cells, those residues necessary for recognition by T-cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors.
  • the amino acid residues of an epitope need not be contiguous/consecutive.
  • an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, B-cell receptor or T-cell receptor or HLA molecule.
  • B-cell epitope or “target epitope” (e.g., HPV L2), refers to a feature of a peptide or protein that is recognized by a B-cell receptor in the immunogenic response to the peptide comprising that antigen (e.g., an HPV L2 epitope (immunogen or target epitope)).
  • antigen e.g., an HPV L2 epitope (immunogen or target epitope)
  • helper T-cell epitope or “Th epitope” means a feature of a peptide or protein that is recognized by a T-cell receptor in the initiation of an immunologic response to the peptide comprising that antigen.
  • Recognition of a T-cell epitope by a T-cell is generally believed to be via a mechanism wherein T-cells recognize peptide fragments of antigens which are bound to class I or class II Major Histocompatibility Complex (MHC) molecules expressed on antigen-presenting cells.
  • MHC Major Histocompatibility Complex
  • the epitopes or epitopic fragments identified as described herein find use in the detection of antigen presenting cells having MHC molecules capable of binding and displaying the epitopes or fragments.
  • HPV human papillomavirus
  • HPV human papillomavirus
  • tropism genetic/mucosal and cutaneous groups
  • virus “types” or “strains” e.g., HPV16, HPV18, HPV31, HPV32, etc.
  • HPV types that are associated with genital infection and malignancy on either mucosal or cutaneous (skin) tissues, as well as those that produce benign papillomas, both at mucosa and skin, resulting in morbidity to the patient.
  • VLP refers to a formulation which contains 1, 2, 3, 4, 5, or more VLP compositions of the present invention.
  • the VLP-based bivalent vaccine composition s will typically be in a form that is capable of being administered to a subject and induces a protective or therapeutic immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another anti-HPV therapy or prophylactic.
  • VLP-based bivalent vaccine composition comprise a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved, although administration of dry powder, for example by inhalation, and even formulation with an additional adjuvant, such as aluminum hydroxide or aluminum phosphate (alum) or CpG 1018, and/or another pharmaceutically acceptable carrier, is also contemplated.
  • the virus-like particle (VLP)-based bivalent vaccine composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat a HPV infection.
  • an immunogenic composition of the invention e.g., a vaccine
  • an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.
  • a response will be cross reactive between, or among, various types of papillomavirus, including, but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more, of the HPV types described herein. Particular cross reactive HPV types are discussed elsewhere herein.
  • prophylactic and “preventive” vaccines, antibodies or immune sera are vaccines, antibodies or immune sera that are designed and administered to prevent infection, disease, and/or any related sequela(e) caused by or associated with a pathogenic organism, particularly HPV.
  • therapeutic vaccines are vaccines that are designed and administered to patients already infected with a pathogenic organism such as at least one HPV strain.
  • Therapeutic vaccines e.g., therapeutic HPV vaccines
  • inhibiting when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result, such as inhibiting, reducing, or preventing viral infection, viral spread, viral growth, or viral transmission.
  • a “subject,” as used herein, includes any human or animal (e.g., a mammal) that has been infected with, or is at risk of, or is susceptible, to being infected with, a papillomavirus.
  • a “susceptible” subject, or cell is one that possesses receptors that can bind a particular papillomaviral virion or pseudo-virion particle, when the susceptible subject, or cell, is exposed to, or in contact with the particle. If genetic material is present in the particle, a viral replication process and/or disease process can ensue in the susceptible subject or cell.
  • Suitable subjects include laboratory animals (such as mouse, rat, rabbit, guinea pig or pig), farm animals (such as cattle), sporting animals (such as dogs or horses) and domestic animals or pets (such as a horse, dog or cat).
  • laboratory animals such as mouse, rat, rabbit, guinea pig or pig
  • farm animals such as cattle
  • sporting animals such as dogs or horses
  • domestic animals or pets such as a horse, dog or cat.
  • Non-human primates and human patients are included.
  • proteins proteins, polypeptide, oligopeptide, “peptide,” as used herein, are not restricted to any particular number of amino acid residues; these terms are sometimes used interchangeably herein.
  • the properties and amino acid sequences of the proteins of the invention, and of the nucleic acids encoding them, are well-known and can be determined routinely, as well as downloaded from various known databases. See, e.g., the NCBI GenBank databases. Some sequences are provided herein. This information is accurate as of the date of filing of this application. However, some sequence information is routinely updated (e.g. to correct mistakes in the previous entries), so updated (corrected) information about the proteins and nucleic acids encoding them is included in this application.
  • an “analogous protein segment” is a polypeptide or oligopeptide comprised within a particular first protein, which polypeptide or oligo peptide shares a conformational motif and/or functional homology, to a protein segment on a second protein, and is, thus, an “analogous protein segment” with respect to the protein segment on the second protein.
  • an “analogous protein segment” See, e.g., Samson et al., Protein segment finder: an online search engine for segment motifs in the PDB, Nucleic Acids Research, 2009, Vol. 37, Database issue doi:10.1093/nar/gkn833, 30 October 2008).
  • chimeric proteins discussed herein are sometimes referred to herein as “proteins of the invention.”
  • a chimeric “virus-like particle” or “VLP” of the invention refers to an empty viral capsid which is composed of papillomavirus LI protein molecules, into which are inserted a peptide of the minor viral capsid, L2.
  • the invention involves a human papilloma virus 16 (HPV16) LI protein (SEQ ID NO: 12) into which is inserted, into a DE loop of the HPV16 LI protein, a surface- displayed HPV16 L2 peptide, wherein the surface-displayed HPV16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 16).
  • HPV16 human papilloma virus 16 LI protein
  • the term “surface-displayed” means that a particular region or domain of the protein, e.g., the inserted L2 peptide, has a thermodynamic affinity for water, and, thus, as a hydrophilic moiety, this region or domain tends be disposed or reside on the exterior surface of the chimeric protein and/or VLP, in contact with the aqueous environment.
  • the amino acid sequence of the wild type HPV16 LI protein (SEQ ID NO: 12) is the following:
  • the surface-displayed HPV16 L2 peptide having the amino acid sequence of SEQ ID NO: 16 is inserted into the DE loop of the HPV16 LI protein between amino acid positions 136 and 137 of SEQ ID NO: 12. This yields a chimeric protein having the amino acid sequence of SEQ ID NO: 15 (underlined residues show placement of SEQ ID NO: 16 in the primary structure):
  • a useful coding sequence for SEQ ID NO: 15 is the following nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence degenerate thereto:
  • HPV16 L2 peptide is inserted into a suitable region of the HPV16
  • the L2 peptide is inserted in the DE loop of LI, e.g., between amino acid residue positions 136 and 137 of HPV16 LI (SEQ ID NO: 12), i.e., inserted into SEQ ID NO: 12 at AA136/137.
  • an HPV18 LI protein (SEQ ID NO: 13) has inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide; the inserted peptide comprises one or more epitopes (e.g., neutralizing epitopes) that are cross -reactive with a broad spectrum of HPV types.
  • epitopes e.g., neutralizing epitopes
  • the term “synthetic,” with respect to the surface-displayed HPV L2 peptide that is inserted into a DE loop of the HPV18 LI protein, means that this recombinantly inserted protein segment is of exogenous origin, i.e., has an amino acid sequence obtained from an HPV type other than a native or wild type HPV 18 L2 peptide sequence.
  • the surface-displayed HPV L2 peptide is an analogous protein segment from a HPV type selected from HPV1, HPV4, HPV5, HPV8, HPV23, HPV38, or HPV76.
  • the L2 peptide inserted into the HPV 18 LI comprises amino acid residues 17-36 of the HPV16 L2 protein (SEQ ID NO: 16), or an equivalent sequence, or analogous protein segment, of amino acid residues from another HPV type.
  • the recombinantly inserted L2 protein segment of exogenous origin is derived through bioinformatic design, e.g., through the use of bioinformatic data and suitable artificial intelligence platforms.
  • the L2 peptide is inserted in the DE loop of HPV18 LI, e.g. between amino acid residue positions 134 and 135 of SEQ ID NO: 13, i.e., inserted into SEQ ID NO: 13 at AA134/135.
  • the amino acid sequence of wild type HPV18 LI protein is the following SEQ ID NO: 13: MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGG GNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPL GVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHW AKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDIC QSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTG MRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTT RSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLC
  • analogous protein segment inserted into the DE loop of HPV18 LI protein can be an amino acid sequence variant of any one of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34.
  • some useful embodiments of the human papilloma virus 18 (HPV18) LI protein into which is inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide include chimeric proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11, as shown in Table 1 (below).
  • Table 1 (next page). Non-limiting examples of chimeric HPV18 LI protein sequences into which is inserted into a DE loop of the HPV18 LI protein (SEQ ID NO: 13), a synthetic surface-displayed HPV L2 peptide. Underlined residues show placement of the synthetic surface-displayed HPV L2 peptide in the primary structure of the chimeric protein.
  • amino acid sequence variants will have an amino acid sequence having at least 80% amino acid sequence identity with the original amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, more preferably at least 85% identity, even more preferably at least 90% identity, and most preferably at least 95% identity, including for example, 80%>, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • Identity or homology with respect to the amino acid sequence is defined herein as the percentage of amino acid residues in the sequence that are identical with the original sequence, after aligning the sequences and candidate introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the analogous protein segment inserted into the DE loop of HPV18 LI protein can be the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, but having 1, 2, 3, or 4 conservative amino acid substitutions.
  • Amino acid residues are commonly categorized according to different chemical and/or physical characteristics. For example amino acid residues can be in D- or L- form, but for the purposes of the present invention the L-form of the amino acid is intended.
  • acidic amino acid residue refers to amino acid residues in having side chains comprising acidic groups. Exemplary acidic residues include aspartic acid and glutamic acid residues.
  • basic amino acid residue refers to amino acid residues having side chains comprising basic groups. Exemplary basic amino acid residues include histidine, lysine, homolysine, ornithine, or arginine residues.
  • hydrophilic amino acid residue refers to amino acid residues having side chains comprising polar groups.
  • hydrophilic residues include cysteine, serine, threonine, histidine, lysine, asparagine, aspartate, glutamate, and glutamine residues.
  • lipophilic amino acid residue refers to amino acid residues having sidechains comprising uncharged, aliphatic or aromatic groups.
  • Exemplary lipophilic sidechains include phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine.
  • Alanine is amphiphilic — it is capable of acting as a hydrophilic, or lipophilic (i.e., hydrophobic), residue.
  • Alanine therefore, is included within the definition of both “lipophilic” (i.e., "hydrophobic") residue and “hydrophilic” residue.
  • nonfunctional or “neutral” amino acid residue refers to amino acid residues in having side chains that lack acidic, basic, or aromatic groups. Exemplary neutral amino acid residues include methionine, glycine, alanine, valine, isoleucine, leucine, and norleucine residues.
  • aromatic amino acid residue refers to amino acid residues having side chains comprising aromatic groups. Exemplary aromatic residues include tryptophan and tyrosine.
  • a “conservative amino acid substitution” involves a substitution of a native amino acid residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • a “conservative amino acid substitution” includes any native residue in the polypeptide substituted with alanine, as has been previously described for "alanine scanning mutagenesis” (see, for example, MacLennan et al, Acta Physiol. Scand. SuppL, 643:55-67 (1998); Sasaki et al, 1998, Adv. Biophys. 35: 1-24 (1998), which discuss alanine scanning mutagenesis).
  • the chimeric HPV16 LI proteins and HPV18 LI proteins assemble spontaneously into VLP and resemble native virions in both structure and immunogenicity, yet lack nucleic acid and thus are non-oncogenic and non-infectious.
  • the term “assemble spontaneously” means that the chimeric proteins of the invention form into complete VLP structures without enzymatic involvement or ATP expenditure, based on favorable thermodynamic values for VLP formation under the reaction conditions such as McCarthy et al., Journal of Virology 1998 Jan; 72(1): 32-41 ; Mukherjee et al., 2008 Journal of Molecular Biology, 381( b:229-37. Mach et ak, 2006 Journal of Pharmaceutical sciences 95( 10):2195-
  • One aspect of the invention is a method for making a VLP (or the polypeptide component thereof) of the invention.
  • HPV epitopes are synthesized using conventional methods as modified for the particular amino acid sequences. Such techniques include, e.g., methods well known to those skilled in the art of peptide synthesis, e.g., solution phase synthesis [see Finn et al. in Proteins, 3rd Ed., Neurath and Hill (Eds), Academic Press, NY, 2, 105-253, 1976], or solid phase synthesis [see Barany et al.
  • Fmoc amino acids can be obtained from various vendors, e.g., Chem-Impex International (Wood Dale, Ill., USA), Merck Biosciences (Nottingham, UK), and Bachem UK Ltd. (St. Helens, UK).
  • a polypeptide of the invention can be prepared recombinantly.
  • the present invention provides recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors.
  • Expression vectors comprising DNA may be used to prepare the polypeptides or polypeptide fragments of the invention encoded by a DNA.
  • a method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture.
  • Polypeptides of the invention can include various leader sequences that direct trafficking or assist in purification.
  • the vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
  • suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence.
  • a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence.
  • An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
  • Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al In: Cloning Vectors: A Laboratory Manual , Elsevier, N Y, 1985. Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein.
  • the methods of the present invention include prevention and/or treatment for a disease or condition caused by or related to papillomavirus infection (e.g., HPV infection).
  • HPV infection e.g., HPV infection
  • An immunogenic HPV peptide and/or antibody that binds the same can be given to induce or provide a protective and/or therapeutic response in a subject infected with or suspected of having been exposed to or at risk of becoming infected with HPV.
  • Methods may be employed with respect to individuals who have tested positive for exposure to HPV or who are deemed to be at risk for infection based on possible exposure.
  • the treatment is administered in the presence of adjuvants or carriers or other antigens, either HPV antigens or antigens from other pathogens.
  • treatment comprises administration of other agents commonly used against viral infection, such as one or more anti-virals.
  • VLP compositions can be enhanced by the use of additional non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions such as alum.
  • adjuvants can be used to enhance an antibody response against a VLP described herein.
  • Adjuvants can be used to (1) trap the antigen in the body to cause a slow release; (2) attract cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of the antigen throughout the subject's body.
  • Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL- 12, y-interferon, GM-CSF, BCG, aluminum salts, such as aluminum hydroxide, aluminum phosphate, or other aluminum salt or compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), CpG-1018, lipid A, and monophosphoryl lipid A (MPL), or inactivated microbial agents.
  • IL-1 IL-2, IL-4, IL-7, IL- 12, y-interferon, GM-CSF, BCG
  • aluminum salts such as aluminum hydroxide, aluminum phosphate, or other aluminum salt or compound
  • MDP compounds such as thur-MDP and nor-MDP,
  • RIB I which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used.
  • Other adjuvants or methods are exemplified in U.S. Pat. Nos. 6,814,971, 5,084,269, 6,656,462, each of which is incorporated herein by reference).
  • Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or aluminum phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (CARBOPOL®) used as an about 0.25% solution, aggregation of a protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101° C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells (e.g., C.
  • Fab pepsin-treated
  • parvum endotoxins or lipopolysaccharide components of Gram-negative bacteria
  • emulsion in physiologically acceptable oil vehicles e.g., mannide mono-oleate (Aracel A)
  • emulsion with a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used as a block substitute may also be employed to produce an adjuvant effect.
  • a typical adjuvant is complete Freund's adjuvant (containing killed Mycobacterium tuberculosis'), incomplete Freund's adjuvants, and aluminum hydroxide.
  • adjuvants for administration to humans, a variety of suitable adjuvants will be evident to a skilled worker. These include, e.g., Alum-MPL as adjuvant, or the comparable formulation, ASO4, which is used in the approved HPV LI vaccine Cervarix®, AS03, AS02, MF59, montanide, saponin-based adjuvants such as GPL0100, CpG-based adjuvants (e.g., CpG- 1018), or imiquimod. In embodiments of the invention, an adjuvant is physically coupled to the VLP, or encapsulated by the VLP, rather than simply mixed with them.
  • BRM biologic response modifiers
  • BRMs have been shown to upregulate T cell immunity or downregulate suppresser cell activity.
  • BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m2 ) (Johnson/Mead, NJ) and cytokines such as y-interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.
  • these genes are encapsulated by the VLP to facilitate their delivery into a subject.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally by injection, inhalation of a powder, via transcutaneous patch, via vaginal instillation and the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.
  • vaccines that contain polypeptide or peptide sequence(s) as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
  • vaccines are formulated with a combination of substances, as described in U.S. Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.
  • Vaccines may be administered by inhalation.
  • a vaccine can be administered as an aerosol.
  • aerosol or “aerosolized composition” refers to a suspension of solid or liquid particles in a gas. The terms may be used generally to refer to a composition that has been vaporized, nebulized, or otherwise converted from a solid or liquid form to an inhalable form including suspended solid or liquid drug particles.
  • aerosols can be used to deliver a vaccine via the respiratory system.
  • respiratory system refers to the system of organs in the body responsible for the intake of oxygen and the expiration of carbon dioxide. The system generally includes all the air passages from the nose to the pulmonary alveoli.
  • delivery of a vaccine to the respiratory system indicates that a drug is delivered to one or more of the air passages of the respiratory system, in particular to the lungs.
  • Additional formulations which are suitable for other modes of administration include suppositories (for anal or vaginal application) and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
  • the VLP-based bivalent vaccine compositions may be formulated into a vaccine as neutral or salt forms.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • T1 it will be desirable to have multiple administrations of the vaccine, usually at most, at least, or not exceeding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more vaccinations including all ranges there between.
  • the vaccinations will normally be at 1, 2, 3, 4, 5, 6, to 5, 6, 7, 8, 9, 10, 11, to 12 week/month/year intervals, including all values and ranges there between, more usually from three to five week intervals.
  • periodic boosters at intervals of 1-15 years, usually ten years will be desirable to maintain protective levels of the antibodies.
  • the course of the immunization may be followed by assays for antibodies against the antigens, as described supra, U.S. Pat. Nos. 3,791,932;
  • compositions and related methods of the present invention may also be used in combination with the administration of traditional HPV screening and/or other vaccines, including, e.g., antibodies or antibody fragments, Pap smears, PCR, Southern blotting, administering CERVARIXTM, GARDASILO9TM, vaccines for HPV or other infectious agents, ablative therapy of HPV lesions, immunomodulatory therapies for HPV lesions (e.g. AldaraTM), or the like.
  • traditional HPV screening and/or other vaccines including, e.g., antibodies or antibody fragments, Pap smears, PCR, Southern blotting, administering CERVARIXTM, GARDASILO9TM, vaccines for HPV or other infectious agents, ablative therapy of HPV lesions, immunomodulatory therapies for HPV lesions (e.g. AldaraTM), or the like.
  • compositions are administered to a subject.
  • Different aspects of the present invention involve administering an effective amount of a composition to a subject.
  • a VLP comprising an HPV L2 epitope is administered to the patient to protect against or treat infection by one or more HPV pathogens.
  • Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • the term “pharmaceutically acceptable” or “pharmacologically acceptable” refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
  • Pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.
  • the active compounds of the present invention can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.
  • other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the VLP-based bivalent vaccine compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions according to the present invention will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, respiratory, or intravenous administration. In certain embodiments, a vaccine composition may be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • aqueous solutions for parenteral administration in an aqueous solution
  • the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in isotonic NaCl solution and either added to hypodermoclysis fluid or injected at the proposed site of infusion (see for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • an effective amount of therapeutic or prophylactic composition is determined based on the intended goal.
  • An “effective amount” is an amount that is effective to bring about a desired outcome (e.g., the induction of a measurable amount of an immune response, the immunization of a subject, etc.).
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.
  • compositions also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. [000114]
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • VLPs are administered to subjects by administering an effective amount of a recombinant attenuated bacterium (such as a salmonella bacterium) which encodes a chimeric polypeptide of the invention.
  • a recombinant attenuated bacterium such as a salmonella bacterium
  • the VLPs are then produced by the gut in vivo, where the bacteria replicate.
  • bacterial vectors For guidance for carrying out methods using such bacterial vectors, see, e.g., Nardelli-Haefliger (2007) Clin Vaccine Immunol 14, 1285-1295, which is incorporated by reference specifically for such disclosure.
  • Methods for generating recombinant constructs that can be expressed in bacteria are conventional; some typical methods are described elsewhere herein.
  • Lyophilized bacteria can be easily shipped to developing countries, where they can then be resuspended and administered to subjects. Such a mode of administration is advantageous in a country to lacks refrigeration capabilities that might be required for other formulations of VLPs.
  • VLPs are administered in an attenuated virus, such as an attenuated Adenovirus, or other viral vectors which are well-known to those of skill in the art. Methods for producing suitable recombinant nucleic acids that can be expressed in a viral host are conventional, and some such methods are discussed elsewhere herein.
  • the present invention includes compositions for preventing or ameliorating HPV infections.
  • the invention contemplates vaccines for use in both active and passive immunization embodiments.
  • One embodiment of the invention is a method of preparing an immunoglobulin for use in prevention or treatment of HPV infection comprising the steps of immunizing a recipient with a vaccine of the invention and isolating immunoglobulin or antibodies from the recipient, and/or recombinantly producing such immunoglobulins or fragments thereof.
  • An immunoglobulin prepared by this method is a further aspect of the invention.
  • a pharmaceutical composition comprising the immunoglobulin of the invention and a pharmaceutically acceptable carrier is a further aspect of the invention which could be used in the manufacture of a medicament for the treatment or prevention of HPV infection.
  • a method for treatment or prevention of HPV infection comprising a step of administering to a patient an effective amount of the pharmaceutical preparation of the invention is a further aspect of the invention.
  • Inocula for polyclonal antibody production are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition.
  • An immuno stimulatory amount of inoculum is administered to a mammal, e.g., a human, and the inoculated subject is then maintained for a time sufficient for the antigenic composition to induce protective antibodies.
  • the antibodies can be isolated to the extent desired by well known techniques such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual 1988).
  • Antibodies can include antiserum preparations from a variety of commonly used animals, e.g., goats, primates, donkeys, swine, horses, guinea pigs, rats, or man. The animals are bled and serum recovered.
  • An immunoglobulin produced in accordance with the present invention can include whole antibodies, antibody fragments or subfragments.
  • Antibodies can be whole immunoglobulins of any class, e.g., IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with dual specificity to two or more antigens of the invention. They may also be fragments, e.g., F(ab')2, Fab', Fab, Fv and the like including hybrid fragments.
  • An immunoglobulin can also include natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex.
  • An HPV composition or vaccine of the present invention can be administered to a recipient who then acts as a source of immunoglobulin, produced in response to challenge from the HPV composition.
  • a subject thus treated would donate plasma from which hyperimmune globulin would be obtained via conventional plasma fractionation methodology.
  • the hyperimmune globulin would be administered to another subject in order to impart resistance against or treat HPV infection.
  • Hyperimmune globulins of the invention are particularly useful for treatment or prevention of HPV infection in infants, immune compromised individuals or where treatment is required and there is no time for the individual to produce antibodies in response to vaccination.
  • An additional aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising one or more monoclonal antibodies (or fragments thereof; preferably human or humanized) reactive against constituents of the immunogenic composition of the invention, which could be used to treat or prevent infection by multiple HPV types.
  • monoclonal antibodies are well known in the art and can include the fusion of splenocytes with myeloma cells (Kohler et al. (1975) Nature 256, 495; Harlow et al. Antibodies: A Laboratory Manual, 1988).
  • monoclonal Fv fragments can be obtained by screening a suitable phage display library (Vaughan et al. (1998) Nat. Biotech. 16, 535-539).
  • Monoclonal antibodies may be human, humanized, or partly humanized by known methods.
  • kits for vaccination or treatment comprises a vial and optionally a package insert with administration instructions, the vial comprises a VLP composition or vaccine for administration according to the methods of the present invention.
  • kits Any of the compositions described herein may be included in a kit.
  • reagents for preparing a VLP and/or administering a VLP, or antibodies generated by vaccination with VLP can be included in a kit.
  • the kit may further include reagents for assessing the activity of the VLP both in vitro and in vivo.
  • the kits will thus comprise, in suitable container, a VLP composition.
  • the kit can include reagents and/or devices for administration, e.g., inhaler or nebulizer. It may also include one or more buffers, compounds, or devices for preparing the composition for administration.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
  • kits may also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • kits of the invention are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the preparation and/or administration of a VLP vaccine of the invention. [000130] Among other uses, kits of the invention can be used in experimental applications. A skilled worker will recognize components of kits suitable for carrying out a method of the invention.
  • Embodiment 1 A virus-like particle (VLP)-based bivalent vaccine composition, comprising:
  • a human papilloma virus 16 (HPV16) LI protein into which is inserted, into a DE loop of the HPV16 LI protein, a surface-displayed HPV16 L2 peptide, wherein the surface-displayed HPV16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 16);
  • a human papilloma virus 18 (HPV18) LI protein into which is inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide;
  • Embodiment 2 The VLP-based bivalent vaccine composition of Embodiment 1, wherein the HPV16 LI protein comprises the amino acid sequence of SEQ ID NO: 12, and wherein the surface-displayed HPV 16 L2 peptide is inserted into the DE loop of the HPV 16 LI protein between amino acid residue positions 136 and 137 of SEQ ID NO: 12.
  • Embodiment 3 The VLP-based bivalent vaccine composition of any of Embodiments 1-2, wherein the surface-displayed HPV L2 peptide is an analogous protein segment from a HPV type selected from the group consisting of HPV1, HPV4, HPV5, HPV8, HPV23, HPV38, and HPV76.
  • Embodiment 4 The VLP-based bivalent vaccine composition of any of Embodiments 1-3, wherein the HPV 18 LI protein comprises the amino acid sequence of SEQ ID NO: 13.
  • Embodiment 5 The VLP-based bivalent vaccine composition of any of Embodiments 1-4, wherein the synthetic surface-displayed HPV L2 peptide is inserted into the DE loop of the HPV18 LI protein between amino acid residue positions 134 and 135 of SEQ ID NO: 13.
  • Embodiment 6 The VLP-based bivalent vaccine composition of any of Embodiments 1-5, wherein the synthetic surface-displayed HPV L2 peptide, inserted into the DE loop of the HPV 18 LI protein, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.
  • Embodiment 7 The VLP-based bivalent vaccine composition of any of Embodiments 1-6, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV 18 LI protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, or the amino acid sequence of any these members with 1-4 conservative amino acid substitutions.
  • Embodiment 8 The VLP-based bivalent vaccine composition of any of Embodiments 1-7, wherein the HPV18 LI protein comprises the amino acid sequence of SEQ ID NO: 13; wherein the synthetic surface-displayed HPV L2 peptide is inserted into the DE loop of the HPV18 LI protein between amino acid residue positions 134 and 135 of SEQ ID NO: 13; and wherein the synthetic surface-displayed HPV L2 peptide comprises an amino acid sequence selected from the group consisting SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:27 , or the amino acid sequence of any these members with 1-4 conservative amino acid substitutions.
  • Embodiment 9 The VLP-based bivalent vaccine composition of any of Embodiments 1-8, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV 18 LI protein comprises the amino acid sequence of HIYQSCKASGTCPPDVINKVE (SEQ ID NO:24), or the amino acid sequence of SEQ ID NO:24 with 1-4 conservative amino acid substitutions.
  • Embodiment 10 The VLP-based bivalent vaccine composition of any of Embodiments 1-9, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV18 LI protein comprises an amino acid sequence variant at least 80, 85, 90 or 95% identical to the amino acid sequence of SEQ ID NO:24.
  • Embodiment 11 The VLP-based bivalent vaccine composition of any of Embodiments 1-10, further comprising an adjuvant.
  • Embodiment 12 The VLP-based bivalent vaccine composition of Embodiment 11, wherein the adjuvant comprises aluminum hydroxide and/or aluminum phosphate.
  • Embodiment 13 The VLP-based bivalent vaccine composition of any of Embodiments 1-12, further comprising a pharmaceutically acceptable carrier.
  • Embodiment 14 The VLP-based bivalent vaccine composition of any of Embodiments 1-13, wherein the composition is formulated for administration by inhalation, ingestion, or in a viral or bacterial vector.
  • Embodiment 15 The VLP-based bivalent vaccine composition of any of Embodiments 1-14, wherein the composition is in a formulation for intramuscular injection.
  • Embodiment 16 The VLP-based bivalent vaccine composition of any of Embodiments 1-15, wherein the composition is packaged in a glass vial for single or multiple-dose use.
  • Embodiment 17 The VLP-based bivalent vaccine composition of any of Embodiments 1-16, which is an immunogenic composition.
  • Embodiment 18 The VLP-based bivalent vaccine composition of any of Embodiments 1-17, which is immunogenic against mucosal high-risk or low- risk, cutaneous low risk, and/or cutaneous beta-type papillomaviruses.
  • Embodiment 19 The VLP-based bivalent vaccine composition of any of Embodiments 1-18, which is immunogenic against one or more papillomaviruses selected from the group consisting of HPV1, HPV2, HPV4, HPV5, HPV16, HPV18, HPV26, HPV35, HPV38, HPV39, HPV45, HPV52, HPV58, HPV68, HPV76, and HPV92.
  • papillomaviruses selected from the group consisting of HPV1, HPV2, HPV4, HPV5, HPV16, HPV18, HPV26, HPV35, HPV38, HPV39, HPV45, HPV52, HPV58, HPV68, HPV76, and HPV92.
  • Embodiment 20 The VLP-based bivalent vaccine composition of any of Embodiments 1-19, wherein the composition is effective in preventing human papillomavirus infection in a susceptible subject.
  • Embodiment 21 The VLP-based bivalent vaccine composition of
  • Embodiment 20 which prevents co-infection in the subject by the following human papillomavirus types: HPV 6, 16, 31, 45, 52, 58, 35, 39 and 59.
  • Embodiment 22 The VLP-based bivalent vaccine composition of any of Embodiments 20-21, which prevents co-infection in the subject by the following human papillomaviruse types: HPV 5, 11, 18, 26, 51, 56, 66 ,68 and 73.
  • Embodiment 23 The VLP-based bivalent composition of any of Embodiments 20-22, which prevents co-infection in the subject by the following human papillomaviruse types: HPV 6, 11, 16, 18, 26, 31, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 73.
  • Embodiment 24 A method for immunizing or vaccinating a subject against a HPV, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1-23.
  • Embodiment 25 The method of Embodiment 24, wherein administering the effective amount of the VLP-based bivalent vaccine composition comprises a 2-dose or 3- dose vaccination schedule.
  • Embodiment 26 A method for inducing an immune response against HPV in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1-23.
  • Embodiment 27 A method for treating a HPV infection in a subject having a HPV infection or at risk of being exposed to HPV, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1- 23.
  • Embodiment 28 A method for preventing HPV- associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer, in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1-23.
  • Embodiment 29 A kit comprising the VLP-based bivalent composition of any of Embodiments 1-23.
  • Embodiment 30 The VLP-based bivalent composition of any of Embodiments 1-23, for treating a HPV infection in a subject having a HPV infection.
  • Embodiment 31 The VLP-based bivalent composition of any of Embodiments 1-23, for preventing HPV-associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer in a subject.
  • Example 1 Epitope selection and Recombinant HPV 18 RG-1 VLP protein design
  • VLP virus-like particle
  • Candidates 1-3 contained HPV minor capsid protein L2 epitopes corresponding to the amino acid residue positions 56-75 region from wildtype HPV types 16, 1, and 4 respectively.
  • Candidates 4 and 5 contained a bio-informatically designed epitope corresponding respectively to the amino acid residue positions 17-36 region or amino acid residue positions 55-75 region of minor capsid protein L2 from HPV types 1, 4 and 38; these three HPV types are known to be genetically distant from one another and cause hand/foot warts, flat warts and non-melanoma skin cancer, respectively.
  • Candidates 6, 7, and 8 each contained a bio-informatically designed epitope corresponding to amino acid residue positions 17-36 , 55-75, or 65-85 regions, respectively, of minor capsid protein L2 from 36 HPV types known to be associated with all known clinically relevant HPV-associated clinical disease or cancer.
  • 36 HPV types are HPVs: 1, 3, 4, 5, 6, 8, 11, 16, 18, 23, 26, 27, 31, 32, 33, 34, 35, 38, 39, 40, 42, 43, 44, 45, 51, 52, 53, 56, 57, 58, 59, 66, 68, 70, 73, and 76.
  • Candidates 9 and 10 contained a bio-informatically designed epitope corresponding to the amino acid residue positions 14-34 and 17-36 regions, respectively, of minor capsid protein L2 from the major seven HPV types most known to be associated with cutaneous HPV disease as well as cutaneous cancers, i.e., HPV types 1, 4, 5, 8, 23, 38 and 76. These HPV types are commonly called “HPV beta types.”
  • the cell lysates were then chilled, and the NaCl concentration of the lysates was adjusted to 0.8 M.
  • the cell lysates were then clarified by ultra-centrifugation for 16 hours at 40,000 x g.
  • the VLPs were purified from the clarified lysate on a 27%/33%/39% OptiPrepTM gradient. Following these expression and purification steps, only Candidates 4, 6, 7, 9 and 10 demonstrated detectable protein expression, as assessed by SDS-PAGE, and only these five candidates were able to form VLPs (50-60nm in diameter), which was verified using transmission electron microscopy (data not shown).
  • Example 2 Candidate 10 has the broadest cross-reactive immunogenicity towards 22 HPV types.
  • HPV pseudovirus neutralization assay against HPV18 was conducted, as previously described by Pastrana et al Virology, 321(2):205-16. The results are shown in Figure 1 and demonstrate comparable HPV 18 neutralization titers among all the Candidates tested, as well as Gardasil® or Cervarix®. These results show that the insertion of additional epitopes into the DE loop region of HPV18 LI protein did not compromise the other HPV18 type-specific epitopes.
  • RGlx22 a concatenated protein that contains the amino acid residue positions 17-36 region of L2 from 22 different HPV types. Approximately 500 ng in 100 pL PBS/well of RGlx22 was added and incubated overnight at 4°C. The next day, plates were blocked with PBS/1% BSA for 1 hour at 37°C. Samples of each of the sera were then diluted 1:50 in PBS/1% BSA and were then added to the 96-well plates for 1 hour at 37°C.
  • mice were vaccinated on days 1 and 28, and a three-dose schedule in which mice were vaccinated on days 1, 14 and 28. All mice were bled 2 weeks after the final vaccination (i.e., day 42) to obtain sera for immunogenicity testing.
  • Example 4 Two dose regimen of HPV16RG-1 VLP co-formulated with candidate 10 is as immunogenic as a three-dose regimen.
  • Example 5 Evaluation of in vitro cross-neutralization capabilities of HPV16 RG1-VLP co-formulated with either Candidate 9 or Candidate 10
  • HPV18 Beta 1 a co-formulation of HPV16 RG1-VLP with Candidate 9
  • HPV18 Beta 2 a co-formulaton of HPV16 RG1-VLP with Candidate 10
  • Figure 5 Sera from mice vaccinated against two doses or three doses of Cervarix® were also tested as negative controls. As expected, the negative control sera did not provide any cross -protection regardless of whether a two- or three-dose regimen had been employed.
  • Example 6 Design and development of a HPV 18 chimeric VLP containing both HPV18 Beta 1 and Beta 2 epitopes
  • HPV 18 Beta 1 or HPV18 Beta 2 The co-formulation of HPV 16 RG1-VLP with either HPV 18 Beta 1 or HPV18 Beta 2 showed that there can be synergy between different types of L2 epitopes derived from the amino acid residue positions 14-34 and 17-36 regions.
  • HPV 18 chimeric VLP containing both the Beta 1 and Beta 2 epitopes. This consists of a 25 amino acid peptide that has both Beta 1 and Beta 2 epitopes overlapping and inserted within the DE loop of HPV 18 LI. This construct was called “HPV18 Beta 3” ( Figure 6).
  • the Beta 3 construct was produced by small scale expression and purification is as described in Example 1, above.
  • Example 7 HPV18 Beta 3 shows the greatest antibody cross-reactivity to multiple HPV types.
  • ELISA assays were performed to assess the cross -reactivity of the antibody responses produced by Beta 1, Beta 2 and Beta 3, and results were compared to control vaccinated sera such as HPV16RG1-VLP (known to be very cross-reactive) and Gardasil®9 (negative control).
  • ELIS As were performed as described in Example 2 but with different peptides consisting of the amino acid residue positions 17-36 region of HPV L2 of types 1, 2, 4 , 5, 8, 38, 76 and 92. Results are summarized in Figure 8) and show that HPV18 Beta 3 had the broadest cross-reactivity in the ELISA, followed by HPV16 RG-1VLP, HPV18 Beta 2 and HPV18 Beta 1.
  • Gardasil®9 negative control
  • Example 8 In vivo synergistic cross -protection of HPV 16RG1 -VLPs with
  • HPV 18 Beta 2 against co-infection of 9 different HPV types in a single animal As described in Example 5 (above), ELISA assays were used to measure the overall antibody response. However, the ELISA system employed does not distinguish whether the detected antibody response can functionally neutralize a HPV virus or inhibit infection in vivo. To address this, a murine HPV pseudo-virion (PsV) challenge model was utilized. This is a HPV surrogate infection model, which involves the generation of HPV PsVs. These HPV PsVs are HPV capsids that encapsidate a luciferase reporter plasmid (PMID: 16350417).
  • pseudo-virions can infect either the mouse’s vagina or skin, and infectivity is measured via luciferase activity as a surrogate marker of infection and can be quantified using imaging software.
  • challenging these HPV pseudo-virions into naive mice or mice that received a vaccine, or passively transferred vaccinated sera can be used to assess prevention of HPV infection and, therefore, determine the promise of any HPV prophylactic vaccine candidate.
  • mice Four days before vaginal challenge, Balb/c mice (8-10 weeks old; purchased from Charles Rivers Laboratories) were subcutaneously injected with 3 mg of medroxyprogesterone (Depo-Provera, Pfizer, New York NY). The day before challenge, 100 pL of rabbit serum was passively transferred into mice by the intraperitoneal route, followed by HPV PsV challenge containing a luciferase reporter; amounts were standardized based on the LI protein content.
  • medroxyprogesterone Depo-Provera, Pfizer, New York NY
  • mice were challenged with 2 pg of PsV (based on stock virus with LI content of 0.2 pg/pL), which was pre-mixed with 3% (w/v) carboxymethyl cellulose (CMC).
  • HPV PsVs were directly instilled into the mouse’s vaginal vault, before and after cytobrush treatment (15-20 rotations, alternating directions), while the mice were under isoflurane anesthesia.
  • the mice were anesthetized by isoflurane 72 hours after challenge, and 20 pL of luciferin substrate (7.8mg/ml, Promega, Madison WI) was then delivered into the vaginal vault before imaging.
  • HPV PsVs Five different HPV PsVs that corresponded to HPV types 5, 6, 11, 51 and 68 were assessed. Unexpectedly, the best in vivo cross neutralizing protection from HPV pseudo-infection was conferred by vaccinated serum from HPV18 Beta 2. This was despite the ELISA results described in Example 7 (above), showing that HPV18 Beta 3 had the broadest antibody cross -reactivity towards many HPVs.
  • HPV 16 RGl-VLPs is able to cross-neutralize over 27 HPVs in vivo, however, those studies were done on a single infection level. (See, Schellenbacher et al., 2013 J Invest Dermatology 133(12): 2706-2713. ). In clinical populations, co-infection of multiple HPV types in a single patient is frequent. Therefore, a HPV vaccine that can prevent co-infection of a diverse multitude of HPV viruses would be a great benefit for public health.
  • the rabbits were again anesthetized, and a 50-pl suspension of cottontail rabbit papillomavirus (CRPV) virion stock (control) or HPV capsid/CRPV genome quasi- virus stock in phosphate buffered saline (PBS) (standardized by genome content for all capsid types) was applied to the pre-scarified site using the edge of a syringe needle.
  • CCPV rabbit papillomavirus
  • PBS phosphate buffered saline
  • Transdermal fentanyl (ear) patches (Duragesic®; 12.5 pg/h) or subcutaneous injections of buprenorphine (0.02 to 0.05 mg/kg twice daily for 3 days) were administered as analgesics post-scarification and post-infection, as needed. The sizes and frequencies of lesions were evaluated at 2 to 8 weeks post-challenge. In the absence of a lesion, a diameter of 0 was assigned. In a given treatment group, full protection was defined as the absence of papillomas in all animals, and partial protection was defined as the presence of any break-out papillomas measured on the rabbits.
  • Results are shown in Figures 9A-E.
  • the no-vaccine group (“Alum control”) showed every single HPV type infected into the rabbits caused a papillomavirus disease. This demonstrates that every single HPV type used in this model can cause disease either independently or in a co-infection of multiple HPVs setting (see, Figure 10A-C).
  • the results show that the combination of HPV16RG1-VLP and HPV18 Beta 2 (“RG1 Bivalent”) showed complete full protection against both infection and disease caused by all 18 HPV viruses.
  • Gardasil®9 is an FDA-approved HPV vaccine for the prevention of HPV genital warts caused by HPV6 and HPV 11, as well as the prevention of HPV-associated cancers caused by HPVs 16, 18, 31, 33, 45 , 52 and 58.
  • Our rabbit studies showed, however, that vaccination was not able to confer full protection against HPV6, HPV16, HPV31, HPV45, and HPV52. This cannot be attributed to a lack of immunogenicity or antibody repertoire since the formulation of Gardasil®9 contains 270 pg of 9 different VLPs, all of which produce nine type-specific neutralizing antibodies against HPV types 6, 11, 16, 18, 31 ,33 ,45 ,52 and 58. As such, the results show that even currently approved vaccines can be overwhelmed in a co-inf ection setting (Figure 10B-C).
  • HPV16RG1-VLP with HPV 18 Beta 2 contains only 40 pg of each respective VLP and produces only four types of antibodies: Two type specific antibodies against HPV16 and HPV18 as well as two cross-neutralizing antibodies.
  • results described herein demonstrate that this combination of HPV16RG1-VLP and HPV 18 Beta 2 can confer full protection in vivo and extend protection beyond what Gardasil®9 is able to cover.
  • the co-formulation of HPV16RG1- VLP with HPV 18 Beta 2 is better and has the potential to provide broad cross protection against multiple HPV infections, coinfections, and diseases.

Landscapes

  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Description

BIVALENT VIRUS-LIKE PARTICLE COMPOSITIONS AND METHODS OF USE
BACKGROUND OF THE INVENTION
[0001 ] 1. Field of the Invention
[0002] This invention relates to virus-like particle (VLP)-based bivalent vaccine compositions for therapeutic and prophylactic uses.
[0003] 2. Discussion of the Related Art
[0004] The more than one hundred types of human papillomaviruses (HPV) identified to date (de Villiers et al. (2004), Virology 22, 670-80) are the etiological agents of skin and mucosal papillomas or warts. Persistent infection with high-risk mucosal types, most often HPV16 and HPV18, cause cervical cancer, which constitutes the second leading fatal cancer in women worldwide, causing about 274,000 deaths per year. Substantial morbidity results from other non-cervical HPV-related conditions, such as anogenital warts, vulval, vaginal, penile, anal or oropharyngeal cancer.
[0005] The development of current prophylactic papillomavirus vaccines was launched by observations that recombinantly expressed major capsid protein LI self-assembles into viruslike particles (VLP). These empty viral capsids are composed of 360 LI molecules and resemble native virions in both structure and immunogenicity, yet they are non-oncogenic and non-infectious. Moreover, VLPs cannot replicate because the cells in which VLP are made contain only LI and no other papillomavirus genes. These VLP vaccines induce high- titer and type-restricted antibody responses to conformational LI epitopes (Christensen et al. (1990), J. Virol 64, 3151-3156); Kimbauer et al. (1992), Proc Natl Acad Sci USA 89, 12180-12184; Rose et al. (1994) J Gen Virol 75, 2445-9; Suzich et al. (1995) Proc Natl Acad Sci USA 92, 11553-11557).
[0006] When applied to women prior to infection, available vaccines targeting the most prevalent high-risk types, HPV16 and HPV18, such as Cervarix® (human papillomavirus bivalent (types 16 and 18) vaccine, recombinant; GlaxoSmithKline, FDA-approved in 2009), have demonstrated 98-100% efficacy against persistent infection and associated disease caused by HPV 16 and 18. However, limited efficacy was observed against other oncogenic HPV types such as HPV31, 33, 45 and 58. Thus, Cervarix® is potentially able to prevent about 70% of cervical high grade dysplasias and probably cancers. Therefore, use of currently licensed LI vaccines necessitates continuation of cytological cervical screening of women. The prevention of approximately 80% of cervical cancer would require immunity to 7 high-risk HPV types (16/18/31/33/45/52/58) (Munoz et al. (2004), Int J Cancer 111, 278- 85) and the development of more highly multivalent (and presumably costly) LI VLP vaccines due to an individual fermentation and purification process required for each VLP followed by co-formulation into a single dose.
[0007] Gardasil®9 (human papillomavirus 9-valent vaccine, recombinant; GlaxoSmithKline), which was approved by the FDA in 2018, contains nine different VLPs, all of which produce nine type-specific neutralizing antibodies against HPV types 6, 11, 16, 18, 31 ,33 ,45 ,52 and 58. The extent of protection against these specific HPV types from clinical trials has shown between 98-100% efficacy. However, important questions remain such as extent of vaccine cross protection against all HPV oncogenic types especially those not covered by Garasil-9, the extent of HPV type replacement and the potential role caused by HPV latent infections. [0008] Recent evidence has suggested that HPV type replacement is a possibility although the subject still remains highly controversial. In two studies by Gray et al ( Int. J.
Cancer 2018, 142( 12):2491 -2500). evaluated pre- and post-vaccination HPV type prevalence in Finnish women. It was concluded that, whilst there was no definitive evidence for type replacement, further study of types 39 and 51 was warranted. A subsequent study by the same group reinforced this conclusion by showing that higher levels of HPV types 51 and 52 were found in more promiscuous vaccinated versus unvaccinated women (Int. J.
Cancer 2019, 145, 785-796).
[0009] In another study focusing on the American population, McClung et al. evaluated the prevalence of CIN2 and associated HPV types in American women pre- and post-vaccination with Cervarix or Gardasil between 2008 and 2016. A very significant reduction in the prevalence of CIN2 was observed in women aged 18-19 and 20-24 years although there was also a pronounced trend towards increasing prevalence of HP V types not covered by the vaccines in vaccinated women aged > 24 years (MMWR Morb. Mortal. Wkly. Rep. 2019, 68, 337-343). Taken together HPV cancers not covered by current vaccines continue to persist and can contribute to HPV associated cancers.
[00010] Solid organ and bone marrow transplants are life-saving modalities. With improvements in immune-suppression and surgical techniques, post-transplant survival has improved and the focus has shifted to improving quality of life after transplantation(Moloney et al Br J Dermatol. 2005). However, the life-long post-transplant immunosuppression causes organ transplant recipients (OTRs) to face elevated risk of HPV infections and HPV-related cancers. Studies have demonstrated elevated incidence of HPV mucosal “high-risk” infections (xl7) and HPV-cancers (x28) in transplant recipients compared to healthy populations (Stockfleth Dermatol Surg. 2004 ;Shamanin et al J Natl Cancer Inst. 1996 ; Boyle et al Lancet. 1984; Andersson Am J Epidemiol. 2012). The burden of benign or malignant cutaneous HPV disease among OTRs is also substantial, with numerous studies reporting varying prevalence (35-37%) of HPV-linked benign warts post-transplant that increases with the duration of immune-suppression. While non-life threatening initially, these conditions severely affect the quality of life for patients. Furthermore, there is a growing association between keratotic lesions and SCC that are induced by P-HPV infection in adjunct with UV radiation in immune-compromised populations. Importantly, current HPV vaccines are unable to protect immune-compromised patients from the plethora of HPV infections and diseases.
[00011] The search for alternative broader-spectrum immunogens drew attention to the minor capsid protein L2, which is immunogenically subdominant in the context of coexpressed LI plus L2 capsids (Roden et al. (2000), Virology 270, 254-257). Immunization of animals with just the amino (N)-terminal peptide of L2 demonstrated its ability to elicit low- titer neutralizing antibodies that protect against in vivo challenge with cognate papillomavirus (PV) types and also cross-neutralize heterologous PV types. The subdominance of the L2 immunogen could be improved upon displaying regions of the L2 immunogen on the surface of virus-like particles or other carrier proteins (See, Roden et al., Papillomavirus-like particles (VLP) as broad spectrum human papillomavirus (HPV) vaccines, US9149503B2, US10046026B2; Embers et al. (2002), J. Virol. 76, 9798-805; Gaukroger et al. (1996), J. Gen. Virol. 77 (Pt 7), 1577-83, Kawana et al. (1999), J. Virol. 73, 6188-90; Pastrana et al. (2005a) Virology 337, 365-72; Roden et al. (2000) Virology 270, 254-257; (Gambhira et al. (2007a) J. Virol. 81, 11585-92).
[00012] Given the public health burden and continued risk of HPV disease, there is a need to develop cost-effective single valent or bi-valent immunogens or vaccinogens that exhibit efficacy against disease and provide cross -protection against multiple HPVs even in a co-infection setting than has been previously available in the art, and, preferably requiring lesser VLPs (to reduce manufacturing costs) and or fewer dosing administrations, in order to improve patient acceptance and compliance. [00013] The present invention provides cost-effective, simple vaccine formulations that address the inadequacies of the currently available HPV vaccines, and which can be administered in fewer doses than the current HPV vaccine solutions.
SUMMARY OF THE INVENTION
[00014] The present invention relates to a virus-like particle (VLP)-based bivalent vaccine composition, which includes:
[00015] a human papilloma virus 16 (HPV 16) LI protein into which is inserted, into a DE loop of the HPV 16 LI protein, a surface-displayed HPV 16 L2 peptide, wherein the surface-displayed HPV 16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 12); and
[00016] a human papilloma virus 18 (HPV18) LI protein into which is inserted, into a DE loop of the HPV 18 LI protein, a synthetic surface-displayed HPV L2 peptide;
[00017] wherein the surface-displayed HPV16 L2 peptide and the synthetic surface- displayed HPV L2 proteins are incorporated into the surface of their respective LI proteins, and
[00018] wherein the HPV16 and HPV18 LI proteins spontaneously form virus-like particles. The VLP-based bivalent vaccine compositions of the invention can be provided in a kit, e.g., for convenient clinical use.
[00019] Another embodiment of the invention is a method for immunizing or vaccinating a subject against a HPV, which involves administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
[00020] Still another embodiment of the invention is a method for inducing an immune response against HPV in a subject, involving administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
[00021] Also, an inventive method for treating a HPV infection in a subject having a HPV infection, or at risk of being exposed to HPV, is provided which comprises administering to the subject an effective amount of the VLP-based bivalent vaccine composition.
[00022] Because of the known association of certain cancers with HPV infection, the invention includes a method for preventing HPV-associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer, in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition. BRIEF DESCRIPTION OF THE DRAWINGS
[00023] Figure 1 demonstrates comparable HPV18 neutralization titers among all tested Candidates 4, 6, 7, 9 and 10 (see, Table 2 and Example 2, herein), as well as Gardasil® or Cervarix®. These results show that the insertion of additional epitopes into the DE loop region of HPV18 LI protein did not compromise the other HPV18 type-specific epitopes. [00024] Figure 2 shows an ELISA assay using a concatenated protein called “RGlx22” to evaluate the cross-antibody reactivity generated by the HPV18 chimeric VLP candidates (Candidate 4, 6, 7, 9 and 10; see, Table 2 and Example 2, herein) following vaccination. A positive control known to react with the RG1X22 peptide was included: HPV16 RG1VLP. Negative controls were wild type HPV 18 VLP with no peptide inserted into the DE-loop, and Cervarix®, both of which contain no L2 peptide inserts in the DE-loops of their VLPs.
[00025] Figure 3 shows results from ELISA assays using a concatenated protein called “RGlx22” to evaluate the cross-antibody reactivity generated by HPV16RG1-VLP (“RGVax prototype”) co-formulated with either HPV18 chimeric VLP Candidate 4, 9 or 10, following vaccination. Vaccinated mouse sera from Cervarix® which contains no L2 peptides in the DE-loops of their VLPs was used as a negative control.
[00026] Figures 4A-C show neutralization assay results comparing sera from mice vaccinated with either 2 or 3 doses of Cervarix® or HPV16RG1-VLP, co-formulated with HPV18 chimeric VLP Candidate 10 (“RGVax prototype + #10”). No detectable difference was observed in HPV16 LI titers (Figure 4A), 18 LI titers (Figure 4B), and L2 reactive antibody response (Figure 4C) between a 2-dose and 3-dose vaccination schedule, offering an advantage of fewer doses with the inventive vaccine composition, compared to previously known vaccine formulations.
[00027] Figure 5 show the results of neutralization assays against multiple HPV types using sera from either a 2-dose or 3-dose vaccination of HPV16 RG1 VLP, co-formulated with either HPV18-Beta 1-VLP, HPV18-Beta 2 VLP or Cervarix®. Pooled sera from each group of 5 mice were tested (triplicate studies).
[00028] Figure 6 shows a schematic alignment illustrating how Beta 1 and Beta 2 epitopes were designed. Both Beta 1 and Beta 2 epitopes contain a bio-informatically designed oligopeptide insert corresponding, respectively, to the amino acid residue positions 14-34 or positions 17-36 region of minor capsid protein L2 from the major seven HPV types 1, 4, 5, 8, 23, 38 and 76, well known to be associated with cutaneous HPV disease as well as cutaneous cancers; such pathogenic HPV types are commonly called “HPV beta types.” The Beta 3 epitope is designed by overlapping Beta 1 and Beta 2. Figure 6 shows, in the hash- bordered box, the alignment at amino acid residue positions 14-38 (in descending order): “HPV1-L2 SEQ1” (DIYPSCKISNTCPPDIQNKKIEHTTF/SEQ ID NO:28); “HPV4-L2 SEQ2” (NLYAKCQLSGNCLPDVKNKVEADTL//SEQ ID NO:29); “HPV5-L2 SEQ3” (HIYQTCKQAGTCPPDVINKVEQTTV//SEQ ID NO:30); “HPV8-L2 SEQ4” (HIYQTCKQAGTCPPDVINKVEQTTV//SEQ ID NO:31); “HPV23-L2 SEQ5” (DIYKGCKASGTCPPDVLNKVEQNTL//SEQ ID NO:32); “HPV38-L2 SEQ6” (DIYRGCKASNTCPPDVINKVEQSTE/SEQ ID NO:33); and “HPV76-L2 SEQ7” (HIYQSCKAAGTCPPDVLNKVEQTTI//SEQ ID NO:34).
[00029] Figures 7A-C shows transmission electron microscopy (TEM) imaging of HPV 18 Beta 1 (Figure 7A), Beta 2 (Figure 7B), or Beta 3 Figure 7C), following expression and purification. [00030] Figure 8 summarizes the results from ELISA assays testing mouse vaccinated sera from HPV18 Beta 1, HPV18 Beta2, or HPV18 Beta3, as well as HPV16 RG1-VLP and Gardasil®9 against HPV types 1, 2/27 (HPV2 and HPV27 having the same amino acid sequence), 4, 5/8 (HPV5 and HPV8 having the same amino acid sequence), 38, 76, 92. Reactivity is determined by “+” as the minimal whereby O.D600 is between 0.1 to 0.3, “++” as OD600 is between 0.3-0.99 and “+++” as O.D600 more than 1.0. O.D600 values that were less than 0.1 absorbance were considered non- reactive and denoted as
[00031] Figures 9A-E show results from passive transfer of rabbit vaccinated sera with
Gardasil®9, HPV16RG1-VLP, HPV18 Beta 1, HPV18 Beta 2 or HPV18 Beta 3 into (n = 3 mice per group) followed by vaginal challenge of HPV pseudo-virion (PsV) 5 (Figure 9A), PsV 6 (Figure 9B), PsV 11 (Figure 9C), PsV 51 (Figure 9D), or PsV 68 (Figure 9E).
[00032] Figures 10A-C illustrates the structure and results of a vaccine study evaluating HPV16 RG-1VLP (“16 RG1 monovalent”) or HPV16 RG1-VLP+ HPV18 Beta 2 (“RG1 Bi-valent”) or Gardasil®9 against in vivo co-infection and disease caused by 18 different HPV types. Figure 10A shows the experimental scheme of this vaccine study. Ten rabbits were vaccinated per group at months 0, 1 and 2. As a negative control, ten rabbits were also vaccinated with just aluminum hydroxide (Alhydrogel® alone; “Alum control”). Two weeks after the third vaccination, 5 rabbits (“Batch A”) were randomly selected from each treatment/control group (n = 10) for challenge with HPV 6,16, 31, 45, 52, 58, 35, 39, and 59 (results shown in Figure 10B). The remaining 5 rabbits (“Batch B”) were challenged with HPV 5, 11, 18, 26, 51, 56, 66, 68, and 73 (results shown in Figure 10C). In all rabbits, CRPV (cotton rabbit papillomavirus wildtype) was also tested as a technical control. Results showed that only Rabbits vaccinated with HPV16RG1-VLP + HPV18 Beta 2 (“RG1 Bivalent”) were able to achieve full protection from any papillomavirus infection and disease. DETAILED DESCRIPTION OF EMBODIMENTS
[00033] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[00034] Definitions
[00035] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. For example, reference to "a protein" includes one protein or a plurality of proteins; reference to "a peptide" includes one or a plurality of peptides.
[00036] As used herein, the term “antigen” is a molecule capable of being bound by an antibody, B-cell or T-cell receptor. An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the stimulation of B- and/or T- lymphocytes. The structural aspect of an antigen that gives rise to a biological response is referred to herein as an “antigenic determinant” or “epitope” and are synonymous. B- lymphocytes respond to foreign antigenic determinants via antibody production, whereas T- lymphocytes are the mediator of cellular immunity. Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies, B-cell receptors, or in the context of an MHC (both canonical and non-canonical), by T-cell receptors. An antigenic determinant or epitope need not be a contiguous/consecutive sequence or segment of protein and may include various sequences that are not immediately adjacent to one another.
[00037] With regard to a particular amino acid sequence, an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, B-cell receptor or in the context of T-cells, those residues necessary for recognition by T-cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. The amino acid residues of an epitope need not be contiguous/consecutive. In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, B-cell receptor or T-cell receptor or HLA molecule. Throughout this disclosure, “epitope” and “peptide” are often used interchangeably. [00038] As used herein, “B-cell epitope” or “target epitope” (e.g., HPV L2), refers to a feature of a peptide or protein that is recognized by a B-cell receptor in the immunogenic response to the peptide comprising that antigen (e.g., an HPV L2 epitope (immunogen or target epitope)).
[00039] As used herein “helper T-cell epitope” or “Th epitope” means a feature of a peptide or protein that is recognized by a T-cell receptor in the initiation of an immunologic response to the peptide comprising that antigen. Recognition of a T-cell epitope by a T-cell is generally believed to be via a mechanism wherein T-cells recognize peptide fragments of antigens which are bound to class I or class II Major Histocompatibility Complex (MHC) molecules expressed on antigen-presenting cells. In some embodiments of the present invention, the epitopes or epitopic fragments identified as described herein find use in the detection of antigen presenting cells having MHC molecules capable of binding and displaying the epitopes or fragments.
[00040] As used herein, “HPV” and “human papillomavirus” (used interchangeably herein) refer to the members of the family Papillomavirus that are capable of infecting humans. There are two major groups of HPVs defined by their tropism (genital/mucosal and cutaneous groups), each of which contains multiple virus “types” or “strains” (e.g., HPV16, HPV18, HPV31, HPV32, etc.). Of particular interest in the present invention are the HPV types that are associated with genital infection and malignancy on either mucosal or cutaneous (skin) tissues, as well as those that produce benign papillomas, both at mucosa and skin, resulting in morbidity to the patient.
[00041] The term “vaccine” refers to a formulation which contains 1, 2, 3, 4, 5, or more VLP compositions of the present invention. The VLP-based bivalent vaccine composition s will typically be in a form that is capable of being administered to a subject and induces a protective or therapeutic immune response sufficient to induce immunity to prevent and/or ameliorate an infection and/or to reduce at least one symptom of an infection and/or to enhance the efficacy of another anti-HPV therapy or prophylactic. Typically, some embodiments of the inventive VLP-based bivalent vaccine composition comprise a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved, although administration of dry powder, for example by inhalation, and even formulation with an additional adjuvant, such as aluminum hydroxide or aluminum phosphate (alum) or CpG 1018, and/or another pharmaceutically acceptable carrier, is also contemplated. The virus-like particle (VLP)-based bivalent vaccine composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat a HPV infection. Upon introduction into a host, an immunogenic composition of the invention (e.g., a vaccine) is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses. Typically, such a response will be cross reactive between, or among, various types of papillomavirus, including, but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more, of the HPV types described herein. Particular cross reactive HPV types are discussed elsewhere herein.
[00042] As used herein, “prophylactic” and “preventive” vaccines, antibodies or immune sera are vaccines, antibodies or immune sera that are designed and administered to prevent infection, disease, and/or any related sequela(e) caused by or associated with a pathogenic organism, particularly HPV.
[00043] As used herein, “therapeutic” vaccines are vaccines that are designed and administered to patients already infected with a pathogenic organism such as at least one HPV strain. Therapeutic vaccines (e.g., therapeutic HPV vaccines) are used to prevent and/or treat the development of benign or malignant tumors in these infected individuals.
[00044] The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result, such as inhibiting, reducing, or preventing viral infection, viral spread, viral growth, or viral transmission.
[00045] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
[00046] It is contemplated that one or more members of a list provided herein may be specifically excluded from or included in a claimed invention.
[00047] A “subject,” as used herein, includes any human or animal (e.g., a mammal) that has been infected with, or is at risk of, or is susceptible, to being infected with, a papillomavirus. A “susceptible” subject, or cell, is one that possesses receptors that can bind a particular papillomaviral virion or pseudo-virion particle, when the susceptible subject, or cell, is exposed to, or in contact with the particle. If genetic material is present in the particle, a viral replication process and/or disease process can ensue in the susceptible subject or cell. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, guinea pig or pig), farm animals (such as cattle), sporting animals (such as dogs or horses) and domestic animals or pets (such as a horse, dog or cat). Non-human primates and human patients are included.
[00048] The terms “protein,” “polypeptide,” “oligopeptide,” “peptide,” as used herein, are not restricted to any particular number of amino acid residues; these terms are sometimes used interchangeably herein. The properties and amino acid sequences of the proteins of the invention, and of the nucleic acids encoding them, are well-known and can be determined routinely, as well as downloaded from various known databases. See, e.g., the NCBI GenBank databases. Some sequences are provided herein. This information is accurate as of the date of filing of this application. However, some sequence information is routinely updated (e.g. to correct mistakes in the previous entries), so updated (corrected) information about the proteins and nucleic acids encoding them is included in this application.
Information provided in the sequence databases discussed herein is incorporated by reference in the present application. An “analogous protein segment” is a polypeptide or oligopeptide comprised within a particular first protein, which polypeptide or oligo peptide shares a conformational motif and/or functional homology, to a protein segment on a second protein, and is, thus, an “analogous protein segment” with respect to the protein segment on the second protein. (See, e.g., Samson et al., Protein segment finder: an online search engine for segment motifs in the PDB, Nucleic Acids Research, 2009, Vol. 37, Database issue doi:10.1093/nar/gkn833, 30 October 2008).
[00049] The chimeric proteins discussed herein are sometimes referred to herein as “proteins of the invention.” A chimeric “virus-like particle” or “VLP” of the invention, as used herein, refers to an empty viral capsid which is composed of papillomavirus LI protein molecules, into which are inserted a peptide of the minor viral capsid, L2. In a particular embodiment, the invention involves a human papilloma virus 16 (HPV16) LI protein (SEQ ID NO: 12) into which is inserted, into a DE loop of the HPV16 LI protein, a surface- displayed HPV16 L2 peptide, wherein the surface-displayed HPV16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 16).
[00050] The term “surface-displayed” means that a particular region or domain of the protein, e.g., the inserted L2 peptide, has a thermodynamic affinity for water, and, thus, as a hydrophilic moiety, this region or domain tends be disposed or reside on the exterior surface of the chimeric protein and/or VLP, in contact with the aqueous environment. [00051] The amino acid sequence of the wild type HPV16 LI protein (SEQ ID NO: 12) is the following:
MSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILV PKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISG HPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPC TNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYP DYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLASS NYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLC AAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWN FGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLD QFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL//SEQ ID NO: 12. [00052] In one embodiment, the surface-displayed HPV16 L2 peptide having the amino acid sequence of SEQ ID NO: 16 is inserted into the DE loop of the HPV16 LI protein between amino acid positions 136 and 137 of SEQ ID NO: 12. This yields a chimeric protein having the amino acid sequence of SEQ ID NO: 15 (underlined residues show placement of SEQ ID NO: 16 in the primary structure):
MSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILV PKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISG HPLLNKLDDTENASAYAOLYKTCKQAGTCPPDIIPKVANAGVDNRECISMDYKQTQ LCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTT LQANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGE NVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICW GNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITL T AD VMTYIHSMNS TILED WNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDP LKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTT AKRKKRKL//SEQ ID NO: 15.
[00053] A useful coding sequence for SEQ ID NO: 15 is the following nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence degenerate thereto:
CATATGAGCGAGGCGACCGTTTACCTGCCGCCGGTGCCGGTTAGCAAAGTGGTT AGCACCGACGAATATGTGGCGCGTACCAACATCTACTATCATGCGGGTACCAGC CGTCTGCTGGCGGTTGGTCACCCGTACTTCCCGATCAAGAAACCGAACAACAAC AAAATTCTGGTTCCGAAAGTGAGCGGCCTGCAGTATCGTGTGTTCCGTATCCACC TGCCGGACCCGAACAAATTCGGTTTTCCGGACACCAGCTTTTACAACCCGGATAC CCAACGTCTGGTTTGGGCGTGCGTGGGCGTTGAAGTGGGTCGTGGTCAACCGCTG GGTGTGGGTATCAGCGGTCACCCGCTGCTGAACAAGCTGGACGATACCGAGAAC GCGAGCGCGTACGCGCAGCTGTATAAAACCTGCAAGCAAGCGGGTACCTGCCCG CCGGACATCATTCCGAAAGTTGCGAACGCGGGTGTGGACAACCGTGAGTGCATC AGCATGGATTACAAGCAGACCCAACTGTGCCTGATCGGCTGCAAACCGCCGATT GGTGAACACTGGGGCAAGGGCAGCCCGTGCACCAACGTTGCGGTGAACCCGGGT GATTGCCCGCCGCTGGAGCTGATCAACACCGTTATTCAGGACGGTGATATGGTGG ACACCGGTTTCGGCGCGATGGATTTTACCACCCTGCAAGCGAACAAGAGCGAAG TGCCGCTGGACATCTGCACCAGCATTTGCAAATACCCGGATTATATTAAGATGGT TAGCGAGCCGTACGGCGACAGCCTGTTCTTTTATCTGCGTCGTGAACAGATGTTC GTGCGTCACCTGTTTAACCGTGCGGGTGCGGTTGGTGAAAACGTGCCGGACGATC TGTACATCAAAGGTAGCGGCAGCACCGCGAACCTGGCGAGCAGCAACTATTTCC CGACCCCGAGCGGTAGCATGGTGACCAGCGACGCGCAGATCTTTAACAAGCCGT ACTGGCTGCAGCGTGCGCAAGGTCACAACAACGGCATTTGCTGGGGTAACCAAC TGTTCGTTACCGTGGTTGATACCACCCGTAGCACCAACATGAGCCTGTGCGCGGC GATTAGCACCAGCGAGACCACCTATAAAAACACCAACTTTAAGGAATACCTGCG TCACGGCGAGGAATATGACCTGCAGTTCATCTTTCAACTGTGCAAAATTACCCTG ACCGCGGATGTTATGACCTACATCCACAGCATGAACAGCACCATTCTGGAGGATT GGAACTTTGGTCTGCAGCCGCCGCCGGGTGGTACCCTGGAAGATACCTATCGTTT TGTGACCAGCCAGGCGATTGCGTGCCAAAAACATACCCCGCCGGCGCCGAAGGA AGACCCGCTGAAGAAATACACCTTCTGGGAAGTGAACCTGAAGGAAAAATTTAG CGCGGACCTGGATCAGTTCCCGCTGGGTCGTAAGTTTCTGCTGCAAGCGGGTCTG AAGGCGAAACCGAAGTTCACCCTGGGTAAACGTAAAGCGACCCCGACCACCAGC AGCACCAGCACCACCGCGAAGCGTAAGAAACGTAAACTGTAAGGATCC//SEQ ID N0:14.
[00054] The inserted HPV16 L2 peptide is inserted into a suitable region of the HPV16
LI protein (SEQ ID NO: 12) so that it is displayed on the surface of the VLP. In some embodiments of the invention, the L2 peptide is inserted in the DE loop of LI, e.g., between amino acid residue positions 136 and 137 of HPV16 LI (SEQ ID NO: 12), i.e., inserted into SEQ ID NO: 12 at AA136/137.
[00055] In another aspect of the inventive, an HPV18 LI protein (SEQ ID NO: 13) has inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide; the inserted peptide comprises one or more epitopes (e.g., neutralizing epitopes) that are cross -reactive with a broad spectrum of HPV types. The term “synthetic,” with respect to the surface-displayed HPV L2 peptide that is inserted into a DE loop of the HPV18 LI protein, means that this recombinantly inserted protein segment is of exogenous origin, i.e., has an amino acid sequence obtained from an HPV type other than a native or wild type HPV 18 L2 peptide sequence. For example, the surface-displayed HPV L2 peptide is an analogous protein segment from a HPV type selected from HPV1, HPV4, HPV5, HPV8, HPV23, HPV38, or HPV76. In one embodiment, the L2 peptide inserted into the HPV 18 LI comprises amino acid residues 17-36 of the HPV16 L2 protein (SEQ ID NO: 16), or an equivalent sequence, or analogous protein segment, of amino acid residues from another HPV type. Optionally, the recombinantly inserted L2 protein segment of exogenous origin is derived through bioinformatic design, e.g., through the use of bioinformatic data and suitable artificial intelligence platforms. In some embodiments of the invention, the L2 peptide is inserted in the DE loop of HPV18 LI, e.g. between amino acid residue positions 134 and 135 of SEQ ID NO: 13, i.e., inserted into SEQ ID NO: 13 at AA134/135.
[00056] Other examples of the useful analogous protein segment inserted into the DE loop of HPV18 LI protein (SEQ ID NO: 13) include peptides with the following amino acid sequences:
[00057] GGLGIGTGSGTGGRTGYIPL(SEQ ID NO: 17),
[00058] GGLGIGTARGSGGRIGYTPL (SEQ ID NO: 18),
[00059] GGLGIGTGRGSGGSTGYNPI (SEQ ID NO: 19),
[00060] DIYASCKISNTCPPDVQNKV (SEQ ID NO:20),
[00061] GGLGIGTGRGSGGATGYVPI (SEQ ID NO:21),
[00062] GTGGRTGYVPLGTRPPTVVD (SEQ ID NO:22),
[00063] QSCKASGTCPPDVINKVEQT (SEQ ID NO:23),
[00064] HIYQSCKASGTCPPDVINKVE (SEQ ID NO:24),
[00065] HIYQSCKASGTCPPDVINKVEQT (SEQ ID NO:25),
[00066] QLYQTCKAAGTCPPDVIPKV (SEQ ID NO:26),
[00067] GGLGIGTGSGTGGRTGYVPL (SEQ ID NO:27),
[00068] DIYPSCKISNTCPPDIQNKKIEHTTI (SEQ ID NO:28),
[00069] NLYAKCQLSGNCLPDVKNKVEADTL (SEQ ID NO:29),
[00070] HIYQTCKQAGTCPPDVINKVEQTTV (SEQ ID NO:30),
[00071] HIYQTCKQAGTCPPDVINKVEQTTV (SEQ ID NOG 1),
[00072] DIYKGCKASGTCPPDVLNKVEQNTL (SEQ ID NO:32), [00073] DIYRGCKASNTCPPDVINKVEQSTI (SEQ ID NO:33), or
[00074] HIYQSCKAAGTCPPDVLNKVEQTTI (SEQ ID NO:34).
[00075] For example, the amino acid sequence of wild type HPV18 LI protein is the following SEQ ID NO: 13: MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGG GNKQDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPL GVGLSGHPFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHW AKGTACKSRPLSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDIC QSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTG MRASPGSCVYSPSPSGSIVTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTT RSTNLTICASTQSPVPGQYDATKFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSM NSSILEDWNFGVPPPPTTSLVDTYRFVQSVAITCQKDAAPAENKDPYDKLKFWNVDL KEKFSLDLDQYPLGRKFLVQAGLRRKPTIGPRKRSAPSATTSSKPAKRVRVRARK//SE Q ID NO: 13.
[00076] In some embodiments of the inventive VLP-based bivalent vaccine composition, analogous protein segment inserted into the DE loop of HPV18 LI protein (SEQ ID NO: 13) can be an amino acid sequence variant of any one of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34.
[00077] For example, some useful embodiments of the human papilloma virus 18 (HPV18) LI protein into which is inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide, include chimeric proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11, as shown in Table 1 (below).
[00078] Table 1 (next page). Non-limiting examples of chimeric HPV18 LI protein sequences into which is inserted into a DE loop of the HPV18 LI protein (SEQ ID NO: 13), a synthetic surface-displayed HPV L2 peptide. Underlined residues show placement of the synthetic surface-displayed HPV L2 peptide in the primary structure of the chimeric protein.
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
[00079] Ordinarily, amino acid sequence variants will have an amino acid sequence having at least 80% amino acid sequence identity with the original amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, more preferably at least 85% identity, even more preferably at least 90% identity, and most preferably at least 95% identity, including for example, 80%>, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Identity or homology with respect to the amino acid sequence is defined herein as the percentage of amino acid residues in the sequence that are identical with the original sequence, after aligning the sequences and candidate introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alternatively, the analogous protein segment inserted into the DE loop of HPV18 LI protein (SEQ ID NO: 13) can be the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, but having 1, 2, 3, or 4 conservative amino acid substitutions.
[00080] Amino acid residues are commonly categorized according to different chemical and/or physical characteristics. For example amino acid residues can be in D- or L- form, but for the purposes of the present invention the L-form of the amino acid is intended. The term "acidic amino acid residue" refers to amino acid residues in having side chains comprising acidic groups. Exemplary acidic residues include aspartic acid and glutamic acid residues. The term "basic amino acid residue" refers to amino acid residues having side chains comprising basic groups. Exemplary basic amino acid residues include histidine, lysine, homolysine, ornithine, or arginine residues. The term "hydrophilic amino acid residue" refers to amino acid residues having side chains comprising polar groups. Exemplary hydrophilic residues include cysteine, serine, threonine, histidine, lysine, asparagine, aspartate, glutamate, and glutamine residues. The terms "lipophilic amino acid residue" refers to amino acid residues having sidechains comprising uncharged, aliphatic or aromatic groups. Exemplary lipophilic sidechains include phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine. Alanine is amphiphilic — it is capable of acting as a hydrophilic, or lipophilic (i.e., hydrophobic), residue. Alanine, therefore, is included within the definition of both "lipophilic" (i.e., "hydrophobic") residue and "hydrophilic" residue. The term "nonfunctional" or "neutral" amino acid residue refers to amino acid residues in having side chains that lack acidic, basic, or aromatic groups. Exemplary neutral amino acid residues include methionine, glycine, alanine, valine, isoleucine, leucine, and norleucine residues. The term "aromatic amino acid residue" refers to amino acid residues having side chains comprising aromatic groups. Exemplary aromatic residues include tryptophan and tyrosine. A "conservative amino acid substitution" involves a substitution of a native amino acid residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, a “conservative amino acid substitution” includes any native residue in the polypeptide substituted with alanine, as has been previously described for "alanine scanning mutagenesis" (see, for example, MacLennan et al, Acta Physiol. Scand. SuppL, 643:55-67 (1998); Sasaki et al, 1998, Adv. Biophys. 35: 1-24 (1998), which discuss alanine scanning mutagenesis).
[00081] The chimeric HPV16 LI proteins and HPV18 LI proteins assemble spontaneously into VLP and resemble native virions in both structure and immunogenicity, yet lack nucleic acid and thus are non-oncogenic and non-infectious. The term “assemble spontaneously” means that the chimeric proteins of the invention form into complete VLP structures without enzymatic involvement or ATP expenditure, based on favorable thermodynamic values for VLP formation under the reaction conditions such as McCarthy et al., Journal of Virology 1998 Jan; 72(1): 32-41 ; Mukherjee et al., 2008 Journal of Molecular Biology, 381( b:229-37. Mach et ak, 2006 Journal of Pharmaceutical sciences 95( 10):2195-
[00082] One aspect of the invention is a method for making a VLP (or the polypeptide component thereof) of the invention. In one embodiment of the invention, HPV epitopes are synthesized using conventional methods as modified for the particular amino acid sequences. Such techniques include, e.g., methods well known to those skilled in the art of peptide synthesis, e.g., solution phase synthesis [see Finn et al. in Proteins, 3rd Ed., Neurath and Hill (Eds), Academic Press, NY, 2, 105-253, 1976], or solid phase synthesis [see Barany et al. In: The Peptides , Gross and Meienhofer (Eds.), Academic Press, NY, 3-284, 1979], or stepwise solid phase synthesis as reported by Merrifield et al. (1963) J. Am. Chem. Soc. 85, 2149- 2154], the contents of each of which are incorporated herein by reference. Other references to peptide synthesis techniques include peptides synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al. (1981) J. Org. Chem. 46, 3433, peptides synthesized using an Fmoc/tBu procedure (Atherton et al. In: Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989). Fmoc amino acids can be obtained from various vendors, e.g., Chem-Impex International (Wood Dale, Ill., USA), Merck Biosciences (Nottingham, UK), and Bachem UK Ltd. (St. Helens, UK).
[00083] Alternatively, a polypeptide of the invention can be prepared recombinantly. The present invention provides recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors. Expression vectors comprising DNA may be used to prepare the polypeptides or polypeptide fragments of the invention encoded by a DNA. A method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture. The skilled artisan will recognize that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is membrane-bound or a soluble form that is secreted from the host cell. Polypeptides of the invention can include various leader sequences that direct trafficking or assist in purification.
[00084] Any suitable expression system may be employed. The vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
[00085] Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al In: Cloning Vectors: A Laboratory Manual , Elsevier, N Y, 1985. Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein. In general, molecular biology methods referred to herein are well-known in the art and are described, e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & sons, New York, N.Y.
[00086] Methods for allowing polypeptides to assemble into VLPs are well-known and conventional, as are methods for purifying them for use in subjects. For “suitable conditions for self-assembly,” see, e.g., the methods described in the Examples herein, or in Kirnbauer et al. (1993) J Virol 67, 6929-6936; Volpers et al. (1994) Virology 200, 504-512; or J Mol Biol 2001 Mar. 16; 307(1): 173-82, all of which are incorporated by reference for the descriptions of such methods.
[00087] The methods of the present invention include prevention and/or treatment for a disease or condition caused by or related to papillomavirus infection (e.g., HPV infection). An immunogenic HPV peptide and/or antibody that binds the same, can be given to induce or provide a protective and/or therapeutic response in a subject infected with or suspected of having been exposed to or at risk of becoming infected with HPV. Methods may be employed with respect to individuals who have tested positive for exposure to HPV or who are deemed to be at risk for infection based on possible exposure.
[00088] In some embodiments, the treatment is administered in the presence of adjuvants or carriers or other antigens, either HPV antigens or antigens from other pathogens. Furthermore, in some examples, treatment comprises administration of other agents commonly used against viral infection, such as one or more anti-virals.
[00089] The immunogenicity of VLP compositions can be enhanced by the use of additional non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions such as alum.
[00090] A number of adjuvants can be used to enhance an antibody response against a VLP described herein. Adjuvants can be used to (1) trap the antigen in the body to cause a slow release; (2) attract cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of the antigen throughout the subject's body.
[00091] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL- 12, y-interferon, GM-CSF, BCG, aluminum salts, such as aluminum hydroxide, aluminum phosphate, or other aluminum salt or compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), CpG-1018, lipid A, and monophosphoryl lipid A (MPL), or inactivated microbial agents. RIB I, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used. Other adjuvants or methods are exemplified in U.S. Pat. Nos. 6,814,971, 5,084,269, 6,656,462, each of which is incorporated herein by reference).
[00092] Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or aluminum phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (CARBOPOL®) used as an about 0.25% solution, aggregation of a protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101° C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of Gram-negative bacteria; emulsion in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsion with a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used as a block substitute may also be employed to produce an adjuvant effect. A typical adjuvant is complete Freund's adjuvant (containing killed Mycobacterium tuberculosis'), incomplete Freund's adjuvants, and aluminum hydroxide.
[00093] For administration to humans, a variety of suitable adjuvants will be evident to a skilled worker. These include, e.g., Alum-MPL as adjuvant, or the comparable formulation, ASO4, which is used in the approved HPV LI vaccine Cervarix®, AS03, AS02, MF59, montanide, saponin-based adjuvants such as GPL0100, CpG-based adjuvants (e.g., CpG- 1018), or imiquimod. In embodiments of the invention, an adjuvant is physically coupled to the VLP, or encapsulated by the VLP, rather than simply mixed with them.
[00094] In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) to enhance immune responses. BRMs have been shown to upregulate T cell immunity or downregulate suppresser cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m2 ) (Johnson/Mead, NJ) and cytokines such as y-interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7. In embodiments of the invention, these genes are encapsulated by the VLP to facilitate their delivery into a subject.
[00095] The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally by injection, inhalation of a powder, via transcutaneous patch, via vaginal instillation and the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.
[00096] The preparation of vaccines that contain polypeptide or peptide sequence(s) as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines. In specific embodiments, vaccines are formulated with a combination of substances, as described in U.S. Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.
[00097] Vaccines may be administered by inhalation. In certain embodiments a vaccine can be administered as an aerosol. As used herein the term “aerosol” or “aerosolized composition” refers to a suspension of solid or liquid particles in a gas. The terms may be used generally to refer to a composition that has been vaporized, nebulized, or otherwise converted from a solid or liquid form to an inhalable form including suspended solid or liquid drug particles. Such aerosols can be used to deliver a vaccine via the respiratory system. As used herein, “respiratory system” refers to the system of organs in the body responsible for the intake of oxygen and the expiration of carbon dioxide. The system generally includes all the air passages from the nose to the pulmonary alveoli. In mammals it is generally considered to include the lungs, bronchi, bronchioles, trachea, nasal passages, and diaphragm. For purposes of the present disclosure, delivery of a vaccine to the respiratory system indicates that a drug is delivered to one or more of the air passages of the respiratory system, in particular to the lungs.
[00098] Additional formulations which are suitable for other modes of administration include suppositories (for anal or vaginal application) and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
[00099] The VLP-based bivalent vaccine compositions may be formulated into a vaccine as neutral or salt forms. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
T1 [000100] In many instances, it will be desirable to have multiple administrations of the vaccine, usually at most, at least, or not exceeding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more vaccinations including all ranges there between. The vaccinations will normally be at 1, 2, 3, 4, 5, 6, to 5, 6, 7, 8, 9, 10, 11, to 12 week/month/year intervals, including all values and ranges there between, more usually from three to five week intervals. Typically, periodic boosters at intervals of 1-15 years, usually ten years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies against the antigens, as described supra, U.S. Pat. Nos. 3,791,932;
4,174,384 and 3,949,064, which are illustrative of these types of assays.
[000101] The compositions and related methods of the present invention, particularly administration of a VLP comprising an HPV L2 epitope to a patient/subject, may also be used in combination with the administration of traditional HPV screening and/or other vaccines, including, e.g., antibodies or antibody fragments, Pap smears, PCR, Southern blotting, administering CERVARIX™, GARDASILO9™, vaccines for HPV or other infectious agents, ablative therapy of HPV lesions, immunomodulatory therapies for HPV lesions (e.g. Aldara™), or the like.
[000102] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects of the present invention involve administering an effective amount of a composition to a subject. In some embodiments of the present invention, a VLP comprising an HPV L2 epitope is administered to the patient to protect against or treat infection by one or more HPV pathogens. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
[000103] As used herein, the term “pharmaceutically acceptable” or “pharmacologically acceptable” refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. The term “pharmaceutically acceptable carrier,” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
Pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. [000104] The active compounds of the present invention can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. In addition to the compounds formulated for aerosol or parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules.
[000105] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[000106] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
[000107] The VLP-based bivalent vaccine compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
[000108] The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[000109] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[000110] Administration of the compositions according to the present invention will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, respiratory, or intravenous administration. In certain embodiments, a vaccine composition may be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
[000111] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in isotonic NaCl solution and either added to hypodermoclysis fluid or injected at the proposed site of infusion (see for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
[000112] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. An “effective amount” is an amount that is effective to bring about a desired outcome (e.g., the induction of a measurable amount of an immune response, the immunization of a subject, etc.). The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.
[000113] Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. [000114] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
[000115] In one embodiment of the invention, VLPs are administered to subjects by administering an effective amount of a recombinant attenuated bacterium (such as a salmonella bacterium) which encodes a chimeric polypeptide of the invention. The VLPs are then produced by the gut in vivo, where the bacteria replicate. For guidance for carrying out methods using such bacterial vectors, see, e.g., Nardelli-Haefliger (2007) Clin Vaccine Immunol 14, 1285-1295, which is incorporated by reference specifically for such disclosure. Methods for generating recombinant constructs that can be expressed in bacteria (bacterial vectors) are conventional; some typical methods are described elsewhere herein. Lyophilized bacteria can be easily shipped to developing countries, where they can then be resuspended and administered to subjects. Such a mode of administration is advantageous in a country to lacks refrigeration capabilities that might be required for other formulations of VLPs. In another embodiment, VLPs are administered in an attenuated virus, such as an attenuated Adenovirus, or other viral vectors which are well-known to those of skill in the art. Methods for producing suitable recombinant nucleic acids that can be expressed in a viral host are conventional, and some such methods are discussed elsewhere herein.
[000116] The present invention includes compositions for preventing or ameliorating HPV infections. As such, the invention contemplates vaccines for use in both active and passive immunization embodiments.
[000117] One embodiment of the invention is a method of preparing an immunoglobulin for use in prevention or treatment of HPV infection comprising the steps of immunizing a recipient with a vaccine of the invention and isolating immunoglobulin or antibodies from the recipient, and/or recombinantly producing such immunoglobulins or fragments thereof. An immunoglobulin prepared by this method is a further aspect of the invention. A pharmaceutical composition comprising the immunoglobulin of the invention and a pharmaceutically acceptable carrier is a further aspect of the invention which could be used in the manufacture of a medicament for the treatment or prevention of HPV infection. A method for treatment or prevention of HPV infection comprising a step of administering to a patient an effective amount of the pharmaceutical preparation of the invention is a further aspect of the invention.
[000118] Inocula for polyclonal antibody production are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition. An immuno stimulatory amount of inoculum is administered to a mammal, e.g., a human, and the inoculated subject is then maintained for a time sufficient for the antigenic composition to induce protective antibodies. The antibodies can be isolated to the extent desired by well known techniques such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual 1988).
[000119] Antibodies can include antiserum preparations from a variety of commonly used animals, e.g., goats, primates, donkeys, swine, horses, guinea pigs, rats, or man. The animals are bled and serum recovered.
[000120] An immunoglobulin produced in accordance with the present invention can include whole antibodies, antibody fragments or subfragments. Antibodies can be whole immunoglobulins of any class, e.g., IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with dual specificity to two or more antigens of the invention. They may also be fragments, e.g., F(ab')2, Fab', Fab, Fv and the like including hybrid fragments. An immunoglobulin can also include natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex.
[000121] An HPV composition or vaccine of the present invention can be administered to a recipient who then acts as a source of immunoglobulin, produced in response to challenge from the HPV composition. A subject thus treated would donate plasma from which hyperimmune globulin would be obtained via conventional plasma fractionation methodology. The hyperimmune globulin would be administered to another subject in order to impart resistance against or treat HPV infection. Hyperimmune globulins of the invention are particularly useful for treatment or prevention of HPV infection in infants, immune compromised individuals or where treatment is required and there is no time for the individual to produce antibodies in response to vaccination.
[000122] An additional aspect of the invention is a pharmaceutical composition comprising one or more monoclonal antibodies (or fragments thereof; preferably human or humanized) reactive against constituents of the immunogenic composition of the invention, which could be used to treat or prevent infection by multiple HPV types.
[000123] Methods of making monoclonal antibodies are well known in the art and can include the fusion of splenocytes with myeloma cells (Kohler et al. (1975) Nature 256, 495; Harlow et al. Antibodies: A Laboratory Manual, 1988). Alternatively, monoclonal Fv fragments can be obtained by screening a suitable phage display library (Vaughan et al. (1998) Nat. Biotech. 16, 535-539). Monoclonal antibodies may be human, humanized, or partly humanized by known methods.
[000124] Another aspect of the invention is a kit for vaccination or treatment according to the present invention. In one embodiment, the kit comprises a vial and optionally a package insert with administration instructions, the vial comprises a VLP composition or vaccine for administration according to the methods of the present invention.
[000125] Any of the compositions described herein may be included in a kit. In a nonlimiting example, reagents for preparing a VLP and/or administering a VLP, or antibodies generated by vaccination with VLP can be included in a kit. The kit may further include reagents for assessing the activity of the VLP both in vitro and in vivo. The kits will thus comprise, in suitable container, a VLP composition. In certain aspects, the kit can include reagents and/or devices for administration, e.g., inhaler or nebulizer. It may also include one or more buffers, compounds, or devices for preparing the composition for administration. [000126] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
[000127] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
[000128] A kit may also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
[000129] It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the preparation and/or administration of a VLP vaccine of the invention. [000130] Among other uses, kits of the invention can be used in experimental applications. A skilled worker will recognize components of kits suitable for carrying out a method of the invention.
[000131] By way of further illustration, the following numbered embodiments are encompassed by the present invention:
[000132] Embodiment 1: A virus-like particle (VLP)-based bivalent vaccine composition, comprising:
[000133] a human papilloma virus 16 (HPV16) LI protein into which is inserted, into a DE loop of the HPV16 LI protein, a surface-displayed HPV16 L2 peptide, wherein the surface-displayed HPV16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 16); [000134] a human papilloma virus 18 (HPV18) LI protein into which is inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide;
[000135] wherein the surface-displayed HPV16 L2 peptide and the synthetic surface- displayed HPV L2 proteins are incorporated into the surface of their respective LI proteins, and
[000136] wherein the HPV16 and HPV18 LI proteins spontaneously form virus-like particles.
[000137] Embodiment 2: The VLP-based bivalent vaccine composition of Embodiment 1, wherein the HPV16 LI protein comprises the amino acid sequence of SEQ ID NO: 12, and wherein the surface-displayed HPV 16 L2 peptide is inserted into the DE loop of the HPV 16 LI protein between amino acid residue positions 136 and 137 of SEQ ID NO: 12.
[000138] Embodiment 3: The VLP-based bivalent vaccine composition of any of Embodiments 1-2, wherein the surface-displayed HPV L2 peptide is an analogous protein segment from a HPV type selected from the group consisting of HPV1, HPV4, HPV5, HPV8, HPV23, HPV38, and HPV76.
[000139] Embodiment 4: The VLP-based bivalent vaccine composition of any of Embodiments 1-3, wherein the HPV 18 LI protein comprises the amino acid sequence of SEQ ID NO: 13.
[000140] Embodiment 5: The VLP-based bivalent vaccine composition of any of Embodiments 1-4, wherein the synthetic surface-displayed HPV L2 peptide is inserted into the DE loop of the HPV18 LI protein between amino acid residue positions 134 and 135 of SEQ ID NO: 13.
[000141] Embodiment 6: The VLP-based bivalent vaccine composition of any of Embodiments 1-5, wherein the synthetic surface-displayed HPV L2 peptide, inserted into the DE loop of the HPV 18 LI protein, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.
[000142] Embodiment 7: The VLP-based bivalent vaccine composition of any of Embodiments 1-6, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV 18 LI protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, or the amino acid sequence of any these members with 1-4 conservative amino acid substitutions.
[000143] Embodiment 8: The VLP-based bivalent vaccine composition of any of Embodiments 1-7, wherein the HPV18 LI protein comprises the amino acid sequence of SEQ ID NO: 13; wherein the synthetic surface-displayed HPV L2 peptide is inserted into the DE loop of the HPV18 LI protein between amino acid residue positions 134 and 135 of SEQ ID NO: 13; and wherein the synthetic surface-displayed HPV L2 peptide comprises an amino acid sequence selected from the group consisting SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:27 , or the amino acid sequence of any these members with 1-4 conservative amino acid substitutions.
[000144] Embodiment 9: The VLP-based bivalent vaccine composition of any of Embodiments 1-8, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV 18 LI protein comprises the amino acid sequence of HIYQSCKASGTCPPDVINKVE (SEQ ID NO:24), or the amino acid sequence of SEQ ID NO:24 with 1-4 conservative amino acid substitutions.
[000145] Embodiment 10: The VLP-based bivalent vaccine composition of any of Embodiments 1-9, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV18 LI protein comprises an amino acid sequence variant at least 80, 85, 90 or 95% identical to the amino acid sequence of SEQ ID NO:24.
[000146] Embodiment 11: The VLP-based bivalent vaccine composition of any of Embodiments 1-10, further comprising an adjuvant.
[000147] Embodiment 12: The VLP-based bivalent vaccine composition of Embodiment 11, wherein the adjuvant comprises aluminum hydroxide and/or aluminum phosphate.
[000148] Embodiment 13: The VLP-based bivalent vaccine composition of any of Embodiments 1-12, further comprising a pharmaceutically acceptable carrier.
[000149] Embodiment 14: The VLP-based bivalent vaccine composition of any of Embodiments 1-13, wherein the composition is formulated for administration by inhalation, ingestion, or in a viral or bacterial vector.
[000150] Embodiment 15: The VLP-based bivalent vaccine composition of any of Embodiments 1-14, wherein the composition is in a formulation for intramuscular injection. [000151] Embodiment 16: The VLP-based bivalent vaccine composition of any of Embodiments 1-15, wherein the composition is packaged in a glass vial for single or multiple-dose use.
[000152] Embodiment 17: The VLP-based bivalent vaccine composition of any of Embodiments 1-16, which is an immunogenic composition.
[000153] Embodiment 18: The VLP-based bivalent vaccine composition of any of Embodiments 1-17, which is immunogenic against mucosal high-risk or low- risk, cutaneous low risk, and/or cutaneous beta-type papillomaviruses.
[000154] Embodiment 19: The VLP-based bivalent vaccine composition of any of Embodiments 1-18, which is immunogenic against one or more papillomaviruses selected from the group consisting of HPV1, HPV2, HPV4, HPV5, HPV16, HPV18, HPV26, HPV35, HPV38, HPV39, HPV45, HPV52, HPV58, HPV68, HPV76, and HPV92.
[000155] Embodiment 20: The VLP-based bivalent vaccine composition of any of Embodiments 1-19, wherein the composition is effective in preventing human papillomavirus infection in a susceptible subject.
[000156] Embodiment 21: The VLP-based bivalent vaccine composition of
Embodiment 20, which prevents co-infection in the subject by the following human papillomavirus types: HPV 6, 16, 31, 45, 52, 58, 35, 39 and 59.
[000157] Embodiment 22: The VLP-based bivalent vaccine composition of any of Embodiments 20-21, which prevents co-infection in the subject by the following human papillomaviruse types: HPV 5, 11, 18, 26, 51, 56, 66 ,68 and 73.
[000158] Embodiment 23: The VLP-based bivalent composition of any of Embodiments 20-22, which prevents co-infection in the subject by the following human papillomaviruse types: HPV 6, 11, 16, 18, 26, 31, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 73.
[000159] Embodiment 24: A method for immunizing or vaccinating a subject against a HPV, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1-23.
[000160] Embodiment 25: The method of Embodiment 24, wherein administering the effective amount of the VLP-based bivalent vaccine composition comprises a 2-dose or 3- dose vaccination schedule.
[000161] Embodiment 26: A method for inducing an immune response against HPV in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1-23. [000162] Embodiment 27: A method for treating a HPV infection in a subject having a HPV infection or at risk of being exposed to HPV, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1- 23.
[000163] Embodiment 28: A method for preventing HPV- associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer, in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of any of Embodiments 1-23.
[000164] Embodiment 29: A kit comprising the VLP-based bivalent composition of any of Embodiments 1-23.
[000165] Embodiment 30: The VLP-based bivalent composition of any of Embodiments 1-23, for treating a HPV infection in a subject having a HPV infection.
[000166] Embodiment 31 : The VLP-based bivalent composition of any of Embodiments 1-23, for preventing HPV-associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer in a subject.
[000167] In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
[000168]
[000169] The following working examples are illustrative and not to be construed in any way as limiting the scope of the invention.
[000170]
EXAMPLES
[000171] Example 1. Epitope selection and Recombinant HPV 18 RG-1 VLP protein design
[000172] Ten chimeric HPV18 virus-like particle (VLP) candidates were designed to have a particular peptide sequence (or “epitope”) inserted into AA134/135 of the DE loop region of HPV18 major capsid protein LI (i.e., between amino acid residue positions 134 and 135 of SEQ ID NO: 13). The conserved L2 epitopes selected are in Table 2 (below).
[000173] Table 2. Summary of 10 different chimeric HPV18 VLPs candidates with a specific HPV minor capsid L2 peptide sequence inserted between residues 134/135 of the DE loop region of HPV18 major capsid protein LI (SEQ ID NO: 13). The ability of these Candidates 1-10 to form 50- to 60-nm VLPs upon expression and purification is indicated (Y = yes, N = no).
Figure imgf000040_0001
Figure imgf000041_0001
[000174] Candidates 1-3 (Table 2) contained HPV minor capsid protein L2 epitopes corresponding to the amino acid residue positions 56-75 region from wildtype HPV types 16, 1, and 4 respectively.
[000175] Candidates 4 and 5 (Table 2) contained a bio-informatically designed epitope corresponding respectively to the amino acid residue positions 17-36 region or amino acid residue positions 55-75 region of minor capsid protein L2 from HPV types 1, 4 and 38; these three HPV types are known to be genetically distant from one another and cause hand/foot warts, flat warts and non-melanoma skin cancer, respectively.
[000176] Candidates 6, 7, and 8 (Table 2), each contained a bio-informatically designed epitope corresponding to amino acid residue positions 17-36 , 55-75, or 65-85 regions, respectively, of minor capsid protein L2 from 36 HPV types known to be associated with all known clinically relevant HPV-associated clinical disease or cancer. These 36 HPV types are HPVs: 1, 3, 4, 5, 6, 8, 11, 16, 18, 23, 26, 27, 31, 32, 33, 34, 35, 38, 39, 40, 42, 43, 44, 45, 51, 52, 53, 56, 57, 58, 59, 66, 68, 70, 73, and 76.
[000177] Candidates 9 and 10 (Table 2) contained a bio-informatically designed epitope corresponding to the amino acid residue positions 14-34 and 17-36 regions, respectively, of minor capsid protein L2 from the major seven HPV types most known to be associated with cutaneous HPV disease as well as cutaneous cancers, i.e., HPV types 1, 4, 5, 8, 23, 38 and 76. These HPV types are commonly called “HPV beta types.”
[000178] The ten candidate recombinant LI sequences (shown in Table 2) were each separately cloned into a mammalian expression vector pcDNA™ 3.1(+) (Thermofisher Scientific). A small-scale expression analysis was then performed for each candidate construct utilizing the Expi293™ Expression system (Thermofisher Scientific) per the manufacturer’s protocols. Following expression, cell lysates were harvested and the respective lysates were placed in 37°C for approximately 16 hours in Dulbecco’s phosphate- buffered saline with calcium and magnesium supplemented with 9.5 mM MgCh, 0.25% Brij58 (P5884, Sigma) and 0.1% benzonase (E1014, Sigma). After the 16-hour incubation, the cell lysates were then chilled, and the NaCl concentration of the lysates was adjusted to 0.8 M. The cell lysates were then clarified by ultra-centrifugation for 16 hours at 40,000 x g. The VLPs were purified from the clarified lysate on a 27%/33%/39% OptiPrep™ gradient. Following these expression and purification steps, only Candidates 4, 6, 7, 9 and 10 demonstrated detectable protein expression, as assessed by SDS-PAGE, and only these five candidates were able to form VLPs (50-60nm in diameter), which was verified using transmission electron microscopy (data not shown).
[000179] The results disclosed above demonstrate that, although the insertion of oligopeptide epitopes into the DE loop of papillomavirus LI major capsid protein with the intention to form VLPs has been generally proposed, the actual ability to form VLPs must be empirically determined, since it is known that the ability of the LI peptides to self-assemble into a VLP is very sensitive to the amino acid sequence and length of the LI peptides; even slight changes in LI peptide sequence and/or length can prevent self-assembly (see, e.g., “Kimbauer R, et al Proc Natl Acad Sei U SA. 1992;89(24): 12180-12184).
[000180] Example 2. Candidate 10 has the broadest cross-reactive immunogenicity towards 22 HPV types.
[000181] To assess the immunogenicity of Candidates 4, 6, 7, 9 and 10 (Table 2), vaccination studies were performed. Briefly, 2 pg of each respective candidate was coformulated with 50 pg aluminum hydroxide (Alhdyrogel®; Invivogen). Next, groups of female BALB/c mice (6-8 weeks old; n = 5 per group) were each vaccinated with either one of Candidates 4, 6, 7, 9, or 10, or with a one-tenth human dose of Gardasil® or Cervarix® on days 1, 14 and 28. All mice were bled 2 weeks after the final vaccination (i.e., day 42) to obtain sera for immunogenicity testing. An HPV pseudovirus neutralization assay against HPV18 was conducted, as previously described by Pastrana et al Virology, 321(2):205-16. The results are shown in Figure 1 and demonstrate comparable HPV 18 neutralization titers among all the Candidates tested, as well as Gardasil® or Cervarix®. These results show that the insertion of additional epitopes into the DE loop region of HPV18 LI protein did not compromise the other HPV18 type-specific epitopes.
[000182] To determine which of the Candidates 4, 6, 7, 9, or 10 was able to produce the greatest cross-reactive antibody response, we subjected the vaccinated sera from the 5 respective Candidates to an ELISA assay. In the ELISA assay, negative controls were either a wildtype HPV18 VLP with no peptide insert in its DE-loop or with Cervarix®. Mouse vaccinated sera against a HPV16 RG1-VLP containing the wildtype HPV16 RG-1 epitope in its DE loop was used as a positive control as it reacts with RGlx22. It was shown that this HPV16 RG-1 oligopeptide epitope QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 16), when displayed in the DE-loop of a HPV16 LI VLP, can neutralize over 27 HPV types including many of the types in RGlx22. (Schellenbacher et al., 2013 J Invest Dermatology 133(12): 2706-2713).
[000183] Briefly, Nunc® MaxiSorp™ microtiter 96-well plates (ThermoFisher Scientific, Waltham MA) were coated with a concatenated protein called “RGlx22”. This protein contains the amino acid residue positions 17-36 region of L2 from 22 different HPV types. Approximately 500 ng in 100 pL PBS/well of RGlx22 was added and incubated overnight at 4°C. The next day, plates were blocked with PBS/1% BSA for 1 hour at 37°C. Samples of each of the sera were then diluted 1:50 in PBS/1% BSA and were then added to the 96-well plates for 1 hour at 37°C. Following this, plates underwent 3 washes with washing buffer (0.01% Tween 20 in PBS) before HRP-sheep anti-mouse IgG diluted 1:5000 in 1% BSA was added to each well, and plates were incubated for 1 hour at 37°C. After 3 further washes, 100 pL of ABTS solution, 2,2’Azinobis [3 -ethylbenzothiazoline-6- sulfonic acid] (Roche, Basel Switzerland) was added to each well for development, and absorbance at 405nm was measured using a Benchmark Plus Microplate Reader (Bio Rad, Hercules CA). [000184] ELISA results (in Figure 2) showed that Candidate 10 demonstrated the broadest cross reactivity and was almost as cross reactive as sera from HPV16RG1-VLP vaccination despite their epitopes sharing only 57% sequence identity, when compared using CLUSTAL-W. Candidates 9, 6 and 4 (in descending order) showed less cross reactivity than Candidate 10. As expected, the vaccinated sera from wildtype HPV18 VLP or Cervarix® vaccine did not show any reactivity, as these vaccines contained HPV VLPs that did not have any additional cross-neutralizing epitopes on their DE loops. [000185] Example 3. Evaluation of synergistic HPV cross-reactivity by co-formulating HPV16 RG-1 VLP with Candidate 4, 9 or 10
[000186] With a view to relevant FDA guidelines and market standards concerning protection against both HPV16 and HPV18, and ensuring broad protection against additional HPVs, we decided to co-formulate HPV16 RG-1 VLP with either Candidates 4 , 9 or 10 (see, Table 2). Briefly, 2 pg of each respective candidate and 2 pg of HPV16 RG1-VLP was coformulated with 50pg aluminum hydroxide (Alhdyrogel®, Invivogen). Next, groups of female BALB/c mice (6-8 weeks old; n = 5 per group) were each vaccinated with a formulation of one of Candidates 4, 9, or 10, or with a one-tenth human dose of Cervarix®. Two different types of vaccination schedules were tested. A two-dose schedule in which mice were vaccinated on days 1 and 28, and a three-dose schedule in which mice were vaccinated on days 1, 14 and 28. All mice were bled 2 weeks after the final vaccination (i.e., day 42) to obtain sera for immunogenicity testing.
[000187] ELISAs were first performed using RGlx22 as the antigen as previously described in Example 2. Results (shown in Figure 3) showed that the best synergistic crossreactivity was demonstrated with Candidate 10 co-formulated with HPV16 RG-1 VLP. This was followed by Candidates 9 and 4. Candidate 6 was not assessed, because its epitope is a bio-informatically designed epitope corresponding respectively to the amino acid residue positions 17-36 region of minor capsid protein L2 from 36 HPV types (as described in Example 1). We anticipated that Candidate 6 would be additive to, and not synergistic with, the other candidates. Based on the results from Figure 3, the best HPV18 chimeric VLP for co-formulation with HPV16 RG1-VLP was Candidate 10.
[000188] Example 4. Two dose regimen of HPV16RG-1 VLP co-formulated with candidate 10 is as immunogenic as a three-dose regimen.
[000189] To assess whether there is immune interference against HPV VLP typespecific antibodies when HPV16RG1-VLP is co-formulated with HPV 18 chimeric VLP Candidate 10 (designated “RGVax prototype + #10”; see, also, Table 2), sera from the vaccinated mice from both the two-dose regimen and three-dose regimen (see, Example 3, above) were collected for neutralization assays as previously described (Pastrana et al Virology, 32l(2):205-16). No detectable difference was observed in HPV16 LI titers (Figure 4A), HPV18 LI titers (Figure 4B), and L2 reactive antibody responses (Figure 4C) between a 2-dose and 3-dose vaccination schedule. Thus, the inventive vaccine compositions offer the advantage of fewer doses (and greater patient acceptance and compliance), compared to previously known vaccine formulations.
[000190] Example 5. Evaluation of in vitro cross-neutralization capabilities of HPV16 RG1-VLP co-formulated with either Candidate 9 or Candidate 10
[000191] The ELISA assays employed measure the overall antibody response. However, this ELISA assay system does not distinguish whether a detected antibody response can functionally neutralize a HPV virus. To demonstrate and assess functional cross-neutralization responses following the two-dose or three-dose regimens for HPV16 RG1-VLP, with either Candidate 9- or Candidate 10- vaccinated mice (see, Example 3, above), we conducted in vitro HPV neutralization assays utilizing sera from those mice. In particular, a co-formulation of HPV16 RG1-VLP with Candidate 9 (designated “HPV18 Beta 1”) and a co-formulaton of HPV16 RG1-VLP with Candidate 10 (designated “HPV18 Beta 2”) were tested against a variety of HPV types. (Figure 5). Sera from mice vaccinated against two doses or three doses of Cervarix® were also tested as negative controls. As expected, the negative control sera did not provide any cross -protection regardless of whether a two- or three-dose regimen had been employed. The co-formulation of HPV 16 RG-1 VLP with HPV18 Beta 1 showed some cross neutralization with three doses but failed to cross- neutralize any HPV types, when two doses had been administered. In contrast, two or three doses of HPV16 RG-1 VLP co-formulated with HPV18 Beta 2 showed the best crossneutralization breadth in vitro (Figure 5). The results demonstrated the functional crossneutralization potential of HPV18 Beta 2, even with the administration of only two doses of vaccine.
[000192] Example 6. Design and development of a HPV 18 chimeric VLP containing both HPV18 Beta 1 and Beta 2 epitopes
[000193] The co-formulation of HPV 16 RG1-VLP with either HPV 18 Beta 1 or HPV18 Beta 2 showed that there can be synergy between different types of L2 epitopes derived from the amino acid residue positions 14-34 and 17-36 regions. To further incorporate more L2 epitopes within the DE loop region of HPV 18 LI, we designed a HPV 18 chimeric VLP containing both the Beta 1 and Beta 2 epitopes. This consists of a 25 amino acid peptide that has both Beta 1 and Beta 2 epitopes overlapping and inserted within the DE loop of HPV 18 LI. This construct was called “HPV18 Beta 3” (Figure 6). The Beta 3 construct was produced by small scale expression and purification is as described in Example 1, above. Insertion of this 25-amino acid residue Beta 3 peptide into the DE loop of HPV18 LI did not impair the assembly and formation of VLPs (see, Figure 7A-C). Although insertion of a 25-amino acid residue oligopeptide into the DE loop of HPV18 LI was tolerated and led to the formation of a chimeric VLPs, other peptides of shorter amino acid length were not tolerated.
[000194] Example 7. HPV18 Beta 3 shows the greatest antibody cross-reactivity to multiple HPV types.
[000195] To further evaluate HPV18 Beta l(“Betal”), HPV18 Beta2 (“Beta2”), and HPV18 Beta3 (“Beta3), we administered to New Zealand white rabbits (n = 3 per vaccinated group) three intra-muscular vaccinations on Days 0, 14 and 28 of either Beta 1, Beta 2, Beta 3 or HPV16RG1-VLP. Each dose consisted of 27 pg of VLPs formulated with 50 pg aluminum hydroxide (Alhydrogel®). In addition to these groups, a Gardasil®9 (1/10 human dose), or no vaccine (50 pg of Alhydrogel®) control group was also vaccinated. All rabbits were bled 2 weeks post-vaccination (i.e., Day 42).
[000196] ELISA assays were performed to assess the cross -reactivity of the antibody responses produced by Beta 1, Beta 2 and Beta 3, and results were compared to control vaccinated sera such as HPV16RG1-VLP (known to be very cross-reactive) and Gardasil®9 (negative control). ELIS As were performed as described in Example 2 but with different peptides consisting of the amino acid residue positions 17-36 region of HPV L2 of types 1, 2, 4 , 5, 8, 38, 76 and 92. Results are summarized in Figure 8) and show that HPV18 Beta 3 had the broadest cross-reactivity in the ELISA, followed by HPV16 RG-1VLP, HPV18 Beta 2 and HPV18 Beta 1. Gardasil®9 (negative control) showed no cross -reactivity. These results show that by combining the Beta 1 and Beta 2’s epitopes, a greater antibody cross -reactivity can be achieved.
[000197] Example 8. In vivo synergistic cross -protection of HPV 16RG1 -VLPs with
HPV 18 Beta 2 against co-infection of 9 different HPV types in a single animal [000198] As described in Example 5 (above), ELISA assays were used to measure the overall antibody response. However, the ELISA system employed does not distinguish whether the detected antibody response can functionally neutralize a HPV virus or inhibit infection in vivo. To address this, a murine HPV pseudo-virion (PsV) challenge model was utilized. This is a HPV surrogate infection model, which involves the generation of HPV PsVs. These HPV PsVs are HPV capsids that encapsidate a luciferase reporter plasmid (PMID: 16350417). These pseudo-virions can infect either the mouse’s vagina or skin, and infectivity is measured via luciferase activity as a surrogate marker of infection and can be quantified using imaging software. Importantly, challenging these HPV pseudo-virions into naive mice or mice that received a vaccine, or passively transferred vaccinated sera, can be used to assess prevention of HPV infection and, therefore, determine the promise of any HPV prophylactic vaccine candidate.
[000199] The protocol of the mouse PsV challenge was based on that described by Robert’s et al. 2007 Nature Medicine 13(7):857-61. Four days before vaginal challenge, Balb/c mice (8-10 weeks old; purchased from Charles Rivers Laboratories) were subcutaneously injected with 3 mg of medroxyprogesterone (Depo-Provera, Pfizer, New York NY). The day before challenge, 100 pL of rabbit serum was passively transferred into mice by the intraperitoneal route, followed by HPV PsV challenge containing a luciferase reporter; amounts were standardized based on the LI protein content. Each mouse was challenged with 2 pg of PsV (based on stock virus with LI content of 0.2 pg/pL), which was pre-mixed with 3% (w/v) carboxymethyl cellulose (CMC). To execute the virus challenge, HPV PsVs were directly instilled into the mouse’s vaginal vault, before and after cytobrush treatment (15-20 rotations, alternating directions), while the mice were under isoflurane anesthesia.The mice were anesthetized by isoflurane 72 hours after challenge, and 20 pL of luciferin substrate (7.8mg/ml, Promega, Madison WI) was then delivered into the vaginal vault before imaging. Bioluminescence was acquired for 10 min with a Xenogen IVIS 100 (Caliper Life Sciences, Hopkinton MA) imager, and analysis was accomplished with Living Image 2.0 software. Luminescence data were expressed as a ratio of the radiance signal (in photons [p] per second per square centimeter) in the vaginal region to the radiance signal in the thoracic region. In a given treatment group, full protection was defined as a geometric mean luminescence signal that is statistically significantly lower than that in the control group. Because of the large variability in the luminescence signals and the small group sizes, partial protection was defined as a geometric mean luminescence signal that is <17% of that in the control group and a relative difference that is not statistically significant. The cutoff of 17% was used to define partial protection because it represented the highest relative geometric mean luminescence signal in which full protection was observed among all of the evaluations conducted.
[000200] Five different HPV PsVs that corresponded to HPV types 5, 6, 11, 51 and 68 were assessed. Unexpectedly, the best in vivo cross neutralizing protection from HPV pseudo-infection was conferred by vaccinated serum from HPV18 Beta 2. This was despite the ELISA results described in Example 7 (above), showing that HPV18 Beta 3 had the broadest antibody cross -reactivity towards many HPVs.
[000201] Example 9. In vivo cross-protection of HPV 18 chimeric VLPs
[000202] It has been demonstrated that HPV 16 RGl-VLPs is able to cross-neutralize over 27 HPVs in vivo, however, those studies were done on a single infection level. (See, Schellenbacher et al., 2013 J Invest Dermatology 133(12): 2706-2713. ). In clinical populations, co-infection of multiple HPV types in a single patient is frequent. Therefore, a HPV vaccine that can prevent co-infection of a diverse multitude of HPV viruses would be a great benefit for public health.
[000203] To assess the in vivo cross -neutralization ability of the HPV chimeric VLP candidates, the cotton tail rabbit papillomavirus model was utilized;this is a model that is able to demonstration protection against actual papillomavirus disease. (See, Mejia et al. 2006 Journal of Virology, 80(24); 12393-7). Disease is produced via cutaneous administration of the CRPV viral genome packaged within clinically relevant HPVs. Parameters that are used to assess virus activity included: (1) the number of papillomas that develop from a series of infection sites (usually 10-14 sites per rabbit) and (2) growth rate and size of papillomas over a 6-8-week period.
[000204] Briefly, New Zealand white rabbits (n = 10 per vaccine group) were administered three intra-muscular vaccinations of (i) HPV16-RG1 (80 pg), or (ii) HPV16- RG1 and HPV18 Beta 2 (40 pg each to make 80 pg), or a human dose of Gardasil®9 (270pg of VLPs per dose), or no vaccine (i.e., aluminum hydroxide; Alhydrogel® only). Rabbits were vaccinated at months 0, 1 and 2. Two weeks following final vaccination, each group of ten rabbits was divided into two groups (n = 5 rabbits in each group). In the first group, in vivo protection was assessed against co-infection of HPV types 6, 16, 31, 45, 52, 58, 35 ,39 and 59. In the second group, in vivo protection was assessed against co-infection of HPV types 5, 11, 18, 26, 51, 56, 66 ,68 and 73. (See, Figures 9A-E). In all rabbits and groups, coinfection with wild type cotton rabbit papillomavirus (CRPV) was also demonstrated as a technical control (data not shown).
[000205] Rabbits were anesthetized with a mixture of ketamine hydrochloride (40 mg/kg) and xylazine (5 mg/kg). After the animals' backs were shaved, 0.5-cm-diameter sites on the left and right flanks were scarified with a scalpel. Three days after scarification, the rabbits were again anesthetized, and a 50-pl suspension of cottontail rabbit papillomavirus (CRPV) virion stock (control) or HPV capsid/CRPV genome quasi- virus stock in phosphate buffered saline (PBS) (standardized by genome content for all capsid types) was applied to the pre-scarified site using the edge of a syringe needle. Each HPV type was challenged at two different sites in each rabbit. Transdermal fentanyl (ear) patches (Duragesic®; 12.5 pg/h) or subcutaneous injections of buprenorphine (0.02 to 0.05 mg/kg twice daily for 3 days) were administered as analgesics post-scarification and post-infection, as needed. The sizes and frequencies of lesions were evaluated at 2 to 8 weeks post-challenge. In the absence of a lesion, a diameter of 0 was assigned. In a given treatment group, full protection was defined as the absence of papillomas in all animals, and partial protection was defined as the presence of any break-out papillomas measured on the rabbits.
[000206] Results are shown in Figures 9A-E. The no-vaccine group (“Alum control”) showed every single HPV type infected into the rabbits caused a papillomavirus disease. This demonstrates that every single HPV type used in this model can cause disease either independently or in a co-infection of multiple HPVs setting (see, Figure 10A-C). The results show that the combination of HPV16RG1-VLP and HPV18 Beta 2 (“RG1 Bivalent”) showed complete full protection against both infection and disease caused by all 18 HPV viruses. Full protection against many HPV types was also seen in rabbits who were vaccinated with HPV16 RG1-VLP alone, however unexpectedly, some partial protection was observed against HPV31, HPV45, HPV58 and HPV66 (Figure 10B-C). Previously, complete protection was observed against HPV types under a single infection setting (Schellenbacher et al., 2013 J Invest Dermatology 133(12): 2706-2713). Thus, in a co-infection setting, it is possible that the broad cross-neutralizing antibody response by HPV16 RG1-VLP maybe overwhelmed.
[000207] Unexpectedly also, although Gardasil®9 is an FDA-approved HPV vaccine for the prevention of HPV genital warts caused by HPV6 and HPV 11, as well as the prevention of HPV-associated cancers caused by HPVs 16, 18, 31, 33, 45 , 52 and 58. Our rabbit studies showed, however, that vaccination was not able to confer full protection against HPV6, HPV16, HPV31, HPV45, and HPV52. This cannot be attributed to a lack of immunogenicity or antibody repertoire since the formulation of Gardasil®9 contains 270 pg of 9 different VLPs, all of which produce nine type-specific neutralizing antibodies against HPV types 6, 11, 16, 18, 31 ,33 ,45 ,52 and 58. As such, the results show that even currently approved vaccines can be overwhelmed in a co-inf ection setting (Figure 10B-C).
[000208] In contrast, the co-formulation of HPV16RG1-VLP with HPV 18 Beta 2 contains only 40 pg of each respective VLP and produces only four types of antibodies: Two type specific antibodies against HPV16 and HPV18 as well as two cross-neutralizing antibodies. However, the results described herein demonstrate that this combination of HPV16RG1-VLP and HPV 18 Beta 2 can confer full protection in vivo and extend protection beyond what Gardasil®9 is able to cover. Taken together, the co-formulation of HPV16RG1- VLP with HPV 18 Beta 2, despite a lower vaccine dose and antibody repertoire, is better and has the potential to provide broad cross protection against multiple HPV infections, coinfections, and diseases.
[000209] The foregoing are merely exemplary, and the skilled practitioner of the present invention can easily vary the components and operating parameters as needed for a particular recombinant therapeutic protein drug substance of interest.

Claims

We claim:
1. A virus-like particle (VLP)-based bivalent vaccine composition, comprising: a human papilloma virus 16 (HPV16) LI protein into which is inserted, into a DE loop of the HPV16 LI protein, a surface-displayed HPV16 L2 peptide, wherein the surface- displayed HPV16 L2 peptide has the amino acid sequence QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 16); a human papilloma virus 18 (HPV18) LI protein into which is inserted, into a DE loop of the HPV18 LI protein, a synthetic surface-displayed HPV L2 peptide; wherein the surface-displayed HPV 16 L2 peptide and the synthetic surface-displayed HPV L2 proteins are incorporated into the surface of their respective LI proteins, and wherein the HPV16 and HPV18 LI proteins spontaneously form virus-like particles.
2. The VLP-based bivalent vaccine composition of claim 1, wherein the HPV16 LI protein comprises the amino acid sequence of SEQ ID NO: 12, and wherein the surface- displayed HPV 16 L2 peptide is inserted into the DE loop of the HPV 16 LI protein between amino acid residue positions 136 and 137 of SEQ ID NO: 12.
3. The VLP-based bivalent vaccine composition of claim 1, wherein the surface- displayed HPV L2 peptide is an analogous protein segment from a HPV type selected from the group consisting of HPV1, HPV4, HPV5, HPV8, HPV23, HPV38, and HPV76.
4. The VLP-based bivalent vaccine composition of claim 1, wherein the HPV18 LI protein comprises the amino acid sequence of SEQ ID NO: 13.
5. The VLP-based bivalent vaccine composition of claim 4, wherein the synthetic surface-displayed HPV L2 peptide is inserted into the DE loop of the HPV 18 LI protein between amino acid residue positions 134 and 135 of SEQ ID NO: 13.
6. The VLP-based bivalent vaccine composition of claim 1, wherein the synthetic surface-displayed HPV L2 peptide, inserted into the DE loop of the HPV18 LI protein, comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.
7. The VLP-based bivalent vaccine composition of claim 1, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV18 LI protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, or the amino acid sequence of any these members with 1-4 conservative amino acid substitutions.
8. The VLP-based bivalent vaccine composition of claim 1, wherein the HPV 18 LI protein comprises the amino acid sequence of SEQ ID NO: 13; wherein the synthetic surface-displayed HPV L2 peptide is inserted into the DE loop of the HPV 18 LI protein between amino acid residue positions 134 and 135 of SEQ ID NO: 13; and wherein the synthetic surface-displayed HPV L2 peptide comprises an amino acid sequence selected from the group consisting SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:27, or the amino acid sequence of any these members with 1-4 conservative amino acid substitutions.
9. The VLP-based bivalent vaccine composition of claim 1, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV 18 LI protein comprises the amino acid sequence of HIYQSCKASGTCPPDVINKVE (SEQ ID NO:24), or the amino acid sequence of SEQ ID NO:24 with 1-4 conservative amino acid substitutions.
10. The VLP-based bivalent vaccine composition of claim 1, wherein the synthetic surface-displayed HPV L2 peptide inserted into the DE loop of the HPV 18 LI protein comprises an amino acid sequence variant at least 80, 85, 90 or 95% identical to the amino acid sequence of SEQ ID NO:24.
11. The VLP-based bivalent vaccine composition of claim 1, further comprising an adjuvant.
12. The VLP-based bivalent vaccine composition of claim 11, wherein the adjuvant comprises aluminum hydroxide or aluminum phosphate.
13. The VLP-based bivalent vaccine composition of claim 11, further comprising a pharmaceutically acceptable carrier.
14. The VLP-based bivalent vaccine composition of claim 11, wherein the composition is formulated for administration by inhalation, ingestion, or in a viral or bacterial vector.
15. The VLP-based bivalent vaccine composition of claim 11, wherein the composition is in a formulation for intramuscular injection.
16. The VLP-based bivalent vaccine composition of claim 11, wherein the composition is packaged in a glass vial for single or multiple-dose use.
17. The VLP-based bivalent vaccine composition of claim 1, which is an immunogenic composition.
18. The VLP-based bivalent vaccine composition of claim 1, which is immunogenic against mucosal high-risk or low- risk, cutaneous low risk, and/or cutaneous beta-type papillomaviruses.
19. The VLP-based bivalent vaccine composition of claim 1, which is immunogenic against one or more papillomaviruses selected from the group consisting of HPV1, HPV2, HPV4, HPV5, HPV16, HPV18, HPV26, HPV35, HPV38, HPV39, HPV45, HPV52, HPV58, HPV68, HPV76, and HPV92.
20. The VLP-based bivalent vaccine composition of claim 1, wherein the composition is effective in preventing human papillomavirus infection in a susceptible subject.
21. The VLP-based bivalent vaccine composition of claim 20, which prevents coinfection in the subject by the following human papillomavirus types: HPV 6, 16, 31, 45, 52, 58, 35, 39 and 59.
22. The VLP-based bivalent vaccine composition of claim 20, which prevents coinfection in the subject by the following human papillomaviruse types: HPV 5, 11, 18, 26, 51, 56, 66 ,68 and 73.
23. The VLP-based bivalent composition of claim 20, which prevents co-inf ection in the subject by the following human papillomaviruse types: HPV 6, 11, 16, 18, 26, 31, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 73.
24. A method for immunizing or vaccinating a subject against a HPV, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of claim 1.
25. The method of claim 24, wherein administering the effective amount of the VLP-based bivalent vaccine composition comprises a 2-dose or 3-dose vaccination schedule.
26. A method for inducing an immune response against HPV in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of claim 1.
27. A method for treating a HPV infection in a subject having a HPV infection or at risk of being exposed to HPV, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of claim 1.
28. A method for preventing HPV-associated cervical, anogenital, oropharyngeal cancer, skin cancer or a precancer, in a subject, comprising administering to the subject an effective amount of the VLP-based bivalent vaccine composition of claim 1.
29. A kit comprising the VLP-based bivalent composition of claim 1.
PCT/US2023/037041 2022-11-09 2023-11-08 Bivalent virus-like particle compositions and methods of use WO2024102417A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263423984P 2022-11-09 2022-11-09
US63/423,984 2022-11-09

Publications (1)

Publication Number Publication Date
WO2024102417A2 true WO2024102417A2 (en) 2024-05-16

Family

ID=91033685

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/037041 WO2024102417A2 (en) 2022-11-09 2023-11-08 Bivalent virus-like particle compositions and methods of use

Country Status (1)

Country Link
WO (1) WO2024102417A2 (en)

Similar Documents

Publication Publication Date Title
US10046026B2 (en) Papillomavirus-like particles (VLP) as broad spectrum human papillomavirus (HPV) vaccines
JP6029631B2 (en) Multiple HPV peptide compositions and methods for the treatment or prevention of human papillomavirus infections
US11944677B2 (en) Chimeric virus-like particles and uses thereof as antigen-specific redirectors of immune responses
JP2012530505A (en) Novel human papillomavirus (HPV) protein constructs and their use in the prevention of HPV disease
US8039001B2 (en) Therapeutic and prophylactic vaccine for the treatment and prevention of papillomavirus infection
Kalnin et al. Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes
US9138470B2 (en) Multi-component L2 vaccine for prevention of human papilloma virus infection
WO2024102417A2 (en) Bivalent virus-like particle compositions and methods of use
US20210299238A1 (en) Compositions, methods and uses for thermally stable broad-spectrum human papillomavirus formulations
JP6916115B2 (en) Vaccine for HPV infection containing HPVL2 peptide / HBs chimeric protein as an active ingredient
WO2012158639A2 (en) Recombinant fusion proteins and methods for use thereof for treatment or prevention of papillomavirus infection
CN117430674A (en) Recombinant proteins and their use in HPV prophylaxis
BRPI1003749B1 (en) HYBRID PROTEIN, RECOMBINANT NUCLEIC ACID SEQUENCE, VECTOR / PLASMIDE, ITS USES, AND PHARMACEUTICAL AND / OR VETERINARY FORMULATION FOR THE CONTROL OF TUMORS INDUCED BY HUMAN PAPILLOMA VIRUSES AND / OR INFANT DISEASES OR HEALTH DISEASES