CA2597878A1 - Capsid protein and use therefore - Google Patents

Abstract

The present invention relates to the compositions comprise macro-molecules and methods for preparing such macro-molecules. The compositions are used as antigen carriers in prophylactic and therapeutic applications by inducing immune responses against carried antigen, preferably a cell-mediated immune response against carried antigen. The compositions are also used as immunogens for prophylactic and therapeutic applications.

Description

Capsid Proteins and Uses Therefore TECHNICAL FIELD

The present invention relates to the development of antigen carriers for inducing immune responses, specifically for inducing a cell-mediated immune response against the antigen for prophylactic and therapeutic applications. The present invention also relates to the development of virus capsid protein based vaccines for prophylactic and therapeutic applications.

BACKGROUND OF THE INVENTION

A variety of virus capsid proteins have the intrinsic ability to self-assemble into highly organized particles. By using recombinant DNA techniques, capsid proteins can be recombinantly produced from different hosts such as mammalian cells, insect cells, yeast and E.coli. Often, the produced capsid proteins can self-assemble into particles in the hosts that closely resemble virions. The resulted particles are called virus-like particles (VLPs). Because of lacking viral genome, VLPs are nonreplicating and noninfectious (1-14).

There are numerous documented research work and granted patents in the area of using VLPs prepared from virus capsid proteins as vaccines or using VLPs as antigen carriers or antigen delivery systems (or vehicles) to carry desired epitopes or antigens, in efforts to enhance the immunogenicity of the carried epitopes or antigens, and to prime in vivo class I-restricted cytotoxic responses (1-14, 67).

There is no doubt that VLPs can be expressed abundantly in a variety of expression systems by recombinant DNA techniques. There are very little doubts to the prophylactic or therapeutic potentials of using VLPs as vaccines or using them as antigen carriers for eliciting enhanced immune responses, particularly cell-mediated immune response against carried antigens or epitopes. Due to their particulate nature, VLPs usually can be purified in particles by methods such as salt precipitation with ammonium sulfate, density gradient centrifugation, and gel filtration.
However, to use this technology to produce medicines, in particular for use in humans, there are still unsolved problems related to the economically and reproducibly preparing intact homogeneous particles from expression host systems with well defined compositions able to withstand Iong-term storage (8).

When produced by recombinant DNA technology, VLPs like many other recombinant proteins will be contaminated with host proteins, lipids, nucleic acids et al.
These contaminations have to be removed to very low levels to meet the requirements for medical application. However, the removal of the contaminations from VLPs is complicated due to the fact that when VLPs are expressed and assembled in the expression systems, the host proteins and lipids can be incorporated into the VLPs and host nucleic acids can be packaged into the VLPs (15-20). Purification of whole VLPs wiIl not be able to remove these incorporated or packaged contaminations. More ever, VLPs are super-molecular structures with molecular weight normally exceeds 10,00Kd, Possibly due to the poor mass transfer in chromatographic processes because of the VLPs' massive sizes compare to monomer proteins or other small molecules, when the separations are conducted by using absorbent resins, the binding, elution and fractionation are not as effective and efficient as smaller molecules.
The importance of being able to purifying totally dissembled capsid proteins are noted in US patent No. 6962777 and others (8, 21). VLPs' assembly requires correctly-folded capsid proteins to start with. Under non-denaturing conditions, the in vitro method for the quantitative disassembly and subsequent reassembly of VLPs is highly specific for each individual capsid protein and US patent 6962777 might be the only published work dealt with this issue with VLPs prepared from human papillomavirus (HPV) L1 major capsid protein. Many factors significant for VLPs formation and stability have not been well elucidated. It is generally known that VLPs' disassembly and assembly can be affected by numerous factors. For example, pH, ionic strength, post-translational modifications of viral capsid proteins, disulfide bonds, and divalent cation bonding. To make this issue even more complicated is that the VLPs' disassembly and assembly often require chaperones participation and for some VLPs formation, certain specific structure nucleic acids are required (8, 21-36).
Thus, there are numerous interrelated factors which may affect capsid stability, assembly and disassembly in vitro, which vary widely even for related viruses.
Further more, the tendency of forming aggregates by partially dissembled or totally dissembled capsid proteins is another major obstacles in the process of producing homogenous and stable VLPs effectively and efficiently in vitro (8) .

To simply dissemble VLPs, high concentration of Chaotropic agents such as urea or guanidine hydrochloride (Gu.HCI) can be used, as these agents will disrupt non-covalent forces such as hydrogen bonding, Van der Waals interactions, and the hydrophobic effect in capsid proteins, and the disulfide bonds in capsid proteins can be disrupted simply by reducing agents or oxidative sulfitolysis process. If just for producing pure capsid proteins, then to subject VLPs in high concentration of Gu.HC1 and urea, plus necessary agents to disrupt disulfide bonds in the purification process would be advantageous because of the following reasons. (1) Capsid proteins are much less likely to form aggregates in high concentration of urea or Gu.HCI, so the purification process can be much more efficient and scalable; (2) High concentration of urea or Gu.HCI can weak the interactions (hydrogen bonding, Van der Waals interactions, and the hydrophobic effect) between capsid proteins and contaminations;
so the purification process can be much more effective in terms at removing contaminations; (3) VLPs are disintegrated in the high concentration of urea or Gu.HCI, so the capsid proteins exhibit more homogenous properties in the purification process; (4) disintegrated VLPs are much more likely to release or expose incorporated or packaged contaminations to the purification forces for removing them.

However, the chaotropic agents such as urea or Gu.HCI are also strong protein denaturants, proteins are denatured after the treatment with high concentration of urea or Gu.HCI, and there is still lack of knowledge on how to correctly refold denatured capsid proteins. If denatured capsid proteins are not correctly refolded, they often form aggregates instead of self-assemble into VLPs (8, 21).

Therefore, there exists a need in the art for a general method, which would conduct the purification of recombinantly expressed capsid proteins in one or more steps in high concentration of chaotropic agents (denaturing conditions) plus necessary agents to disrupt disulfide bonds, then refold and reassemble of purified homogenous capsid proteins.

SUMMARY OF THE INVENTION

The present invention provides compositions for use as antigen or epitope carriers and methods to prepare such compositions. In one or more steps of preparation of the said compositions, high concentration of chaotropic agents such as urea or Gu.HC1 is (are) used and the subunits of the compositions are separated and purified in denatured forms. The denatured subunits of the said compositions, which contain capsid proteins of viruses, are refolded and self-assembled into the compositions, which are macro-molecules containing multi-subunits, by a process involving gradually removing out chaotropic agents presented in the denatured subunits by dialysis or ultra-filtration. The compositions can be used as antigen or epitope carriers in prophylactic or therapeutic applications in the way that authentic VLPs are used and may have advantages over authentic VLPs due to their different morphology.
The invention further provides a method for using the compositions as delivery vehicles for desired moieties. The compositions can also be related to prophylactic or therapeutic applications.

As the refolding and reassembly of capsid proteins are affected by many factors, most of them are not well defined and some of the factors are still not unknown, it will be difficult to refold and reassemble denatured capsid proteins if some of the related factors are unknown or not well defined. To overcome this problem, a fusion capsid-chaperone protein (FCCP) has been designed based on the following reasons:
(1) virus capsid proteins have the intrinsic ability to self-assemble into highly organized particles, (2) virus capsid proteins can accommodate certain length of peptide fused to its N-terminal or C-terminal and still retain the ability to self-assemble into VLPs (12, 36-37); (3) chaperones can bind and prevent non-native or denatured proteins from aggregations and facilitate their folding (38-59), (4) when exogenous antigens are particulate in nature, they are presented 1,000 or 10,000-fold more efficiently than soluble antigen in both class I and class II pathways (5, 60-68).
The FCCP is composed with a capsid protein and a chaperone protein, the chaperone protein is linked to capsid protein to its N-terminal or C-terminal via a peptide bond to form a single molecule. The objects of this designed FCCP are (1) when the fusion protein is recombinantly expressed in a host system, the separation and purification process can be conducted in high concentration of chaotropic agents in one or more steps, such as up to IOM urea or Gu.HCI can be used in the purification process, preferably from 4M-8M for urea and 3-6M for Gu.HCI; (2) the purified homogenous FCCP will be refolded with a process involving gradually removing out chaotropic agents presented in the purified sample by dialysis or ultra-filtration; (3) in the refolding process, FCCP will self-assemble into macro-molecules containing multi-FCCP subunits, the assembled macro-molecules can be a VLP structure or another structure with totally different morphology; (4) the macro-molecules can be used as antigen or epitope delivery systems, such as protein or peptide based antigens or epitopes can be fused to the FCCP by recombinant DNA method to produce peptide bond linked fusion proteins, or can be chemically linked or conjugated to the FCCP; the purified FCCP can be used to incorporate desired moieties, e.g., nucleic acids, proteins, peptides, hormones, anti-cancer agents and antiviral agents into the macro-molecules during reassembly. In the FCCP molecules, capsid protein is the component mainly responsible for the formation of macro-molecule structures because of its intrinsic self-assembly ability, chaperone protein is the component providing the possibility to process FCCP in denatured form with the incorporation of high concentration of chaotropic agents in the purification process and subsequently refolding and reassembly. The denatured capsid protein alone often forms aggregates in the refolding process, and very often, the denatured capsid protein can not stay in non-denaturing solution in soluble forms because of the formation of the aggregates.
The possible mechanism might be related to the hydrophobic patches in capsid proteins. Hydrophobic patches in capsid proteins are critical in self-assembling and maintaining the VLP structures, and they are buried inside of the capsid proteins in the VLP structures (69-74). In the high concentration of denaturant solutions, such as urea or Gu.HC1 solutions, capsid proteins are denatured and the buried hydrophobic patches are exposed to the solutions. When denaturants are gradually removed from the solutions in the refolding process, the gradually increased interaction among the exposed hydrophobic patches make the capsid proteins forming aggregates. When FCCP subjected to the high concentration of urea or Gu.HC1, the hydrophobic patches in capsid protein are exposed to the solutions too, but when denaturants are gradually removed from the solutions in the refolding process, the exposed hydrophobic patches in capsid proteins can be protected by fused chaperone protein, and the FCCP
can stay in solutions to take refolding and self-assembling process to form soluble macro-molecules. The FCCP is a different molecule compare to capsid protein, the refolding and in vitro assembly process are conducted by gradually removing out chaotropic agents presented in the denatured preparation of FCCP, which are fundamentally different from the nature process of capsid protein's folding and self-assembly into VLPs. Because of above reasons, the morphology of the macro-molecules prepared from this invention may totally differ from authentic VLPs.

One of this invention's findings is that the macro-molecules formed by the self-assembling of FCCP without the morphology of authentic VLPs can be very immunogenic, and may have the following advantages over authentic VLPs: (1) pre-existing immunity to authentic VLPs might be circumvented by the use of macro-molecules with different morphology; (2) existing immune tolerance to authentic capsid proteins might be circumvented by the use of the macro-molecules with different morphology; (3) the use of macro-molecules with different morphology might circumvent the problem of interference with commercial anti-capsid protein assays; (4) macro-molecules are much more stable in the solutions. The strong immunogenicity of the macro-molecules could be due to (1) when exogenous antigens are particulate in nature, they are presented 1,000 or 10,000-fold more efficiently than soluble antigen in both class I and class II pathways, and macro-molecule structures might have features of particle antigens, (2) innate immunity might be able to recognize some conserved sequences in capsid protein in FCCP, which can work synergistically to generate strong, lasting immunological responses.
Furthermore the capsid protein in the FCCP can be utilized to package nucleic acids. Some nucleic acids such as double strand RNA and unmethylated CpG-DNA are well known for their ability to greatly enhance the immune responses (75-83).

Those skilled in the art will recognize and appreciate that in the FCCP
molecule, capsid protein can be a whole protein, part of the whole protein, science mutated or variant of capsid proteins which still retain the ability of self-assembly;
chaperone protein can be a full length protein, a functional equivalent, such as, a fragment of whole chaperone protein, a science mutated or a variant of chaperone protein.
Many chaperones are heat shock proteins, that is, proteins expressed in response to elevated temperatures or other cellular stresses (38-59). The reason for this behaviour is that protein folding is severely affected by heat and, therefore, some chaperones act to repair the potential damage caused by misfolding. Other chaperones are involved in folding newly made proteins as they are extruded from the ribosome.
There are many different families of chaperones; each family acts to aid protein folding in a different way. In bacteria like E. coli, many of these proteins are highly expressed under conditions of high stress, for example, when placed in high temperatures. For this reason, the term "heat shock protein" has historicaIly been used to name these chaperones. The prefix "Hsp" designates that the protein is a heat shock protein.
Some of the common chaperone familys are Hsp60, Hsp70, Hsp90, HsplOO and small moleculae weight family of Hsp proteins (38-59). Chaperones are not limited to Hsp proteins and those skilled in the art will recognize that present unknown chaperones can be used to produce FCCP in the method provideed in this invention when they are discovered. In this invention, the idea is to have a peptide or a protein joined to a capsid protein via a peptide bond, then the fused protein can be processed in one or more steps in high concentration of chaotrapic agents such as urea or Gu.HCI
solution, the purified fusion protein then can be subjected to refold and self assemble process by gradually removing chaotrapoic agents out from the samples. After the purification, refolding and assembling process, the chaperone protein might be cliped off from FCCP with a chemical method, or by an enzyemtic method, such as, a specific enzyme cleavage site can be designed at the joint of the capsid protein and chaperone protein . For example, asp asp asp asp lys can be recognized by enterokinase, and this sequence can be introduced into the joint of the capsid protein and chaperone protein, after refolding and reassembling, the enterokinase can be used to clip off the chaperone protein.
Thus, the objects of the invention are to solve the problems of the prior art and provide a novel method for utilizing viral capsid proteins.

More specifically, it is an object of the invention to provide a novel method for utilizing capsid proteins to produce macro-molecules, which are capsid protein based vaccines. The macro-molecules are self-assembled by FCCP, which is a capsid protein fused to a chaperone protein or their functional fragments fused together.

Still more specifically, it is an object of the invention to use the macro-molecules as antigen carriers for eliciting enhanced immune responses, particularly cell-mediated immune response against carried antigens or epitopes. Such as, protein or peptide based antigens or epitopes can be fused to the FCCP by recombinant DNA method, or desired antigens or epitopes can be chemically linked or conjugated to the FCCP.

It is also an object of the invention to provide a method which enables the purification of FCCP in one or more steps in denatured form using high concentration of chaotropic agents such as urea or Gu.HCI, the purified homogenous FCCP
subsequently refolded and reassembled into macro-molecules by gradually removing chaotropic agents out from the denatured FCCP sample.

It is another object of the invention to provide a method for packaging or encapsulating desired moieties in macro-molecules, e.g., therapeutic or diagnostic agents.

It is still another object of the invention to provide a novel delivery system to incorporate desired moieties, e.g., nucleic acids, proteins, peptides, hormones, anti-cancer agents and antiviral agents into the macro-molecules during reassembly.

It is still another object of the invention to provide a novel method with the ability to prepare homogenous and well defined capsid protein based vaccines in a scalable process.

It is still another object of the invention to provide a novel method to prepare capsid protein based vaccines with improved quality, e.g., improved homogeneity, immunogenicity, and stability.

It is still another object of the invention to provide a novel method to prepare capsid protein based vaccines with the potential to circumvent pre-existing inununity to authentic VLPs.

It is still another object of the invention to provide a novel method to prepare capsid protein based vaccines with the potential to circumvent existing immune tolerance to authentic capsid proteins.

It is still another object of the invention to provide a novel method to prepare capsid protein based vaccines with the potential to circumvent the problem associated with authentic VLPs of interference with commercial anti-capsid protein based assays.

The following examples are provided in order to demonstrate and further illustrate the present invention, and are not to be construed as limiting the scope thereof.
SEQUENCE LISTS

Sequence 1: The amino acid sequence of the fusion protein of E7-Core-Hsp65 in example 1.

Sequence 2: The DNA sequence of Ankegens 2479bp for E7-Core-Hsp65 in example 1.

EXAMPLES
Example 1 FCCP molecule carrying an HPV antigen A chaperone protein-Hsp65 derived from Mycobacterium bovis BCG hsp65 gene (84) is fused to the C-terminal of the nucleocapsid protein (core antigen) of Hepatitis B virus (HBV) subtype ADW2 (85-86) to form a FCCP molecule. An E7 protein from human papillomavirus type 16 (87) is fused to the N-terminal of the FCCP molecule.
The amino acid sequence of the fusion protein of E7-Core-Hsp65 is listed in attached Sequence 1. According to the amino acid sequence, the DNA sequence was designed and synthesized.

The synthesized DNA sequence was named Ankegens 2479bp and cloned into Smal digested pBluescript II SK (+/-) from Stratagene (88) to produce pBSK-Ankegens-2479bp. The DNA sequence of Ankegens 2479bp for E7-Core-Hsp65 is listed in attached Sequence 2.

Example 2 Expression and purification of E7-Core-Hsp65 fusion protein E7-Core-BCG65 DNA fragment was cut from pBSK-Ankegens-2479bp by Ndel and EcoRl then subcloned into pET-23a (89) corresponding sites to produce pET-23a-2479. The pET-23a-2479 was transformed into Rosetta-gami(DE3) from Novagen. E7-Core-BCG65 fusion protein was expressed in E. coil cells by fermentation and induction of transformed Rosetta-gami(DE3) cells with 0.5mM
isopropyl-thio-galatopyranoside according to Novagen's pET System Manual.
After fermentation, cells were harvested by centrifugation. Cells were washed once by suspending 100g cell paste in 1000m1 of buffer A (100mM Tris-Hcl pH 9.0; 5mM
EDTA) then centrifuging at 8500rpm for 30 minutes. Discarded the supernatant then re-suspended the pelleted cells with 1000m1 of buffer B(50mM sodium acetate;
2mM
EDTA). The suspended cells were ruptured by homogenization process with pressure at 760bar, and then centrifuged at 8500rpm for 30 minutes. The supernatant was collected and the volume was measured. Urea was added to the supernatant according to 0.7g urea for lmi supernatant, and then sodium chloride was added to final concentration at 100 mM, L-Cysteine was added to final concentration at 20mM.
The solution was stirred at room temperature to have all the urea dissolved then stirred at 4 `C for overnight. After overnight stirring, the sample was applied to an XK-50 column (GE Health) containing 300ml of SP-Sepharose resin (GE Health), which was previously washed with 1 M sodium chloride and equilibrated with buffer C(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine). After sample loading, the column was washed with 10 column-volumes of buffer D(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine; 2.5%
Triton-X-100) overnight to remove endotoxin. After overnight washing with buffer D, the column was washed with 5 column-volumes of buffer C to remove Triton-X-100, and then the column was washed with 3 column-volumes of buffer E(50mM
sodium acetate; 300mM NaCl; 2mM EDTA; 8M urea; 10mM L-Cysteine) to remove contaminations. E7-Core-BCG65 fusion protein was eluted from the column with buffer D (50mM sodium acetate; 800mM NaCI; 2mM EDTA; 8M urea; 10mM
L-Cysteine). Pooled eluted protein was dialyzed against 4X 40 volumes of buffer F
(50mM sodium acetate, 6Murea) to remove NaCl and L-Cysteine. After dialysis, Oxidative sulfitolysis was performed by adding sodium sulfite and sodium tetrathionate to final concentrations of 200mM and 50mM respectively and incubating for overnight at room temperature. The sulfitolyzed sample was diluted 5 volumes with buffer F then applied to an XK-50 column with 150m1 of Q-Sepharose resin (GE
Health), which was previously washed with 1M NaCI and equilibrated with buffer F.
After sample loading, the column was washed with 2 column-volumes of 95%
buffer F and 5% buffer G (50mM sodium acetate; 1M NaCI; 6Murea), and then E7-Core-BCG65 fusion protein was eluted with a lineal gradient from 95% buffer F
and 5% buffer G to 50% buffer F and 50% buffer G over 8 column-volumes. Eluted E7-Core-BCG65 fusion protein was pooled, and then dialyzed against 1X40 volumes of Tris.HCl pH9.0, 1X40 volumes of Tris.HCl pH7.5 with 100mM NaCI to remove urea and refold E7-Core-BCG65 fusion protein. The endotoxin levels in the final preparations (E7-Core-BCG65 in Tris.HCl pH7.5 with 100mM NaCI) were below 5EU/mg protein.

Example 3 Therapeutic and prophylactic effects of E7-Core-BCG65 treatment in mice The E7-Core-BCG65 is an FCCP carrying an E7 antigen from HPV type 16, and the E7 expressing TC-1 tumor cells were used to evaluate the therapeutic and prophylactic applications of E7-Core-BCG65 on mice bearing TC-1 tumor or being challenged with TC-1 tumor.

Female C57BL/6 mice, six to eight weeks old (20.0 2.0g) were purchased from SHANGHAI SLAC LABORATORY ANIMAL CO. LTD. Quality Control No.:
SCXK(Shanghai)2003-0003 0 TC-1 cell line expressing HPV16 E7 protein was derived from primary lung cells of C57BU6 mice by immortalization and transformation with HPV16 E7 gene and an activated human C-Ha-ras gene as described in Lin et al. (90). TC-1 cells were grown in RPMI1640 medium supplemented with 10% fetal calf serum, 2mM nonessential amino acids, 2mM L-glutamine, 1mM pyruvate, Penicillin/Streptomycin, and the cells were harvested by trypsinization, the cells were washed three times with PBS
then re-suspended in PBS. 1X105 TC-1 cells were inoculated subcutaneously into the mice and the mice were treated with E7-Core-BCG65 or saline subcutaneously according to their experiment groups.

Animal experiment groups:
Groups Mice Dose Time of E7-Core-BCG65 Treatment Treatment Therapeutic 8 500ug 48h and 16 days after inoculation of TC-1 Application E7-Core-BCG6 8 100ug 48h and 16 days after inoculation of TC-1 E7-Core-BCG6 8 20ug 48h and 16 days after inoculation of TC-1 E7-Core-BCG6 Prophylactic 8 100ug Two treatments with 14 days in between Application E7-Core-BCG6 Inoculation of TC-1 14 days after second 5 treatment 8 20ug Two treatments with 14 days in between E7-Core-BCG6 Inoculation of TC-1 14 days after second 5 treatment Control 6 Saline 48h and 16 days after inoculation of TC-1 The mice were monitored for the presence or absence of tumor by palpation and the volume of the tumor was measured with Vernier Caliber by 2 orthogonal dimensions twice a week; these measurements were extrapolated to mm3 and are presented as average tumor volume standard error of the mean. The life span of the mice was recorded.

In control group, the presence of the tumor was observed 4 days after TC-1 inoculation; the average volume of the tumor was grown to 40mm3 10 day after inoculation and 7499.84mm3 36 days after inoculation. All mice in the control group died within 60 days after inoculation.

In therapeutic group, mice were treated with E7-Core-BCG65 48h and 16 days after TC-1 inoculation; the average volume of the tumor was grown to 181.89mm3 (500ug), 671.34mm3 (IOOug) and 2148.57mm3 (20ug) 36 days after inoculation. All mice were alive 60 days after inoculation.

In the prophylactic group, mice were treated with E7-Core-BCG65 twice in 14 days, and after second treatment, mice were inoculated with TC-1; the average volume of the tumor was grown to22.43mm3 (100ug) and 89.08mm3 (20ug) 36 days after inoculation. All mice were alive 60 days after inoculation.

Table 1 The average tumor volume in different experiment groups (mm) (z s) 'Iherapeutic Group Prophylactic Group Control Date (day) SOOug 100ug 20ug 100ug 20ug Group (n=8) (n=8) (n=8) (n=8) (n=8) (n=6) 8.65 5.40 16.33 8.83 42.54 24.55* 2.32 1.06 5.56 2.91 39.00 19.28 13 41.70 20.90 51.01 20.37 84.72-+36.72* 1.97 2.44 10.58 25.56 133.57 69.64 16 31.91 12.26 49.96 20.62 189.07 91.07* 1.97 1.42 5.24 2.08 320.20 149.14 19 35.69 10.52 156.28 46.49 208.49 85.46 2.85 1.49 25.65 10.43 782.65 257.69 22 43.89 21.13 224.71 107.46 357.47 159.47 2.44 1.98 35.24 80.41 1033.81 594.12 25 109.04 47.41 257.01 107.19 756.40 258.40 4.52 2.78 17.38 6.76 2414.19 1201.87 28 127.68 56.24 395.56 128.72 892.54 364.47 18.81 5.42 69.63 24.46 4432.67 1824.46 31 156.124-49.46 525.81 152.94 1527.21 510.46 20.57 10.46 85.57 25.85 6024.54 2465.46 34 181.89 75.53 671.34 301.28 2048.57 1050.57 22.42 18.60 89.08 43.09 7499.84 3722.56 37 250.52 120.59 785.69 268.85 3051.65 1253.32 56.83 25.56 173.59 89.41 9483.58 4565.74 40 396.88+~08.12 921.87 368.03 3887.08 1889.08 59.81 26.79 173.92 86.39 13141.43 5077.39 Those skilled in the art will recognize, or be able to ascertain that the basic construction in this invention can be altered to provide other embodiments which utilize the process of this invention. Therefore, it will be appreciated that the scope of this invention is to be defmed by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.

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HMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT
FCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPMDIDPYKEFGATVEL
LSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWV
GNNLEDPASRDLWNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTP
PAYRPPNAPILSTLPETTWRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCM
AKTIAYDEEARRGLERGLNALADAVKVTLGPKGRNWLEKKWGAPTITNDGVSIAKE
IELEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGL
KRGIEKAVEKVTETLLKGAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGV
ITVEESNTFGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVK
DLLPLLEKVIGAGKPLLIIAEDVEGEALSTLWNKIRGTFKSVAVKAPGFGDRRKAM
LQDMAILTGGQVISEEVGLTLENADLSLLGKARKVVVTKDETTIVEGAGDTDAIAGR
VAQIRQEIENSDSDYDREKLQERLAKLAGGVAVIKAGAATEVELKERKHRIEDAVRN
AKAAVEEGIVAGGGVTLLQAAPTLDELKLEGDEATGANIVKVALEAPLKQIAFNSGL
EPGWAEKVRNLPAGHGLNAQTGVYEDLLAAGVADPVKVTRSALQNAASIAGLFLTT
EAVVADKPEKEKASVPGGGDMGGMDF

Sequence 1: The amino acid sequence of the fusion protein of E7-Core-Hsp65 V
CA 0259787:8 2007-09-13 catatgcatggagatacacctacattgcatgaatatatgttagatttgcaaccagagaca actgatctctactgttatgagcaattaaatgacagctcagaggaggaggatgaaatagat ggtccagctggacaagcagaaccggacagagcccattacaatattgtaaccttttgttgc aagtgtgactctacgcttcggttgtgcgtacaaagcacacacgtagacattcgtactttg gaagacctgttaatgggcacactaggaattgtgtgccccatctgttctcagaaaccaatg gacattgacccttataaagaatttggagctactgtggagttactctcgtttttgccttct gacttctttccttccgtcagagatctcctagacaccgcctcagctctgtatcgggaagcc ttagagtctcctgagcattgctcacctcaccatactgcactcaggcaagccattctctgc tggggggaattgatgactctagctacctgggtgggtaataatttggaagatccagcatcc agggatctagtagtcaattatgttaatactaacatgggtttaaagatcaggcaactattg tggtttcacatatcttgccttacttttggaagagagactgtacttgaatatttggtatct ttcggagtgtggattcgcactcctccagcctatagaccaccaaatgcccctatcttatca acacttccggaaactactgttgttagacgacgggaccgaggcaggtcccctagaagaaga actccctcgcctcgcagacgcagatctcaatcgccgcgtcgcagaagatctcaatctcgg gaatctcaatgtatggccaagacaattgcgtacgacgaagaggcccgtcgcggcctcgag cggggcttgaacgccctcgccgatgcggtaaaggtgacattgggccccaagggccgcaac gtcgtcctggaaaagaagtggggtgcccccacgatcaccaacgatggtgtgtccatcgcc aaggagatcgagctggaggatccgtacgagaagatcggcgccgagctggtcaaagaggta gccaagaagaccgatgacgtcgccggtgacggcaccacgacggccaccgtgctggcccag gcgttggttcgcgagggcctgcgcaacgtcgcggccggcgccaacccgctcggtctcaaa cgcggcatcgaaaaggccgtggagaaggtcaccgagaccctgctcaagggcgccaaggag gtcgagaccaaggagcagattgcggccaccgcagcgatttcggcgggtgaccagtccatc ggtgacctgatcgccgaggcgatggacaaggtgggcaacgagggcgtcatcaccgtcgag gagtccaacacctttgggctgcagctcgagctcaccgagggtatgcggttcgacaagggc itacatctcggggtacttcgtgaccgacccggagcgtcaggaggcggtcctggaggacccc tacatcctgctggtcagctccaaggtgtccactgtcaaggatctgctgccgctgctcgag aaggtcatcggagccggtaagccgctgctgatcatcgccgaggacgtcgagggcgaggcg ctgtccaccctggtcgtcaacaagatccgcggcaccttcaagtcggtggcggtcaaggct cccggcttcggcgaccgccgcaaggcgatgctgcaggatatggccattctcaccggtggt caggtgatcagcgaagaggtcggcctgacgctggagaacgccgacctgtcgctgctaggc aaggcccgcaaggtcgtggtcaccaaggacga.gaccaccatcgtcgagggcgccggtgac accgacgccatcgccggacgagtggcccagatccgccaggagatcgagaacagcgactcc gactacgaccgtgagaagctgcaggagcggctggccaagctggccggtggtgtcgcggtg atcaaggccggtgccgccaccgaggtcgaact.caaggagcgcaagcaccgcatcgaggat gcggttcgcaatgccaaggccgccgtcgagga.gggcatcgtcgccggtgggggtgtgacg ctgttgcaagcggccccgaccctggacgagctgaagctcgaaggcgacgaggcgaccggc gccaacatcgtgaaggtggcgctggaggcccc:gctgaagcagatcgccttcaactccggg ctggagccgggcgtggtggccgagaaggtgcgcaacctgccggctggccacggactgaac gctcagaccggtgtctacgaggatctgctcgc:tgccggcgttgctgacccggtcaaggtg acccgttcggcgctgcagaatgcggcgtccat.cgcggggctgttcctgaccaccgaggcc gtcgttgccgacaagccggaaaaggagaaggc:ttccgttcccggtggcggcgacatgggt ggcatggatttctgaattc Sequence 2: DNA Sequence of Ankegens 2479bp for E7-Core-Hsp65 Fusion Protein

Claims (43)

1. An immunogenic composition comprising macro-molecules.

2. The macro-molecules of Claim 1 comprising a fusion protein.

3. The fusion protein of claim 2 comprising a virus capsid (or nucleocapsid) protein joined via a peptide bond to a chaperone protein.

4. A method of preparation of the macro-molecules of claim 1, comprising the steps of:
A. recombinantly producing the fusion protein of claim 2 by an expression system;
B. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCl solutions or buffered solutions in the separation and purification process, preferably 2-8M

for urea and 1-6M for Gu.HCl;
C. refolding and reassembling of the fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;
D. the final macro-molecules comprising many subunits of the fusion protein.

5. An immunogenic composition comprising macro-molecules.

6. The macro-molecules of Claim 5 comprising a virus capsid protein.

7. A method of preparation of the macro-molecules of Claim 5, comprising the steps of:
A. designing an unique enzyme cleavage site at the joint of a fusion protein comprising a capsid protein and a chaperone protein, the unique enzyme cleavage site is a thrombin cleavage site or an enterokinase cleavage site, or any other unique enzyme cleavage site;
B. recombinantly producing the said fusion protein containing a specific enzyme cleavage site at the joint by an expression system;
C. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCl solutions or buffered solutions in the separation and purification process, preferably 2-8M

for urea and 1-6M for Gu.HCl;
D. refolding and reassembling of the said fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;
E. clipping off the chaperone protein from the macro-molecules by using the desired enzyme, such as using thrombin or enterokinase;
F. separating macro-molecules from the clipped off chaperone protein;
G. the final macro-molecules comprising many subunits of mostly capsid protein.

8. An immunogenic composition comprising macro-molecules.

9. The macro-molecules of Claim 8 comprising a fusion protein.

10. The fusion protein of claim 9 comprising a foreign antigen, a virus capsid (or nucleocapsid) protein and a chaperone protein joined together via peptide bonds.

11. A method of preparation of the macro-molecules of claim 8, comprising the steps of:
A. recombinantly producing the fusion protein of claim 10 by an expression system;
B. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCl solutions or buffered solutions in the separation and purification process, preferably 2-8M

for urea and 1-6M for Gu.HCl;
C. refolding and reassembling of the fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;
D. the final macro-molecules comprising many subunits of the fusion protein.

12. An immunogenic composition comprising macro-molecules.

13. The macro-molecules of Claim 12 comprising chemically linked or conjugated foreign antigen and fusion protein.

14. The fusion protein of claim 13 comprising a virus capsid (or nucleocapsid) protein joined via a peptide bond to a chaperone protein.

15. A method of preparation of the macro-molecules of claim 12, comprising the steps of:
A. recombinantly producing the fusion protein of claim 13 by an expression system;
B. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCl solutions or buffered solutions in the separation and purification process, preferably 2-8M

for urea and 1-6M for Gu.HCI;
C. refolding and reassembling of the fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;
D. chemically linking or conjugating foreign antigen with macro-molecules.

16. An immunogenic composition comprising macro-molecules.

17. The macro-molecules of Claim 16 comprising a fusion protein.

18. The fusion protein of claim 17 comprising mostly a virus capsid protein linked to a foreign antigen via a peptide bond.

19. A method of preparation of the macro-molecules of Claim 16, comprising the steps of:
A. Designing a fusion protein with the capsid protein linked to a foreign antigen and a chaperone protein via peptide bonds, designing an unique enzyme cleavage site at the joint of the capsid protein and the chaperone protein, the unique enzyme cleavage site is a thrombin cleavage site or an enterokinase cleavage site, or any other unique enzyme cleavage site;

B. recombinantly producing the said fusion protein containing a specific enzyme cleavage site at the joint of the capsid protein and the chaperone protein by an expression system;
C. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCl solutions or buffered solutions in the separation and purification process, preferably 2-8M

for urea and 1-6M for Gu.HCl;
D. refolding and reassembling of the said fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;
E. clipping off the chaperone protein from the macro-molecules by using the desired enzyme, such as using thrombin or enterokinase;
F. separating macro-molecules from the clipped off chaperone protein;
G. the final macro-molecules comprising many subunits of mostly capsid protein fused to foreign antigen.

20. An immunogenic composition comprising macro-molecules.

21. The macro-molecules of Claim 20 comprising foreign antigen chemically linked or conjugated with a capsid protein (mostly capsid protein).

22. A method of preparation of the macro-molecules of Claim 20, comprising the steps of:
A. designing an unique enzyme cleavage site at the joint of a fusion protein comprising a capsid protein and a chaperone protein, the unique enzyme cleavage site is a thrombin cleavage site or an enterokinase cleavage site, or any other unique enzyme cleavage site;
B. recombinantly producing the said fusion protein containing a specific enzyme cleavage site at the joint by an expression system;
C. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCl solutions or buffered solutions in the separation and purification process, preferably 2-8M

for urea and 1-6M for Gu.HCl;
D. refolding and reassembling of the said fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;
E. clipping off the chaperone protein from the macro-molecules by using the desired enzyme, such as using thrombin or enterokinase;
F. separating macro-molecules from the clipped off chaperone protein;
G. chemically linking or conjugating foreign antigen with said macro-molecules.

23. The immunogenic composition of any one of claims, wherein said capsid protein is the protein shell of a virus. It consists of one or several monomeric subunits made of protein, and in some cases, is named as nucleocapsid.

24. The immunogenic composition of any one of claims, wherein said capsid protein is a full length protein, part of the full length protein, science mutated or a variant of a capsid protein, which still retains the ability of self-assembly.

25. The immunogenic composition of any one of claims, wherein saidcapsid protein is HBV core antigen.

26. The immunogenic composition of any one of claims, wherein said chaperone protein is a member of one of the chaperone families.

27. The immunogenic composition of any one of claims, wherein the chaperone protein is a full length protein, a functional equivalent, such as, a fragment of a whole chaperone protein, a science mutated or a variant of a chaperone protein.

28. The immunogenic composition of any one of claims, wherein the chaperone protein is a protein or any polypeptide when joined to a capsid protein via a peptide bond to form a fusion protein, wherein the said fusion protein gains the feature of being able to refold and reassemble into macro-molecules after being denatured with the treatment of high concentration of chaotropic agents, such as being denatured in urea or guanidine hydrochloride (Gu.HCl) solutions or buffered solutions up to 10M concentration, preferably 2-8M for urea and 1-6M
for Gu.HCl.

29. The immunogenic composition of any one of claims, wherein the chaperone protein is an M. bovis BCG hsp65 protein.

30. The immunogenic composition of any one of claims 8, 12, 16 and 20, wherein said foreign antigen is derived from the group consisting of :(a) viruses; (b) bacteria; (c) parasites; (d) prions; (e) tumors; (f) self-molecules ;(g) non-peptide hapten molecules (h) allergens; (i) hormones and antigenic fragments of any of the antigens from (a) to (i).

31. The immunogenic composition of any one of claims 8, 12, 16 and 20, wherein said foreign antigen is epitope or epitopes derived from the group consisting of :
(a) viruses; (b) bacteria; (c) parasites; (d) prions; (e) tumors; (f) self-molecules; (g) allergens and (h) hormones

32. The immunogenic composition of any one of claims 8, 12, 16 and 20, wherein said foreign antigen is a E7 antigen from HPV type 16.

33. An immunogenic composition comprising (a) composition of any one of claims 1, 5; (b) at least one immunostimulatory substance; wherein said immunostimulatory substance is bound or packaged to said composition, and wherein said immunostimulatory substance is an unmethylated CpG- containing oligonucleotide, or double strand RNA.

34. An immunogenic composition comprising (a) composition of any one of claims 8, 12, 16 and 20; (b) at least one immunostimulatory substance; wherein said immunostimulatory substance is bound or packaged to said composition, and wherein said immunostimulatory substance is an umnethylated CpG-DNA, or double strand RNA.

35. A method of having the double strand RNA or unmethylated CpG-DNA in claim 34 packaged into the composition of any one of claims 1, 5, 8, 12, 16 and 20 comprises: (a) removing contaminated nucleic acids from the fusion protein containing capsid protein and chaperone protein by purification process using solutions with high concentration of chaotropic agents, preferably in 4-8M
urea solutions or buffered solutions; (b) adding said double strand RNA or unmethylated CpG-DNA into denatured fusion protein containing capsid protein and chaperone protein; (c) refolding and reassembling said fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample.

36. A method of generating immune responses in a host, comprising the step of administering an effective amount of the composition of any one of claims 1, 5 and 33 to the host.

37. A method of prophylactic or therapeutic application for a condition, comprising administering to a host in need of treatment an effective amount of the any one of claims 1, 5 and 33.

38. A method of generating immune responses in a host against foreign antigen of any one of claims 30-32, comprising the step of administering an effective amount of the composition of any one of claims 8, 12, 16, 20 and 34 to the host.

39. The method of claim 38, wherein the immune responses comprise a cell-mediated immune response.

40. A method of prophylactic or therapeutic application for a condition, comprising administering to a host in need of treatment an effective amount of the composition of any one of claims 8, 12, 16, 20 and 34, wherein prevention of the condition or therapeutic treatment of the condition relateing to immune resonses against said foreign antigen of any one of the claims of 30-32.

41. A method of circumventing pre-existing immunity to authentic VLPs by using composition of any one of claims 1, 5, 8, 12, 16, 20, 33 and 34 with different morphology compare to authentic VLPs to administer a host.

42. A method of circumventing existing immune tolerance to authentic VLPs by using composition of any one of claims 1, 5, 8, 12, 16, 20, 33 and 34 with different morphology compare to authentic VLPs to administer a host.

43. A method of circumventing the problem associated with authentic VLPs of interference with commercial anti-capsid protein based assays by using composition of any one of claims 1, 5, 8, 12, 16, 20, 33 and 34 with different morphology compare to authentic VLPs to administer a host.