Fusion proteins for use as immunogenic enhancers for inducing antigen-specific T cell responses

Abstract

A vaccine composition comprising a fusion protein for inducing enhanced pathogen antigen-specific T cell responses is disclosed. The fusion protein comprises: (a) an antigen-presenting cell (APC)-binding domain or a CD91 receptor-binding domain, located at the N-terminus of the fusion protein; (b) a translocation peptide of 34-112 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, 2, 3, or 6, located at the C-terminus of the APC-binding domain or the CD91 receptor-binding domain; and (c) an antigen of a pathogen, located at the C-terminus of the translocation peptide; (d) a nuclear export signal, comprising the amino acid sequence of SEQ ID NO: 13; and (e) an endoplasmic reticulum retention sequence, located at the C-terminus of the fusion protein.

Claims

1. A fusion protein comprising: (a) an antigen-presenting cell (APC)-binding domain or a CD91 receptor-binding domain, located at the N-terminus of the fusion protein; (b) a translocation peptide of 34-112 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, 2, 3, or 6, located at the C-terminus of the APC-binding domain or the CD91 receptor-binding domain; and (c) an antigen of a pathogen or a cancer cell; (d) an endoplasmic reticulum retention sequence, located at the C-terminus of the fusion protein; and (e) a nuclear export signal, located between the antigen and the endoplasmic reticulum retention sequence or between the translocation peptide and the antigen, the nuclear export signal comprising the amino acid sequence of SEQ ID NO: 13, in which the C-terminal amino acid of the SEQ ID NO: 13 is alanine; wherein: the pathogen is at least one selected from the group consisting of human papillomavirus (HPV), porcine reproductive and respiratory syndrome virus (PRRSV), human immunodeficiency virus-1 (HIV-1), dengue virus, hepatitis C virus (HCV), hepatitis B virus (HBV), porcine circovirus 2 (PCV2), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), Newcastle disease virus (NDV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), influenza virus, pseudorabies virus, parvovirus, swine vesicular disease virus (SVDV), poxvirus, rotavirus, Mycoplasma pneumonia, herpes virus, infectious bronchitis virus, and infectious bursal disease virus; and further wherein: the cancer cell is at least one selected from the group consisting of non-small cell lung cancer, breast carcinoma, melanoma, lymphomas, colon carcinoma, and hepatocellular carcinoma.

2. The fusion protein of claim 1, wherein the antigen of the pathogen is at least one selected from the group consisting of PCV2 ORF2, CSFV E2, and human papillomavirus (HPV)E7 proteins.

3. The fusion protein of claim 2, wherein the PCV2 ORF2 protein comprises the amino acid sequence of SEQ ID NO: 20.

4. The fusion protein of claim 2, wherein the antigen of the pathogen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

5. The fusion protein of claim 2, wherein the CSFV E2 protein comprises the amino acid sequence of SEQ ID NO: 21.

6. The fusion protein of claim 2, wherein the antigen of the pathogen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

7. The fusion protein of claim 1, wherein the antigen is a fusion antigen of foot-and-mouth disease virus protein VP1 (FMDV VP1) and Foot-and-mouth disease virus protein 3A (FMDV 3A).

8. The fusion protein of claim 7, wherein the fusion antigen comprises the amino acid sequence of SEQ ID NO: 24.

9. The fusion protein of claim 7, wherein the fusion antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24.

10. The fusion protein of claim 1, wherein the antigen is a fusion antigen of Newcastle disease virus (NDV) F peptide and Newcastle disease virus hemagglutinin-neuraminidase (NDV HN) protein.

11. The fusion protein of claim 10, wherein the fusion antigen comprises the amino acid sequence of SEQ ID NO: 27.

12. The fusion protein of claim 10, wherein the fusion antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 27.

13. The fusion protein of claim 2, wherein the HPV E7 protein comprises the amino acid sequence of SEQ ID NO: 28.

14. The fusion protein of claim 13, wherein the antigen of the pathogen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.

15. The fusion protein of claim 1, wherein the antigen is a fusion antigen of two or more antigenic peptides from a pathogen.

16. A fusion protein comprising: (a) an antigen-presenting cell (APC)-binding domain or a CD91 receptor-binding domain, located at the N-terminus of the fusion protein; (b) a translocation peptide of 34-61 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, 2, or 3, located at the C-terminus of the APC-binding domain or the CD91 receptor-binding domain; and (c) an antigen of a pathogen or cancer cell; (d) an endoplasmic reticulum retention sequence, located at the C-terminus of the fusion protein; and (e) a nuclear export signal, located between the antigen and the endoplasmic reticulum retention sequence or between the translocation peptide and the antigen, the nuclear export signal comprising the amino acid sequence of SEQ ID NO: 13; wherein: the pathogen is at least one selected from the group consisting of human papillomavirus (HPV), porcine reproductive and respiratory syndrome virus (PRRSV), human immunodeficiency virus-1 (HIV-1), dengue virus, hepatitis C virus (HCV), hepatitis B virus (HBV), porcine circovirus 2 (PCV2), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), Newcastle disease virus (NDV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), influenza virus, pseudorabies virus, parvovirus, swine vesicular disease virus (SVDV), poxvirus, rotavirus, Mycoplasma pneumonia, herpes virus, infectious bronchitis virus, and infectious bursal disease virus; and further wherein; the cancer cell is at least one selected from the group consisting of non-small cell lung cancer, breast carcinoma, melanoma, lymphomas, colon carcinoma, and hepatocellular carcinoma.

17. The fusion protein of claim 16, wherein the antigen of the pathogen is at least one selected from the group consisting of PCV2 ORF2, CSFV E2, and human papillomavirus (HPV) E7 proteins.

18. The fusion protein of claim 16, wherein the antigen is a fusion antigen of foot-and-mouth disease virus protein VP1 (FMDV VP1) and Foot-and-mouth disease virus protein 3A (FMDV 3A), or a fusion antigen of Newcastle disease virus (NDV) F peptide and Newcastle disease virus hemagglutinin-neuraminidase (NDV HN) protein.

19. A vaccine composition comprising the fusion protein of claim 1 and an adjuvant.

20. A vaccine composition comprising the fusion protein of claim 16 and an adjuvant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is a schematic drawing showing a full-length Pseudomonas aeruginosa exotoxin A (PE), and partial fragment of PE.

(2) FIGS. 1B-C show vector maps.

(3) FIGS. 2-5 are graphs showing fusion proteins according to the invention eliciting enhanced CD8.sup.+/IFN-.sup.+ T cell (FIGS. 2A-5A) and CD4.sup.+/IFN-.sup.+ T cell (FIGS. 2B-5B) mediated immunogenicities, respectively.

(4) FIG. 6 shows animal groups, vaccines and dosage used for immunizing the animals, and immunization schedules.

(5) FIGS. 7-8 are graphs showing tumor size curves and percentage of tumor-free mice in the animal groups vaccinated with various fusion proteins or placebo, respectively.

DETAILED DESCRIPTION OF THE INVENTION

(6) The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like components throughout the views. As used in the description herein and throughout the claims that follow, the meaning of a, an, and the includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of in includes in and on unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention. Additionally, some terms used in this specification are more specifically defined below.

DEFINITIONS

(7) The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

(8) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

(9) The term an antigen-presenting cell (APC) or accessory cell refers to a cell that displays foreign antigens complexed with major histocompatibility complexes (MHC's) on their surfaces. T-cells may recognize these complexes using their T-cell receptors (TCRs). These cells process antigens and present them to T-cells. Main types of professional antigen-presenting cell are dendritic cells (DCs), macrophages, which are also CD4+ and are therefore also susceptible to infection by HIV; monocytes, and certain B-cells.

(10) The term an antigen-presenting cell (APC)-binding domain refers to a domain (which is a polypeptide) that can bind to an antigen-presenting cell (APC). The APC-binding domain may be a polypeptide comprising an amino acid sequence that is at least 90% identical to the sequence selected from the group consisting of SEQ ID NOs: 1 and 8-11. An APC-binding domain is a ligand that recognizes and binds to a receptor on APC.

(11) Cluster of differentiation 91 (CD91) is a protein that forms a receptor in the membrane of cells and is involved in receptor-mediated endocytosis.

(12) The term PE.sub.t refers to a translocation peptide (or a translocation domain) with 34-112 amino acid residues in length. PE.sub.t may comprises the amino acid sequence that is at least 90% identical to SEQ ID NO: 2-4 and 6. For example, the amino acid sequence of PE.sub.t may be a fragment of a.a. 280-a.a. 313 (SEQ ID NO: 4), a.a. 268-a.a. 313 (SEQ ID NO: 3), a.a. 253-a.a. 313 (SEQ ID NO: 2), or a.a. 253-a.a. 364 (SEQ ID NO: 6) of PE. That is, the amino acid sequence of PE.sub.t may contain any region of the PE domain 11 (a.a. 253 to a.a. 364; SEQ ID NO: 6) as long as it comprises a.a. 280-a.a. 313 (SEQ ID NO: 4) essential sequence (i.e., the essential fragment).

(13) The PE.sub.407 (SEQ ID NO. 7) is described in prior patent (U.S. Pat. No. 7,335,361 B2) as PE(III).

(14) The term minimum translocation peptide refers to PE.sub.253-313 (SEQ ID NO. 2), which can translocate an antigen into the cytoplasm of a target cell.

(15) The term an endoplasmic reticulum (ER) retention sequence refers to a peptide whose function is to assist translocation of an antigen from the cytoplasm into ER and retains the antigen in the lumen of the ER. An ER retention sequence comprises the sequence of Lys Asp Glu Leu (KDEL; SEQ ID NO: 15) or RDEL. An ER retention sequence may comprise the sequence KDEL, RDEL, KDELKDELKDEL (K3; SEQ ID NO: 16), KKDLRDELKDEL (K3; SEQ ID NO: 17), KKDELRDELKDEL (K3; SEQ ID NO: 18), or KKDELRVELKDEL (K3; SEQ ID NO: 19).

(16) A nuclear export signal (NES) refers to a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. The NES is recognized and bound by exportins. The most common spacing of the hydrophobic residues to be L.sub.xxKL.sub.xxL.sub.xL.sub.x (SEQ ID NO. 13), where L is leucine, K is lysine and x is any naturally occurring amino acid. For example, an artificial NES may comprise the sequence Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Ala (LQKKLEELELA; SEQ ID NO: 14).

(17) The term NESK refers to a fusion peptide of a NES and an ER retention signal (i.e., a NES fused to an ER retention signal). It is an artificial peptide possessing the function of a nuclear export signal (NES) and an ER retention sequence. Thus, it can export an antigen from the cell nucleus to the cytoplasm through the nuclear pore complex, and assist translocation of an antigen from the cytoplasm to ER and retain the antigen in the lumen of the ER. For example, the amino acid sequence of NESK may be LQKKLEELELAKDEL (SEQ ID NO: 12).

(18) An antigen may be a pathogenic protein, polypeptide or peptide that is responsible for a disease caused by the pathogen, or is capable of inducing an immunological response in a host infected by the pathogen, or tumor-associated antigen (TAA) which is a polypeptide specifically expressed in tumor cells. The antigen may be selected from a pathogen or cancer cells including, but not limited to, Human Papillomavirus (HPV), Porcine reproductive and respiratory syndrome virus (PRRSV), Human immunodeficiency virus-1 (HIV-1), Dengue virus, Hepatitis C virus (HCV), Hepatitis B virus (HBV), Porcine Circovirus 2 (PCV2), classical Swine Fever Virus (CSFV), Foot-and-mouth disease virus (FMDV), Newcastle disease virus (NDV), transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV). Influenza virus, pseudorabies virus, Parvovirus, Pseudorabies virus, Swine vesicular disease virus (SVDV), Poxvirus, Rotavirus, Mycoplasma pneumonia, Herpes virus, infectious bronchitis, or infectious bursal disease virus, non-small cell lung cancer, breast carcinoma, melanoma, lymphomas, colon carcinoma, hepatocellular carcinoma and any combination thereof. For example, HPV E7 protein (E7), HCV core protein (HCV core), HBV X protein (HBx) were selected as antigens for vaccine development. The antigen may be a fusion antigen from a fusion of two or more antigens selected from one or more pathogenic proteins. For example, a fusion antigen of PRRSV ORF6 and ORF5 fragments, or a fusion of antigenic proteins from PRRSV and PCV2 pathogens.

(19) The term treating or treatment refers to administration of an effective amount of the fusion protein to a subject in need thereof, who has cancer or infection, or a symptom or predisposition toward such a disease, with the purpose of cure, alleviate, relieve, remedy, ameliorate, or prevent the disease, the symptoms of it, or the predisposition towards it. Such a subject can be identified by a health care professional based on results from any suitable diagnostic method.

(20) The term an effective amount refers to the amount of an active compound that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on rout of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

(21) The invention relates to fusion proteins for enhancing antigen delivery and modulating cell-mediated immune response. The fusion protein comprises: (a) an antigen-presenting cell (APC)-binding domain or a CD91 receptor-binding domain, located at the N-terminus of the fusion protein; (b) a translocation peptide of 34-112 amino acid residues in length, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 2-4 and 6 and located at the C-terminus of the APC-binding domain or the CD91 receptor-binding domain; and (c) an antigen of a pathogen, located at the C-terminus of the translocation peptide; (d) a nuclear export signal (NES); and (e) an endoplasmic reticulum (ER) retention sequence, the ER retention sequence being located at the C-terminus of the fusion protein, wherein the NES comprises the amino acid sequence of SEQ ID NO: 13.

(22) Using the fusion protein PE.sub.313-ORF2-NESK as an example, the strategy is that the fusion protein of the invention stimulates the production and activation of T cells that can recognize the antigen Porcine Circovirus Type 2 (PCV2) capsid protein ORF2. The fusion protein comprises, from N-terminus to C-terminus, a PE domain I (APC-binding domain), a translocation peptide of 34-112 amino acid residues in length (e.g., a.a. 253-313 of the PE domain II), a truncated PCV2 ORF2 protein (N-terminal nuclear localization signal removed), a NES signal and an ER retention sequence (KDEL). The underlying mechanisms of eliciting enhanced ORF2-specific T cell immune responses by PE.sub.313-ORF2-NESK involve the following steps: a) binding to dendritic cell (or antigen-presenting cell) surface receptor (CD91); b) internalization by endocytosis; c) transporting to the ER and proteolytic hydrolysis by furin in front of the translocation peptide; d) processing and presenting by MHC I complex; and e) activating antigen-specific CD4+ and CD8+ T cells. CD4+ Th1 cells are able to efficiently stimulate and enhance cytotoxic CD8+ T cell immune response. Together, these two arms of the adaptive immune system have the specificity and potency to kill PCV2 and PCV2-infected cells.

(23) The fusion protein PE.sub.313-ORF2-NESK here is distinguishable from the fusion protein vaccine PE.sub.407-Ag-K3 disclosed by Lai in U.S. Pat. No. 7,335,361 in several aspects. Firstly, the length of PE.sub.313 (SEQ ID NO: 5) is 94 amino acid residues shorter than PE.sub.407 (SEQ ID NO: 7), the advantage of which is that unwanted humoral response elicited by the presence of an extra fragment of PE is minimized or eliminated. Secondly, the ER retention sequence is shortened. Instead of K3 (that is, 3 of KDER), only one KDER or RDER is needed. Thirdly, only cytosolic antigen can be processed and presented by MHC type I pathway, so the addition of a NES signal into the fusion protein is beneficial to enhance pathogen antigen-specific T cell responses because increasing the opportunity of translocation of antigen into cytosol. Antigens of a pathogen may be imported into the cell nucleus. By incorporating a NES signal, the antigen imported into the cell nucleus can be exported to the cytoplasm by the NES signal of the fusion protein.

EXAMPLES

(24) Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

Example 1

Construction of Expression Vectors

(25) FIG. 1A shows PE contains 3 domains (1, II, and III). PE.sub.407 is the region from a.a. 1 to a.a 407 of PE. PE.sub.407 does not contain the cytotoxic domain III and thus contains domains I and II. PE.sub.313 is the region from a.a. 1 to a.a. 313 of PE. Thus, PE.sub.313 contains only domain II and a partial N-terminal region of domain II of PE.

(26) FIGS. 1B-C show constructions of expression vectors, each of which comprises an antigen-presenting cell (APC)-binding domain, a translocation peptide, an antigen, with (bottom panel) or without (top panel) a nuclear export signal (NES); and an endoplasmic reticulum (ER) retention sequence (top panel. K3 or bottom panel, K), the ER retention sequence being located at the C-terminus of the fusion protein. The plasmids pTac-2-PE.sub.313-NESK, pTac-2-PE.sub.407-K3, pTac-2-RAP1-PE.sub.268-313-NESK and pTac-2-RAP1-PE.sub.268-313-K3 were generated as follows: The .sup.NdeIPE.sub.313-.sup.(EcoRI,XhoI)-NESK.sup.XhoI, .sup.NdeIPE.sub.407-.sup.(EcoRI,XhoI)-K3.sup.XhoI, .sup.NdeIRAP1-.sup.(EcoRI)-PE.sub.268-313-.sup.(EcoRI,XhoI)-NESK.sup.XhoI and .sup.NdeIRAP1-.sup.(EcoRI)-PE.sub.268-313-.sup.(EcoRI,XhoI)-K3.sup.XhoI fragments were synthesized by a PCR method and then ligated into a pUC18 back bond with kanamycin resistance gene to obtain respective plasmids.

(27) A target DNA encoding an antigen or a fusion antigen of a pathogen of interest may then be inserted into the aforementioned plasmids to generate an expression vector for expression of a fusion protein. For example, DNA fragments encoding antigens of Porcine Circovirus Type 2 (PCV2) ORF2 (SEQ ID NO: 20), Classical Swine Fever Virus (CSFV) E2 (SEQ ID NO: 21), Foot-and-mouth disease virus (FMDV) VP1-3A (SEQ ID NO: 24) and Newcastle disease virus (NDV) FUN (SEQ ID NO: 27) were synthesized and inserted into the plasmids pTac-2-PE.sub.313-NESK and pTac-2-PE.sub.407-K3, respectively, to generate the following expression vectors: (1) PE.sub.313-ORF2-NESK; (2) PE.sub.407-ORF2-K3; (3) PE.sub.313-E2-NESK; (4) PE.sub.407-E2-K3; (5) PE.sub.313-VP1-3A-NESK; (6) PE.sub.407-VP1-3A-K3; (7) PE.sub.313-FHN-NESK; and (8) PE.sub.407-FHN-K3. DNA fragments encoding antigen of Human Papillomavirus Type 16 E7 (SEQ ID NO: 28) were synthesized and inserted into the plasmids pTac-2-PE.sub.407-K3, pTac-2-RAP1-PE.sub.238-313-NESK and pTac-2-RAP1-PE.sub.268-313-K3, respectively, to generate the following expression vectors: (9) PE.sub.407-E7-K3, (10) RAP1-PE.sub.268-313-E7-NESK and (11) RAP1-PE.sub.268-313-E7-K3.

Example 2

Protein Expression

(28) E. coli BL21 cells harboring plasmids for expression of fusion proteins (1) PE.sub.313-ORF2-NESK; (2) PE.sub.407-ORF2-K3; (3) PE.sub.313-E2-NESK; (4) PE.sub.407-E2-K3; (5) PE.sub.313-VP1-3A-NESK; (6) PE.sub.407-VP1-3A-K3; (7) PE.sub.313-FHN-NESK; (8) PE.sub.407-FHN-K3; (9) PE.sub.407-E7-K3; (10) RAP1-PE.sub.268-313-E7-NESK and (11) RAP1-PE.sub.268-313-E7-K3 were respectively cultured in Luria Bertani broth containing 25 ppm of kanamycin at 37 C. When the culture reaching early log phase, (A600=0.1 to 0.4), isopropyl-1-thio--D-galactopyranoside (IPTG) was added with a final concentration of 0.5 to 2 mM for induction. Cells were harvested after induction after 4 hours and immediately stored at 70 C. The fusion proteins were purified by urea extraction as described previously (Liao et al., 1995. Appl. Microbiol. Biotechnol. 43: 498-507) and then were refolded by dialysis method against 50 volume of TNE buffer (50 mM Tris. 50 mM NaCl and 1 mM EDTA) at 4 C. for overnight. The refolded proteins were subjected to SDS-PAGE analyses and quantitative analyses performed using Bradford Protein Assay Kit (Pierce). The results indicated that most of the refolded proteins were monomers under a non-reduced condition, indicating that the fusion proteins refolded easily and were not aggregated.

Example 3

PCV2 Subunit Vaccines Immunogenicity Assay

(29) Mice were vaccinated with 0.1 ml PCV2 subunit vaccine containing 40 g of PE.sub.313-ORF2-NESK or PE.sub.407-ORF2-K3 with aluminum phosphate (a protein absorbent for slow release of the fusion protein; 10% v/v) and 10 g of saponin (an adjuvant extracted from Quillaja saponaria) via s.c. injection once a week for 3 weeks. The control group (placebo) was injected with adjuvant only without the fusion protein. All mice were sacrificed 14 days after the last immunization, and the spleens were harvested. The splenocytes were isolated and cultured in 6-well plate (10.sup.8 cells/2 ml/well) with or without the recombinant ORF2 protein in the presence of 1 g/ml GolgiPlug (BD Pharmingen. San Diego, Calif.) at 37 C. for 16 hr. The stimulated splenocytes were then washed with FACScan buffer and stained with phycoerythrin-conjugated monoclonal rat anti-mouse CD8a and AF700-conjugated monoclonal rat anti-mouse CD4 antibodies. Cells were intracellular cytokine stained using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Pharmingen). Intracellular IFN- was stained with AF488-conjugated rat anti-mouse IFN- to measure the immune response and cytokine levels. Flow cytometry analyses were performed using Gallios flow cytometry with Kaluza analysis software (Beckman Coulter).

(30) FIGS. 2A-B show the numbers of CD8 and CD4 positive IFN- T cells in the splenocytes from mice vaccinated with a placebo (adjuvant only without the fusion protein) or fusion proteins, respectively. The IFN- production by CD4+ and CD8+ T cells in splenocytes stimulated with ORF2 was detected by intracelluar staining via flow cytometry. Bar graphs show the numbers of ORF2-specific IFN-+CD4+ T cells (FIG. 2B) and IFN-+CD8+ T cells (FIG. 2A) from each group with (grey bars) or without (black bars) stimulation by the ORF2 peptide. The results indicated that the mice that had been vaccinated with PE.sub.313-ORF2-NESK had more ORF2-specific CD4+IFN-+ and CD8+IFN-+ T cells stimulated by the ORF2 peptide than the mice that had been vaccinated with PE.sub.407-ORF2-K3 group.

Example 4

CSFV Subunit Vaccines Immunogenicity Assay

(31) Using the same immunization schedule and dosage, mice were vaccinated with CSFV subunit vaccines containing PE.sub.313-E2-NESK or PE.sub.407-E2-K3, and splenocytes isolated, cultured and assayed by a flow cytometry method as described above, except that the recombinant E2 protein was added to stimulate the splenocytes in the culture.

(32) FIGS. 3A-B show the numbers of CD8 and CD4 positive IFN- T cells in the splenocytes from mice vaccinated with a placebo (adjuvant only without the fusion protein) or fusion proteins, respectively. The IFN- production by CD4+ and CD8+ T cells in splenocytes stimulated with E2 was detected by intracelluar staining via flow cytometry. Bar graphs show the numbers of E2-specific IFN-+CD4+ T cells (FIG. 3B) and IFN-+CD8+ T cells (FIG. 3A) from each group with (grey bars) or without (black bars) stimulation by the E2 peptide. The results indicated that the mice that had been vaccinated with PE.sub.313-E2-NESK had more E2-specific CD4+IFN-+ and CD8+IFN-+ T cells stimulated by the E2 peptide than the mice that had been vaccinated with PE.sub.407-E2-K3 group.

Example 5

FMDV Subunit Vaccines Immunogenicity Assay

(33) Using the same immunization schedule and dosage, mice were vaccinated with FMDV subunit vaccines containing PE.sub.313-VP1-3A-NESK or PE.sub.407-VP1-3A-K3, and splenocytes isolated, cultured and assayed by a flow cytometry method as described above, except that the recombinant VP1-3A protein was added to stimulate the splenocytes in the culture.

(34) FIGS. 4A-B show the numbers of CD8 and CD4 positive IFN- T cells in the splenocytes from mice vaccinated with a placebo or fusion proteins. The IFN- production by CD4+ and CD8+ T cells in splenocytes stimulated with VP1-3A was detected by intracelluar staining via flow cytometry. Bar graphs show the numbers of VP1-3A-specific IFN-+CD4+ T cells (FIG. 4B) and IFN-+CD8+ T cells (FIG. 4A) from each group with (grey bars) or without (black bars) stimulation by the VP1-3A peptide. The results indicated that the mice that had been vaccinated with PE.sub.313-VP1-3A-NESK had more VP1-3A-specific CD4+IFN-+ and CD8+IFN-+ T cells stimulated by the VP1-3A peptide than the mice that had been vaccinated with PE.sub.407-VP1-3A-K3 group.

Example 6

NDV Subunit Vaccines Immunogenicity Assay

(35) Using the same immunization schedule and dosage, mice were vaccinated with FMDV subunit vaccines containing PE.sub.313-FHN-NESK or PE.sub.407-FHN-K3, and splenocytes isolated, cultured and assayed by a flow cytometry method as described above, except that the recombinant FHN protein was added to stimulate the splenocytes in the culture.

(36) FIGS. 5A-B show the numbers of CD8 and CD4 positive IFN- T cells in the splenocytes from mice vaccinated with a placebo or fusion proteins. The IFN- production by CD4+ and CD8+ T cells in splenocytes stimulated with FHN was detected by intracelluar staining via flow cytometry. Bar graphs show the numbers of FHN-specific IFN-+CD4+ T cells (FIG. 5B) and IFN-+CD8+ T cells (FIG. 5A) from each group with (grey bars) or without (black bars) stimulation by the FHN peptide. The results indicated that the mice that had been vaccinated with PE.sub.313-FHN-NESK had more FHN specific CD4+IFN-+ and CD8+IFN-7+ T cells stimulated by the FHN peptide than the mice that had been vaccinated with PE.sub.407-FHN-K3 group.

Example 7

Enhanced Inhibition of Tumor Growth Induced by Human Papilloma Virus Type 16 E7 Protein

(37) The fusion proteins PE.sub.407-K3, RAP1-PE.sub.268-313-E7-K3 and RAP1-PE.sub.268-313-E7-NESK were expressed and refolded using similar methods as described above. Mice were challenged with 210.sup.3 TC-01 cells (mouse lung epithelia cell harboring HPV type 16 E7 gene) via s.c. injection to induce HPV-16 type carcinoma. Twelve days after the TC-01 cell challenge, mice were vaccinated via s.c. with placebo (PBS), PE.sub.407-K3 (100 mg/dose), RAP1-PE.sub.268-313-E7-K3 (100 g/dose) or RAP1-PE.sub.268-313-E7-NESK (100 g/dose) with AS04C (GlaxoSmithKline) as an adjuvant once per week for 3 weeks (FIG. 6). AS04C, which is a cytotoxic T lymphocyte-enhancing adjuvant, comprises MPL (monophosphoryl lipid A, an immune potentiator) and aluminum phosphate (a protein absorbent for antigen delivery). The term K3 refers to an ER retention sequence comprising KDEL. For example, K3 may be the amino acid sequence KDELKDELKDEL (SEQ ID NO: 16). The term NESK refers to a fusion peptide comprising a nuclear export signal and an ER retention sequence. In one embodiment of the invention, the NESK is the amino acid sequence LQKKLEELELAKDEL (SEQ ID NO: 12). The size of tumors and the number of tumor-free animals in each group were recorded (FIGS. 7 and 8). The tumor growth was significantly suppressed by vaccines PE.sub.407-E7-K3, RAP1-PE.sub.268-313-E7-K3 and RAP1-PE.sub.268-313-E7-NESK with AS04C as an adjuvant. However, the vaccine RAP1-PE.sub.268-313-E7-NESK was superior to PE.sub.407-E7-K3 and better than RAP1-PE.sub.268-313-E7-K3 in suppressing tumor growth and increasing the percentage of tumor-free animals.

Example 8

(38) The following fusion proteins are generated: PE.sub.313-NES-antigen-K, PE.sub.1-252-PE.sub.268-313-NES-antigen-K, PE.sub.1-252-PE.sub.280-313-NES-antigen-K. In addition, the fragment of PE domain Ia (PE.sub.1-252) of the fusion protein PE.sub.313-antigen-NESK is replaced by RAP1 domain 3 (SEQ ID NO: 8), A2M minimum (SEQ ID NO: 9), HIV-Tat minimum (SEQ ID NO: 10) or HSPs minimum (SEQ ID NO: 11) to generate the fusion proteins RAP1 domain 3-PE.sub.253-313-antigen-NESK. A2M-PE.sub.253-313-antigen-NESK, Tat-PE.sub.253-313-antigen-NESK and HSP-PE.sub.253-313-antigen-NESK, RAP1 domain 3-PE.sub.268-313-antigen-NESK, A2M-PE.sub.268-313-antigen-NESK, Tat-PE.sub.268-313-antigen-NESK and HSP-PE.sub.268-313-antigen-NESK vaccines, RAP1 domain 3-PE.sub.280-313-antigen-NESK, A2M-PE.sub.280-313-antigen-NESK, Tat-PE.sub.280-313-antigen-NESK and HSP-PE.sub.280-313-antigen-NESK, respectively. RAP1 domain 3-PE.sub.253-313-NES-antigen-K, A2M-PE.sub.253-313-NES-antigen-K, Tat-PE.sub.253-313-NES-antigen-K and HSP-PE.sub.253-313-NES-antigen-K, RAP1 domain 3-PE.sub.268-313-NES-antigen-K, A2M-PE.sub.268-313-NES-antigen-K, Tat-PE.sub.238-313-NES-antigen-K and HSP-PE.sub.268-313-NES-antigen-K vaccines, RAP1 domain 3-PE.sub.280-313-NES-antigen-K, A2M-PE.sub.280-313-NES-antigen-K, Tat-PE.sub.280-313-NES-antigen-K and HSP-PE.sub.280-313-NES-antigen-K. The cell mediated immune responses enhanced by these vaccines are examined using similar methods as described above.

(39) Table 1 shows SEQ ID NOs. of peptides used for making various fusion proteins.

(40) TABLE-US-00001 TABLE1 SEQ amino Component IDNO: acidresidues MinimumPseudomonasexotoxinA(PE)bindingdomain 1 252 Ia(APC-bindingdomain,a.a.1-a.a.252ofPE) PE.sub.253-313 2 61 PE.sub.268-313(translocationdomain) 3 46 PE.sub.tCore(PEtranslocationdomaincore;a.a.280-a.a. 4 34 313ofPE) PE.sub.313(a.a.1-a.a.313ofPE) 5 313 PE.sub.253-364 6 112 PE.sub.407(a.a.1-a.a.407ofPE) 7 407 RAP1Minimum(domainIIIofRAP1) 8 104 A2MMinimum 9 153 HIV-TatMinimum 10 24 HSPsMinimum 11 641 NESKisLQKKLEELELAKDEL* 12 15 NESconsensussequenceisL.sub.xxKL.sub.xxL.sub.xL.sub.x,whereinL 13 11 isleucine,K islysineandx isanynaturally occurringaminoacid. NESisLQKKLEELELA 14 11 KDEL 15 4 KDELKDELKDEL(K3) 16 12 KKDLRDELKDEL(K3) 17 12 KKDELRDELKDEL(K3) 18 13 KKDELRVELKDEL(K3) 19 13 PCV2ORF2(PorcineCircovirustype2OpenReading 20 192 Frame2) CSFVE2(ClassicalSwineFeverVirusEnvelope 21 328 glycoproteinE2) FMDVVP1peptide(viralcapsidproteina.a.127-a.a. 22 50 176ofVP1) FMDV3Apeptide(a.a.21-35of3A) 23 15 FMDV(Foot-and-MouthDiseaseVirus)VP1-3A 24 65 peptide** NDVFpeptide(a.a.65-a.a.82ofFusionprotein) 25 18 NDVHNpeptide(a.a.101-a.a.111ofHemagglutinin- 26 11 Neuraminidase) NDVFHNpeptide*** 27 29 HPV(HumanPapillomavirus)Type16E7 28 98 FulllengthPE(ExotoxinA,Pseudomonasaeruginosa) 29 613 *The bold letters represents the amino acid sequence of an artificial nuclear exporting signal; the underlined letters represents the amino acid sequence of and endoplasmic reticulum retention signal. **The VP1-3A peptide is a fusion antigen composed of a.a. 127-a.a. 176 of VP1 and a.a. 21-a.a. 35 of 3A; i.e., of a fusion of FMDV VP1 peptide (SEQ ID NO: 22) and FMDV 3A peptide (SEQ ID NO: 23). ***The FHN peptide is a fusion antigen composed of a.a. 65-a.a. 82 of fusion protein and (a.a. 101-a.a. 111 of Hemagglutinin-Neuraminidase; i.e., a fusion of NDV F peptide (SEQ ID NO: 25) and NDV HN peptide (SEQ ID NO: 26)

(41) In summary, the results have proved that a fusion protein containing an APC-binding domain at the N-terminal end, a translocation domain, followed by an antigen of a pathogen, and then a fusion peptide of NESK at the carboxyl terminal end is an improved design over the PE-fusion protein that is without the fusion peptide of NESK at the carboxyl terminus in terms of enhancing cell-mediated immune response, suppressing tumor growth, and/or increasing the percentage of tumor-free animals.

(42) While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. It is intended that the present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are within the scope as defined in the appended claims.

(43) The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

(44) Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.