Immunogenic HPV L2-containing VLPs and related compositions, constructs, and therapeutic methods

Abstract

The invention provides immunotherapeutic and prophylactic bacteriophage viral-like particle (VLPs) which are useful in the treatment and prevention of human papillomavirus (HPV) infections and related disorders, including cervical cancer and persistent infections associated with HPV. Related compositions (e.g. vaccines), nucleic acid constructs, and therapeutic methods are also provided. VLPs and related compositions of the invention induce high titer antibody responses against HPV L2 and protect against HPV challenge in vivo. VLPs, VLP-containing compositions, and therapeutic methods of the invention induce an immunogenic response against HPV infection, confer immunity against HPV infection, protect against HPV infection, and reduce the likelihood of infection by HPV infection.

Claims

1. A PP7 RNA bacteriophage virus-like particle comprising a PP7 RNA bacteriophage single chain coat polypeptide dimer having an immunogenic papillomavirus (PV) L2 peptide insertion in the AB loop, the peptide insertion consisting essentially of the amino acid sequence corresponding to amino acids 17-31 of HPV16 L2 protein, which is displayed on the surface of the virus-like particle.

2. The RNA bacteriophage virus-like particle of claim 1, wherein the L2 peptide insertion further comprises a second L2 peptide sequence from a second PV.

3. The RNA bacteriophage virus-like particle of claim 1, further comprising a second RNA bacteriophage single chain coat polypeptide dimer having a second immunogenic papillomavirus (PV) L2 peptide insertion.

4. The RNA bacteriophage virus-like particle of claim 1, wherein the second L2 peptide sequence corresponds to amino acids 17-31 of HPV16 is selected from HPV1, HPV5, HPV8, HPV35, HPV31, HPV33, HPV58, HPV52, HPV73, HPV6, HPV11, HPV18, HPV45, HPV39, HPV68, HPV59, HPV51, HPV56, HPV 66, HPV2, CPRV and BPV1.

5. An immunogenic composition comprising a bacteriophage virus-like particle of claim 1.

6. A viral-like particle comprising a PP7 bacteriophage single chain coat polypeptide dimer having an HPV L2 peptide insertion consisting essentially of amino acids of SEQ ID NO:24, wherein the HPV L2 peptide is displayed on the virus-like particle.

7. A method for inducing an immune response to HPV in a subject comprising administering to the subject an RNA bacteriophage virus-like particle of claim 1, wherein the composition induces a HPV-immunogenic response in the patient.

8. The RNA bacteriophage virus-like particle of claim 1, wherein the immunogenic papillomavirus (PV) L2 peptide insertion has an amino acid sequence that is identical to amino acids 17-31 of HPV16 L2 protein.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Plasmids for expression of recombinant PP7 coat protein, including pP7K, p2P7K32, pETP7K, and pET2P7K32.

(2) FIG. 2. Nucleotide sequences of the plasmids p2P7K32 and pET2P7K32. SEQ ID No. 1

(3) FIG. 3, The N-terminal sequence of the downstream copy of coat protein encoded by p2P7K32, SEQ ID No. 2. Below this is a list of selected forward primers used to clone the listed sequences into PP7 coat (shown 5 to 3). A similar strategy was used to clone sequences from HPV1, 5/8, 6, 11/33, 16, 18, 33, 39/45, and 52/58 L2 into the PP7 coat protein. The KpnI restriction site is shown in italics and the peptide insertion is shown in bold text. All of these primers yield an insertion which is flanked by a thr residue on the N-terminal side and a glu (the wild-type position 11 amino acid) on the C-terminal side.

(4) FIG. 4. Amino acid sequence of L2 (17-31, or equivalent) from selected STI/carcinogenic/cutaneous HPV and animal papillomavirus types, adapted from (1). PP7 VLPs displaying a subset of these sequences were constructed. The following sequence ID Nos. apply to the disclosed amino acid sequences: HPV1 (SEQ ID No. 12), HPV5 (SEQ ID No. 13), HPV8 (SEQ ID No. 14), HPV16 (SEQ ID No. 15), HPV35 (SEQ ID No. 16), HPV31 (SEQ ID No. 17), HPV33 (SEQ ID No. 18), HPV58 (SEQ ID No. 19), HPV52 (SEQ ID No. 20), HPV73 (SEQ ID No. 21), HPV6 (SEQ ID No. 22), HPV11 (SEQ ID No. 23), HPV18 (SEQ ID No. 24), HPV45 (SEQ ID No. 25), HPV39 (SEQ ID No. 26), HPV68 (SEQ ID No. 27), HPV59 (SEQ ID No. 28), HPV51 (SEQ ID No. 29), HPV56 (SEQ ID No. 30), HPV66 (SEQ ID No. 31), HPV2 (SEQ ID No. 32), CPRV (SEQ ID No. 33) and BPV1 (SEQ ID No. 34).

(5) FIG. 5. Agarose gel electrophoresis of purified wild-type single-chain dimer (lane 1), 16L2 (lane 2), 45L2 (lane 3), and 58L2 (lane 4) VLPs. Variations in electrophoretic mobility reflect charge differences conferred by the inserted peptides.

(6) FIG. 6. Binding of an anti-16L2 monoclonal antibody against (RG-1) to recombinant VLPs. (A) Dilutions of the mAb was reacted with 500 ng/well of wild-type PP7, 16L2, and Flag-VLPs. Binding was detected using a horseradish peroxidase-labeled goat anti-mouse IgG secondary followed by development with ABTS. Reactivity was determined by measurement of the absorbance at 405 nm (OD 405). (B) Reactivity of a 1:5000 dilution of RG-1 mAb to the eight recombinant L2 VLPs constructed, or to wild-type PP7 VLPs.

(7) FIG. 7. IgG antibody responses in groups of mice immunized with wild-type PP7 VLPs, 1L2-VLPs, 5L2-VLPs, 6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. End-point dilution ELISA titers against a peptides representing amino acids 14-40 from the appropriate HPV L2 (shown in the key) conjugated to streptavidin. 10 g of VLPs were administered intramuscularly in the presence of incomplete Freund's adjuvant. Results are from sera obtained three to four weeks after the second vaccination. Each datum point represents the antibody titer from an individual mouse. Lines represent the geometric mean titer for each group.

(8) FIG. 8. Mice immunized with PP7 16L2-VLPs are protected from vaginal challenge with HPV16 or HPV45 pseudovirions. Groups of five mice were immunized two times with 10 g 16L2-VLPs, wild-type PP7 VLPs, or HPV16 L1-VLPs formulated in incomplete Freund's adjuvant (IFA). As an additional control, mice were immunized with IFA alone. Three weeks after the second immunization mice were intravaginally challenged with 10.sup.8 IU of HPV16 pseudovirus (left panel) or HPV45 pseudovirus (right panel) containing a luciferase reporter. As a control, a group of five mice were not infected. Luciferase activity was quanititated 48 hours after infection by taking images 3 min post-installation of luciferin at medium binning with a 30-s exposure. Images were then analyzed by drawing an equally sized region of interest for each mouse and measuring average radiance (photons/second/cm.sup.2/sr) within this region. Results shown are the mean average radiance for each group of five mice. Error bars represent the standard error of the mean. Lines above pairs of data indicate the percent reduction of signal in mice immunized with 16L2-VLPs relative to wild-type PP7 VLPs or the IFA control. All comparisons shown here are statistically significant (p<0.01) as calculated by T-test.

(9) FIG. 9. Immunization with a mixture of L2-PP7 VLPs induces broad anti-L2 IgG responses. Mice were immunized three times without adjuvant with 10 g (total) of a mixture of equal amounts of 1L2-VLPs, 5L2-VLPs, 6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. Two weeks after the final immunization, sera was taken as tested for reactivity to HPV L2 peptides representing L2 amino acids 14-40 from HPV1, HPV5, HPV6, HPV16, and HPV18 sequences.

(10) FIG. 10. Immunization with a mixture of L2-PP7 VLPs protects from genital infection with HPV5, HPV6, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 pseudovirions. Groups of five mice were immunized three times with 10 g (total) of a mixture of equal amounts of 1L2-VLPs, 5L2-VLPs, 6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. Two weeks after the final immunization, mice were challenged with 10.sup.6-10.sup.8 IU of the indicated pseudovirus containing a luciferase reporter. As controls for each pseudovirus infection, groups of five mice were also immunized with wild-type PP7 VLPs. Luciferase activity was quanititated 48 hours after infection as described in FIG. 8. The extent of protection was determined by comparing the luciferase signal in the group immunized with control VLPs with the group immunized with mixed L2-VLPs.

(11) FIG. 11. Antibody responses in mice immunized with VLPs displaying HPV16 L2 amino acids 35-50 or 51-65. Mice were immunized three times and then sera was taken and tested for reactivity with synthetic peptides representing A) HPV16 L2 amino acids 34-52, and B) HPV16 L2 amino acids 49-71.

DETAILED DESCRIPTION OF THE INVENTION

(12) In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual; Ausubel, ed., 1994, Current Protocols in Molecular Biology Volumes I-III; Celis, ed., 1994, Cell Biology: A Laboratory Handbook Volumes I-III; Coligan, ed., 1994, Current Protocols in Immunology Volumes I-III; Gait ed., 1984, Oligonucleotide Synthesis; Hames & Higgins eds., 1985, Nucleic Acid Hybridization; Hames & Higgins, eds., 1984, Transcription And Translation; Freshney, ed., 1986, Animal Cell Culture; IRL Press, 1986, Immobilized Cells And Enzymes; Perbal, 1984, A Practical Guide To Molecular Cloning.

(13) Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

(14) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

(15) It must be noted that as used herein and in the appended claims, the singular forms a, and and the include plural references unless the context clearly dictates otherwise.

(16) Furthermore, the following terms shall have the definitions set out below.

(17) The term patient or subject is used throughout the specification within context to describe an animal, generally a mammal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the immunogenic compositions and/or vaccines according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a human patient of either or both genders.

(18) The term effective is used herein, unless otherwise indicated, to describe a number of VLP's or an amount of a VLP-containing composition which, in context, is used to produce or effect an intended result, whether that result relates to the prophylaxis and/or therapy of an HPV-induced or HPV-related disorder or disease state or as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms (including the term therapeutically effective) which are otherwise described or used in the present application.

(19) As used herein, the term polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, such as coding regions, and non-coding regions such as regulatory sequences (e.g., promoters or transcriptional terminators). A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.

(20) As used herein, the term polypeptide refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term polypeptide also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

(21) The term single-chain dimer refers to a normally dimeric protein whose two subunits have been genetically (chemically, through covalent bonds) fused into a single polypeptide chain. Specifically, in the present invention single-chain dimer versions of PP7 coat proteins were constructed. Each of these proteins is naturally a dimer of identical polypeptide chains. In the PP7 coat protein dimers the N-terminus of one subunit lies in close physical proximity to the C-terminus of the companion subunit. Single-chain coat protein dimers were produced using recombinant DNA methods by duplicating the DNA coding sequence of the coat proteins and then fusing them to one another in tail to head fashion. The result is a single polypeptide chain in which the coat protein amino acid appears twice, with the C-terminus of the upstream copy covalently fused to the N-terminus of the downstream copy. Normally (wild-type) the two subunits are associated only through noncovalent interactions between the two chains. In the single-chain dimer these noncovalent interactions are maintained, but the two subunits have additionally been covalently tethered to one another. This greatly stabilizes the folded structure of the protein and confers to it its high tolerance of peptide insertions as described above.

(22) This application makes frequent reference to coat protein's AB-loop. The RNA phage coat proteins possess a conserved tertiary structure. The PP7 coat proteins, for example, possess a structure wherein each of the polypeptide chains is folded into of a number of -strands. The -strands A and B form a hairpin with a three-amino acid loop connecting the two strands at the top of the hairpin, where it is exposed on the surface of the VLP. As evidenced in the present application, peptides inserted into the AB-loop are exposed on the surface of the VLP and are strongly immunogenic.

(23) The amino acid residues described herein are preferred to be in the L isomeric form. However, residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired functional is retained by the polypeptide. NH.sub.2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.

(24) The term valency is used to describe the density of L2 peptide display on VLPs according to the present invention. Valency in the present invention may range from low valency to high valency, about less than 1 to more than about 180, preferably about 90 to 180. Immunogenic compositions according to the present invention comprise VLPs which are preferably high valency and comprise VLPs which display at least 50-60 up to about 180 or more L2 peptides.

(25) The term coding sequence is defined herein as a portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5-end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3-end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

(26) A heterologous region of a recombinant cell is an identifiable segment of nucleic acid within a larger nucleic acid molecule that is not found in association with the larger molecule in nature.

(27) An origin of replication, used within context, normally refers to those DNA sequences that participate in DNA synthesis by specifying a DNA replication initiation region. In the presence of needed factors (DNA polymerases, and the like) an origin of replication causes or facilitates DNA associated with it to be replicated. By way of a non-limiting example, the ColE1 replication origin endows many commonly used plasmid cloning vectors with the capacity to replicate independently of the bacterial chromosome. Another example is the p15A replication origin. The presence on a plasmid of an additional origin of replication from phage M13 confers the additional ability to replicate using that origin when E. coli cells are infected with a so-called helper phage (e.g. M13CM1) which provides necessary protein factors. M13 replicates intracellularly as double-stranded circular DNA, but also produces a single-stranded circular form, which it packages within the phage particle. These particles provide a convenient source of single-stranded circular DNA for plasmids.

(28) A promoter sequence is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 direction) coding sequence. For purposes of defining the present invention, the promoter sequence includes the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found DNA sequences responsible for the binding of RNA polymerase and any of the associated factors necessary for transcription initiation. In bacteria promoters normally consist of 35 and 10 consensus sequences and a more or less specific transcription initiation site. Eukaryotic promoters will often, but not always, contain TATA boxes and CAT boxes. Bacterial expression vectors (usually plasmids or phages) typically utilize promoters derived from natural sources, including those derived from the E. coli Lactose, Arabinose, Tryptophan, and ProB operons, as well as others from bacteriophage sources. Examples include promoters from bacteriophages lambda, T7, T3 and SP6.

(29) In bacteria, transcription normally terminates at specific transcription termination sequences, which typically are categorized as rho-dependent and rho-independent (or intrinsic) terminators, depending on whether they require the action of the bacterial rho-factor for their activity. These terminators specify the sites at which RNA polymerase is caused to stop its transcription activity, and thus they largely define the 3-ends of the RNAs, although sometimes subsequent action of ribonucleases further trims the RNA.

(30) An antibiotic resistance gene refers to a gene that encodes a protein that renders a bacterium resistant to a given antibiotic. For example, the kanamycin resistance gene directs the synthesis of a phosphotransferase that modifies and inactivates the drug. The presence on plasmids of a kanamycin resistance gene provides a mechanism to select for the presence of the plasmid within transformed bacteria. Similarly, the chloramphenicol resistance gene allows bacteria to grow in the presence of the drug by producing an acetyltransferase enzyme that inactivates the antibiotic through acetylation.

(31) The term PCR refers to the polymerase chain reaction, a technique used for the amplification of specific DNA sequences in vitro. The term PCR primer refers to DNA sequences (usually synthetic oligonucleotides) able to anneal to a target DNA, thus allowing a DNA polymerase (e.g. Taq DNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are used in the polymerase chain reaction to initiate DNA synthesis on each of the two strands of a DNA and to thus amplify the DNA segment between two primers. Representative PCR primers which used in the present invention are those which are presented in FIG. 3 hereof. Additional PCR primers may be obtained for the various HPV L2 peptides which are presented herein.

(32) Examples of primers used for PCR are given in FIG. 3 as described above and the following.

(33) E3.2: 5 CGG GCT TTG TTA GCA GCC GG 3(SEQ ID No. 35) serves as the 3 (reverse)-primer in PCR reactions to amplify coat protein.

(34) An expression control sequence is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is under the control of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. Transcriptional control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. Translational control sequences determine the efficiency of translation of a messenger RNA, usually by controlling the efficiency of ribosome binding and translation initiation. For example, as discussed elsewhere in this application, the coat proteins of the RNA phages are well-known translational repressors of the phage replicase. As coat protein accumulates to a sufficiently high concentration in the infected cell, it binds to an RNA hairpin that contains the translation initiation region (Shine-Dalgarno and initiator AUG) of the phage's replicase gene. This prevents ribosome binding and shuts off replicase synthesis at a time in the viral life cycle where the transition from replication to virus assembly occurs.

(35) A cell has been transformed by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid, which normally replicate independently of the bacterial chromosome by virtue of the presence on the plasmid of a replication origin. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.

(36) A signal sequence can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

(37) It should be appreciated that also within the scope of the present invention are nucleic acid sequences encoding the polypeptide(s) of the present invention, which code for a polypeptide having the same amino acid sequence as the sequences disclosed herein, but which are degenerate to the nucleic acids disclosed herein. By degenerate to is meant that a different three-letter codon is used to specify a particular amino acid.

(38) As used herein, epitope refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 5 such amino acids, and more usually, consists of at least about 8-10 up to about 20 or more such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

(39) As used herein, a mimotope is a peptide that mimics an authentic antigenic epitope.

(40) As used herein, the term coat protein(s) refers to the protein(s) of a bacteriophage or a RNA-phage capable of being incorporated within the capsid assembly of the bacteriophage or the RNA-phage.

(41) As used herein, a coat polypeptide as defined herein is a polypeptide fragment of the coat protein that possesses coat protein function and additionally encompasses the full length coat protein as well or single-chain variants thereof.

(42) As used herein, the term immune response refers to a humoral immune response and/or cellular immune response leading to the activation or proliferation of B- and/or T-lymphocytes and/or antigen presenting cells. In some instances, however, the immune responses may be of low intensity and become detectable only when using at least one substance in accordance with the invention. Immunogenic refers to an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system are increased and directed towards the immunogenic agent. An immunogenic polypeptide is a polypeptide that elicits a cellular and/or humoral immune response, whether alone or linked to a carrier in the presence or absence of an adjuvant. Preferably, antigen presenting cell may be activated.

(43) As used herein, the term vaccine refers to a formulation which contains the composition of the present invention and which is in a form that is capable of being administered to an animal and provides a measure of protection (protective effect) against a disease state or condition for which the vaccine is administered. The term prevention or prophylaxis is used in context synonymously with the term reducing the likelihood of or inhibiting wherein the measure of prevention (of a disease state or condition) is one of degree of effect. Vaccines according to the present invention may also instill immunity in a patient or subject against a disease state or condition and such immunity is consistent with the use of the term prevention or prophylaxis as used above.

(44) As used herein, the term virus-like particle of a bacteriophage refers to a virus-like particle (VLP) resembling the structure of a bacteriophage, being non-replicative and noninfectious, and lacking at least the gene or genes encoding for the replication machinery of the bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.

(45) This definition should, however, also encompass virus-like particles of bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and noninfectious virus-like particles of a bacteriophage.

(46) VLP of RNA bacteriophage coat protein: The capsid structure formed from the self-assembly of one or more subunits of RNA bacteriophage coat protein and optionally containing host RNA is referred to as a VLP of RNA bacteriophage coat protein. In a particular embodiment, the capsid structure is formed from the self assembly of 90-180 subunits.

(47) A hybrid VLP refers to a VLP that displays two or more heterologous amino acid sequences on its surface, such as, for example, amino acids 17-31 from HPV16 and HPV18. Such a hybrid VLP could be formed by coexpression of two recombinant coat proteins in the same expression strain of bacteria. Alternatively, hybrid VLPs could be generated in vitro by disassembly of two separate recombinant VLPs into coat protein dimers. Following disassembly, the recombinant coat protein dimers may be mixed together and then reassembled. Methods for assembly and disassembly of PP7 VLPs are described by Caldeira and Peabody (6).

(48) A nucleic acid molecule is operatively linked to, or operably associated with, an expression control sequence when the expression control sequence controls and regulates the transcription and translation of nucleic acid sequence. The term operatively linked includes having an appropriate start signal (e.g., ATG) in front of the nucleic acid sequence to be expressed and maintaining the correct reading frame to permit expression of the nucleic acid sequence under the control of the expression control sequence and production of the desired product encoded by the nucleic acid sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

(49) HPV-Induced Disorders, Immunogenicity, and Prophylactic Efficacy

(50) HPV-induced disorders or HPV-related disorders include, but are not limited to, the disorders identified in this section. Immunogenicity and prophylactic efficacy (e.g. whether a composition is immunotherapeutic and prophylactic for HPV-induced disorders) may be evaluated either by the techniques and standards mentioned in this section, or through other methodologies that are well-known to those of ordinary skill in the art.

(51) Over 100 different HPV types have been identified and are referred to by number. Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 are among the high-risk sexually transmitted HPVs and may lead to the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), vaginal cancer, penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). Several types of HPV, particularly type 16, have been found to be associated with oropharyngeal squamous-cell carcinoma, a form of head and neck cancer. HPV (e.g., HPV 6 and 11) cause genital warts, and HPV 6 and 11 can cause recurrent respiratory papillomatosis. HPV may cause epidermodysplasia verruciformis in immunocompromised individuals. Other HPV types, such as HPV1, can causes cutaneous warts.

(52) High-risk human HPV infection of the cervical epithelium is causally linked with the generation of cervical cancer. HPV16 is associated with premalignant and malignant diseases of the genito-urinary tract, and in particular with carcinoma of the cervix. Papillomavirus prophylactic immunogenic compositions or vaccines target the systemic immune system for induction of neutralizing antibodies that protect the basal cells against infection. Because the carcinogenic HPVs are susceptible to neutralization by antibodies for 9-48 hours after reaching the basal cells, both low and high titered HPV type-specific antibodies induced by HPV L2-based vaccines should prove highly efficacious.

(53) To assess immunogenicity (e.g. whether a composition has induced a high titer antibody responses against HPV L2, an anti-HPV 16 L2 geometric mean titer (GMT) can be measured, e.g. after a few weeks of treatment (e.g. 3 or 4 weeks) and after administration of a few dosages (e.g. 3 or 4). The percentage of subjects who seroconverted for HPV 16 after a few weeks of treatment (e.g. 3 or 4 weeks) and after administration of a few dosages (e.g. 3 or 4) and the magnitude of these responses can also be determined to assess immunogenicity.

(54) HPV L2

(55) HPV L2 as used herein includes the L2 capsid proteins of all human papillomaviruses.

(56) Production of Virus-Like Particles

(57) The present invention is directed to virus-like phage particles as well as methods for producing these particles in vivo or in vitro. The methods typically include producing virus-like particles (VLPs) and recovering the VLPs. As used herein, producing VLPs in vitro refers to producing VLPs outside of a cell, for instance, in a cell-free system, while producing virions in vivo refers to producing VLPs inside a cell, for instance, an Eschericia coli or Pseudomonas aeruginosa cell.

(58) Bacteriophages

(59) The system envisioned here is based on the properties of single-strand RNA bacteriophages [RNA Bacteriophages, in The Bacteriophages. Calendar, R L, ed. Oxford University Press. 2005]. The known viruses of this group attack bacteria as diverse as E. coli, Pseudomonas and Acinetobacter. Each possesses a highly similar genome organization, replication strategy, and virion structure. In particular, the bacteriophages contain a single-stranded (+)-sense RNA genome, contain maturase, coat and replicase genes, and have small (<300 angstrom) icosahedral capsids. These preferably include but are not limited to PP7, Q, R17, SP, PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNA bacteriophages. PP7 bacteriophages are used in preferred aspects of the present invention.

(60) PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Q, which also may be used in the present invention. PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Q phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles have been established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nM. Using a genetic system in which coat protein represses translation of a replicase--galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified (28).

(61) The coat proteins of several single-strand RNA bacteriophages are known translational repressors. They shut off viral replicase synthesis by binding an RNA hairpin that contains the replicase ribosome binding site. X-ray structure determination of RNA phages shows that homologies evident from comparisons of coat protein amino acid sequences are reflected in their tertiary structures. The coat protein dimer, which is both the repressor and the basic building block of the virus particle, consists of two intertwined monomers that together form a large -sheet surface upon which the RNA is bound. Each of the coat proteins uses a common structural framework to bind different RNAs, thereby presenting an opportunity to investigate the basis of specific RNA-protein recognition. We have described the RNA binding properties of the coat protein of PP7, an RNA bacteriophage of Pseudomonas aeroginosa whose coat protein shows only 13% amino acid sequence identity to that of MS2. We have also presented the following findings. 1) The coat protein of PP7 is a translational repressor. 2) An RNA hairpin containing the PP7 replicase translation initiation site is specifically bound by PP7 coat protein both in vivo and in vitro, indicating that this structure represents the translational operator. 3) The RNA binding site resides on the coat protein -sheet.

(62) By way of comparison, the genome of MS2 comprises a single strand of (+)-sense RNA 3569 nucleotides long, encoding only four proteins, two of which are structural components of the virion. The viral particle is comprised of an icosahedral capsid made of 180 copies of coat protein and one molecule of maturase protein together with one molecule of the RNA genome. Coat protein is also a specific RNA binding protein. Assembly may possibly be initiated when coat protein associates with its specific recognition target an RNA hairpin near the 5-end of the replicase cistron. The virus particle is then liberated into the medium when the cell bursts under the influence of the viral lysis protein. The formation of an infectious virus requires at least three components, namely coat protein, maturase and viral genome RNA, but experiments show that the information required for assembly of the icosahedral capsid shell is contained entirely within coat protein itself. For example, purified coat protein can form capsids in vitro in a process stimulated by the presence of RNA [Beckett et al., 1988, J. Mol Biol 204: 939-47]. Moreover, coat protein expressed in cells from a plasmid assembles into a virus-like particle in vivo (37).

(63) Examples of PP7 coat polypeptides include but are not limited to the various chains of PP7 Coat Protein Dimer in Complex With RNA Hairpin (e.g. Genbank Accession Nos. 2QUXR; 2QUXO; 2QUX_L; 2QUX_I; 2QUX_F; and 2QUX_C). See also Example 1 herein and (29).

(64) PP7 Coat Polypeptide

(65) The coat polypeptides useful in the present invention also include those having similarity with one or more of the coat polypeptide sequences disclosed above. The similarity is referred to as structural similarity. Structural similarity may be determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate amino acid sequence can be isolated from a single stranded RNA virus, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, two amino acid sequences are compared using the BESTFIT algorithm in the GCG package (version 1 0.2, Madison Wis.), or the Blastp program of the BLAST 2 search algorithm available on the worldwide web at URL ncbi.nlm.nih.gov/blast/bl2seq/bl2.html. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap xdropoff=50, expect=10, wordsize=3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as identities. Preferably, a coat polypeptide also includes polypeptides with an amino acid sequence having at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity to one or more of the amino acid sequences disclosed above. Preferably, a coat polypeptide is active. Whether a coat polypeptide is active can be determined by evaluating the ability of the polypeptide to form a capsid and package a single stranded RNA molecule. Such an evaluation can be done using an in vivo or in vitro system, and such methods are known in the art and routine. Alternatively, a polypeptide may be considered to be structurally similar if it has similar three dimensional structure as the recited coat polypeptide and/or functional activity.

(66) The HPV L2 peptide sequence may be present at the amino-terminal end of a coat polypeptide, at the carboxy-terminal end of a coat polypeptide, or it may be present elsewhere within the coat polypeptide. Preferably, the HPV L2 peptide sequence is present at a location in the coat polypeptide such that the insert sequence is expressed on the outer surface of the capsid. In a particular embodiment, the HPV L2 peptide sequence may be inserted into the AB loop regions of the above-mentioned coat polypeptides, preferably in the downstream subunit of the single-chain dimer of the coat polypeptide. Examples of such locations include, for instance, insertion or replacement of the insert sequence into a coat polypeptide in accordance with the examples presented hereinafter. Insertion or replacement, preferably insertion, of the L2 peptide sequence into the AB loop region at amino acid units 8-11 of the AB loop, preferably in the downstream subunit of the single-chain dimer coat polypeptide, is preferred.

(67) Alternatively, the HPV L2 peptide sequence may be inserted at the N-terminus or C-terminus of the coat polypeptide, preferably in the downstream subunit of the dimer coat polypeptide.

(68) The HPV L2 peptide sequence preferably includes but is not limited to amino acid sequences of, at least, five, ten, fifteen, twenty amino, twenty five or thirty amino acids derived from the minor capsid protein L2 of human Papillomavirus types 1-100, preferably 16 (HPV16), 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 or 59, preferably 16 (HPV16).

(69) In another particular embodiment, the L2 peptide sequence includes amino acid sequences with at least 75%, 80%, 85%, 90%, or 95% homology to L2 sequences derived from HPV strains representing the five clades of the virus (HPV1, HPV5, HPV6, HPV16, and HPV18).

(70) In order to determine a corresponding position in a structurally similar coat polypeptide, the amino acid sequence of this structurally similar coat polypeptide is aligned with the sequence of the named coat polypeptide as specified above.

(71) In a particular embodiment, the coat polypeptide is a single-chain dimer containing an upstream and downstream subunit. Each subunit contains a functional coat polypeptide sequence. The HPV L2 peptide sequence may be inserted in the upstream and/or downstream subunit at the sites mentioned hereinabove, e.g., AB loop region of downstream subunit, preferably at amino acid units 8-11 and as otherwise specified in the examples which are described hereinbelow. In a particular embodiment, the coat polypeptide is a single chain dimer of a PP7 coat polypeptide and the L2 peptide sequence is inserted in the AB loop region of the downstream subunit.

(72) Preparation of Transcription Unit

(73) The transcription unit of the present invention comprises an expression regulatory region, (e.g., a promoter), a sequence encoding a single chain of a coat polypeptide which includes a HPV L2 peptide encoding sequence and a transcription terminator. The RNA polynucleotide may optionally include a coat recognition site (also referred to a packaging signal, translational operator sequence, coat recognition site). Alternatively, the transcription unit may be free of the translational operator sequence. The promoter, coding region, transcription terminator, and, when present, the coat recognition site, are generally operably linked. Operably linked or operably associated with refer to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is operably linked to, or operably associated with, a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. The coat recognition site, when present, may be at any location within the RNA polynucleotide provided it functions in the intended manner.

(74) The invention is not limited by the use of any particular promoter, and a wide variety of promoters are known. The promoter used in the invention can be a constitutive or an inducible promoter. Preferred promoters are able to drive high levels of RNA encoded by me coding region encoding the coat polypeptide Examples of such promoters are known in the art and include, for instance, the lac promoter, T7, T3, and SP6 promoters.

(75) The nucleotide sequences of the coding regions encoding coat polypeptides described herein are readily determined. These classes of nucleotide sequences are large but finite, and the nucleotide sequence of each member of the class can be readily determined by one skilled in the art by reference to the standard genetic code. Furthermore, the coding sequence of an RNA bacteriophage single chain dimer coat polypeptide comprises a site for insertion of HPV L2 peptide-encoding sequences. In a particular embodiment, the site for insertion of the HPV L2 peptide-encoding sequence is a restriction enzyme site.

(76) In a particular embodiment, the coding region encodes a single-chain dimer of the coat polypeptide, preferably a PP7 coat polypeptide. In a most particular embodiment, the coding region encodes a modified single chain coat polypeptide dimer, where the modification comprises an insertion of a coding sequence at least four amino acids at the insertion site. The transcription unit may contain a bacterial promoter, such as a lac promoter or it may contain a bacteriophage promoter, such as a T7 promoter and optionally a T7 transcription terminator.

(77) In addition to containing a promoter and a coding region encoding a fusion polypeptide, the RNA polynucleotide typically includes a transcription terminator, and optionally, a coat recognition site. A coat recognition site is a nucleotide sequence that forms a hairpin when present as RNA. This is also referred to in the art as a translational operator, a packaging signal, and an RNA binding site. Without intending to be limiting, this structure is believed to act as the binding site recognized by the translational repressor (e.g., the coat polypeptide), and initiate RNA packaging. The nucleotide sequences of coat recognition sites are known in the art. Other coat recognition sequences have been characterized in the single stranded RNA bacteriophages R17, GA, Q, SP, and PP7, and are readily available to the skilled person. Essentially any transcriptional terminator can be used in the RNA polynucleotide, provided it functions with the promoter. Transcriptional terminators are known to the skilled person, readily available, and routinely used.

(78) Synthesis

(79) As will be described in further detail below, the VLPs of the present invention may be produced in vivo by introducing transcription units into bacteria, especially if transcription units contain a bacterial promoter Alternatively VLPs synthesized in vitro in a coupled cell-free transcription/translation system.

(80) Assembly of VLPs Encapsidating Heterologous Substances

(81) As noted above, the VLPs of the present invention display a HPV L2 peptide-encoding sequence. These VLPs may be assembled by performing an in vitro VLP assembly reaction. Specifically, purified coat protein subunits are obtained from VLPs that have been disaggregated with a denaturant (usually acetic acid). The protein subunits are mixed with a heterologous substance. In a particular embodiment, the substance has some affinity for the interior of the VLP and is preferably negatively charged. This substance could include an adjuvant, including, but not limited to RNA, bacterial DNA (CpG oligonucleotides), cholera toxin subunit B, or E. coli lymphotoxin,

(82) Synthesis

(83) In a particular embodiment, the populations of the present invention may be synthesized in a coupled in vitro transcription/translation system using procedures known in the art (see, for example, U.S. Pat. No. 7,008,651, relevant portions of which are incorporated by reference herein). In a particular embodiment, bacteriophage T7 (or a related) RNA polymerase is used to direct the high-level transcription of genes cloned under control of a T7 promoter in systems optimized to efficiently translate the large amounts of RNA thus produced.

(84) Uses of VLPs and VLP Populations

(85) There are a number of possible uses for the VLPs and VLP populations of the present invention. As will be described in further detail below, the VLPs may be used as immunogenic compositions, particularly vaccines.

(86) Immunogenic Compositions

(87) As noted above, the VLPs of the present invention may be used to formulate immunogenic compositions, particularly vaccines. The vaccines should be in a form that is capable of being administered to an animal. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved. In this form, the composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat a condition or disorder. Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.

(88) Optionally, the vaccine of the present invention additionally includes an adjuvant which can be present in either a minor or major proportion relative to the compound of the present invention. The term adjuvant as used herein refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine of the present invention provide for an even more enhanced immune response. A variety of adjuvants can be used. Examples include complete and incomplete Freund's adjuvant, aluminum hydroxide, and modified muramyl dipeptide. Squalene has also been used as an adjuvant.

(89) Optionally, the vaccine of the present invention additionally includes an adjuvant which can be present in either a minor or major proportion relative to the compound of the present invention.

EXAMPLES

(90) The invention may be better understood by reference to the following non-limiting examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention. References corresponding to numerical reference citations are listed after the examples.

(91) Materials and Methods

(92) Bacteriophages PP7 and MS2.

(93) MS2 and PP7 coat protein single-chain dimers are highly tolerant of peptide insertions and produce correctly assembled VLPs displaying the peptide insertion on the surface of VLP in a highly dense, repetitive array. These VLPs are highly immunogenic and confer this high immunogenicity to heterologous peptides displayed on their surfaces. Here we describe VLPs displaying a peptide antigens derived from the Human Papillomavirus (HPV) minor capsid protein, L2. Such recombinant VLPs serve as a prophylactic vaccine to prevent infection by diverse HPV strains.

(94) The vaccines described below induced high titer antibody responses against L2 and protected against HPV challenge in a mouse model of infection. Similar techniques could also be used to construct MS2 VLPs that display L2 peptides.

(95) The Plasmids pP7K and p2P7K32.

(96) Overview of Plasmid Construction.

(97) Two general kinds of plasmid were constructed for the synthesis of PP7 coat protein in E coli (see FIGS. 1 and 2). The first (pP7K and p2P7K32) expresses coat protein from the lac promoter and is used (in combination with pRZP7see below) to assay for coat protein's tolerance of peptide insertions using translational repressor and VLP assembly assays. The second plasmid type (pETP7K and pET2P7K32) expresses the protein from the T7 promoter and transcription terminator. These plasmids produce large amounts of coat protein that assembles correctly into a VLP. They also produce coat-specific mRNA with discrete 5- and 3-termini for encapsidation into VLPs.

(98) Design of the Peptide Insertion Site.

(99) The three-dimensional structure of the PP7 capsid shows that it is comprised of a coat protein whose tertiary structure closely mimics that of MS2, even though the amino acid sequences of the two proteins show only about 12% sequence identity (47). The PP7 protein possesses an AB-loop into which peptides may be inserted following a scheme similar to the one we described previously for MS2 (38). As in the MS2 case, mutation of the PP7 coat sequence to contain a site for the restriction endonuclease KpnI facilitates insertion of foreign sequences in the plasmids called pP7K and pETP7K (FIG. 1). This modification resulted in the amino acid substitution (E11T) shown in FIG. 3. This substitution was well tolerated, since the mutant coat protein represses translation and assembles correctly into a VLP. Again following the MS2 example, it was assumed that the folding of a single chain dimer version of PP7 coat protein would be more resistant to AB-loop insertions than the conventional dimer. Its construction was described previously (6). The single-chain dimer was modified to contain a KpnI site only in the downstream copy of the coding sequence, producing p2P7K32 and pET2P7K32 (FIG. 1). In this design, peptides can be inserted at amino acid 11, but it should be noted that other specific insertion sites are possible, including in the amino terminus, in the carboxy terminus or anywhere within the AB-loop of the coat polypeptide, preferably at amino acids 8-11 of the AB loop. Insertion or replacement of amino acids within the AB-loop (or the amino acid or carboxy terminus of the coat polypeptide, preferably within the downstream subunit of the coat polypeptide) may be used to accommodate the L2 peptide.

Example 1

Design of L2-Displaying PP7 VLPs

(100) The construction of the expression plasmids p2P7K32 and pET2P7K32 have been described above. As explained, these plasmids code for the expression of a version of PP7 coat protein in which two copies of coat protein are genetically fused into a single-chain dimer. p2P7K32 and pET2P7K32 also contain a unique KpnI sites that allow for genetic insertion of sequences at amino acid 11 of the downstream copy of coat. To create the VLPs that display L2 peptides we designed PCR primers (shown in FIG. 2) that allowed us to clone L2-derived sequences into the AB-loop of PP7 coat. These sequences represented L2 amino acids 17-31 from different HPV isolates, including HPV16 (QLYKTCKQAGTCPPD) SEQ ID No. 15, HPV45 (DLYRTCKQSGTCPPD) SEQ ID No. 25, and HPV58 (QLYQTCKASGTCPPD) SEQ ID No. 19. This strategy was used to insert the corresponding L2 amino acids from other HPV types (shown in FIG. 2) into the PP7 single-chain dimer. The sequence of this region of L2 is relatively conserved across diverse HPV isolates (FIG. 4).

(101) The functionality of coat protein encoded by the resulting plasmids (including, for example, p2P7-16L2 p2P7-45L2, and p2P7-58L2) was tested by two assays. First, we assessed the translational repression activity of recombinant PP7 coat proteins. PP7 coat normally functions as a translational repressor, shutting off synthesis of the viral replicase by binding to a specific RNA hairpin structure containing its ribosome-binding site (the translational operator). We described previously the construction of pRZP7, a plasmid that fuses the PP7 translational operator to the E. coli lacZ gene, thus placing -galactosidase synthesis under control of coat protein's translational repressor activity (28). Because it confers resistance to a different antibiotic (chloramphenicol), and because it comes from a different incompatibility group (i.e. it uses the p15A replication origin), it can easily be maintained in the same E. coli strain as p2P7K32, which confers resistance to ampicillin and uses a colE1 origin. The expression of PP7 coat protein from p2P7K32 represses translation of -galactosidase expressed from pRZP7. This makes it easy to determine whether a given peptide insertion has interfered with the ability of coat protein to correctly fold, since defective coat proteins give blue colonies on plates containing the -galactosidase chromogenic substrate known as X-gal, whereas a properly functioning coat protein yields white colonies. All three recombinant coat proteins produced white colonies, indicating that the L2-recombinant coat proteins were functional.

(102) Second, we assessed the presence of VLPs in lysates of cells expressing a peptide-coat protein recombinant by electrophoresis on agarose gel of cells lysed by sonication. Ethidium bromide staining detects the RNA-containing VLP, whose presence can be confirmed by western blot analysis using anti-PP7 serum. Electrophoresis of the VLPs in an agarose gel shows that each construct contains RNA (it stains with ethidium bromide) and exhibits an altered electrophoretic mobility due to charge differences conferred by the inserted peptides (FIG. 5). Thus, all three recombinant single-chain dimer coat proteins formed VLPs.

Example 2

L2 Peptides Displayed on PP7 VLPs are Displayed to the Immune System and are Immunogenic

(103) Overview.

(104) In order to demonstrate that L2 peptides inserted into the PP7 AB-loop were indeed displayed on the surface of VLPs, we assessed the ability of a monoclonal antibody (mAb) RG-1; (20)) specific for the HPV16 L2 sequence to bind to recombinant 16L2-PP7 VLPs by ELISA. As shown in FIG. 6a, mAb RG-1 bound to 16L2-VLPs, but not to wild-type PP7 VLPs or PP7 VLPs that were modified to display the FLAG epitope (FLAG-VLPs). Moreover, as shown in FIG. 6b, mAb RG-1 bound to all eight of the L2-VLPs we produced, but not to wild-type PP7 VLPs.

(105) To test the immunogenicity of the VLPs, groups of three to nine mice were immunized with L2 displaying-VLPs or wild-type PP7 VLPs by intramuscular injection. Groups of three to nine mice were immunized intramuscularly with 10 g of VLPs plus incomplete Freunds Adjuvant (IFA). All mice were boosted with the same amount of VLPs two weeks later. Sera were collected before each inoculation and weekly for three to four weeks after the boost. Sera from the mice were tested, by end-point dilution ELISA, for IgG antibodies specific for synthetic L2 peptides representing HPV1, 5, 6, 16, or 18 (FIG. 7). Mice immunized with 1L2-, 5L2-, 6L2-, 11L2-, 16L2-, 18L2-, 45L2-, and 58L2-VLPs generated high-titer (geometric mean titer typically >10.sup.4) IgG responses against the corresponding peptide whereas no antibodies were detected in control mice. Thus, L2 peptides displayed on the surface of PP7 single-chain dimer VLPs display the high immunogenicity that is characteristic of other VLP-displayed antigens.

Example 3

PP7 VLPs Displaying a HPV16 L2 Peptide can Induce Neutralizing Antibodies that Protect Mice from Homologous and Heterologous Genital HPV Pseudovirus Challenge

(106) Overview.

(107) The 16L2-VLP vaccine we designed contains amino acids 17-31 from HPV16 L2, a region shown to contain one or more highly cross-reactive neutralizing epitopes (1, 20), suggesting that the 16L2 VLPs could potentially protect against HPV challenge. We demonstrated that 16L2-VLPs could protect mice from HPV challenge using a HPV pseudovirus/mouse genital challenge model, first reported by Roberts and colleagues (40).

(108) Additional Description.

(109) The 16L2-VLP vaccine we designed contains amino acids 17-31 from HPV16 L2, a region shown to contain one or more highly cross-reactive neutralizing epitopes (1, 20), suggesting that the 16L2 VLPs could potentially protect against HPV challenge. We assessed whether 16L2-VLPs could protect mice from HPV challenge using a HPV pseudovirus/mouse genital challenge model, first reported by Roberts and colleagues (40). Groups of five Balb/c mice were given two intramuscular injections of HPV16 L1-VLPs, wild type PP7 VLPs, or 16L2-VLPs, or adjuvant (IFA) alone, and then, three weeks after the boost, challenged intravaginally with a high dose (10.sup.8 IU) of HPV pseudovirus carrying a luciferase reporter. As a negative control, mice were mock-challenged with PBS. Infection was detected as a bioluminescent signal two days after the administration of pseudovirions, immediately after intravaginal instillation of the challenged mice with the reporter substrate, luciferin.

(110) As shown in FIG. 8, mice immunized with 16L2-VLPs were strongly (90%) protected from infection with the homologous pseudovirus, HPV16, whereas mice immunized with wild-type PP7 VLPs were not protected. We also tested whether vaccination with 16L2-VLPs could protect mice from genital infection with a heterologous HPV type. We chose HPV45 pseudovirus because it is not closely related to HPV16 and because its L2(17-31) sequence varies from the HPV16 sequence at three of the fifteen amino acid positions. Immunization with HPV16 L1 VLPs did not protect mice from HPV45 challenge. However, 16L2-VLP-immunized mice were protected (83%) from genital infection with HPV45 pseudovirus. Thus, 16L2-VLPs have potential as a pan-HPV vaccine.

Example 4

A Mixture of PP7 VLPs Displaying HPV L2 Peptides can Induce Antibodies that Protect Mice from Homologous and Heterologous Genital HPV Pseudovirus Challenge

(111) We also tested the immunogenicity of a combination vaccine consisting of all eight L2-PP7 VLPs that were constructed. Groups of mice were immunized with a mixture of equal amounts of 1L2-VLPs, 5L2-VLPs, 6L2-VLPs, 11L2-VLPs, 16L2-VLPs, 18L2-VLPs, 45L2-VLPs, and 58L2VLPs. Mice were immunized three times at two-week intervals with a 10 g dose and without exogenous adjuvant. Sera were collected before each inoculation and weekly for three to four weeks after the boost. Sera from the mice were tested, by end-point dilution ELISA, for IgG antibodies specific for synthetic L2 peptides representing HPV1, 5, 6, 16, or 18 (FIG. 9). Mice immunized with the mixture of L2-VLPs produced high-titer antibodies reactive with peptides representing 1L2, 5L2, 6L2, 11L2, 16L2, and 18L2. Thus, a mixture of L2-VLPs displayed on the surface of PP7 single-chain dimer VLPs is also highly immunogenic.

(112) We assessed whether the mixed L2-VLP vaccination could protect mice from HPV challenge using the HPV pseudovirus/mouse genital challenge model described above. Following immunization with the mixed L2-VLPs (as described above) or, as a negative control, wild-type PP7 VLPs, mice were challenged intravaginally with a high dose (10.sup.7-10.sup.8 IU) of HPV5, 6, 16, 18, 31, 45, 52, or 58 pseudovirus carrying a luciferase reporter. As shown in FIG. 10, immunization with mixed PP7 L2-VLPs protected mice from HPV5 pseudovirus infection (98.2% reduction in signal), HPV6 pseudovirus infection (98.6% reduction in signal), HPV16 pseudovirus infection (99.7% reduction in signal), HPV18 pseudovirus infection (99.1% reduction in signal), HPV31 pseudovirus infection (99.9% reduction), HPV45 pseudovirus infection (99.2% reduction), HPV52 pseudovirus infection (98.5% reduction in signal), and HPV58 pseudovirus infection (93.1% reduction in signal). Thus, mixed L2-VLPs also have potential as a pan-HPV vaccine.

Example 5

Other Regions of HPV L2 can be Displayed on the Surface of Bacteriophage VLPs and are Immunogenic

(113) Using the methods described elsewhere in this application, we generated recombinant PP7 VLPs that display HPV16 L2 amino acids 35-50 (amino acid sequence: KVEGKTIADQILQYGS) SEQ ID No. 36 and amino acids 51-65 (sequence: MGVFFGGLGIGTGSG), SEQ ID No. 37. These VLPs were used to immunize mice, and, following two immunizations, antibodies against a synthetic peptides representing A) HPV16 L2 amino acids 34-52 or B) HPV16 L2 amino acids 49-71 were measured by end-point dilution ELISA. As shown in FIG. 11, both recombinant VLPs induced high-titer IgG antibodies that recognized the L2 peptides.

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