WO2012158639A2 - Recombinant fusion proteins and methods for use thereof for treatment or prevention of papillomavirus infection - Google Patents

Recombinant fusion proteins and methods for use thereof for treatment or prevention of papillomavirus infection Download PDF

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WO2012158639A2
WO2012158639A2 PCT/US2012/037831 US2012037831W WO2012158639A2 WO 2012158639 A2 WO2012158639 A2 WO 2012158639A2 US 2012037831 W US2012037831 W US 2012037831W WO 2012158639 A2 WO2012158639 A2 WO 2012158639A2
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fla
fusion
hpv
fusion product
seq
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PCT/US2012/037831
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French (fr)
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WO2012158639A3 (en
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Kirill Kalnin
Harold Kleanthous
Yanhua Yan
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Sanofi Pasteur Biologics, Llc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus.
  • the present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
  • Genital-tropic human papillomavirus (HPV) infections are considered the most common sexually transmitted infections in the United States (CDC Report to Congress, Prevention of Genital Human Papillomavirus Infection, January, 2004).
  • the major manifestations of anogenital HPV include genital warts (condyloma acuminatum) and intraepithelial neoplasia of the vulva, cervix, anus, or penis.
  • a small fraction of persistent high-risk HPV infections if left untreated, progresses into one or more various forms of cancer, including, cervical, anogenital, laryngea, tongue, head and neck, and some types of non-melanoma skin cancers.
  • Warts may exist in different forms depending on the HPV type responsible and the epithelium involved.
  • Common warts usually occur on the hands, as flesh-colored to brown exophytic and hyperkeratotic papules.
  • Plantar warts (verruca plantaris) occur on the soles of the feet and can be quite painful. These warts can be differentiated from calluses by removing the surface layer to reveal thrombosed capillaries.
  • Flat or planar warts (verruca plana) are most common among children and can occur on the face, neck, chest and flexor surfaces of the forearms and legs.
  • HPV infection can also lead to morbidity in the form of cervical cancer.
  • HPV Preventing HPV infection is more challenging than prevention of most other sexually transmitted disease due to the fact that women infected with the HPV are generally
  • HPV is also believed to be relatively easily transmitted between partners. In fact, at least 50 percent of sexually active men and women have been infected with genital HPV at some point in their lives.
  • HPV Approximately 35 of the more than 100 types of HPV are specific for the anogenital epithelium and have varying potentials for malignant transformation. Furthermore, about 15 types of HPV are considered oncogenic genital HPV types, with HPV16 being the most common, followed by HPV18 and HPV45 (causing about 50%, about 20%, and about 10% of cervical cancer cases, respectively).
  • HPV infection is considered a necessary factor for the development and persistence of cervical cancer. Overall, the presence of HPV DNA has been reported in 99.7% of cervical carcinomas worldwide, suggesting that HPV infection is a necessary cause of this type of cancer and that this disease can be prevented by prophylactic HPV vaccination.
  • HPV high-risk
  • HPV types include, HPV16, 18, 26, 30, 31 , 34, 35, 39, 45, 51 , 52, 53, 56, 58, 59, 61 , 66, 67, 68, 69, 70, and 73.
  • Other types such as HPV types 2, 3, 6, 7, 10, 13, 32, 40, 42, 43, 44, 55, 54 and 57, are categorized as being relatively "low-risk” because of their lower potential for progression to malignancy.
  • the HPV has a circular double-stranded genome that is about 8 kbp in length.
  • the genome of all known HPV types contains open reading frames (ORFs), which are DNA regions coding for proteins having similar properties, that are divided into two major regions: the early (E) region, and late (L) region.
  • ORFs open reading frames
  • the early region of about 4.5 kbp codes for genes which are associated with functions including viral DNA replication (i.e., E1 ), induction or suppression of the action of DNA encoding a protein inducing malignant transformation of host cells (i.e., E2), the synthesis of proteins responsible for the growth of host cells and viruses (i.e., E4), stimulation of the activity of epidermal growth factor (EGF) and colony stimulator factor (CSF) receptors (i.e., E5), and malignant transformation through the permanent survival of cells, activation of oncogenes (E6), and inactivation of tumor suppressor genes (i.e., E7).
  • E1 viral DNA replication
  • E2 induction or suppression of the action of DNA encoding a protein inducing malignant transformation of host cells
  • E4 the synthesis of proteins responsible for the growth of host cells and viruses
  • E4 the synthesis of proteins responsible for the growth of host cells and viruses
  • E5 the synthesis of proteins responsible for the growth of host cells and viruses
  • the oncogenic E6 and E7 proteins which are expressed after HPV infects the epithelial cells of a host, bind to tumor suppressor proteins of host cells, p53 and pRB, respectively, and thus inhibiting the function of these suppressor proteins, resulting in the neoplastic transformation of infected cells.
  • the late region of 2.5 kbp comprises genes coding for viral major (i.e., L1 ) and minor (i.e., L2) capsid proteins and a non-coding region 1 kbp long, which is called the long control region (i.e., LCR) which regulates the transcription and translation of the two late genes.
  • HPV genome is surrounded by a 60-nm, non-enveloped icosahedral capsid skeleton (Baker et al., Biophysical J., 60(6):1445-1456 (1991 )) that contains the two genetically unrelated major capsid L1 proteins and the minor L2 capsid protein.
  • the HPV capsid is thought to comprise 72 L1 protein pentamers (capsomeres) and 12 L2 minor capsid protein molecules bound thereto. Both terminals of the L2 protein are located in the capsid, but part of the N- terminal region is located on the surface of the capsid (L2 surface region).
  • VLPs virus-like particles
  • VLPs virus-like particles
  • VLPs virus-like particles
  • VPs virus like particles
  • L1 VLP-based vaccines are generally protective against infection corresponding to the papillomavirus type used to derive the immunogen (homologous vaccine), but are ineffective against all but the most closely related HPV types (Roden et al., Virology,
  • HPV vaccines i.e., L1 based VLPs
  • L1 based VLPs L1 based VLPs
  • HPV16 L2 truncations have been tested. (Gambhira, J. Virology, 81 (24):13927-13931 (2007); Kando et al., Virology, 358:266- 272 (2007); and Rubio et al., Vaccine, 27:1949-1956 (2009)).
  • HPV16 L2 truncations using AA2-200, AA1 1 -200, AA1 1 -88, AA1 1 -38, AA17-36 (some of these fragments are presented as multimers and some as monomers) showed varying degrees of efficacy as determined by pseudovirus neutralization assays. (Rubio et al., 1949-1956).
  • HPV L1 VLP vaccines require refrigeration during handling and storage which renders them impractical for use in low resource and remote areas where they are needed most. Furthermore, because the existing HPV L1 vaccines are ineffective against a significant number of oncogenic HPV types, costly cytologic screening programs remain necessary and a significant public health disease burden is still present.
  • HPV vaccines should be safe and effective and broadly effective against a greater number of HPV types.
  • the invention provides fusion products including at least a portion of a flagellin sequence of substantially the same amino acid sequence as a flagellin sequence described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305, and at least a portion of a human papillomavirus (HPV) L2 sequence of substantially the same amino acid sequence as an HPV L2 sequence described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305.
  • HPV human papillomavirus
  • Such fusion products can, in various embodiments, product activates Toll-like Receptor 5 (TLR5).
  • TLR5 Toll-like Receptor 5
  • the portions noted above are at least about 10, 20, 30, 40, 50, 75, or 100 amino acids in length.
  • the portion of the flagellin sequence includes a deletion of or within domain D2 and/or domain D3.
  • the fusion products include at least a portion of two or more (e.g., 5-10) different HPV L2 sequences of substantially the same amino acid sequence as at least two (e.g., 5-10) or more HPV L2 sequences described in one or more of SEQ I D NOs: 101 -199, 202-204, or 303-305.
  • At least one of the different HPV L2 sequences is substantially the same as that of HPV16 and/or HPV18, as described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305. Further, in other embodiments, at least one of the different HPV L2 sequences is substantially the same as that of HPV31 , HPV39, HPV52, HPV58, HPV35, HPV45, and/or HPV6B, as described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305.
  • the invention also includes nucleic acid molecules including a sequence encoding a fusion product as described herein.
  • the nucleic acid molecules can include a portion of a flagellin nucleotide sequence of substantially the same nucleotide sequence of a flagellin sequence described in one or more of SEQ ID NOs: 1 -99, 100, 200, 201 , or 300-302, and at least a portion of an HPV L2 nucleotide sequence of substantially the same nucleotide sequence of an HPV L2 described in one or more of SEQ I D NOs: 1 -99, 100, 200, 201 , or 300- 302.
  • the invention includes vectors containing the nucleic acid molecules described herein, as well as cells including the vectors.
  • the invention also includes methods of inducing an immune response in a subject, including administering to a subject an effective amount of one or more fusion products described herein, or a modification(s) thereof.
  • the invention includes methods of preventing an infection in a subject by the human papillomavirus including administering to the subject an effective amount of one or more of the fusion products described herien, or a modification(s) thereof.
  • the invention includes methods of modulating an immune response in a subject having a pathological condition, including administering to the subject an effective amount of one or more of the fusion products described herein, or a modification(s) thereof.
  • the pathological condition results from infection of the subject by a human papillomavirus.
  • the invention includes methods of inducing an antigen-specific immune response in a subject involving administering to the subject an effective amount of one or more of the fusion products described herein, or a modification(s) thereof.
  • the invention further includes methods of preventing an infection in a subject by one or more human papillomavirus involving administering to the subject an effective amount of one or more fusion products as described herein, or a modification(s) thereof.
  • the invention also includes uses of one or more fusion products as described herein, or a modification(s) thereof, in the preparation of a medicament for inducing an immune response in a subject.
  • the present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus.
  • the present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
  • the present invention describes the design, expression, purification, formulation, and in certain embodiments, demonstrates the efficacy of immunological compositions useful for eliciting an immune response in a subject.
  • the immunological compositions are based on recombinant fusion proteins (e.g., Fla-L2) containing derivatives of conservative cross-protective epitopes of HPV m inor protein L2 and one or more portions of Flagellin.
  • Flagellin is a pathogen-associated molecular pattern (PAMP) recognized by Toll-
  • TLR-5 Like Receptor 5 (TRL-5). TLR-5 is a member of a family of receptors involved in TLR-5.
  • Toll-like receptors recognize PAMPs that distinguish infectious agents from self and mediating the production of
  • immunomodulatory molecules such as cytokines, necessary for the development of effective adaptive immunity (Aderem and Ulevitch, Nature, 406:782-787 (2000) and
  • TLR-2 recognizes various fungal, Gram-positive, and mycobacterial
  • TLR-4 recognizes the Gram-negative product lipopolysaccharide (LPS)
  • TLR-9 recognizes nucleic acids such as CpG repeats in bacterial DNA.
  • TLR-5 has been identified as a receptor for bacterial Flagellin.
  • Flagellin induces an innate immune response by binding to and activating TLR-5. Activation of TLR-5 by binding to Flagellin induces the production of immunomodulatory molecules, such as cytokines and co-stimulatory molecules, by a TLR-5 expressing cell. For example, activation of TLR-5 in macrophages results in the expression of the
  • cytokines TNF-a, IL-1 , and IL-6. These cytokines directly and indirectly alter the activities of immune system cells that participate in both humoral (TH2) and cell-mediated (TH1 ) adaptive immune responses. In this manner, in certain embodiments, an
  • immunomodulatory Flagellin based fusion product peptide, polypeptide or modification thereof, can act as an adjuvant to stimulate a general immune response.
  • Flagellin is known as TLR-5 agonist.
  • Patent No. 7,915,381 discloses a number of TLR-5 agonists, and in particular, Flagellin related compositions (e.g., sequences), methods, and formulations the disclosure of this patent is incorporated herein by reference in its entirety.
  • an immunomodulatory Flagellin based fusion product, polypeptide, peptide, or modifications thereof can be used to induce an immune response in an individual having a pathological condition (e.g., PV or HPV related
  • the present invention contemplates that by stimulating an innate immune response, the
  • immunomodulatory Flagellin based fusion products promote humoral and cell-mediated responses to antigens on foreign cells or pathologically aberrant cells, such as cancer cells.
  • a vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved.
  • the vaccine can be a solid (e.g., powdered or lyophilized formulation).
  • the compositions of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat an infection. Upon introduction into a host, certain compositions are 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.
  • Flagellin derivatives as vaccine vehicles for delivery of L2-protective epitopes.
  • Flagellin has been shown in preclinical studies to be an efficacious adjuvant in immunological compositions. For example, preclinical studies of Flagellin as a component of compositions directed against diseases caused by
  • Flagellin can be an effective adjuvant.
  • flagellins from different bacterial species can be used as sources for HPV-L2 fusion in a fashion described herein.
  • Flagellin homologs are exemplified but not limited by the list below: (1 ) fliC gene product of Salmonella enterica subsp. enterica serovar typhimurium str. 798; accession gene bank number YP_005397322 ; (2) flagellin of Salmonella enterica subsp. enterica serovar hadar str. RI_05P066; accession gene bank number ZP_02684103; (3) phase-1 flagellin
  • accession gene bank number YP_003229794 Flagellin Escherichia hermannii NBRC 105704; accession gene bank number ZP 09806483; (7)Flagellin Escherichia fergusonii ATCC 35469; accession gene bank number YP_002382331 ; (8) Flagellin Citrobacter youngae ATCC 29220; accession gene bank number ZP_06352418; (9) Flagellin Edwardsiella tarda ATCC 23685; accession gene bank number ZP_06715189; (10) FliC gene product Bordetella petrii DSM 12804; accession gene bank number YP_001630704; (1 1 ) Flagellin Enterobacter hormaechei ATCC 49162; accession gene bank number ZP 08498671 .
  • the present invention provides Fla-L2 fusions
  • epitopes within the first 200 AA of L2 that are broadly cross-protective against disease and/or infection by multiple (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) HPV types.
  • the present invention demonstrates that therapeutic and/or prophylactic fusion product molecules can be expressed in high levels in available expression systems and can subsequently be purified from these expression systems.
  • Fla-L2 fusion product molecules are expressed in bacterial expression systems.
  • the present invention utilizes Escherichia coli (E. coli) based expression systems.
  • E. coli Escherichia coli
  • the present invention is not limited to expression in bacterial expression systems.
  • suitable protein expression systems include those derived from prokaryotic cells (e.g., bacteria or archea) or eukaryotic cells (e.g., fungi, including, but not limited to yeast cells, algae, mammalian cells, and insect cells).
  • prokaryotic cells e.g., bacteria or archea
  • eukaryotic cells e.g., fungi, including, but not limited to yeast cells, algae, mammalian cells, and insect cells.
  • Preferred methods and compositions of the present invention provide highly protective immune responses in animals against HPV challenge.
  • the present invention is not intended to be limited to the list of constructs depicted in Table 1 , but rather is extended to any Flagellin and L2 fusions and mixtures of portions, repeats, and/or mutations of HPV L2 with portions, repeats, and/or mutations of Flagellin used with or without adjuvant(s) in any combination for prophylactic and/or therapeutic purposes.
  • the present fusion products and methods contemplated by the present invention are useful as additional components in a further prophylactic and/or therapeutic sense when administered in conjunction (e.g., 1 , 2, 3, 4, or more, doses administered sequentially or concomitantly) with one or more doses of commercially available HPV vaccine(s).
  • One or more of the fusion products can be useful as a vaccine composition for the prophylaxis, treatment, or prevention of papillomavirus infection.
  • the compositions can be combined with a pharmaceutical carrier.
  • the vaccine composition is administered to an individual prior to papillomavirus exposure to minimize or prevent papillomavirus infection, or is administered after a patient has been infected to reduce the severity of infection and retard/halt progression of the disease, or to prevent transmission of a papillomavirus from the infected host to another individual who does not have a papillomavirus infection.
  • the invention also contemplates methods of purification as described herein, as well as animal models that can be used, e.g., for testing constructs as described herein, such as those described below.
  • Figure 1 shows the expression of Fla-02 (HPV16 L2 2-200 AA) and Fla-07 (HPV16 L2 of
  • Figure 2 shows the general purification scheme of Fla-02 and Fla-07 fusions as well as 2 exemplary SDS PAGE analyses of the chromatographic elution fractions for Fla-02 and Fla-07.
  • Figure 3 shows purified lots of Fla-01 ((1 -506 AA)x6xHIS), Fla-02, and Fla-07 fusion products used for subsequent in vivo studies.
  • Figure 4 shows projected 3-D models of HPV16 L2 (Panel A) and the Fla-07 fusion D1 , D2, and D3 domains (Panel B).
  • FIG. 5 shows bioactivity results of the TLR-5 of Fla-07 and the Flagellin backbone (Fla-01 ).
  • TLR Toll-like Receptor
  • Bioactivity results were as follows: Fla-01 > Fla-07 » Flagellin of S. typhimurium.
  • Figure 6 shows at, Panel A, a 3D model of Fla-31 or Fla-32 as modeled using Accelrys software (Accelrys, Inc., San Diego, CA); Panel B, provides an SDS gel showing expression of Fla-31 and Fla-32 at the specified culture conditions.
  • Accelrys software Accelrys, Inc., San Diego, CA
  • Figure 7 shows at, Panel A, the comparative purification results for Fla-01 , Fla-62, Fla- 02, Fla-31 and Fla-32;
  • Panel B shows characteristics of Flagellin or various Fla-L2 fusion variants used for preclinical studies.
  • release tests demonstrated high purity, low endotoxin level (below 0.1 Eu ⁇ g) and high yield of purified antigens.
  • Assay conditions were: 1 ) Protein concentration: BSA; 2) Purity: SDS PAGE; 3) Endotoxin level: Endosafe-PTS; and 4) Specificity: Western blot with anti L2 mAbs RG1 .
  • Figure 8 shows the TLR-5 bioactivity of various Fla-L2 fusions in vitro.
  • Figure 9 shows SDS gels demonstrating the expression of monomeric Fla-L2 fusions (C- terminus fusion; full-length Fla) for HPV L2 1 1 -200AA of type 6, 1 1 , 18, and 31 . Conditions were as shown on the left of the figure.
  • Figure 10 shows SDS gels demonstrating the expression of monomeric FlaAD3-L2 fusions (D3-replacements) containing L2 of HPV types 39 (1 1 -199 AA), 18 (1 1 -200 AA), 6 (10- 198), 31 (1 1 -195) and 45 (1 1 -200AA) epitopes. Conditions were as shown on the left of the figure.
  • FIG. 1 Panels A, B, and C, shows 3D models of Fla-07, Fla-32, and Fla-69, respectively.
  • the models in Panels A and B were built using Accelrys software while the model in Panel C is an approximate structure shown for comparison purposes.
  • Figure 12 shows some of the characteristics of L2 multimeric fusion constructs used for expression, purification, and in vivo studies.
  • Panel A shows the scope of L2 epitopes and HPV serotypes used therein.
  • Panel B shows some selected characteristics of selected Fla-L2 fusion products and their use for in vivo (e.g., rabbit) studies.
  • FIG 13 in Panel A, provides data demononstrating the Fla-69 and Fla-70 fusions are highly soluble after microfluidization (MF) (microfluidization conditions were as described in the panel).
  • Panel B provides a physical map of the multimeric structures of Fla-69 and Fla-70 fusions, respectively.
  • Figure 14 shows the general purification scheme for multimeric Fla-L2 fusions Fla-69
  • the purification scheme provides for a high recovery and good separation at each step, high yield and high purity of the recovered fusions products, high recovery after refolding, is robust, highly scalable, and cost effective.
  • FIG. 15 Panel A, shows SDS gel results for release test purification steps for the Fla-
  • Panel A shows that the fusion product was recovered in high purity, with low residual endotoxin levels, and at a high yield of purified antigens ( ⁇ 90 mg/1 L culture).
  • Panel provides SEC-HPLC product characterization results for the Fla-62 (flagellinAD3 control), Fla-69 (5XL2) and Fla-32 (HPV16L2; 1 1 -200 AA) fusion products, respectively.
  • Figure 16 shows purification results and certain physical characteristics for fusion products Fla-65 and Fla-76, respectively.
  • FIG 17 shows a comparison between mono- and multimeric -L2 Fla-L2 fusions using a TLR-5 bioactivity in vitro assay.
  • TLR5 binding of two lots of Fla-69 is comparable to that of Fla-62, Fla-32, or WT Flagellin (Fla) from S. typhimurium.
  • Figure 18 shows a comparison between various multimeric -L2 Fla-L2 fusions using a TLR-5 bioactivity in vitro assay.
  • Fla-SM denotes that a S. typhimurium Flagellin control was used for comparison testing.
  • Figure 19 shows the schedule used in an exemplary animal challenge study.
  • the animals challenged were rabbits and the study was labeled "Rabbit Study No. 3."
  • Test animals were challenged with Cottontail Rabbit Papillomavirus (CRPV) and quazi-viruses which have CRPV genome packaged within HPV L1/L2 shell of serotype 6, 16, 18, or 58.
  • CRPV Cottontail Rabbit Papillomavirus
  • Figure 20 shows the results of an exemplary animal study measuring end point titers at days 21 , 42, 63, and 102 of the study.
  • the animals challenged were rabbits and the study was labeled "Rabbit Study No. 3.”
  • immunogenicity results of Fla-02 (L2 2- 200 AA) and Fla-07 (L2 1 1 -200 AA) fusions, respectively, are shown.
  • Figure 21 shows the results of an exemplary animal study measuring end point titers at days 21 , 42, 63, and 102 of the study.
  • the animals challenged were rabbits and the study was labeled "Rabbit Study No. 3."
  • immunogenicity results of Fla-31 (L2 2-200 AA) and Fla-32 (L2 1 1 -200 AA) fusions, respectively, are shown. Both D3-L2 replacements
  • Figure 22 shows the results of an exemplary immunogenicity study of Fla-L2 fusions at day 63 post immunization with pre-challenge sera.
  • the animals challenged were rabbits and the study was labeled "Rabbit Study No. 3.”
  • Figure 22 provides a comparison of 50% neutralizing titers of against pseudovirus (PsV16 (Panel A) and 18 (Panel B), respectively, at Day 63 (circular dots; after two immunizations) and Pre-Challenge Sera (d102) (square dots; after four immunizations). Neutralizing titers were boosted by the 4th immunization for both PsV16 and 18.
  • Figure 23 shows in multiple panels (Panels A, B, C, D, and E) the efficacy of various Fla- L2 fusions in a wart analysis study eight weeks post challenge with quazi-virus 6 (Panel A), quazi-virus 16 (Panel B), quazi-virus 18 (Panel C), quazi-virus 58 (Panel D), and CRPV (Panel E), respectively.
  • the results of this exemplary study show that the Fla-32 fusion was highly cross protective against all of the challenge virus in this study and the adjuvant used did not significantly increase protection.
  • Figure 24 shows the vaccine L2-types and quazi-viruses used in an exemplary animal study.
  • Panel A shows the HPV types used in a 5X vaccine formulation (Fla-69).
  • Panel B shows the quazi-virus types used in the subsequent challenge study(ies).
  • Panel C depicts an L-2- dendrogram based on 1 1 -200 AA L2 epitopes.
  • Figure 25 shows the design of an exemplary animal study.
  • Study animals were scheduled for intramuscular immunization with the indicated fusion formulations either three times (Days: 14, 35, and 56) or four times (Days: 0, 21 , 35, and 56) and subsequently challenged on Day 77 each with each of quazi-virions 6, 16, 18, 58 (three sites per virus) and CRPV (two sites per virus).
  • Figure 26 shows results of an exemplary in vitro neutralization assays in rabbits against PsV16. These assays were part of Rabbit Study No. 6.
  • the figure shows the 50% neutralization titers against PsV16 of individual serum samples assayed using a conventional neutralization method.
  • Panel A shows the individual 50% neutralization data.
  • Panel B shows the experimental schedule (immunization and bleeding days are shown by arrows and red boxes respectively; challenge day is depicted by callout).
  • Figure 27 shows results of an exemplary in vitro neutralization assays in rabbits against
  • PsV18 are part of Rabbit Study No. 6.
  • the figure shows the 50% neutralization titers against PsV16 of individual serum samples assayed using a conventional neutralization method.
  • Panel A shows the individual 50% neutralization data.
  • Panel B shows the experimental schedule (immunization and bleeding days are shown by arrows and red boxes respectively; challenge day is depicted by callout).
  • Figure 28 shows in multiple panels (Panels A, B, C, D, and E) the efficacy of various Fla- L2 fusions in a wart analysis study eight weeks post challenge with quazi-virus (QV) 6 (Panel A), QV 16 (Panel B), QV 18 (Panel C), QV 58 (Panel D), and wild type CRPV (Panel E), respectively.
  • QV quazi-virus
  • Figure 29 shows results of an exemplary in vitro neutralization assays in rabbits.
  • Figure provides the 50% neutralization titers of pooled serum samples generated in Rabbit Study No. 6 under one of two methods as specified.
  • the first method is labeled the "novel methodology” and data therein is based on spatiotemporal separation of L2 epitope exposure on the base membrane (HSPG) and binding to the secondary receptor on the HSPG- epithelial cell surface (stages for viral HSPG binding, furine cleavage and binding/internalization to/into HSPG- cells are spatiotemporaly separated).
  • the second method is labeled the "Convention methodology” and data therein was generated by exploiting viral binding/internalization to/into HSPG+/Secondary receptor+ cells (data shown in parentheses).
  • Figure 30 shows the general scheme used for passive immunization experiments with pooled serum samples obtained from various arms of Rabbit Study No. 6.
  • Figure 31 shows the vaccine L2-types and quazi-viruses used in an exemplary passive immunization animal study.
  • Panel A shows the HPV types used in a 5X vaccine formulation (Fla-69).
  • Panel B shows the quazi-virus types used in the subsequent challenge study(ies).
  • Panel C depicts an L-2-dendrogram based on 1 1 -200 AA L2 epitopes.
  • Figure 32 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples (anti-Fla-32, anti-Cervarix ® , anti-Fla-69, and anti-Gardasil ® ) obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 6.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil ® HPV vaccine (Merck, Whitehouse Station, NJ) and the Cervarix ® HPV vaccine (GlaxoSmithKlein, London, United Kingdom) in dilution series against the specified quazi-virus (QV 6) : Panel A (Fla-32); Panel B (Cervarix ® ); Panel C (Fla-69); Panel D (Gardasil ® ).
  • Figure 33 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 16.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil ® HPV vaccine and the Cervarix ® HPV vaccine in dilution series against the specified quazi-virus (QV 16): Panel A (Fla-32); Panel B (Cervarix ® ); Panel C (Fla-69); Panel D
  • Figure 34 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 18.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil ® HPV vaccine and the Cervarix ® HPV vaccine in dilution series against the specified quazi-virus (QV 18): Panel A (Fla-32); Panel B (Cervarix ® ); Panel C (Fla-69); Panel D
  • Figure 35 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 31 .
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the
  • Figure 36 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 45.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil ® HPV vaccine and the Cervarix ® HPV vaccine in dilution series against the specified quazi-virus (QV 45): Panel A (Fla-32); Panel B (Cervarix ® ); Panel C (Fla-69); Panel D
  • Figure 37 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 58.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil ® HPV vaccine and the Cervarix ® HPV vaccine in dilution series against the specified quazi-virus (QV 58): Panel A (Fla-32); Panel B (Cervarix ® ); Panel C (Fla-69); Panel D
  • Figure 38 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with CRPV.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the
  • Gardasil ® HPV vaccine and the Cervarix ® HPV vaccine in dilution series against CRPV Panel A (Fla-32); Panel B (Cervarix ® ); Panel C (Fla-69); Panel D (Gardasil ® ).
  • Figures 40A, 40B, and 40C show ELISA results using pooled Fla-69 antiserum obtained from Rabbit Study No. 6 with L2- peptide libraries covering 1 1 -200 AA of L2 of HPV types 6 (FIG. 40A, Panel A), 16 (FIG. 40A, Panel B), 18 (FIG. 40B, Panel A), 31 (FIG. 40B, Panel B), 45 (FIG. 40C, Panel A), and 58 (FIG. 40C, Panel B), respectively.
  • the location of RG1 epitopes is indicated by arrows.
  • Figures 41 A, 41 B, and 41 C show ELISA results using pooled Fla-32 antiserum obtained from Rabbit Study No. 6 with L2- peptide libraries covering 1 1 -200 AA of L2 of HPV types 6 (FIG. 41 A, Panel A), 16 (FIG. 41 A, Panel B), 18 (FIG. 41 B, Panel A), 31 (FIG. 41 B, Panel B), 45 (FIG. 41 C, Panel A), and 58 (FIG. 41 C, Panel B), respectively.
  • the location of RG1 epitopes is indicated by arrows.
  • Figure 42 shows a summary of peptide library analysis of HPV types 6, 1 1 , 1 6, 18, 31 39, 45, 52, 58, 73 with anti-Fla-32 and anti-Fla-69 serum samples obtained from Rabbit Study No. 6.
  • Figure 43 shows exemplary RG1 epitope end-point ELISA titers of pooled serum samples obtained from rabbits immunized with Fla-32 and Fla-69 (Rabbit Study No. 6) (Panel A) , and RG1 epitopes alignment (Panel B). Sequences shown on Panel B correspond to synthetic serotype-specific RG1 - peptides used for ELISAs.
  • Figure 44A Panel A, shows the fermentation parameters used for production of Fla-69 fusions. These parameters were computer controlled by a set of Proportional Integral Derivative algorithms which utilize periodic measurements (based on the parameter being measured) to make adjustments necessary to maintain the indicated set point.
  • Figure 44B, Panel A provides a expression time course of Fla-69 fermentation. Panel B of Figure 44B provides capture ELISA data.
  • Figure 45 Panel B, shows the design of an exemplary animal study.
  • Study animals were scheduled for intramuscular immunization with the indicated fusion formulations four times (Days: 0, 21 , 42, and 63) and subsequently challenged on Day 91 with each of quazi-virions 6, 1 6, 18, 31 , 45 (two sites per virus) , and CRPV (two sites per virus) .
  • Panel A provides an illustration of Fla-32, Fla-69, Fla-76, and Fla-65 fusions constructs used in Rabbit Study No. 5.
  • Figure 46 shows in multiple panels (Panels A, B, C, D, E, and F) the efficacy of various
  • QV quazi-virus
  • Figure 47 shows results of an exemplary in vitro neutralization titer assay in rabbits.
  • the Figure provides neutralization titers of pooled serum samples generated in Rabbit Study No. 5 (4 doses) and Rabbit Study No. 6 (3 doses) as determ ined by "novel methodology" described infra.
  • Figure 48 Panel A, shows exemplary RG-1 epitope end-point ELISA titers of pooled serum samples obtained from animals (Rabbit Study No. 5) immunized with Fla-32, Fla-69, Fla- 65, Fla-76 fusions, respectively.
  • RG-1 Mab ELISAs Demonstrated Specificity to HPV types 1 6, 18, and 45. Fla-69, Fla-76 and Fla-65 fusions elicited comparable RG-1 type-specific immunogenicity as assayed by RG-1 -peptide ELISAs (HPV types 16, 18, and 45 were relatively dominant to other HPV subtypes).
  • Panel B of the Figure 48, provides an alignment of HPV subtype-specific RG1 -peptides used for ELISAs.
  • Figure 49 shows the general scheme used for passive immunization experiments with pooled serum samples obtained from various arms of Rabbit Study No. 5.
  • Figure 50 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples (anti-Fla-32, anti-Fla-69, anti-Fla-65, and anti-Fla-76) obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 6.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi-virus (QV 6): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 51 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 16.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 16): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 52 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 18.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 18): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 53 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 31 .
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 31 ): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 54 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 45.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 45): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 55 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 58.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 58): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 56 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus CRPV.
  • the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified CRPV: Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
  • Figure 57 shows results of an exemplary end-point protection titers assay of pooled serum samples from Rabbit Study No. 5 (4 doses) & Rabbit Study No. 6 (3 doses) as determined via passive immunization (see Examples).
  • Figure 58 Panel A, illustrates the phylogenetic relationships between 5XL2 fusion vaccine candidate (Fla-69) and challenge (HPV56) L2 types as used in "Mouse Study No. 1 .”
  • Figure 59 shows results from an exemplary 50% neutralization titer assay of individual serum samples obtained from animals in Mouse Study No. 1 .
  • Animals were immunized as described infra with HPV pseudoviruses of types 16 (Panel A), 18 (Panel B), and 45 (Panel C). Serum samples were derived from mice two (2) weeks after the third immunization.
  • Figures 60A and 60B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 .
  • Mice were immunized with Cervarix ® HPV vaccine and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
  • Figures 61 A and 61 B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 .
  • Mice were immunized with Gardasil ® HPV vaccine and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
  • Figures 62A and 62B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with PBS and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
  • Figures 63A and 63B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with the Fla-32 fusion and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
  • Figures 64A and 64B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with Fla-69 fusion and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
  • Figure 65 show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 .
  • Figure 65 shows presents background data (PBS challenge) in the study animals. Bioluminescence values are indicated in the callout boxes.
  • Figure 66 shows the results of an exemplary assay.
  • Figure 66 provides a quantitative representation of efficacy data (following PsV56 challenge) from Mouse Study No. 1 .
  • Panel A provides individual relative bioluminiscence unit (RBU) values obtained following immunization with: Cervarix ® HPV vaccine, Gardasil ® HPV vaccine, PBS (control), Fla-69 fusion, Fla-32 fusion, and of Background (no immunization), respectively.
  • Panel B provides average RBU values obtained following immunization with: Cervarix ® HPV vaccine, Gardasil ® HPV vaccine, PBS (control), Fla-69 fusion, Fla-32 fusion, and of Background (no immunization), respectively.
  • This data demonstrates that superior protection against PsV56 challenge was provided by the Fla-69 fusion (P ⁇ 0.05).
  • Figure 67 provides an expression plasmid encoding the Fla-01 fusion product.
  • the nucleic acid sequence of the fusion (SEQ ID NO: 1 ) as well as the amino acid sequence (SEQ ID NO: 101 ) of the fusion product.
  • the following domains are indicated by labeling: one or more HPV domains ⁇ e.g., full length, portions, derivatives, and mutations thereof, etc.); one or more flagellin molecules (e.g., full length, portions, derivatives, and mutations thereof, etc.); one or more amino acid (e.g., naturally occurring or modified) as linkers between any of the constituents of the fusion product; and specific mutations in of the above fusion products constituents.
  • Figure 68 provides an expression plasmid encoding the Fla-02 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 2) as well as the amino acid sequence (SEQ ID NO: 102) of the fusion product.
  • Figure 69 provides an expression plasmid encoding the Fla-03 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 3) as well as the amino acid sequence (SEQ ID NO: 103) of the fusion product.
  • Figure 70 provides an expression plasmid encoding the Fla-04 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 4) as well as the amino acid sequence (SEQ ID NO: 104) of the fusion product.
  • Figure 71 provides an expression plasmid encoding the Fla-05 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 5) as well as the amino acid sequence (SEQ ID NO: 105) of the fusion product.
  • Figure 72 provides an expression plasmid encoding the Fla-06 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 6) as well as the amino acid sequence (SEQ ID NO: 106) of the fusion product.
  • Figure 73 provides an expression plasmid encoding the Fla-07 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 7) as well as the amino acid sequence (SEQ ID NO: 107) of the fusion product.
  • Figure 74 provides an expression plasmid encoding the Fla-8 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 8) as well as the amino acid sequence (SEQ ID NO: 108) of the fusion product.
  • Figure 75 provides an expression plasmid encoding the Fla-09 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 9) as well as the amino acid sequence (SEQ ID NO: 109) of the fusion product.
  • Figure 76 provides an expression plasmid encoding the Fla-10 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 10) as well as the amino acid sequence (SEQ ID NO: 1 10) of the fusion product.
  • Figure 77 provides an expression plasmid encoding the Fla-1 1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 1 1 ) as well as the amino acid sequence (SEQ ID NO: 1 1 1 ) of the fusion product.
  • Figure 78 provides an expression plasmid encoding the Fla-12 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 12) as well as the amino acid sequence (SEQ ID NO: 1 12) of the fusion product.
  • Figure 79 provides an expression plasmid encoding the Fla-13 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 13) as well as the amino acid sequence (SEQ ID NO: 1 13) of the fusion product.
  • Figure 80 provides an expression plasmid encoding the Fla-14 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 14) as well as the amino acid sequence (SEQ ID NO: 1 14) of the fusion product.
  • Figure 81 provides an expression plasmid encoding the Fla-15 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 15) as well as the amino acid sequence (SEQ ID NO: 1 15) of the fusion product.
  • Figure 82 provides an expression plasmid encoding the Fla-16 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 16) as well as the amino acid sequence (SEQ ID NO: 1 16) of the fusion product.
  • Figure 83 provides an expression plasmid encoding the Fla-17 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 17).
  • Figure 84 provides an expression plasmid encoding the Fla-18 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 18) as well as the amino acid sequence (SEQ ID NO: 1 18) of the fusion product.
  • Figure 85 provides an expression plasmid encoding the Fla-19 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 19) as well as the amino acid sequence (SEQ ID NO: 1 19) of the fusion product.
  • Figure 86 provides an expression plasmid encoding the Fla-20 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 20) as well as the amino acid sequence (SEQ ID NO: 120) of the fusion product.
  • Figure 87 provides an expression plasmid encoding the Fla-21 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 21 ) as well as the amino acid sequence (SEQ ID NO: 121 ) of the fusion product.
  • Figure 88 provides an expression plasmid encoding the Fla-22 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 22) as well as the amino acid sequence (SEQ ID NO: 122) of the fusion product.
  • Figure 89 provides an expression plasmid encoding the Fla-23 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 23) as well as the amino acid sequence (SEQ ID NO: 123) of the fusion product.
  • Figure 90 provides an expression plasmid encoding the Fla-24 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 24) as well as the amino acid sequence (SEQ ID NO: 124 of the fusion product.
  • Figure 91 provides an expression plasmid encoding the Fla-25 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 25) as well as the amino acid sequence (SEQ ID NO: 125) of the fusion product.
  • Figure 92 provides an expression plasmid encoding the Fla-26 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 26) as well as the amino acid sequence (SEQ ID NO: 126) of the fusion product.
  • Figure 93 provides an expression plasmid encoding the Fla-27 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 27) as well as the amino acid sequence (SEQ ID NO: 127) of the fusion product.
  • Figure 94 provides an expression plasmid encoding the Fla-28 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 28) as well as the amino acid sequence (SEQ ID NO: 128) of the fusion product.
  • Figure 95 provides an expression plasmid encoding the Fla-29 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 29) as well as the amino acid sequence (SEQ ID NO: 129) of the fusion product.
  • Figure 96 provides an expression plasmid encoding the Fla-30 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 30) as well as the amino acid sequence (SEQ ID NO: 130) of the fusion product.
  • Figure 97 provides an expression plasmid encoding the Fla-31 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 31 ) as well as the amino acid sequence (SEQ ID NO: 131 ) of the fusion product.
  • Figure 98 provides an expression plasmid encoding the Fla-32 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 32) as well as the amino acid sequence (SEQ ID NO: 132) of the fusion product.
  • Figure 99 provides an expression plasmid encoding the Fla-33 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 33) as well as the amino acid sequence (SEQ ID NO: 133) of the fusion product.
  • Figure 100 provides an expression plasmid encoding the Fla-34 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 34) as well as the amino acid sequence (SEQ ID NO: 134) of the fusion product.
  • Figure 101 provides an expression plasmid encoding the Fla-35 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 35) as well as the amino acid sequence (SEQ ID NO: 135) of the fusion product.
  • Figure 102 provides an expression plasmid encoding the Fla-36 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 36) as well as the amino acid sequence (SEQ ID NO: 136) of the fusion product.
  • Figure 103 provides an expression plasmid encoding the Fla-37 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 37) as well as the amino acid sequence (SEQ ID NO: 137) of the fusion product.
  • Figure 104 provides an expression plasmid encoding the Fla-38 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 38) as well as the amino acid sequence (SEQ ID NO: 138) of the fusion product.
  • Figure 105 provides an expression plasmid encoding the Fla-39 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 39) as well as the amino acid sequence (SEQ ID NO: 139) of the fusion product.
  • Figure 106 provides an expression plasmid encoding the Fla-40 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 40) as well as the amino acid sequence (SEQ ID NO: 140) of the fusion product.
  • Figure 107 provides an expression plasmid encoding the Fla-41 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 41 ) as well as the amino acid sequence (SEQ ID NO: 141 ) of the fusion product.
  • Figure 108 provides an expression plasmid encoding the Fla-42 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 42) as well as the amino acid sequence (SEQ ID NO: 142) of the fusion product.
  • Figure 109 provides an expression plasmid encoding the Fla-43 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 43) as well as the amino acid sequence (SEQ ID NO: 143) of the fusion product.
  • Figure 1 10 provides an expression plasmid encoding the Fla-44 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 44) as well as the amino acid sequence (SEQ ID NO: 144) of the fusion product.
  • Figure 1 1 1 provides an expression plasmid encoding the Fla-45 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 45) as well as the amino acid sequence (SEQ ID NO: 145) of the fusion product.
  • Figure 1 12 provides an expression plasmid encoding the Fla-46 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 46) as well as the amino acid sequence (SEQ ID NO: 146) of the fusion product.
  • Figure 1 13 provides an expression plasmid encoding the Fla-47 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 47) as well as the amino acid sequence (SEQ ID NO: 147) of the fusion product.
  • Figure 1 14 provides an expression plasmid encoding the Fla-48 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 48) as well as the amino acid sequence (SEQ ID NO: 148) of the fusion product.
  • Figure 1 15 provides an expression plasmid encoding the Fla-49 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 49) as well as the amino acid sequence (SEQ ID NO: 149) of the fusion product.
  • Figure 1 16 provides an expression plasmid encoding the Fla-50 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 50) as well as the amino acid sequence (SEQ ID NO: 150) of the fusion product.
  • Figure 1 17 provides an expression plasmid encoding the Fla-51 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 51 ) as well as the amino acid sequence (SEQ ID NO: 151 ) of the fusion product.
  • Figure 1 18 provides an expression plasmid encoding the Fla-56 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 56) as well as the amino acid sequence (SEQ ID NO: 156) of the fusion product.
  • Figure 1 19 provides an expression plasmid encoding the Fla-57 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 57) as well as the amino acid sequence (SEQ ID NO: 157) of the fusion product.
  • Figure 120 provides an expression plasmid encoding the Fla-58 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 58) as well as the amino acid sequence (SEQ ID NO: 158) of the fusion product.
  • Figure 121 provides an expression plasmid encoding the Fla-59 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 59) as well as the amino acid sequence (SEQ ID NO: 159) of the fusion product.
  • Figure 122 provides an expression plasmid encoding the Fla-60 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 60) as well as the amino acid sequence (SEQ ID NO: 160) of the fusion product.
  • Figure 123 provides an expression plasmid encoding the Fla-61 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 61 ) as well as the amino acid sequence (SEQ ID NO: 161 ) of the fusion product.
  • Figure 124 provides an expression plasmid encoding the Fla-62 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 62) as well as the amino acid sequence (SEQ ID NO: 162) of the fusion product.
  • Figure 125 provides an expression plasmid encoding the Fla-63 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 63) as well as the amino acid sequence (SEQ ID NO: 163) of the fusion product.
  • Figure 126 provides an expression plasmid encoding the Fla-64 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 64) as well as the amino acid sequence (SEQ ID NO: 164) of the fusion product.
  • Figure 127 provides an expression plasmid encoding the Fla-65 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 65) as well as the amino acid sequence (SEQ ID NO: 165) of the fusion product.
  • Figure 128 provides an expression plasmid encoding the Fla-66 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 66) as well as the amino acid sequence (SEQ ID NO: 166) of the fusion product.
  • Figure 129 provides an expression plasmid encoding the Fla-67 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 67) as well as the amino acid sequence (SEQ ID NO: 167) of the fusion product.
  • Figure 130 provides an expression plasmid encoding the Fla-68 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 68) as well as the amino acid sequence (SEQ ID NO: 168) of the fusion product.
  • Figure 131 provides an expression plasmid encoding the Fla-69 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 69) as well as the amino acid sequence (SEQ ID NO: 169) of the fusion product.
  • Figure 132 provides an expression plasmid encoding the Fla-70 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 70) as well as the amino acid sequence (SEQ ID NO: 170) of the fusion product.
  • Figure 133 provides an expression plasmid encoding the Fla-71 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 71 ) as well as the amino acid sequence (SEQ ID NO: 171 ) of the fusion product.
  • Figure 134 provides an expression plasmid encoding the Fla-72 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 72) as well as the amino acid sequence (SEQ ID NO: 172) of the fusion product.
  • Figure 135 provides an expression plasmid encoding the Fla-73 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 73) as well as the amino acid sequence (SEQ ID NO: 173) of the fusion product.
  • Figure 136 provides an expression plasmid encoding the Fla-74 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 74) as well as the amino acid sequence (SEQ ID NO: 174) of the fusion product.
  • Figure 137 provides an expression plasmid encoding the Fla-75 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 75) as well as the amino acid sequence (SEQ ID NO: 175) of the fusion product.
  • Figure 138 provides an expression plasmid encoding the Fla-76 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 76) as well as the amino acid sequence (SEQ ID NO: 176) of the fusion product.
  • Figure 139 provides an expression plasmid encoding the Fla-77 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 77) as well as the amino acid sequence (SEQ ID NO: 177) of the fusion product.
  • Figure 140 provides an expression plasmid encoding the Fla-78 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 78) as well as the amino acid sequence (SEQ ID NO: 178) of the fusion product.
  • Figure 141 provides an expression plasmid encoding the Fla-79 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 79) as well as the amino acid sequence (SEQ ID NO: 179) of the fusion product.
  • Figure 142 provides an expression plasmid encoding the Fla-80 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 80) as well as the amino acid sequence (SEQ ID NO: 180) of the fusion product.
  • Figure 143 provides an expression plasmid encoding the Fla-81 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 81 ) as well as the amino acid sequence (SEQ ID NO: 181 ) of the fusion product.
  • Figure144 provides an expression plasmid encoding the Fla-82 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 82) as well as the amino acid sequence (SEQ ID NO: 182) of the fusion product.
  • Figure 145 provides an expression plasmid encoding the Fla-84 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 84) as well as the amino acid sequence (SEQ ID NO: 184) of the fusion product.
  • Figure 146 provides an expression plasmid encoding the Fla-85 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 85) as well as the amino acid sequence (SEQ ID NO: 185) of the fusion product.
  • Figure 147 provides an expression plasmid encoding the Fla-86 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 86) as well as the amino acid sequence (SEQ ID NO: 186) of the fusion product.
  • Figure 148 provides an expression plasmid encoding the Fla-87 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 87) as well as the amino acid sequence (SEQ ID NO: 187) of the fusion product.
  • Figure 149 provides an expression plasmid encoding the Fla-88 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 88) as well as the amino acid sequence (SEQ ID NO: 188) of the fusion product.
  • Figure 150 provides an expression plasmid encoding the Fla-89 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 89) as well as the amino acid sequence (SEQ ID NO: 189) of the fusion product.
  • Figure 151 provides an expression plasmid encoding the Fla-90 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 90) as well as the amino acid sequence (SEQ ID NO: 190) of the fusion product.
  • Figure 152 provides an expression plasmid encoding the Fla-91 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 91 ) as well as the amino acid sequence (SEQ ID NO: 191 ) of the fusion product.
  • Figure 153 provides an expression plasmid encoding the Fla-92 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 92) as well as the amino acid sequence (SEQ ID NO: 192) of the fusion product.
  • Figure 154 provides an expression plasmid encoding the Fla-93 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 93) as well as the amino acid sequence (SEQ ID NO: 193) of the fusion product.
  • Figure 155 provides an expression plasmid encoding the Fla-94 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 94) as well as the amino acid sequence (SEQ ID NO: 194) of the fusion product.
  • Figure 156 provides an expression plasmid encoding the Fla-95 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 95) as well as the amino acid sequence (SEQ ID NO: 195) of the fusion product.
  • Figure 157 provides an expression plasmid encoding the Fla-96 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 96) as well as the amino acid sequence (SEQ ID NO: 196) of the fusion product.
  • Figure 158 provides an expression plasmid encoding the Fla-97 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 97) as well as the amino acid sequence (SEQ ID NO: 197) of the fusion product.
  • Figure 159 provides an expression plasmid encoding the Fla-99 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 99) as well as the amino acid sequence (SEQ ID NO: 199) of the fusion product.
  • Figure 160 provides an expression plasmid encoding the Fla-100 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 100) as well as the amino acid sequence (SEQ I D NO: 202) of the fusion product.
  • Figure 161 provides an expression plasmid encoding the Fla-200 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 200) as well as the amino acid sequence (SEQ I D NO: 203) of the fusion product.
  • Figure 162 provides an expression plasmid encoding the Fla-201 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 201 ) as well as the amino acid sequence (SEQ I D NO: 204) of the fusion product.
  • Figure 163 provides an expression plasmid encoding the Fla-62M1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 300) as well as the amino acid sequence (SEQ I D NO: 303) of the fusion product.
  • Figure 164 provides an expression plasmid encoding the Fla-32M1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 301 ) as well as the amino acid sequence (SEQ I D NO: 304) of the fusion product.
  • Figure 165 provides an expression plasmid encoding the Fla-65M1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 302) as well as the amino acid sequence (SEQ I D NO: 305) of the fusion product.
  • antigen or "immunogenic polypeptide/peptide” is a molecule capable of being bound by an antibody or T-cell receptor.
  • An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production 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.”
  • 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, or in the context of an MHC, by T-cell receptors.
  • an antigen will be a peptide derived from a protein expressed by a pathogenic organism (e.g., HPV).
  • An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another.
  • an antigenic determinant is an PV polypeptide segment, PV peptide.
  • an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, 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.
  • 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, T-cell receptor, or HLA molecule.
  • epitope and “peptide” are often used interchangeably.
  • B-cell epitope or “target epitope” 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 segment or sub region thereof).
  • HPV and “human papillomavirus” refer to the members of the genus Papillomavirus (PV) that are capable of infecting humans.
  • HPVs genital and cutaneous groups
  • virus “types” or “strains” e.g., HPV 16, HPV 18, HPV 31 , HPV 32, etc.
  • the HPV types that are associated with genital infection and malignancy.
  • vaccine refers to a formulation which contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more oligomeric and/or multimeric HPV peptide compositions of the present invention.
  • the multimeric HPV peptide compositions typically will 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 and/or to attenuate HPV infection and/or attenuate transmissibility of HPV.
  • prophylactic and “preventive” fusion products, vaccines, or compositions are compositions designed and administered to prevent infection, disease, and/or any related sequelae caused by or associated with a pathogenic organism, particularly PV, and more particularly HPV.
  • therapeutic fusion products, vaccines, or compositions are compositions designed and administered to subjects already infected with a pathogenic organism such as at least one HPV strain.
  • Therapeutic vaccines e.g., therapeutic HPV vaccines
  • infectious disease refers to a disorder arising from the invasion of a host, superficially, locally, or systemically, by an infectious organism. Infectious organisms include bacteria, viruses, parasites, fungi, and protozoa.
  • amino acid is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics.
  • Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example.
  • Non- naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like.
  • Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids.
  • Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivitization of the amino acid.
  • Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the epsilon-amino group of the side chain of the naturally occurring Arg amino acid.
  • Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
  • amino acid analogs and mimetics can be found described in, for example, Roberts and Vellaccio, The Peptides: Analysis Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341 , Academic Press, Inc., New York, N.Y. (1983), the entire volume of which is incorporated herein by reference.
  • Other examples include peralkylated amino acids, particularly permethylated amino acids. See, for example, Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 1 1 , p. 235, John Wiley & Sons Inc., New York, N.Y. (1997), the entire book of which is incorporated herein by reference.
  • Yet other examples include amino acids whose amide portion (and, therefore, the amide backbone of the resulting peptide) has been replaced, for example, by a sugar ring, steroid, benzodiazepine or carbo cycle. See, for instance, Burger's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of which is incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptidomimetics and proteins are well known in the art (See, for example, U.S. Pat. No. 5,420,109; M.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules.
  • nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given peptide. Such nucleic acid variations are silent variations, which are one species of conservatively modified variations.
  • AUG which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a peptide is implicit in any described amino acid sequence.
  • individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • TLR-5 agonist refers to a composition that selectively activates or increases normal signal transduction through TLR-5.
  • Nucleic acid sequence refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • a “deletion”, as used herein, refers to a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent.
  • insertion or “addition”, as used herein, refer to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • Amplification refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY (1995)).
  • hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • antigenic determinant refers to that portion of a molecule that makes contact with a particular antibody (i.e., an epitope).
  • a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • protein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • the term includes naturally occurring proteins and peptides as well as those which are recombinantly or synthetically synthesized.
  • fusion product(s) or "fusion protein(s)” as used herein refers to protein constructs that are the result of combining multiple protein domains or linker regions for the purpose of gaining function of the combined functions of the domains or linker regions. This is most often accomplished by molecular cloning of the nucleotide sequences to result in the creation of a new polynucleotide sequence that codes for the desired protein. Alternatively, creation of a fusion protein may be accomplished by chemically joining two proteins together.
  • linker refers to stretches of polynucleotide or polypeptide sequence that are used in the construction of a cloning vector or fusion protein. Functions of a linker region can include introduction of cloning sites into the nucleotide sequence, introduction of a flexible component or space-creating region between two protein domains, or creation of an affinity tag for specific molecule interaction. A linker region may be introduced into a fusion protein without a specific purpose, but results from choices made during cloning.
  • expression vectors or "expression plasmid”, and similar terms, are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host.
  • Such vectors can be used to express eukaryotic genes in a variety of hosts including, but not limited to, bacteria, for example, E. coli, blue-green algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells.
  • mucosal refers to having an affinity for a mucous membrane.
  • cutaneous refers to having an affinity for non-mucosal, skin epithelial cells.
  • comparison of a sequence to a reference sequence is applicable to sequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, and at least about 99% sequence identity to the reference sequence.
  • the determination of percent identity between two sequences can be determined using standard methods and algorithms including, e.g., BLASTN (NCBI ; Schaffer et al., NAR 29:2994-3005, 2001 ), BLASTX (NCBI ; Schaffer et al., NAR 29:2994-3005, 2001 ), ALIGN (GCG, Accelrys), and FASTA (Pearson et al.,
  • amino acid sequences of the invention include those having, e.g., 5, 7, 10, 20, 30, 40, 50, 75, or 100 consecutive amino acids that are 100% identical to the reference sequences.
  • the present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus.
  • the present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
  • the present invention provides various genetic constructs, nucleic acid molecules, that contain or encode for either full length, portions and fragments thereof, or deletions, of the Flagellin (Fla) molecule fused to various versions of additional nucleic acid molecules that contain or encode one or more HPV-L2 peptides, or portions and fragments thereof, presented in mono- or multimeric forms, representing either single or multiple HPV types/subtypes.
  • Detailed descriptions of exemplary constructs are presented in Table 1 below. The present invention is not intended however to be limited to the constructs presented in Table 1 .
  • compositions and methods described herein form the basis for design and construction of additional compositions (e.g., nucleic acid and/or amino acid sequences) and therapeutic and/or prophylactic methods within the sprit of the invention but not specifically recited herein.
  • additional compositions e.g., nucleic acid and/or amino acid sequences
  • therapeutic and/or prophylactic methods within the sprit of the invention but not specifically recited herein.
  • immunmodulating agents e.g., adjuvants
  • adjuvants may be added to, or substituted in place of, Flagellin in the formulations contemplated by the present invention.
  • Fla-L2 fusion product constructs that provide: (1 ) broadly cross-neutralizing properties of various epitopes within the N-terminal portion of HPV L2 proteins (e.g., comprising amino acids (AA) 1 1 -200); and/or (2)
  • L2-peptide fusions and/or (3) one or more strong adjuvants (e.g., Flagellin, or polypeptides of Flagellin, TLR).
  • strong adjuvants e.g., Flagellin, or polypeptides of Flagellin, TLR
  • agonists/antagonists include aluminum adjuvants, oil-in-water based adjuvants including
  • Adjuvants are known immune response potentiators and have been widely applied for many years to increase a subject's immune response to antigenic compositions. (See, WO 2007/1 10409 A1 incorporated by reference herein in its entirety). Examples of adjuvants that have been used for many years and that are approved for human applications, are mainly those based on aluminum (also referred to as "alum") : aluminum hydroxide and aluminum phosphate. In recent years, many new adjuvant compounds have been found, or developed.
  • the main mechanisms in which adjuvants are thought to work are seen as: 1 ) retaining the antigen at the site of injection; 2) causing a mild inflammation at the site of injection; 3) causing the recruitment of dendritic cells towards the site of injection; 4) inducing the uptake of antigen by the dendritic cells, and 5) promoting the maturation of dendritic cells, or combinations of two or more of the above.
  • the present invention is not however intended to be limited to any particular mechanism(s) or theories concerning adjuvants or adjuventation of the fusion products.
  • adjuvants include, but are not limited to, compounds from the following categories: mineral containing compositions, mineral salts, such as, aluminum salts and calcium salts, hydroxides (e.g., aluminum oxyhydroxides), phosphates (e.g., aluminum
  • the antigen becomes more or less adsorbed onto the adjuvant.
  • Oil-emulsion compositions suitable for use as adjuvants include squalene-water emulsions, such as MF59 (See, WO 1990/14837 incorporated by reference herein in its entirety) or submicron oil-in-water emulsions based on MF59. Other submicron oil-in-water emulsions are MF75 (or SAF) and Covaccine HT.
  • squalene-water emulsions such as MF59 (See, WO 1990/14837 incorporated by reference herein in its entirety) or submicron oil-in-water emulsions based on MF59.
  • Other submicron oil-in-water emulsions are MF75 (or SAF) and Covaccine HT.
  • Saponins are a group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots, and flowers of wide variety of plant species. Saponins from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Other saponins are those from Silax ornate, Gypsophilla paniculata and Saponaria officinalis.
  • Saponin adjuvant formulations include purified formulations such as QS7, QS17, QS18, and QS21 (See, U.S. Pat. No. 5,057,540; and WO 1996/33739 each of which is incorporated herein by reference in its entirety), QH-A, QH-B and QH-C, and lipid formulations such as
  • ISCOMs Immunostimulating Complexes
  • ISCOMs typically include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine, and may include one or more of Quill A, QH- A and QH-C.
  • bacterial or microbial derivatives provide useful adjuvants, for example, monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), RC-529, OM-I74, and CpG-motif containing oligonucleotides.
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • RC-529 OM-I74
  • CpG-motif containing oligonucleotides CpG-motif containing oligonucleotides.
  • ADP-ribosylating bacterial toxins may be applied, for example, E. coli heat labile enterotoxin LT, cholera toxin CT, pertussis toxin PT, diphtheria toxoid, tetanus toxoid TT. Mutants of such toxins may also be applicable, for example, LT-K63, LT-R72, LTR192G, and
  • adjuvants that may also be used in vaccine compositions include bioadhesives and mucoadhesives, liposomes, polyoxyethylene ethers and -esters, polyoxyethylene sorbitan ester surfactants in combination with an octoxynol, as well as polyoxyethylene alkyl esters or ester surfactants in combination with at least one additional non-ionic surfactant such as octoxynol.
  • PCPP formulations Suitable adjuvants comprise PCPP formulations, muramyl peptides and imidazoquinolone compounds.
  • cytokines have been applied to stimulate immune responses and in this regard may additionally be useful as adjuvants in combination with the fusion products of the present invention, for instance, the interleukins: IL-I ; IL-2; IL- 4; IL-5; IL-6; IL-7; and IL-12; interferon- ⁇ ; and tumor necrosis factor (TNF).
  • interleukins IL-I ; IL-2; IL- 4; IL-5; IL-6; IL-7; and IL-12
  • interferon- ⁇ interferon- ⁇
  • TNF tumor necrosis factor
  • fusion product constructs may comprise one or more linkers
  • the HPV L2 sequences used within particular fusion products correspond to those presented in WO 2006/083984 A1 .
  • the present invention provides polypeptide
  • compositions for prevention of infection by various papillomavirus (PV) types, and especially human papillomavirus types (HPV).
  • PV papillomavirus
  • HPV human papillomavirus types
  • a multimeric HPV peptide composition is a non-naturally occurring polypeptide comprising two or more PV protein segments or immunogenic peptides from different PV types
  • HPV L2 polypeptide can comprise all or part of the amino acid sequence of an L2 protein of a virus in the family papovavirus;
  • polyomavirus papillomavirus
  • papillomavirus and/or a papillomavirus within the a genus, or the genera ⁇ , Y, ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ (See, de Villiers et al., Virology, 324(1 ):17-27
  • HPV1 human papillomaviruses: HPV1 , HPV2, HPV3, HPV4, HPV5, HPV6,
  • HPV7, HPV8, HPV9, HPV10 HPV1 1 , HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21 , HPV22, HPV23, HPV24, HPV25, HPV26, HPV27,
  • HPV107, HPV108, HPV109, HPV1 10, HPV1 1 1 ; and/or animal papillomaviruses e.g., bovine papillomavirus type 1 (BPV1 ), bovine papillomavirus type 2 (BPV2), bovine
  • BPV4 BPV4
  • CPPV rabbit papillomavirus
  • DSV European elk papillomavirus
  • EEPV European elk papillomavirus
  • COV canine oral papillomavirus
  • RhPV Rhesus monkey papillomavirus
  • ROPV rabbit oral papillomavirus
  • any one or more nucleic acid sequence disclosed, or incorporated by reference, herein e.g., the HPV L2 sequences within SEQ I D NOs: 1 -99, 1 00, 200, 201 , and 300-302).
  • H PV1 Cutaneous
  • HPV2 Cutaneous
  • HPV3 Cutaneous
  • HPV4 Cutaneous
  • H PV5 Cutaneous
  • HPV 6 Mocosal
  • HPV7 Cutaneous
  • HPV8 HPV8
  • RhPV Mocosal
  • RoPV Mocosal
  • the Human Papillomaviruses Compendium On Line compiles and publishes relevant molecular data concerning the human papillomaviruses (HPV) and related animal papillomaviruses.
  • HPV human papillomaviruses
  • the scope of the compendium and database comprises: (I) HPV and animal PV Nucleotide Sequences; (II) Amino Acid and Nucleotide Sequence Alignments; (I II) Analyses; (IV) Related Host Sequences; and (V) Database
  • a PV (e.g., H PV) antigen, or epitope, or peptide, or polypeptide segment of the invention can comprise from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 1 1 , 12, 13, 14, 1 5, 16, 1 7, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 1 0, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 501 , 502, 503, 504, 505, 506, 550, 600, or more, contiguous amino acids, including all values and ranges there between, of a papillomavirus L2 polypeptide (e.g., the HPV L2 sequences within SEQ I D NOs: 101 -199), or more, con
  • a polypeptide segment can comprise at most, at least, or about amino acid position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15,
  • L2 polypeptides can be found in publicly available protein databases such as GenBank (gb), SwissPro (sp), EMBL, and the like.
  • Representative database entries, listed by HPV type with accession number in parenthesis, include, but are not limited to: HPV2 (gb/AAY86489, gb/ABN49461 , gb/ABN49469, gb/AB014925, gb/NP_077121 ) ; HPV3 (sp/P36744); HPV7 (gb/NP_041858.1 ); HPV10
  • HPV16 gb/AA085414, gb/AA015703, gb/AA01571 1 , gb/AAQ10726, gb/AAV91650
  • HPV18 gb/AAF14009, gb/ABP99710, gb/ABP99718, gb/ABP99726, gb/ABP99742, gb/ABP99766, gb/ABP99774, gb/ABP99782, gb/ABP99790, gb/ABP99798, gb/ABP99806, gb/NP_040316
  • HPV26 gb/NP_041 786.1
  • HPV27 dbj/BAE16268, sp/P36755)
  • HPV28 sp/P50799
  • HPV29 sp/P50800
  • HPV36756 sp/P36756 ; HPV33 (sp/P06418) ; HPV39 (gb/AAA47055) ; HPV40 (sp/P36760) ; HPV43 (sp/Q705H5); HPV45 (gb/AAY86493) ; HPV45 (gb/ABP99814, gb/ABP99854, gb/ABP99862, gb/ABP99870, gb/ABP99878, gb/ABP99894, gb/ABP99902, sp/P36761 ) ; HPV51 (sp/P26539) ; HPV52 (sp/P36763) ; H PV53 (gb/ABU54103, gb/ABU541 17, gb/ABU54131 , gb/ABU54152, gb/ABU54159, gb/ABU541 73, gb/N P_
  • HPV77 emb/CAA75467
  • HPV81 emb/CAF05697
  • HPV83 gb/AAK28455, sp/Q91 R53
  • HPV84 gb/AAK09276
  • HPV85 gb/AAD24187
  • HPV86 gb/AAL06740
  • HPV87 emb/CAC17717
  • HPV90 (ref/NP_671 508); HPV91 (gb/AAM89135) ; HPV94
  • HPV97 gb/AAZ39505, gb/AB027082
  • HPV102 gb/AAZ39525)
  • H PV106 gb/AAZ395178. Disclosure of additional HPV L2 and/or Flagellin related compositions (e.g., sequences), methods, and formulations can be found, for example, in the following publications: U.S. Pat. No. 7,915,381 ; WO
  • each amino/nucleic acid sequence represented by the accession number throughout the disclosure is incorporated herein by reference as of the filing date of this application.
  • L2 peptides from at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 1 7, 18, 19, 20, 21 , 22, 25, 30, or more HPV types are coupled together to form a multimeric HPV polypeptide (e.g., portion of a fusion product).
  • Coupling of the segments can be by expression or synthesis of a fusion protein, or by chemical conjugation of the peptides to each other, or chemical conjugation of the peptides to a common substrate or polymer, or via one or more linkers.
  • the present invention includes compositions for preventing or ameliorating PV infections, in particular, HPV infections.
  • the invention contemplates vaccines for use in both active and passive immunization embodiments.
  • Immunogenic compositions proposed to be suitable for use as vaccine(s), may be prepared from multitype HPV polypeptide(s) comprising segments of HPV L2 protein.
  • multitype HPV L2 polypeptides can be used in combination with other HPV proteins or segments thereof, such as: E1 , E2, E3, E4, E5, E6, E7, E8, and/or L1 protein.
  • E1 , E2, E3, E4, E5, E6, E7, E8, and/or L1 protein See, for example, U.S. Pat. Nos. 7,425,438; 7,416,846; 7,416,732; 7,407,807; 7,374,767; 7,201 ,908;
  • the present invention provides a number of monomeric or oligomeric Fla-L2 constructs containing one or more L2 epitopes from a single HPV subtype fused to, or inserted into, a full length Flagellin (Fla) molecule or a portion thereof in various fashions. Additionally, further embodiments, provide a number of multimeric Fla-L2 constructs containing one or more L2 epitope repeats from a single HPV subtype fused to, or inserted into, one or more full length Flagellin molecules or a portions thereof in various fashions.
  • Flagellin molecules As summarized in Table 1 , three types of Flagellin molecules were tested: (1 ) full length Flagellin molecules; (2) D3 domain deletions; or (3) D2-D3 deletion (D2D3 replacement) variants. However, the present invention is not intended to be limited to these three primary classes of Flagellin molecules. Moreover, the present invention contemplates L2 mutants, derivatives, and fragments etc. Table 1 provides exemplary embodiments of certain monomeric and multimeric expression plasmids and certain characteristics of particular corresponding expressed fusion products. TABLE1
  • Fla-41 Fla (1-506 AA)/CT (11-200 AA of HPV16)
  • Fla-42 Fla (1-506 AA)/CT (11-88 AAof HPV16)
  • Fla-60 Fla Fla-60 Fla (1-506 AA)/CT (11-200 AA of HPV16)
  • Fla-63 Fla (1-187; 355- Deleted flagellin AA188- N
  • Fla-66 Fla (1-187; 297- (11-88 AA of HPV16)x
  • Fla-67 Fla (1-187; 297- Fla-34 plus C-terminal
  • Fla-78 Fla (1-180; 402- Fla-65 plus C-terminal
  • Fla-79 Fla (1-187; 355- Fla-74 plus C-terminal
  • Fla-81 Fla Fla-81 Fla (1-187; 297- 11-88AA of HPV16X6HIS
  • Fla-84 Fla (1-187; 297- 10-199AA of HPV39
  • Fla-85 Fla (1-187; 297- 11-200AA of HPV18
  • Fla-86 Fla (1-187; 297- 10-198AA of HPV6
  • Fla-87 Fla (1-187; 297- 11-195AA of HPV31
  • Fla-88 Fla (1-187; 297- 11-200AA of HPV45
  • Fla (1-187; 297- (11-88 AA of HPV16)x 506 AA)/CT (10-88 AA of HPV18)X
  • Fla (1-187; 297- (11-88 AA of HPV16)x 506 AA)/CT (10-88 AA of HPV18)X
  • Fla-96 Fla (1-187; 297- (11-88 AA of HPV39)x 506 AA)/CT (11-88 AA of HPV59X
  • Fla-97 Fla (1-187; 297- (11-88 AA of HPV16)x 506 AA)/CT (10-88 AA of HPV18)X
  • Fla- Fla- Fla (1 -187; 355- (17-38 AA of HPV31 )X 200
  • Fla- Fla- Fla (1 -187; 297- Deleted flagellin AA188- 62M1
  • Fla- Fla- Fla (1 -187; 297- 1 1 -200 AA of HPV16 32M1
  • 6xHIS C-terminal 6 histidine tag
  • Fla 1 -506 AA of structural flagella protein fljB of Salmonella typhimurium LT2 (Gene Bank accession* NP_461698; SEQ ID. No. 21 1 as below:
  • CT C-terminal fusion to Fla
  • D2-D3 domains (188-354aa), or D2-D3 domains
  • NT N-terminal L2 fusion to Fla
  • FCS Furin Cleavage Site mutant
  • WT wild type of L2 (indicated only if used in fusions with mutant L2 sequence, (if not indicated WT sequence area used by default);
  • X used to signify fusion between different L2 sequences or 6xHis tag.
  • Fla-02 and Fla-07 fusion products were purified using purification schemes briefly depicted in FIG. 2 to high homogeneity. (See, FIG. 3).
  • the L2 based fusion products of the present invention do not form virus like particles but rather soluble expression products.
  • Flagellin molecule looks somewhat like an aircraft with two wings and a short body, each wing being about 70A long, 25A wide, and 20 A thick. (See, Samatey et ai,
  • the Flagellin structure was divided into three domains labeled D1 , D2, and D3.
  • Domain D1 comprises an N-terminal segment from Asn 56 to Gin 176, and a C-terminal segment from Thr 402 to Arg 450.
  • Domain D2 also comprises two segments: Lys 177 to Gly 189, and Ala 284 to Ala 401 .
  • a central segment from Tyr 190 to Val 283 makes up Domain D3.
  • the domains are connected by short stretches of two chains in both cases.
  • a cross ⁇ -motif motif ties up the two ends of Domain D1 and connects it to D2, where two hydrogen bonds are formed between Asn 173 and Thr 404.
  • the relatively conservative D1 Domain contains the TLR-5 binding motif, while the immunodominant hypervariable D2-D3 Domains have been shown to play an important role in the observed activity as a mucosal adjuvant. (Ivison et al., Inflamm. Bowel Dis. , 16:401 -409 (2001 ); and Liu et al., Biochem. Biophys. Res. Commun., 392:582-587 (2010)).
  • FIG. 4 supports the proposition that the Fla-L2 fusion products of the present invention (e.g., Fla-07) comprise L2 peptide(s) that are preferably fused or otherwise closely attached to the C-terminal portion of the flagellin domain D1 .
  • This particular structure linkage has been found to only minimally, if at all, interfere with Flagellin's TLR-5-binding motif or its accompanying immunodominant domains (Domains D2/D3).
  • preferred embodiments of the present invention provide fusion products wherein the immunological properties of any, or all, of the domains of the adjuvant molecule (e.g., Flagellin: Domains D1 , and/or D2, and/or D3) are accessible and immunologically active.
  • the immunological activity of the adjuvant (e.g., Flagellin) molecule in the fusion product likewise does not substantially diminish the immunological activity of the L2 sequences (or other PV/HPV polypeptides) therein.
  • various embodiments of the present fusion products comprise a Flagellin backbone molecule that is not significantly impacted (e.g., immunological activity substantially diminished) by the presence of L2 peptides (i.e., Fla-02 or Fla-07) (See, FIG. 5 and FIG. 16). Furthermore, it was shown that the immunogenicity of the immunodominant Flagellin epitopes were not impacted (e.g., immunological activity substantially diminished) in actual Fla-L2 constructs when compared to the immunogenicity of the same epitopes in wild-type Flagellin.
  • the Flagellin-specific ELISA titers of anti-Fla- 02 or anti-Fla-07 rabbit serum showed flagellin-biased responses.
  • additional embodiments further provide Flagellin D3, or D2-D3 Domain deletion variants in certain Fla-L2 fusions products.
  • FIG. 6A depicts one such construct, Fla-32.
  • the Fla-32 fusion product contains a D3 replacement for HPV16L2 (1 1 -200 AA).
  • Fla-31 contains a D3 replacement for HPV16L2 (2-200 AA). Both the Fla-31 and Fla-32 constructs were expressed in soluble bacterial fractions (FIG. 6B) and were purified to high homogeneity for subsequent preclinical studies. (FIG. 7).
  • Fla-31 and Fla-32 fusion products both showed significant L2- immunogenicity.
  • the L2-immunogenicity of the Fla-31 and Fla-32 were greater than that of certain full length fusion product variants.
  • the TLR-5-binding activities of Fla-31 and Fla-32 were comparable. (FIG. 8).
  • both wild type and deletion mutants of Flagellin retain good solubility as well as TLR-5 binding activity when fused to long HPV L2 peptides.
  • Figures 9 and 10 show the expression of other selected monomeric Fla-L2 fusion constructs further representing other HPV serotypes.
  • the present invention contemplates certain monomeric fusions products described herein will be useful as vaccines, immunogenic compositions, or as therapeutics.
  • the aforementioned fusion products are further contemplated to be useful components in the preparation and/or administration of multi-serotype cocktail vaccines, immunogenic compositions, or therapeutic formulations.
  • VLPs virus like particles
  • B-cell receptors will recognize concatenated neutralization epitopes of HPV L2 (e.g., complete sequences, or portions, of one, two, three, or more different HPV L2 genotypes such as, but not limited to: 1 , 6, 1 1 , 15, 16, 18, 31 , 33, 35, 39, 45, 52, 58, 59, or 65 etc.
  • the B-cell receptors will be preferentially activated by the concatenated epitopes as compared to L2 type specific B-cell receptors. It is still further contemplated that the B-cells presenting concatenated HPV epitopes will be more readily activated and will bias the global repertoire of the neutralizing antibody response to cross- reactive epitopes.
  • additional embodiments provide multiple concatenated neutralization epitopes (or portions and mutations thereof) of the same or different HPV L2 genotypes, such that a fusion product can comprise multiple concatenated L2 epitopes from, for example, HPV16, or from HPV18, or from HPV31 , etc.
  • various other fusion products may comprise multiple concatenated L2 epitopes from two, three, four, five, six, seven, eight, or more different types of HPV, for example, one, two, three, or more, L2 epitopes from HPV type 16, and/or one, two, three, or more, L2 epitopes from HPV type 18, and/or one, two, three, or more, L2 epitopes from HPV type 31 , etc.
  • certain embodiments of the present invention validate the above contemplations by providing fusion products comprising concatenated multimeric L2 epitopes that exhibit enhanced breadth and titer of cross-neutralizing antibody generation as compared to certain other monotypic L2 immunogen fusion products.
  • a number of multimeric concatenated fusion products were constructed comprising one, two, three, or more, stretches of multiple HPV L2 peptides representing a single, or different, HPV type(s).
  • Flagellin D3, (e.g., Fla-62) or -D2-D3 (e.g., Fla-64) deletion variants were used as the fusion backbone.
  • Fla-69 construct comprising the combined AD3 property of Fla-32 and the C- terminal L2 fusion site of Fla-07
  • FIG. 1 1 is shown in FIG. 1 1 .
  • FIG. 12 shows some of the characteristics of four different multimeric fusion constructs used for expression, purification, and/or subsequent in vivo studies.
  • Fla-69 and Fla-70 contain 1 1 -88 AA L2 peptides representing HPV5 and 8, respectively, while Fla-65 and Fla-76 contain 17-38 AA L2 peptides from 5 types of HPV plus longer L2 epitopes of HPV16 of either 1 1 -200 AA (Fla-76) or 1 1 -88 AA (Fla-65).
  • the expression, solubility, and a brief purification process, for fusion products Fla-69 and Fla-70 is shown in FIGs. 13 and 14, respectively.
  • FIG. 15 shows SEC HPLC assay results for purified Fla-69 fusion product along with some of its comparative characteristics including the Fla-32 and Fla-62 backbone. Fla-69 fusion product forms soluble 7-8-mer oligomers.
  • the purification of Fla-65 and Fla-76 fusion products is shown in FIG. 16.
  • the purification scheme used for Fla-65 and Fla-76 fusion products was the same as that used for Fla-69 and Fla-70 as shown, briefly, in FIG. 13.
  • FIGs. 15 and 16 show that the fusion products of the present invention (e.g., Fla-65 and Fla-76) can be purified to high homogeneity.
  • fusion products were highly expressed (e.g., in bacterial expression systems, including but not limited to E. coli. BL21 (DE3) or
  • BLR(DE3) BLR(DE3)), soluble, or solulizable (e.g., using standard solubilization techniques, including, but not limited to, microfluidization techniques), and can be readily purified to high homogeneity (e.g., >10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, or 99.0%, or greater, purity).
  • the present invention provides methods of designing and producing clinical quality fusion products suitable for research, preclinical, clinical, and public health uses.
  • the L2 based fusion products of the present invention do not form virus like particles but rather are soluble expression product proteins.
  • the TLR-5 binding activity of two exemplary lots of Fla-69 fusion product were comparable with that of the Fla-32 and/or Fla-62 backbone fusions; furthermore, the TLR-5 binding activity of Fla-69 and/or Fla-76 were comparable with that of Fla-65 backbone fusion.
  • the invention encompasses Flagellin D2 and/or D2-3 mutants
  • compositions and methods of the present invention provide effective fusion product constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs against a broad spectrum of HPV types/subtypes.
  • HPV animal models A number of useful HPV animal models are known to those skilled in the art. For example, in certain embodiments, the efficacy of fusion products of the present invention was evaluated in both mouse and rabbit HPV animal models.
  • One such animal model exploits the ability of HPV L1/L2 virus-like particles to encapsidate foreign DNA, representing either heterologous PV genome (i.e., Cotton-Rabbit Papilloma Virus (CRPV) (Christensen, Antivir. Chem. Chemother. , 16:355-362 (2005); and Culp et al., J.
  • CRPV Cotton-Rabbit Papilloma Virus
  • hybrid pseudoviruses are able to induce immune responses related to either: 1 ) a disease related to the encapsidated genome (e.g., the rabbit model used; CRPV); or 2) the infection which might be detected by expression of reporter genes (e.g., the mouse model used).
  • the exemplary rabbit model used herein exploits "quazi-viruses” that encapsidate the CRPV genome (classified as type I virus under the "Baltimore scheme") which possess a nonsegmented double strand-DNA genome. (Baltimore, D., Bacteriol. Rev., 35:235-241 (1971 )). Quazi-virus infects rabbits, causing keratinous carcinomas that are histologically indistinguishable from tumors caused by wild type CRPV. (Culp et al., J. Virol., 80:1 1381 -1 1384 (2006)).
  • the exemplary mouse model exploits vaginal infection by pseudovirions expressing luciferase, or red, or green, fluorescent proteins which can be detected in vivo. (Alphs et al., Proc. Natl. Acad. Sci. U.S.A. 105:5850-5855 (2008)).
  • various fusion product compositions of the present invention were tested head-to-head against commercially available HPV L1 vaccines (i.e., Gardasil ® HPV vaccine, Merck, Whitehouse Station, NJ, and Cervarix ® HPV vaccine, GlaxoSmithKlein, London, United Kingdom) in preclinical animals studies.
  • HPV L1 vaccines i.e., Gardasil ® HPV vaccine, Merck, Whitehouse Station, NJ, and Cervarix ® HPV vaccine, GlaxoSmithKlein, London, United Kingdom
  • the head-to-head studies included assessment of the respective efficacy of the candidates in mice and rabbits via active and passive immunizations as well as analysis of the immunogenicity of target antigens therein as assayed by in vitro neutralization assays, L2-specific ELISAs, RG1 -specific ELISAs, and PepSet ® Peptide Libraries.
  • L2 vaccines recombinant concatenated L2 (1 1 -88 AA) fusion products e.g., vaccine candidates
  • monomeric fusion product formulations administered with adjuvant(s) in the aforementioned rabbit and mouse models were tested in comparison with monomeric fusion product formulations administered with adjuvant(s) in the aforementioned rabbit and mouse models.
  • TLR-5 agonists e.g., Flagellin
  • Rabbit Study No. 3 was designed to compare the immunogenicity and/or protective efficacy of monomeric fusion product constructs comprising: 1 ) C-terminal L2 polypeptides fused to full-length Flagellin (e.g., Fla-02 and Fla-07); and 2) Flagellin D3-L2 replacements (e.g., Fla-31 or Fla-32) containing HPV16 L2 peptides.
  • Flagellin e.g., Fla-02 and Fla-07
  • Flagellin D3-L2 replacements e.g., Fla-31 or Fla-32 containing HPV16 L2 peptides.
  • FIG. 19 shows test animals were immunized with either: 1 ) placebo ("None”); or 2) full length Flagellin (e.g., Fla-01 ); or 3) D3 deletion Flagellin backbone (e.g., Fla-62); or 4) full length Fla-L2 fusion product (e.g., Fla-02 or Fla-07); or 5) D3- L2 Flagellin replacements (e.g., Fla-31 or Fla-32).
  • 1 placebo
  • full length Flagellin e.g., Fla-01
  • D3 deletion Flagellin backbone e.g., Fla-62
  • full length Fla-L2 fusion product e.g., Fla-02 or Fla-07
  • D3- L2 Flagellin replacements e.g., Fla-31 or Fla-32).
  • Vaccinations were performed 4 times, 21 Days apart and selected animals were challenged on Day 21 after the last immunization with quizi- viruses 6, 16, 18, and 58, and wild type CRPV. Serum samples generated at various time points over the course of study were assessed for the polyclonal immune response(s) against either recombinant L2-antigen (rl_2) (HPV16) or Flagellin (full length Fla-01 antigen) via standard ELISA assays. Results are shown as End Point ELISA titers in FIGs. 20 and 21 , respectively.
  • FIG. 21 shows the results for full-length Flagellin-L2 fusions.
  • FIG. 21 further shows results for D3-L2 replacements.
  • FIG. 21 shows individual anti-rl_2 titers (Panel A) were about ten times lower than corresponding anti-Flagellin titers (Panel B) for all time points depicted. D3-L2 replacements demonstrated a significant shift towards L2 antigen (Panel A versus Panel B) and the appearance of a "Boosting Effect" from sequential immunizations was more evident than with full-length Flagellin constructs (FIG. 21 ).
  • FIG 22 shows the homologous neutralizing immune response (anti-Psv-16) (Panel A) was just slightly increased after 4th dose when compared with a 2 dose regimen for all vaccine formulations, while differences between the 2 and 4 doses for heterologous responses (anti-Psv-18) (Panel B) were more pronounced.
  • anti-Psv-16 homologous neutralizing immune response
  • Analogous responses anti-Psv-18
  • Psv-18 heterologous responses
  • both PsVs tested the neutralizing responses to D-3-L replacements (Fla-32 or Fla-31 ) were significantly higher than responses to the corresponding full-length-L2 fusions (Fla-02 or Fla-07).
  • an adjuvant e.g., AF04 adjuvant
  • AF04 adjuvant was shown to significantly increase neutralization potency of certain fusion products, such as, Fla-32, but only marginally or not appreciable increase the potency of other certain fusion products, such as, Fla-31 .
  • FIG. 23 shows results from one of these studies. Briefly, FIG. 23 provides results 8 weeks post challenge as described above; the figure shows that both full-length Fla-L2 fusions as well as Fla-32 were fully protective against challenge with QV6, 16, 18, and 58. In this embodiment, the presence of AF04 adjuvant in the particular formulations of Fla-31 and/or Fla-32 tested reduced protection in test animals against challenge with either QV6 (Fla-31 ) or QV18 (Fla-32).
  • the present invention contemplates broadly cross protective fusion products.
  • representative multimeric construct Fla-69 comprising 1 1 -200 AA L2 epitopes from HPV6, 16, 18, 31 , and 39 (FIG. 24) was designed and subsequently tested in an exemplary animal study to access its immunogenicity and/or protectiveness in rabbits challenged with QV6, 16, 18, 58 as well as wild-type CRPV.
  • FIG. 25 L2 epitopes of three quazi- virions (6, 16, and 18) used to challenge test animals were presented in certain fusion product vaccine formulations while QV58 was not.
  • the L2-dendrogram of FIG. 24 reveals a close relationship between QV16 and 58.
  • FIG. 25 presents the study design including immunization and challenge dosages and regimes.
  • commercially available human HPV vaccines Gardasil ® and Cervarix ® were administered four times at human dosage levels.
  • the immunogenicity of candidate vaccine formulations were assessed under two types of neutralization methodologies ((1 ) "conventional methodology”: Buck et al., Methods in Mol. Med., 445-461 (Monograph) (2004); and (2) “novel methodology”: Day et al., Cell Host. Microbe., 8:260-270 (2010)) as well as under serotype specific RG1 -epitope ELISAs and peptide library studies as described below.
  • FIGs. 26 and 27 depict the results of a first type of neutralization methodology test ("conventional methodology") of individual serum samples against PsV16 and 18, respectively. Serum samples from two time-points, Days 56 and 77, were compared. Neutralization titers of pre-challenge serum samples of Fla-32 and Fla- 69 against HPV16 were comparable to each other and were about 10 fold lower than corresponding titers for commercial L1 vaccines. (See, FIG. 26). A "boosting effect” (i.e., an increase in titer after the third immunization) was observed and more pronounced for Fla-69 than for Fla-32.
  • a "boosting effect” i.e., an increase in titer after the third immunization
  • the present invention is not intended to be limited to any particular mechanism(s), it is thought the results can be explained by the size difference in HPV16 L2 epitopes (1 1 -88 versus 1 1 -200 AA).
  • the Fla-69 fusion product is superior to the Fla-32 fusion product based on observed heterologous neutralization results (PsV18). Fla-32 antiserum showed no detectable level of neutralization, while pre-challenge titers of Fla-69 serum samples showed titers comparable to those against PsV16. (See, FIG. 27).
  • pseudoviruses 293TT cells, which contains both primary (HSPG) and secondary (unknown) receptors.
  • HSPG primary
  • secondary (unknown) receptors One of the features of 293TT cells allows them to quickly internalize target pseudoviruses. This feature prevents proper virus maturation and therefore results in only weak surface exposure of L2 neutralizing domain(s) on the cells.
  • the proffered in vivo mechanism for HP (Ps) virus infection of 293TT cells involves HPV binding to HSPG receptors on the base membrane for a relatively long period of time (e.g., from 6 to 24 hours) whereupon the HPV undergoes conformational changes and subsequent furin cleavage.
  • L2-neutralizing epitope(s) are thought to allow L2-neutralizing epitope(s) to be more fully exposed on the viral surface and to thus better interact with L2-antibodies.
  • the present invention is not intended to be limited to any particular mechanism(s) of action, despite the recitation of the potential mechanisms by Day et al.
  • Still other embodiments provide additional exemplary neutralization assays and tests.
  • the present invention employs a second type of neutralization methodology test referred to as the "novel methodology".
  • the "novel methodology” used was as described by Day et al. (Day et al., Cell Host Microbe, 8:260-270 (2010)). Briefly, this methodology is based on the proposed spatiotemporal separation of L2 epitope(s) exposed on the base membrane (HSPG) and the subsequent/concomitant binding of HPV to secondary receptor(s) on the HSPG-epithelial cell surface.
  • the "novel methodology” is thought to increase sensitivity of the assay more than 30 fold when compared to the “conventional methodology.” Again, the present invention is not intended to be limited to any particular mechanism(s) of action, despite the recitation of potential mechanisms by Day et al. In certain embodiments, tests done using the "novel methodology” revealed significant neutralization differences between proposed fusion product formulations. (See, FIG. 29). Nevertheless, the differences observed among fusion product candidates did not correlate well to the actual protectiveness of the candidates since all candidates showed sufficient to good protection against challenge viruses.
  • fusion product formulations e.g., vaccine candidates
  • a passive immunization study was devised using pooled serum samples from Rabbit Study No. 6. (See, FIG. 30). Briefly, pooled fusion product samples were serially diluted (in five fold increments) in preimmune rabbit serum as indicated in FIG. 30. The resulting mixtures were used for the intravenous immunization of study animals (e.g., rabbits) at two animals per described dilution followed by challenge with a broad spectrum of quazi-viruses (QV 6, 16, 18, 31 , 45, and 58) and CRPV. (FIG. 30 and FIG.
  • FIGs. 32-38 demonstrate the protective efficacy of exemplary fusion product dilutions against each of the noted challenge quazi-virions at 8 weeks post challenge. Efficacy results from this study are represented as end-point protective titers shown in
  • FIG. 39 shows the Fla-69 fusion product candidate provided good cross- protective antibody responses against QV6, 16, 18, 31 , and 58 as well as a lower but significant response against QV45 (even at 100 fold serum dilution level) and a modest response against CRPV.
  • the protective properties of the commercially available L1 vaccines (FIG. 39) approximates results observed in respective human clinical trials, notably: Gardasil ® protected against QV6, 16, and 18, Cervarix ® protected against QV16 and 18 and somewhat against QV31 as well (although at a lower level than the Fla-69 fusion product). Cervarix ® did not significantly protect against QV45.
  • the exemplary multimeric-L2 fusion product, Fla-69 provided protection comparable to commercially available HPV L1 vaccines against homologous challenges and furthermore demonstrated superior cross-protection as shown in this passive immunization study.
  • Non- Cleavable PepSet ® Peptide Libraries comprising: 1 ) L2 amino acids 2 to 200; 2) 15 residues with an offset of 4 residues representing HPV types 6, 1 1 , 16, 18, 31 , 39, 45, 51 , 52, 58, 59, and 73; 3) a Rabbit Oral Papillomavirus (ROPV); and 4) a Cottontail Rabbit Papillomavirus (CRPV) were generated by Mimotopes, Inc., (St. Paul, MN) as described in Example 1 1 and used in subsequent additional studies.
  • ROPV Rabbit Oral Papillomavirus
  • Results from two pooled serum samples from Rabbit Study No. 6 were applied to these peptide libraries via ELISA.
  • Results of ELISA with anti- Fla-32 (HPV 16 L2 1 1 -200 AA) serum pool (diluted to 1/1 ,000) against L2-PepSet ® Peptide Libraries of HPV types 6, 16, 18, 45 and 58 used for challenge of rabbits during active immunization phase are shown in FIGs. 40A-40C, Panels A-B, respectively, therein.
  • FIGs. 40A-40C Panels A-B, respectively, therein.
  • each bar shown represents individual peptide signal intensity measured at OD 405 nm and each library is presented by about 47 overlapping 15 AA peptide sequences each of which being off-set by 4 residues.
  • the homologous immune response depicted against HPV16 (FIG. 40A, Panel B) demonstrated the broadest range of signal intensity across the tested L2 sequence.
  • peptides 4, 5, and 6, constituting the RG1 neutralizing epitope were dominant across all tested types.
  • signals for HPV16 group were higher than for HPV18, 45, or 6.
  • the condolyma type HPV6 is depicted with lowest signal intensity, but broad cross-reactivity with an emphasis on the "RG1 " domain.
  • FIGs. 41 A-41 C show a second example of an HPV L2 type specific PepSet ®
  • FIGs. 41 A-41 C show signal intensity of multimeric L2 construct, Fla-69, against all HPV types is much stronger than for monomeric constructs in this embodiment. (See, FIGs. 40A-40C).
  • FIGs. 41 A- 41 C show the immunoreactivity of test peptides NOs. 4, 5, and 6. Downstream regions in each Panel also show immunoreactivity with anti-Fla-69 serum, although not as intense as RG1 . (FIGs. 41 A-41 C). Little to no reactivity was seen for peptides from the middle to the C-terminal end of each HPV type L2 epitope.
  • FIG. 42, Panels A and B show the intensity and specificity of rabbit anti-Fla-32 and anti-Fla-69 serum pools (diluted 1/1 ,000), respectively, as assayed with HPV L2 type PepSet ® Peptide Libraries.
  • serotype specific RG1 -End Point ELISA titers were evaluated (FIG. 43, Panels A and B) to compare RG1 -specific immune responses elicited by two exemplary fusion product vaccine candidates (e.g., Fla-32 and Fla-69).
  • RG1 peptides corresponding to HPV6, 16, 18, 31 , and 45 as well as CRPV were synthesized (FIG. 43, Panel B; RG1 epitope alignment of synthetic serotype specific RG1 peptides used in ELISAs) and subsequently used as probes in determining titers in pooled serum samples from rabbits immunized with either Fla-32 or Fla-69.
  • FIG. 43, Panels A and B RG1 epitope alignment of synthetic serotype specific RG1 peptides used in ELISAs
  • Fla-69 fusion product was from 3 to 20 fold more immunoreactive with all tested RG1 sequences than was Fla-32.
  • Fla-69 induced anti- HPV45 and anti-HPV58 titers that were 20 times higher than those of Fla-32 for HPV45 and 58.
  • PepSet ® Peptide Libraries were used to discriminate Fla- 32 (L2-monomeric) from Fla-69 (L2- multimeric) antigens by L2-linear-epitope-specific immunogenicity. Consistent with results from exemplary passive immunization studies (See, FIG. 39), Fla-69 elicited broader immune responses than Fla-32. The elicited responses were mainly directed to the immunodominant RG-1 epitope.
  • FIG. 44A shows parameter set points and control methods for a low temperature (26 °C) cultivation at 20% dissolved oxygen held at pH 6.8 prior to induction with 1 mM IPTG; 2) FIG. 44A, Panel A, shows parameter set points and control methods for a low temperature (26 °C) cultivation at 20% dissolved oxygen held at pH 6.8 prior to induction with 1 mM IPTG; 2) FIG. 44A, Panel A, shows parameter set points and control methods for a low temperature (26 °C) cultivation at 20% dissolved oxygen held at pH 6.8 prior to induction with 1 mM IPTG; 2) FIG.
  • FIG. 44A Panel B, shows an exemplary cultivation profile showing a cell density target at 20 OD 600 nm prior to induction and a glucose concentration with a minimum of 20 g/L(left side) and cell density and specific replication rate (right side) (the shaded area on right panel indicates an optimal range of cell density and a specific growth rate versus glucose concentration) ;
  • FIG. 44B, Panel A shows pre- and post-induction samples treated with EasyLyse® Bacterial Protein
  • Extraction Solution (Epicentre Technologies, Inc, Madison, Wl) run on SDS/PAGE to evaluate soluble (S) and insoluble (P) fractions as compared to the insoluble fraction from a shake flask as positive control (e.g., microfluidization yields a 1 , 5, 1 0, 20, 30, 40, 50, 60, 70, 80, or 90%, or greater, soluble fraction of target fusion product protein) ; and FIG.
  • Panel B shows quantization by capture ELISA (ELISA wells coated with rabbit polyclonal serum raised against Fla-62 (AD3 deletion)) to capture post induction Fla-69 as detected by Mab RG1 directed against the putative neutralizing domain of HPV16 L2 (AA 1 7-36).
  • exemplary production processes for fusion products of the present invention were shown to be scalable and
  • capture-ELISA methods were developed for Fla- L2 fusions that were successfully applied to monitoring exemplary production processes.
  • fermentation conditions were developed that yielded from about 1 .0-0.1 g/L of target fusion product proteins, and preferably, about 0.6g/L of target fusion product proteins.
  • Fla-65 and Fla-76 fusion products containing mainly RG1 peptides of various HPV types, were constructed and evaluated in exemplary animal model studies (Rabbit Study No. 5) (FIG. 45). I n various studies with Fla-65 and Fla-78 the Fla-32 and Fla-69 constructs and fusion products were used as controls.
  • Fla-65 and Fla-78 fusion products were designed to examine the protectiveness of concatenated RG1 s fused to either 1 1 -200 AA (e.g., Fla-78) or 1 1 -88 AA (e.g., Fla-65) of HPV1 6 L2 used as the source of putative T-helper epitopes.
  • the present invention contemplates that in some embodiments, the addition of one or more T-helper epitopes (e.g., promiscuous T helper epitope(s)) to the fusion products increases the protective efficacy of RG1 -based vaccines in branched or unbranched configurations.
  • T-helper epitopes e.g., promiscuous T helper epitope(s)
  • several putative T-helper epitopes have been identified at the N terminus of the L2 protein.
  • FIGs. 50-56 show an exemplary preferred embodiment the Fla-76 fusion product.
  • various multi-RG1 fusion products/constructs of the present invention provided similar protection profiles as compared to that of Fla-69 against all tested viral challenges. Accordingly, in some of these embodiments, it is contemplated that one long peptide (e.g., HPV16-L2 1 1 -88 or 1 1 -200 AA) is sufficient for effective presentation of 1 , 2, 3, 4, 5, or more, other concatenated protective epitopes (RG1 s).
  • one long peptide e.g., HPV16-L2 1 1 -88 or 1 1 -200 AA
  • HP16-L2 1 1 -200 AA peptide demonstrated slight superiority over HP16-L2 1 1 -88 AA peptide (Fla-65). While the present invention is not intended to be limited to any particular mechanism(s) or sequence(s), it is thought that this result suggests the presence of T-helper epitopes in the AA 88-200 L2-region.
  • the present invention contemplates, broadly cross-neutralizing and/or protective (e.g., efficacious prophylactic vaccine compositions and/or therapeutic compositions against from about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, or more, types of HPV) L2 based fusion products comprising a number (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, or more, sequences) of short RG1 -epitopes in one formulation, or multiple separate formulations, administered to a subject.
  • protective e.g., efficacious prophylactic vaccine compositions and/or therapeutic compositions against from about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, or more, types of HPV
  • L2 based fusion products comprising a number (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, or more, sequences) of short RG1 -epit
  • the art provides useful murine cervicovaginal challenge model(s) of papillomavirus virus (PV) transmission.
  • PV papillomavirus virus
  • the murine model of Roberts et al. employs high titer PV pseudoviruses, which are authentic PV capsids composed of the L1 major and L2 minor structural proteins that have encapsidated a non-PV plasm id encoding a quantifiable reporter gene to monitor successful infection.
  • high titer PV pseudoviruses which are authentic PV capsids composed of the L1 major and L2 minor structural proteins that have encapsidated a non-PV plasm id encoding a quantifiable reporter gene to monitor successful infection.
  • viral challenge of mice involved in certain studies was delayed for a period of time (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 24, or more, months) after the last immunization received in order to study the durability of immune responses. In certain preferred embodiments, viral challenge of the mice was delayed for a period of 4 months after the last immunization.
  • FIG. 59 shows that, in certain embodiments, the Fla-32 fusion product elicited a slightly higher immune response to HPV16, as detected in the 2nd week after final immunization than did Fla-69. While the present invention is not intended to be limited to any particular mechanism(s), it is thought that this result can be explained by the difference in length of the L2 epitope (AA 11 -200 for Fla-32 and AA 1 1 -88 for Fla-69) .
  • mice immunized with either commercial HPV vaccine were not protected against PsV56 challenge.
  • multimeric variants e.g., Fla-69
  • Quantification of these data is expressed in Relative Bioluminescence Unites in FIG. 66.
  • the fusion products described herein generally contain from about 1 , 2, 3, 4, 5,6, 7, 8, 9, 10, 100, 200, 300, 400, to about 500 or more amino acid residues.
  • products/polypeptides can be prepared using any of a number of chemical peptide synthesis techniques well known to those of ordinary skill in the art including both solution methods and solid phase methods.
  • chemical peptide synthesis techniques well known to those of ordinary skill in the art including both solution methods and solid phase methods.
  • the following description briefly provides an overview of various synthetic polypeptide production methodologies applicable to certain embodiments of the present invention.
  • solid phase synthesis in which the C-terminal amino acid of the polypeptide sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred synthetic method for preparing the polypeptides.
  • Techniques for solid phase synthesis are described by Merrifield et al., J. Am. Chem. Soc, 85:2149-2154 (1963). Many automated systems for performing solid phase peptide synthesis are commercially available.
  • Solid phase synthesis is started from the carboxy-terminal end (i.e., the C-terminus) of the polypeptide by coupling a protected amino acid via its carboxyl group to a suitable solid support.
  • the solid support used is not a critical feature provided that it is capable of binding to the carboxyl group while remaining substantially inert to the reagents utilized in the peptide synthesis procedure.
  • a starting material can be prepared by attaching an amino- protected amino acid via a benzyl ester linkage to a chloromethylated resin or a hydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin.
  • halomethyl resins such as chloromethyl resin or bromomethyl resin
  • hydroxymethyl resins such as phenol resins, such as 4-(a-[2,4- dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin
  • tert-alkyloxycarbonyl-hydrazidated resins such as tert-alkyloxycarbonyl-hydrazidated resins
  • the acid form of the peptides may be prepared by the solid phase peptide synthesis procedure using a benzyl ester resin as a solid support.
  • the corresponding amides may be produced by using benzhydrylamine or methylbenzhydrylamine resin as the solid support.
  • the a-amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive a-amino function.
  • Certain amino acids also contain reactive side-chain functional groups (e.g., sulfhydryl, amino, carboxyl, hydroxyl, etc.) which must also be protected with appropriate protecting groups to prevent chemical reactions from occurring at those sites during the peptide synthesis.
  • Protecting groups are well known to those of skill in the art. (See, for example, The Peptides: Analysis, Synthesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, N.Y. (1981 )).
  • a properly selected a-amino protecting group will render the .alpha. -amino function inert during the coupling reaction, will be readily removable after coupling under conditions that will not remove side chain protecting groups, will not alter the structure of the peptide fragment, and will prevent racemization upon activation immediately prior to coupling.
  • side chain protecting groups must be chosen to render the side chain functional group inert during the synthesis, must be stable under the conditions used to remove the a-amino protecting group, and must be removable after completion of the peptide synthesis under conditions that will not alter the structure of the peptide.
  • Coupling of the amino acids may be accomplished by a variety of techniques known to those of skill in the art. Typical approaches involve either the conversion of the amino acid to a derivative that will render the carboxyl group more susceptible to reaction with the free N- terminal amino group of the peptide fragment, or use of a suitable coupling agent such as, for example, ⁇ , ⁇ '-dicyclohexylcarbodimide (DCC) or ⁇ , ⁇ '-diisopropylcarbodiimide (DIPCDI).
  • DCC ⁇ , ⁇ '-dicyclohexylcarbodimide
  • DIPCDI ⁇ , ⁇ '-diisopropylcarbodiimide
  • hydroxybenzotriazole HABt
  • HABt hydroxybenzotriazole
  • synthesis of the peptide is commenced by first coupling the C-terminal amino acid, which is protected at the N-amino position by a protecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solid support.
  • Fmoc fluorenylmethyloxycarbonyl
  • Fmoc-amino acid can, for example, be coupled to the 4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin using ⁇ , ⁇ '- dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) at about 25 °C for about two hours with stirring.
  • DCC dicyclohexylcarbodimide
  • HOBt hydroxybenzotriazole
  • the remaining Fmoc-protected amino acids are coupled stepwise in the desired order.
  • Appropriately protected amino acids are commercially available from a number of suppliers.
  • appropriately protected peptide fragments consisting of more than one amino acid may also be coupled to the "growing" peptide. Selection of an appropriate coupling reagent, as explained above, is well known to those of skill in the art.
  • Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in excess and the coupling is carried out in a medium of dimethylformamide (DMF), methylene chloride (CH 2 CI 2 ), or mixtures thereof. If coupling is incomplete, the coupling reaction may be repeated before deprotection of the N-amino group and addition of the next amino acid. Coupling efficiency may be monitored by a number of means well known to those of skill in the art. A preferred method of monitoring coupling efficiency is by the ninhydrin reaction. Peptide synthesis reactions may be performed automatically using a number of commercially available peptide synthesizers such as the Applied Biosystems ABI 433A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA).
  • the peptide can be cleaved and the protecting groups removed by stirring the insoluble carrier or solid support in anhydrous, liquid hydrogen fluoride (HF) in the presence of anisole and dimethylsulfide at about 0 °C. for about 20 to 90 minutes, preferably 60 minutes; by bubbling hydrogen bromide (HBr) continuously through a 1 mg/10 ml suspension of the resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting groups selected; or by incubating the solid support inside the reaction column used for the solid phase synthesis with 90% trifluoroacetic acid, 5% water and 5% triethylsilane for about 30 to 60 minutes.
  • HF liquid hydrogen fluoride
  • TFA trifluoroacetic acid
  • Other deprotection methods well known to those of skill in the art may also be used.
  • the peptides can be isolated and purified from the reaction mixture by means of peptide purification well known to those of skill in the art.
  • the peptides may be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution. 5. Recombinant Fusion Products, Polypeptides, and/or Proteins
  • the fusion products described herein generally contain from about 1 , 2, 3, 4, 5,6, 7, 8, 9, 10, 100, 200, 300, 400, to about 500 or more amino acid residues.
  • products/polypeptides can be prepared using any of a number of recombinant techniques well known to those of ordinary skill in the art.
  • the following description briefly provides an overview of various recombinant polypeptide production methodologies applicable to certain
  • polypeptides can also be prepared by other means including, for example, recombinant techniques.
  • appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., Molecular Cloning-A Laboratory, Manual (2nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (1989).
  • the polypeptides described herein are derived from papillomavirus L2 protein.
  • the nucleotide sequence of the nucleic acid that encodes L2 is known. Accordingly, the known nucleic acid sequence can be used to make the polypeptides recombinantly or a nucleic acid encoding the desired polypeptide can be derived from the amino acid sequence.
  • this involves creating a nucleic acid sequence that encodes the polypeptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the polypeptide in a host, isolating the expressed polypeptide and, if required, renaturing the polypeptide.
  • Techniques sufficient to guide one of skill through such procedures are found in Sambrook et ai, supra.
  • nucleic acids that encode the polypeptide. This is because the genetic code requires that each amino acid residue in a peptide is specified by at least one triplet of nucleotides in a nucleic acid which encodes the peptide. Due to the degeneracy of the genetic code, many amino acids are equivalently coded by more than one triplet of nucleotides. For instance, the triplets CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.
  • nucleic acid has any of the triplets which encode arginine.
  • One of skill is thoroughly familiar with the genetic code and its use. An introduction to the subject is found in, for example, Chapter 15 of Watson et al., Molecular Biology, of the Gene (Fourth Edition, The
  • any nucleic acid triplet or codon which encodes an amino acid can be used to specify the position of the amino acid in a peptide, certain codons are preferred. It is desirable to select codons for elevated expression of an encoded peptide, for example, when the peptide is purified for use as an immunogenic reagent. Codons are selected by reference to species codon bias tables, which show which codons are most typically used by the organism in which the peptide is to be expressed. The codons used frequently by an organism are translated by the more abundant t-RNAs in the cells of the organism.
  • Codon bias tables are available for most organisms. For an introduction to codon bias tables, see, e.g., Watson et ai, supra.
  • fusion products/polypeptides described herein and the nucleic acid molecules encoding such immunogenic polypeptides can be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non conservative, where such changes might provide for certain advantages in their use, e.g., to increase biological activity.
  • nucleic acid constructs yield a functionally identical construct.
  • silent substitutions i.e., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded peptide
  • amino acid sequence which encodes an amino acid.
  • one of skill will recognize many ways of generating alterations in a given nucleic acid construct.
  • Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Sambrook et ai., supra.
  • Modifications to nucleic acids are evaluated by routine screening techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of encoded peptides can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a complementary nucleic acid, redox or thermal stability of encoded proteins, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
  • Antibodies that bind with specificity to the fusion products/polypeptides described herein are also contemplated.
  • the antibodies include individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these polypeptides in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill.
  • the antibodies are useful as research tools for the isolation of additional quantities of the antigenic polypeptides and for studying the pathogenesis of papillomavirus in general.
  • the antibodies may also be useful therapeutically for passive immunization of an HPV-infected patient.
  • the antibodies include neutralization antibodies.
  • Methods for screening antibodies for neutralization are known in the art. Several specific exemplary in vitro neutralization assays are described in Dvoretsky et al., Virology, 103:369-375 (1980); Roden et al., J. Virol., 70:5875- 5883 (1996); and Pastrana et al., Virology, 321 :205-216 (2004).
  • a number of immunogens are used to produce antibodies specifically reactive with polypeptides.
  • recombinant or synthetic polypeptides of at least 10 amino acids in length, or greater, selected from the polypeptides disclosed herein are the preferred polypeptide immunogens for the production of monoclonal or polyclonal antibodies.
  • an immunogenic polypeptide conjugate is also included as an immunogen.
  • the polypeptides are used either in pure, partially pure or impure form.
  • Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques.
  • the polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies.
  • Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
  • an immunogen preferably a purified peptide, a peptide coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is mixed with an adjuvant and animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the peptide of interest.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the peptide is performed where desired.
  • Antibodies, including binding fragments and single chain recombinant versions thereof, against the polypeptides are raised by immunizing animals, e.g., using immunogenic conjugates comprising a polypeptide covalently attached (conjugated) to a carrier protein as described above.
  • the immunogen of interest is a polypeptide of at least about 10 amino acids, in another embodiment the polypeptide is 20 amino acids in length, and in another embodiment, the fragment is about 30 amino acids in length and comprises amino acids acid residues 1 through 200 from the N-terminal or C-terminal of the papillomavirus L2 protein.
  • immunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.
  • a carrier protein e.g., as a fusion protein
  • immunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides, or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a K D of at least about 0.1 mM, more usually at least about 50 mM, and most preferably at least about 1 mM or better. Often, specific monoclonal antibodies bind with a K D of 0.1 mM or better.
  • this method proceeds by injecting an animal with an immunogen, e.g., an immunogenic peptide of the present invention either alone or optionally linked to a carrier protein. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro.
  • an immunogen e.g., an immunogenic peptide of the present invention either alone or optionally linked to a carrier protein.
  • the animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells.
  • the result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro.
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen.
  • the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host.
  • the polypeptides and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies.
  • Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. (See, for example, Huse et al., Science, 246:1275-1281 (1989); and Ward et al., Nature, 341 :544-546 (1989)).
  • the polypeptide or antibody will be labeled by joining, either covalently or noncovalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
  • the antibodies provided herein can be used in affinity
  • chromatography for isolating additional amounts of the polypeptides identified herein.
  • Columns are prepared, e.g., with the antibodies linked to a solid support, e.g. particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified polypeptides are released.
  • the antibodies can be used to screen expression libraries for particular expression products, for example, papillomavirus proteins.
  • the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
  • antibodies raised against the immunogenic polypeptides described herein can also be used to raise anti-idiotypic antibodies. Such antibodies are useful for detecting or diagnosing various pathological or resistance conditions related to the presence of the respective antigens.
  • both the fusion products/polypeptides described herein and the antibodies that bind with specificity to the fusion products/polypeptides are useful as reagents, as capture agents or labeling agents, in assays to detect a target peptide or antibody.
  • the target molecule can be quantified by a variety of immunoassay methods.
  • the immunoassays can be performed in any of several configurations.
  • Immunoassays often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a labeled peptide or a labeled anti-peptide antibody.
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/peptide complex, or to a modified capture group (e.g., biotin) which is covalently linked to the peptide or anti- peptide antibody.
  • the labeling agent can be a streptavidin molecule which has a fluorescent dye on it and onto which are captured the peptides complexed with MHC (HLA) molecules.
  • HLA MHC
  • These reagents can be used to count single T cells specific for the peptides using commonly used equipment such as flow cytometers, thus providing precise quantitation and phenotype information on the immune response as described by Altman et al., Science, 274:94-96 (1996).
  • the labeling agent is an antibody that specifically binds to the capture agent.
  • agents are well known to those of skill in the art, and most typically comprise labeled antibodies that specifically bind antibodies of the particular animal species from which the capture agent is derived, such as an anti-idiotypic antibody, or antibodies against a peptide when the peptide is the capture agent.
  • the label agent may be a goat anti-mouse IgG, e.g., an antibody specific to the constant region of the mouse antibody.
  • proteins capable of specifically binding immunoglobulin constant regions such as streptococcal protein A or protein G are also used as the labeling agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong nonimmunogenic reactivity with immunoglobulin constant regions from a variety of species.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about five seconds to several hours, preferably from about five minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays are carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 5 °C to 45 °C.
  • Non competitive assay formats such as "sandwich” assays, where the captured analyte
  • analyte e.g., immunogenic peptide or antibody to an immunogenic peptide
  • a capture agent e.g., an antibody or peptide
  • assay formats such as Western blot analysis may also be utilized.
  • various components, including the immunogenic polypeptide or anti- peptide antibody may be bound to a solid surface ("solid phase" assay).
  • the present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus.
  • the present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
  • the present invention includes compositions and methods for preventing or ameliorating PV infections, especially, HPV infections.
  • the invention contemplates immunogenic compositions (e.g., vaccines) for use in active and passive immunization embodiments, methodologies, and administration regimes.
  • Immunogenic compositions may be prepared from multimeric HPV polypeptide(s) comprising segments of HPV L2 protein and carrier/backbone molecule(s).
  • multimeric HPV L2 polypeptides can be used in combination with other HPV proteins or segments thereof, such as E1 , E2, E3, E4, E5, E6, E7, E8, and/or L1 protein.
  • E1 , E2, E3, E4, E5, E6, E7, E8, and/or L1 protein See, for example: U.S. Pat. Nos. 7,425,438; 7,416,846; 7,416,732; 7,407,807; 7,374,767; 7,201 ,908; 7,189,513; and 7,288,258 each of which is incorporated herein by reference in its entirety).
  • vaccines are administered in a manner compatible with a vaccine formulation, and in such amount as will be therapeutically effective and/or immunogenic.
  • the quantity to be administered depends on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Typically, 0.1 , 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, to 100 ng, ⁇ g, or mg may be administered per vaccination or administration.
  • the compositions of the present invention may be administered in a pharmaceutically effective amount.
  • the accurate dosage may vary depending on the antigen(s) to be administered and the desired effect to be obtained, and may be readily determined by those skilled in the art according to factors known in medicine and vaccinology, including the patients age, weight, health state, gender and sensitivity to any components of the intended administration(s), administration routes, and various administration methods.
  • the compositions are intended to be administered in a single dose or in several divided doses.
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the interval(s) between any two or more administration points are constant (e.g., of equal duration).
  • the interval(s) between any two or more administration points are varied (e.g., not of equal duration).
  • Varied intervals can be either random or repeating and formulaic.
  • Exemplary routes of administration to the human body can be through the eyes
  • suitable routes of administration include, for example, oral or transmucosal administration as well as parenteral delivery (e.g., intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration).
  • parenteral delivery e.g., intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients and generally 10% to 95% of active ingredient and more preferably at a concentration of 25% to 75% of active ingredient.
  • exemplary techniques and reagents for solidification/semi- solidification of the particular preparation(s) may be found in, for example, U.S. pat. Nos.
  • the fusion products/polypeptides are preferably supplied in finely divided form along with a surfactant and propellant.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • a carrier can also be included, as desired, such as the inclusion of lecithin for intranasal delivery.
  • injectable preparations may be prepared using physiological saline, aqueous solutions such as Ringers solution, and non-aqueous solutions, such as vegetable oils, high fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzylalcohol, propylene glycol and glycerin, etc.).
  • physiological saline aqueous solutions such as Ringers solution
  • non-aqueous solutions such as vegetable oils, high fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzylalcohol, propylene glycol and glycerin, etc.).
  • aqueous solutions such as Ringers solution
  • non-aqueous solutions such as vegetable oils, high fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzyl
  • a stabilizer for preventing degeneration e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.
  • reagents, and techniques for facilitating solidification/semi-solidification of the preparation(s) (e.g., foam drying, freeze-foam drying, spray drying (atomization), spray-freeze-drying, evaporative drying, percolative drying, vacuum drying, lyophilization, micropelleting, prilling, and variations thereof, etc.), an emulsifier(s), an excipient(s), a buffering agent for pH adjustment, and a preservative for inhibiting contamination, including but not limited to, microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzylalcohol, etc.), and other appropriate microbial growth (e.g., phen
  • compositions and methods of present invention intended for treating a pathological condition additionally can be practiced in conjunction with other therapies.
  • the methods of the invention can be practiced prior to, during, or subsequent to conventional cancer treatments such as surgery, chemotherapy, including administration of small molecule drugs (e.g., oncolytics, anti-angiogenics, etc.) cytokines and growth factors, radiation, surgical interventions, or other methods known in the art.
  • small molecule drugs e.g., oncolytics, anti-angiogenics, etc.
  • the methods of the invention can be practiced prior to, during, or subsequent to conventional treatments, such as antibiotic and/or antiviral administration against infectious agents or other methods known in the art.
  • Treatment of pathological conditions of autoimmune disorders also can be accomplished by combining the methods of the invention for inducing an immune response with conventional treatments for the particular autoimmune diseases.
  • Conventional treatments include, for example, chemotherapy, steroid therapy, insulin and other growth factor and cytokine therapy, passive immunity and inhibitors of T-cell receptor binding.
  • the methods of the invention can be administered in conjunction with these or other methods known in the art and at various times prior, during, or subsequent to initiation of conventional treatments.
  • administration of a compound, immunomodulatory Flagellin fusion products, Flagellin fusion polypeptide, or modification thereof can be, for example, simultaneous with or delivered in alternative administrations with the conventional therapy, including multiple administrations.
  • Simultaneous administration can be, for example, together in the same formulation or in different formulations delivered at about the same time, or immediately in sequence, or following some period of rest.
  • Alternating administrations can be, for example, delivering a Flagellin fusion peptide or polypeptide formulation and a conventional therapeutic treatment in temporally separate administrations.
  • the temporally separate administrations of a compound, immunomodulatory Flagellin peptide, polypeptide or modification thereof, and conventional therapy can similarly use different modes of delivery and routes.
  • Flagellin B (GenBank Gl:16445344, SEQ ID. NO. 210) sequence was amplified using standard PCR techniques from a genomic DNA template isolated directly from Salmonella typhimurium (ATCC # 700720). Codon-optimized HPV L2 sequences were synthesized either by Retrogen, Inc., (San Diego, CA) or Blue Heron Biotechnology, Inc. (Bothell, WA). Derivatives of the Flagellin gene or synthesized L2 sequence were generated by PCR. For expression of Fla-L2 inserts the pET24a+ vector supplied by Novagen, Inc., (Madison, Wl) was used. Clones were grown under kanamycin (Kan) selection and tested via PCR for presence of the inserts. Fla-L2 inserts of selected clones were confirmed by sequencing on a Beckman Coulter CEQ 8800 Genetic Analysis System according to established protocols. A list of all constructs produced is provided at Table 1 supra.
  • Flagellin fusion product constructs were grown in 1 Liter (L) cultures in 4 L baffled shaker flasks for expression and characterization. Briefly, -80 °C glycerol stocks were stabbed and seeded into 125 ml baffled flasks containing 50 ml of Animal Free Luria Broth (AFLB; Teknova, Inc., Hollister, CA) supplemented with Kan at a final concentration of 30 ⁇ g/ml. Seed cultures were grown to stationary phase overnight at 200 rpm in an Innova shaking incubator (see above) at 30 °C.
  • AFLB Animal Free Luria Broth
  • the production cultivation conditions were set to 250 rpm at 37 °C with hourly sampling.
  • a pre-induction sample was taken and the culture was induced with 1 millimolar (mM) of Isopropyl ⁇ -D-l thiogalactopyranoside (IPTG).
  • IPTG Isopropyl ⁇ -D-l thiogalactopyranoside
  • Fla-69 Fermentation of Fla-69 was performed at 4 L culture size in a 5 L Bioflo-3000 device (New Brunswick Scientific, Inc., Edison, NJ) Briefly, -80 °C glycerol bacterial E. coli BL21 (DE3)/ Fla-69 stocks were stabbed and seeded into 150 ml baffled flasks containing 50 ml of AFLB supplemented with Kanamycin at a final concentration of 30 ⁇ g/ml.
  • Seed cultures were grown to stationary phase overnight at 200 rpm in an Annova 44 shaking incubator (see above) at 30 °C; the overnight seed cultures were inoculated into fermentation vessels in accordance with New Brunswick Fermentation preparation manual (See, Jorgensen et ai, Microbiology, 153:1963-1973 (2007)), containing 4 L of AFLB supplemented with 30 ⁇ g/mL of Kan, 5 g/L
  • PID Proportional Integral Derivative
  • the cultivation temperature was set at 26 °C (a cooling ice bath water loop was required to maintain temperature below 30 °C) (Horn et ai, Appl.
  • IPTG Isopropyl ⁇ -D-l thiogalactopyranoside
  • the production phase was allowed to carry on for 4 hours at which time the production culture was harvested by centrifugation in pre-weighed 450 ml bottles at 8,000 rpm for 10 minutes at 4 °C in RC4 Sorvall centrifuge and Ultralight 3,000 rotor. Supernatant was discarded and cell pellets were stored at -80 °C for purification. Biomass yields (g/L wet cell weights (wcw) were calculated based on gross weight (bottle + wet cell pellet) minus bottle tare weight.
  • the post induction samples were analyzed for target protein expression via SDS/PAGE gel electrophoresis and Western Blot with anti-penta-hisitidine antibody or monoclonal antibody directed against the RG-1 domain of HPV16 L2 to verify target antigen (according to the methods of (1 ) Burnette W.N., (Analytical Biochemistry, 1 12(2) : 195-203 ((1981 )); and (2) Milan Bier (ed.) (Electrophoresis. Theory, Methods and Applications, 3rd printing ed., Academic Press. (1959)).
  • Cells were pelletized by centrifugation and the cell paste was stored at -80 s C. Prior to purification, the cell paste was thawed and diluted to 10% solids in 50 mM Tris-HCI, 150 mM NaCI, pH 8.0 with addition of Complete EDTA free protein inhibitors cocktail tablets from Roche, Inc., (Basel, Switzerland) according to included instructions. The cell suspension was passed twice through a macrofluidizer at an average running pressure of 18,000 psi then cooled to 4-8 2 C. The resultant lysate was clarified from cell debris and the insoluble material was removed by centrifugation at 29,000 X g for 40-60 min at 4 to 8 2 C.
  • the first step in the separation of the soluble contaminates from the target protein was achieved by precipitation of the target protein with a 30% of saturation of ammonium sulfate obtained by adding a 3 M ammonium sulfate solution drop wise into the clarified cell lysate on a stir plate with continued stirring for 90 - 120 min at 4 2 C.
  • Precipitated target protein was collected by centrifugation at 29,000 X g for 40-60 min at 4 to 8 2 C.
  • the resulting pellet was resuspended in the same volume of 50 mM TrisHCI, pH 8.0 with the addition of Complete EDTA free protein inhibitors cocktail tablets from Roche, Inc.
  • the collected pellets could be also stored at -80 2 C and processed later.
  • target material was clarified from insoluble materials by centrifugation at 29,000 X g for 40 min at 4 to 8 2 C.
  • the soluble fraction was applied to 5 ml HiTrap Q HP pre-packed columns (attached together for total column volume of 10 ml) and pre-equilibrated with 50 mM Tris-HCI, pH 8.0 (anion exchange chromatography starting buffer). Differences in utilized column volume sizes depend on the required amount of purified material. Larger scale up activities utilized HiTrap FF media in XK 50/30 (GE Healthcare Life Sciences, Piscataway, NJ) with column volumes of 150 -180 ml.
  • Bound protein was eluted with a linear salt gradient of NaCL from 0 to 1 .0 M in 50 mM Tris-HCI, pH 8.0. For final polishing, peak elution fractions were pooled, 3 M ammonium sulfate solution was added into pool up to 1 M followed by filtration through 0.45 ⁇ filter and loaded onto HiTrap Phenyl HP 5 ml pre-packed column pre- equilibrated with 50 mM sodium phosphate, 1 M ammonium sulfate, pH 8.0 binding buffer. Bound protein was eluted with a liner gradient from of Ammonium Sulfate 1 .0 M to 0 M in 50 mM sodium phosphate, pH 8.0.
  • This example describes an alteration to the purification scheme for Fla-69 HPV L2 flagellin fusions described in Example 5. Specifically, the anion exchange chromatography (AEC) and hydrophobic interaction chromatography (HIC) steps from Example 5 were changed as follows. Modification one, post-ammonium sulfate precipitation pellets containing target protein were resuspended in 50 mM Tris-HCI, 8 M urea, pH 8.6 buffer and clarified from insoluble materials by centrifugation at 29,000 X g for 40 min at 4 to 8 2 C.
  • AEC anion exchange chromatography
  • HIC hydrophobic interaction chromatography
  • the soluble fraction was applied to 5 ml HiTrap Q HP pre-packed column or to 180 ml of HiTrap FF resin packed in XK 50 /30 column (GE Healthcare Life Sciences) pre-equilibrated with the same buffer containing 8 M urea at pH 8.6. Bound protein was eluted in the same fashion at new conditions. Modification two, similar modifications have been applied to HIC polishing step - addition of 8 M urea in HiTrap Phenyl HP resin binding (50 mM Tris-HCI, 1 M Ammonium Sulfate) and elution (50 mM Tris-HCI) buffers at pH 8.6.
  • the cell suspension was passed twice through a macrofluidizer at an average running pressure 18,000 psi, cooled to 4-8 2 C.
  • the resultant lysate was clarified from cell debris and insoluble material by centrifugation at 29,000 X g for 15-30 min at 4 to 8 2 C.
  • the soluble fraction was applied to a 5 ml HiTrap Q HP pre-packed column pre-equilibrated with 50 mM Tris-HCI, 8 M urea, pH 8.0 (anion exchange chromatography starting buffer).
  • peak elution fractions were pooled and a 3 M ammonium sulfate solution was added into the pool up to 1 M followed by filtration through 0.45 ⁇ filter and loaded onto HiTrap Phenyl HP 5 ml pre-packed column pre- equilibrated with 50 mM sodium pPhosphate, 1 M ammonium sulfate, 8 M urea, pH 8.0 binding buffer. Flowthrough fractions of target protein separated from bound contaminates were pooled.
  • the target protein underwent a refolding procedure by extensive dialysis against 50 mM Tris-HCI, 150 mM NaCI, 5 mM EDTA, pH 8.0 formulation buffer followed by sterile filtration (0.2 ⁇ ) and stored at -80 2 C.
  • the cell suspension was passed twice through a macrofluidizer at an average running pressure of 18,000 psi, cooled to 4-8 2 C.
  • the resultant lysate was clarified from cell debris and insoluble material by centrifugation at 29,000 X g for 15- 30 min at 4 to 8 2 C.
  • the soluble fraction was applied to two 5 ml HiTrap Q HP pre-packed columns (total CV 10 ml) attached together that were pre-equilibrated with 50 mM Tris-HCI, 8 M urea, pH 7.5 (anion exchange chromatography starting buffer).
  • Bound protein was eluted at the beginning of a linear salt gradient from 0 to 1.0 M NaCI in 50 mM Tris-HCI, 8 M urea, pH 7.5.
  • the pool of peak elution fractions of target protein was extensively dialyzed from urea against 50 mM Tris-HCI, pH 8.0 buffer using Slide-A-Lyzer dialysis cassette from Pierce (Rockford, IL) with a 20K MWCO membrane.
  • samples were clarified from insoluble contaminates by centrifugation at 29,000 X g for 15-30 min at 4 to 8 2 C and the soluble fraction was applied to two 5 ml HiTrap Q HP.
  • Example 9 Bioactivity of Fla-L2 Fusions hTLR5 BioAssay
  • the flagella binding assay utilizes a stable cell line of primary human embryonal kidney cells (HEK293) expressing the transforming gene of adenovirus 5. Assays were performed at InvivoGen Inc., (San Diego, CA). The principle of the bioassay is based on the ability of HEK293 cells stably transfected with plasmid (HEK293pNiFty-SEAP) expressing human Toll- like receptor 5 (hTLR-5) to be induced by binding TLR-5 agonist (flagellin).
  • SEAP soluble secretory embryonic alkaline phosphotase
  • the broadly cross-neutralizing epitope of Human Papillomavirus 16 L2 has previously been described (Gambhira et al., Virology, 81 :13927-13931 (2007); Kando et al., Virology, 358:266-272 (2007); and Rubio et al., Vaccine, 27:1949-1956 (2009)) and is referred to as either the RG-1 neutralizing domain or the RG-1 epitope.
  • RG-1 epitope may also be broadly cross-protective in the quazi-virus challenge of HPV types 6, 16, 18, 31 , 45 and 58 as well as the rabbit type Cottontail Rabbit Papillomavirus (CRPV) in the New Zealand White (NZW) rabbit quazi-virus dermal challenge model.
  • the model epitope resides within amino acids 17-36 for HPV 16 L2: QLYKTCKQAGTCPPDIIPKV (SEQ ID NO. 205, Accession # ACS92698).
  • the RG-1 End Point Elisa procedure contained the following steps: 1 ) the synthetic peptides were solubilized at a concentration of 2 mg/ml in dimethyl sulfoxide (DMSO) (Sigma) and coated onto Corning Costar (#3369, high binding) (Corning Life Sciences, Kennebunk, ME) 96 well ELISA plates in carbonate bi-carbonate buffer (Sigma) at a concentration of 0.5 ⁇ g/well/100 ⁇ and incubated at 4 °C overnight on a level surface; 2) the diluent and blocking solution (Block) used was 1 xPBS+ 0.05% TWEEN 20 (Sigma) with 1 % bovine serum albumin (BSA) (Sigma) and 0.05% Sodium Azide (NaN 3 , Sigma) in all antibody solutions; 3) plates were removed from 4 °C and allowed to warm to room temperature for 30 minutes followed by washing with 1x PBS + 0.05% TWEEN 20; 4) 200 ⁇ of Block was added
  • Non-Cleavable PepSet ® Peptide Libraries (Mimotopes, Inc., St. Paul, MN) consisted of L2 amino acids (AA) 2 to 200, are15 residues in length with an offset of 4 residues between neighboring peptides.
  • Each PepSet ® Peptide Library represented one of the following HPV types: 6, 1 1 , 16, 18, 31 , 39, 45, 51 , 52, 58, 59 and 73 and two rabbit species specific types, Rabbit Oral Papillomavirus ("ROPV”) and Cottontail Rabbit Papillomavirus ("CRPV”).
  • the approximate number of peptides produced per each HPV PepSet ® Peptide Libraries is 47, allowing for two HPV PepSet ® Peptide Libraries on each Block (96-well plate), i.e., 16/6, 18/1 1 , etc. This allowed for multi-library screens with a single serum sample.
  • the Multipin ® peptide technology (Mimotopes, Inc.) involved synthesis of non-cleavable peptides which remained covalently bound at the C- terminus to the modular resin on SynPhase Gears (Mimotopes, Inc.) attached by removable stems to holders ("Blocks") that were compatible with a standard 96-well ELISA plate footprint.
  • the "Blocks” sat on a reservoir or on top of a 96-well plate for processing.
  • PepSet ® Peptide Libraries were removed from the blocking buffer and washed 3 times in 200 ml of washing buffer, followed by incubation in the diluted test serum sample for 1 hour at 60 rpm at room temperature; 6) PepSet ® Peptide Libraries were washed 3 times and then placed in Goat a-Rabbit IgG-AP (labeled secondary antibody, Southern Biotech, Inc.) in blocking solution at a dilution of 1 /1 ,000; 7) PepSet ® Peptide Libraries were washed 4 times in 200 ml of wash buffer; 8) alkaline phosphate substrate buffer (0.1 M glycine buffer pH 10.4, 1 mM MgCI 2 , 1 mM ZnCI 2 , (Sigma)) was used to prepare a 1 mg/ml solution of 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma) ; 9) 100 ⁇ pNPP solution was added to a 96 well high-bind
  • Quazi-virions were provided by Dr. N. Christensen (Pennsylvania State University). Briefly, 293TT producer cells were transfected with circular CRPV genomes together with plasmids expressing codon-modified L1 and L2. Two after transfection the cells were lysed with Brij-58, incubated to allow particle maturation, and treated with Benzonase (Sigma) and Plasmid Safe (Epicenter Biotechnologies, Madison, Wl) to destroy unprotected DNA. Cell lysates were centrifuged on an Optiprep density gradient (Accurate Chemical & Scientific Corp., Westbury, NY).
  • Generation of pseudoviruses using the codon-modified L1 and L2 genes of BPV1 (plasmid pSheLL) (Buck et al., J. Virol. , 78:751 -757 (2004); Buck et al., Methods in Mol. Med., 445-461 (Monograph) (2004)), HPV16 (plasmids p16L1 h and p16L2h) (Leder et al., J.
  • HPV18 plasmids peLl fB and peL2bhb
  • HPV6 and CRPV pseudoviruses were produced using expression plasmids carrying L1 and L2 genes that were entirely codon-modified using a previously described strategy (Buck et ai, J. Virol., 78:751 -757 (2004)). Pseudovirions were produced as previously described (Buck et al., (2004); and Pastrana et al., (2004)) with minor modifications.
  • plasmids encoding L1 and L2 genes were cotransfected into 293TT cells along with a reporter plasmid encoding secreted alkaline phosphatase (pYSEAP). After 48 h, cells were lysed with 0.2% Brij-58, 9.5 mM MgCI 2 , 0.1-0.2% Benzonase (Sigma) and 0.1 % plasmid safe (Epicentre Biotechnologies) and incubated at 37 °C for 15 min.
  • pYSEAP reporter plasmid encoding secreted alkaline phosphatase
  • the entire volume was transferred to corresponding well of the plate which had been seeded with 293TT cells a day before. Plates were returned to the incubator for 67 hours. Upon completion culture supernatant was analyzed for the presence of SEAP.
  • the in vitro neutralization titer was defined as the reciprocal of the highest dilution of serum that reduces the SEAP activity by at least 50% in comparison to the reactivity in the wells that received no PsV sample without antiserum.
  • the method described in the example is based on (Day et ai, Cell Host Microbe, 8:260- 270 (2010)).
  • Flat bottom 96-well cell culture plate were coated with Extra Cellular Matrix (200 ⁇ g per well), covered with neutralization medium (DMEM without phenol red, 10% FBS, 1 % non-essential amino acids, 1 % GlutaMax) and incubated the plate at 37 °C, 5% C0 2 culture incubator for 4 hours. Plates were washed three times with PBS and 80 ⁇ of the diluted PsV prepared in Delta Furin CHO conditional Medium were added to each well. Plates were incubated overnight at 37 °C.
  • the in vitro neutralization titer is defined as the reciprocal of the highest dilution of serum that reduces the Luciferase activity by at least 50% in comparison to the reactivity in the wells that received PsV but no anti-serum.
  • Example 16 Challenge Rabbits with Quazi-virions (Rabbits)
  • quaizivirions are L1/L2 HPV virus like particles encapsidating the CRPV genome.
  • CRPV particles were also used for challenge. These reagents are infectious only for rabbits due to species restriction of CRPV genome in rabbits. Animals were monitored for 10 weeks after challenge. Papilloma volumes were charted as height x width x depth. Additionally, the back of each rabbit was individually photographed.
  • mice Female BALB/c mice aged 6-8 weeks were immunized three times with Fla-69, Fla-32, Gardasil ® (Merck, Whitehouse Station, NJ), Cervarix ® (GlaxoSmithKlein, London, United Kingdom) and placebo control three times biweekly as described in FIG. 58.
  • Mice received 3 mg of medroxyprogesterone (Depo-provera) (Pfizer, NY, NY) diluted in 100 ⁇ of sterile PBS in a subcutaneous injection 4 days prior to HPV56 pseudovirus challenge.
  • Depo-provera medroxyprogesterone
  • the pseudovirus inoculum was a 20 ⁇ dose composed of purified HPV56 pseudovirus carrying the luciferase reporter gene with a titer of about 10 l U/ml mixed in 2% carboxymethyl cellulose (CMC) (Sigma, C5013).
  • the virus was delivered in two doses. Half the virus was deposited into the mouse's vagina using an M50 positive-displacement pipette (Gilson, Inc., Middleton, Wl). A cytobrush cell collector was inserted in the vagina and twirled clockwise and counter-clockwise 10 times, and the remaining 10 ⁇ was introduced. Three days later, the mice were anesthetized
  • luciferin (20 ⁇ at concentration of 7.8 mg/ml) was deposited intravaginally, and images were acquired for 10 min using an Xenogen I VIS 200 (Caliper Life Sciences, Hopkinton, MA) as previously described by Johnson et al., J. Virology, 83:2067-2074 (2009). The average radiance within the region of interest was determined. Data are representative of 10 mice per group, and experiments were performed in duplicate. Statistical analysis was performed with GraphPad Prism Software (GraphPad Software, Inc., La Jolla, CA), in which a one-tailed, unpaired t-test was used to determine p values.
  • the present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus.
  • the present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
  • the methods and compositions disclosed herein may be effective in preventing and/or treating diseases caused by one or more types of the roughly 100 or more known types of Human papillomavirus.

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Abstract

The present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus. The present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.

Description

RECOMBINANT FUSION PROTEINS AND METHODS OF USE THEREOF FOR TREATMENT OR PREVENTION OF PAPILLOMAVIRUS INFECTION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Serial No. 61 /519,014, filed May 14, 201 1 , the contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus. The present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
BACKGROUND OF THE INVENTION
Genital-tropic human papillomavirus (HPV) infections are considered the most common sexually transmitted infections in the United States (CDC Report to Congress, Prevention of Genital Human Papillomavirus Infection, January, 2004). The major manifestations of anogenital HPV include genital warts (condyloma acuminatum) and intraepithelial neoplasia of the vulva, cervix, anus, or penis. A small fraction of persistent high-risk HPV infections, if left untreated, progresses into one or more various forms of cancer, including, cervical, anogenital, laryngea, tongue, head and neck, and some types of non-melanoma skin cancers. In addition to genital warts and certain forms of cancer, HPV infection can result in common warts, plantar warts, or planar warts. Warts may exist in different forms depending on the HPV type responsible and the epithelium involved. Common warts (verruca vulgaris) usually occur on the hands, as flesh-colored to brown exophytic and hyperkeratotic papules. Plantar warts (verruca plantaris) occur on the soles of the feet and can be quite painful. These warts can be differentiated from calluses by removing the surface layer to reveal thrombosed capillaries. Flat or planar warts (verruca plana) are most common among children and can occur on the face, neck, chest and flexor surfaces of the forearms and legs.
While the various types of warts caused by HPV infect can cause significant pain, social distress, and morbidity, HPV infection can also lead to morbidity in the form of cervical cancer.
Cervical cancer claims many lives in the United States and abroad and the economic and societal burdens from HPV infection and the accompanying disease manifestations are large. (National Institutes of Health, Cervical Cancer, NIH Consensus Statement, 14(1 ):1— 38 (1996); U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999-2007 Incidence and Mortality Web-based Report. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute (2010); http ://www.cdc .qov/u scs) . Notably, according to U.S. Government statistics 12,280 women were diagnosed with cervical cancer in the United States in 2007 (the most recent data available). That same year in the United States alone 4,021 women died from cervical cancer. While the number of deaths resulting from cervical cancer has decreased significantly in recent years, largely because of initiatives to promote regular screening measures (i.e., Papanicolaou (PAP) tests) that can detect precancerous cellular changes (dysplasia) before progression into invasive cancer, tragically, cervical cancer and HPV related morbidity remains a major public health issue in the United States and the rest of the world.
Infection with the HPV virus presents a major public health issue in the developing world. (Sherris, West J. Med., 175(4) :231-233 (2001 )). Indeed, nearly 80% of cases of cervical cancers occur in women in the developing world where it the third most common form of cancer and the leading cause of cancer related death accounting for about 190,000 deaths each year. (Pisani et al., Int. J. Cancer, 83(6):870-873 (1999)). Cervical cancer rates are highest in Central America, sub-Saharan Africa, and parts of Oceania.
Despite the significant morbidity and mortality related to HPV infection and the various types of cancer that may arise from infection, fortunately, most cases of cervical cancer can be prevented by preventing infection with the HPV virus. The human papillomavirus infects the cells of the cervix and slowly causes precancerous cellular changes (dysplasia). These pre- dysplastic cellular changes can be relatively mild and fortunately often do not progress further and may even regress. (Walboomers et al., J. Pathol. ,189:12-19 (1999). However, more severe dysplasias are more likely to progress to cervical cancer. (Nasiell et al., Obstet.
Gynecol., 67:665-669 (1986); and Holowaty et al, J. Natl. Cancer Inst., 91 :252-258 (1999)).
Sexually active women in their early adulthood are most commonly infected with the
HPV. Preventing HPV infection is more challenging than prevention of most other sexually transmitted disease due to the fact that women infected with the HPV are generally
asymptomatic and thus may unknowingly harbor the infection and pass the virus on to their sexual partners. Compounding the potential problem, infected women can harbor an HPV infection for up to 20 years before developing cervical cancer. (Sherris, West J. Med.,
175(4) :231-233 (2001 )). HPV is also believed to be relatively easily transmitted between partners. In fact, at least 50 percent of sexually active men and women have been infected with genital HPV at some point in their lives.
Approximately 35 of the more than 100 types of HPV are specific for the anogenital epithelium and have varying potentials for malignant transformation. Furthermore, about 15 types of HPV are considered oncogenic genital HPV types, with HPV16 being the most common, followed by HPV18 and HPV45 (causing about 50%, about 20%, and about 10% of cervical cancer cases, respectively).
HPV infection is considered a necessary factor for the development and persistence of cervical cancer. Overall, the presence of HPV DNA has been reported in 99.7% of cervical carcinomas worldwide, suggesting that HPV infection is a necessary cause of this type of cancer and that this disease can be prevented by prophylactic HPV vaccination.
Certain types of HPV are classified as being "high-risk" types because they have high potential for progression to cancer, including, cervical cancer. High-risk HPV types include, HPV16, 18, 26, 30, 31 , 34, 35, 39, 45, 51 , 52, 53, 56, 58, 59, 61 , 66, 67, 68, 69, 70, and 73. Other types, such as HPV types 2, 3, 6, 7, 10, 13, 32, 40, 42, 43, 44, 55, 54 and 57, are categorized as being relatively "low-risk" because of their lower potential for progression to malignancy.
The HPV has a circular double-stranded genome that is about 8 kbp in length. The genome of all known HPV types contains open reading frames (ORFs), which are DNA regions coding for proteins having similar properties, that are divided into two major regions: the early (E) region, and late (L) region. The early region of about 4.5 kbp codes for genes which are associated with functions including viral DNA replication (i.e., E1 ), induction or suppression of the action of DNA encoding a protein inducing malignant transformation of host cells (i.e., E2), the synthesis of proteins responsible for the growth of host cells and viruses (i.e., E4), stimulation of the activity of epidermal growth factor (EGF) and colony stimulator factor (CSF) receptors (i.e., E5), and malignant transformation through the permanent survival of cells, activation of oncogenes (E6), and inactivation of tumor suppressor genes (i.e., E7). In particular, the oncogenic E6 and E7 proteins, which are expressed after HPV infects the epithelial cells of a host, bind to tumor suppressor proteins of host cells, p53 and pRB, respectively, and thus inhibiting the function of these suppressor proteins, resulting in the neoplastic transformation of infected cells. The late region of 2.5 kbp comprises genes coding for viral major (i.e., L1 ) and minor (i.e., L2) capsid proteins and a non-coding region 1 kbp long, which is called the long control region (i.e., LCR) which regulates the transcription and translation of the two late genes.
The HPV genome is surrounded by a 60-nm, non-enveloped icosahedral capsid skeleton (Baker et al., Biophysical J., 60(6):1445-1456 (1991 )) that contains the two genetically unrelated major capsid L1 proteins and the minor L2 capsid protein. The HPV capsid is thought to comprise 72 L1 protein pentamers (capsomeres) and 12 L2 minor capsid protein molecules bound thereto. Both terminals of the L2 protein are located in the capsid, but part of the N- terminal region is located on the surface of the capsid (L2 surface region). Recombinant L1 self-assembles into virus-like particles (VLPs) which are morphologically and immunologically similar to native virions (virus like particles "VLPs") (Kirnbauer et al., PNAS, 89(24):12180- 12184 (1992)). L1 VLP-based vaccines are generally protective against infection corresponding to the papillomavirus type used to derive the immunogen (homologous vaccine), but are ineffective against all but the most closely related HPV types (Roden et al., Virology,
270(2) :254-257 (2000).
As shown above, there are a large number of HPV types that are of public health concern. Currently licensed HPV vaccines (i.e., L1 based VLPs) have attempted to address some of these health concerns by designing limited multivalent vaccine preparations, for example, the Cervarix® HPV vaccine, GlaxoSmithKline, London, United Kingdom, contains L1 VLP derived from HPV types 16 and 18 which cause about 70% of cervical cancer cases.
(MMWR, 59(20) :626-629, (2010)). The recently licensed Gardasil® HPV vaccine, Merck & Co., Inc., Whitehouse Station, New Jersey, contains L1 VLP derived from HPV types 16 and 18 as well as L1 VLPs from types 6 and 1 1 for the prevention of benign genital warts. (MMWR, 56 (RR-2):1-24 (2000); and MMWR, 59(20) :630-632 (2000)).
While L1 HPV vaccines provide protection to designated HPV types, evidence suggests that immunization with several different HPV L2 polypeptides induces broader cross-neutralizing antibodies compared to the type-restricted neutralizing serum antibodies and immunity generated by L1 VLP vaccines. (Baltimore, Bacteriol. Rev., 35:235-241 (1971 ); Kawana et al., J. Virol., 73:6188-6190 (1999); Roden et al., Virology, 270:254-257 (2000); Kawana et al., Vaccine, 19:1496-1502 (2001 ); and Pastrana et al., Virology, 337:365-372 (2005)). Further, in animal models, immunization with HPV minor capsid protein L2 protects subjects from experimental papillomavirus infection at both mucosal and cutaneous sites. (Embers et al., J. Virol., 76:9798-9805 (2002)). Protection is thought to be mediated by neutralizing antibodies, and the work of several laboratories has identified cross-neutralizing epitopes. (Christensen et al., Virology, 181 :572-579 (1991 ); Kawana et al., J. Virol., 75:2331 -2336 (2001 ); Fleury et al., Arch. Virol., 151 :151 1 -1523 (2006); Gambhira et al., J. Virol., 81 :1 1585-1 1592 (2007); and Gambhira et al., J. Virol., 81 :13927-13931 (2007)).
The field of HPV research is unsettled as to the specific optimal amino acid length of the L2 molecules best suited for efficacious vaccine candidates. For example, Gambhira (13927- 13931 ) describe the use of a full length HPV16 L2 protein molecule as the antigen presented. However, they observed difficulties with the solubility and purification of their candidate.
Recent efforts to truncate the L2 amino acid sequence have been bolstered by the identification of a neutralizing monoclonal antibody, RG-1 , that maps to the 17-38 AA epitope of HPV16 L2 minor capsid protein.
Additionally, various C-terminal truncations of the HPV16 L2 protein molecule have been tested. (Gambhira, J. Virology, 81 (24):13927-13931 (2007); Kando et al., Virology, 358:266- 272 (2007); and Rubio et al., Vaccine, 27:1949-1956 (2009)). In still other work, HPV16 L2 truncations, using AA2-200, AA1 1 -200, AA1 1 -88, AA1 1 -38, AA17-36 (some of these fragments are presented as multimers and some as monomers) showed varying degrees of efficacy as determined by pseudovirus neutralization assays. (Rubio et al., 1949-1956).
Although there have been attempts to create an efficacious HPV L2 peptide-based vaccine (See, Kawana et al., Vaccine, 21 :4256-4260 (2003)), the L2 protein molecule is less immunogenic than L1 VLPs (Alphs et al, Proc. Natl. Acad. Sci. U.S.A. 105:5850-5855 (2008)) these attempts have meet with only limited success.
Finally, existing HPV L1 VLP vaccines require refrigeration during handling and storage which renders them impractical for use in low resource and remote areas where they are needed most. Furthermore, because the existing HPV L1 vaccines are ineffective against a significant number of oncogenic HPV types, costly cytologic screening programs remain necessary and a significant public health disease burden is still present.
To realize the full potential of HPV prevention globally, the next generation of HPV vaccines should be safe and effective and broadly effective against a greater number of HPV types.
SUMMARY OF THE INVENTION
The invention provides fusion products including at least a portion of a flagellin sequence of substantially the same amino acid sequence as a flagellin sequence described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305, and at least a portion of a human papillomavirus (HPV) L2 sequence of substantially the same amino acid sequence as an HPV L2 sequence described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305. Such fusion products can, in various embodiments, product activates Toll-like Receptor 5 (TLR5). In certain embodiments, the portions noted above are at least about 10, 20, 30, 40, 50, 75, or 100 amino acids in length.
In certain embodiments, the portion of the flagellin sequence includes a deletion of or within domain D2 and/or domain D3.
In other embodiments, the fusion products include at least a portion of two or more (e.g., 5-10) different HPV L2 sequences of substantially the same amino acid sequence as at least two (e.g., 5-10) or more HPV L2 sequences described in one or more of SEQ I D NOs: 101 -199, 202-204, or 303-305.
In certain other embodiments, at least one of the different HPV L2 sequences is substantially the same as that of HPV16 and/or HPV18, as described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305. Further, in other embodiments, at least one of the different HPV L2 sequences is substantially the same as that of HPV31 , HPV39, HPV52, HPV58, HPV35, HPV45, and/or HPV6B, as described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305.
The invention also includes nucleic acid molecules including a sequence encoding a fusion product as described herein. For example, the nucleic acid molecules can include a portion of a flagellin nucleotide sequence of substantially the same nucleotide sequence of a flagellin sequence described in one or more of SEQ ID NOs: 1 -99, 100, 200, 201 , or 300-302, and at least a portion of an HPV L2 nucleotide sequence of substantially the same nucleotide sequence of an HPV L2 described in one or more of SEQ I D NOs: 1 -99, 100, 200, 201 , or 300- 302.
Further, the invention includes vectors containing the nucleic acid molecules described herein, as well as cells including the vectors.
The invention also includes methods of inducing an immune response in a subject, including administering to a subject an effective amount of one or more fusion products described herein, or a modification(s) thereof.
Also, the invention includes methods of preventing an infection in a subject by the human papillomavirus including administering to the subject an effective amount of one or more of the fusion products described herien, or a modification(s) thereof.
Further, the invention includes methods of modulating an immune response in a subject having a pathological condition, including administering to the subject an effective amount of one or more of the fusion products described herein, or a modification(s) thereof. In various embodiments, the pathological condition results from infection of the subject by a human papillomavirus.
In addition, the invention includes methods of inducing an antigen-specific immune response in a subject involving administering to the subject an effective amount of one or more of the fusion products described herein, or a modification(s) thereof.
The invention further includes methods of preventing an infection in a subject by one or more human papillomavirus involving administering to the subject an effective amount of one or more fusion products as described herein, or a modification(s) thereof.
The invention also includes uses of one or more fusion products as described herein, or a modification(s) thereof, in the preparation of a medicament for inducing an immune response in a subject.
Thus, in summary, the present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus. The present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
The present invention describes the design, expression, purification, formulation, and in certain embodiments, demonstrates the efficacy of immunological compositions useful for eliciting an immune response in a subject. In certain embodiments, the immunological compositions are based on recombinant fusion proteins (e.g., Fla-L2) containing derivatives of conservative cross-protective epitopes of HPV m inor protein L2 and one or more portions of Flagellin.
Flagellin is a pathogen-associated molecular pattern (PAMP) recognized by Toll-
Like Receptor 5 (TRL-5). TLR-5 is a member of a family of receptors involved in
mediating the innate immune response. Toll-like receptors recognize PAMPs that distinguish infectious agents from self and mediating the production of
immunomodulatory molecules, such as cytokines, necessary for the development of effective adaptive immunity (Aderem and Ulevitch, Nature, 406:782-787 (2000) and
Brightbill, Immunology, 101 :1 -10 (2000)). Members of the toll-like receptor family
recognize a variety of antigen types and can discriminate between pathogens. For example, TLR-2 recognizes various fungal, Gram-positive, and mycobacterial
components, TLR-4 recognizes the Gram-negative product lipopolysaccharide (LPS), and TLR-9 recognizes nucleic acids such as CpG repeats in bacterial DNA. TLR-5 has been identified as a receptor for bacterial Flagellin.
Flagellin induces an innate immune response by binding to and activating TLR-5. Activation of TLR-5 by binding to Flagellin induces the production of immunomodulatory molecules, such as cytokines and co-stimulatory molecules, by a TLR-5 expressing cell. For example, activation of TLR-5 in macrophages results in the expression of the
cytokines TNF-a, IL-1 , and IL-6. These cytokines directly and indirectly alter the activities of immune system cells that participate in both humoral (TH2) and cell-mediated (TH1 ) adaptive immune responses. In this manner, in certain embodiments, an
immunomodulatory Flagellin based fusion product, peptide, polypeptide or modification thereof, can act as an adjuvant to stimulate a general immune response.
As mentioned above, Flagellin is known as TLR-5 agonist. Exemplary, U.S.
Patent No. 7,915,381 discloses a number of TLR-5 agonists, and in particular, Flagellin related compositions (e.g., sequences), methods, and formulations the disclosure of this patent is incorporated herein by reference in its entirety.
In certain embodiments, an immunomodulatory Flagellin based fusion product, polypeptide, peptide, or modifications thereof, can be used to induce an immune response in an individual having a pathological condition (e.g., PV or HPV related
infection), promoting the individual's own immune system to function more effectively and thereby ameliorate the pathological condition. An individual's immune system may not recognize cancer cells and other types of pathologically aberrant cells as foreign because the particular antigens are not different enough from those of normal cells to cause an immune reaction. In addition, the immune system may recognize cancer cells, but induce a response insufficient to destroy the cancer. In certain embodiments, the present invention contemplates that by stimulating an innate immune response, the
immunomodulatory Flagellin based fusion products, peptide, polypeptide or modification thereof, promote humoral and cell-mediated responses to antigens on foreign cells or pathologically aberrant cells, such as cancer cells.
Typically, a vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved. In other aspects the vaccine can be a solid (e.g., powdered or lyophilized formulation). The compositions of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat an infection. Upon introduction into a host, certain compositions are 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.
Additional embodiments provide Flagellin derivatives as vaccine vehicles for delivery of L2-protective epitopes. Flagellin has been shown in preclinical studies to be an efficacious adjuvant in immunological compositions. For example, preclinical studies of Flagellin as a component of compositions directed against diseases caused by
Yersinia pestis, Influenza, and Malaria, have shown that Flagellin can be an effective adjuvant. (See, for example, Honko et al., Infect. Immun., 74:1 1 13-1 120 (2006); Bargieri et al., Vaccine, 26:6132-6142 (2008); Huleatt et al., Vaccine, 26:201 -214 (2008); Mizel et al., Clin. Vaccine Immunol., 16:21 -28 (2009); Skountzou et al., Vaccine, 333:347-368
(2009); and Song et al., Vaccine, 27:5875-84 (2009)).
Since N- and C-termini of many flagellin types are highly conserved, the flagellins from different bacterial species can be used as sources for HPV-L2 fusion in a fashion described herein.
Flagellin homologs are exemplified but not limited by the list below: (1 ) fliC gene product of Salmonella enterica subsp. enterica serovar typhimurium str. 798; accession gene bank number YP_005397322 ; (2) flagellin of Salmonella enterica subsp. enterica serovar hadar str. RI_05P066; accession gene bank number ZP_02684103; (3) phase-1 flagellin
Salmonella enterica subsp. enterica serovar Dublin str. CT_02021853; accession gene bank number YP_002215129; (4) Hypothetical protein ykris0001_38030 Yersinia kristensenii ATCC 33638; accession gene bank number ZP_04625826; (5) Flagellar filament structural protein Shigella sonnei Ss046; accession gene bank number YP 310149; (6) Flagellin Escherichia coli 026:H1 1 str. 1 1368; accession gene bank number YP_003229794; (6) Flagellin Escherichia hermannii NBRC 105704; accession gene bank number ZP 09806483; (7)Flagellin Escherichia fergusonii ATCC 35469; accession gene bank number YP_002382331 ; (8) Flagellin Citrobacter youngae ATCC 29220; accession gene bank number ZP_06352418; (9) Flagellin Edwardsiella tarda ATCC 23685; accession gene bank number ZP_06715189; (10) FliC gene product Bordetella petrii DSM 12804; accession gene bank number YP_001630704; (1 1 ) Flagellin Enterobacter hormaechei ATCC 49162; accession gene bank number ZP 08498671 .
In preferred embodiments, the present invention provides Fla-L2 fusions
containing epitopes within the first 200 AA of L2 that are broadly cross-protective against disease and/or infection by multiple (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) HPV types.
The present invention demonstrates that therapeutic and/or prophylactic fusion product molecules can be expressed in high levels in available expression systems and can subsequently be purified from these expression systems. In certain embodiments, Fla-L2 fusion product molecules are expressed in bacterial expression systems. In some preferred embodiments, where bacterial expression systems are utilized for production of the instant compositions, the present invention utilizes Escherichia coli (E. coli) based expression systems. However, the present invention is not limited to expression in bacterial expression systems. A number of expression systems are suitable for use in preparing the compositions of the present invention, for example, suitable protein expression systems include those derived from prokaryotic cells (e.g., bacteria or archea) or eukaryotic cells (e.g., fungi, including, but not limited to yeast cells, algae, mammalian cells, and insect cells).
Preferred methods and compositions of the present invention provide highly protective immune responses in animals against HPV challenge.
The present invention is not intended to be limited to the list of constructs depicted in Table 1 , but rather is extended to any Flagellin and L2 fusions and mixtures of portions, repeats, and/or mutations of HPV L2 with portions, repeats, and/or mutations of Flagellin used with or without adjuvant(s) in any combination for prophylactic and/or therapeutic purposes. In still further embodiments, the present fusion products and methods contemplated by the present invention are useful as additional components in a further prophylactic and/or therapeutic sense when administered in conjunction (e.g., 1 , 2, 3, 4, or more, doses administered sequentially or concomitantly) with one or more doses of commercially available HPV vaccine(s). One or more of the fusion products (e.g., polypeptides) can be useful as a vaccine composition for the prophylaxis, treatment, or prevention of papillomavirus infection. In certain aspects the compositions can be combined with a pharmaceutical carrier. Preferably, the vaccine composition is administered to an individual prior to papillomavirus exposure to minimize or prevent papillomavirus infection, or is administered after a patient has been infected to reduce the severity of infection and retard/halt progression of the disease, or to prevent transmission of a papillomavirus from the infected host to another individual who does not have a papillomavirus infection.
The invention also contemplates methods of purification as described herein, as well as animal models that can be used, e.g., for testing constructs as described herein, such as those described below.
Other embodiments and advantages of the invention are described as follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the expression of Fla-02 (HPV16 L2 2-200 AA) and Fla-07 (HPV16 L2 of
1 1 -200 AA). Both fusions showed high solubility when expressed in conditions shown on the left from the gel.
Figure 2 shows the general purification scheme of Fla-02 and Fla-07 fusions as well as 2 exemplary SDS PAGE analyses of the chromatographic elution fractions for Fla-02 and Fla-07.
Figure 3 shows purified lots of Fla-01 ((1 -506 AA)x6xHIS), Fla-02, and Fla-07 fusion products used for subsequent in vivo studies.
Figure 4 shows projected 3-D models of HPV16 L2 (Panel A) and the Fla-07 fusion D1 , D2, and D3 domains (Panel B).
Figure 5 shows bioactivity results of the TLR-5 of Fla-07 and the Flagellin backbone (Fla-01 ). In particular, Toll-like Receptor (TLR) stimulation was tested by assessing NF-kB activation in HEK293 cells expressing human TLR-5 (InvivoGen Inc., San Diego, CA).
Bioactivity results were as follows: Fla-01 > Fla-07 » Flagellin of S. typhimurium.
Figure 6 shows at, Panel A, a 3D model of Fla-31 or Fla-32 as modeled using Accelrys software (Accelrys, Inc., San Diego, CA); Panel B, provides an SDS gel showing expression of Fla-31 and Fla-32 at the specified culture conditions.
Figure 7 shows at, Panel A, the comparative purification results for Fla-01 , Fla-62, Fla- 02, Fla-31 and Fla-32; Panel B, shows characteristics of Flagellin or various Fla-L2 fusion variants used for preclinical studies. As summarized in Panels A and B, release tests demonstrated high purity, low endotoxin level (below 0.1 Eu^g) and high yield of purified antigens. Assay conditions were: 1 ) Protein concentration: BSA; 2) Purity: SDS PAGE; 3) Endotoxin level: Endosafe-PTS; and 4) Specificity: Western blot with anti L2 mAbs RG1 . Figure 8 shows the TLR-5 bioactivity of various Fla-L2 fusions in vitro.
Figure 9 shows SDS gels demonstrating the expression of monomeric Fla-L2 fusions (C- terminus fusion; full-length Fla) for HPV L2 1 1 -200AA of type 6, 1 1 , 18, and 31 . Conditions were as shown on the left of the figure.
Figure 10 shows SDS gels demonstrating the expression of monomeric FlaAD3-L2 fusions (D3-replacements) containing L2 of HPV types 39 (1 1 -199 AA), 18 (1 1 -200 AA), 6 (10- 198), 31 (1 1 -195) and 45 (1 1 -200AA) epitopes. Conditions were as shown on the left of the figure.
Figure 1 1 , Panels A, B, and C, shows 3D models of Fla-07, Fla-32, and Fla-69, respectively. The models in Panels A and B were built using Accelrys software while the model in Panel C is an approximate structure shown for comparison purposes.
Figure 12 shows some of the characteristics of L2 multimeric fusion constructs used for expression, purification, and in vivo studies. In particular, Panel A shows the scope of L2 epitopes and HPV serotypes used therein. Panel B shows some selected characteristics of selected Fla-L2 fusion products and their use for in vivo (e.g., rabbit) studies.
Figure 13, in Panel A, provides data demononstrating the Fla-69 and Fla-70 fusions are highly soluble after microfluidization (MF) (microfluidization conditions were as described in the panel). Panel B provides a physical map of the multimeric structures of Fla-69 and Fla-70 fusions, respectively.
Figure 14 shows the general purification scheme for multimeric Fla-L2 fusions Fla-69
(5XL2) and Fla-70 (8XL2). In preferred embodiments, as shown infra the purification scheme provides for a high recovery and good separation at each step, high yield and high purity of the recovered fusions products, high recovery after refolding, is robust, highly scalable, and cost effective.
Figure 15, Panel A, shows SDS gel results for release test purification steps for the Fla-
69 (5XL2) fusion. Panel A shows that the fusion product was recovered in high purity, with low residual endotoxin levels, and at a high yield of purified antigens (~90 mg/1 L culture). Panel provides SEC-HPLC product characterization results for the Fla-62 (flagellinAD3 control), Fla-69 (5XL2) and Fla-32 (HPV16L2; 1 1 -200 AA) fusion products, respectively.
Figure 16 shows purification results and certain physical characteristics for fusion products Fla-65 and Fla-76, respectively.
Figure 17 shows a comparison between mono- and multimeric -L2 Fla-L2 fusions using a TLR-5 bioactivity in vitro assay. TLR5 binding of two lots of Fla-69 is comparable to that of Fla-62, Fla-32, or WT Flagellin (Fla) from S. typhimurium. Figure 18 shows a comparison between various multimeric -L2 Fla-L2 fusions using a TLR-5 bioactivity in vitro assay. Fla-SM denotes that a S. typhimurium Flagellin control was used for comparison testing.
Figure 19 shows the schedule used in an exemplary animal challenge study. In this case, the animals challenged were rabbits and the study was labeled "Rabbit Study No. 3." Test animals were challenged with Cottontail Rabbit Papillomavirus (CRPV) and quazi-viruses which have CRPV genome packaged within HPV L1/L2 shell of serotype 6, 16, 18, or 58.
Figure 20 shows the results of an exemplary animal study measuring end point titers at days 21 , 42, 63, and 102 of the study. Day of bleed is depicted using different shapes (see lower left panel); d21 = circle, d42 = square, d63 = triangl, d102 = inverted triangle (this shape convention is maintained in Figure 21 infra). The animals challenged were rabbits and the study was labeled "Rabbit Study No. 3." In particular, immunogenicity results of Fla-02 (L2 2- 200 AA) and Fla-07 (L2 1 1 -200 AA) fusions, respectively, are shown. In this study, the immunogenicity to the Fla-backbone was about x10 higher than to the recombinant L2 (*Fla = Fla-01 or recombinant Flagellin as used herein). Both full-length-flagellin fusions demonstrated strong flagellin-biased responses. Individual anti-RL2 (recombinant HPV16 L2 protein) and anti-Flagellin (Fla-01 End Point ELISA Titers) are shown on the left (Panel A) and right panels (Panel B), respectively.
Figure 21 shows the results of an exemplary animal study measuring end point titers at days 21 , 42, 63, and 102 of the study. The animals challenged were rabbits and the study was labeled "Rabbit Study No. 3." In particular, immunogenicity results of Fla-31 (L2 2-200 AA) and Fla-32 (L2 1 1 -200 AA) fusions, respectively, are shown. Both D3-L2 replacements
demonstrated shifted response towards the L2 epitope (*Fla = Fla62 = r-FlagellinAD3).
Individual anti-RL2 (recombinant HPV16 L2 protein) and anti-Flagellin (Fla-01 End Point ELISA Titers are shown on the left (Panel A) and right panel (Panel B), respectively.
Figure 22 shows the results of an exemplary immunogenicity study of Fla-L2 fusions at day 63 post immunization with pre-challenge sera. The animals challenged were rabbits and the study was labeled "Rabbit Study No. 3." In particular, Figure 22 provides a comparison of 50% neutralizing titers of against pseudovirus (PsV16 (Panel A) and 18 (Panel B), respectively, at Day 63 (circular dots; after two immunizations) and Pre-Challenge Sera (d102) (square dots; after four immunizations). Neutralizing titers were boosted by the 4th immunization for both PsV16 and 18.
Figure 23 shows in multiple panels (Panels A, B, C, D, and E) the efficacy of various Fla- L2 fusions in a wart analysis study eight weeks post challenge with quazi-virus 6 (Panel A), quazi-virus 16 (Panel B), quazi-virus 18 (Panel C), quazi-virus 58 (Panel D), and CRPV (Panel E), respectively. The results of this exemplary study show that the Fla-32 fusion was highly cross protective against all of the challenge virus in this study and the adjuvant used did not significantly increase protection.
Figure 24 shows the vaccine L2-types and quazi-viruses used in an exemplary animal study. The subject animal study contemplated using rabbits and was labeled "Rabbit Study No. 6." Panel A shows the HPV types used in a 5X vaccine formulation (Fla-69). Panel B shows the quazi-virus types used in the subsequent challenge study(ies). Panel C depicts an L-2- dendrogram based on 1 1 -200 AA L2 epitopes.
Figure 25 shows the design of an exemplary animal study. The study contemplated using rabbits and was labeled "Rabbit Study No. 6." Study animals were scheduled for intramuscular immunization with the indicated fusion formulations either three times (Days: 14, 35, and 56) or four times (Days: 0, 21 , 35, and 56) and subsequently challenged on Day 77 each with each of quazi-virions 6, 16, 18, 58 (three sites per virus) and CRPV (two sites per virus).
Figure 26 shows results of an exemplary in vitro neutralization assays in rabbits against PsV16. These assays were part of Rabbit Study No. 6. In particular, the figure shows the 50% neutralization titers against PsV16 of individual serum samples assayed using a conventional neutralization method. Panel A shows the individual 50% neutralization data. Panel B shows the experimental schedule (immunization and bleeding days are shown by arrows and red boxes respectively; challenge day is depicted by callout).
Figure 27 shows results of an exemplary in vitro neutralization assays in rabbits against
PsV18. These assays were part of Rabbit Study No. 6. In particular, the figure shows the 50% neutralization titers against PsV16 of individual serum samples assayed using a conventional neutralization method. Panel A shows the individual 50% neutralization data. Panel B shows the experimental schedule (immunization and bleeding days are shown by arrows and red boxes respectively; challenge day is depicted by callout).
Figure 28 shows in multiple panels (Panels A, B, C, D, and E) the efficacy of various Fla- L2 fusions in a wart analysis study eight weeks post challenge with quazi-virus (QV) 6 (Panel A), QV 16 (Panel B), QV 18 (Panel C), QV 58 (Panel D), and wild type CRPV (Panel E), respectively.
Figure 29 shows results of an exemplary in vitro neutralization assays in rabbits. The
Figure provides the 50% neutralization titers of pooled serum samples generated in Rabbit Study No. 6 under one of two methods as specified. The first method is labeled the "novel methodology" and data therein is based on spatiotemporal separation of L2 epitope exposure on the base membrane (HSPG) and binding to the secondary receptor on the HSPG- epithelial cell surface (stages for viral HSPG binding, furine cleavage and binding/internalization to/into HSPG- cells are spatiotemporaly separated). The second method is labeled the "Convention methodology" and data therein was generated by exploiting viral binding/internalization to/into HSPG+/Secondary receptor+ cells (data shown in parentheses).
Figure 30 shows the general scheme used for passive immunization experiments with pooled serum samples obtained from various arms of Rabbit Study No. 6.
Figure 31 shows the vaccine L2-types and quazi-viruses used in an exemplary passive immunization animal study. The animal study contemplated using rabbits and was labeled "Rabbit Study No. 6." Panel A shows the HPV types used in a 5X vaccine formulation (Fla-69). Panel B shows the quazi-virus types used in the subsequent challenge study(ies). Panel C depicts an L-2-dendrogram based on 1 1 -200 AA L2 epitopes.
Figure 32 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples (anti-Fla-32, anti-Cervarix®, anti-Fla-69, and anti-Gardasil®) obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 6. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil® HPV vaccine (Merck, Whitehouse Station, NJ) and the Cervarix® HPV vaccine (GlaxoSmithKlein, London, United Kingdom) in dilution series against the specified quazi-virus (QV 6) : Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D (Gardasil®).
Figure 33 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 16. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil® HPV vaccine and the Cervarix® HPV vaccine in dilution series against the specified quazi-virus (QV 16): Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D
(Gardasil®).
Figure 34 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 18. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil® HPV vaccine and the Cervarix® HPV vaccine in dilution series against the specified quazi-virus (QV 18): Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D
(Gardasil®).
Figure 35 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 31 . In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the
Gardasil® HPV vaccine and the Cervarix® HPV vaccine in dilution series against the specified quazi-virus (QV 31 ): Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D
(Gardasil®).
Figure 36 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 45. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil® HPV vaccine and the Cervarix® HPV vaccine in dilution series against the specified quazi-virus (QV 45): Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D
(Gardasil®).
Figure 37 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 58. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the Gardasil® HPV vaccine and the Cervarix® HPV vaccine in dilution series against the specified quazi-virus (QV 58): Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D
(Gardasil®).
Figure 38 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 6 conducted in rabbits at eight (8) weeks post-challenge with CRPV. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates as well as comparative data using the
Gardasil® HPV vaccine and the Cervarix® HPV vaccine in dilution series against CRPV: Panel A (Fla-32); Panel B (Cervarix®); Panel C (Fla-69); Panel D (Gardasil®).
Figure 39 shows end-point protection titers calculated on the basis of efficacy data from an exemplary passive immunization study (Rabbit Study No. 6) eight (8) weeks post-challenge (End Point Protection Titer (EPPT) = Highest Serum Dilution Provided Complete Protection; EPPT was calculated relative to total rabbit blood volume).
Figures 40A, 40B, and 40C (each Figure having a Panel A and Panel B, respectively) show ELISA results using pooled Fla-69 antiserum obtained from Rabbit Study No. 6 with L2- peptide libraries covering 1 1 -200 AA of L2 of HPV types 6 (FIG. 40A, Panel A), 16 (FIG. 40A, Panel B), 18 (FIG. 40B, Panel A), 31 (FIG. 40B, Panel B), 45 (FIG. 40C, Panel A), and 58 (FIG. 40C, Panel B), respectively. The location of RG1 epitopes is indicated by arrows.
Figures 41 A, 41 B, and 41 C (each Figure having a Panel A and Panel B, respectively) show ELISA results using pooled Fla-32 antiserum obtained from Rabbit Study No. 6 with L2- peptide libraries covering 1 1 -200 AA of L2 of HPV types 6 (FIG. 41 A, Panel A), 16 (FIG. 41 A, Panel B), 18 (FIG. 41 B, Panel A), 31 (FIG. 41 B, Panel B), 45 (FIG. 41 C, Panel A), and 58 (FIG. 41 C, Panel B), respectively. The location of RG1 epitopes is indicated by arrows. Figure 42 shows a summary of peptide library analysis of HPV types 6, 1 1 , 1 6, 18, 31 39, 45, 52, 58, 73 with anti-Fla-32 and anti-Fla-69 serum samples obtained from Rabbit Study No. 6.
Figure 43 shows exemplary RG1 epitope end-point ELISA titers of pooled serum samples obtained from rabbits immunized with Fla-32 and Fla-69 (Rabbit Study No. 6) (Panel A) , and RG1 epitopes alignment (Panel B). Sequences shown on Panel B correspond to synthetic serotype-specific RG1 - peptides used for ELISAs.
Figure 44A, Panel A, shows the fermentation parameters used for production of Fla-69 fusions. These parameters were computer controlled by a set of Proportional Integral Derivative algorithms which utilize periodic measurements (based on the parameter being measured) to make adjustments necessary to maintain the indicated set point. I n Figure 44A, Panel B, Cultivation Profile, the specific replication rate per hour was measured for the doubling time of an actively growing culture of Fla-69 fusions. The rate is defined by the following formulae: specific replication rate [iR =(1 /X(t))(AX(t)/At) = (h-1 ), where X= OD600nm and t =time. The rate is used to monitor glucose consumption and acetate production which are known to be produced at potentially toxic levels when specific replication rates are high. Figure 44B, Panel A, provides a expression time course of Fla-69 fermentation. Panel B of Figure 44B provides capture ELISA data.
Figure 45, Panel B, shows the design of an exemplary animal study. The study contemplated using rabbits and was labeled "Rabbit Study No. 5." Study animals were scheduled for intramuscular immunization with the indicated fusion formulations four times (Days: 0, 21 , 42, and 63) and subsequently challenged on Day 91 with each of quazi-virions 6, 1 6, 18, 31 , 45 (two sites per virus) , and CRPV (two sites per virus) . Panel A, provides an illustration of Fla-32, Fla-69, Fla-76, and Fla-65 fusions constructs used in Rabbit Study No. 5.
Figure 46 shows in multiple panels (Panels A, B, C, D, E, and F) the efficacy of various
Fla-L2 fusions in a wart analysis study eight weeks post challenge with quazi-virus (QV) 6 (Panel A), QV 16 (Panel B), QV 18 (Panel C), QV 31 (Panel D), QV 58 (Panel E), and wild type CRPV (Panel E) , respectively.
Figure 47 shows results of an exemplary in vitro neutralization titer assay in rabbits. The Figure provides neutralization titers of pooled serum samples generated in Rabbit Study No. 5 (4 doses) and Rabbit Study No. 6 (3 doses) as determ ined by "novel methodology" described infra.
Figure 48, Panel A, shows exemplary RG-1 epitope end-point ELISA titers of pooled serum samples obtained from animals (Rabbit Study No. 5) immunized with Fla-32, Fla-69, Fla- 65, Fla-76 fusions, respectively. RG-1 Mab ELISAs Demonstrated Specificity to HPV types 1 6, 18, and 45. Fla-69, Fla-76 and Fla-65 fusions elicited comparable RG-1 type-specific immunogenicity as assayed by RG-1 -peptide ELISAs (HPV types 16, 18, and 45 were relatively dominant to other HPV subtypes). Panel B, of the Figure 48, provides an alignment of HPV subtype-specific RG1 -peptides used for ELISAs.
Figure 49 shows the general scheme used for passive immunization experiments with pooled serum samples obtained from various arms of Rabbit Study No. 5.
Figure 50 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples (anti-Fla-32, anti-Fla-69, anti-Fla-65, and anti-Fla-76) obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 6. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi-virus (QV 6): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
Figure 51 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 16. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 16): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
Figure 52 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 18. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 18): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
Figure 53 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 31 . In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 31 ): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
Figure 54 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 45. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 45): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
Figure 55 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus 58. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified quazi- virus (QV 58): Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76). Figure 56 shows efficacy data from an exemplary passive immunization experiment with pooled serum samples obtained from Rabbit Study No. 5 conducted in rabbits at eight (8) weeks post-challenge with quazi-virus CRPV. In particular, the respective panels demonstrate the protective efficacy of various vaccine candidates in dilution series against the specified CRPV: Panel A (Fla-32); Panel B (Fla-69); Panel C (Fla-65); Panel D (Fla-76).
Figure 57 shows results of an exemplary end-point protection titers assay of pooled serum samples from Rabbit Study No. 5 (4 doses) & Rabbit Study No. 6 (3 doses) as determined via passive immunization (see Examples).
Figure 58, Panel A, illustrates the phylogenetic relationships between 5XL2 fusion vaccine candidate (Fla-69) and challenge (HPV56) L2 types as used in "Mouse Study No. 1 ." Panel B, shows the design of an exemplary animal study, Mouse Study 1 . Study animals were scheduled for intramuscular immunization with the indicated fusion formulations three times (Days: 0, 14, and 28) and subsequently challenged on Day 184 with PsV56 (0.1 H = 0.1 of Human Dose Group).
Figure 59 shows results from an exemplary 50% neutralization titer assay of individual serum samples obtained from animals in Mouse Study No. 1 . Animals were immunized as described infra with HPV pseudoviruses of types 16 (Panel A), 18 (Panel B), and 45 (Panel C). Serum samples were derived from mice two (2) weeks after the third immunization.
Figures 60A and 60B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with Cervarix® HPV vaccine and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
Figures 61 A and 61 B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with Gardasil® HPV vaccine and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
Figures 62A and 62B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with PBS and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
Figures 63A and 63B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with the Fla-32 fusion and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
Figures 64A and 64B show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . Mice were immunized with Fla-69 fusion and challenged with PsV56. Bioluminescence values are indicated in the callout boxes.
Figure 65 show the efficacy results of an exemplary immunization study conducted in mice as part of Mouse Study No. 1 . In particular, Figure 65 shows presents background data (PBS challenge) in the study animals. Bioluminescence values are indicated in the callout boxes.
Figure 66 shows the results of an exemplary assay. In particular, Figure 66 provides a quantitative representation of efficacy data (following PsV56 challenge) from Mouse Study No. 1 . Panel A provides individual relative bioluminiscence unit (RBU) values obtained following immunization with: Cervarix® HPV vaccine, Gardasil® HPV vaccine, PBS (control), Fla-69 fusion, Fla-32 fusion, and of Background (no immunization), respectively. Panel B provides average RBU values obtained following immunization with: Cervarix® HPV vaccine, Gardasil® HPV vaccine, PBS (control), Fla-69 fusion, Fla-32 fusion, and of Background (no immunization), respectively. This data demonstrates that superior protection against PsV56 challenge was provided by the Fla-69 fusion (P<0.05).
Figure 67 provides an expression plasmid encoding the Fla-01 fusion product. The nucleic acid sequence of the fusion (SEQ ID NO: 1 ) as well as the amino acid sequence (SEQ ID NO: 101 ) of the fusion product. As used in Figure 67, and subsequent Figures through Figure 165, the following domains are indicated by labeling: one or more HPV domains {e.g., full length, portions, derivatives, and mutations thereof, etc.); one or more flagellin molecules (e.g., full length, portions, derivatives, and mutations thereof, etc.); one or more amino acid (e.g., naturally occurring or modified) as linkers between any of the constituents of the fusion product; and specific mutations in of the above fusion products constituents.
Figure 68 provides an expression plasmid encoding the Fla-02 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 2) as well as the amino acid sequence (SEQ ID NO: 102) of the fusion product.
Figure 69 provides an expression plasmid encoding the Fla-03 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 3) as well as the amino acid sequence (SEQ ID NO: 103) of the fusion product.
Figure 70 provides an expression plasmid encoding the Fla-04 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 4) as well as the amino acid sequence (SEQ ID NO: 104) of the fusion product.
Figure 71 provides an expression plasmid encoding the Fla-05 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 5) as well as the amino acid sequence (SEQ ID NO: 105) of the fusion product.
Figure 72 provides an expression plasmid encoding the Fla-06 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 6) as well as the amino acid sequence (SEQ ID NO: 106) of the fusion product. Figure 73 provides an expression plasmid encoding the Fla-07 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 7) as well as the amino acid sequence (SEQ ID NO: 107) of the fusion product.
Figure 74 provides an expression plasmid encoding the Fla-8 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 8) as well as the amino acid sequence (SEQ ID NO: 108) of the fusion product.
Figure 75 provides an expression plasmid encoding the Fla-09 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 9) as well as the amino acid sequence (SEQ ID NO: 109) of the fusion product.
Figure 76 provides an expression plasmid encoding the Fla-10 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 10) as well as the amino acid sequence (SEQ ID NO: 1 10) of the fusion product.
Figure 77 provides an expression plasmid encoding the Fla-1 1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 1 1 ) as well as the amino acid sequence (SEQ ID NO: 1 1 1 ) of the fusion product.
Figure 78 provides an expression plasmid encoding the Fla-12 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 12) as well as the amino acid sequence (SEQ ID NO: 1 12) of the fusion product.
Figure 79 provides an expression plasmid encoding the Fla-13 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 13) as well as the amino acid sequence (SEQ ID NO: 1 13) of the fusion product.
Figure 80 provides an expression plasmid encoding the Fla-14 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 14) as well as the amino acid sequence (SEQ ID NO: 1 14) of the fusion product.
Figure 81 provides an expression plasmid encoding the Fla-15 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 15) as well as the amino acid sequence (SEQ ID NO: 1 15) of the fusion product.
Figure 82 provides an expression plasmid encoding the Fla-16 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 16) as well as the amino acid sequence (SEQ ID NO: 1 16) of the fusion product.
Figure 83 provides an expression plasmid encoding the Fla-17 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 17).
Figure 84 provides an expression plasmid encoding the Fla-18 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 18) as well as the amino acid sequence (SEQ ID NO: 1 18) of the fusion product. Figure 85 provides an expression plasmid encoding the Fla-19 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 19) as well as the amino acid sequence (SEQ ID NO: 1 19) of the fusion product.
Figure 86 provides an expression plasmid encoding the Fla-20 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 20) as well as the amino acid sequence (SEQ ID NO: 120) of the fusion product.
Figure 87 provides an expression plasmid encoding the Fla-21 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 21 ) as well as the amino acid sequence (SEQ ID NO: 121 ) of the fusion product.
Figure 88 provides an expression plasmid encoding the Fla-22 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 22) as well as the amino acid sequence (SEQ ID NO: 122) of the fusion product.
Figure 89 provides an expression plasmid encoding the Fla-23 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 23) as well as the amino acid sequence (SEQ ID NO: 123) of the fusion product.
Figure 90 provides an expression plasmid encoding the Fla-24 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 24) as well as the amino acid sequence (SEQ ID NO: 124 of the fusion product.
Figure 91 provides an expression plasmid encoding the Fla-25 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 25) as well as the amino acid sequence (SEQ ID NO: 125) of the fusion product.
Figure 92 provides an expression plasmid encoding the Fla-26 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 26) as well as the amino acid sequence (SEQ ID NO: 126) of the fusion product.
Figure 93 provides an expression plasmid encoding the Fla-27 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 27) as well as the amino acid sequence (SEQ ID NO: 127) of the fusion product.
Figure 94 provides an expression plasmid encoding the Fla-28 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 28) as well as the amino acid sequence (SEQ ID NO: 128) of the fusion product.
Figure 95 provides an expression plasmid encoding the Fla-29 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 29) as well as the amino acid sequence (SEQ ID NO: 129) of the fusion product.
Figure 96 provides an expression plasmid encoding the Fla-30 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 30) as well as the amino acid sequence (SEQ ID NO: 130) of the fusion product. Figure 97 provides an expression plasmid encoding the Fla-31 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 31 ) as well as the amino acid sequence (SEQ ID NO: 131 ) of the fusion product.
Figure 98 provides an expression plasmid encoding the Fla-32 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 32) as well as the amino acid sequence (SEQ ID NO: 132) of the fusion product.
Figure 99 provides an expression plasmid encoding the Fla-33 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 33) as well as the amino acid sequence (SEQ ID NO: 133) of the fusion product.
Figure 100 provides an expression plasmid encoding the Fla-34 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 34) as well as the amino acid sequence (SEQ ID NO: 134) of the fusion product.
Figure 101 provides an expression plasmid encoding the Fla-35 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 35) as well as the amino acid sequence (SEQ ID NO: 135) of the fusion product.
Figure 102 provides an expression plasmid encoding the Fla-36 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 36) as well as the amino acid sequence (SEQ ID NO: 136) of the fusion product.
Figure 103 provides an expression plasmid encoding the Fla-37 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 37) as well as the amino acid sequence (SEQ ID NO: 137) of the fusion product.
Figure 104 provides an expression plasmid encoding the Fla-38 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 38) as well as the amino acid sequence (SEQ ID NO: 138) of the fusion product.
Figure 105 provides an expression plasmid encoding the Fla-39 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 39) as well as the amino acid sequence (SEQ ID NO: 139) of the fusion product.
Figure 106 provides an expression plasmid encoding the Fla-40 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 40) as well as the amino acid sequence (SEQ ID NO: 140) of the fusion product.
Figure 107 provides an expression plasmid encoding the Fla-41 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 41 ) as well as the amino acid sequence (SEQ ID NO: 141 ) of the fusion product.
Figure 108 provides an expression plasmid encoding the Fla-42 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 42) as well as the amino acid sequence (SEQ ID NO: 142) of the fusion product. Figure 109 provides an expression plasmid encoding the Fla-43 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 43) as well as the amino acid sequence (SEQ ID NO: 143) of the fusion product.
Figure 1 10 provides an expression plasmid encoding the Fla-44 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 44) as well as the amino acid sequence (SEQ ID NO: 144) of the fusion product.
Figure 1 1 1 provides an expression plasmid encoding the Fla-45 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 45) as well as the amino acid sequence (SEQ ID NO: 145) of the fusion product.
Figure 1 12 provides an expression plasmid encoding the Fla-46 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 46) as well as the amino acid sequence (SEQ ID NO: 146) of the fusion product.
Figure 1 13 provides an expression plasmid encoding the Fla-47 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 47) as well as the amino acid sequence (SEQ ID NO: 147) of the fusion product.
Figure 1 14 provides an expression plasmid encoding the Fla-48 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 48) as well as the amino acid sequence (SEQ ID NO: 148) of the fusion product.
Figure 1 15 provides an expression plasmid encoding the Fla-49 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 49) as well as the amino acid sequence (SEQ ID NO: 149) of the fusion product.
Figure 1 16 provides an expression plasmid encoding the Fla-50 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 50) as well as the amino acid sequence (SEQ ID NO: 150) of the fusion product.
Figure 1 17 provides an expression plasmid encoding the Fla-51 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 51 ) as well as the amino acid sequence (SEQ ID NO: 151 ) of the fusion product.
Figure 1 18 provides an expression plasmid encoding the Fla-56 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 56) as well as the amino acid sequence (SEQ ID NO: 156) of the fusion product.
Figure 1 19 provides an expression plasmid encoding the Fla-57 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 57) as well as the amino acid sequence (SEQ ID NO: 157) of the fusion product.
Figure 120 provides an expression plasmid encoding the Fla-58 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 58) as well as the amino acid sequence (SEQ ID NO: 158) of the fusion product. Figure 121 provides an expression plasmid encoding the Fla-59 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 59) as well as the amino acid sequence (SEQ ID NO: 159) of the fusion product.
Figure 122 provides an expression plasmid encoding the Fla-60 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 60) as well as the amino acid sequence (SEQ ID NO: 160) of the fusion product.
Figure 123 provides an expression plasmid encoding the Fla-61 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 61 ) as well as the amino acid sequence (SEQ ID NO: 161 ) of the fusion product.
Figure 124 provides an expression plasmid encoding the Fla-62 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 62) as well as the amino acid sequence (SEQ ID NO: 162) of the fusion product.
Figure 125 provides an expression plasmid encoding the Fla-63 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 63) as well as the amino acid sequence (SEQ ID NO: 163) of the fusion product.
Figure 126 provides an expression plasmid encoding the Fla-64 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 64) as well as the amino acid sequence (SEQ ID NO: 164) of the fusion product.
Figure 127 provides an expression plasmid encoding the Fla-65 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 65) as well as the amino acid sequence (SEQ ID NO: 165) of the fusion product.
Figure 128 provides an expression plasmid encoding the Fla-66 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 66) as well as the amino acid sequence (SEQ ID NO: 166) of the fusion product.
Figure 129 provides an expression plasmid encoding the Fla-67 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 67) as well as the amino acid sequence (SEQ ID NO: 167) of the fusion product.
Figure 130 provides an expression plasmid encoding the Fla-68 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 68) as well as the amino acid sequence (SEQ ID NO: 168) of the fusion product.
Figure 131 provides an expression plasmid encoding the Fla-69 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 69) as well as the amino acid sequence (SEQ ID NO: 169) of the fusion product.
Figure 132 provides an expression plasmid encoding the Fla-70 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 70) as well as the amino acid sequence (SEQ ID NO: 170) of the fusion product. Figure 133 provides an expression plasmid encoding the Fla-71 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 71 ) as well as the amino acid sequence (SEQ ID NO: 171 ) of the fusion product.
Figure 134 provides an expression plasmid encoding the Fla-72 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 72) as well as the amino acid sequence (SEQ ID NO: 172) of the fusion product.
Figure 135 provides an expression plasmid encoding the Fla-73 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 73) as well as the amino acid sequence (SEQ ID NO: 173) of the fusion product.
Figure 136 provides an expression plasmid encoding the Fla-74 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 74) as well as the amino acid sequence (SEQ ID NO: 174) of the fusion product.
Figure 137 provides an expression plasmid encoding the Fla-75 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 75) as well as the amino acid sequence (SEQ ID NO: 175) of the fusion product.
Figure 138 provides an expression plasmid encoding the Fla-76 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 76) as well as the amino acid sequence (SEQ ID NO: 176) of the fusion product.
Figure 139 provides an expression plasmid encoding the Fla-77 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 77) as well as the amino acid sequence (SEQ ID NO: 177) of the fusion product.
Figure 140 provides an expression plasmid encoding the Fla-78 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 78) as well as the amino acid sequence (SEQ ID NO: 178) of the fusion product.
Figure 141 provides an expression plasmid encoding the Fla-79 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 79) as well as the amino acid sequence (SEQ ID NO: 179) of the fusion product.
Figure 142 provides an expression plasmid encoding the Fla-80 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 80) as well as the amino acid sequence (SEQ ID NO: 180) of the fusion product.
Figure 143 provides an expression plasmid encoding the Fla-81 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 81 ) as well as the amino acid sequence (SEQ ID NO: 181 ) of the fusion product.
Figure144 provides an expression plasmid encoding the Fla-82 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 82) as well as the amino acid sequence (SEQ ID NO: 182) of the fusion product. Figure 145 provides an expression plasmid encoding the Fla-84 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 84) as well as the amino acid sequence (SEQ ID NO: 184) of the fusion product.
Figure 146 provides an expression plasmid encoding the Fla-85 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 85) as well as the amino acid sequence (SEQ ID NO: 185) of the fusion product.
Figure 147 provides an expression plasmid encoding the Fla-86 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 86) as well as the amino acid sequence (SEQ ID NO: 186) of the fusion product.
Figure 148 provides an expression plasmid encoding the Fla-87 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 87) as well as the amino acid sequence (SEQ ID NO: 187) of the fusion product.
Figure 149 provides an expression plasmid encoding the Fla-88 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 88) as well as the amino acid sequence (SEQ ID NO: 188) of the fusion product.
Figure 150 provides an expression plasmid encoding the Fla-89 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 89) as well as the amino acid sequence (SEQ ID NO: 189) of the fusion product.
Figure 151 provides an expression plasmid encoding the Fla-90 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 90) as well as the amino acid sequence (SEQ ID NO: 190) of the fusion product.
Figure 152 provides an expression plasmid encoding the Fla-91 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 91 ) as well as the amino acid sequence (SEQ ID NO: 191 ) of the fusion product.
Figure 153 provides an expression plasmid encoding the Fla-92 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 92) as well as the amino acid sequence (SEQ ID NO: 192) of the fusion product.
Figure 154 provides an expression plasmid encoding the Fla-93 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 93) as well as the amino acid sequence (SEQ ID NO: 193) of the fusion product.
Figure 155 provides an expression plasmid encoding the Fla-94 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 94) as well as the amino acid sequence (SEQ ID NO: 194) of the fusion product.
Figure 156 provides an expression plasmid encoding the Fla-95 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 95) as well as the amino acid sequence (SEQ ID NO: 195) of the fusion product. Figure 157 provides an expression plasmid encoding the Fla-96 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 96) as well as the amino acid sequence (SEQ ID NO: 196) of the fusion product.
Figure 158 provides an expression plasmid encoding the Fla-97 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 97) as well as the amino acid sequence (SEQ ID NO: 197) of the fusion product.
Figure 159 provides an expression plasmid encoding the Fla-99 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 99) as well as the amino acid sequence (SEQ ID NO: 199) of the fusion product.
Figure 160 provides an expression plasmid encoding the Fla-100 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 100) as well as the amino acid sequence (SEQ I D NO: 202) of the fusion product.
Figure 161 provides an expression plasmid encoding the Fla-200 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 200) as well as the amino acid sequence (SEQ I D NO: 203) of the fusion product.
Figure 162 provides an expression plasmid encoding the Fla-201 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 201 ) as well as the amino acid sequence (SEQ I D NO: 204) of the fusion product.
Figure 163 provides an expression plasmid encoding the Fla-62M1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 300) as well as the amino acid sequence (SEQ I D NO: 303) of the fusion product.
Figure 164 provides an expression plasmid encoding the Fla-32M1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 301 ) as well as the amino acid sequence (SEQ I D NO: 304) of the fusion product.
Figure 165 provides an expression plasmid encoding the Fla-65M1 fusion product, the nucleic acid sequence of the fusion (SEQ ID NO: 302) as well as the amino acid sequence (SEQ I D NO: 305) of the fusion product.
DEFINITIONS
The use of the word "a" or "an" when used in conjunction with the term
"comprising" in the claims and/or the specification may mean "one," but it is also
consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the
disclosure supports a definition that refers to only alternatives and "and/or." It is also contemplated that anything listed using the term "or" may also be specifically excluded. As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
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.
It is contemplated that one or more members of a list provided herein may be specifically excluded from or included in a claimed invention.
As used herein, the term "antigen" or "immunogenic polypeptide/peptide" is a molecule capable of being bound by an antibody or T-cell receptor. An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production 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." 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, or in the context of an MHC, by T-cell receptors. Typically, an antigen will be a peptide derived from a protein expressed by a pathogenic organism (e.g., HPV). An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another. In certain aspects an antigenic determinant is an PV polypeptide segment, PV peptide.
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, 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. 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, T-cell receptor, or HLA molecule. Throughout this disclosure, "epitope" and "peptide" are often used interchangeably.
As used herein, "B-cell epitope" or "target epitope" 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 segment or sub region thereof). As used herein, "HPV" and "human papillomavirus" refer to the members of the genus Papillomavirus (PV) that are capable of infecting humans. There are two major groups of HPVs (genital and cutaneous groups), each of which contains multiple virus "types" or "strains" (e.g., HPV 16, HPV 18, HPV 31 , HPV 32, etc.). Of particular interest in the present invention are the HPV types that are associated with genital infection and malignancy.
The term "vaccine" refers to a formulation which contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more oligomeric and/or multimeric HPV peptide compositions of the present invention. The multimeric HPV peptide compositions typically will 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 and/or to attenuate HPV infection and/or attenuate transmissibility of HPV.
As used herein, "prophylactic" and "preventive" fusion products, vaccines, or compositions are compositions designed and administered to prevent infection, disease, and/or any related sequelae caused by or associated with a pathogenic organism, particularly PV, and more particularly HPV.
As used herein, "therapeutic" fusion products, vaccines, or compositions are compositions designed and administered to subjects 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.
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.
An "infection" or "infectious disease", as used herein, refers to a disorder arising from the invasion of a host, superficially, locally, or systemically, by an infectious organism. Infectious organisms include bacteria, viruses, parasites, fungi, and protozoa.
As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non- naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivitization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the epsilon-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
Specific examples of amino acid analogs and mimetics can be found described in, for example, Roberts and Vellaccio, The Peptides: Analysis Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341 , Academic Press, Inc., New York, N.Y. (1983), the entire volume of which is incorporated herein by reference. Other examples include peralkylated amino acids, particularly permethylated amino acids. See, for example, Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 1 1 , p. 235, John Wiley & Sons Inc., New York, N.Y. (1997), the entire book of which is incorporated herein by reference. Yet other examples include amino acids whose amide portion (and, therefore, the amide backbone of the resulting peptide) has been replaced, for example, by a sugar ring, steroid, benzodiazepine or carbo cycle. See, for instance, Burger's Medicinal Chemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of which is incorporated herein by reference. Methods for synthesizing peptides, polypeptides, peptidomimetics and proteins are well known in the art (See, for example, U.S. Pat. No. 5,420,109; M. Bodanzsky, Principles of Peptide Synthesis (1 st ed. & 2d rev. ed.), Springer- Verlag, New York, N.Y. (1984 & 1993), See, Chapter 7; Stewart and Young, Solid Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, III. (1984), each of which is incorporated herein by reference).
"Amino acid sequence" as used herein, refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules.
As used herein, "conservative variations" or "conservative modified variations" of a particular sequence refers to amino acids encoded by nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given peptide. Such nucleic acid variations are silent variations, which are one species of conservatively modified variations. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques.
Accordingly, each silent variation of a nucleic acid which encodes a peptide is implicit in any described amino acid sequence. Furthermore, one of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
As used herein, the term "TLR-5 agonist" refers to a composition that selectively activates or increases normal signal transduction through TLR-5.
"Nucleic acid sequence" as used herein, refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
A "deletion", as used herein, refers to a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent.
An "insertion" or "addition", as used herein, refer to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule.
A "substitution", as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
The term "substantially purified", as used herein, refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. "Amplification" as used herein, refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY (1995)).
The term "hybridization", as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The term "antigenic determinant", as used herein, refers to that portion of a molecule that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "protein", as used herein, is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term includes naturally occurring proteins and peptides as well as those which are recombinantly or synthetically synthesized.
The terms "fusion product(s)" or "fusion protein(s)" as used herein refers to protein constructs that are the result of combining multiple protein domains or linker regions for the purpose of gaining function of the combined functions of the domains or linker regions. This is most often accomplished by molecular cloning of the nucleotide sequences to result in the creation of a new polynucleotide sequence that codes for the desired protein. Alternatively, creation of a fusion protein may be accomplished by chemically joining two proteins together.
The terms "linker," "linker region," or "linker domain," or similar, such descriptive terms as used herein refers to stretches of polynucleotide or polypeptide sequence that are used in the construction of a cloning vector or fusion protein. Functions of a linker region can include introduction of cloning sites into the nucleotide sequence, introduction of a flexible component or space-creating region between two protein domains, or creation of an affinity tag for specific molecule interaction. A linker region may be introduced into a fusion protein without a specific purpose, but results from choices made during cloning.
As used herein, "expression vectors" or "expression plasmid", and similar terms, are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts including, but not limited to, bacteria, for example, E. coli, blue-green algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells.
The term "mucosal" refers to having an affinity for a mucous membrane. The term "cutaneous" refers to having an affinity for non-mucosal, skin epithelial cells.
The term "substantially the same" when used herein with respect to the
comparison of a sequence to a reference sequence is applicable to sequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, and at least about 99% sequence identity to the reference sequence. The determination of percent identity between two sequences can be determined using standard methods and algorithms including, e.g., BLASTN (NCBI ; Schaffer et al., NAR 29:2994-3005, 2001 ), BLASTX (NCBI ; Schaffer et al., NAR 29:2994-3005, 2001 ), ALIGN (GCG, Accelrys), and FASTA (Pearson et al.,
Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448, 1988) programs, which may employ default settings. In various specific examples, as described herein, amino acid sequences of the invention include those having, e.g., 5, 7, 10, 20, 30, 40, 50, 75, or 100 consecutive amino acids that are 100% identical to the reference sequences.
DESCRIPTION OF THE INVENTION
The present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus. The present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs.
In particular, the present invention provides various genetic constructs, nucleic acid molecules, that contain or encode for either full length, portions and fragments thereof, or deletions, of the Flagellin (Fla) molecule fused to various versions of additional nucleic acid molecules that contain or encode one or more HPV-L2 peptides, or portions and fragments thereof, presented in mono- or multimeric forms, representing either single or multiple HPV types/subtypes. Detailed descriptions of exemplary constructs are presented in Table 1 below. The present invention is not intended however to be limited to the constructs presented in Table 1 . Those skilled in the art will appreciate that the compositions and methods described herein form the basis for design and construction of additional compositions (e.g., nucleic acid and/or amino acid sequences) and therapeutic and/or prophylactic methods within the sprit of the invention but not specifically recited herein. One of the difficulties faced in the field of HPV L2 vaccines is that the target
epitopes often suffer poor immunogenicity and low solubility due to the relatively high inherent hydrophobicity of the L2 proteins and polypeptides. Therefore advantageous selection of one or more agents that increase the immunogenicity and/or solubility (e.g., adjuvants and delivery vehicles) of candidate HPV vaccine formulations comprising L2 proteins and/or polypeptides is an important consideration. Various preferred
embodiments of the present invention demonstrate that Flagellin satisfies both of the above-mentioned formulation requirements. It is important to note however that the present invention is not intended to be limited to formulations comprising Flagellin. Those skilled in the art will appreciate that other suitable polypeptide based, or otherwise,
immunmodulating agents (e.g., adjuvants) may be added to, or substituted in place of, Flagellin in the formulations contemplated by the present invention. It is further
appreciated that various delivery vehicles or solubility enhancing agents, as used within the present invention, could likewise be added to, or substituted in place of, Flagellin in the formulations contemplated by the present invention.
Certain preferred embodiments comprise Fla-L2 fusion product constructs that provide: (1 ) broadly cross-neutralizing properties of various epitopes within the N-terminal portion of HPV L2 proteins (e.g., comprising amino acids (AA) 1 1 -200); and/or (2)
extended cross-neutralization properties provided by multiple L2-peptide fusions; and/or (3) one or more strong adjuvants (e.g., Flagellin, or polypeptides of Flagellin, TLR
agonists/antagonists, aluminum adjuvants, oil-in-water based adjuvants including
squalene, etc.).
Adjuvants are known immune response potentiators and have been widely applied for many years to increase a subject's immune response to antigenic compositions. (See, WO 2007/1 10409 A1 incorporated by reference herein in its entirety). Examples of adjuvants that have been used for many years and that are approved for human applications, are mainly those based on aluminum (also referred to as "alum") : aluminum hydroxide and aluminum phosphate. In recent years, many new adjuvant compounds have been found, or developed.
Generally, the main mechanisms in which adjuvants are thought to work are seen as: 1 ) retaining the antigen at the site of injection; 2) causing a mild inflammation at the site of injection; 3) causing the recruitment of dendritic cells towards the site of injection; 4) inducing the uptake of antigen by the dendritic cells, and 5) promoting the maturation of dendritic cells, or combinations of two or more of the above. The present invention is not however intended to be limited to any particular mechanism(s) or theories concerning adjuvants or adjuventation of the fusion products. Examples of adjuvants include, but are not limited to, compounds from the following categories: mineral containing compositions, mineral salts, such as, aluminum salts and calcium salts, hydroxides (e.g., aluminum oxyhydroxides), phosphates (e.g., aluminum
hydroxyphosphates, orthophosphates), and sulfates, or mixtures thereof. Depending on numerous factors, but also on the phosphorylation status of the antigen, the antigen becomes more or less adsorbed onto the adjuvant.
Oil-emulsion compositions (or oil-in-water compositions, as also used herein) suitable for use as adjuvants include squalene-water emulsions, such as MF59 (See, WO 1990/14837 incorporated by reference herein in its entirety) or submicron oil-in-water emulsions based on MF59. Other submicron oil-in-water emulsions are MF75 (or SAF) and Covaccine HT.
Saponins are a group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots, and flowers of wide variety of plant species. Saponins from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Other saponins are those from Silax ornate, Gypsophilla paniculata and Saponaria officinalis.
Saponin adjuvant formulations include purified formulations such as QS7, QS17, QS18, and QS21 (See, U.S. Pat. No. 5,057,540; and WO 1996/33739 each of which is incorporated herein by reference in its entirety), QH-A, QH-B and QH-C, and lipid formulations such as
Immunostimulating Complexes (ISCOMs). ISCOMs typically include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine, and may include one or more of Quill A, QH- A and QH-C.
In certain other embodiments, bacterial or microbial derivatives provide useful adjuvants, for example, monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), RC-529, OM-I74, and CpG-motif containing oligonucleotides. Also ADP-ribosylating bacterial toxins may be applied, for example, E. coli heat labile enterotoxin LT, cholera toxin CT, pertussis toxin PT, diphtheria toxoid, tetanus toxoid TT. Mutants of such toxins may also be applicable, for example, LT-K63, LT-R72, LTR192G, and CRM197.
Other adjuvants that may also be used in vaccine compositions include bioadhesives and mucoadhesives, liposomes, polyoxyethylene ethers and -esters, polyoxyethylene sorbitan ester surfactants in combination with an octoxynol, as well as polyoxyethylene alkyl esters or ester surfactants in combination with at least one additional non-ionic surfactant such as octoxynol.
Still other suitable adjuvants comprise PCPP formulations, muramyl peptides and imidazoquinolone compounds.
Certain human proteins, such as cytokines, have been applied to stimulate immune responses and in this regard may additionally be useful as adjuvants in combination with the fusion products of the present invention, for instance, the interleukins: IL-I ; IL-2; IL- 4; IL-5; IL-6; IL-7; and IL-12; interferon-γ; and tumor necrosis factor (TNF).
Additionally, the fusion product constructs may comprise one or more linkers
(e.g., one, two, three, four, five, or more, contiguous amino acid residues, or other
moieties) between fusion product constituents.
In certain preferred embodiments, the HPV L2 sequences used within particular fusion products correspond to those presented in WO 2006/083984 A1 . The WO
2006/083984 patent application is hereby incorporated by reference in its entirety.
In additional embodiments, the present invention provides polypeptide
compositions (e.g., fusion products) for prevention of infection by various papillomavirus (PV) types, and especially human papillomavirus types (HPV). In certain aspects, a multimeric HPV peptide composition is a non-naturally occurring polypeptide comprising two or more PV protein segments or immunogenic peptides from different PV types
configured as a linear (concatamer) or branched polypeptide structure providing a
multimeric/multitype HPV L2 polypeptide. The HPV L2 peptide can comprise all or part of the amino acid sequence of an L2 protein of a virus in the family papovavirus;
polyomavirus; papillomavirus; and/or a papillomavirus within the a genus, or the genera β, Y, δ, ε, ζ, η, θ, ι, κ, λ, μ, ν, ξ, ο, or π (See, de Villiers et al., Virology, 324(1 ):17-27
(2004)); and/or human papillomaviruses: HPV1 , HPV2, HPV3, HPV4, HPV5, HPV6,
HPV7, HPV8, HPV9, HPV10, HPV1 1 , HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21 , HPV22, HPV23, HPV24, HPV25, HPV26, HPV27,
HPV28, HPV29, HPV30, HPV31 , HPV32, HPV33, HPV34, HPV35, HPV36, HPV37,
HPV38, HPV39, HPV40, HPV41 , HPV42, HPV43, HPV44, HPV45, HPV46, HPV47,
HPV48, HPV49, HPV50, HPV51 , HPV52, HPV53, HPV54, HPV55, HPV56, HPV57,
HPV58, HPV59, HPV60, HPV61 , HPV62, HPV63, HPV64, HPV65, HPV66, HPV67,
HPV68, HPV69, HPV70, HPV71 , HPV72, HPV73, HPV74, HPV75, HPV76, HPV77,
HPV78, HPV79, HPV80, HPV81 , HPV82, HPV83, HPV84, HPV85, HPV86, HPV87,
HPV88, HPV89, HPV90, HPV91 , HPV92, HPV93, HPV94, HPV95, HPV96, HPV97,
HPV98, HPV99, HPV100, HPV101 , HPV102, HPV103, HPV104, HPV105, HPV106,
HPV107, HPV108, HPV109, HPV1 10, HPV1 1 1 ; and/or animal papillomaviruses (e.g., bovine papillomavirus type 1 (BPV1 ), bovine papillomavirus type 2 (BPV2), bovine
papillomavirus type 4 (BPV4), cottontail rabbit papillomavirus (CRPV), deer
papillomavirus (DPV), European elk papillomavirus (EEPV), canine oral papillomavirus (COPV), Rhesus monkey papillomavirus (RhPV) and rabbit oral papillomavirus (ROPV));
and/or portions or mutations of any one or more nucleic acid sequence disclosed, or incorporated by reference, herein (e.g., the HPV L2 sequences within SEQ I D NOs: 1 -99, 1 00, 200, 201 , and 300-302).
The following list describes the affinities of some of the 1 18 human and animal papillomavirus types : H PV1 (Cutaneous), HPV2 (Cutaneous), HPV3 (Cutaneous) , HPV4 (Cutaneous), H PV5 (Cutaneous), HPV 6 (Mucosal), HPV7 (Cutaneous), HPV8
(Cutaneous), H PV9 (Cutaneous), HPV10 (Cutaneous), HPV1 1 (Mucosal), HPV12 (Cutaneous), H PV13 (Mucosal), HPV14 (Cutaneous), HPV15 (Cutaneous), HPV16 (Mucosal), HPV17 (Cutaneous), HPV18 (Mucosal), HPV19 (Cutaneous), H PV20 (Cutaneous), H PV21 (Cutaneous), HPV22 (Cutaneous) , HPV23 (Cutaneous), HPV24 (Cutaneous), H PV25 (Cutaneous), HPV26 (Cutaneous) , HPV27 (Cutaneous), HPV28 (Cutaneous), H PV29 (Cutaneous), HPV30 (Mucosal) , HPV31 (Mucosal), H PV32 (Mucosal), HPV33 (Mucosal), HPV34 (Mucosal and Cutaneous), HPV35 (Mucosal), HPV36 (Cutaneous), HPV37 (Cutaneous), HPV38 (Cutaneous), H PV39 (Mucosal) , HPV40 (Mucosal), HPV41 (Cutaneous), HPV42 (Mucosal), HPV43 (Mucosal), HPV44 (Mucosal), HPV45 (Mucosal), HPV46 (Cutaneous), HPV47 (Cutaneous) , H PV48 (Cutaneous), H PV49 (Cutaneous), HPV50 (Cutaneous) , HPV51 (Mucosal), HPV52 (Mucosal), HPV53 (Mucosal), HPV 54 (Mucosal), HPV55 (Mucosal), HPV56 (Mucosal), HPV57 (Cutaneous and Mucosal), HPV58 (Mucosal), H PV59 (Mucosal), HPV60
(Cutaneous), H PV61 (Mucosal), HPV62 (Mucosal), HPV63 (Cutaneous) , H PV64 (Mucosal), HPV65 (Cutaneous and Mucosal), HPV66 (Mucosal), HPV67 (Mucosal), HPV68 (Mucosal), HPV69 (Mucosal), HPV70 (Mucosal), HPV71 (Mucosal), H PV72 (Mucosal), HPV73 (Mucosal), BPV1 (Cutaneous), BPV2 (Cutaneous), BPV4
(Cutaneous), CRPV (Cutaneous), DPV (Cutaneous), EEPV (Cutaneous), COPV
(Mucosal), RhPV (Mucosal) , and RoPV (Mucosal).
The Human Papillomaviruses Compendium On Line compiles and publishes relevant molecular data concerning the human papillomaviruses (HPV) and related animal papillomaviruses. The scope of the compendium and database comprises: (I) HPV and animal PV Nucleotide Sequences; (II) Amino Acid and Nucleotide Sequence Alignments; (I II) Analyses; (IV) Related Host Sequences; and (V) Database
Communications. The compendium is accessed on the internet at (hpv- web.1 an1 .gov/stdgen/virus/hpv/compendium/htdocs/HTML FILES/HPVcompin- tro4.html)
In various preferred embodiments, a PV (e.g., H PV) antigen, or epitope, or peptide, or polypeptide segment of the invention can comprise from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 1 1 , 12, 13, 14, 1 5, 16, 1 7, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 1 0, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 501 , 502, 503, 504, 505, 506, 550, 600, or more, contiguous amino acids, including all values and ranges there between, of a papillomavirus L2 polypeptide (e.g., the HPV L2 sequences within SEQ I D NOs: 101 -199, 202-204, and 303-305).
In still further preferred embodiments, a polypeptide segment can comprise at most, at least, or about amino acid position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15,
Figure imgf000039_0001
56, 57, 58, 59 60, 61 , 62, 63 64, 65, 66, 67 68, 69, 70, 71 72, 73, 74, 75 76, 77, 78, 89, 80, 81 , 82 83, 84, 85, 86 87, 88, 89, 90 91 , 92, 93, 93 94, 95, 96, 97 98, 99, 100, 101 , 102, 103 104, 105, 106 107, 108, 109 1 10, 1 1 1 , 1 12 1 13, 1 14, 1 15 1 16, 1 17, 1 19 120 121 , 122 123 124 , 125 126 127 , 128 129 130 , 131 132 133 134, 136 137 138 , 139 140 141 , 142 143 144 , 145 146 147 , 148 149 150 151 , _, 153 154 155 , 156 157 158 , 159 160 161 , 162 163 164 , 165 166 167 168, ), 170 171 172 , 173 174 175 , 176 177 178 , 189 180 181 , 182 183 184 185, 187 188 189 , 190 191 192 , 193 193 194 , 195 196 197 , 198 199 200 201 , I, 203 204 205 , 206 207 208 , 209 210 21 1 , 212 213 214 , 215 216 217 218, ), 220 221 222 , 223 224 225 , 226 227 228 , 229 230 231 , 232 233 234 235, 237 238 239 , 240 241 242 , 243 244 245 , 246 247 248 , 249 250 251 252, 254 255 256 , 257 258 259 , 260 261 262 , 263 264 265 , 266 267 268 269, 271 272 273 , 274 275 276 , 277 278 289 , 280 281 282 , 283 284 285 286, , 288 289 290 , 291 292 293 , 293 294 295 , 296 297 298 , 299 300 301 302, 304 305 306 , 307 308 309 , 310 31 1 312 , 313 314 315 , 316 317 318 319, 321 322 323 , 324 325 326 , 327 328 329 , 330 331 332 , 333 334 335 336, r, 338 339 340 , 341 342 343 , 344 345 346 , 347 348 349 , 350 351 352 353, 1, 355 356 357 , 358 359 360 , 361 362 363 , 364 365 366 , 367 368 369 370, . , 372 373 374 , 375 376 377 , 378 389 380 , 381 382 383 , 384 385 386 387, 389 390 391 , 392 393 393 , 394 395 396 , 397 398 399 , 400 401 402 403, . , 405 406 407 , 408 409 410 , 41 1 412 413 , 414 415 416 , 417 418 419 420, . , 422 423 424 , 425 426 427 , 428 429 430 , 431 432 433 , 434 435 436 437, 439 440 441 , 442 443 444 , 445 446 447 , 448 449 450 , 451 452 453 454, 3, 456 457 458 , 459 460 461 , 462 463 464 , 465 466 467 , 468 469 470 471 , I, 473 474 475 , 476 477 478 , 489 480 481 , 482 483 484 , 485 486 487 488, ), 490 491 492 , 493 494 495 , 496 497 498 , 499 500 501 , 502 503 504 505, 3, 550 600 or more, of an L2 polypeptide (e.g., the HPV L2 sequences within SEQ ID NOs: 101 -199, 202-204, and 303-305).
Examples of L2 polypeptides can be found in publicly available protein databases such as GenBank (gb), SwissPro (sp), EMBL, and the like. Representative database entries, listed by HPV type with accession number in parenthesis, include, but are not limited to: HPV2 (gb/AAY86489, gb/ABN49461 , gb/ABN49469, gb/AB014925, gb/NP_077121 ) ; HPV3 (sp/P36744); HPV7 (gb/NP_041858.1 ); HPV10
(gb/NP_041745); HPV16 (gb/AA085414, gb/AA015703, gb/AA01571 1 , gb/AAQ10726, gb/AAV91650); HPV18 (gb/AAF14009, gb/ABP99710, gb/ABP99718, gb/ABP99726, gb/ABP99742, gb/ABP99766, gb/ABP99774, gb/ABP99782, gb/ABP99790, gb/ABP99798, gb/ABP99806, gb/NP_040316) ; HPV26 (gb/NP_041 786.1 ); HPV27 (dbj/BAE16268, sp/P36755) ; HPV28 (sp/P50799); HPV29 (sp/P50800) ; HPV30
(sp/P36756) ; HPV33 (sp/P06418) ; HPV39 (gb/AAA47055) ; HPV40 (sp/P36760) ; HPV43 (sp/Q705H5); HPV45 (gb/AAY86493) ; HPV45 (gb/ABP99814, gb/ABP99854, gb/ABP99862, gb/ABP99870, gb/ABP99878, gb/ABP99894, gb/ABP99902, sp/P36761 ) ; HPV51 (sp/P26539) ; HPV52 (sp/P36763) ; H PV53 (gb/ABU54103, gb/ABU541 17, gb/ABU54131 , gb/ABU54152, gb/ABU54159, gb/ABU541 73, gb/N P_041847) ; HPV56 (gb/AB076808, gb/AB076815, gb/AB076822, gb/AB076829, sp/P36765); HPV57 (dbj/BAF80485, sp/P22164) ; HPV58 (sp/P26538) ; HPV59 (emb/CAA54855) ; HPV61 (ref/NP_043449) ; H PV62 (sp/Q676U7) ; HPV66 (gb/AB076836, gb/AB076843, gb/AB076857, gb/AB076864, gb/AB076885, gb/AB076892, gb/AB076899, sp/Q80960) ; HPV68a (gb/AAZ39497); HPV 69 (sp/Q9JH45) ; HPV70 (gb/AAC54856) ; HPV71
(gb/AAQ95182, gb/AAQ95189, gb/AAQ95203, ref/NP_597937) ; H PV72
(emb/CAA63878) ; HPV77 (emb/CAA75467); HPV81 (emb/CAF05697); HPV82
(gb/AAK28455, sp/Q91 R53) ; HPV83 (gb/AAD38973) ; HPV84 (gb/AAK09276) ; HPV85 (gb/AAD24187) ; HPV86 (gb/AAL06740); HPV87 (emb/CAC17717) ; HPV89
(gb/AAM921 56) ; HPV90 (ref/NP_671 508); HPV91 (gb/AAM89135) ; HPV94
(dbj/BAD89178, emb/CAF05714) ; HPV97 (gb/AAZ39505, gb/AB027082); HPV102 (gb/AAZ39525) ; or H PV106 (gb/AAZ39518). Disclosure of additional HPV L2 and/or Flagellin related compositions (e.g., sequences), methods, and formulations can be found, for example, in the following publications: U.S. Pat. No. 7,915,381 ; WO
2005/042564 A1 ; WO 2004/022092 A2; WO 2005/1 07381 A2; WO 2009/082440 A2; WO 2006/066214 A2; WO 1993/20846 A1 ; WO 2007103322 A2; and WO 2000032228 A2, each of which is incorporated herein by reference in its entirety.
Each amino/nucleic acid sequence represented by the accession number throughout the disclosure is incorporated herein by reference as of the filing date of this application. In certain aspects at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more, L2 peptides from at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 1 7, 18, 19, 20, 21 , 22, 25, 30, or more, HPV types are coupled together to form a multimeric HPV polypeptide (e.g., portion of a fusion product). Coupling of the segments can be by expression or synthesis of a fusion protein, or by chemical conjugation of the peptides to each other, or chemical conjugation of the peptides to a common substrate or polymer, or via one or more linkers.
The present invention includes compositions for preventing or ameliorating PV infections, in particular, HPV infections. As such, the invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as vaccine(s), may be prepared from multitype HPV polypeptide(s) comprising segments of HPV L2 protein. In other embodiments, multitype HPV L2 polypeptides can be used in combination with other HPV proteins or segments thereof, such as: E1 , E2, E3, E4, E5, E6, E7, E8, and/or L1 protein. (See, for example, U.S. Pat. Nos. 7,425,438; 7,416,846; 7,416,732; 7,407,807; 7,374,767; 7,201 ,908;
7,189,513; and 7,288,258; and WO 2009/059325 A2 each of which is incorporated herein by reference in its entirety).
1. Mono- or Oligomeric Fla-L2 Fusion Constructs
The present invention provides a number of monomeric or oligomeric Fla-L2 constructs containing one or more L2 epitopes from a single HPV subtype fused to, or inserted into, a full length Flagellin (Fla) molecule or a portion thereof in various fashions. Additionally, further embodiments, provide a number of multimeric Fla-L2 constructs containing one or more L2 epitope repeats from a single HPV subtype fused to, or inserted into, one or more full length Flagellin molecules or a portions thereof in various fashions.
As summarized in Table 1 , three types of Flagellin molecules were tested: (1 ) full length Flagellin molecules; (2) D3 domain deletions; or (3) D2-D3 deletion (D2D3 replacement) variants. However, the present invention is not intended to be limited to these three primary classes of Flagellin molecules. Moreover, the present invention contemplates L2 mutants, derivatives, and fragments etc. Table 1 provides exemplary embodiments of certain monomeric and multimeric expression plasmids and certain characteristics of particular corresponding expressed fusion products. TABLE1
Figure imgf000042_0001
Fla-05 Fla (1-506 AA)/CT 11-200 AA of HPV16
Fla-06 Fla (1-506 AA)/CT 2-200 AA of HPV16
FCS
Fla-07 Fla (1-506 AA)/CT 11 -200 AA of + + + +
HPV16X6HIS
Fla-08 Fla (1-506 AA)/CT 2-200 AA of
HPV16X6XHIS
FCS
Fla-09 Fla (1-506 AA)/CT 2-473 AA of HPV16
Fla-10 Fla (1-506 AA)/CT
Fla-11 Fla (1-506 AA)/CT 2-200 AA of
HPV16X6XHIS
Fla-12 Fla (1-506 AA)/CT 2-195 AA of HPV31
Fla-13 Fla (1-506 AA)/CT 2-195 AA of HPV31
FCS
Fla-15 Fla (1-506 AA)/CT 11-195 AA of HPV31
N
+ + D
Fla-16 Fla (1-506 AA)/CT 17-36 AA of HPV16
Fla-17 Fla (1-506 AA)/CT 2-88 AAof HPV16
Fla-18 Fla (1-506 AA)/CT 10-199 AA of HPV18 N
+ + D
Fla-19 Fla (1-506 AA)/CT 11-200 AA of HPV6 N
+ + D
Fla-20 Fla (1-506 AA)/CT 11-200 AA of HPV11 N
+ + D
Fla-21 Fla (1-506 AA)/CT 9-216 AAof ROPV
Fla-22 Fla (1-506 AA)/CT 9-215 AAof CRPV
Fla-23 Fla (1-506 AA)/CT 8-235 AA of HPV5
Fla-24 Fla (1-506 AA)/CT 10-199 AA of HPV45
Fla-25 Fla (1-506 AA)/CT 10-200 AA of HPV52
Fla-26 Fla (1-506 AA)/CT (11-200 AAof HPV16)
X(10-199 AAof HPV18)
X(10-197 AAof HPV6)X
(9-196 AA of HPV11)
Figure imgf000044_0001
Fla-40 Fla (1-506 AA)/CT (10-200 AAof HPV58 WT)
Fla-41 Fla (1-506 AA)/CT (11-200 AA of HPV16)
X(11-200 AAof HPV18)
X(11-200 AAof HPV6
WT)
N
X6XHIS
+ + D
Fla-42 Fla (1-506 AA)/CT (11-88 AAof HPV16)
X(11-88 AA of HPV18)
X(11-88 AA of HPV6)
N
X6XHIS
+ + D
Fla-43 Fla (1-506 AA)/CT (11-88 AAof HPV16)
X(11-88 AA of HPV18)
X(11-88 AA of HPV1)
X(11-88 AA of HPV5)
X(11-88 AA of HPV6)X
N
6XHIS
+ + D
Fla-44 Fla (2-506 AA)/NT (11-200 AAof HPV16)
X(11-200 AAof HPV18)
X(11-200 AAof HPV6)
Fla-45 Fla (2-506 AA)/NT (11-88 AAof HPV16)
X(11-88 AA of HPV18)
X(11-88 AA of HPV6 WT)
Fla-46 Fla (2-506 AA)/NT (11-88 AA of HPV16)
X(11-88 AA of HPV18)
X(11-88 AA of HPV1)
X(11-88 AA of HPV5)
X(11-88 AA of HPV6)
Fla-47 Fla (2-506 AA)/NT 11-200 AA of HPV16
Fla-48 Fla (2-506 AA)/NT 11-200 AA of HPV18
Fla-49 Fla (2-506 AA)/NT 11-200 AA of HPV6
Fla-50 Fla (2-506 AA)/NT 11-200 AA of HPV31 Fla51 Fla (2-506 AA)/NT 11-200 AA of HPV11
Fla-56 Fla (1-187; 297- (11-200 AA of HPV16)x
506 AA)/RP (11-200 AA of HPV18)X
N
(11-200 AA of HPV6)
+ D
Fla-57 Fla (1-187; 297- (11-88 AA of HPV16)x
506 AA)/RP (11-88 AA of HPV18)X
N
(11-88 AA of HPV6)
+ + D
Fla-58 Fla (1-187; 297- (11-88 AA of HPV16)x
506 AA)/RP (11-88 AA of HPV18)X
(11-88 AA of HPV1)X
(11-88 AA of HPV5)X
(11-88 AA of HPV6)X
Fla-59 Fla (1-187; 297- (17-38 AA of HPV16)x
506 AA)/RP (17-38 AA of HPV18)X
(17-38 AA of HPV1)X
(17-38 AA of HPV5)X
(17-38 AA of HPV6)X
Fla-60 Fla (1-506 AA)/CT (11-200 AA of HPV16)
X(11-200 AAof HPV18)
X(11-200 AA of HPV31)
X(11-200AA of HPV52)
X(11-200 AAof
HPV6)x6HIS
Fla-61 Fla (1-506 AA)/CT (11-200 AAof HPV16)
X(11-200 AAof HPV18)
X(11-200 AAof HPV1)
X(11-200AA of HPV5)
X(11-200 AAof
HPV6)x6HIS
Fla-62 Fla (1-187; 297- Deleted flagellin AA188-
506 AA) 296
+ + + +
Fla-63 Fla (1-187; 355- Deleted flagellin AA188- N
+ + D 506 AA) 354
Fla-64 Fla (1-180; 402- Deleted flagellin AA181-
N
506 AA) 401
+ + D
Fla-65 Fla (1-180; 402- (17-38 AA of HPV6)x
506 AA)/RP (17-38 AA of HPV18)X
(11-88 AA of HPV16)x
(17-38 AA of HPV31)X
(17-38 AA of HPV39)X
(17-38 AA of HPV52)
+ + + +
Fla-66 Fla (1-187; 297- (11-88 AA of HPV16)x
506 AA)/CT (11-88 AA of HPV18)X
(11-88 AA of HPV1)X
(11-88 AA of HPV5)X
(11-88 AA of HPV6)
N
X6HIS
+ D
Fla-67 Fla (1-187; 297- Fla-34 plus C-terminal
506 AA) fusion of
/RP and CT (11-88 AA of HPV16)x
(11-88 AA of HPV18)X
(11-88 AA of HPV1)X
(11-88 AA of HPV5)X
(11-88 AA of HPV6)
N
X6HIS
+ D
Fla-68 Fla (1-180; 402- (11-88 AA of HPV16)x
506 AA)/RP (11-88 AA of HPV18)X
N
(11-88 AA of HPV6)
+ D
Fla-69 Fla (1-187; 297- (10-88 AA of HPV6B)x
506 AA)/CT (11-88 AA of HPV16)X
(10-88 AA of HPV18)X
(11-88 AA of HPV31)X
(10-88 AA of HPV39)
X6HIS
+ + + +
Fla-70 Fla (1-187; 297- (10-88 AA of HPV6B)x - + N 506 AA)/CT (11-88 AA of HPV16)X D (10-88 AA of HPV18)X
(11-88 AA of HPV31)X
(10-88 AA of HPV39)X
(10-88 AA of HPV51)x
(10-88 AA of HPV56)X
(11-88 AA of HPV73)
X6HIS
Fla-71 Fla (1-506 AA)/CT (10-88 AA of HPV6B)x
(11-88 AA of HPV16)X
(10-88 AA of HPV18)X
(11-88AAof HPV31)X
(10-88 AA of HPV39)X
(10-88 AA of HPV51)x
(10-88 AA of HPV56)X
(11-88 AA of HPV73)
X6HIS
Fla-72 Fla (1-506 AA)/CT ( 0-88 AA of HPV6B)x
(11-88 AA of HPV16)X
(10-88 AA of HPV18)X
(11-88 AA of HPV31)X
(10-88 AA of HPV39)
X6HIS
Fla-73 Fla (1-187; 355- (11-88 AA of HPV6B)x
506 AA)/CT (11-88 AA of HPV16)X
(11-88 AA of HPV18)X
(11-88 AA of HPV31)X
(11-88 AA of HPV39)
X6HIS
Fla-74 Fla (1-187; 355- (17-38 AA of HPV6)x
506 AA)/RP (17-38 AA of HPV18)X
(11-88 AA of HPV16)x
N
(17-38 AA of HPV31)X
+ + D (17-38 AA of HPV39)X
(17-38 AA of HPV52)
Fla-75 Fla (1-187; 355- (11-88 AA of HPV16)x
506 AA)/RP (11-88 AA of HPV18)X
N
(11-88 AA of HPV6)
+ D
Fla-76 Fla (1-180; 402- (17-38 AA of HPV6)x
506 AA)/RP (17-38 AA of HPV18)X
(11-200 AA of HPV16)x
(17-38 AA of HPV31)X
(17-38 AA of HPV39)X
(17-38 AA of HPV52)
+ + + +
Fla-77 Fla (1-187; 355- (17-38 AA of HPV6)x
506 AA)/RP (17-38 AA of HPV18)X
(11-200 AA of HPV16)x
(17-38 AA of HPV31)X
(17-38 AA of HPV39)X
N
(17-38 AA of HPV52)
+ + D
Fla-78 Fla (1-180; 402- Fla-65 plus C-terminal
506 AA) /RP/CT fusion of
(10-88 AA of HPV6B)x
(11-88 AA of HPV16)X
(10-88 AA of HPV18)
N
X6HIS
+ D
Fla-79 Fla (1-187; 355- Fla-74 plus C-terminal
506 AA) /RP/CT fusion of
(10-88 AA of HPV6B)x
(11-88 AA of HPV16)X
(10-88 AA of
N
HPV18)X6H!S
+ D
Fla-80 Fla (1-187; 297- 11-88AA of HPV16X6HiS
N
506 AA)/RP
+ + D
Fla-81 Fla (1-187; 297- 11-88AA of HPV16X6HIS
N
506 AA)/CT
+ + D Fla-82 Fla (1-187; 297- 11-200AA of
N
506 AA)/CT HPV16X6HIS
+ + D
Fla-84 Fla (1-187; 297- 10-199AA of HPV39
N
506 AA)/RP
+1- + D
Fla-85 Fla (1-187; 297- 11-200AA of HPV18
N
506 AA)/RP
+ + D
Fla-86 Fla (1-187; 297- 10-198AA of HPV6
N
506 AA)/RP
+ + D
Fla-87 Fla (1-187; 297- 11-195AA of HPV31
N
506 AA)/RP
+ + D
Fla-88 Fla (1-187; 297- 11-200AA of HPV45
N
506 AA)/RP
+ + D
Fla-89 Fla (1-180; 402- (17-38 AA of HPV18)x
506 AA)/RP (17-38 AA of HPV31)X
(17-38 AA of HPV33)x
(11-88 AA of HPV16)X
(17-38 AA of HPV35)X
(17-38 AA of HPV45)X
(17-39 AA of HPV58(52))X
(17-39 AA of HPV39)
Fla-90 Fla (1-180; 402- (17-38 AA of HPV31)X
506 AA)/CT (17-38 AA of HPV33)x
(11-88 AA of HPV16)X
(17-38 AA of HPV35)X
(17-38 AA of HPV45)x
(10-88 AA of HPV18)X
(17-38 AA of HPV58(52))X
(17-38 AA of HPV39)x
(17-39 AA of HPV59)X
(10-88 AA of
HPV6b)X6HIS
Fla-91 Fla (1-180; 402- (17-38 AA of HPV18)x
506 AA)/RP (17-38 AA of HPV31)X 17-38 AA of HPV33)x 17-38 AA of HPV16)X 17-38 AA of HPV35)X 17-38 AA of HPV45)X 17-39 AA of HPV58(52))X 17-39 AA of HPV39)
Fla (1-180; 402- 17-38 AA of HPV18)x 506 AA)/RP 17-38 AA of HPV33)x
11-88 AA of HPV16)X 17-38 AA of HPV35)X 17-38 AA of HPV45)X 17-39 AA of HPV58(52))
Fla (1-180; 402- 17- ■38 AA of HPV31)X 506 AA)/CT 17- ■38 AA of HPV33)x
11- ■88 AA of HPV16)X
17- ■38 AA of HPV35)X
17- ■38 AA of HPV45)x
10- ■88 AA of HPV18)X
17- ■38 AA of HPV58(52))X
17- ■38 AA of HPV39)x
17- " .* 0* AA of
HPV59)X6HIS
Fla (1-187; 297- (11-88 AA of HPV16)x 506 AA)/CT (10-88 AA of HPV18)X
(11-88 AA of HPV58)X (11-88 AA of HPV33)X (11-88 AA of HPV31) X6HIS
Fla (1-187; 297- (11-88 AA of HPV16)x 506 AA)/CT (10-88 AA of HPV18)X
(11-88 AA of HPV58)X (11-88 AA of HPV33)X (11-88 AA of HPV31)X (11-88 AA of HPV45)X (11-88 AA of HPV35)X (10-88 AA of
HPV6b)X6HIS
Fla-96 Fla (1-187; 297- (11-88 AA of HPV39)x 506 AA)/CT (11-88 AA of HPV59X
(11-88 AA of HPV45)X (11-88 AA of HPV35)X (10-88 AA of
HPV6b)X6H!S
Fla-97 Fla (1-187; 297- (11-88 AA of HPV16)x 506 AA)/CT (10-88 AA of HPV18)X
(11-88 AA of HPV58)X (11-88 AA of HPV33)X (11-88 AA of HPV31)X (10-88 AA of HPV6b)X 6HIS
Fla-99 Fla (1-180; 402- (17-38 AA of HPV18)x 506 AA)/RP (17-38 AA of HPV31)X
(17-38 AA of HPV33)x (11-200 AA of HPV16)X (17-38 AA of HPV35)X (17-38 AA of HPV45)X (17-39 AA of HPV58(52))X (17-39 AA of HPV39)
Fla- Fla (1-180; 402- (11-88 AA of HPV16)x 100
506 AA)/CT (10-88 AA of HPV18)X
(11-88 AA of HPV58)X (11-88 AA of HPV33)X (11-88 AA of HPV31)X (11-88 AA of HPV45)X (11-88 AA of HPV35)X (10-88 AA of HPV6b)X6HIS
Fla- Fla (1 -187; 355- (17-38 AA of HPV31 )X 200
506 AA)/RP (17-38 AA of HPV33)x
(1 1 -88 AA of HPV16)X (17-38 AA of HPV35)X (17-38 AA of HPV45)x (10-88 AA of HPV18)X (17-38 AA of HPV58(52))X (17-38 AA of HPV39)x (17-39 AA of HPV59)X (10-88 AA of HPV6b)
Fla- Fla (1 -180; 402- (17-38 AA of HPV31 )X 201
506 AA)/RP (17-38 AA of HPV33)x
(1 1 -88 AA of HPV16)X (17-38 AA of HPV35)X (17-38 AA of HPV45)x (10-88 AA of HPV18)X (17-38 AA of HPV58(52))X (17-38 AA of HPV39)x (17-39 AA of HPV59)X (17-38 AA of HPV6b)
Fla- Fla (1 -187; 297- Deleted flagellin AA188- 62M1
506 AA), mutation 296
I423A
Fla- Fla (1 -187; 297- 1 1 -200 AA of HPV16 32M1
506 AA)/RP
mutation I423A
Fla- Fla (1 -180; 402- (17-38 AA of HPV6)x 65M1
506 AA)/RP (17-38 AA of HPV18)X mutation I423A (1 1 -88 AA of HPV16)x
(17-38 AA of HPV31 )X (17-38 AA of HPV39)X (17-38 AA of HPV52)
S†= Solubility;
E* = Expression;
ΡΔ = Purification;
l§ = In Vivo
6xHIS =C-terminal 6 histidine tag;
Fla =1 -506 AA of structural flagella protein fljB of Salmonella typhimurium LT2 (Gene Bank accession* NP_461698; SEQ ID. No. 21 1 as below:
maqvintnslslltqnnlnksqsalgtaierlssglrinsakddaagqaianrftanikgltqasrnandgisiaqttegalneinnnlqrvrel avqsanstnsqsdldsiqaeitqrlneidrvsgqtqfngvkvlaqdntltiqvgandgetididlkqinsqtlgldslnvqkaydvkdtavtt kayanngttldvsglddaaikaatggtngtasvtggavkfdadnnkyfvtiggftgadaakngdyevnvatdgtvtlaagatkttmpag attktevqelkdtpavvsadaknaliaggvdatdangaelvkmsytdkngktieggyalkagdkyyaadydeatgaikakttsytaa dgttktaanqlggvdgktevvtidgktynaskaaghdfkaqpelaeaaakttenplqkidaalaqvdalrsdlgavqnrfnsaitnlgnt vnnlsearsriedsdyatevsnmsraqilqqagtsvlaqanqvpqnvlsllr
Type of L2 fusions: CT = C-terminal fusion to Fla;
RP = Replacement Fla mutant: D3 domain of Fla (188-296aa)
or D2-D3 domains (188-354aa), or D2-D3 domains
(181 -401 aa) is replaced by L2 sequence(s) ; and
NT = N-terminal L2 fusion to Fla;
SS = L2 mutants with cysteine residues replaced by serine residues;
FCS = Furin Cleavage Site mutant;
WT = wild type of L2 (indicated only if used in fusions with mutant L2 sequence, (if not indicated WT sequence area used by default);
X = used to signify fusion between different L2 sequences or 6xHis tag.
Various fusions products were constructed using full length Flagellin molecules fused at the C-terminus (C-terminally) to either HPV16 L2 2-200 or HPV16 L2 1 1 -200 AA (Fla-02 and or Fla-07, respectively).
Expression of the Table 1 fusion products/constructs in E. coll revealed high
levels of soluble protein products are produced using the methods of the present
invention. (See, FIG. 1 ). Both Fla-02 and Fla-07 fusion products were purified using purification schemes briefly depicted in FIG. 2 to high homogeneity. (See, FIG. 3). In certain particularly preferred embodiments, the L2 based fusion products of the present invention do not form virus like particles but rather soluble expression products.
To predict possible functional changes in Flagellin structure, 3D models of Fla-02 and Fla-07 were generated using Accelrys software (Accelrys, Inc., San Diego, CA) starting from the Flagellin structure described by Samatey (Samatey et ai, Nature,
410:331 -337 (2001 )) and the predicted L2 structure. (See, FIG. 4). The overall shape of the Flagellin molecule looks somewhat like an aircraft with two wings and a short body, each wing being about 70A long, 25A wide, and 20 A thick. (See, Samatey et ai,
(2001 )). The Flagellin structure was divided into three domains labeled D1 , D2, and D3.
Domain D1 comprises an N-terminal segment from Asn 56 to Gin 176, and a C-terminal segment from Thr 402 to Arg 450. Domain D2 also comprises two segments: Lys 177 to Gly 189, and Ala 284 to Ala 401 . A central segment from Tyr 190 to Val 283 makes up Domain D3. The domains are connected by short stretches of two chains in both cases. A cross β-motif motif ties up the two ends of Domain D1 and connects it to D2, where two hydrogen bonds are formed between Asn 173 and Thr 404. The two chains connecting Domains D2 and D3, Gly 189 to Thr 193, and Val 281 to Asn 285, form a short β-strand. The relatively conservative D1 Domain contains the TLR-5 binding motif, while the immunodominant hypervariable D2-D3 Domains have been shown to play an important role in the observed activity as a mucosal adjuvant. (Ivison et al., Inflamm. Bowel Dis. , 16:401 -409 (2001 ); and Liu et al., Biochem. Biophys. Res. Commun., 392:582-587 (2010)).
While the compositions and methods of the present invention are not limited to any mechanism or particular structural configuration, FIG. 4 supports the proposition that the Fla-L2 fusion products of the present invention (e.g., Fla-07) comprise L2 peptide(s) that are preferably fused or otherwise closely attached to the C-terminal portion of the flagellin domain D1 . This particular structure linkage has been found to only minimally, if at all, interfere with Flagellin's TLR-5-binding motif or its accompanying immunodominant domains (Domains D2/D3). Accordingly, preferred embodiments of the present invention provide fusion products wherein the immunological properties of any, or all, of the domains of the adjuvant molecule (e.g., Flagellin: Domains D1 , and/or D2, and/or D3) are accessible and immunologically active. In even more preferred embodiments, the immunological activity of the adjuvant (e.g., Flagellin) molecule in the fusion product likewise does not substantially diminish the immunological activity of the L2 sequences (or other PV/HPV polypeptides) therein.
Consistent with structural predictions of Flagellin's TLR-5 activity, various embodiments of the present fusion products comprise a Flagellin backbone molecule that is not significantly impacted (e.g., immunological activity substantially diminished) by the presence of L2 peptides (i.e., Fla-02 or Fla-07) (See, FIG. 5 and FIG. 16). Furthermore, it was shown that the immunogenicity of the immunodominant Flagellin epitopes were not impacted (e.g., immunological activity substantially diminished) in actual Fla-L2 constructs when compared to the immunogenicity of the same epitopes in wild-type Flagellin. Indeed, in certain embodiments, the Flagellin-specific ELISA titers of anti-Fla- 02 or anti-Fla-07 rabbit serum showed flagellin-biased responses. (See, FIG. 22). Based on this finding, additional embodiments further provide Flagellin D3, or D2-D3 Domain deletion variants in certain Fla-L2 fusions products. For example, FIG. 6A, depicts one such construct, Fla-32. The Fla-32 fusion product contains a D3 replacement for HPV16L2 (1 1 -200 AA). In another example, of a similar fusion product, Fla-31 contains a D3 replacement for HPV16L2 (2-200 AA). Both the Fla-31 and Fla-32 constructs were expressed in soluble bacterial fractions (FIG. 6B) and were purified to high homogeneity for subsequent preclinical studies. (FIG. 7).
The Fla-31 and Fla-32 fusion products both showed significant L2- immunogenicity. In some embodiments, the L2-immunogenicity of the Fla-31 and Fla-32 were greater than that of certain full length fusion product variants. The TLR-5-binding activities of Fla-31 and Fla-32 were comparable. (FIG. 8). Thus, in certain embodiments, both wild type and deletion mutants of Flagellin retain good solubility as well as TLR-5 binding activity when fused to long HPV L2 peptides.
Figures 9 and 10 show the expression of other selected monomeric Fla-L2 fusion constructs further representing other HPV serotypes.
The present invention contemplates certain monomeric fusions products described herein will be useful as vaccines, immunogenic compositions, or as therapeutics. The aforementioned fusion products are further contemplated to be useful components in the preparation and/or administration of multi-serotype cocktail vaccines, immunogenic compositions, or therapeutic formulations.
2. Multimeric Fla-L2 fusion constructs
Previous studies have shown that cross-linking of B-cell receptors by arrays of epitopes on virus like particles (VLPs) or by polymers potentiates B-cell activation.
(Baltimore, Bacteriol. Rev., 35:235-241 (1971 ); Bachmann and Zinkernagel, Annu. Rev. Immunol., 15:235-270 (1997); and Govan et al., Virol. J. ,5:45 (2008)). The present invention extends this understanding, by contemplating that B-cell receptors will recognize concatenated neutralization epitopes of HPV L2 (e.g., complete sequences, or portions, of one, two, three, or more different HPV L2 genotypes such as, but not limited to: 1 , 6, 1 1 , 15, 16, 18, 31 , 33, 35, 39, 45, 52, 58, 59, or 65 etc. Furthermore, it is contemplated that the B-cell receptors will be preferentially activated by the concatenated epitopes as compared to L2 type specific B-cell receptors. It is still further contemplated that the B-cells presenting concatenated HPV epitopes will be more readily activated and will bias the global repertoire of the neutralizing antibody response to cross- reactive epitopes. Importantly, additional embodiments provide multiple concatenated neutralization epitopes (or portions and mutations thereof) of the same or different HPV L2 genotypes, such that a fusion product can comprise multiple concatenated L2 epitopes from, for example, HPV16, or from HPV18, or from HPV31 , etc., It is still further understood that various other fusion products may comprise multiple concatenated L2 epitopes from two, three, four, five, six, seven, eight, or more different types of HPV, for example, one, two, three, or more, L2 epitopes from HPV type 16, and/or one, two, three, or more, L2 epitopes from HPV type 18, and/or one, two, three, or more, L2 epitopes from HPV type 31 , etc.
Accordingly, certain embodiments of the present invention validate the above contemplations by providing fusion products comprising concatenated multimeric L2 epitopes that exhibit enhanced breadth and titer of cross-neutralizing antibody generation as compared to certain other monotypic L2 immunogen fusion products.
To increase the neutralizing capabilities and/or the broad cross subtype protectiveness of preferred Fla-L2 fusion products, a number of multimeric concatenated fusion products were constructed comprising one, two, three, or more, stretches of multiple HPV L2 peptides representing a single, or different, HPV type(s). In certain of these embodiments, Flagellin D3, (e.g., Fla-62) or -D2-D3 (e.g., Fla-64) deletion variants were used as the fusion backbone.
An exemplary comparison of certain monomeric and multimeric fusion constructs
(e.g., Fla-69 construct comprising the combined AD3 property of Fla-32 and the C- terminal L2 fusion site of Fla-07) is shown in FIG. 1 1 .
FIG. 12 shows some of the characteristics of four different multimeric fusion constructs used for expression, purification, and/or subsequent in vivo studies. In particular, Fla-69 and Fla-70 contain 1 1 -88 AA L2 peptides representing HPV5 and 8, respectively, while Fla-65 and Fla-76 contain 17-38 AA L2 peptides from 5 types of HPV plus longer L2 epitopes of HPV16 of either 1 1 -200 AA (Fla-76) or 1 1 -88 AA (Fla-65). The expression, solubility, and a brief purification process, for fusion products Fla-69 and Fla-70 is shown in FIGs. 13 and 14, respectively.
FIG. 15 shows SEC HPLC assay results for purified Fla-69 fusion product along with some of its comparative characteristics including the Fla-32 and Fla-62 backbone. Fla-69 fusion product forms soluble 7-8-mer oligomers. The purification of Fla-65 and Fla-76 fusion products is shown in FIG. 16. The purification scheme used for Fla-65 and Fla-76 fusion products was the same as that used for Fla-69 and Fla-70 as shown, briefly, in FIG. 13. FIGs. 15 and 16 show that the fusion products of the present invention (e.g., Fla-65 and Fla-76) can be purified to high homogeneity.
In preferred embodiments, fusion products were highly expressed (e.g., in bacterial expression systems, including but not limited to E. coli. BL21 (DE3) or
BLR(DE3)), soluble, or solulizable (e.g., using standard solubilization techniques, including, but not limited to, microfluidization techniques), and can be readily purified to high homogeneity (e.g., >10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, or 99.0%, or greater, purity). The present invention provides methods of designing and producing clinical quality fusion products suitable for research, preclinical, clinical, and public health uses. In certain particularly preferred embodiments, the L2 based fusion products of the present invention do not form virus like particles but rather are soluble expression product proteins.
As shown in FIGs. 17 and 18, the TLR-5 binding activity of two exemplary lots of Fla-69 fusion product were comparable with that of the Fla-32 and/or Fla-62 backbone fusions; furthermore, the TLR-5 binding activity of Fla-69 and/or Fla-76 were comparable with that of Fla-65 backbone fusion.
In certain embodiments, the invention encompasses Flagellin D2 and/or D2-3 mutants
(e.g., deletions, additions, and/or substitutions) fused to various L2 oligomers purified to high homogeneity (e.g., >60, 65, 70, 75, 80, 85, 90, 95, or 99.0%, or greater, purity) that provide TLR-5 binding activity comparable to monomeric Fla-L2 fusions products. Various compositions and methods of the present invention provide effective fusion product constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs against a broad spectrum of HPV types/subtypes.
3. Preclinical Studies
A number of useful HPV animal models are known to those skilled in the art. For example, in certain embodiments, the efficacy of fusion products of the present invention was evaluated in both mouse and rabbit HPV animal models. One such animal model exploits the ability of HPV L1/L2 virus-like particles to encapsidate foreign DNA, representing either heterologous PV genome (i.e., Cotton-Rabbit Papilloma Virus (CRPV) (Christensen, Antivir. Chem. Chemother. , 16:355-362 (2005); and Culp et al., J. Virol., 80:1 1381 -1 1384 (2006)) or a plasmid containing reporter genes expressed from strong a constitutive promoter (Roden et al., J. Virol., 70:5875-5883 (1996); Day, et al., Cell Host. Microbe., 8:260-270 (2010); and Ishii et al., Virology, 406:181 -188 (2010)). In some of the animal models, hybrid pseudoviruses are able to induce immune responses related to either: 1 ) a disease related to the encapsidated genome (e.g., the rabbit model used; CRPV); or 2) the infection which might be detected by expression of reporter genes (e.g., the mouse model used).
The exemplary rabbit model used herein exploits "quazi-viruses" that encapsidate the CRPV genome (classified as type I virus under the "Baltimore scheme") which possess a nonsegmented double strand-DNA genome. (Baltimore, D., Bacteriol. Rev., 35:235-241 (1971 )). Quazi-virus infects rabbits, causing keratinous carcinomas that are histologically indistinguishable from tumors caused by wild type CRPV. (Culp et al., J. Virol., 80:1 1381 -1 1384 (2006)). The exemplary mouse model exploits vaginal infection by pseudovirions expressing luciferase, or red, or green, fluorescent proteins which can be detected in vivo. (Alphs et al., Proc. Natl. Acad. Sci. U.S.A. 105:5850-5855 (2008)).
Both animal models have been validated in previous studies as useful in demonstrating the highly protective properties of various L2-based antigens. (See, Pastrana et al., Virology, 337:365-372 (2005); Culp et al., 1 1381 -1 1384 (2006); Mejia et al., J. Virol., 8012393-12397 (2006); Slupetzky et al., Vaccine, 25:2001 -2010 (2007); Day et al., Virology, 82:4638-4646 (2008); and Schiller et al., Gynecol. Oncol., 1 18:S12-S17 (2010)).
In still further embodiments, various fusion product compositions of the present invention were tested head-to-head against commercially available HPV L1 vaccines (i.e., Gardasil® HPV vaccine, Merck, Whitehouse Station, NJ, and Cervarix® HPV vaccine, GlaxoSmithKlein, London, United Kingdom) in preclinical animals studies. The head-to-head studies included assessment of the respective efficacy of the candidates in mice and rabbits via active and passive immunizations as well as analysis of the immunogenicity of target antigens therein as assayed by in vitro neutralization assays, L2-specific ELISAs, RG1 -specific ELISAs, and PepSet® Peptide Libraries.
The head-to-head studies allowed for quantification of the protective antibody responses elicited by candidate Fla-L2 fusion products versus L1 vaccines in vivo; as well as the opportunity to define the dominant linear immunogen within the L2-antigen.
Results of the exemplary animal studies show that: 1 ) despite certain Fla-2 fusion products (e.g., vaccine candidates) having neutralization titers lower than those of the commercial HPV vaccines tested (e.g., Gardasil® and Cervarix®) certain other Fla-2 fusion products showed comparable, or superior, protective efficacy against homologous or heterologous HPV challenge; 2) certain multimeric Fla-2 fusion products demonstrated greater efficacy than certain other monomeric Fla-2 fusion products in either, or both, animal models presented; 3) peptide libraries and/or RG-1 ELISAs analyses of the L2 immune responses in rabbits to certain Fla-L2 fusion products showed RG-1 is an immunodominant epitope consistent with protection profiles; and 4) in certain embodiments, the Flagellin TLR-5 agonist is a good vaccine delivery vehicle for multiple concatenated HPV L2-antigens an that it provides high protective adjuvancy in both exemplary animal models tested.
3.1 Exemplary Rabbit studies
Use of rabbit models provides the opportunity to study protective immunity to both cutaneous and mucosotropic types of papillomaviruses because rabbits can be simultaneously challenged with both CRPV and ROPV. (Christensen, Antivir. Chem. Chemother., 16:355-362 (2005); and Govan et al., Virol. J. , 5:45 (2008)). In some embodiments, the in vivo protection and/or the cross-protection potentials of L2 vaccines recombinant concatenated L2 (1 1 -88 AA) fusion products (e.g., vaccine candidates) representing various HPV types were tested in comparison with monomeric fusion product formulations administered with adjuvant(s) in the aforementioned rabbit and mouse models. More particularly, in several rabbit studies, the utility of various TLR-5 agonists (e.g., Flagellin) as vaccine carrier molecules for either monomeric or multimeric concatenated L2 fusion products were evaluated.
One particular animal study, Rabbit Study No. 3, as shown in FIG. 19, was designed to compare the immunogenicity and/or protective efficacy of monomeric fusion product constructs comprising: 1 ) C-terminal L2 polypeptides fused to full-length Flagellin (e.g., Fla-02 and Fla-07); and 2) Flagellin D3-L2 replacements (e.g., Fla-31 or Fla-32) containing HPV16 L2 peptides.
Briefly, concerning Rabbit Study No. 3, FIG. 19 shows test animals were immunized with either: 1 ) placebo ("None"); or 2) full length Flagellin (e.g., Fla-01 ); or 3) D3 deletion Flagellin backbone (e.g., Fla-62); or 4) full length Fla-L2 fusion product (e.g., Fla-02 or Fla-07); or 5) D3- L2 Flagellin replacements (e.g., Fla-31 or Fla-32). In some embodiments,
further studies were done to evaluate the immunogenicity of various fusion products (e.g., Fla- 31 or fla-32) administered in combination with proprietary Sanofi Pasteur, Inc., adjuvant AF04. The AF04 adjuvant, and similar adjuvants, used in these studies are described in, for example, FR Pat. No. 0600309; U.S. 7,344,720; U.S. 2004202669; U.S. 2007/0191314; EP 1904099 B1 ; and EP 1696954 B1 , all of which are herein incorporated by reference in their entirety, incorporated herein by reference in its entirety. Vaccinations were performed 4 times, 21 Days apart and selected animals were challenged on Day 21 after the last immunization with quizi- viruses 6, 16, 18, and 58, and wild type CRPV. Serum samples generated at various time points over the course of study were assessed for the polyclonal immune response(s) against either recombinant L2-antigen (rl_2) (HPV16) or Flagellin (full length Fla-01 antigen) via standard ELISA assays. Results are shown as End Point ELISA titers in FIGs. 20 and 21 , respectively. FIG. 21 shows the results for full-length Flagellin-L2 fusions. FIG. 21 further shows results for D3-L2 replacements. Both full-length-flagellin-L2 fusions demonstrated strong Flagellin-biased responses (FIG. 20). FIG. 21 shows individual anti-rl_2 titers (Panel A) were about ten times lower than corresponding anti-Flagellin titers (Panel B) for all time points depicted. D3-L2 replacements demonstrated a significant shift towards L2 antigen (Panel A versus Panel B) and the appearance of a "Boosting Effect" from sequential immunizations was more evident than with full-length Flagellin constructs (FIG. 21 ).
Additional animal studies (i.e., Rabbit Studies Nos. 1 and 2) provided data showing certain fusion products of the present invention elicited significant Flagellin-biased immune responses. In still further embodiments, the appearance of a "boosting effect" was assessed by in vitro neutralization assays performed with selected serum samples and
pseudoviruses (PsV) 16 and 18 (FIG. 22). FIG 22 shows the homologous neutralizing immune response (anti-Psv-16) (Panel A) was just slightly increased after 4th dose when compared with a 2 dose regimen for all vaccine formulations, while differences between the 2 and 4 doses for heterologous responses (anti-Psv-18) (Panel B) were more pronounced. In certain of these embodiments, in both PsVs tested the neutralizing responses to D-3-L replacements (Fla-32 or Fla-31 ) were significantly higher than responses to the corresponding full-length-L2 fusions (Fla-02 or Fla-07). In still further of these embodiments, addition of an adjuvant (e.g., AF04 adjuvant) was shown to significantly increase neutralization potency of certain fusion products, such as, Fla-32, but only marginally or not appreciable increase the potency of other certain fusion products, such as, Fla-31 .
In still further embodiments, additional rabbits were challenged with quazi-virus (QV) 6, 16, 18, 58 and wild type CRPV (FIG. 23). FIG. 23 shows results from one of these studies. Briefly, FIG. 23 provides results 8 weeks post challenge as described above; the figure shows that both full-length Fla-L2 fusions as well as Fla-32 were fully protective against challenge with QV6, 16, 18, and 58. In this embodiment, the presence of AF04 adjuvant in the particular formulations of Fla-31 and/or Fla-32 tested reduced protection in test animals against challenge with either QV6 (Fla-31 ) or QV18 (Fla-32).
In still further embodiments, the present invention contemplates broadly cross protective fusion products. In this regard, representative multimeric construct Fla-69, comprising 1 1 -200 AA L2 epitopes from HPV6, 16, 18, 31 , and 39 (FIG. 24), was designed and subsequently tested in an exemplary animal study to access its immunogenicity and/or protectiveness in rabbits challenged with QV6, 16, 18, 58 as well as wild-type CRPV. (See, Rabbit Study No. 6; FIG. 25). L2 epitopes of three quazi- virions (6, 16, and 18) used to challenge test animals were presented in certain fusion product vaccine formulations while QV58 was not. However, the L2-dendrogram of FIG. 24 reveals a close relationship between QV16 and 58. Briefly, FIG. 25 presents the study design including immunization and challenge dosages and regimes. In this animal study, commercially available human HPV vaccines Gardasil® and Cervarix® were administered four times at human dosage levels.
In yet other embodiments, the immunogenicity of candidate vaccine formulations (fusion products) were assessed under two types of neutralization methodologies ((1 ) "conventional methodology": Buck et al., Methods in Mol. Med., 445-461 (Monograph) (2004); and (2) "novel methodology": Day et al., Cell Host. Microbe., 8:260-270 (2010)) as well as under serotype specific RG1 -epitope ELISAs and peptide library studies as described below.
For example, FIGs. 26 and 27 depict the results of a first type of neutralization methodology test ("conventional methodology") of individual serum samples against PsV16 and 18, respectively. Serum samples from two time-points, Days 56 and 77, were compared. Neutralization titers of pre-challenge serum samples of Fla-32 and Fla- 69 against HPV16 were comparable to each other and were about 10 fold lower than corresponding titers for commercial L1 vaccines. (See, FIG. 26). A "boosting effect" (i.e., an increase in titer after the third immunization) was observed and more pronounced for Fla-69 than for Fla-32. While the present invention is not intended to be limited to any particular mechanism(s), it is thought the results can be explained by the size difference in HPV16 L2 epitopes (1 1 -88 versus 1 1 -200 AA). In certain embodiments, the Fla-69 fusion product is superior to the Fla-32 fusion product based on observed heterologous neutralization results (PsV18). Fla-32 antiserum showed no detectable level of neutralization, while pre-challenge titers of Fla-69 serum samples showed titers comparable to those against PsV16. (See, FIG. 27).
However, in still further neutralization tests, contrasting results showed certain fusion product constructs provided comparable levels of protection against all quazi-virus challenges. (See, FIG. 28). Moreover, the "conventional" neutralization method has been criticized by some as not being particularly relevant to predicting in vivo protection. (See, Day et ai., Cell Host Microbe, 8:260-270 (2010)). Day et al. discuss the 293TT cell substrate used in the "conventional methodology" assay for propagation of
pseudoviruses, 293TT cells, which contains both primary (HSPG) and secondary (unknown) receptors. One of the features of 293TT cells allows them to quickly internalize target pseudoviruses. This feature prevents proper virus maturation and therefore results in only weak surface exposure of L2 neutralizing domain(s) on the cells. In contrast to the situation for pseudoviruses, the proffered in vivo mechanism for HP (Ps) virus infection of 293TT cells involves HPV binding to HSPG receptors on the base membrane for a relatively long period of time (e.g., from 6 to 24 hours) whereupon the HPV undergoes conformational changes and subsequent furin cleavage. These changes are thought to allow L2-neutralizing epitope(s) to be more fully exposed on the viral surface and to thus better interact with L2-antibodies. As mentioned previously, the present invention is not intended to be limited to any particular mechanism(s) of action, despite the recitation of the potential mechanisms by Day et al.
Still other embodiments provide additional exemplary neutralization assays and tests. In this regard, the present invention employs a second type of neutralization methodology test referred to as the "novel methodology". The "novel methodology" used was as described by Day et al. (Day et al., Cell Host Microbe, 8:260-270 (2010)). Briefly, this methodology is based on the proposed spatiotemporal separation of L2 epitope(s) exposed on the base membrane (HSPG) and the subsequent/concomitant binding of HPV to secondary receptor(s) on the HSPG-epithelial cell surface. The "novel methodology" is thought to increase sensitivity of the assay more than 30 fold when compared to the "conventional methodology." Again, the present invention is not intended to be limited to any particular mechanism(s) of action, despite the recitation of potential mechanisms by Day et al. In certain embodiments, tests done using the "novel methodology" revealed significant neutralization differences between proposed fusion product formulations. (See, FIG. 29). Nevertheless, the differences observed among fusion product candidates did not correlate well to the actual protectiveness of the candidates since all candidates showed sufficient to good protection against challenge viruses.
Additionally, further assays, tests, and studies were conducted to further clarify the protective thresholds of certain fusion product formulations (e.g., vaccine candidates). In one such embodiment, a passive immunization study was devised using pooled serum samples from Rabbit Study No. 6. (See, FIG. 30). Briefly, pooled fusion product samples were serially diluted (in five fold increments) in preimmune rabbit serum as indicated in FIG. 30. The resulting mixtures were used for the intravenous immunization of study animals (e.g., rabbits) at two animals per described dilution followed by challenge with a broad spectrum of quazi-viruses (QV 6, 16, 18, 31 , 45, and 58) and CRPV. (FIG. 30 and FIG. 31 ). FIGs. 32-38 demonstrate the protective efficacy of exemplary fusion product dilutions against each of the noted challenge quazi-virions at 8 weeks post challenge. Efficacy results from this study are represented as end-point protective titers shown in
FIG. 39. Briefly, FIG. 39 shows the Fla-69 fusion product candidate provided good cross- protective antibody responses against QV6, 16, 18, 31 , and 58 as well as a lower but significant response against QV45 (even at 100 fold serum dilution level) and a modest response against CRPV. The protective properties of the commercially available L1 vaccines (FIG. 39) approximates results observed in respective human clinical trials, notably: Gardasil® protected against QV6, 16, and 18, Cervarix® protected against QV16 and 18 and somewhat against QV31 as well (although at a lower level than the Fla-69 fusion product). Cervarix® did not significantly protect against QV45. In certain preferred embodiments, the exemplary multimeric-L2 fusion product, Fla-69, provided protection comparable to commercially available HPV L1 vaccines against homologous challenges and furthermore demonstrated superior cross-protection as shown in this passive immunization study.
In still other embodiments, to further elucidate L2-immunogenicity and
characterize specific dominant epitopes within the L2-protective antigen, a set of Non- Cleavable PepSet® Peptide Libraries comprising: 1 ) L2 amino acids 2 to 200; 2) 15 residues with an offset of 4 residues representing HPV types 6, 1 1 , 16, 18, 31 , 39, 45, 51 , 52, 58, 59, and 73; 3) a Rabbit Oral Papillomavirus (ROPV); and 4) a Cottontail Rabbit Papillomavirus (CRPV) were generated by Mimotopes, Inc., (St. Paul, MN) as described in Example 1 1 and used in subsequent additional studies.
Results from two pooled serum samples from Rabbit Study No. 6 (anti-Fla-32 and anti-Fla-69) were applied to these peptide libraries via ELISA. Results of ELISA with anti- Fla-32 (HPV 16 L2 1 1 -200 AA) serum pool (diluted to 1/1 ,000) against L2-PepSet® Peptide Libraries of HPV types 6, 16, 18, 45 and 58 used for challenge of rabbits during active immunization phase are shown in FIGs. 40A-40C, Panels A-B, respectively, therein. In FIGs. 40A-40C, each bar shown represents individual peptide signal intensity measured at OD405nm and each library is presented by about 47 overlapping 15 AA peptide sequences each of which being off-set by 4 residues. The homologous immune response depicted against HPV16 (FIG. 40A, Panel B) demonstrated the broadest range of signal intensity across the tested L2 sequence. However, peptides 4, 5, and 6, constituting the RG1 neutralizing epitope, were dominant across all tested types.
Intensity of the signals correlated with divergence of HPV types: signals for HPV16 group (HPV31 and 58) were higher than for HPV18, 45, or 6. The condolyma type HPV6 is depicted with lowest signal intensity, but broad cross-reactivity with an emphasis on the "RG1 " domain.
FIGs. 41 A-41 C show a second example of an HPV L2 type specific PepSet®
Peptide Library ELISA (same libraries as shown in FIGs. 40A-40C, Panels A-B, respectively, therein) with anti-Fla-69 (multimer HPV 6, 16, 18, 31 , 39 L2s AA 1 1 -88) serum pool (diluted 1/1 ,000). Briefly, FIGs. 41 A-41 C show signal intensity of multimeric L2 construct, Fla-69, against all HPV types is much stronger than for monomeric constructs in this embodiment. (See, FIGs. 40A-40C). Each of the Panels of FIGs. 41 A- 41 C show the immunoreactivity of test peptides NOs. 4, 5, and 6. Downstream regions in each Panel also show immunoreactivity with anti-Fla-69 serum, although not as intense as RG1 . (FIGs. 41 A-41 C). Little to no reactivity was seen for peptides from the middle to the C-terminal end of each HPV type L2 epitope.
A summary of cross-reactivity with a larger set of HPV type L2 PepSet® Peptide
Libraries is shown in FIG. 42 (OD4o5nm simplified color scale (burgundy <=3.0; red <2.5; light red <2.0; salmon <1.5; pink <1 .0; white <0.5; and blue <0). Briefly, FIG. 42, Panels A and B, show the intensity and specificity of rabbit anti-Fla-32 and anti-Fla-69 serum pools (diluted 1/1 ,000), respectively, as assayed with HPV L2 type PepSet® Peptide Libraries. The PepSet® Peptide Library studies further showed that there are "hot spots" for B-cell epitope antigenicity and immunogenicity, notably: 1 ) homologous (HPV16) anti- Fla-32 reactivity was broad and intense, while cross- reactivity was directed against the RG1 epitope and much less intense in the area of peptide No. 18; 2) anti-Fla-69 pooled serum samples demonstrated a more pronounced dominancy for RG1 epitope than Fla- 32 and also increased immunoreactivity to the area of peptide No. 18; 3) there was notable cross-reactivity of the RG-1 epitopes for HPV1 1 , 45, 73, 52, and 58; and 4) the Fla-69 fusion product showed a broad and intense reactivity against HPV6, 16, 18, 31 , and 39.
In still other embodiments, serotype specific RG1 -End Point ELISA titers were evaluated (FIG. 43, Panels A and B) to compare RG1 -specific immune responses elicited by two exemplary fusion product vaccine candidates (e.g., Fla-32 and Fla-69). To that end, RG1 peptides corresponding to HPV6, 16, 18, 31 , and 45 as well as CRPV were synthesized (FIG. 43, Panel B; RG1 epitope alignment of synthetic serotype specific RG1 peptides used in ELISAs) and subsequently used as probes in determining titers in pooled serum samples from rabbits immunized with either Fla-32 or Fla-69. FIG. 43, Panel A, shows the Fla-69 fusion product was from 3 to 20 fold more immunoreactive with all tested RG1 sequences than was Fla-32. For example, Fla-69 induced anti- HPV45 and anti-HPV58 titers that were 20 times higher than those of Fla-32 for HPV45 and 58.
In other embodiments, PepSet® Peptide Libraries were used to discriminate Fla- 32 (L2-monomeric) from Fla-69 (L2- multimeric) antigens by L2-linear-epitope-specific immunogenicity. Consistent with results from exemplary passive immunization studies (See, FIG. 39), Fla-69 elicited broader immune responses than Fla-32. The elicited responses were mainly directed to the immunodominant RG-1 epitope.
Production of fusion product vaccine candidate Fla-69 was evaluated in small- scale production settings. The results of cultivation of Fla-69 at 4 L culture size in 5 L Bioflo-3000 fermentor devices (New Brunswick Scientific, Inc., Edison, NJ) (Example 4) in semi-defined complex media are shown in FIG. 44A, Panels A and B and FIG. 44B, Panels A and B, as follows: 1 ) FIG. 44A, Panel A, shows parameter set points and control methods for a low temperature (26 °C) cultivation at 20% dissolved oxygen held at pH 6.8 prior to induction with 1 mM IPTG; 2) FIG. 44A, Panel B, shows an exemplary cultivation profile showing a cell density target at 20 OD600nm prior to induction and a glucose concentration with a minimum of 20 g/L(left side) and cell density and specific replication rate (right side) (the shaded area on right panel indicates an optimal range of cell density and a specific growth rate versus glucose concentration) ; FIG. 44B, Panel A, shows pre- and post-induction samples treated with EasyLyse® Bacterial Protein
Extraction Solution (Epicentre Technologies, Inc, Madison, Wl) run on SDS/PAGE to evaluate soluble (S) and insoluble (P) fractions as compared to the insoluble fraction from a shake flask as positive control (e.g., microfluidization yields a 1 , 5, 1 0, 20, 30, 40, 50, 60, 70, 80, or 90%, or greater, soluble fraction of target fusion product protein) ; and FIG.
44B, Panel B, shows quantization by capture ELISA (ELISA wells coated with rabbit polyclonal serum raised against Fla-62 (AD3 deletion)) to capture post induction Fla-69 as detected by Mab RG1 directed against the putative neutralizing domain of HPV16 L2 (AA 1 7-36).
In one embodiment, exemplary production processes for fusion products of the present invention (e.g., Fla-69 fusion products) were shown to be scalable and
reproducible. In further embodiments, capture-ELISA methods were developed for Fla- L2 fusions that were successfully applied to monitoring exemplary production processes. In still further embodiments, fermentation conditions were developed that yielded from about 1 .0-0.1 g/L of target fusion product proteins, and preferably, about 0.6g/L of target fusion product proteins.
In additional embodiments, Fla-65 and Fla-76 fusion products, containing mainly RG1 peptides of various HPV types, were constructed and evaluated in exemplary animal model studies (Rabbit Study No. 5) (FIG. 45). I n various studies with Fla-65 and Fla-78 the Fla-32 and Fla-69 constructs and fusion products were used as controls. While the present inventions is not limited to any particular mechanism(s) or particular rational(s) for development, in one sense, the Fla-65 and Fla-78 fusion products were designed to examine the protectiveness of concatenated RG1 s fused to either 1 1 -200 AA (e.g., Fla-78) or 1 1 -88 AA (e.g., Fla-65) of HPV1 6 L2 used as the source of putative T-helper epitopes.
The present invention contemplates that in some embodiments, the addition of one or more T-helper epitopes (e.g., promiscuous T helper epitope(s)) to the fusion products increases the protective efficacy of RG1 -based vaccines in branched or unbranched configurations. In this regard, several putative T-helper epitopes have been identified at the N terminus of the L2 protein.
As shown in FIG. 46 all tested vaccine candidates (e.g., fusion products) demonstrated high protectiveness against all tested quazi-virus challenge types (QV6, 16, 18, 58, and 31 ) in active immunization studies. Neutralization titers of pooled pre-challenge serum samples against five HPV types showed mostly insignificant variations between multi-RG1 (Fla-65, and Fla-76) and L2-multimeric (e.g., Fla-69) constructs, except for HPV18 where Fla-69 showed a 6 fold higher immunogenicity than Fla-65 or Fla-76. (FIG. 47). In some embodiments, the neutralization potency of exemplary L2-Monomeric Fla-fusions (e.g., Fla-32) was shown to be inferior to that of L2-concatenated variants.
FIGs. 50-56, show an exemplary preferred embodiment the Fla-76 fusion product. Fla-
76 provided good protection against challenge with QV6, 16, 18, 31 , and 58. In still further embodiments, various multi-RG1 fusion products/constructs of the present invention provided similar protection profiles as compared to that of Fla-69 against all tested viral challenges. Accordingly, in some of these embodiments, it is contemplated that one long peptide (e.g., HPV16-L2 1 1 -88 or 1 1 -200 AA) is sufficient for effective presentation of 1 , 2, 3, 4, 5, or more, other concatenated protective epitopes (RG1 s). For example, in one preferred embodiment, HP16-L2 1 1 -200 AA peptide (Fla-76) demonstrated slight superiority over HP16-L2 1 1 -88 AA peptide (Fla-65). While the present invention is not intended to be limited to any particular mechanism(s) or sequence(s), it is thought that this result suggests the presence of T-helper epitopes in the AA 88-200 L2-region.
The present invention contemplates, broadly cross-neutralizing and/or protective (e.g., efficacious prophylactic vaccine compositions and/or therapeutic compositions against from about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, or more, types of HPV) L2 based fusion products comprising a number (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, or more, sequences) of short RG1 -epitopes in one formulation, or multiple separate formulations, administered to a subject.
3.2 Exemplary Mouse Studies
The art provides useful murine cervicovaginal challenge model(s) of papillomavirus virus (PV) transmission. (See, Roberts et al., Nat. Med., 13:857-861 (2007) ; and Johnson et al., J. Virol., 83:2067-2074 (2009)). Briefly, the murine model of Roberts et al., employs high titer PV pseudoviruses, which are authentic PV capsids composed of the L1 major and L2 minor structural proteins that have encapsidated a non-PV plasm id encoding a quantifiable reporter gene to monitor successful infection. (See also, Buck and Thompson, Curr. Protoc. Cell Biol., Chapter 26, Unit 26.1 (2007)).
In view of data generated from the various animal studies (e.g., various Rabbit Studies) described herein as well other sources, concerning the efficacy of particular fusion products against quazi-virions representing HPV16 and 18, additional animal studies in mice involving challenge with more distantly related HPV subtype 56 were conducted. (FIG. 58). Murine challenge studies with HPV56 were also undertaken on the basis of the reported prevalence of this HPV subtype in Asia. (See, Shahmahmoudi et al., Cancer Lett., 247:72-76 (2007); and Chen et al., Int. J. Cancer, 128(5):1 192-1203 (2010)). In certain embodiments, viral challenge of mice involved in certain studies was delayed for a period of time (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 24, or more, months) after the last immunization received in order to study the durability of immune responses. In certain preferred embodiments, viral challenge of the mice was delayed for a period of 4 months after the last immunization.
FIG. 59 shows that, in certain embodiments, the Fla-32 fusion product elicited a slightly higher immune response to HPV16, as detected in the 2nd week after final immunization than did Fla-69. While the present invention is not intended to be limited to any particular mechanism(s), it is thought that this result can be explained by the difference in length of the L2 epitope (AA 11 -200 for Fla-32 and AA 1 1 -88 for Fla-69) .
As shown in FIGs. 60-64 mice immunized with either commercial HPV vaccine were not protected against PsV56 challenge. In contrast, multimeric variants (e.g., Fla-69) provided significant levels of protection against PsV56 challenge. Quantification of these data is expressed in Relative Bioluminescence Unites in FIG. 66.
4. Synthetic Polypeptide Production
The fusion products described herein generally contain from about 1 , 2, 3, 4, 5,6, 7, 8, 9, 10, 100, 200, 300, 400, to about 500 or more amino acid residues. The fusion
products/polypeptides can be prepared using any of a number of chemical peptide synthesis techniques well known to those of ordinary skill in the art including both solution methods and solid phase methods. The following description briefly provides an overview of various synthetic polypeptide production methodologies applicable to certain embodiments of the present invention.
In particular, solid phase synthesis in which the C-terminal amino acid of the polypeptide sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred synthetic method for preparing the polypeptides. Techniques for solid phase synthesis are described by Merrifield et al., J. Am. Chem. Soc, 85:2149-2154 (1963). Many automated systems for performing solid phase peptide synthesis are commercially available.
Solid phase synthesis is started from the carboxy-terminal end (i.e., the C-terminus) of the polypeptide by coupling a protected amino acid via its carboxyl group to a suitable solid support. The solid support used is not a critical feature provided that it is capable of binding to the carboxyl group while remaining substantially inert to the reagents utilized in the peptide synthesis procedure. For example, a starting material can be prepared by attaching an amino- protected amino acid via a benzyl ester linkage to a chloromethylated resin or a hydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin. Materials suitable for use as solid supports are well known to those of skill in the art and include, but are not limited to, the following: halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenol resins, such as 4-(a-[2,4- dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl-hydrazidated resins; and the like. Such resins are commercially available and their methods of preparation are known to those of ordinary skill in the art.
The acid form of the peptides may be prepared by the solid phase peptide synthesis procedure using a benzyl ester resin as a solid support. The corresponding amides may be produced by using benzhydrylamine or methylbenzhydrylamine resin as the solid support. Those skilled in the art will recognize that when the BHA or MBHA resin is used treatment with anhydrous hydrofluoric acid to cleave the peptide from the solid support produces a peptide having a terminal amide group.
The a-amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive a-amino function. Certain amino acids also contain reactive side-chain functional groups (e.g., sulfhydryl, amino, carboxyl, hydroxyl, etc.) which must also be protected with appropriate protecting groups to prevent chemical reactions from occurring at those sites during the peptide synthesis. Protecting groups are well known to those of skill in the art. (See, for example, The Peptides: Analysis, Synthesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, N.Y. (1981 )).
A properly selected a-amino protecting group will render the .alpha. -amino function inert during the coupling reaction, will be readily removable after coupling under conditions that will not remove side chain protecting groups, will not alter the structure of the peptide fragment, and will prevent racemization upon activation immediately prior to coupling. Similarly, side chain protecting groups must be chosen to render the side chain functional group inert during the synthesis, must be stable under the conditions used to remove the a-amino protecting group, and must be removable after completion of the peptide synthesis under conditions that will not alter the structure of the peptide.
Coupling of the amino acids may be accomplished by a variety of techniques known to those of skill in the art. Typical approaches involve either the conversion of the amino acid to a derivative that will render the carboxyl group more susceptible to reaction with the free N- terminal amino group of the peptide fragment, or use of a suitable coupling agent such as, for example, Ν,Ν'-dicyclohexylcarbodimide (DCC) or Ν,Ν'-diisopropylcarbodiimide (DIPCDI).
Frequently, hydroxybenzotriazole (HOBt) is employed as a catalyst in these coupling reactions.
Generally, synthesis of the peptide is commenced by first coupling the C-terminal amino acid, which is protected at the N-amino position by a protecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solid support. Prior to coupling of an Fmoc-amino acid, the Fmoc residue has to be removed from the polymer. Fmoc-amino acid can, for example, be coupled to the 4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin using Ν,Ν'- dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) at about 25 °C for about two hours with stirring. Following the coupling of the Fmoc protected amino acid to the resin support, the a-amino protecting group is removed using 20% piperidine in DMF at room temperature.
After removal of the a-amino protecting group, the remaining Fmoc-protected amino acids are coupled stepwise in the desired order. Appropriately protected amino acids are commercially available from a number of suppliers. As an alternative to the stepwise addition of individual amino acids, appropriately protected peptide fragments consisting of more than one amino acid may also be coupled to the "growing" peptide. Selection of an appropriate coupling reagent, as explained above, is well known to those of skill in the art.
Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in excess and the coupling is carried out in a medium of dimethylformamide (DMF), methylene chloride (CH2CI2), or mixtures thereof. If coupling is incomplete, the coupling reaction may be repeated before deprotection of the N-amino group and addition of the next amino acid. Coupling efficiency may be monitored by a number of means well known to those of skill in the art. A preferred method of monitoring coupling efficiency is by the ninhydrin reaction. Peptide synthesis reactions may be performed automatically using a number of commercially available peptide synthesizers such as the Applied Biosystems ABI 433A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA).
The peptide can be cleaved and the protecting groups removed by stirring the insoluble carrier or solid support in anhydrous, liquid hydrogen fluoride (HF) in the presence of anisole and dimethylsulfide at about 0 °C. for about 20 to 90 minutes, preferably 60 minutes; by bubbling hydrogen bromide (HBr) continuously through a 1 mg/10 ml suspension of the resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting groups selected; or by incubating the solid support inside the reaction column used for the solid phase synthesis with 90% trifluoroacetic acid, 5% water and 5% triethylsilane for about 30 to 60 minutes. Other deprotection methods well known to those of skill in the art may also be used.
The peptides can be isolated and purified from the reaction mixture by means of peptide purification well known to those of skill in the art. For example, the peptides may be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution. 5. Recombinant Fusion Products, Polypeptides, and/or Proteins
The fusion products described herein generally contain from about 1 , 2, 3, 4, 5,6, 7, 8, 9, 10, 100, 200, 300, 400, to about 500 or more amino acid residues. The fusion
products/polypeptides can be prepared using any of a number of recombinant techniques well known to those of ordinary skill in the art. The following description briefly provides an overview of various recombinant polypeptide production methodologies applicable to certain
embodiments of the present invention.
It will be understood by those of ordinary skill in the art that the polypeptides can also be prepared by other means including, for example, recombinant techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., Molecular Cloning-A Laboratory, Manual (2nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (1989). Product information from manufacturers of biological reagents and experimental equipment, such as the SIGMA Chemical Company (Saint Louis, MO), also provide information useful in known biological methods.
The polypeptides described herein are derived from papillomavirus L2 protein. The nucleotide sequence of the nucleic acid that encodes L2 is known. Accordingly, the known nucleic acid sequence can be used to make the polypeptides recombinantly or a nucleic acid encoding the desired polypeptide can be derived from the amino acid sequence.
Generally, this involves creating a nucleic acid sequence that encodes the polypeptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the polypeptide in a host, isolating the expressed polypeptide and, if required, renaturing the polypeptide. Techniques sufficient to guide one of skill through such procedures are found in Sambrook et ai, supra.
Provided with the polypeptide sequences described herein, one of skill will recognize a variety of equivalent nucleic acids that encode the polypeptide. This is because the genetic code requires that each amino acid residue in a peptide is specified by at least one triplet of nucleotides in a nucleic acid which encodes the peptide. Due to the degeneracy of the genetic code, many amino acids are equivalently coded by more than one triplet of nucleotides. For instance, the triplets CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is to be encoded by a nucleic acid triplet, the nucleic acid has any of the triplets which encode arginine. One of skill is thoroughly familiar with the genetic code and its use. An introduction to the subject is found in, for example, Chapter 15 of Watson et al., Molecular Biology, of the Gene (Fourth Edition, The
Benjamin/Cummings Company, Inc., Menlo Park, CA (1987)), and the references cited therein. Although any nucleic acid triplet or codon which encodes an amino acid can be used to specify the position of the amino acid in a peptide, certain codons are preferred. It is desirable to select codons for elevated expression of an encoded peptide, for example, when the peptide is purified for use as an immunogenic reagent. Codons are selected by reference to species codon bias tables, which show which codons are most typically used by the organism in which the peptide is to be expressed. The codons used frequently by an organism are translated by the more abundant t-RNAs in the cells of the organism. Because the t-RNAs are abundant, translation of the nucleic acid into a peptide by the cellular translation machinery is facilitated. Codon bias tables are available for most organisms. For an introduction to codon bias tables, see, e.g., Watson et ai, supra.
In addition, it will be readily apparent to those of ordinary skill in the art that the fusion products/polypeptides described herein and the nucleic acid molecules encoding such immunogenic polypeptides can be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non conservative, where such changes might provide for certain advantages in their use, e.g., to increase biological activity.
One of skill will appreciate that many conservative variations of nucleic acid constructs yield a functionally identical construct. For example, due to the degeneracy of the genetic code, silent substitutions (i.e., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded peptide) are an acceptable feature of every nucleic acid sequence which encodes an amino acid. In addition, one of skill will recognize many ways of generating alterations in a given nucleic acid construct. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Sambrook et ai., supra.
Modifications to nucleic acids are evaluated by routine screening techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of encoded peptides can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a complementary nucleic acid, redox or thermal stability of encoded proteins, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
Similarly, conservative amino acid substitutions, in one or a few amino acids in an amino acid sequence of a protein are substituted with different amino acids with highly similar properties (see the definitions section, supra), are also readily identified as being highly similar to a disclosed construct. 6. Antibody Production
Antibodies that bind with specificity to the fusion products/polypeptides described herein are also contemplated. The antibodies include individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these polypeptides in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill. The antibodies are useful as research tools for the isolation of additional quantities of the antigenic polypeptides and for studying the pathogenesis of papillomavirus in general. The antibodies may also be useful therapeutically for passive immunization of an HPV-infected patient.
The antibodies include neutralization antibodies. Methods for screening antibodies for neutralization are known in the art. Several specific exemplary in vitro neutralization assays are described in Dvoretsky et al., Virology, 103:369-375 (1980); Roden et al., J. Virol., 70:5875- 5883 (1996); and Pastrana et al., Virology, 321 :205-216 (2004).
The following discussion is presented as a general overview of the techniques available for the production of antibodies; however, one of skill will recognize that many variations upon the following methods are known.
A number of immunogens are used to produce antibodies specifically reactive with polypeptides. In preferred embodiments, recombinant or synthetic polypeptides of at least 10 amino acids in length, or greater, selected from the polypeptides disclosed herein are the preferred polypeptide immunogens for the production of monoclonal or polyclonal antibodies. In one class of preferred embodiments, an immunogenic polypeptide conjugate is also included as an immunogen. The polypeptides are used either in pure, partially pure or impure form.
Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified peptide, a peptide coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the peptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the peptide is performed where desired.
Antibodies, including binding fragments and single chain recombinant versions thereof, against the polypeptides are raised by immunizing animals, e.g., using immunogenic conjugates comprising a polypeptide covalently attached (conjugated) to a carrier protein as described above. Typically, the immunogen of interest is a polypeptide of at least about 10 amino acids, in another embodiment the polypeptide is 20 amino acids in length, and in another embodiment, the fragment is about 30 amino acids in length and comprises amino acids acid residues 1 through 200 from the N-terminal or C-terminal of the papillomavirus L2 protein. The
immunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.
Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides, or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a KD of at least about 0.1 mM, more usually at least about 50 mM, and most preferably at least about 1 mM or better. Often, specific monoclonal antibodies bind with a KD of 0.1 mM or better.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in Kohler and Milstein, Nature, 256:495-497 (1975). Summarized briefly, this method proceeds by injecting an animal with an immunogen, e.g., an immunogenic peptide of the present invention either alone or optionally linked to a carrier protein. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The polypeptides and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies. Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. (See, for example, Huse et al., Science, 246:1275-1281 (1989); and Ward et al., Nature, 341 :544-546 (1989)).
Frequently, the polypeptide or antibody will be labeled by joining, either covalently or noncovalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
As mentioned above, the antibodies provided herein can be used in affinity
chromatography for isolating additional amounts of the polypeptides identified herein. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g. particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified polypeptides are released. In addition, the antibodies can be used to screen expression libraries for particular expression products, for example, papillomavirus proteins. Usually, the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
Moreover, antibodies raised against the immunogenic polypeptides described herein can also be used to raise anti-idiotypic antibodies. Such antibodies are useful for detecting or diagnosing various pathological or resistance conditions related to the presence of the respective antigens.
7. Immunoassays
In certain embodiments, both the fusion products/polypeptides described herein and the antibodies that bind with specificity to the fusion products/polypeptides are useful as reagents, as capture agents or labeling agents, in assays to detect a target peptide or antibody. In general, the target molecule can be quantified by a variety of immunoassay methods.
Moreover, the immunoassays can be performed in any of several configurations.
Immunoassays often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled peptide or a labeled anti-peptide antibody. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/peptide complex, or to a modified capture group (e.g., biotin) which is covalently linked to the peptide or anti- peptide antibody.
Alternatively, the labeling agent can be a streptavidin molecule which has a fluorescent dye on it and onto which are captured the peptides complexed with MHC (HLA) molecules. These reagents can be used to count single T cells specific for the peptides using commonly used equipment such as flow cytometers, thus providing precise quantitation and phenotype information on the immune response as described by Altman et al., Science, 274:94-96 (1996).
In a preferred embodiment, the labeling agent is an antibody that specifically binds to the capture agent. Such agents are well known to those of skill in the art, and most typically comprise labeled antibodies that specifically bind antibodies of the particular animal species from which the capture agent is derived, such as an anti-idiotypic antibody, or antibodies against a peptide when the peptide is the capture agent. Thus, for example, where the capture agent is a mouse derived anti-peptide antibody, the label agent may be a goat anti-mouse IgG, e.g., an antibody specific to the constant region of the mouse antibody.
Other proteins capable of specifically binding immunoglobulin constant regions, such as streptococcal protein A or protein G are also used as the labeling agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong nonimmunogenic reactivity with immunoglobulin constant regions from a variety of species.
Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about five seconds to several hours, preferably from about five minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays are carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 5 °C to 45 °C.
Non competitive assay formats such as "sandwich" assays, where the captured analyte
(e.g., anti-peptide antibody) is directly measured may be used. In competitive assays, the amount of analyte (e.g., immunogenic peptide or antibody to an immunogenic peptide) present in the sample is measured indirectly by measuring the amount of added (exogenous) analyte displaced (or competed away) from a capture agent (e.g., an antibody or peptide) by the analyte present in the sample. Other assay formats such as Western blot analysis may also be utilized. Depending on the assay, various components, including the immunogenic polypeptide or anti- peptide antibody may be bound to a solid surface ("solid phase" assay).
8. Formulations and Administrations
The present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus. The present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs. The present invention includes compositions and methods for preventing or ameliorating PV infections, especially, HPV infections. As such, the invention contemplates immunogenic compositions (e.g., vaccines) for use in active and passive immunization embodiments, methodologies, and administration regimes. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared from multimeric HPV polypeptide(s) comprising segments of HPV L2 protein and carrier/backbone molecule(s). In other embodiments, multimeric HPV L2 polypeptides can be used in combination with other HPV proteins or segments thereof, such as E1 , E2, E3, E4, E5, E6, E7, E8, and/or L1 protein. (See, for example: U.S. Pat. Nos. 7,425,438; 7,416,846; 7,416,732; 7,407,807; 7,374,767; 7,201 ,908; 7,189,513; and 7,288,258 each of which is incorporated herein by reference in its entirety).
Typically, vaccines are administered in a manner compatible with a vaccine formulation, and in such amount as will be therapeutically effective and/or immunogenic. The quantity to be administered depends on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Typically, 0.1 , 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, to 100 ng, μg, or mg may be administered per vaccination or administration.
In particular embodiments, the compositions of the present invention may be administered in a pharmaceutically effective amount. The term "immunologically effective amount", as used herein, refers to an amount sufficient for eliciting an immunological effect but not causing side effects or severe or excessive immune responses. The accurate dosage may vary depending on the antigen(s) to be administered and the desired effect to be obtained, and may be readily determined by those skilled in the art according to factors known in medicine and vaccinology, including the patients age, weight, health state, gender and sensitivity to any components of the intended administration(s), administration routes, and various administration methods. In some of the embodiments, the compositions are intended to be administered in a single dose or in several divided doses.
Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations. In some embodiments, from 1 , 2, 3, 4, 5, . . . 10, . . . 20, . . . 35, . . . 55, . . . 100, . . . 1 ,000, . . . 10,000, or more, units of time (e.g., minutes, hours, days, weeks, etc.) pass between the first administration of a composition and subsequent administration(s). In some of these embodiments, the interval(s) between any two or more administration points are constant (e.g., of equal duration). In still other embodiments, the interval(s) between any two or more administration points are varied (e.g., not of equal duration). Varied intervals can be either random or repeating and formulaic. Those skilled in the art will appreciate the steps necessary for designing and adjusting the dosing schedules and/or the dosing order of any one or more fusion products and any additional administered agents (e.g., additional HPV vaccines or other vaccines against sexually transmitted disease, or vaccines against cancer(s)) or therapies as mentioned herein.
Exemplary routes of administration to the human body can be through the eyes
(opthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, etc., by injection (e.g., intravenously, subcutaneously, intratumorally,
intraperitoneally, etc.) and the like. In specific embodiments, suitable routes of administration include, for example, oral or transmucosal administration as well as parenteral delivery (e.g., intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration). A brief review of methods for drug delivery is provided by Langer, Science, 249:1527-1533 (1990).
Generally, techniques for formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton, PA).
Exemplary pharmaceutical formulations and methods of producing pharmaceuticals are described in U.S. 2003021 1046A1 ; U.S. 20030004182A1 ; U.S. 2002060356384; U.S.
20020015728A1 ; U.S. 6,51 1 ,660; U.S. 6,406,745; U.S. 6,346,269; U.S. 6,039,977; U.S.
5,858,408; U.S. 5,631 ,023; U.S. 5,476,667; 5,044,091 ; U.S. 4,867,970; and WO 0028969A2 each of which is incorporated herein by reference in its entirety.
In certain embodiments, for solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients and generally 10% to 95% of active ingredient and more preferably at a concentration of 25% to 75% of active ingredient.
In certain embodiments, exemplary techniques and reagents for solidification/semi- solidification of the particular preparation(s) may be found in, for example, U.S. pat. Nos.
5,307,640; U.S. 5,897,852; U.S. 6,106,836; U.S. 6,458,363; U.S. 7,836,606; U.S.
20080060213; U.S. 12/397,140; U.S. 12/500,156; EP 0 689 867B1 ; EP 0 799 613B1 ; EP 1 140 152B1 ; EP 1 794 524B1 ; WO 2003/072016; WO 2004/073652; WO 2006/008006; FR 1054443; and FR 1056961 , each of which is incorporated herein by reference in its entirety.
In still other embodiments, for aerosol administration, the fusion products/polypeptides are preferably supplied in finely divided form along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, such as the inclusion of lecithin for intranasal delivery.
Certain injectable preparations may be prepared using physiological saline, aqueous solutions such as Ringers solution, and non-aqueous solutions, such as vegetable oils, high fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzylalcohol, propylene glycol and glycerin, etc.). The injectable preparation(s) may be supplemented with
pharmaceutical carriers, which are exemplified by a stabilizer for preventing degeneration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), or reagents, and techniques, for facilitating solidification/semi-solidification of the preparation(s) (e.g., foam drying, freeze-foam drying, spray drying (atomization), spray-freeze-drying, evaporative drying, percolative drying, vacuum drying, lyophilization, micropelleting, prilling, and variations thereof, etc.), an emulsifier(s), an excipient(s), a buffering agent for pH adjustment, and a preservative for inhibiting contamination, including but not limited to, microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzylalcohol, etc.), and other appropriate reagents Generally Regarded as Safe (i.e., GRAS reagents).
Certain compositions and methods of present invention intended for treating a pathological condition additionally can be practiced in conjunction with other therapies. For example, for treating cancer, the methods of the invention can be practiced prior to, during, or subsequent to conventional cancer treatments such as surgery, chemotherapy, including administration of small molecule drugs (e.g., oncolytics, anti-angiogenics, etc.) cytokines and growth factors, radiation, surgical interventions, or other methods known in the art.
Similarly, for treating pathological conditions which include infectious disease, the methods of the invention can be practiced prior to, during, or subsequent to conventional treatments, such as antibiotic and/or antiviral administration against infectious agents or other methods known in the art. Treatment of pathological conditions of autoimmune disorders also can be accomplished by combining the methods of the invention for inducing an immune response with conventional treatments for the particular autoimmune diseases. Conventional treatments include, for example, chemotherapy, steroid therapy, insulin and other growth factor and cytokine therapy, passive immunity and inhibitors of T-cell receptor binding. The methods of the invention can be administered in conjunction with these or other methods known in the art and at various times prior, during, or subsequent to initiation of conventional treatments. For a description of treatments for pathological conditions characterized by aberrant cell growth See, for example, The Merck Manual, Sixteenth Ed, (Berkow, R., Editor) Rahway, NJ, (1992). Depending on the condition being treated, preferred embodiments of the present invention are formulated and administered systemically or locally.
As described above, administration of a compound, immunomodulatory Flagellin fusion products, Flagellin fusion polypeptide, or modification thereof can be, for example, simultaneous with or delivered in alternative administrations with the conventional therapy, including multiple administrations. Simultaneous administration can be, for example, together in the same formulation or in different formulations delivered at about the same time, or immediately in sequence, or following some period of rest. Alternating administrations can be, for example, delivering a Flagellin fusion peptide or polypeptide formulation and a conventional therapeutic treatment in temporally separate administrations. As described previously, the temporally separate administrations of a compound, immunomodulatory Flagellin peptide, polypeptide or modification thereof, and conventional therapy can similarly use different modes of delivery and routes. EXAMPLES
Example 1 Constructing Fla-L2 Expression Plasmids
Cloning procedures were performed via standard molecular biology techniques such as plasmid and chromosomal DNA isolation, restriction digestion, DNA ligation, and competent cell transformation as described in (Sambrook et ai, Molecular Cloning-A Laboratory, Manual (2nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (1989)). The
Flagellin B (GenBank Gl:16445344, SEQ ID. NO. 210) sequence was amplified using standard PCR techniques from a genomic DNA template isolated directly from Salmonella typhimurium (ATCC # 700720). Codon-optimized HPV L2 sequences were synthesized either by Retrogen, Inc., (San Diego, CA) or Blue Heron Biotechnology, Inc. (Bothell, WA). Derivatives of the Flagellin gene or synthesized L2 sequence were generated by PCR. For expression of Fla-L2 inserts the pET24a+ vector supplied by Novagen, Inc., (Madison, Wl) was used. Clones were grown under kanamycin (Kan) selection and tested via PCR for presence of the inserts. Fla-L2 inserts of selected clones were confirmed by sequencing on a Beckman Coulter CEQ 8800 Genetic Analysis System according to established protocols. A list of all constructs produced is provided at Table 1 supra.
Example 2 Mini-Expression and Primary Solubility-Test for Fla-L2 Fusions
Selected clones were grown in 5 ml Luria-Bretani Medium (LB) medium
(http://www.bcm.edu/physio/lab_pages/pedersen/protocols.html) with Kanamycin (Invitrogen, Inc., Carlsbad, CA) at a concentration of 50 microgram ^g) per milliliter (ml) in 50 ml Falcon tubes in an Innova shaking incubator (INNOVA 44R, New Brunswick Scientific, New Jersey, USA) at 200 revolutions per minute (rpm) overnight at 34 <€. The next day, samples were diluted in fresh LB medium (1 :10) and grew to OD600nm= 0.6-0.8 before inducing with IPTG for 3 hours at 37 °C or 5.5 hours at 28 °C. After incubation samples were harvested and bacterial pellets were frozen at -20 °C. To evaluate solubility of target proteins the pellets were lysed with EasyLyse® Bacterial Protein Extraction Solution (Epicentre Technologies, Inc., Madison, Wl) per kit directions. Briefly, cell lysates were centrifuged to separate soluble and insoluble fractions of cell debris (at 14,000 rmp for 2 min in a bench top Eppendorf centrifuge, Model 5424). SDS reducing sample buffer and TrisGlycing or Bis-Tris SDS gel (Invitrogen, Inc.) were used for protein gel electrophoreses. A number of Fla-L2 fusion constructs predicted to be totally insoluble using this procedure were later found to be soluble when macrofluidization procedures were applied.
Example 3 Shake Flask Cultures
Flagellin fusion product constructs were grown in 1 Liter (L) cultures in 4 L baffled shaker flasks for expression and characterization. Briefly, -80 °C glycerol stocks were stabbed and seeded into 125 ml baffled flasks containing 50 ml of Animal Free Luria Broth (AFLB; Teknova, Inc., Hollister, CA) supplemented with Kan at a final concentration of 30 μg/ml. Seed cultures were grown to stationary phase overnight at 200 rpm in an Innova shaking incubator (see above) at 30 °C. Production cultures consisting of 1 L AFLB/Kan(30μg/ml) in 4L baffled flasks were inoculated with the overnight seed culture to a starting cell density of OD600 nm = 0.1 The production cultivation conditions were set to 250 rpm at 37 °C with hourly sampling. Upon achieving a cell density of OD600nm = 0.8, a pre-induction sample was taken and the culture was induced with 1 millimolar (mM) of Isopropyl β-D-l thiogalactopyranoside (IPTG). The induction temperature was reduced to 28 °C at the time of induction; agitation remained set at 250 rpm. Samples were taken at 1 hour intervals for 4 to 6 hours post-induction, at which time the cultures were harvested by centrifugation in pre-weighed 450 mL bottles at 8,000 rpm for 10 minutes at 4 °C in an RC4 Sorvall centrifuge (Thermo Fisher Scientific, Inc., Asheville, NC) and Ultra Light 3,000 rotor at 7,000 rpm. The supernatant was discarded and cell pellets were stored at -80 °C for purification. Biomass yields (g/L) wet cell weights (wcw) were calculated based on gross weight (bottle + wet cell pellet) minus bottle tare weight. The post induction samples were analyzed for target protein expression via SDS/PAGE gel
electrophoresis and Western Blot with penta-Histag antibody (Qiagen Sciences, Inc.,
Germantown, MD, Catalog No. 34660) or monoclonal antibody directed against the RG-1 domain of HPV16 L2 to verify target antigen according to known procedures (RG-1 antibody reference: Gambhira, R., J. Virology, 81 (24):13927-13931 (2007)); method reference: Proc. Natl. Acad. Sci. USA., 105(15):5850-5855 (2008)). Example 4 Fermentation of Fla-69
Fermentation of Fla-69 was performed at 4 L culture size in a 5 L Bioflo-3000 device (New Brunswick Scientific, Inc., Edison, NJ) Briefly, -80 °C glycerol bacterial E. coli BL21 (DE3)/ Fla-69 stocks were stabbed and seeded into 150 ml baffled flasks containing 50 ml of AFLB supplemented with Kanamycin at a final concentration of 30 μg/ml. Seed cultures were grown to stationary phase overnight at 200 rpm in an Annova 44 shaking incubator (see above) at 30 °C; the overnight seed cultures were inoculated into fermentation vessels in accordance with New Brunswick Fermentation preparation manual (See, Jorgensen et ai, Microbiology, 153:1963-1973 (2007)), containing 4 L of AFLB supplemented with 30 μg/mL of Kan, 5 g/L
Potassium Phosphate (Sigma-Aldrich, Inc., St. Louis, MO), 10 mM Magnesium Sulfate (Sigma- Aldrich), Trace Metal Solution and with 40 g/L of glucose (Sigma-Aldrich), at a starting cell density of OD600nm = 0.1 (Diaz-Ricci et ai, Biotechnol. Prog., 6:326-332 (1990); Konstantinov et ai, Biotechnol. Bioeng., 36:750-758 (1990); Riesenberg et ai, J. Biotechnol., 20:17-27 (1991 ); Korz et ai, J. Biotechnol., 39:59-65 (1995) ; Horn et ai, Appl. Microbiol. Biotechnol., 46:524-532 (1996); Shiloach et ai, Biotechnol. Bioeng., 49:421 -428 (1996); Lee, Trends Biotechnol., 14:98-105 (1996); Shiloach and Fass, Biotechnol. Adv., 23:345-357 (2005); and Jorgensen et ai, Microbiology, 153:1963-1973 (2007)) and the cultivation and expression parameters and set points were controlled by Proportional Integral Derivative (PID) algorithms (BioFlo 300 instruction, BioProcessing Software; Benchtop Fermentor, New Brunswick
Scientific, Inc.). Briefly, in this regard, the cultivation temperature was set at 26 °C (a cooling ice bath water loop was required to maintain temperature below 30 °C) (Horn et ai, Appl.
Microbiol. Biotechnol., 46:524-532 (1996)), pH at 6.8 (pH was controlled dropwise using ammonium hydroxide (NH4OH)), aeration was set at 1 volume per volume per minute (vvm) using house air (no oxygen supplementation), and dissolved oxygen (%D02) at 20% of saturation (Korz et ai, J. Biotechnol., 39:59-65 (1995); Horn et ai, Appl. Microbiol. Biotechnol., 46:524-532 (1996)) controlled by the agitation cascade set from 300 rpm to 1 ,000 rpm ; and the fermentor was monitored hourly during the cultivation period for cell density (OD 600nm) as well as glucose depletion.
Upon consumption of glucose below a concentration of 20 g/L, a bolus of 50% glucose stock solution (Horn et ai, Appl. Microbiol. Biotechnol., 46:524-532 (1996); Shiloach et ai, Biotechnol. Bioeng., 49:421 -428 (1996); Lee, Trends Biotechnol., 14:98-105 (1996)) was added to the fermentor to re-establish an in situ concentration of 40 g/L. Upon achieving a cell density of OD 600nm = 20, a pre-induction sample was taken and the culture was induced with 1 mM Isopropyl β-D-l thiogalactopyranoside (IPTG) (Jorgensen et ai, Microbiology, 153:1963-1973 (2007); Lee, Trends Biotechnol., 14:98-105 (1996)). Hourly post induction samples were taken for SDS/PAGE, Western Blot, and Capture Elisa for quantification and characterization of the product. The production phase was allowed to carry on for 4 hours at which time the production culture was harvested by centrifugation in pre-weighed 450 ml bottles at 8,000 rpm for 10 minutes at 4 °C in RC4 Sorvall centrifuge and Ultralight 3,000 rotor. Supernatant was discarded and cell pellets were stored at -80 °C for purification. Biomass yields (g/L wet cell weights (wcw) were calculated based on gross weight (bottle + wet cell pellet) minus bottle tare weight. The post induction samples were analyzed for target protein expression via SDS/PAGE gel electrophoresis and Western Blot with anti-penta-hisitidine antibody or monoclonal antibody directed against the RG-1 domain of HPV16 L2 to verify target antigen (according to the methods of (1 ) Burnette W.N., (Analytical Biochemistry, 1 12(2) : 195-203 ((1981 )); and (2) Milan Bier (ed.) (Electrophoresis. Theory, Methods and Applications, 3rd printing ed., Academic Press. (1959)).
Example 5 Purification of Fla-02, Fla-07, Fla-31 , Fla-32 as well as Fla-01 and Fla-62
Controls
Cells were pelletized by centrifugation and the cell paste was stored at -80s C. Prior to purification, the cell paste was thawed and diluted to 10% solids in 50 mM Tris-HCI, 150 mM NaCI, pH 8.0 with addition of Complete EDTA free protein inhibitors cocktail tablets from Roche, Inc., (Basel, Switzerland) according to included instructions. The cell suspension was passed twice through a macrofluidizer at an average running pressure of 18,000 psi then cooled to 4-8 2C. The resultant lysate was clarified from cell debris and the insoluble material was removed by centrifugation at 29,000 X g for 40-60 min at 4 to 8 2C. The first step in the separation of the soluble contaminates from the target protein was achieved by precipitation of the target protein with a 30% of saturation of ammonium sulfate obtained by adding a 3 M ammonium sulfate solution drop wise into the clarified cell lysate on a stir plate with continued stirring for 90 - 120 min at 4 2C. Precipitated target protein was collected by centrifugation at 29,000 X g for 40-60 min at 4 to 8 2C. The resulting pellet was resuspended in the same volume of 50 mM TrisHCI, pH 8.0 with the addition of Complete EDTA free protein inhibitors cocktail tablets from Roche, Inc. The collected pellets could be also stored at -80 2C and processed later. Upon complete resuspension, target material was clarified from insoluble materials by centrifugation at 29,000 X g for 40 min at 4 to 8 2C. The soluble fraction was applied to 5 ml HiTrap Q HP pre-packed columns (attached together for total column volume of 10 ml) and pre-equilibrated with 50 mM Tris-HCI, pH 8.0 (anion exchange chromatography starting buffer). Differences in utilized column volume sizes depend on the required amount of purified material. Larger scale up activities utilized HiTrap FF media in XK 50/30 (GE Healthcare Life Sciences, Piscataway, NJ) with column volumes of 150 -180 ml. Bound protein was eluted with a linear salt gradient of NaCL from 0 to 1 .0 M in 50 mM Tris-HCI, pH 8.0. For final polishing, peak elution fractions were pooled, 3 M ammonium sulfate solution was added into pool up to 1 M followed by filtration through 0.45 μιη filter and loaded onto HiTrap Phenyl HP 5 ml pre-packed column pre- equilibrated with 50 mM sodium phosphate, 1 M ammonium sulfate, pH 8.0 binding buffer. Bound protein was eluted with a liner gradient from of Ammonium Sulfate 1 .0 M to 0 M in 50 mM sodium phosphate, pH 8.0. Peak fractions free from contaminates were pooled, dialyzed against 1 x PBS, 5 mM EDTA, pH 8.0, formulation buffer followed by sterile filtration (0.2 μιη) and stored at -80 2C. Example 6 Modified Purification Procedures for Fla-69
This example describes an alteration to the purification scheme for Fla-69 HPV L2 flagellin fusions described in Example 5. Specifically, the anion exchange chromatography (AEC) and hydrophobic interaction chromatography (HIC) steps from Example 5 were changed as follows. Modification one, post-ammonium sulfate precipitation pellets containing target protein were resuspended in 50 mM Tris-HCI, 8 M urea, pH 8.6 buffer and clarified from insoluble materials by centrifugation at 29,000 X g for 40 min at 4 to 8 2C. The soluble fraction was applied to 5 ml HiTrap Q HP pre-packed column or to 180 ml of HiTrap FF resin packed in XK 50 /30 column (GE Healthcare Life Sciences) pre-equilibrated with the same buffer containing 8 M urea at pH 8.6. Bound protein was eluted in the same fashion at new conditions. Modification two, similar modifications have been applied to HIC polishing step - addition of 8 M urea in HiTrap Phenyl HP resin binding (50 mM Tris-HCI, 1 M Ammonium Sulfate) and elution (50 mM Tris-HCI) buffers at pH 8.6. Modification three, the peak elution fractions containing denatured target protein underwent a refolding procedure by extensive dialysis against 50 mM Tris-HCI, 150 mM NaCI, 5 mM EDTA, pH 8.0 formulation buffer followed by sterile filtration (0.2 μιη) and stored at -80 2C.
Example 7 Purification Procedure for Fla-65
Cell paste was thawed and diluted to 10% solids in 50 mM Tris-HCI, 8 M urea, pH 8.0 with addition of Complete EDTA free protein inhibitors cocktail tablets from Roche, Inc.
(according to instructions). To disrupt cells, the cell suspension was passed twice through a macrofluidizer at an average running pressure 18,000 psi, cooled to 4-8 2C. The resultant lysate was clarified from cell debris and insoluble material by centrifugation at 29,000 X g for 15-30 min at 4 to 8 2C. The soluble fraction was applied to a 5 ml HiTrap Q HP pre-packed column pre-equilibrated with 50 mM Tris-HCI, 8 M urea, pH 8.0 (anion exchange chromatography starting buffer). The flow-through and first linear gradient fractions from 0 to 1 .0 M NaCI in 50 mM Tris-HCI, 8 M urea, pH 8.0 contained target protein separated from the majority of bound contaminates. These fractions were then pooled together. The pH of collected material was carefully adjusted to 8.6-8.8 pH by drop wise addition of 5 M NaOH. Fractions with adjusted pH were loaded onto HiTrap Q HP pre-packed columns pre-equilibrated with 50 mM Tris-HCI, 8 M urea, pH 8.6-8.8 buffer. Bound target protein was eluted with a linear salt gradient from 0 to 1 .0 M NaCI in 50 mM Tris-HCI, 8 M urea, pH 8.0. For final polishing, peak elution fractions were pooled and a 3 M ammonium sulfate solution was added into the pool up to 1 M followed by filtration through 0.45 μιη filter and loaded onto HiTrap Phenyl HP 5 ml pre-packed column pre- equilibrated with 50 mM sodium pPhosphate, 1 M ammonium sulfate, 8 M urea, pH 8.0 binding buffer. Flowthrough fractions of target protein separated from bound contaminates were pooled. The target protein underwent a refolding procedure by extensive dialysis against 50 mM Tris-HCI, 150 mM NaCI, 5 mM EDTA, pH 8.0 formulation buffer followed by sterile filtration (0.2 μιη) and stored at -80 2C.
Example 8 Purification Procedures for Fla-76
Cell paste was thawed and diluted to 10% solids in 50 mM Tris-HCI, 8 M urea, pH 7.5 with addition of Complete EDTA free protein inhibitors cocktail tablets from Roche, Inc.
(according to instructions). To disrupt cells, the cell suspension was passed twice through a macrofluidizer at an average running pressure of 18,000 psi, cooled to 4-8 2C. The resultant lysate was clarified from cell debris and insoluble material by centrifugation at 29,000 X g for 15- 30 min at 4 to 8 2C. The soluble fraction was applied to two 5 ml HiTrap Q HP pre-packed columns (total CV 10 ml) attached together that were pre-equilibrated with 50 mM Tris-HCI, 8 M urea, pH 7.5 (anion exchange chromatography starting buffer). Bound protein was eluted at the beginning of a linear salt gradient from 0 to 1.0 M NaCI in 50 mM Tris-HCI, 8 M urea, pH 7.5. The pool of peak elution fractions of target protein was extensively dialyzed from urea against 50 mM Tris-HCI, pH 8.0 buffer using Slide-A-Lyzer dialysis cassette from Pierce (Rockford, IL) with a 20K MWCO membrane. Upon completion of dialysis, samples were clarified from insoluble contaminates by centrifugation at 29,000 X g for 15-30 min at 4 to 8 2C and the soluble fraction was applied to two 5 ml HiTrap Q HP. Example 9 Bioactivity of Fla-L2 Fusions (hTLR5 BioAssay)
The flagella binding assay utilizes a stable cell line of primary human embryonal kidney cells (HEK293) expressing the transforming gene of adenovirus 5. Assays were performed at InvivoGen Inc., (San Diego, CA). The principle of the bioassay is based on the ability of HEK293 cells stably transfected with plasmid (HEK293pNiFty-SEAP) expressing human Toll- like receptor 5 (hTLR-5) to be induced by binding TLR-5 agonist (flagellin). Later binding initiates a signaling cascade leading to the translocation of transcription factor NF-κΒ which in turn induces expression of reporter gene, encoding a soluble secretory embryonic alkaline phosphotase (SEAP) controlled by the NF-KB-dependent promoter. The SEAP gene has been provided in trans from a second plasmid. SEAP catalysed hydrolysis of pNitrphenyl phosphate produces a detectable colorometric change (in the range of 405nm to 650nm) in the culture media or to various added reagents.
Purified Fla-L2 samples were prepared in equimolar concentration of the flagellin portion forwarded in a blinded manner to InvivoGen, Inc. Briefly, the detection procedure has been performed as follows: 1 ) 96-well plates (200μΙ_ total volume) containing 25,000-50,000 cells/well of H E K293/pN i Fty- SEAP and a) 20μΙ_ of protein Fla-L2 sample; or b) 20 μΙ_ of positive control ligands were prepared; 2) each Fla-fusion well (or control well) was assayed in the
concentration range of 100 ng/ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml. Each sample was titrated two fold in triplicate and added to the wells; 3) after a 16-20 hr incubation the supernatant was removed and the plate was centrifuged to pellet cell debris; 4) 10 μΙ of the clarified culture supernatant was transferred to a clean 96 well tray and heated at 65 °C for 5-10 minutes to inhibit endogenous alkaline phosphatase activity; 5) 50 μΙ of 1 X Dilition Buffer (InvioGen, Inc), 100 μΙ of 1 X Assay Buffer, 20 μΙ of 100mM L-Homoarginine, and 20 μΙ of water was added to each well; 6) the plates were incubated at 37 °C for 10 minutes and 20 μΙ of Staining Solution (InvioGen, Inc.) was added; and 7) the plates were incubated for an additional 10 minutes at 37 °C in the dark and then the OD was read at 405nm to 650nm.
Example 10 RG-1 End Point Elisa
The broadly cross-neutralizing epitope of Human Papillomavirus 16 L2 has previously been described (Gambhira et al., Virology, 81 :13927-13931 (2007); Kando et al., Virology, 358:266-272 (2007); and Rubio et al., Vaccine, 27:1949-1956 (2009)) and is referred to as either the RG-1 neutralizing domain or the RG-1 epitope. This same RG-1 epitope may also be broadly cross-protective in the quazi-virus challenge of HPV types 6, 16, 18, 31 , 45 and 58 as well as the rabbit type Cottontail Rabbit Papillomavirus (CRPV) in the New Zealand White (NZW) rabbit quazi-virus dermal challenge model. The model epitope resides within amino acids 17-36 for HPV 16 L2: QLYKTCKQAGTCPPDIIPKV (SEQ ID NO. 205, Accession # ACS92698).
Sequence alignments for HPV type L2s were performed to identify the heterologous amino acid range for these other HPV type's "RG-1 epitope" belonging to the oncogenic group HPV16 (HPV types 33, 35, 58, 31 , 52), group HPV18 (HPV types 18, 45, 39, 59, 68), group HPV18 (HPV types 26, 51 , 82, 69), group HPV53 (HPV types 53, 56, 66, 30) and others HPV types of 5, 6 and 1 1 belonging to condonalyma (anal cancers). Synthetic peptides of the "RG- 1 " putative protective domain (all above) were made by Bio-Synthesis, Inc., (Lewisville, TX) at a 5 milligram scale with a minimum purity of 80%.
Briefly, the RG-1 End Point Elisa procedure contained the following steps: 1 ) the synthetic peptides were solubilized at a concentration of 2 mg/ml in dimethyl sulfoxide (DMSO) (Sigma) and coated onto Corning Costar (#3369, high binding) (Corning Life Sciences, Kennebunk, ME) 96 well ELISA plates in carbonate bi-carbonate buffer (Sigma) at a concentration of 0.5 μg/well/100 μΙ and incubated at 4 °C overnight on a level surface; 2) the diluent and blocking solution (Block) used was 1 xPBS+ 0.05% TWEEN 20 (Sigma) with 1 % bovine serum albumin (BSA) (Sigma) and 0.05% Sodium Azide (NaN3, Sigma) in all antibody solutions; 3) plates were removed from 4 °C and allowed to warm to room temperature for 30 minutes followed by washing with 1x PBS + 0.05% TWEEN 20; 4) 200 μΙ of Block was added to each well and plates were incubated at 37 °C for 60 minutes; 5) during this incubation step, the serum from individual test rabbits was diluted in Block starting at a dilution of 1/50 and titrated three fold to 1/109K in duplicate in 2 ml x 96 well deep well dilution trays; 6) positive control Rabbit anti-HPV16L2 AA1 1 -200 rabbit hyper-immune serum was diluted 3 fold in duplicate starting at 1/1 ,000 and ending at 1/27,000; 7) negative control rabbit serum (placebo) was diluted 10 fold in duplicate starting at 1/100 and ending at 1/100,000; 8) after 60 minutes of incubation in Block, plates were washed and 100 μΙ of primary test rabbit antiserum dilutions and controls serums were added to the wells of the each 96 well plate (Note: Step 8 was repeated for each HPV type synthetic peptide coated on individual plates); 9) plates were returned to 37 °C C for 60 minutes and than washed 3 times; 10) 100 μΙ of the detecting antibody, goat anti-rabbit IgG-AP, (Southern Biotech, Birmingham, Alabama) diluted to 1/1 ,000 in Block per well were added and plates incubated at 37 °C for 60 minutes; 1 1 ) alkaline phosphate substrate buffer (0.1 M glycine buffer pH 10.4, 1 mM MgCI2, 1 mM ZnCI2 (Sigma) was used to prepare 1 mg/ml solution of 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma); 12) plates were washed 4 times and tapped dry; 13) 100 μΙ of pNPP solution was added per well; and 14) plates were incubated at room temperature for 30 minutes followed by reading at OD 405nm. Example 11 Mimotopes PepSet® Non-Cleavable Peptide Library ELISA
This example describes an assay technique to characterize the antigenicity and strength of the immune response to various HPV L2 truncations and multimer vaccine candidates disclosed herein. A more detailed analysis of host immune responses was initiated through B- cell epitope mapping with the aide of Non-Cleavable PepSet® Peptide Libraries. Non-Cleavable PepSet® Peptide Libraries (Mimotopes, Inc., St. Paul, MN) consisted of L2 amino acids (AA) 2 to 200, are15 residues in length with an offset of 4 residues between neighboring peptides. Each PepSet® Peptide Library represented one of the following HPV types: 6, 1 1 , 16, 18, 31 , 39, 45, 51 , 52, 58, 59 and 73 and two rabbit species specific types, Rabbit Oral Papillomavirus ("ROPV") and Cottontail Rabbit Papillomavirus ("CRPV"). The approximate number of peptides produced per each HPV PepSet® Peptide Libraries is 47, allowing for two HPV PepSet® Peptide Libraries on each Block (96-well plate), i.e., 16/6, 18/1 1 , etc. This allowed for multi-library screens with a single serum sample. The Multipin® peptide technology (Mimotopes, Inc.) involved synthesis of non-cleavable peptides which remained covalently bound at the C- terminus to the modular resin on SynPhase Gears (Mimotopes, Inc.) attached by removable stems to holders ("Blocks") that were compatible with a standard 96-well ELISA plate footprint. The "Blocks" sat on a reservoir or on top of a 96-well plate for processing.
An adapted ELISA procedure was used to qualify the strength of the immune response to various HPV L2 truncations and multimeric vaccine candidates, briefly: 1 ) the PepSet® Peptide Library Gears were washed in a bulk volume (tray) of 1 xPBS+ 0.05% TWEEN 20 (Sigma) (hereinafter referred to as wash buffer) overnight at 4 °C on a rotating platform set at 60 rpm ; 2) the diluent and blocking solution (blocking buffer) used was 1 xPBS+ 0.05% TWEEN 20 (Sigma) with 1 % bovine serum albumin (BSA) (Sigma) and 0.05% Sodium Azide (NaN3, Sigma) for all antibody solutions; 3) the following morning the gears were placed in 200 μΙ of blocking buffer to reduce non-specific binding and incubated at room temperature on a rotating platform set at 60 rpm for 1 hour; 4) test (primary antibodies) anti-sera were diluted to 1/1 ,000 in 80 ml of blocking buffer per PepSet® Peptide Library "Block" during this blocking step; 5)
PepSet® Peptide Libraries were removed from the blocking buffer and washed 3 times in 200 ml of washing buffer, followed by incubation in the diluted test serum sample for 1 hour at 60 rpm at room temperature; 6) PepSet® Peptide Libraries were washed 3 times and then placed in Goat a-Rabbit IgG-AP (labeled secondary antibody, Southern Biotech, Inc.) in blocking solution at a dilution of 1 /1 ,000; 7) PepSet® Peptide Libraries were washed 4 times in 200 ml of wash buffer; 8) alkaline phosphate substrate buffer (0.1 M glycine buffer pH 10.4, 1 mM MgCI2, 1 mM ZnCI2, (Sigma)) was used to prepare a 1 mg/ml solution of 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) (Sigma) ; 9) 100 μΙ pNPP solution was added to a 96 well high-binding flat bottomed ELISA plate; 10) PepSet® Peptide Library pins were placed into the wells for 20 minutes in the dark at room temperature; and 1 1 ) OD405nm signal from 96 well plates was read within 10 minutes after removal of the PepSet® Peptide Library pins from the 96 well plates.
Example 12 Quazi-virion Production for Rabbit Challenge Studies
Quazi-virions were provided by Dr. N. Christensen (Pennsylvania State University). Briefly, 293TT producer cells were transfected with circular CRPV genomes together with plasmids expressing codon-modified L1 and L2. Two after transfection the cells were lysed with Brij-58, incubated to allow particle maturation, and treated with Benzonase (Sigma) and Plasmid Safe (Epicenter Biotechnologies, Madison, Wl) to destroy unprotected DNA. Cell lysates were centrifuged on an Optiprep density gradient (Accurate Chemical & Scientific Corp., Westbury, NY). 300 μΙ fractions were collected dropwise from the bottom of the column and analyzed for the presence of capsid proteins by immunoblotting and/or nondenaturing enzyme-linked immunosorbent assay. Fractions with appropriate densities and positive data for capsid proteins were assayed for their infectivity in RK13 cells by quantitative reverse transcription- PCR (QRT-PCR) measuring viral E1 -E4 transcripts. (Culp and Christensen, Virology, 319:152- 161 (2004)). Those fractions capable of generating viral transcripts in inoculated cells were retested for their ability to be neutralized by monoclonal antibodies (MAbs) (CRPV.1 A or
H16.V5). Fractions (and particles) termed "infectious" both induced the production of E1 or E4 transcripts in the in vitro infection assay and lost this ability in the presence of a specific MAb (Christensen and Kreider, Virus Res., 21 :169-179 (1991 ); Culp and Christensen, Virology, 319:152-161 (2004); and Culp et al., J. Virol., 80:1 1381 -1 1384 (2006)).
Example 13 Pseudovirus Production for Neutralization Studies
Maps of plasmids used for generation of high-titer pseudoviruses are available at the website http://www.ccr.cancer.qov/Staff/links.asp?profileid=5637. Generation of pseudoviruses using the codon-modified L1 and L2 genes of BPV1 (plasmid pSheLL) (Buck et al., J. Virol. , 78:751 -757 (2004); Buck et al., Methods in Mol. Med., 445-461 (Monograph) (2004)), HPV16 (plasmids p16L1 h and p16L2h) (Leder et al., J. Virol., 75:9201 -9209 (2001 )), and HPV18 (plasmids peLl fB and peL2bhb) (Pastrana et al., Virology, 321 :205-216 (2004)) have been described previously. HPV6 and CRPV pseudoviruses were produced using expression plasmids carrying L1 and L2 genes that were entirely codon-modified using a previously described strategy (Buck et ai, J. Virol., 78:751 -757 (2004)). Pseudovirions were produced as previously described (Buck et al., (2004); and Pastrana et al., (2004)) with minor modifications. Briefly, plasmids encoding L1 and L2 genes were cotransfected into 293TT cells along with a reporter plasmid encoding secreted alkaline phosphatase (pYSEAP). After 48 h, cells were lysed with 0.2% Brij-58, 9.5 mM MgCI2, 0.1-0.2% Benzonase (Sigma) and 0.1 % plasmid safe (Epicentre Biotechnologies) and incubated at 37 °C for 15 min. The resulting pseudovirions were then matured by overnight incubation of the lysates at 25 °C (BPV1 , HPV16 and HPV18) or 37 °C (HPV31 , CRPV, and HPV6) overnight (Buck et al, J. Virology, 79:2839-2846 (2005)). The mature pseudovirions were solubilized by addition of 0.17 volumes of 5 M NaCI, then clarified by low speed (1 ,500 g) centrifugation. Pseudoviruses were purified on a pre-formed 27, 33, 39% Optiprep (Sigma) step gradient. Optiprep fractions containing SEAP-transducing activity were pooled and frozen. Example 14 HPV-Pseudovirus in vitro Neutralization Assay ("Conventional
Methodology")
The method described in this example is based on Buck et al., Methods in Mol. Med., 445-461 (Monograph) (2004). Briefly, 293TT cells were seed at 1.5X104 per well in a flat bottom 96-well cell culture plate with neutralization medium (DMEM without phenol red, 10% FBS, 1 % non-essential amino acids, 1 % GlutaMax, 10mM HEPES) and incubated at 37 °C culture 5% C02 incubator overnight. Anti-serum samples were serially diluted with the neutralization medium in a 96-well plate. 50 μΙ of diluted serum were mixed with 50 μΙ of PsV working solution in a round bottom 96-well plate and incubated at 37 °C for 2 hours. The entire volume was transferred to corresponding well of the plate which had been seeded with 293TT cells a day before. Plates were returned to the incubator for 67 hours. Upon completion culture supernatant was analyzed for the presence of SEAP. The in vitro neutralization titer was defined as the reciprocal of the highest dilution of serum that reduces the SEAP activity by at least 50% in comparison to the reactivity in the wells that received no PsV sample without antiserum.
Example 15 HPV-Pseudovirus in vitro Neutralization Assay ("Novel Methodology")
The method described in the example is based on (Day et ai, Cell Host Microbe, 8:260- 270 (2010)). Flat bottom 96-well cell culture plate were coated with Extra Cellular Matrix (200 μg per well), covered with neutralization medium (DMEM without phenol red, 10% FBS, 1 % non-essential amino acids, 1 % GlutaMax) and incubated the plate at 37 °C, 5% C02 culture incubator for 4 hours. Plates were washed three times with PBS and 80 μΙ of the diluted PsV prepared in Delta Furin CHO conditional Medium were added to each well. Plates were incubated overnight at 37 °C. Plates were carefully washed three times with PBS and 80 μΙ of the neutralization medium was added to each well. Twenty (20) μΙ of serially diluted anti-serum (in neutralization medium) was added to each well and the plates were incubated overnight at 37 °C. Upon completion of the incubation period, 100 μΙ of 104 pgsA-745 cells were added to each well. Cells were then incubated at 37 °C for 72 hours and cell supernatants were analyzed for luminescence by using the New England BioLabs BioLux Gaussia Luciferase
Assay Kit (NEB#E3300L, New England Biolabs, Boston, MA), using 15 μΙ supernatant and 50 μΙ of GLuc assay solution provided in the kit. The assay was conducted per kit directions.
The in vitro neutralization titer is defined as the reciprocal of the highest dilution of serum that reduces the Luciferase activity by at least 50% in comparison to the reactivity in the wells that received PsV but no anti-serum. Example 16 Challenge Rabbits with Quazi-virions (Rabbits)
To evaluate the infectivity of quasivirions in vivo, several sites of scarified rabbit skin were exposed to 5 μΙ aliquots of stock preparations as previously described in Gambhira et al., J. Virol., 81 :13927-13931 (2007); and Gambhira et al., J. Virol. , 81 :1 1585-1 1592 (2007).
Briefly, quaizivirions are L1/L2 HPV virus like particles encapsidating the CRPV genome.
CRPV particles were also used for challenge. These reagents are infectious only for rabbits due to species restriction of CRPV genome in rabbits. Animals were monitored for 10 weeks after challenge. Papilloma volumes were charted as height x width x depth. Additionally, the back of each rabbit was individually photographed.
Example 17 Vaginal HPV Challenge in Mice
Female BALB/c mice aged 6-8 weeks were immunized three times with Fla-69, Fla-32, Gardasil® (Merck, Whitehouse Station, NJ), Cervarix® (GlaxoSmithKlein, London, United Kingdom) and placebo control three times biweekly as described in FIG. 58. Mice received 3 mg of medroxyprogesterone (Depo-provera) (Pfizer, NY, NY) diluted in 100 μΙ of sterile PBS in a subcutaneous injection 4 days prior to HPV56 pseudovirus challenge. The pseudovirus inoculum was a 20 μΙ dose composed of purified HPV56 pseudovirus carrying the luciferase reporter gene with a titer of about 10 l U/ml mixed in 2% carboxymethyl cellulose (CMC) (Sigma, C5013). The virus was delivered in two doses. Half the virus was deposited into the mouse's vagina using an M50 positive-displacement pipette (Gilson, Inc., Middleton, Wl). A cytobrush cell collector was inserted in the vagina and twirled clockwise and counter-clockwise 10 times, and the remaining 10 μΙ was introduced. Three days later, the mice were anesthetized
(anesthesia induction was accomplished within 3-5 min by using a chamber filled with 2.5% isoflurane (Baxter, Deerfield, IL)), luciferin (20 μΙ at concentration of 7.8 mg/ml) was deposited intravaginally, and images were acquired for 10 min using an Xenogen I VIS 200 (Caliper Life Sciences, Hopkinton, MA) as previously described by Johnson et al., J. Virology, 83:2067-2074 (2009). The average radiance within the region of interest was determined. Data are representative of 10 mice per group, and experiments were performed in duplicate. Statistical analysis was performed with GraphPad Prism Software (GraphPad Software, Inc., La Jolla, CA), in which a one-tailed, unpaired t-test was used to determine p values.
INDUSTRIAL APPLICABILITY
The present invention relates to methods and compositions for production of fusion protein constructs useful for the treatment and/or prevent of infection, disease, and/or any related sequelae caused by or associated with transmission of one or more of the various types of human papillomavirus. The present invention further provides nucleic acid sequences that encode said compositions and fusion protein constructs as well as methods of therapeutic use and/or prophylactic use of said compositions and fusion protein constructs. In particular, the methods and compositions disclosed herein may be effective in preventing and/or treating diseases caused by one or more types of the roughly 100 or more known types of Human papillomavirus.
Additionally, particular embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention. Other objects, features, and advantages of the present invention will become apparent. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.

Claims

We claim :
1 . A fusion product comprising at least a portion of a flagellin sequence of substantially the same amino acid sequence as a flagellin sequence described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305, and at least a portion of a human papillomavirus (HPV) L2 sequence of substantially the same amino acid sequence as an HPV L2 sequence described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305.
2. The fusion product of claim 1 , wherein the fusion product activates Toll-like Receptor 5 (TLR5).
3. The fusion product of claim 1 , wherein said portion of said flagellin sequence is at least about 10 amino acids in length, and said portion of said HPV L2 sequence is at least about 10 amino acids in length.
4. The fusion product of claim 3, wherein said portion of said flagellin sequence is at least about 20 amino acids in length, and said portion of said HPV L2 sequence is at least about 20 amino acids in length.
5. The fusion product of claim 1 , wherein said portion of said flagellin sequence comprises a deletion of or within domain D2 and/or domain D3.
6. The fusion product of claim 1 , wherein said fusion product comprises at least a portion of two or more different HPV L2 sequences of substantially the same amino acid sequence as at least two or more HPV L2 sequences described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305.
7. The fusion product of claim 6, comprising 5-10 of said different HPV L2 sequences.
8. The fusion product of claim 6 or 7, wherein at least one of said different HPV L2 sequences is substantially the same as that of HPV16 and/or HPV18, as described in one or more of SEQ ID NOs: 101 -199, 202-204, or 303-305.
9. The fusion product of any one of claims 6-8, wherein at least one of said different HPV L2 sequences is substantially the same as that of HPV31 , HPV39, HPV52, HPV58, HPV35, HPV45, and/or HPV6B, as described in one or more of SEQ ID NOs: 101 -199, 202-204, or SOS- SOS.
10. A nucleic acid molecule comprising a sequence encoding a fusion product of one of claims 1 -9.
1 1 . The nucleic acid molecule of claim 10, comprising at least a portion of a flagellin nucleotide sequence of substantially the same nucleotide sequence of a flagellin sequence described in one or more of SEQ ID NOs: 1 -99, 100, 200, 201 , or 300-302, and at least a portion of an HPV L2 nucleotide sequence of substantially the same nucleotide sequence of an HPV L2 described in one or more of SEQ ID NOs: 1 -99, 100, 200, 201 , or 300-302.
12. A vector comprising a nucleic acid molecule of claim 10 or 1 1 .
13. A cell comprising a vector of claim 12.
14. A method of inducing an immune response in a subject, comprising administering to a subject an effective amount of the fusion product of claim 1 , or a modification thereof.
15. A method of preventing an infection in a subject by the human papillomavirus comprising administering to said subject an effective amount of the fusion product of claim 1 , or a modification thereof.
16. A method of modulating an immune response in a subject having a pathological condition, comprising administering to said subject an effective amount of said fusion product of claim 1 , or a modification thereof.
17. The method of claim 16, wherein said pathological condition results from infection of said subject by a human papillomavirus.
18. A method of inducing an antigen-specific immune response in a subject comprising administering to said subject an effective amount of said fusion product of claim 1 , or a modification thereof.
19. A method of preventing an infection in a subject by one or more human papillomavirus comprising administering to said subject an effective amount of the fusion product of claim 1 , or a modification thereof.
20. Use of a fusion product of claim 1 , or a modification thereof, in the preparation of a medicament for inducing an immune response in a subject.
PCT/US2012/037831 2011-05-14 2012-05-14 Recombinant fusion proteins and methods for use thereof for treatment or prevention of papillomavirus infection WO2012158639A2 (en)

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