WO2015068101A1

WO2015068101A1 - Papillomavirus vaccine formulations

Info

Publication number: WO2015068101A1
Application number: PCT/IB2014/065786
Authority: WO
Inventors: Semburakkiannan Jaganathan KILVANI; Vijayarangam DAMOTHARAN
Original assignee: Shantha Biotechnics Pvt Ltd
Current assignee: Shantha Biotechnics Pvt Ltd
Priority date: 2013-11-06
Filing date: 2014-11-04
Publication date: 2015-05-14
Anticipated expiration: 2016-05-06

Abstract

The invention relates to stable papillomavirus L2 (minor capsid) protein vaccine compositions or formulations for the prevention and/or treatment of papillomavirus infections. Preferably, the papillomavirus L2 protein comprises of the N-terminal fragments of L2 protein from one or more papillomavirus types, usually in the form of a fusion protein along with pharmaceutically acceptable excipients. The invention also provides a method of detecting antibodies that recognize papillomavirus L2 protein and ELISA kits.

Description

PAPILLOMAVIRUS VACCINE FORMULATIONS

FIELD OF THE INVENTION

The invention relates to the general field of bio-pharmaceuticals. More particularly, the invention provides stable papillomavirus L2 (minor capsid) protein vaccine compositions or formulations for the prevention and/or treatment of papillomavirus infections. The invention also provides method of detecting antibodies that recognize papillomavirus L2 protein.

BACKGROUND

Papillomaviruses have been known to cause infection in a variety of animals, including humans. Human papillomaviruses (HPVs), in particular, are responsible for causing genital warts and intra epithelial neoplasia of the vulva, cervix or anus, which may lead to cervical cancer in women. HPVs are increasingly found to infect men also, although the incidence of their infection has not been extensively studied compared to women. Some oropharyngeal, anal, and penile cancers have been found to be etiologically related to HPV infection in men (Gillison, M. L. et al. Cancer 2008 113 :3036-3046).

Vaccines have been shown to prevent HPV infection. Currently two vaccines are available in the market: Gardasil and Cervarix, which both comprise virus like particles (VLPs) formed from LI (major capsid) protein. Although VLP based HPV vaccines have been demonstrated to be efficacious, they only raise a type specific immune response and hence do not confer protection against many of the HPV types that are known to cause cervical cancer. It is reported that the non vaccine HPV types currently account for about 30% of cervical cancers (Vincenzo, Rosa De et al. Gynecologic Oncology 2013 130:642-651). Efforts are being made to develop vaccines with additional VLPs from HPV types not included in the currently marketed HPV vaccines, but this will only add cost to the already expensive VLP based vaccines making them unaffordable to large segments of population who are susceptible to HPV infection. Further, there is also a possibility that large scale use of the type specific VLP based HPV vaccines may result in a shift in prevailing HPV types currently targeted by the marketed vaccines to less prevalent HPV types, a phenomenon commonly known as "type replacement" (Kahn, Jessica A. et al. Pediatrics 2012 130: 1-8).

In contrast to the predominately type specific response directed against LI based vaccines, the minor capsid protein (L2) of papillomavirus has been shown to elicit antibodies that neutralize both homologous and heterologous HPV types owing to its conserved amino acid sequences (Kawana, Kei et al. Journal of Virology 1999 73 :6188-6190; Roden, Richard et al. Virology 2000 270:254-257). Fragments of L2 peptides are also suitable functional L2 protein derivatives capable of raising an immune response. WO 1994023037 describes a composition having L2 protein or its fragment from bovine papillomavirus (BPV) or its GST fusion protein with aluminium adjuvant for prophylaxis or therapeutic purpose. WO2009059325 describes multitype HPV L2 composition with different adjuvants such as alum, CpG and GPL Such compositions are not suitable for use as vaccines unless they are appropriately formulated. Considering the importance of papillomavirus L2 protein in inducing broadly cross neutralizing antibodies, it is necessary that L2 protein of papillomavirus be formulated such that it remains stable during the shelf life.

Development of stable protein formulations is one of the critical steps in any protein based pharmaceutical to be used as vaccine. Proteins are marginally stable and hence can be easily denatured by various stress encountered in a solution. Various chemical reactions, such as disulfide scrambling, non-disulfide crosslinking, deamidation, peptide cleavage, oxidation, hydrolysis etc., may affect the stability of protein and render them unsuitable for being used as vaccines. Further, development of stable formulation of a fusion protein which is generally constructed by splicing two or more proteins or their domains to obtain new non- natural polypeptide or synthetic fusion protein with combined functionalities of the parental proteins or peptides, for example, the multitype HPV L2 protein, can be a real challenge as such synthetic fusion proteins are known to be potentially unstable because of the coming together of multiple domains and the absence of stabilizing structures, especially at the junctions i.e., the peptide bonds joining two or more peptides or domains since they have not been selected by nature through an evolutionary process (Fast, Jonas L. et al. Biochemistry 2009, 48: 11724-11736).

Therefore, pharmaceutical additives must be carefully tested to ensure that they do not cause instability or have other negative effects on the protein and at the same time are safe when administered to humans. Additionally, ingredients used to stabilize the protein may cause problems with proteins stability over time or with protein stability in changing environments during storage. Therefore, a careful choice should be made with respect to the selection of appropriate excipients to prevent any negative impact the excipients might have on the protein antigen during storage. Although the factors that contribute to the stabilization of protein has provided valuable information for stabilizing proteins, but the structural differences among different proteins are so significant that generalization of a universal stabilization strategies have never been successful. For example, gelatin and β-cyclodextrin have been found to be better stabilizers for tetanus toxoid and diphtheria toxoid, but not for hepatitis B surface antigen (HBsAg) (Chang, An-Cheng et al. Pharmaceutical Sciences 1996, 85: 129-132; Alpar, H.O. et al. Advanced Drug Delivery Reviews 2001, 51 : 173-201; Jaganathan, K.S. 2006, PhD Thesis: Development and Characterization of Novel Delivery System(s) for Mucosal Vaccination through Nasal Route, Dr. H.S. Gour University, Sagar, M.P., India). Similarly, trehalose and sucrose have been found to be better stabilizers for HBsAg when compared to tetanus toxoid and diphtheria toxoid (Jaganathan, K.S. et al. Journal of Pharmacy & Pharmacology 2004, 56: 1243-1250). Very often proteins are needed to be evaluated individually and stabilized on a case to case basis taking into account the nature of each protein. Components used to stabilize protein based vaccine formulations may be known, however, particular components useful to stabilize protein based vaccines must be determined experimentally.

Thus, a need exists for formulations conferring stability to papillomavirus L2 protein for developing effective vaccines against papillomavirus infection. Therefore, in order to develop a commercially viable papillomavirus L2 protein based vaccine, a stable formulation is needed.

SUMMARY OF THE INVENTION

The present invention discloses a stable papillomavirus vaccine formulation. In accordance with the invention, papillomavirus L2 protein or its fragment thereof of any papillomavirus type may be used. Preferably, the papillomavirus L2 protein comprises of the N-terminal fragments of L2 protein from one or more papillomavirus types, usually in the form of a fusion protein. The invention also provides a method of detecting antibodies that recognize papillomavirus L2 protein and ELISA kits.

In one of the embodiments, the invention relates to a papillomavirus vaccine formulation with pharmaceutically acceptable excipients.

In one of the embodiments, the invention relates to a papillomavirus vaccine formulation comprising:

a papillomavirus L2 protein adsorbed onto an aluminum adjuvant,

a buffer providing a pH of about 4.0 to about 7.5,

a stabilizer, and optionally

a tonicity modifier.

In one of the embodiments, the stabilizer is selected from the group consisting of a surfactant, a chelating agent, a sugar, an amino acid or combination thereof. In one of the embodiments, the invention relates to a papillomavirus vaccine formulation comprising:

a papillomavirus L2 protein adsorbed onto an aluminum adjuvant,

a buffer providing a pH of about 4.0 to about 7.5,

a surfactant,

a chelating agent, and optionally

a tonicity modifier.

In one of the embodiments, the papillomavirus L2 protein comprises N-terminal fragments of animal or human papillomavirus (HPV) L2 protein.

In one of the embodiments, the papillomavirus L2 protein comprises N-terminal fragments of human papillomavirus (HPV) L2 protein belonging to one or more HPV types.

In one of the embodiments, the papillomavirus L2 protein comprises a multitype or multimeric human papillomavirus (HPV) L2 protein or polypeptide obtained by joining N-terminal fragments of HPV L2 protein belonging to one or more HPV types such as, but not limited to, HPV 1, HPV2, HPV5, HPV6, HPVl l, HPV16, HPV18, HPV31, HPV33, HPV34, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV 68, HPV68, or HPV73,.

In one of the embodiments, the multitype or multimeric human papillomavirus (HPV) L2 protein or polypeptide is obtained by joining 11-88 amino acids from the N-terminus of HPV L2 protein of at least five different HPV types such as, but not limited to, HPV6, HPV 16, HPV18, HPV31, and HPV39.

a multitype or multimeric papillomavirus L2 protein adsorbed onto an aluminum adjuvant, a buffer providing a pH of 4.0 to 7.5,

a surfactant,

a chelating agent, and optionally

a tonicity modifier.

In one of the embodiments, the buffer is selected from the group consisting of acetate, phosphate, citrate, succinate or combination thereof.

In one of the embodiments, the surfactant is a non-ionic surfactant selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or combination thereof.

In one of the specific embodiments the invention relates to a papillomavirus vaccine formulation comprising:

multitype or multimeric papillomavirus L2 protein adsorbed onto an aluminum adjuvant,

acetate buffer providing a pH of 4.5 to 5.5,

non-ionic surfactant,

EDTA, and

sodium chloride.

In one of the embodiments, the invention relates to a method of making papillomavirus vaccine formulation comprising the steps of:

a. adjusting the concentration of a multitype or multimeric papillomavirus L2 protein to a biologically effective amount using a buffer comprising a stabilizer selected from the group the group comprising a chelating agent, a surfactant, and optionally an amino acid or a sugar, or combination thereof,

b. adsorbing the multitype or multimeric papillomavirus L2 protein of step (a) onto an aluminum adjuvant, c. incubating the adsorbed multitype or multimeric papillomavirus L2 protein of step (b) for maturation, and optionally

d. adjusting the tonicity of the formulation using a tonicity modifier. In certain embodiments the invention also relates to a method of detecting antibodies against papillomavirus L2 protein the method comprising:

a. coating the wells of microtiter plate with papillomavirus L2 protein, b. blocking the wells with a blocking solution,

c. contacting the sample with the papillomavirus L2 protein coated

microtiter plate, and

d. detecting the antibody in the sample using secondary antibody.

In another embodiment the invention provides an ELISA kit comprising:

a. a microtiter plate coated with papillomavirus L2 protein,

b. a blocking solution,

c. a wash buffer,

d. a diluent,

e. a secondary antibody,

f. a substrate and,

g. a stop solution.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate embodiments of the invention and, together with the description, serve to explain the invention. These drawings are offered by way of illustration and not by way of limitation.

Figure la - Effect of different stabilizers (cf. Example 2a) on the stability of papillomavirus L2 protein (11-88 x8) at 37 °C.

Figure lb - Effect of different stabilizers (cf. Example 2a) on the stability of papillomavirus L2 protein (11-88 x8) at 5±3 °C. Figure 2 - SEC-HPLC profile of different papillomavirus L2 protein (11-88 x5) formulations (cf. Example 3) during real time (5±3 °C) accelerated (25±2 °C) and stress (37±2 °C) stability studies.

Figure 3 - Percent adsorption of papillomavirus L2 protein (11-88 x5) onto aluminum adjuvant.

Figure 4 - Immunogenicity of papillomavirus L2 protein (11-88 x5) formulation (cf. Example 4).

Figure 5a - Effect of different buffers (cf. Example 2b) on the stability of papillomavirus L2 protein (11-88 x5) formulations under stress conditions (37 °C).

Figure 5b - Effect of different buffers (cf. Example 2b) on the stability of papillomavirus L2 protein (11-88 x5) formulations under accelerated conditions (25 °C). Figure 5c - Effect of different buffers (cf. Example 2b) on the stability of papillomavirus L2 protein (11-88 x5) formulations under real time conditions (5±3 °C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described with reference to the tables / figures etc. and specific embodiments; this description is not meant to be construed in a limiting sense. Various alternate embodiments of the invention will become apparent to persons skilled in the art, upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of person skill in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.

The singular forms "a", "an" and "the" as used in the specification also include plural aspects unless the context dictates otherwise. Similarly, any singular term used in the specification also mean plural or vice versa unless the context dictates otherwise.

It must be noted that the words "comprising" or any of its forms such as "comprise" or "comprises", "having" or any of its forms such as "have" or "has", "including" or any of its forms such as "include" or includes", or "containing" or any of its forms such as "contain" or "contains" are open-ended and do not exclude additional unrecited elements or method steps.

Wherever any quantity or range is stated one skilled in the art will recognize that quantity or range within 10 or 20 percent of the stated values can also be expected to be appropriate, i.e., where 20% is stated, a range of from 16-18% to 22-24% is implicit and can be appropriate.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed or as described throughout the present specification.

The term "adjuvant", as used herein, refers to agents or substances that modulate the immunogenicity of an antigen. Modulating the immunogenicity includes enhancing the magnitude, and duration of an immune response stimulated by an antigen.

The term "buffer" as used herein, includes those agents that maintain the pH of a solution, e.g., a formulation, in a desired range. In one of the embodiments pH of the buffer is between about 4.0 to 7.5. In another embodiment the buffer used is selected from acetate, citrate, phosphate, succinate, phthalate, or combination thereof, or preferably an acetate buffer. The term "papillomavirus" as used herein means any of the animal or human papillomaviruses such as, but not limited to, bovine papillomavirus (BPV), cottontail rabbit papillomavirus (CRPV), deer papillomavirus (DPV), European elk papillomavirus (EEPV), canine oral papillomavirus (COPV), Rhesus monkey papillomavirus (RhPV), rabbit oral papillomavirus (ROPV), or any of the human papillomavirus (HPV) types.

The term "human papillomavirus", "human papillomavirus types" or "HPV types" as used herein means any of the HPV type that infect human host and includes, but not limited to, HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV 12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV 25, HPV26, HPV27, HPV28, HPV29, 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, HPV110, HPV111, HPV112, HPV113, HPV114, HPV115, HPV116, HPV117, HPV118, HPV119 and HPV120.

The term "L2 protein" or "minor capsid protein" has been used interchangeably herein to refer to the minor protein of the capsid of papillomaviruses that localizes along the inner surface of the virion within the central cavities beneath the LI (major capsid protein) capsomeres.

The term "protein" or "peptide" or "polypeptide" has been used interchangeably herein and means a polymer of amino acids linked through peptide bonds, but does not imply any limitation as to a specific length. The term also includes fusion proteins, muteins, analogs or modified forms.

The term "multitype or multimeric L2 protein" or "multitype or multimeric L2 polypeptide" has been used interchangeably to mean a fusion protein formed by joining fragments of L2 protein obtained from one or more papillomavirus types (animal and/or human papillomavirus types). Each fragment may be as short as ten amino acids in length and may extend up to any length of papillomavirus L2 polypeptide provided that it is not a full length papillomavirus L2 protein. Such multitype or multimeric L2 protein may generally be represented as "peptide x(n)" wherein the "peptide" represents a fragment of L2 protein (generally beginning from 11^th amino acid of the full length L2 protein and extending up to the 300^th amino acid or less such that each such fragment is at least ten amino acids long) while the number "(n)" following "x" represents different "HPV types" (which may usually be two or more) from which the fragments of L2 protein are derived, or alternately the fragment of L2 protein may be repeated as many number of times as represented by the number "(n)" if belonging to only one HPV type (for example, 11-200 x3, 11-88 x5, 11-88 x8, 17-36 x22 and so on, wherein the number following "x" either represents as many different HPV types or number of times the fragment of L2 protein repeats if represented by only one HPV type). Such fragments are typically joined by peptide bonds. In non limiting embodiments the multitype L2 protein or polypeptides may be prepared by the methods described in WO2009059325 and Jagu, Subhashini et al. (Journal of National Cancer Institute 2009, 101 :782-792; and Journal of Virology 2013 87(11):6127-6136).

A "fragment" of a reference polypeptide refers to a contiguous stretch of amino acids from any portion of the reference polypeptide. A fragment may be of any length that is less than the length of the full length reference polypeptide. The term "stable formulation" as used herein means a formulation in which the antigen retains it physical stability, chemical stability and/or biological activity upon storage, for example, shelf life. Stability of the antigen may be measured by techniques known to the person skilled in the art, for example, by SDS PAGE, SEC-HPLC, Dynamic Light Scattering, Mass Spectrometry, or immunogenicity assays.

The term "stabilizer" as used herein means any excipient that is added to the formulation to protect or prevent the papillomavirus L2 protein from undergoing denaturation, degradation, aggregation, oxidation, reduction and/or inactivation to an extent that the formulation still remains suitable for being administered as vaccine.

The term "excipient" as used herein means any component of the formulation other than the antigen and adjuvant, but not limited to, buffering agents, stabilizers, tonicity modifiers, surfactants, chelating agents, salts, anti-oxidant, diluents, and/or preservative or combinations thereof. The term "formulation" or "composition" has been used interchangeably to mean a composition comprising papillomavirus L2 protein adsorbed onto an aluminum adjuvant with pharmaceutically acceptable excipients.

The term "vaccine" as used herein, refers to a pharmaceutically acceptable formulation of at least one antigen. Such pharmaceutically acceptable formulations of an antigen may also include adjuvants, excipients, diluents, etc., that enhance the activity, stability, etc., of a formulation or administration.

The term "antigen" as used herein means a component of the formulation which is responsible for stimulating an immune response i.e., generation of antibodies against it when administered to an animal or human, such as papillomavirus L2 protein as described herein.

The term "biologically effective amount" as used herein means an amount of the antigen, such as papillomavirus L2 protein, that is needed to stimulate, initiate, or induce the desired immunologic, often beneficial result. Description

Any fragment of the papillomavirus L2 protein may be used to prepare the formulation of the invention. The papillomavirus L2 protein of the invention may conveniently be produced by recombinant means. Bacterial cells genetically engineered to express truncated papillomavirus L2 protein may be constructed using N-terminal fragments of papillomavirus L2 protein from any papillomavirus types using recombinant techniques known in the art (Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, Frederick M. et al, Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1998)). Exemplary papillomavirus L2 protein or peptides are described in PCT Publication Nos. WO2006083984 and WO2009059325. Any length of papillomavirus L2 peptide from any papillomavirus type from N-terminus may be used provided that it is not a full length papillomavirus L2 protein. It is advisable to use papillomavirus L2 protein fragments that may at least be about 10 amino acids residues in length having a sequence corresponding to the amino acid sequence from the N-terminal amino acids 11-300 of full length papillomavirus L2 protein, or at least about 10 amino acids residues in length having a sequence corresponding to the amino acid sequence from the N-terminal amino acids 11- 200 of full length papillomavirus L2 protein, or at least about 10 amino acids residues in length having a sequence corresponding to the amino acid sequence from the N-terminal amino acids 11-100 of full length papillomavirus L2 protein, or at least about 10 amino acids residues in length having a sequence corresponding to a sequence from the N-terminal amino acids 11-88 of full length papillomavirus L2 protein. Preferably fragments of L2 protein from two or more papillomavirus types may be cloned in expression vectors and transformed into a suitable host to express as a fusion protein or polypeptide of papillomavirus L2 protein to be used as an antigen of the formulation in accordance with the invention. It is particularly preferred that a multitype HPV L2 polypeptide is expressed as a fusion polypeptide or protein such as those described in WO2009059325 and Jagu, Subhasini et al. (Journal of National Cancer Institute 2009 101 :782-792). Such fusion proteins may also be expressed in insect, yeast or mammalian cells currently used to express heterologous genes.

Standard protein purification techniques generally employed to isolate and purify recombinant proteins can be used to isolate and purify papillomavirus L2 protein as known in the art (Deutscher M.P., Editor. Guide to Protein Purification, Methods Enzymology 182, Academic Press Inc, London; Coligan, John E., Editor. Current Protocols in Protein Science, John Wiley and Sons). For example, the multitype papillomavirus L2 protein may be purified as described by Jagu, Subhasini et al. (Journal of Virology 2013 87(11):6127-6136; WO2009529325), or by other methods known in the art such as the techniques described in WO 199626277. Such L2 protein or polypeptides are ideal for being formulated in accordance with the invention. To prepare the formulation of the invention, papillomavirus L2 protein or peptides are formulated with appropriate excipients and adjuvanted. Excipients

Protein based formulations use a variety of excipients for at least the following purposes: including, for example, as diluents, vehicles, buffering agents, stabilizers, tonicity modifiers, surfactants, anti-oxidants, metal ion sources, chelating agents and/or preservatives. In certain cases some excipients may have dual functions, i.e., one excipient may confer more than one of the above functions onto a formulation of the invention, for example, sugars can act both as stabilizers and tonicity adjusting agents. Moreover, different types of excipients can be combined into a single formulation. Accordingly, a formulation of the invention may contain a single excipient, or may contain two or more different types of excipients. Thus two or more excipients can be included in a formulation of the invention to perform more than one of the above or other functions. For example, an excipient can be included as a component in a formulation of the invention to change, adjust, or optimize the osmolality of the formulation, thereby acting as a tonicity modifier. Similarly, a tonicity modifier and a surfactant can both be included in a formulation of the invention to adjust the osmolality and control aggregation. For example, sucrose and polysorbates can be used to stabilize proteins, adjust tonicity, and control aggregation or prevent adsorption respectively in a formulation. Similarly, citric acid can be used as a buffering agent as well as a chelating agent for trace metal in a dual role in the formulation.

Excipients of the invention may include, for example, polyols such as, but not limited to, sorbitol, mannitol, glycerol, xylitol, maltitol, lactitol, erythritol or threitol; sugars such as, but not limited to, sucrose, lactose, maltose, trehalose, fucose, cellobiose, melibiose, octulose, ribose or dextrose; polymers such as, but not limited to, polyethylene glycol; salts such as, but not limited to, NaCl, KC1 or calcium phosphate; amino acids such as, but not limited to, glycine, alanine, lysine, arginine, cysteine, methionine or glutamic acid; surfactants such as, but not limited to, polysorbates or polyethylene oxide polypropylene oxide block copolymers; buffer salts such as, but not limited to, acetate, phosphate, citrate, succinate; chelating agents, preservatives or combinations thereof, as well as saline and water.

Buffers

A buffer component of a formulation in accordance with the invention can be prepared to demonstrate effective buffering capacity within a pH range of between about pH 4.0 to about pH 7.5. Those skilled in the art will understand that much of the strength of the buffering capacity of a buffer will decrease outside of about one pH unit of its pKa and can easily determine whether inclusion of a particular buffer below a pH of about 4.0 or above a pH of about 7.5 is useful in a formulation of the invention. Non-limiting examples of buffers that can be used in a formulation described herein include acetate buffer, phosphate buffer, citrate buffer, succinate buffer, phthalate buffer or combination thereof. pH ranges of a buffer which can stabilize papillomavirus L2 protein, for example, the multitype or multimeric L2 protein, can include pH ranges between about 4.0- 7.5. Thus the formulation of the invention can be contemplated to have a pH of 7.4, a pH of 7.3, a pH of 7.2, a pH of 7.1, a pH of 7.0, a pH of 6.9, a pH of 6.8, a pH of 6.7, a pH of 6.6, a pH of 6.5, a pH of 6.4, a pH of 6.3, a pH of 6.2, a pH of 6.1, a pH of 6.0, a pH of 5.9, a pH of 5.8, a pH of 5.7, a pH of 5.5, a pH of 5.4, a pH of 5.3, a pH of 5.2, a pH of 5.1, a pH of 5.0, a pH of 4.9, a pH of 4.8, a pH of 4.7, a pH of 4.6, a pH of 4.5, a pH of 4.4, a pH of 4.3, a pH of 4.2, a pH of 4.1, or a pH of 4.0.

In one of the embodiments, the buffer provides a pH range between about pH 4.5 to 7.0, preferably about pH 5.0 to 7.0. In one of the embodiments, the buffer provides a pH range between about pH 4.5 to 6.5, preferably about pH 4.5 to 5.5.

In one of the embodiments, the buffers that may be used to maintain the pH are selected from the group consisting of acetate buffer, succinate buffer, phosphate buffer, citrate buffer or combination thereof.

In one of the preferred embodiments, acetate buffer is used to maintain pH of the formulation.

Surfactants

Surfactants are commonly used excipients in many protein based formulations. The presence of surfactants decreases the surface tension of the solvent, and thus reduces the adsorption of protein molecules to the walls of the container and also prevents any protein denaturation events at the liquid-liquid or liquid-gas interfaces, thereby exercising a stabilizing effect on the protein. Surfactants of the invention may include, but are not limited to, for example, alkyl sulfates such as SDS; alkyltrimethylammonium salts such as CTAB; Polyoxypropylene glycol alkyl ethers; Polyoxyethylene glycol octylphenol ethers such as Triton; Polyoxyethylene glycol sorbitan alkyl esters such as polysorbates/tweens such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, etc.; block copolymers of polyethylene glycol and polypropylene glycol such as poloxamer 188. Appropriate surfactant may be included in the formulation of the invention to enhance or promote the stability of papillomavirus L2 protein, for example the multitype or multimeric L2 protein, by reducing aggregation and/or adsorption. Adsorption to surfaces usually results in a drop in protein concentration or loss of activity of the protein while aggregation can compromise biological activity or may even give rise to undesired immunologic responses. The presence of surfactants in the formulation can overcome such loses and altered immunologic response. Non-ionic surfactants are generally preferred in protein stabilization. Low concentrations of non-ionic surfactant are often sufficient to prevent or reduce protein surface adsorption and/or aggregation due to their relatively low critical micelle concentration. Sorbitan fatty acid esters (polysorbates) are surfactants which exhibits a wide range of hydrophilic and emulsifying characteristics. They can be used individually or in combination with other surfactants to provide the desired stability to the papillomavirus L2 protein. Accordingly polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80) are preferred non-ionic surfactant in accordance with the invention.

In one of the embodiments, polysorbate 80 in a range of at about 0.001% to about 0.01%) is used to protect or reduce papillomavirus L2 protein from adsorption or aggregation. In some other embodiments, polysorbate 80 is used, at about 0.002%> to about 0.008%, or at about 0.003% to about 0.007%, or at about 0.005% to about 0.006%) in accordance with the invention.

Sugars

Sugars are often used to stabilize proteins. In accordance with the invention, sugars such as sucrose, maltose, raffinose, trehalose, lactose and combinations thereof can be useful for imparting stability to papillomavirus L2 protein, for example, the multitype or multimeric L2 protein. The sugar is preferably added in a protein-stabilizing effective amount. On a weight percentage basis the amount of sugar that can be contained in the formulation of the invention is in the range of about 1 to about 30 weight percent. Concentration of sugar that may be used in the formulation may be governed by the viscosity and osmolality desired for the formulation as well as the stability offered to the papillomavirus L2 protein. Additionally, the upper limit of the concentration of sugar can be dictated by the ability of a formulation to undergo required filtration or processing steps. In one of the embodiments sugar is present at a concentration below 25 weight percent. Such a concentration also include concentrations below 20 weight percent, below 15 weight percent, below 10 weight percent, below 9 weight percent, below 8 weight percent, below 7 weight percent, below 6 weight percent, below 5 weight percent, below 4 weight percent, below 3 weight percent, below 2 weight percent, or 1 weight percent.

In one of the embodiments, sugar is present at a concentration between about 1- 20 weight percent, between about 1-15 weight percent, between about 1-10 weight percent, or preferably about 5 weight percent.

Osmolality

In certain embodiments, the composition exhibits a physiological osmolality. As used herein, the phrase "physiological osmolality" means the composition, when administered to a patient does not cause irritation or pain. A suitable range for the osmolality according to certain embodiments is between about 250 and 350 mOsm/kg. In one of the embodiments osmolality of the present formulation is adjusted, for example, by the use of appropriate tonicity modifiers. Suitable tonicity modifiers include glycerin, lactose, mannitol, dextrose, sodium chloride, potassium chloride, sorbitol, trehalose, sucrose, raffinose, maltose and others known to those ordinary skill in the art. In one of the embodiments, the tonicity of the formulation approximates that of the tonicity of blood or plasma.

Useful papillomavirus L2 protein formulations include tonicity modifiers in an amount required to bring osmolality of the composition into an acceptable range (between about 250 and 350 mOsm/kg). Accordingly, the amount and type of tonicity modifier to be included in a formulation of the invention may be selected based on the desired osmolality of the final formulation as well as the amounts and osmolality of other components to be included in the formulation. For example, 0.9% w/v NaCl can achieve an osmolality range of the invention while about 1.19% w/v KC1 is achieved to attain the same osmolality. Similarly, about 0.6% w/v NaCl along with 3-6% sucrose or about 3-6% trehalose can be used to achieve the desired osmolality of the formulation.

Chelating agents

Chelating agents are often used to sequester ions which might have negative impact on the protein antigen, for example metal catalyzed oxidation of protein. Chelating agent such as, but not limited to, EDTA, EGTA, Citric Acid etc., may be used. Chelating agent when used in accordance with the invention may be in the range of about 0.1 mM to about 10 mM, preferably between about 1 mM to about 5 mM, most preferably between about 1 mM to about 3 mM.

In one of the embodiments EDTA at about 3 mM is used a chelating agent.

Table 1 - Different papillomavirus vaccine formulations

Adjuvants

Adjuvants are used to enhance or modulate the intrinsic immunogenicity of an antigen. They are often used to increase the immune response of a vaccine by stimulating the immune system, thereby conferring enhanced immunity against the disease. Many substances, both natural and synthetic, have been shown to function as adjuvants. For example, adjuvants may include, but are not limited to, inorganic salts, squalene mixtures, muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, certain emulsions, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, immunostimulating complexes (ISCOMs), cytokine adjuvants, MF59 adjuvant, lipid adjuvants, mucosal adjuvants, certain bacterial exotoxins and other components, certain oligonucleotides, PLG, and others.

In one of the embodiments the adjuvant used with the papillomavirus L2 protein, for example the multitype or multimeric L2 protein, antigen is an inorganic salt, preferably an aluminium salt such as aluminum hydroxide or aluminum phosphate. A variety of factors may affect the ability of antigens to adsorb to the adjuvants in vaccine compositions, including for example, the charge of both the antigen and the adjuvant, pH, temperature, ionic strength, presence of excipients, and other factors. In one of the embodiments the preferred adjuvant is aluminum phosphate. Before adsorption, it is preferable to adjust the concentration of the papillomavirus L2 protein to a biologically effective amount , either by dilution or concentration (for example, the papillomavirus L2 protein may be diluted in acetate buffer pH 5.0±0.5 to adjust the strength required for immunization).

The papillomavirus L2 protein may be adsorbed onto an adjuvant at 2-37 °C for 15-60 min. In one of the embodiments, the papillomavirus L2 protein is adsorbed at 5±3 °C for about 15 min, about 30 min, or about 60 min. In another embodiment the papillomavirus L2 protein is adsorbed at 25 °C for about 15 min, about 30 min, or about 60 min. In yet another embodiment the papillomavirus L2 protein is adsorbed at 37 °C for about 15 min, about 30 min or about 60 min. In a preferred embodiment the papillomavirus L2 protein is adsorbed at 25±2 °C for 45-60 min. The adsorbed papillomavirus L2 protein may be incubated at 5±3 °C or at room temperature (25±2 °C) for 8-24 h for maturation. Amount of antigen adsorbed onto an adjuvant plays an important role in increasing the immune response of a vaccine. In one of the embodiments the invention provides a papillomavirus L2 protein formulation wherein at least about 90% or more, at least about 80% or more, at least about 70% or more, at least about 60% or more, at least about 50% or more papillomavirus L2 protein is adsorbed onto aluminium adjuvant [Figure 3].

In non-limiting embodiments of papillomavirus L2 protein based vaccine formulations, for example the multitype or multimeric L2 protein based vaccine formulations the papillomavirus L2 protein/polypeptide is present at biologically effective amounts. Biologically effective amount as used herein means an amount of the papillomavirus L2 protein needed to stimulate or initiate the desired immunologic, often beneficial result. In some of the preferred embodiments the biologically effective amount of papillomavirus L2 protein is in the range of about 25 μg to about 1000 μg. In specific embodiments of the invention the papillomavirus L2 protein/polypeptide is present in an amount at least about 25 μg, at least about 50 μg, at least about 75 μg, at least about 100 μg, at least about 125 μg, at least about 150 μg, at least about 150 μg, at least about 175 μg, at least about 200 μg, at least about 225 μg, at least about 250 μg, at least about 275 μg, at least about 300 μg, at least about 325 μg, at least about 350 μg, at least about 375 μg, at least about 400 μg, at least about 425 μg, at least about 450 μg, at least about 475 μg, at least about 500 μg, at least about 525 μg, at least about 550 μg, at least about 575 μg, at least about 600 μg, at least about 625 μg, at least about 650 μg, at least about 675 μg, at least about 700 μg, at least about 725 μg, at least about 750 μg, at least about 775 μg, at least about 800 μg, at least about 825 μg, at least about 850 μg, at least about 875 μg, at least about 900 μg, at least about 925 μg, at least about 950 μg, at least about 975 μg, or at least about 1000 μg. The present invention further provides methods of preventing papillomavirus infection by administering a formulation or composition of the invention to human subjects or use of the formulation or composition in the manufacture of a medicament for the prevention of disease caused by papillomavirus. In one of the embodiment the method involves administering a biologically effective amount of formulation to a human subject, wherein the formulation comprises of papillomavirus L2 protein adsorbed onto an aluminum adjuvant, a buffer providing a pH of 4.0 to 7.5, a stabilizer and optionally a tonicity modifier.

In an another embodiment the method involves administering a biologically effective amount of formulation to a human subject, wherein the formulation comprises of papillomavirus L2 protein adsorbed onto an aluminum adjuvant, a buffer providing a pH of about 4.0 to about 7.5, a surfactant, a chelating agent, and optionally a tonicity modifier.

In yet another embodiment the method involves administering a biologically effective amount of formulation to a human subject, wherein the formulation comprise of multitype or multimeric papillomavirus L2 protein adsorbed onto an aluminum adjuvant, a buffer providing a pH of about 4.0 to about 7.5, a surfactant, a chelating agent, and optionally a tonicity modifier.

Method of detecting antibodies that recognize papillomavirus L2 protein and ELISA Kits

The present invention also provides a method for detecting antibodies in test samples that recognize the papillomavirus L2 protein by means of an immunoassay. The immunoassay is based upon the principle of Enzyme Linked Immunosorbent Assay (ELISA) which involves determining or quantifying the primary antibody reaction that recognize the target antigen by using a detection molecule generally in the form of secondary antibody conjugated to an enzyme. The method can be particularly useful for assessing the antigenicity or potency of papillomavirus L2 protein based vaccine by measuring the antibodies that recognize it in the biological sample, such as serum. The method is also suitable for diagnosing papillomavirus infection by detecting the antibodies that recognize papillomavirus L2 protein. In one of the embodiments the method consists of

c. contacting the sample with the papillomavirus L2 protein coated

microtiter plate, and

d. detecting the antibody in the sample using secondary antibody.

The term "coating" or "coated" has been used interchangeably to mean immobilization of the antigen, for example papillomavirus L2 protein, onto the solid surface such as microtiter plate. Microtiter plates are first coated with papillomavirus L2 protein. The papillomavirus L2 protein comprises of N- terminal fragments of one or more human papillomavirus L2 protein. In preferred embodiments the papillomavirus L2 protein comprises of a multitype or multimeric human papillomavirus (HPV) L2 protein or polypeptide obtained by joining N-terminal fragments of HPV L2 protein belonging to one or more HPV types as described herein. The amount of papillomavirus L2 protein that may be coated onto the wells of microtiter plates may vary between 10 ng to 600 ng per well. In some of the embodiments the papillomavirus L2 protein is coated onto the wells of microtiter plates at an amount of about 10 ng, about 20 ng, about 30 ng, about 40 ng, about 50 ng, about 100 ng, about 150 ng, about 200 ng, about 250 ng, about 300 ng, about 350 ng, about 400 ng, about 450 ng, about 500 ng, about 550 ng or about 600 ng per well. For effective coating of papillomavirus L2 protein the microtiter plates are incubated overnight at 2-8 °C. It is understood in the art that various factors affect the coating of antigen onto microtiter plates, in particular time and temperature. At higher temperature the coating of the antigen can be accomplished in a short time and vice versa. Similarly, antigen concentration may also influence the coating of plates. Those skilled in the art will be able to determine operative and optimal coating conditions by employing routine experimentation. Thus the incubation conditions can vary from overnight at 2-8 °C to about 1-3 hour at 37 °C or room temperature. After coating, the plates are washed about 2-6 times with phosphate buffered saline containing non- ionic surfactant, for example, Tween 20 to remove excess antigen. Other buffers generally known in the art may also be used. It is advisable to use such buffers having a pH value 1 to 2 units higher than the isoelectric point of the protein being coated. To increase the sensitivity of the assay it is desirable to reduce the background noise or non specific binding. By non specific binding it is meant that the binding occurs with an antigen or antibody other than the antigen or antibody for which the ELISA is designed. Thus in order to reduce the background noise or non specific binding, the places unoccupied by papillomavirus L2 protein on microtiter plates may be blocked by blocking solution containing a blocking agent in phosphate buffered saline (PBS). A variety of blocking agents, such as but not limited to, skimmed milk, bovine serum albumin, porcine thyroglobulin, fish gelatin, insect cell lysate or whole normal serum may be used. In some of the embodiments skimmed milk at about 0.5%, at about 1%, at about 2 %, at about 3%), at about 4%, at about 5%, at about 6%, at about 7%, at about 38, at about 9%, at about 10%, at about 11%, at about 12%, at about 13%, at about 14%, or at about 15%) is used to block the unoccupied areas on the microtiter plate.

In one of the embodiments skimmed milk at about 2-12%, preferably about 5- 10%) in PBS is used for blocking. After blocking, the microtiter plates may be washed with phosphate buffered saline (PBS) containing tween 20.

The sample to be assayed may be of any type which is suitable for use with the ELISA method for example, biological sample such as serum. In one of the embodiments the serum sample may be of animal or human origin either obtained by immunizing the animals or humans with papillomavirus L2 protein, or obtained from animals or humans naturally infected with papillomavirus. The serum sample may be contacted with the papillomavirus L2 protein coated microtiter plates under appropriate conditions for the formation of immune complex i.e., a complement formed due to the interaction of antigen with antibody present in the serum sample (for example primary antibody). Under "appropriate conditions" as used herein means conditions suitable for the formation of immune complex such as desired dilutions of the serum sample and incubation conditions. Primary antibody as used herein means the antibody present in the sample which is generated against papillomavirus L2 protein by either immunizing animal or human with papillomavirus L2 protein or generated from natural papillomavirus infection in an animals or humans. In one of the embodiments, it is preferable to bring the sample to an appropriate dilution (for example, 1 : 10 to 1 : 1000000) before contacting the sample with the papillomavirus L2 protein coated microtiter plates. The sample may be allowed to remain in contact with the papillomavirus L2 protein coated microtiter plates by incubating it for about two hours at about 25 ±2 °C in a humidified chamber in order to avoid drying of wells. It is well known in the art that the duration of time and the temperature can influence the formation of immune complex. As such, the duration of time and temperature can be varied so as to allow the binding of the specific antibody (primary antibody) present in the sample to interact with papillomavirus L2 protein coated onto the microtiter plate. This step may be followed by a further step of washing with PBS containing tween 20.

The secondary antibody that may be used to detect the immune complex can be anti-mouse antibody, anti-rat antibody, anti-rabbit antibody, anti-goat antibody, anti-horse antibody, anti- sheep antibody, anti-donkey antibody, or anti-human antibody which is generally conjugated to an enzyme such as horse radish peroxidase (HRP), alkaline phosphatase, beta galactosidase, maleate dehydrogenase etc. The secondary antibodies are generally generated against the immunoglobulins of the species in which the primary antibody is produced, for example goat anti-mouse antibody. The secondary antibody conjugated to an enzyme binds to the immune complex (formed due to the interaction between the papillomavirus L2 protein (antigen) and the primary antibody and is detected by adding the appropriate substrate. For example, if HRP labeled secondary antibody is used the appropriate substrate can be TMB (tetramethylbenzidine), ABTS (2,2 - Azino-di(3-ethyl-benzthiazoline sulfonic acid), OPD (o-phenylenediamine), 3- amino-9-ethyl carbazole, 4-chloro-l -naphthol, and 3,3'-diaminobenzidine tetrahydrochloride. If alkaline phosphatase labeled secondary antibody is used the appropriate substrate can be p-nitrophenyl phosphate. Detection or quantification is achieved by measuring the build-up of coloured product using a spectrophotometer/ELISA reader. In one of the embodiments goat anti-mouse antibody labeled with horse radish peroxidase is used as a secondary antibody. After adding the secondary antibody to the microtiter plate, the plates may be incubated for overnight at 2-8 °C to 1 h at 37 °C and washed 2-5 times with PBS containing tween 20. A person skilled in the art understands that appropriate dilution of secondary antibody and incubation conditions can be varied for effective interaction of secondary antibody with the immune complex. It is preferable to bring the secondary antibody to an appropriate dilution (for example, 1 : 10 to 1 : 1000000) before adding the secondary antibody to the microtiter plate. In one of the embodiment appropriate ranges contemplated for dilution of secondary antibody may be between 1 : 1000 to 1 : 100000. After allowing sufficient time (about 1 hour at 25 ±2 °C) for the binding of secondary antibody with the immune complex appropriate substrate can be added to the microtiter plates. It is preferable to have a washing step before adding the substrate. In one of the embodiment the TMB substrate is added to the wells of the microtiter plates and incubated for about 10-45 min, preferably for about 20 min, at room temperature. Suitable reagents may be used to stop the reaction, such as strong acids or strong bases. In one of the embodiments the reaction is stopped by using 1 N H₂SO₄ to each well and the plates were read in a spectrophotometer/ELISA reader.

ELISA kits

A kit of the invention comprises of

a. a microtiter plate coated with papillomavirus L2 protein,

b. a blocking solution,

c. a wash buffer,

d. a diluent,

e. a secondary antibody,

f. a substrate, and

g a stop solution. In one of the embodiments, component (a) of the ELISA kit can be provided separately (i.e., microtiter plate and papillomavirus L2 protein) when fresh coating of papillomavirus L2 protein is required.

In one of the embodiments, the blocking solution of the invention comprises of skimmed milk dissolved in phosphate buffered saline (PBS).

In one of the embodiments, the wash buffer comprises of phosphate buffered saline containing Tween 20. In one of the embodiments, blocking solution is used as diluent.

In one of the embodiments, the secondary antibody is goat anti- mouse antibody conjugated to horse radish peroxidase (HRP). In one of the embodiments, the substrate is TMB. In one of the embodiments, 1 N sulphuric acid is used as stop solution. Examples:

The present invention is further exemplified by the following non limiting examples. It should be understood that the examples are provided to illustrate the invention. From the description and the exemplified embodiments and examples, on skilled in the art can make various modifications or adaptations to the invention. Such modifications or adaptations are deemed to be within the scope and the spirit of the invention.

Example 1: Preparation of papillomavirus L2 protein formulation

The papillomavirus L2 protein comprising 11-88 amino acids from the N- terminus of five different HPV types (11-88 x5 representing HPV types HPV6, HPV16, HPV18, HPV31, and HPV39) was prepared according to the methods described by Jagu, Subhashini et al. (Journal of Virology 2013 87(11):6127-6136; Journal of National Cancer Institute 2009 101 :782-792; WO2009529325). Purified multitype HPV L2 protein antigen was diluted with 20 mM sodium acetate buffer (pH 5.0) containing 3 mM EDTA, 0.005% Tween80 and 20 mM sodium chloride. The multitype HPV L2 antigen was adsorbed onto aluminum phosphate (1 mg/mL) at 25 ± 2 °C for 30-45 min with stirring at 200 rpm and incubated for maturation at room temperature (25 ± 2 °C) for 10-12 h. The tonicity of the formulation was adjusted to 250-350 mOsm/kg using NaCl. Example 2a: Effect of excipients on stability of papillomavirus L2 protein formulation

Stability of papillomavirus L2 protein (11-88 x8) antigen in sodium acetate buffer pH 5.5 was analyzed in the presence of various excipients, for example, ascorbic acid (5%), EDTA (0.04%), Arginine (2.3%), Cysteine (1.6%), Sorbitol (5%), Sucrose (10%), Polysorbate-80 (0.1%), Glycerol (1%) or combinations thereof at different temperature and time points. The protein profiles were analyzed on SDS PAGE to identify the stability in different excipients and the density of protein bands observed were quantified using the software 'Image J'(Image Processing & Analyzing in Java program) from NIH (available at http://rsbweb.nih.gov/ij/index.html). The results are represented in Figure la and Figure lb. The papillomavirus L2 protein appears to be quite stable in several of the excipients, however, ascorbic acid, arginine and sorbitol appear to offer less stability compared to other excipients. Example 2b: Effect of excipients (buffering agents) on stability of papillomavirus L2 protein formulation

Stability of papillomavirus L2 protein (11-88 x5) was studied in different buffers viz., acetate buffer pH 5.0 (Lane 2), succinate buffer pH 5.8 (Lane 3), phosphate buffer pH 6.8 (Lane 4) and citrate buffer pH 6.1 (Lane 5). Lane 1 represents marker and blank lanes represent supernatant from respective buffers. The protein profiles of papillomavirus L2 protein (11-88 x5) in all the buffers were analyzed on SDS PAGE at different temperature and time points [Figure 5a, 5b and 5c]. Under stress (37 °C) and accelerated (25 °C) conditions the papillomavirus L2 protein appears to be less stable. Based upon the trends observed in stress and accelerated SDS gel profiles, acetate buffer appears to offer maximum stability to papillomavirus L2 protein followed by succinate, phosphate and citrate buffers. However, the papillomavirus L2 protein appears to be quite stable in all the buffers under real time conditions (2-8 °C).

Example 3: SEC-HPLC profile of papillomavirus L2 protein formulation

Stability of papillomavirus L2 formulations (Formulation A, Formulation B, and Formulation C) was analyzed by size exclusion chromatography on TSK 4000 SWx_L column (Tosoh). The column was pre-equilibrated with the mobile phase buffer 20 mM sodium acetate, 0.5 M NaCl, pH 5.0 and flow rate was maintained at 0.5 mL/min before sample injection (100 μΐ.). Elution of the papillomavirus L2 protein was monitored at a wavelength of 214 nm. The papillomavirus L2 protein analyzed in the native condition (day 'zero') eluted in a single peak with a retention time of 19±2 min. The native condition was retained at 2-8 °C for all the months during the stability period. However papillomavirus L2 protein was found to be susceptible to high temperature and eluted early with a lower retention time when stored at 37 °C and 25 °C [Figure 2].

Example 4: Immunogenicity of the papillomavirus L2 protein formulation

Immunogenicity of a papillomavirus L2 protein formulation (Formulation A) was assessed under real time and accelerated stability conditions. Two groups of female Balb/c mice (10 animals per group) were immunized with 25 μg of antigen formulated as described herein at day zero (primary dose) and two boosters on day 14 and day 28 respectively. Sera were collected before immunization on day zero (pre immune) and on day 42 and endpoint antibody titers were determined by an enzyme immunoassay. ELISA plates were coated with 11-88 x5 antigen at a concentration of 50 ng/well and incubated overnight at 4 °C. Skimmed milk (5%) in PBS was used for blocking. Blocking solution (300 μΐ) was added to each well and incubated for one hour at 37 °C. Mice serum samples were added to the designated wells at dilutions starting from 1 :400 to 1 :409600 by performing two fold serial dilutions. Plates were incubated for one hour at 37 °C. Goat anti-mouse secondary antibody conjugated to HRP at 1 : 1500 dilutions was added to each well and plates were incubated for one hour at 37 °C. TMB substrate (100 μΐ) was added to each well and incubated in dark for 10 min at room temperature. The enzyme substrate reaction was stopped by adding 100 μΐ of 1 N H₂SO₄ per each well. The absorbance (optical density - OD) was measured at 450/540 nm on Spectramax M5 (Molecular Devices). The endpoint titer is calculated in the following manner. The endpoint titer of a sample is defined as the reciprocal of the highest dilution that gives a reading above the cutoff. The "cutoff value for each dilution is calculated by taking mean of the OD values and multiplying the standard deviation by 3 (i.e., cutoff = Mean + 3SD). The cutoff value is taken as pre-immune titer. A graph is plotted by taking dilutions on x-axis and ODs on y-axis for both pre-immune (cutoff) and test sample. The dilution at which the titer of the test sample intersects with the cutoff value is taken as an endpoint titer.

The papillomavirus L2 protein formulation could elicit L2 antibody titers up to 12 months when stored at real time conditions (5±3 °C) as compared to pre-immune (baseline). There was no significant decrease in antibody titers up to 6 months compared to initial time point. However slight decrease in antibody titers was observed at 12 month time point. Under accelerated conditions (25 °C) and (37 °C) no significant decrease in antibody titers was observed for up to 6 months and 30 days respectively [Figure 4].

Claims

The Claims:

1. A papillomavirus vaccine formulation comprising:

a. a multitype or multimeric papillomavirus L2 protein adsorbed onto an aluminum adjuvant,

b. a buffer providing a pH of 4.0 to 7.5,

c. a stabilizer, and optionally

d. a tonicity modifier.

2. The formulation as claimed in claim 1, wherein the papillomavirus is selected from animal or human papillomavirus (HPV) types.

3. The formulation as claimed in claim 2, wherein the human papillomavirus types are selected from the group consisting of HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV 25, HPV26, HPV27, HPV28, HPV29, 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, HPV110, HPV111, HPV112, HPV113, HPV114, HPV115, HPV116, HPV117, HPV118, HPV119 and HPV120.

4. The formulation as claimed in claim 3, wherein the L2 protein comprises of N- terminal fragments of one or more human papillomavirus types.

5. The formulation as claimed in claim 4, wherein the multitype or multimeric L2 protein or polypeptide is obtained by joining N-terminal fragments of one or more human papillomavirus types.

6. The formulation as claimed in claim 5, wherein the N-terminal fragments comprises of 11-88 amino acids from the L2 protein of one or more human papillomavirus types.

7. The formulation as claimed in claim 6, wherein the human papillomavirus types are selected from the group consisting of HPV 1, HPV2, HPV5, HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV34, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, or HPV73.

8. The formulation as claimed in claim 7, wherein the human papillomavirus types are selected from the group consisting of HPV 6, HPV 16, HPV 18, HPV 31, HPV39, HPV 51, HPV56, or HPV79.

9. The formulation as claimed in claim 8, wherein the human papillomavirus types are selected from the group consisting of HPV6, HPV16, HPV18, HPV31, or HPV39.

10. The formulation as claimed in claim 9, wherein the aluminum adjuvant is selected from aluminum hydroxide or aluminum phosphate.

11. The formulation as claimed in claim 10, wherein the buffer is selected from the group consisting of acetate, phosphate, citrate, succinate or combination thereof.

12. The formulation as claimed in claim 11, wherein the buffer has a pH of 4.5 to 7.0, preferably between pH 5.0 to 7.0.

13. The formulation as claimed in claim 12, wherein the stabilizer is selected from the group consisting of a surfactant, a chelating agent and optionally an amino acid or a sugar or combination thereof.

14. The formulation as claimed in claim 13, wherein the surfactant is a non-ionic surfactant.

15. The formulation as claimed in claim 14, wherein the non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80 or combination thereof.

16. The formulation as claimed in claim 15, wherein the polysorbate 80 is present in a range of 0.001% to 0.01%

17. The formulation as claimed in claim 13, wherein the chelating agent is present at a concentration of 0.1 mM to 10 mM.

18. The formulation as claimed in claim 17, wherein the chelating agent is selected from the group consisting of EDTA, EGTA, or citric acid.

19. The formulation as claimed in claim 13, wherein the sugar is present at a concentration between 1-20 weight percent, preferably between 1-15 weight percent or between 1-10 weight percent, or most preferably 5 weight percent.

20. The formulation as claimed in claim 19, wherein the sugar is selected from the group consisting of sucrose, trehalose, lactose, or combination thereof.

21. The formulation as claimed in claim 13, wherein the amino acid is selected from the group consisting of cysteine, glycine, lysine or combination thereof.

22. The formulation as claimed in claims 1-21 wherein the multitype or multimeric papillomavirus L2 protein is present in a biologically effective amount, preferably between 25 μg to 1000 μg.

23. The formulation as claimed in claims 1-22, wherein the tonicity modifier is selected from the group consisting of NaCI, sucrose, trehalose, or combination thereof.

24. A method of making papillomavirus vaccine formulation comprising the steps of

a. adjusting the concentration of a multitype or multimeric papillomavirus L2 protein to a biologically effective amount using a buffer comprising a stabilizer selected from the group comprising a chelating agent, a surfactant, and optionally an amino acid or a sugar, or combination thereof,

b. adsorbing the multitype or multimeric papillomavirus L2 protein of step (a) onto an aluminum adjuvant,

c. incubating the adsorbed multitype or multimeric papillomavirus L2 protein of step (b) for maturation, and optionally

d. adjusting the tonicity of the formulation using a tonicity modifier.

25. The method as claimed in claim 24, wherein the buffer has a pH of 4.5 to 7.0, preferably between pH 5.0 to 7.0.

26. The method as claimed in claim 25, wherein the buffer is selected from the group consisting of acetate buffer, succinate buffer, phosphate buffer, citrate buffer or combination thereof.

27. The method as claimed in claim 24, wherein the buffer additionally comprises of a salt selected from the group consisting of NaCI, KCI or calcium phosphate.

28. The method as claimed in claim 24, wherein the aluminum adjuvant is aluminum phosphate or aluminum hydroxide.

29. The method as claimed in claim 28, wherein the multitype or multimeric papillomavirus L2 protein is adsorbed onto the aluminum phosphate adjuvant at 2-37 °C for 15-60 min, preferably at 23-27 °C for 30-45 min.

30. The method as claimed in claim 29, wherein the multitype or multimeric papillomavirus L2 protein adsorbed onto the aluminum phosphate is incubated at 2-27 °C for 8-24 hours, preferably at 23-27 °C for 10-12 hours.

31. A method of preventing or treating papillomavirus infection by administering the formulation of claims 1-30 to a human in need thereof.

32. Use of the formulation of claims 1-30 in the manufacture of a medicament for the prevention of disease caused by papillomavirus.