CN116457009A

CN116457009A - Nucleic acid encoding a polypeptide comprising a modified Fc region of human IgG1 and at least one heterologous antigen

Info

Publication number: CN116457009A
Application number: CN202180073443.1A
Authority: CN
Inventors: 琳达·吉莉安·达兰特; 米雷勒·万科梅贝克; 维多利亚·布伦特维勒; 瑞秋·梅特林汉姆
Original assignee: Scancell Ltd
Current assignee: Scancell Ltd
Priority date: 2020-08-26
Filing date: 2021-08-25
Publication date: 2023-07-18
Also published as: GB202101435D0

Abstract

The present invention relates to nucleic acids and polypeptides encoded by those nucleic acids. In particular, the peptides comprise a modified IgG1Fc region and one or more heterologous epitopes, which may be B-cell epitopes or T-cell epitopes. The nucleic acids of the invention may encode polypeptides comprising: (i) A modified Fc region of human IgG1 and (ii) at least one heterologous antigen, wherein (a) the modified Fc region comprises at least an Fc portion capable of binding CD64 and/or TRIM21, (b) at least one residue of the Fc region is modified to a corresponding residue from a mouse IgG3 antibody, and (c) the modified Fc region has increased affinity for an Fc-gamma receptor (fcγr) as compared to a corresponding wild-type Fc region.

Description

Nucleic acid encoding a polypeptide comprising a modified Fc region of human IgG1 and at least one heterologous antigen

The present invention relates to nucleic acids and peptides encoded by those nucleic acids. In particular, the peptides comprise a modified IgG1Fc region and one or more heterologous epitopes, which may be B-cell epitopes or T-cell epitopes. The nucleic acids and peptides of the invention are useful as vaccines that stimulate high affinity CD 8T cells, th1 CD 4T-cells, and strong antibody responses. For cancer vaccines, it is critical to stimulate potent cellular immunity, including CD4 and CD 8T cell responses. For infectious disease vaccines, it is important to stimulate cellular immunity and neutralize antibodies.

The immune system is a powerful defense mechanism that can be used against harmful pathogens and malignancies. In particular, the adaptive immune system can be trained by vaccination to target altered, mutated or overexpressed autoantigens characteristic of malignant disease and invading foreign pathogens (e.g., viruses).

The concept by which the immune system can recognize and eliminate cancer cells is called immune surveillance [1,2]. Immunological differences between cancer and itself can be detected naturally and lead to the elimination of cancer cells in many individuals. This theory suggests that the immune system is naturally able to detect and eliminate cancer cells. Studies in immunocompetent mice have shown that tumor rejection and the resulting protection can be adoptively transferred to mice by metastatic T cells [3]. The mouse knockout model shows that interferon gamma (ifnγ) and lymphocytes are important for reducing the incidence of carcinogen-induced sarcomas and spontaneous epithelial cancers [4]. Patients with spontaneously regressing melanoma show signs of tumor-specific clonal T cell expansion, providing evidence of immune surveillance [5]. Recent work has shown that mutations caused by tumors during transformation can produce neoantigens that can be targeted efficiently by T cells [6]. This work emphasizes the fact that: the immune system is able to distinguish between tumors and itself, but cancer still occurs in many immunocompromised individuals. This is due to the high mutation frequency of tumors, which can lead to an immune balance, in which case the tumor avoids the original immune response, but the immune response can adapt and still attack the tumor. Eventually, despite the immune response, the tumor escapes and grows in a process called "immune editing" [7].

Immunotherapy seeks to recruit immune cells into the tumor microenvironment and enhance anti-tumor immunity. There are two main reasons that this situation can be prevented from occurring. First, tumors do not provide the correct environmental conditions to elicit an effective immune response. In the absence of co-stimulation induced by signals such as Toll-like receptors (TLRs) or damage related molecular pattern molecules (DAMPS) to stimulate a hazard or to sufficiently present tumor-specific epitopes to elicit a response, high avidity T cells capable of tumor lysis are not produced [8,9]. Stimulation of low avidity T cells that fail to kill tumor cells is believed to be the cause of failure of many early tumor vaccines. Strategies to address the lack of T cell priming and induce tumor-specific immune responses include vaccines. Effective anticancer immunotherapy relies on the efficient targeting of antigens that can be recognized by high affinity T cells. Many antigens expressed on tumors are also expressed on normal tissues and T cells that recognize them undergo thymus tolerance. This leaves numerous low avidity T cells that can be stimulated by high doses of immunogen, but never observed to target tumor cells sufficiently to kill them. The goal is to find antigens that are not thymus expressed and are present in high abundance on tumor cells but not in healthy tissue.

Viruses may be generally classified as "non-enveloped" or "enveloped". Functionally, the viral envelope is capable of allowing the virus to enter its host. Viral glycoproteins on the surface of the envelope recognize and bind to receptor sites on the host cell membrane. This results in fusion of the viral envelope with the host's membrane, entry and release of the viral genome, and infection of the host. At the early stages of viral infection, CD4 and CD 8T cell responses are generated to localize and kill the virus-infected cells, thereby preventing further replication of the virus. In subsequent infections, virus neutralizing antibodies (VNab) are raised to help prevent re-infection. The combination of memory T cell response and VNab is important in preventing new infections with the same or related viruses. The same phenomenon applies to other pathogen infections. Vaccines need to stimulate an immune response prior to exposure to the virus/pathogen, and are designed to enable the host to respond quickly and effectively remove low viral loads and prevent any morbidity associated with the virus. Thus, vaccines require neutralizing antibodies that stimulate recognition of high avidity T cells of low antigen load and prevent virus entry into the cells. T cells recognize viral proteins because they are presented in the context MHC on the cell surface of infected cells. However, neutralizing antibodies need to bind to viral proteins that contact receptors on host cells and allow them to enter the cell. The most effective viral vaccines are attenuated viruses that stimulate potent T cell and antibody responses but are associated with low morbidity. Several licensed attenuated viral vaccines have been available, including smallpox, measles and poliomyelitis. However, many viruses have evolved to evade immune recognition and are therefore unsuitable as attenuated virus vaccines. This can be overcome by using inactivated viruses, which are inactivated using chemicals such as formaldehyde or heat inactivation. Such vaccines can stimulate antibody responses, but require large amounts of virus/pathogen and are poor in stimulating high avidity T cell responses. A similar approach is to use virus-like particles that assemble and look like viruses, but cannot replicate. Also, these vaccines induce strong antibody responses and have been licensed to prevent HPV infection. An alternative to attenuated viruses is to use hybrid viruses. Viruses such as measles or adenovirus are genetically modified to produce proteins from heterologous viruses. Attenuation or incapacitation of these viruses renders them non-pathogenic; they may still replicate in cells or they may have deleted genes so that they cannot replicate. Viral vectors tend to have good safety and ebola virus vaccines have recently been approved. However, the presence of any pre-existing immunity to viral vectors (measles, adenovirus) may affect their effectiveness and limit the ability to boost the attenuated immune response. They can also be used only for single viruses, because once an immune response to a vector virus is established, they cannot be used as vectors for new viruses. Viral vaccines have the advantage that they can produce high levels of protein expression, inducing a strong antibody response. This can also be achieved using protein vaccines. However, protein vaccines also require adjuvants to mimic a potent immune response; in addition, multiple doses are often required. Both protein and heterologous viral vaccines produce high levels of antigen, but this stimulates a low avidity T cell response, which kills only cells with high viral load, which is dangerous because these cells may be lysed by the virus before the low avidity T cells have time to react and cause large amounts of virus to flood the host.

US7067110B1 discloses the use of Fc-antigen fusion proteins, wherein fusion of an intact antigen or antigen domain to the hinge-CH 2-CH3 domain of an antibody enhances antibodies and cellular immunity. WO2002/058728 discloses that targeting FcgammaRI with a human IgG1 Fc polypeptide fused to an antigen can stimulate a high affinity T-cell response. WO2008/116937 discloses nucleic acids comprising a non-specific promoter and at least one sequence encoding a recombinant heavy chain of an immunoglobulin molecule, wherein the heavy chain has at least one heterologous T cell epitope such that the heavy chain is unable to assume its native conformation upon expression of the nucleic acid. Disruption of primary antibody structure, inhibition of folding and/or limiting secretion to only heavy chains or very small amounts of intact antibodies stimulates strong helper and antigen-specific T cell responses.

In a first aspect, the present invention provides a nucleic acid encoding a polypeptide comprising:

(i) A modified Fc region of human IgG1, and

(ii) At least one of the antigens is a heterologous antigen,

wherein (a) the modified Fc region comprises at least an Fc portion capable of binding CD64, compared to a corresponding wild-type Fc region, (b) at least one residue of the modified Fc region is modified to a corresponding residue from a mouse IgG3 antibody, and (c) the modified Fc region has enhanced affinity for an Fc-gamma receptor (fcγr).

In a second aspect, the present invention provides a nucleic acid encoding a polypeptide comprising:

(i) A modified Fc region of human IgG1, and

(ii) At least one of the antigens is a heterologous antigen,

wherein (a) the modified Fc region comprises at least an Fc portion capable of binding TRIM21, (b) at least one residue of the Fc region is modified to a corresponding residue from a mouse IgG3 antibody, and (c) the modified Fc region has increased affinity for an Fc-gamma receptor (fcγr) as compared to a corresponding wild-type Fc region.

In a third aspect, the present invention provides a nucleic acid encoding a polypeptide comprising:

(i) A modified Fc region of human IgG1, and

(ii) At least one of the antigens is a heterologous antigen,

wherein (a) the modified Fc region comprises at least an Fc portion capable of binding CD64 and/or TRIM21, (b) at least one residue of the Fc region is modified to a corresponding residue from a mouse IgG3 antibody, and (c) the modified Fc region has enhanced affinity for an Fc-gamma receptor (fcγr) as compared to a corresponding wild-type Fc region.

In a fourth aspect, the invention provides a vector comprising the nucleic acid of the first aspect.

In a fifth aspect, the invention provides a polypeptide encoded by a nucleic acid of the first aspect or a vector of the second aspect.

The inventors have unexpectedly found that transferring certain mouse IgG3 (mIgG 3) Fc residues to the IgG1Fc region of an antigen-Fc fusion protein increases the immunogenicity of the antigen. MIgG3 is the only isotype in MIgG that forms a non-covalent oligomer, strongly influences its biological activity [10], and enhances functional affinity for multivalent antigens. Non-covalent interactions between adjacent mIgG3 Fc regions are thought to enhance this enhanced functional affinity by extending target occupancy and decreasing dissociation rates [11,12]. Grey et al [13] initially determined the propensity of mIgG3 to oligomerize, indicating that binding to multivalent antigens promotes mIgG3 intermolecular interactions, resulting in enhanced functional affinity for the antigen [14,15], a feature known as "intermolecular synergy" [12,15,16]. This phenomenon was determined to be Fc dependent, since IgG 3F (ab') 2 fragments did not bind synergistically to the antigen [14]. The observation of improved immunogenicity in the present invention is surprising, as no oligomerized hIgG1Fc was expected to bind to FcγRI on dendritic cells. Surprisingly, the inventors demonstrate that Fc modifications according to the invention improve cellular and humoral responses to heterologous antigens. This suggests that enhanced fcyri avidity results in more efficient antigen presentation. Furthermore, since the Fc-modified polypeptides of the invention initially bind in monomeric form, they are unable to bind to the low affinity FcRIIb and FcRIIIb inhibitory receptors that lead to suppression of immune responses. The creation of improved vaccines with enhanced immunogenicity by establishing intermolecular synergistic binding can lead to superior clinical applications.

The modified Fc region may have an enhanced affinity for Fc-gamma receptors (fcγr), preferably fcγri, of at least about 10% as compared to the corresponding wild-type human IgG1 Fc region. The modified Fc region may have an enhanced affinity for Fc-gamma receptors (fcγr), preferably fcγri, of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% compared to the corresponding wild-type human IgG1 Fc region.

The polypeptides of the invention preferably have enhanced immunogenicity and/or non-covalent oligomerization compared to the corresponding peptides comprising an unmodified wild-type human IgG1 region and at least one heterologous antigen.

The immunogenicity and/or non-covalent oligomerization of the polypeptides of the invention may be enhanced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% as compared to a corresponding polypeptide comprising an unmodified wild-type human IgG1 region and at least one heterologous antigen.

In the present invention, the modified Fc region comprises an Fc portion that binds CD64 and/or TRIM 21. The modified Fc region may comprise CH2 and CH3, and optionally may also comprise a hinge region. One or more residues of the human IgG1 CH2 and/or CH3 domain may be substituted with corresponding residues from the mouse IgG3 CH2 and/or CH3 domain. In some aspects of the invention, at least one residue of the Fc region is selected from the CH2 and/or CH3 domains. In some aspects, at least one residue is selected from the CH2 domain. In some aspects, at least one residue is selected from the CH3 domain.

Profiling the combined CH2-CH3 region by subdomain analysis revealed that by transferring non-contiguous segments comprising CH2 and CH3 elements (23 residues total) comprising mIgG3 residues 286-306 and 339-378, it was possible to enhance the affinity of the Fc region of human IgG1 for fcγr. These 23 residues are N286T, K288W, K290Q, A339 342R, P343A, R344Q, E345T, L351I, S354P, D356 357Q, L M, T359 361K, Q K, K T, G F, P38395 375E, D376A, A S. These residues are necessary to increase non-covalent oligomerization by intermolecular synergy due to direct interactions and the combined action of conformational residues, the latter possibly forming a permissive (permissive) framework. Further details of Fc region modifications are described in PCT/EP2020/071724, the contents of which are incorporated by reference in their entirety.

The human IgG1 Fc region may have modifications to one or more of the following Fc region residues: n286, K288, K290, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378. The modification may be one or more of the following: N286T, K288W, K Q, A339P, Q342R, P343A, R344 3834 345T, L I, S354P, D Q, A357Q, L358M, T359S, N361K, Q K, K370 371N, Y373F, P38395 375E, D376A, A378S.

The modified Fc region of human IgG1 can comprise modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 residues selected from the following: n286, K288, K290, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378. Preferably, the modified IgG1 antibody Fc region comprises modifications at all 23 residues.

The modified Fc region of human IgG1 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modifications selected from the group consisting of: N286T, K288W, K Q, A339P, Q342R, P343A, R344 3834 345T, L I, S354P, D Q, A357Q, L358M, T359S, N361K, Q K, K370 371N, Y373F, P38395 375E, D376A, A378S. Preferably, the modified IgG1 antibody Fc region comprises all 23 modifications.

The modified Fc region may comprise modifications to one or more of the following Fc region residues: n286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, a378. The modification may be one or more of the following: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q K, G371N, P S, S375E, D376A, A378S.

The modified Fc region may comprise modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 residues selected from the following: n286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, a378. Preferably, the modified human IgG1 antibody Fc region comprises modifications at all 15 residues.

The modified Fc region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modifications selected from the group consisting of: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q K, G371N, P S, S375E, D376A, A378S. Preferably, the modified Fc region comprises all 15 modifications.

The modified Fc region may comprise modifications to one or more of the following Fc region residues: q342, P343, E345, N361, Q362, P374, D376. The modification may be one or more of Q342R, P343A, E345T, N361K, Q K, P374S, D376A.

The modified Fc region may comprise modifications at 1, 2, 3, 4, 5, 6, or 7 residues selected from the following: q342, P343, E345, N361, Q362, P374, D376. Preferably, the modified Fc region comprises modifications at all 7 residues.

The modified Fc region may comprise 1, 2, 3, 4, 5, 6, or 7 modifications selected from the group consisting of: Q342R, P343A, E345T, N361K, Q K, P374S, D a. Preferably, the modified Fc region comprises all 7 modifications.

The modified Fc region may comprise the amino acid sequence provided in SEQ ID NO. 1, or an amino acid sequence having at least 90% identity to SEQ ID NO. 1. SEQ ID NO. 1 is an amino acid sequence "iv1" of an exemplary modified Fc region (see Table 4).

The structure of the polypeptide of the invention may have the heavy chain sequence of the antibody or a substantial portion thereof. The structure and position of the immunoglobulin domain can be determined by reference to http:// www.imgt.org.

Throughout the specification, residue numbers refer to the standardized IMGT system for antibody sequence numbering, as disclosed by Lefranc et al (2009) [17]. Other suitable numbering systems are known to those skilled in the art. Other suitable numbering systems may be used to identify the corresponding residues between the modified human IgG1 antibody-its antigen fusion protein. Any numbering system that allows the identification of the corresponding residues is suitable for use in the present invention. The numbering system used herein is not limiting to the scope of the invention, but is used only to identify relevant residues that may be modified. The term "corresponding residue" means a residue at a structurally or functionally equivalent position in two or more antibodies or antigen binding fragments thereof that are compared. In some cases, the corresponding residues can be identified by sequence alignment. In some cases, the corresponding residues may be identified by structural comparison.

In some aspects of the invention, the polypeptide may comprise at least 10 amino acid residues of the Fc region, at least 20 amino acid residues of the Fc region, at least 30 amino acid residues of the Fc region, at least 40 amino acid residues of the Fc region, at least 50 amino acid residues of the Fc region, at least 75 amino acid residues of the Fc region, at least 100 amino acid residues of the Fc region, at least 200 amino acid residues of the Fc region, at least 300 amino acid residues of the Fc region, at least 400 amino acid residues of the Fc region, or at least 500 amino acid residues of the Fc region. Preferably, the polypeptide comprises the entire Fc region of human IgG 1.

At least one heterologous antigen may be linked (directly or through a linker) to the N-terminus of the modified Fc region of human IgG 1. It is less preferred if at least one heterologous antigen is linked (directly or via a linker) to the C-terminus of the modified Fc region of human IgG 1. When the heterologous antigen is a polypeptide, it may be linked to the modified Fc region at the N-terminus or more preferably the C-terminus of the heterologous antigen. Where the heterologous antigen is a relatively large molecule, such as a viral or bacterial protein or immunogenic fragment thereof, this form of the polypeptide of the invention may be used. In a preferred such form, the C-terminus of the heterologous antigen is linked to the N-terminus of the modified Fc region, optionally via a linker.

In some aspects of the invention, the at least one heterologous antigen comprises one or more epitopes selected from the epitopes listed in any one of figures 28 to 33.

In some aspects of the invention, the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of the epitopes listed in table 2 or table 3.

In some aspects of the invention, the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)GTGRAMLGTHTMEVTVYH(SEQ ID NO:29)；

(b) SVYDFFVWL (SEQ ID NO: 30); and

(c)WNRQLYPEWTEAQRLD(SEQ ID NO:31)。

(a)GTGRAMLGTHTMEVTVYH(SEQ ID NO:29)；

(b)SVYDFFVWL(SEQ ID NO:30)；

(c) WNRQLYPEWTEAQRLD (SEQ ID NO: 31); and

(d)VPLDCVLYRYGSFSVTLDIVQG(SEQ ID NO:32)。

(a)GTGRAMLGTHTMEVTVYH(SEQ ID NO:29)；

(b)SVYDFFVWL(SEQ ID NO:30)；

(c)WNRQLYPEWTEAQRLD(SEQ ID NO:31)；

(d)VPLDCVLYRYGSFSVTLDIVQG(SEQ ID NO:32)；

(e) ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 33); and

(f)QCTEVRADTRPWSGPYILRNQDDRELWPRKFF(SEQ ID NO:34)。

(a)LLMWITQCF(SEQ ID NO:35)；

(b)SLLMWITQC(SEQ ID NO:36)；

(c) PESRLLEFYLAMPFATPMEAELARRSLAQ (SEQ ID NO: 37); and

(d)PGVLLKEFTVSGNILTIRLTAADHR(SEQ ID NO:38)。

(a)LLMWITQCF(SEQ ID NO:35)；

(b)SLLMWITQC(SEQ ID NO:36)；

(c)PESRLLEFYLAMPFATPMEAELARRSLAQ(SEQ ID NO:37)；

(d)PESRLLEFY(SEQ ID NO:39)；

(e)RLLEFYLAMPFATP(SEQ ID NO:40)；

(f)LEFYLAMPF(SEQ ID NO:41)；

(g)EFYLAMPFATPM(SEQ ID NO:42)；

(h)MPFATPMEA(SEQ ID NO:43)；

(i)LAMPFATPM(SEQ ID NO:44)；

(j)LLEFYLAMPFATPM(SEQ ID NO:45)；

(k)LLEFYLAMPFATPMEAELARRSLAQ(SEQ ID NO:46)；

(l)PGVLLKEFTVSGNILTIRLTAADHR(SEQ ID NO:38)；

(m)LKEFTVSGNILTIRL(SEQ ID NO:47)；

(n)KEFTVSGNILT(SEQ ID NO:48)；

(o)KEFTVSGNILTI(SEQ ID NO:49)；

(p) TVSGNILTIR (SEQ ID NO: 50); and

(q)TVSGNILTI(SEQ ID NO:51)。

(a)KIADYNYKL(SEQ ID NO:52)；

(b)KLPDDFTGCV(SEQ ID NO:53)；

(c)ELLHAPATV(SEQ ID NO:54)；

(d)CPFGEVFNATRFASVTAWNR(SEQ ID NO:55)；

(e)RISNCVADYSVLYNSASFST(SEQ ID NO:56)；

(f)YLYRLFRKSNLKPFERDI(SEQ ID NO:57)；

(g)YQPYRVVVLSFELLHAPATV(SEQ ID NO:58)；

(h)ALNTPKDHI(SEQ ID NO:59)；

(i)LQLPQGTTL(SEQ ID NO:60)；

(j)LLLDRLNQL(SEQ ID NO:61)；

(k)GMSRIGMEV(SEQ ID NO:62)；

(l)ILLNKHIDA(SEQ ID NO:63)；

(m) GNGGDAALALLLLDRLNQLE (SEQ ID NO: 64); and

(n)KHWPQIAQFAPSASAFFGMS(SEQ ID NO:65)。

as used herein, the term "immunogenic fragment" is a portion of an antigen or protein that is smaller than the entire antigen or protein and is capable of eliciting a specific humoral and/or cellular immune response to the fragment in a host animal (e.g., human). Protein fragments may be produced using techniques known in the art, such as by proteolytic digestion, recombination, or by chemical synthesis. An internal fragment or terminal fragment of a polypeptide may be produced by removing one or more nucleotides from one end (for the terminal fragment) or both ends (for the internal fragment) of a nucleic acid encoding the polypeptide.

Linker sequences are generally flexible in that they are composed primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains that may limit flexibility. Alternatively, a joint with greater rigidity is also desirable. The available length or optimal length of the linker sequence can be readily determined. Typically, the linker sequence is less than about 12 amino acids in length, such as less than about 10 amino acids, or 2-10 amino acids in length. The length of the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Examples of suitable linkers that may be used include, but are not limited to: GGGGS (SEQ ID NO: 106), GGGSG (SEQ ID NO: 107), GGSGG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GSGGGP (SEQ ID NO: 110), GGEPS (SEQ ID NO: 111), GGEGGGP (SEQ ID NO: 112), GGEGGGSEGGGS (SEQ ID NO: 113) and GGGSGGGG (SEQ ID NO: 114). Other linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 115), TVELRT (SEQ ID NO: 116), TVSAS (SEQ ID NO: 117) and TVLSSAS (SEQ ID NO: 118). The preferred linker for use in the present invention is an Ig hinge.

Alternatively, the polypeptide of the invention may comprise an antibody variable region into which the or each heterologous antigen is inserted or substituted. The polypeptides of the invention may comprise a human IgG1 heavy chain comprising modifications in its Fc region. It is preferred if the or each heterologous antigen is replaced in one or more CDRs of the variable region. Although all CDRs can be used for substitution of heterologous antigens, the preferred CDR is CDR3. Where the heterologous antigen is, for example, a cancer antigen, this form of the polypeptide of the invention may be used.

The variable antibody may be a heavy chain variable region comprising the following heterologous antigens substituted into CDR1, CDR2 and CDR3, respectively:

(a) GTGRAMLGTHTMEVTVYH (SEQ ID NO: 29), SVYDFFVWL (SEQ ID NO: 30) and VPLDCVLYRYGSFSVTLDIVQG (SEQ ID NO: 32); or (b)

(b) LLMWITQCF (SEQ ID NO: 35), SLLMWITQC (SEQ ID NO: 36) and PESRLLEFYLAMPFATPMEAELARRSLAQ (SEQ ID NO: 37).

The polypeptide encoded by the nucleic acid of the invention may comprise the amino acid sequence provided by SEQ ID NO. 2 or SEQ ID NO. 3. SEQ ID NO. 2 and SEQ ID NO. 3 are the amino acid sequences of the complete antibody heavy chain encoded by the iSCIB1plus vector (see FIG. 31) and the iSCIB2 vector (see FIG. 33), respectively.

The heterologous antigen may be the N protein of a coronavirus or an immunogenic fragment thereof. Preferably, the N protein is derived from SARS-CoV-2. For example, the N protein may be from the SARS-CoV-2 lineage A Wuhan strain, the SARS-CoV-2 variant B.1.351 or the SARS-CoV-2 variant B.1.617.2. The N protein may comprise the amino acid sequence provided by SEQ ID NO. 4 (Wuhan strain). The N protein may comprise the amino acid sequence provided in SEQ ID NO. 5 (B.1.351 variant). The N protein may comprise the amino acid sequence provided in SEQ ID NO. 26 (B.1.1.7 variant).

Preferably, the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided by SEQ ID NO. 6. SEQ ID NO. 6 is the amino acid sequence of the N protein (Wuhan strain) fused to the modified Fc region iv1, encoded by the "SN15" vector (see FIG. 27).

Preferably, the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided by SEQ ID NO. 7. SEQ ID NO. 7 is the amino acid sequence of an N protein (B.1.351 variant) fused to the modified Fc region iv1, encoded by the "SN17" vector (see FIG. 48).

The polypeptide encoded by the nucleic acid of the invention may comprise the amino acid sequence provided by SEQ ID NO. 27. SEQ ID NO. 27 is the amino acid sequence of the N protein (B.1.1.7 variant) fused to the modified Fc region iv1, encoded by the "SN16" vector (see FIG. 47).

The nucleic acids of the invention may be provided in combination (alone or in conjunction) with a second nucleic acid encoding a second polypeptide comprising at least one heterologous antigen. The second polypeptide may be an antibody light chain. The light chain may have one or more heterologous antigens inserted or substituted into it. The or each heterologous antigen may be substituted into one or more CDRs of an antibody light chain. Although all CDRs can be used for substitution of heterologous antigens, the preferred CDR is CDR3.

The antibody light chain encoded by the second nucleic acid may comprise the following heterologous antigens substituted into CDR1, CDR2 and CDR3, respectively: WNRQLYPEWTEAQRLD (SEQ ID NO: 31), ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 33) and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 34).

The antibody light chain encoded by the second nucleic acid may comprise sequence PGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO: 38) substituted into CDR 2.

The antibody light chain encoded by the second nucleic acid may comprise the amino acid sequence provided by SEQ ID NO. 10 or SEQ ID NO. 11. SEQ ID NOS.10 and 11 are the amino acid sequences of the antibody light chains encoded by the iSCIB1plus vector (FIG. 31) and the iSCIB2 vector (FIG. 33), respectively.

Preferably, the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided by SEQ ID NO. 2 and the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided by SEQ ID NO. 10.

Preferably, the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID NO. 3 and the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided by SEQ ID NO. 11.

In some aspects of the invention, the second nucleic acid encodes a receptor binding domain of SARS-Cov-2. The receptor binding domain may comprise the amino acid sequence provided in SEQ ID NO. 8 (Wuhan strain RBD). The receptor binding domain may comprise the amino acid sequence provided in SEQ ID NO 9 (B.1.351 variant RBD). The receptor binding domain may comprise the amino acid sequence provided in SEQ ID NO. 28 (B.1.1.7 variant RBD).

Preferably, the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided by SEQ ID NO. 6 and the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided by SEQ ID NO. 8.

Preferably, the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID NO. 7 and the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided by SEQ ID NO. 9.

The polypeptide encoded by the nucleic acid of the invention may comprise the amino acid sequence provided by SEQ ID NO. 27 and the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided by SEQ ID NO. 28.

The polypeptide encoded by a nucleic acid of the invention may comprise an amino acid sequence that is at least 90% identical to any of the sequences described above. For example, a polypeptide encoded by a nucleic acid of the invention may comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the above sequences.

The present invention provides one or two of the amino acid sequences disclosed by figures 27, 28, 29, 30, 31, 32, 33, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 47, 48, 54, 55, 56, 57, 58 or 59, respectively. Preferred polypeptides according to the invention are listed in fig. 12b (optionally in combination with fig. 12 a), fig. 13b (optionally in combination with fig. 13 a), fig. 14b (optionally in combination with fig. 14 a), fig. 15b (optionally in combination with fig. 15 a), fig. 27b (optionally in combination with fig. 27 a), fig. 29a (optionally in combination with fig. 29 b), fig. 31a (optionally in combination with fig. 31 b) and fig. 33b (optionally in combination with fig. 33 a). Particularly preferred sequences are disclosed in figures 27, 31, 33, 48 and 54 to 59.

The inventors have demonstrated that immunization with SN15 (encoding the protein N and RBD of the martial strain of lineage a) results in a strong VNAb directed against this strain but cross-reactive with both the b.1.351 and b.1.617.2 variants RBD. Similarly, the inventors have demonstrated that immunization with SN17 (encoding the b.1.351 variant N protein and RBD) resulted in a strong VNAb directed against this strain but cross-reactive with both the lineage a wuhan strain and the b.1.617.2 variant RBD.

The avidity of the polypeptides of the invention comprising a modified Fc region and the corresponding polypeptides comprising a wild-type Fc region can be determined by surface plasmon resonance (e.g., biacore 3000/T200, GE Healthcare), for example, by injecting increasing concentrations of the polypeptides of the invention (0.3 nmol/L to 200 nmol/L) onto a CM5 chip comprising a suitable ligand (e.g., fcγri) and fitting the data to a suitable binding model using suitable software (e.g., BIAevaluation 4.1). Corresponding experiments can be performed on corresponding polypeptides having a wild-type Fc region using the same conditions. In some aspects of the invention, surface plasmon resonance indicates that a polypeptide of the invention comprising a modified Fc region exhibits a greater functional affinity for a ligand than a corresponding polypeptide having a wild-type Fc region when the polypeptide of the invention binds more tightly to a ligand-coated CM5 chip. As mentioned, the ligand may comprise an Fc receptor, in particular an fcγ receptor. All fcγ receptors (fcγr) belong to the immunoglobulin superfamily and are the most important Fc receptors for inducing phagocytosis of opsonized (labeled) microorganisms. The family includes several members: fcyri (CD 64), fcyriia (CD 32), fcyriib (CD 32), fcyriiia (CD 16 a), fcyriiib (CD 16 b), which members differ in their molecular structure and in their affinity for antibodies. Fcyri binds IgG more strongly than fcyrii or fcyriii binds IgG. Fcyri also has an extracellular portion consisting of three immunoglobulin (Ig) -like domains, one more domain than fcyrii or fcyriii. This feature allows fcyri to bind to a single IgG molecule (or monomer), but all fcyri receptors must bind to multiple IgG molecules in the immune complex to be activated. In some aspects of the invention, the preferred receptor is fcyri (CD 64). In some aspects of the invention, the preferred receptor is TRIM21. In some aspects of the invention, the polypeptides of the invention are capable of binding CD64 and/or TRIM21.TRIM21 is a cytoplasmic antibody receptor and E3 ubiquitin ligase. It detects antibodies inside cells and mediates their rapid proteasome degradation. If the antibody is modified within its variable region to express a T cell epitope or a heterologous antigen is linked to Fc and administered via a DNA plasmid that can directly transduce antigen presenting cells, the protein will be translated in the cell and targeted by TRIM21.

The Biacore CM5 chip coated with anti-his antibodies contained carboxymethylated dextran covalently attached to the gold surface. The molecules are covalently bound to the sensor surface via amine groups, thiol groups, aldehyde groups or carboxyl groups. Interactions involving small organic molecules (e.g., drug candidates) to macromolecular assemblies or whole viruses can be studied. The high binding capacity produces a high response, facilitating capture assays and interactions involving small molecules. High surface stability provides accuracy and precision and allows repeated analysis of the same surface. Other suitable chips are known to those skilled in the art and the surface plasmon resonance scheme can be tuned by standard techniques known in the art.

The immunogenicity of the polypeptide according to the invention can be determined. The improved properties of a polypeptide can be measured relative to the corresponding properties of a corresponding polypeptide that does not comprise modified Fc region residues. Since the improved functional property is a relative measure, the precise method used to determine the immunogenicity or any other functional property of the polypeptides of the invention does not affect the relative change in that functional property.

While not wishing to be bound by theory, the ability of the polypeptides of the invention to provide enhanced immunogenicity may be a direct result of modified human IgG1 Fc binding to cell surface receptors. The polypeptides and/or nucleic acids of the invention are generally administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the nucleic acid/polypeptide.

The polypeptides of the invention comprise at least one heterologous antigen. As used herein, "heterologous antigen" means an antigen heterologous to the modified Fc region. The antigen may be a T cell antigen or a B cell antigen. Some polypeptides according to the invention comprise both T cell antigens and B cell antigens. The antigen may be contained in a relatively large molecule (e.g., a viral protein or bacterial protein or immunogenic fragment thereof). Alternatively, the antigen may be an amino acid sequence that constitutes an antigen or an epitope within an antigen. Examples of such antigens/epitopes are listed in tables 2 and 3 herein.

The antigen may be from cancer or may be from an infectious disease. In some aspects, the antigen is from cancer. In some aspects, the antigen is from an infectious disease. The antigen may stimulate high affinity CD 8T cells, th1 CD 4T cells, and/or a strong antibody response. When the present invention is used as a cancer vaccine, it is important to stimulate potent cellular immunity, including CD4 and CD 8T cell responses. When the present invention is used as an infectious disease vaccine, it is important to stimulate cellular immunity and neutralize antibodies.

The polypeptides, nucleic acids and vectors of the invention can be used as vaccines against viral infections, in particular against coronavirus infections. Coronaviruses (CoV) are members of the subfamily coronaviridae (coronaviridae; the order of the viruses of the order cover), and are divided into four genera: alpha-coronavirus (alpha-CoV), beta-coronavirus (beta-CoV), gamma-coronavirus (gamma-CoV) and delta-coronavirus (delta-CoV) [18,19]. gamma-CoV and delta-CoV are commonly infected in birds, but some can cause mammalian infections. alpha-CoV and beta-CoV viruses are known to infect and cause disease in humans and animals. SARS-CoV (beta-CoV), 229E (alpha-CoV), HKU1 (beta-CoV), NL63 (alpha-CoV) and OC43 (beta-CoV) viruses all cause human infections [18], often causing upper respiratory tract infections and some relatively mild symptoms [20]. beta-CoV is the most pathogenic virus in humans, and the group also includes SARS-CoV-2, MERS-CoV and SARS-CoV [18,21,22]; these viruses have all exploded in the 21 st century. Among these coronaviruses, SARS-CoV-2 shows the highest homology with SARS-CoV, exhibiting 79% genetic similarity [23]. SARS-CoV-2 is most similar to bats coronavirus RaTG13 with 98% similarity [24].

The genome of CoV is a single stranded sense RNA (+ssrna) with a 5 '-cap structure and a 3' -poly-a tail. The RNA virus genome is typically less than 10kb in length, whereas the CoV genome is known to be the largest, approximately 30kb, in RNA viruses. The genomic viral RNA was used as a template to directly translate polyprotein 1a/1ab, which encodes the nonstructural protein (nsp) to form the replication-transcription complex (RTC) in a Double Membrane Vesicle (DMV) [25]. A nested set of subgenomic RNAs (sgRNAs) was then synthesized by RTC in a discontinuous transcriptional fashion [26]. Subgenomic messenger RNAs (mrnas) have a common 5 '-leader sequence and 3' -terminal sequence. Transcription termination occurs at transcriptional regulatory sequences located between Open Reading Frames (ORFs) and the leader RNA is then obtained. These negative strand sgrnas serve as templates for the production of subgenomic mRNA [27,28]. Typical CoV genomes and subgenomes comprise at least six ORFs. The first ORF (ORF 1 a/b) is approximately two-thirds the length of the entire genome, encoding 16 nsps (nsp 1-16), the other ORFs of the genome near the 3' -end, encoding at least four major structural proteins: spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). In addition to these four major structural proteins, different CoVs encode additional specific structural and accessory proteins, such as HE proteins, 3a/b proteins, and 4a/b proteins.

Like other coronaviruses causing respiratory tract infections, SARS-Cov-2 is transmitted primarily through respiratory tract droplets; the transmission rate of SARS-CoV-2 appears to be higher compared to SARS-CoV and MERS. In some people, the infection is asymptomatic, and these individuals are considered to be potential sources of SARS-CoV-2 infection [29], resulting in rapid spread of SARS-CoV-2. Pneumonia appears to be the most common manifestation in patients with post-infection covd-19, other symptoms also including fever, cough, shortness of breath, and bilateral infiltration with visible chest radiographs [30].

Upon infection with SARS-CoV-2, the median latency period is about 4 to 5 days, but symptoms may appear after 14 days [31-34], with 97.5% of symptomatic patients appearing within 11.5 days [32]. Viral load peaks within 5 to 6 days after symptoms appear; this is significantly earlier than the SARS virus that peaks 10 days after symptoms appear [35-38]. In those patients with acute respiratory distress syndrome, this occurs about 8 to 9 days after onset of symptoms [30,39]. SARS-CoV-2 causes an invasive inflammatory response, resulting in airway damage [40], and the severity of the disease depends not only on the viral infection but also on the host's immune response. The most common cause of death is respiratory failure (70% of cases); in addition, cytokine release induces cytokine storm effects, resulting in secondary infections [41] and sepsis, leading to multiple organ failure and death.

The first step in infection is the binding of the virus to the host cell via its target receptor. SARS-CoV-2 utilizes densely glycosylated spike (S) proteins to enter host cells. The S protein is a trimeric class I fusion protein in a metastable pre-fusion conformation that undergoes structural rearrangement to fuse the viral membrane with the host cell membrane [42,43]; this process is triggered when the S1 subunit binds to a host cell receptor. The SARS-CoV-2 virus binds to angiotensin converting enzyme 2 (ACE 2) receptor [24]; serine protease TMPRSS2 has also been reported to play an important role in host cell entry [44]. Viruses target airway epithelial cells, vascular endothelial cells and macrophages of the lung, all of which express ACE2[24,45]. The S1 subunit consists of an amino-terminal domain and a Receptor Binding Domain (RBD). RBD binds to ACE2, which triggers endocytosis of the SARS-CoV-2 virus, and exposes it to endosomal proteases [46]. The S2 subunit consists of a Fusion Peptide (FP) region and two heptad repeat regions (HR 1 and HR 2) [47,48]. In endosomes, the S1 subunit is cleaved off, exposing the fusion peptide, which inserts itself into the host membrane. The S2 region then folds upon itself bringing the HR1 and HR2 regions together. This results in membrane fusion and release of the viral package to the host cytoplasm.

Once the SARS-CoV-2 virus enters the host cell, four structural proteins are required for viral assembly: s protein, M protein, E protein and N protein. Homotrimers of the S protein constitute spikes on the viral surface that are responsible for attachment to host receptors [49,50], and the M protein shapes and binds the virion to the N protein [51,52]. The E protein plays a role in viral assembly and subsequent release and is important in disease pathology [53,54]. The N protein comprises two domains, each of which can bind to the viral RNA genome by a different mechanism. N protein has been reported to bind nsp3 protein to aid in linking the genome to the Replication Transcription Complex (RTC) and packaging the packaged genome into virions [20,55,56].

The RBDs of SARS-CoV and SARS-CoV-2 have 72% homologous amino acid sequences and highly similar tertiary protein structures. Computational modeling and biophysical measurements indicated that SARS-CoV-2RBD binds ACE2 with higher affinity than RBD from SARS-CoV [57,58]. The SARS-CoV-2S protein also contains a furin-like cleavage site similar to MERS-CoV and human coronavirus OC43, but this cleavage site is absent in SARS-CoV [59]. These features are likely to promote the observed increase in infection with SARS-CoV-2 and ultimately contribute to viral spread.

About 80% of individuals who are infected with SARS-CoV-2 are asymptomatic or mildly infected [30], indicating that effective immunity has been established. As with SARS-CoV and MERS, most patients exhibiting severe symptoms show evidence of peripheral lymphopenia, which is thought to be due to lymphocyte migration from the blood to the site of pulmonary infection [60,61]. Th1 cytokines such as IL-6, IFNγ, IP-10 and MCP-1 produced by CD 4T cells in response to viral infection recruit other T cells and monocytes [60], and in most cases successfully clear the infection. However, in some cases, the infection progresses and appears as a more severe lung pathology. This is thought to be due to immune response dysfunction and subsequent cytokine storm effects, with high levels of pro-inflammatory cytokines including IL-2, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP-1α and TNF α [30,40,62], possibly mediated by macrophages and elevated inflammatory monocyte levels. Such cytokine storms cause most of the associated severe pathologies, but it has not been clear until now whether persistent viral infections are required to drive persistent immune-mediated damage.

Detailed information about the immune response to SARS-CoV-2 has not been collected, but analogy can be derived from SARS-CoV and MERS. Both SARS-CoV and MERS are known to interfere with the response of type I and type III IFN to viral infection, which in turn results in an increase in inflammatory neutrophils and macrophages at the site of infection [63,64]. It is likely that SARS-CoV-2 also has a similar immune-subversion (immune subversion) mechanism. Increased disease severity is associated with increased neutrophils and decreased lymphocytes [65,66]. In the case of MERS, infection by macrophages and DCs can also lead to down-regulation of MHC class I and II, which can lead to a significant reduction in T cell priming at the site of infection [67]. It is currently unclear whether the SARS-CoV-2 virus will have a similar effect and whether this results in persistent infection with the virus that is not controlled.

The reduction of peripheral T lymphocytes in severe SARS-CoV and MERS cases suggests an important role for these cells in antiviral immunity. T cell mediated immune responses have been known to be critical for controlling viral infections for some time [68]. T-lymphopenia associated with severe disease is also associated with reduced versatility of CD 4T cells, particularly those producing IFNγ [69]. The combination of a less than ideal T-cell response with sustained antigen activation can lead to a state of functional exhaustion [70]. Interestingly, SARS-CoV-2 patients showed that disease severity was associated with increased T cell depletion, reduced functional diversity [71] and reduced activation [72], suggesting that impaired T cell responses may in part lead to disease progression and severity. It is likely that this may be an immune subversion caused by viral or virus-driven T cell depletion. The severe cohort is also often characterized by a predominance of Th2 cytokines (IL-4, IL-5 and IL-10) rather than Th1 cytokines, meaning that polarization to a Th1 type response is beneficial [73]. The presence of Th2 cytokine responses also promotes lung eosinophil infiltration and exacerbates pathology [74]. In SARS-CoV infection, strong T cell responses are also associated with higher titers of virus neutralizing antibodies [73]. However, the current literature suggests that although T cell responses are critical for viral clearance, care needs to be taken not to exacerbate lung pathology, as high T cell levels at the site of infection may also be associated with severe disease [75]. Many reports using murine models indicate that strong Th1 responses, as well as virus-specific CD8 responses and virus-specific neutralizing antibodies, are necessary for successful control of SARS-CoV and MERS [76,77]. The same may be the case for SARS-CoV-2. In a murine model of SARS-CoV infection, T cells, particularly CD 4T cells, have been hypothesized to be important for controlling infection. Indeed, in the mouse model, depletion of CD 4T cells resulted in prolonged viral persistent infection and increased lung pathology [78]. Adoptive T-cell transfer studies in mice have also shown the beneficial effects of previous SARS-CoV specific immunity in preventing infection [79].

Longitudinal data on the immune response of the SARS-CoV-2 survivors was also obtained too early, but evidence from recovered SARS-CoV patients suggests that long-term memory T-cell responses to both the S-antigen and the N-antigen exist up to 11 years after infection [80-82], which means that the beta coronavirus may have immune memory. Mouse models have also demonstrated that memory CD 8T cell responses are effective in providing protection from SARS-CoV infection [76]. Several bioinformatics approaches have identified potentially immunogenic regions of the SARS-CoV-2 protein and possible HLA limitations [83-86]. Although T cell epitopes have not been defined so far within SARS-CoV-2, S, N and M viral antigens have been demonstrated to be targets ([ 87] pre-copies) of CD4 and CD 8T cell mediated immunity. Grifoni et al identified responses in SARS-CoV-2 patients, with 100% of patients showing a co-dominant CD4 response to spike protein, M protein and N protein. A Th1 response was mainly observed, these strong Th1 responses as well as CD8 responses correlated well with the antibody response to S protein. About 70% of patients show a dominant CD8 response to S and M proteins, but responses to all other antigens are also observed. Interestingly, in Grifoni et al studies, evidence of cross-reaction of responses between other human coronaviruses was observed, as 40-60% of unexposed individuals showed a response to the SARS-CoV-2 antigen tested ([ 87] preprint).

Studies of SARS and MERS have shown that virus-specific antibodies are detectable in 80-100% of patients 2 weeks after symptoms, but circulating titers are generally short, gradually decreasing and becoming undetectable in most recovered individuals over the years [88,89 ]]. In the case of SARS-CoV-2, antibody Secreting Cells (ASCs), follicular helper T cells (T) _FH Cells), activated CD4+ T cells and CD8+ T cells, and increased SARS-CoV-2IgM and IgG antibodies [90 ]]. Memory B-cell as well as T-cell responses promote long term protection, but the former are not readily maintained and are detected mainly in the circulation and residues in secondary lymphoid tissues [76,88,91 ]]. Similarly, recent kinetic studies for analyzing antibody responsesThe study shows that most SARS-CoV-2 patients are [ ]>95%) seroconversion within 19 days of onset of the disease and antibody was detected at the end of the first week after onset of symptoms [89,92 ]]. In a small cohort of patients who could obtain serum samples 14 days or more after symptoms had developed, the seropositive rate for anti-N IgG (n=15) was 94%, the seropositive rate for anti-N IgM (n=14) was 88%, the seropositive rate for anti-RBD IgG (n=16) was 100%, and the seropositive rate for anti-RBD IgM (n=15) was 94%. IgG titres for N and RBD correlate with virus neutralization titres [93 ] ]。

However, the SARS-CoV-2 antibody response and its effect on disease progression remains unclear worldwide, as demonstrated by reports, indicating that early high serum IgG antibody titers correlate with disease severity [94]. In particular, early elevation of anti-S protein titers has been correlated with worse outcome due to induction of ARDS (acute respiratory distress syndrome) which is characterized by the differentiation of alveolar macrophages to a highly inflammatory rather than wound healing phenotype via antibody effector function mediated pathways [95]. Similarly, during SARS-CoV-1, rapid but transient anti-S and anti-N titers have been shown to correlate with worse results [96]. The recovered patients showed a peak titer on average 20 days after onset of symptoms, while the dead patients showed a peak titer only 14.7 days.

Recent studies have shown that antibodies against nucleocapsid proteins can bind to N proteins released during viral lysis or expressed on the surface of infected cells, and that immune complexes can be taken up by antigen presenting cells, on which TRIM21 targets cytosolic degraded N proteins and produces cytotoxic T cells against N peptides [97]. This is a rapid response, mediating viral clearance prior to establishment of neutralizing antibodies. This may be the primary method of action of a vaccine containing an N-protein linked to a modified Fc, whereby the modified Fc shows enhanced binding to TRIM 21. Furthermore, SN11 expressing an N-protein fused to a modified Fc produced a significantly better T cell response to the N-protein and also produced a stronger response to RBD, S1 and peptide RBD 417-425 than a similar construct expressing the same RBD construct but without N-Fc. This suggests that the modified N-Fc acts as an adjuvant and activates APCs, thereby also enhancing T cell responses to other antigens.

Although non-neutralizing antibodies may mediate intra-conductor protection, neutralizing antibodies are of greatest interest [98]. For example, polyclonal IgG concentrated from serum of vaccinated and challenged non-human primate (NHP) was able to protect naive NHP from Ebola Virus (EV) challenge, and early studies showed that development of antibody responses was related to the survival rate of EV disease [99,100], and finally three monoclonal antibody mixtures against ebola virus were developed by Regeneron. During the SARS/MERS virus life cycle, neutralizing antibodies inhibit virus docking (RBD targeting), membrane fusion (S-protein targeting) or exocytosis (egress) (M-protein targeting). Importantly, antibodies or plasma from convalescent patients are currently evaluated as one of the available treatment options for patients with covd-19—more than 70% of patients recovering from light covd-19 have measurable neutralizing antibodies that persist at return to the hospital—further confirming the idea that SARS-CoV-2 antibody responses play a role in disease resolution [101].

The surface S glycoprotein is the major antigen of coronaviruses and is critical for viral entry by engaging host receptors and mediating virus-host membrane fusion. Other structural proteins include E protein, M protein, and N protein, which are produced earlier and in larger amounts than other structural proteins. Immunization of rabbits with VLPs encoding a combination of all four proteins showed that the S protein and the N protein were more immunogenic than the other two proteins, with the N protein titer rising faster but more transiently than the S protein titer [102]. The SARS-CoV-2 receptor-binding domain (RBD) most tends to cause a potent neutralization reaction in a similar manner as shown by MERS-CoV and SARS-CoV-1RBD [103]. These facts indicate that RBD in the S1 subunit is the main vulnerable (vulnerable) site for antibody recognition and neutralization, which includes a range of conformational epitopes, but this is also the region where most naturally occurring mutations of the S protein occur [103-105]. Thus, cross-reactivity analysis of SARS-CoV-2 serum antibodies showed that most antibodies against the S protein did not cross-react with SARS-CoV-1, while antibodies against the N protein did show some cross-reactivity.

In rare cases, pathogen-specific antibodies can promote pathology, leading to a phenomenon known as extrinsic Antibody Dependence Enhancement (ADE). ADE has been well characterized in vitro studies of several flaviviruses including the Zika (Zika) virus and Dengue (Dengue) virus, as well as Respiratory Syncytial Virus (RSV) [106]. This process appears to be governed by low quality and/or low numbers of non-neutralizing antibodies that bind to viral particles through their Fab domains, while their Fc domains bind to Fc receptors (fcrs) expressed on monocytes or macrophages, thereby promoting viral entry and infection. ADE is mediated by the engagement of Fc receptors (FcR) expressed on different immune cells, including monocytes, macrophages and B cells, enabling entry of viral cells in the absence of their typical receptor or endosomal pH or proteases. For coronaviruses, previous studies have shown that immunization of mice with inactivated whole SARS-COV2, macaques with MVA encoded S protein, and mice with DNA vaccines encoding full-length S protein, can induce ADE or eosinophil mediated immunopathology to some extent [95,107]. The latter phenomenon appears to be driven by a combination of neutralizing antibody quality produced and short-term antibody responses (the faster titers lead to more severe disease), by mechanisms involving shifting alveolar macrophages from the wound healing phenotype to the pro-inflammatory phenotype (IL-8 production) and pro-inflammatory monocyte/macrophage recruitment and accumulation; this process is driven at least in part by fcγ effector functions [95].

Establishing pre-existing immunity against viruses has some value for eliminating viruses before the immune response becomes deregulated. In order to establish a population immunity to SARS-CoV-2, we need to allow the development of "population immunity", or we need to employ vaccination techniques. Both of which rely on the establishment of a long-term memory response. Vaccines for animals against several diseases caused by coronaviruses have been produced, including for avian infectious bronchitis virus, canine coronavirus and feline coronavirus (FCoV) [108]. However, the possibility of vaccine-enhanced disease (VED) hampers the development of vaccines against respiratory viruses, including FCoV. The pathology associated with the VED in FCoV is similar to acute lung injury caused by covd-19 in a small proportion of patients. The currently under development covd-19 vaccine focused on subunit vaccines that mainly encode S protein and stimulate VNAb and T cell responses; there remains a need to determine whether they stimulate a sustained memory response or avoid immune pathology.

Previous projects to develop vaccines affecting human coronaviridae have been directed against SARS and MERS. Vaccines against both SARS 109 and MERS 110 were tested in non-human animal models. There is no cure or protective vaccine shown to be safe and effective against SARS in humans [111].

According to the current information, immunization stimulated by vaccination is likely to require both a potent virus-specific CD8 and CD4T cell response as well as a neutralizing antibody response. Markers for potent antiviral T cell responses include high functional avidity combined with versatility. The regulation of functional avidity involves several factors, such as cytokines IL-12 and IL-15[112,113], CD 8. Alpha. Beta. Expression [114-116], TCR affinity [117], costimulatory molecule levels expressed by antigen presenting cells [112,118], and the maturation state of DCs. Thus, the challenge is to find vaccine approaches that mimic these conditions. Large immune complexes can be cross-presented by low affinity FcgammaRIIa (FcgammaRIV in mice), but only when the inhibitory FcgammaRIIb receptor is blocked or down-regulated [119]. Thus, the challenge is to prepare small genetic complexes that target high affinity fcyri and replace IgG that potentially occupy serum.

An effective vaccine strategy must prove: (i) Being able to prevent heterologous viral variants occurring during independent emergencies-notably, many S-targeting antibodies have significantly reduced neutralizing titers against heterologous spike glycoproteins; (ii) Capable of eliciting a robust immune response in elderly populations that are difficult to immunize and have an increased risk of SARS-COV 2-induced morbidity and mortality; and (iii) avoiding adverse vaccine results, such as showing vaccine-induced immunopathology after vaccination with SARS N protein [120]. Strategies are generally directed to eliciting neutralizing antibodies, which are demonstrated to be associated with in vivo protection; however, this may come at the cost of covering only a narrow range of viral reactivities (i.e., non-extensive neutralization). Antibodies that bind more conserved portions of the virus tend to be less neutralizing (in vitro assays), while relying on FcR engagement to provide in vivo protection, but may cover a wider range of protection. Although carrying more epitopes than RBD, the risk of ADE hampers the use of intact S protein [107]. Structurally, like SARS-CoV-1, SARS-CoV-2RBD is exposed to two known states of S protein trimer, namely a closed state in which each RBD contacts its analog on another protomer symmetrically, and an open state in which at least one RBD domain extends to contact ACE2.RBD also more readily produces and produces higher levels of neutralizing antibodies, most of which are directed against conformational epitopes and are not necessarily associated with ACE2 binding sites [104,105].

Certain polypeptides, nucleic acids and vectors according to the invention are intended to induce high affinity antibodies against RBD, as higher affinity antibodies with stronger neutralizing capacity have been shown to carry reduced risk of ADE [107]. Meanwhile, N protein is the earliest expressed protein and is more abundant than S or M protein [89]. Combining their more conserved nature and thus more likely to be heteroprotectively, are also effective targets of the present invention.

The invention can target S protein key RBD and virus N protein to generate CD 8T cells, CD 4T cells and VNAB. The S protein appears as a trimer upon interaction with ACE2 in order to optimally induce antibodies with a neutralizing phenotype. The present inventors multimerize RBD and this can be used in vaccines for the prevention or treatment of SARS-CoV-2.

One polypeptide of the invention comprises an N protein or immunogenic fragment thereof fused to a modified human IgG3 Fc region. The N protein or immunogenic fragment thereof may comprise amino acids 2-419 or 138-146. The modified human IgG3 Fc region can comprise a hinge-CH 2-CH3 region having the murine IgG3 modifications described herein. An example is shown in fig. 12 b. The polypeptide can be combined with a second polypeptide comprising RBD or an immunogenic fragment thereof. RBDs may contain amino acids 319-541 or 330-525 (accession number YP_ 009724390). The RBD may be (a) alone, (b) linked to a trimerization domain such as a secondary fibrin trimer folding (fibritin trimer fold on) motif or disulfide bridge motif, for example, by a glycine serine linker, or (c) fused in-frame to (i) the hinge-CH 2-CH3 domain of the HuIgG1 constant domain (accession No. P01857) or (ii) the variant hinge-CH 2-CH3 iV1 according to the invention. These examples are shown in table 4 herein.

In addition to infectious diseases, such as viral infections (as described above) and bacterial infections, the present invention can be used to target tumor antigens. Examples of such antigens are listed in table 3 herein.

Tumors accumulate mutations that drive growth and metastasis. These mutations represent unique epitopes that avoid thymus selection. They are called "neoepitopes" and are specific for individual tumors and are not found on normal tissues [121]. Lennerz et al identified responses to 8 antigens in mixed lymphocyte-tumor cell cultures from a single chronically surviving melanoma patient, 5 of which were neoantigens [122]. This is an early study that suggests that the neoantigen is involved in responses in long-term survivors. This has led researchers to develop personalized vaccines against identified neoepitopes. Not all mutations stimulate a T cell response, but there is a correlation between the frequency of mutation and the likelihood of presenting a T cell epitope. Indeed, patients with tumors with higher mutation rates generally showed better responses to checkpoint blocking therapies, suggesting that endogenous neoepitope responses are revealed by checkpoint blocking [123]. Since most mutations do not stimulate an immune response, it may be difficult to select the most appropriate targeting epitope. However, significant progress is being made in the proper selection of candidate epitopes [124].

In human melanoma, mass spectrometry has been used to identify neoepitopes directly from primary tumors, resulting in the identification of many potential targets [125]. Therapies targeting these neoepitopes are being transformed into the clinic and show effective induction of specific immune responses [126-128]. Several groups have treated patients with vaccines targeting multiple neoepitopes. Sahin et al demonstrate that a variety of neoepitope specific responses can be generated in patients following treatment with an intra-node delivered RNA multi-epitope vaccine [127]. They demonstrated a decrease in metastatic events and a sustained progression-free survival. Ott et al also treated 6 melanoma patients with peptide multi-epitope vaccine in combination with the adjuvant Hiltonol (stabilizing poly IC and poly-L-lysine double stranded RNA). They observed potent neoepitope-specific T cell responses with reduced recurrence rates [128]. Furthermore, patients with recurrent disease after vaccination showed complete regression after subsequent treatment with programmed cell death (PD-1), which is related to expansion of neoepitope specific T cell responses. Interestingly, the neoepitope identified in these studies was recognized by both CD8 and CD 4T cells, suggesting an important role for CD 4T cell responses in addition to CD8 responses in humans. This confirms previous data obtained by Kreiter et al in the mouse model [129]. One disadvantage of targeting neoantigens is that they are expensive because they are patient-specific and there can be great variability both within a tumor sample and between tumors of the same patient; this results in tumor growth that no longer expresses the mutation [130]. To overcome key "driver" mutations (e.g., BRAFV 600E) and other common mutations during tumorigenesis, they can be specifically targeted; however, these are relatively rare and do not always stimulate a T cell response.

During development, high-avidity T cells specific for autoantigens are often deleted in the thymus, leaving numerous low-avidity portions. Thus, antigens that show limited normal expression are likely to act as better targets, as they may not be subjected to the same degree of tolerance. Detection of autoantigen-specific T cells in patients with regressive cancer suggests that thymus tolerance is not always complete. Patients with regressive cancer mainly respond to antigens with restricted expression in normal tissues, such as the differentiation antigen TRP-2 and the cancer testis antigen NY-ESO-1[131,132]. Tumor Associated Antigens (TAA) [131,133] including NY-ESO-1 and the melanoma antigen MAGE-1[134] provide good targets for immune responses, indicating that they are subverted immune-tolerance.

Once the appropriate antigen is selected, it is important to consider how best to present it to the immune system. Stimulation of T cells requires processing and presentation of antigen by professional Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs), along with appropriate activating co-stimulatory signals. Activating costimulatory signals include those provided by TLR ligands [ reviewed in 135 ]. Preclinical studies examining the direct attachment of peptide vaccines to TLR ligands have begun to show promise. These are thought to target epitopes and TLRs more effectively to DCs, resulting in increased expression of DC maturation and costimulatory molecules, secretion of cytokines and chemokines, and formation of antigen reservoirs within DCs to allow long term peptide presentation [136,137]. In addition to direct linkage, studies have investigated the use of amphiphilic peptides in combination with TLR ligands assembled into nanostructures and show promise in preclinical studies [138,139]. It is also important to consider the antigen dose provided by the vaccine. The low dose may be sufficient to select the highest affinity T Cell Receptor (TCR) and thus select high affinity CD 8T cells [140], but may be insufficient to stimulate CD4T cells whose epitope targets exhibit lower affinity MHC-II binding.

It is widely accepted that the generation of a high frequency T cell response is not necessarily indicative of the induction of an effective immune response. It is apparent from the work previously disclosed that T cell functional avidity is a better indicator of clinical response [141-145]. The term functional affinity is often confused with affinity. Affinity is generally classified as a measure of the binding strength of peptide MHC molecules to T Cell Receptors (TCRs), whereas functional avidity is a measure of the combination of stimulation by TCRs, costimulatory molecules, adhesion molecules and cytokines, which is indicative of the overall strength of interaction between T cells and targets and their functional consequences [146]. In viral infection models and tumor models, only high avidity Cytotoxic T Lymphocytes (CTLs) mediate viral clearance and tumor eradication [117,141,143,147,148]. During the generation of an immune response in vivo, CTLs exhibit a range of functional affinities at both the cloning and polyclonal levels. Although avidity has been shown to be important in both viral and oncological settings, the mechanism by which high and low avidity CTLs are produced in vivo is still unclear, as TCRs cannot undergo somatic hypermutations. It has been demonstrated in vitro that culturing TCR transgenic CTLs in the presence of high or low doses of antigen results in polarization of low and high avidity responses, respectively [141,143].

In early studies, peptide vaccines encoding tumor epitopes showed promise in animal models, stimulating specific T cell responses and tumor treatment in mice. The transformation of these peptide vaccines into the clinic was less successful, with a short response tentatively with very low clinical efficacy. Early vaccines focused on stimulation of CD 8T cell responses with short peptides (< 15 amino acids). However, recent studies have focused on the use of longer peptide sequences that stimulate both CD4 and CD 8T cell responses to avoid the tolerability problems previously seen with shorter peptide sequences [149]. Longer peptide sequences began to show promising results in clinical studies [150,151]. Peptides encoding neoepitopes also began to show evidence of a certain potential for detection of robust immune responses and overall increased survival [152,153]. Studies by Ott et al (2017) showed an enhancement of neoepitope-specific responses following vaccination with 20 long peptides mixed with TLR3 ligand hiltenol [128].

Synthetic peptides are also used as part of DC-based vaccines. Many studies have been performed in which in vitro cultured DCs are pulsed with peptides, proteins or tumor lysates. These have been shown to stimulate an effective immune response in preclinical studies [ reviewed in 154 ]. Despite stimulation of immune responses, DC vaccines have shown limited efficacy clinically. The only therapeutic autologous cell-based vaccine that has been approved to date is Sipuleucel-TShows moderate survival benefits for 3 months, but production costs and time severely limit its use [155 ]]. This is a major limiting factor for most DC and autologous cell based vaccines. Furthermore, DC vaccines that bind neoepitope peptides show promising expansion of existing and de novo neoepitope-specific responses [126]. The use of immature DCs affects their immunogenicity and leads to tolerance induction, thus requiring close monitoring of the activation state of the DCs used. The rough culture method (extensive culture method) of making DC-based vaccines also affects their immunogenicity in vivo. Recent work focused on ex vivo isolation of DC subpopulations with minimal in vitro manipulations has shown promising results [156 ]]。

The nucleic acid of the invention may be DNA, cDNA or RNA such as mRNA obtained by cloning or produced entirely or in part by chemical synthesis. For therapeutic use, the nucleic acid is preferably in a form that is capable of expression in the subject to be treated. The nucleic acids of the invention may be recombinant or provided as an isolate in isolated and/or purified form. It may be free or substantially free of nucleic acids flanking the gene in the human genome, except that there may be one or more regulatory sequences for expression. Where a nucleic acid according to the invention comprises RNA, reference to a sequence as shown herein should be interpreted as reference to an RNA equivalent, wherein U replaces T.

The nucleic acids of the invention may be readily prepared by one of skill in the art, in view of the availability of nucleic acid sequences and clones, for example, using the information and references contained herein and techniques known in the art (see, e.g., sambrook et al (1989) [157] and Ausubel et al (1992) [158 ]). These techniques include (i) amplification of a sample of such nucleic acid, e.g., of genomic origin, using the Polymerase Chain Reaction (PCR), (ii) chemical synthesis, or (iii) preparation of a cDNA sequence. The DNA encoding the polypeptide may be produced and used in any suitable manner known to those skilled in the art, including by employing the encoding DNA, identifying suitable restriction enzyme recognition sites on either side of the portion to be expressed, and removing the portion from the DNA. This portion is then operably linked to a suitable promoter in standard commercially available expression systems. Another recombinant method is to amplify the relevant part of the DNA using suitable PCR primers. The sequence may be modified, for example using site-directed mutagenesis, resulting in expression of the modified peptide, or taking into account codon preference for the host cell in which the nucleic acid is expressed.

To obtain expression of a nucleic acid sequence, the sequence may be incorporated into a vector having one or more control sequences operably linked to the nucleic acid to control its expression. The vector may comprise other sequences, such as promoters or enhancers that drive expression of the inserted nucleic acid, nucleic acid sequences that allow the polypeptide to be produced as a fusion, and/or nucleic acids that encode secretion signals that allow the polypeptide produced in the host cell to be secreted from the cell. If desired, the polypeptide may be obtained by: transforming the vector into a host cell in which the vector is functional, culturing the host cell to produce the polypeptide, and recovering the polypeptide from the host cell or surrounding medium. Prokaryotic and eukaryotic cells are used for this purpose in the art, including E.coli strains, yeast strains, and eukaryotic cells, such as insect cells and animal cells, e.g., COS, CHO cells, bowes melanoma cells, and other suitable human cells. The present invention relates to nucleic acids encoding the heavy and light chains of an antibody, each of which may be present in the same expression vector (driven by the same promoter or different promoters), or in separate expression vectors.

The nucleic acids of the invention can be used to stimulate an immune response against at least one heterologous antigen in a patient (e.g., a mammal, including a human). May stimulate helper and/or cytotoxic T cell responses. The avidity of the T cell response to a specific epitope obtained in the present invention may be higher than that obtained by immunization with the same epitope as a simple peptide or by immunization with the same epitope encoded in the hospital as a peptide or a nucleic acid. The nucleic acids of the invention may be administered as a combination therapy (i.e., a nucleic acid encoding a light chain and a nucleic acid encoding a heavy chain). The nucleic acid may be administered intravenously, intradermally, intramuscularly, orally or by other routes. Intradermal or intramuscular administration is preferred because these tissues contain dendritic cells.

Another aspect of the invention provides a vector comprising a nucleic acid of the invention. Vectors may be used to express nucleic acids to obtain the polypeptides of the invention, or they may be used as therapeutics (e.g., vaccines) themselves.

Exemplary vectors include iSCIB1plus (see fig. 31), iSCIB2 (see fig. 33), SN15 (see fig. 27), and SN17 (see fig. 48).

The vector of the invention may comprise the nucleotide sequence provided below:

(a) SEQ ID NO. 12 and SEQ ID NO. 13; or (b)

(b) SEQ ID NO. 14 and SEQ ID NO. 15.

SEQ ID NO. 12 and SEQ ID NO. 13 are the nucleotide sequences of the complete iSCIB1plus heavy and light chain expression cassettes, respectively, both of which include the CMV promoter and the BGH polyA signal. SEQ ID NO. 14 and SEQ ID NO. 15 are the nucleotide sequences of the complete iSCIB2 heavy and light chain expression cassettes, respectively, both of which include the CMV promoter and the BGH polyA signal.

The vector of the present invention may comprise the nucleotide sequences provided by SEQ ID NO. 16 and SEQ ID NO. 17.

SEQ ID NO. 16 and SEQ ID NO. 17 are the nucleotide sequences of the complete SN 15N protein-Fc and S protein expression cassettes, respectively, both of which include the CMV promoter and the BGH polyA signal.

The vector of the invention may comprise SEQ ID NO. 18 and SEQ ID NO. 19.

SEQ ID NO. 18 and SEQ ID NO. 19 are the nucleotide sequences of the complete SN 17N protein-Fc and S protein expression cassettes, respectively, both of which include the CMV promoter and the BGH polyA signal.

The vector of the invention may comprise SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22. Preferably, the vector of the invention consists of the nucleotide sequence provided by SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22.

SEQ ID NO. 20 is the complete iSCIB1plus nucleotide sequence in plasmid form. SEQ ID NO. 21 is the complete iSCIB1plus nucleotide sequence in form of doggybone (dbDNA). SEQ ID NO. 22 is the complete iSCIB2 nucleotide sequence in plasmid form.

The vector of the invention may comprise SEQ ID NO. 23. Preferably, the vector of the invention consists of the nucleotide sequence provided by SEQ ID NO. 23. SEQ ID NO. 23 is the complete SN15 vector nucleotide sequence in plasmid form.

The vector of the invention may comprise the nucleotide sequence provided by SEQ ID NO. 24 or SEQ ID NO. 25. Preferably, the vector of the invention consists of the nucleotide sequence provided by SEQ ID NO. 24 or SEQ ID NO. 25. SEQ ID NO. 24 is the complete SN17 vector nucleotide sequence in plasmid form. SEQ ID NO. 25 is the complete SN17 vector nucleotide sequence in the form of doggybone (dbDNA).

The vector of the present invention may be DNA. Plasmid DNA vaccines offer advantages over other vaccine forms because they have inherent adjuvant activity, resulting in the recruitment of large numbers of inflammatory cells to the immune site. The mechanism by which DNA vaccines induce immunity is complex and not yet fully elucidated, but is thought to involve promiscuous and distinctive DNA sensors expressed by APCs (table 1). CpG motifs promote DC activation and maturation through TLR9 signaling [159]. Interestingly, DNA vaccine activity was still observed in TLR9 knockout mice, which involved other endosomes and cytoplasmic DNA sensors that mediate adjuvant activity [160]. DNA sensors such as TBK-1 and STING activate TLR independent pathways and induce type I interferons [161]. Recently, the helicase DDX41 was identified in myeloid dendritic cells as a new DNA sensor [162]. Furthermore, RIG-1 can stimulate type I IFN by sensing cytoplasmic DNA associated with RNA polymerase III [163]. IFN modulating factor DNA dependent activators (DAI/DLM-1/ZBP 1) are cytoplasmic DNA sensors and innate immune activators [164].

TABLE 1 DNA sensor providing powerful adjuvanticity

Nucleic acid	DNA sensor	Reference to the literature
			CpG	TLR9	[159]
dsDNA	STING/TBK1	[161]
			dsDNA	DDX41	[162]
DNA/RNA polymerase III	RIG-1	[163]
			Cytoplasmic DNA	DAI	[164]
Cytoplasmic DNA	AIM2	[160]
			Cytoplasmic DNA	IFI16	[165]
CpG,dsDNA	HMGB-1	[166]

HMGB-1 has emerged as a hybrid sensor for nucleic acid-mediated induction of innate immune responses [166]. HMGB-1 (chromatin binding protein) has a range of functions depending on its subcellular and extracellular localization, redox state and interactions with other cell surface receptors. As an intracellular complex, HMGB-1 related nucleic acids stimulate type I IFN through TLR and cytoplasmic mediated sensors, activating pro-inflammatory cytokines and inducing the inflammasome (albeit AIM 2) (reviewed in [160 ]). Extracellular release of HMGB-1 has a number of consequences, including maintenance of tumor cell autophagy (by competing with bcl-2 for beclin-1 binding and recruitment and activation of immune cells [167 ]). Recently, it has been shown that different redox states of HMGB-1 play an important role in HMGB-1 activity [167]. HMGB-1 contains three cysteines at positions C23, C45, HC106 that can be modified. The reduced holothiolal form of HMGB-1 is a chemoattractant that mediates leukocyte recruitment. The disulfide form has cytokine (but not chemokine) activity. The fully oxidized form of HMGB-1 induced by reactive oxygen species is inactive. HMGB-1 also forms complexes with cytokines and other immune receptors including RAGE, TLR4, TLR2, CD24, TIM-3, thrombospondin and TREM1[168,169]. The HMGB-1/DNA complex binds to RAGE and induces a switch from apoptosis to autophagy [169]. HMGB-1/CXCL12 binds CXCR4 to mediate recruitment of inflammatory cells [170].

Preferably, the vector of the invention is a DNA plasmid or doggybone (dbDNA) vector. Such vectors and methods for their production are well known in the art. In particular, dbDNA vectors and methods for their production are described in WO 2010/086626.

The vector of the invention may be RNA. There are two classes of mRNA vaccines, one that uses non-replicating mRNA and the other that uses self-replicating RNA. Both are contemplated by the present invention. Non-replicating mRNA vaccines contain only transcripts of the antigen of interest, whereas self-replicating RNA vaccines contain transcripts required for mRNA amplification by RNA replication mechanisms in addition to the antigen of interest. Self-replicating RNA vaccines induce large amounts of antigen from only small doses; this has the advantage that the development and manufacture of the vaccine is less complex and much less costly than other platforms. However, RNA vaccines have a short half-life and therefore cannot sustain the production of antigens that produce transient protection. For both non-replicating and self-replicating mRNA vaccines, the mRNA must be formulated to prevent degradation or to be enclosed within a vector that protects the mRNA from nuclease degradation prior to ingestion by the host cell. Many different carriers have been successfully used [171-174], based mainly on lipid-nanoparticles that encapsulate mRNA and promote cellular uptake. The present invention contemplates the use of these mRNA vaccines.

The invention provides

A vaccine comprising a polypeptide, nucleic acid and/or vector of the invention, optionally in combination with an adjuvant,

polypeptides, nucleic acids, vectors and/or vaccines of the present invention for use as a medicament,

the polypeptides, nucleic acids, vectors and/or vaccines of the present invention for the treatment of cancer or infectious diseases,

a method for treating cancer or an infectious disease comprising administering to a subject in need of such treatment a polypeptide, nucleic acid, vector and/or vaccine of the invention.

As used herein, the term "treatment" includes any regimen that may benefit a human or non-human animal. The treatment may be of a genetic disease or an acquired disease. Preferably, the treatment is the treatment of a condition/disorder associated with cell proliferation, such as cancer or an infectious disease. Examples of types of cancers that can be treated with nucleic acids include any solid tumor, colorectal cancer, lung cancer, breast cancer, gastric cancer, ovarian cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, bladder cancer, head and neck cancer, brain cancer, esophageal cancer, pancreatic cancer, and bone tumor, as well as soft tissue cancer and leukemia. Examples of infectious diseases that can be treated with the present invention include bacterial or viral infections (e.g., coronaviruses, HIV, hepatitis c viruses), or any infection that requires T cell immunity to clear and neutralize mabs to prevent re-infection.

In some aspects, the invention provides a nucleic acid, peptide, vector and/or vaccine described herein for preventing or treating cancer in a subject, optionally wherein the cancer is melanoma.

In other aspects, the invention provides a nucleic acid, peptide, vector and/or vaccine as described herein for use in preventing or treating an infectious disease in a subject, optionally wherein the infectious disease is covd-19.

Two or more different peptides and/or vaccines may be administered to a subject. Preferably, the following two nucleic acids are administered to a subject:

(a) A combination of a nucleic acid encoding a polypeptide comprising an amino acid sequence provided in SEQ ID No. 6 and a second nucleic acid encoding a receptor binding domain comprising an amino acid sequence provided in SEQ ID No. 8; and

(b) A combination of a nucleic acid encoding a polypeptide comprising the amino acid sequence provided in SEQ ID No. 7 and a second nucleic acid encoding a receptor binding domain comprising the amino acid sequence provided in SEQ ID No. 9.

The nucleic acid, polypeptide and/or vector may be used in combination with a pharmaceutically acceptable carrier or carriers. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, ethanol, and combinations thereof.

The nucleic acids, polypeptides and/or vectors useful in the invention may be formulated into pharmaceutical compositions. These compositions may comprise, in addition to one or more of the above, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other substance may depend on the route of administration, e.g., intradermal, oral, intravenous, epidermal or subcutaneous, intranasal, intramuscular, intraperitoneal. The formulation is preferably a DNA solution that is precipitated as a stable dry powder on the surface of tiny gold particles and is suitable for injection by a gene gun, or is mixed with GET peptide. The formulation may be suitable for intradermal or intramuscular administration using electroporation. The formulation may be suitable for administration using needleless injection.

The composition comprising or for delivering nucleic acid is preferably administered to the individual in a "therapeutically effective amount" sufficient to show a benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Treatment prescriptions, such as dosages, etc., are often determined by considering, within the responsibility of the general practitioner and other doctors, the condition to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. The nucleic acids of the invention are particularly directed to the treatment of existing cancers and to the prevention of cancer recurrence following initial treatment or surgery. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16 th edition, oslo, a. (ed.), 1980.

Preferably, the nucleic acids of the invention stimulate helper and/or cytotoxic T cells that, when administered to a human in an effective amount, can significantly kill virus-infected cells or produce vnabs to prevent viral entry. The optimal dosage can be determined by the physician based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration. For example, a dose of 1-1000 μg of DNA is sufficient to stimulate helper and cytotoxic T cell responses.

The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. Other cancer treatments include other monoclonal antibodies, other chemotherapeutic agents, other radiation therapy techniques, or other immunotherapies known in the art.

The dosage of nucleic acid depends on the nature of the agent used, such as its binding activity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the route of administration, the site and rate of administration, the clinical tolerance of the patient involved, the pathological condition afflicting the patient, etc., and is within the skill of the physician. For example, a dosage of 200 μg of nucleic acid per patient is preferred, but the amount may be in the range of about 10 μg to 8mg per dose. Different amounts were used during a series of consecutive inoculations; the practitioner can administer an initial inoculation followed by boosting with a relatively small dose of nucleic acid.

Another aspect of the invention provides a host cell comprising a nucleic acid as disclosed herein. The nucleic acids of the invention may be integrated into the genome (e.g., chromosome) of the host cell. Integration may be facilitated by the inclusion of sequences that promote recombination with the genome according to standard techniques. The nucleic acid may be on an extrachromosomal vector within the cell, or otherwise distinguishable from or foreign to the cell.

In yet another aspect, methods of introducing a nucleic acid of the invention into a host cell are provided. Introduction (particularly in vitro introduction) is generally referred to as "transformation" without limitation, and any available technique may be used. Suitable techniques for eukaryotic cells may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction with retroviruses or other viruses (e.g., vaccinia virus), or transduction with baculovirus for insect cells. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection with phage. Alternatively, direct injection of nucleic acid may be used.

Marker genes such as antibiotic resistance or susceptibility genes can be used to identify clones containing the nucleic acid of interest, as is well known in the art.

Following introduction, the nucleic acid may be caused or allowed to express, e.g., by culturing the host cell (which may include the actual transformed cell, but is more likely to be a progeny of the transformed cell) under conditions that express the gene, thereby producing the encoded polypeptide (or peptide). If the polypeptide is expressed coupled to an appropriate signal leader peptide, it may be secreted from the cell into the culture medium. After production by expression, the polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as appropriate, and subsequently used as desired, e.g., in a formulation of a composition that may include one or more additional components, e.g., a pharmaceutical composition that includes one or more pharmaceutically acceptable excipients, vehicles, or carriers (see below).

In addition to the active ingredient, the pharmaceutical composition may also contain pharmaceutically acceptable excipients, diluents, carriers, buffers, stabilizers or other substances well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other substance will depend on the route of administration, which may be oral, or by injection, for example intradermal or intramuscular injection.

It is contemplated that injection will be the primary route of therapeutic administration of the composition, but delivery through a catheter or other surgical tube is also used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal, and intramuscular administration. The liquid formulation may be used after reconstitution from a powder formulation. The preferred route of administration is intradermal or intramuscular.

The nucleic acids, vectors, peptides and/or vaccines of the present invention can be administered using needleless injection. It is known to those skilled in the art that needleless injectors (also known as "ejectors") use a narrow, high pressure flow of liquid to penetrate the outermost layer of the skin (stratum corneum), delivering the composition to the underlying tissue of the epidermis or dermis (i.e., intradermal injection), fat (i.e., subcutaneous injection), or muscle (i.e., intramuscular injection).

For intravenous injection or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen free and has suitable pH, isotonicity and stability. Those skilled in the art are able to prepare suitable solutions, such as sodium chloride injection, ringer's injection, lactated ringer's injection, using, for example, isotonic solvents. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included if desired.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. May include physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When the formulation is a liquid, it may be, for example, a physiological saline solution containing a non-phosphate buffer at a pH of 6.8-7.6, or a lyophilized powder.

The composition may also be administered via microspheres, liposomes, other microparticle delivery systems, or sustained release formulations for placement in certain tissues, including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g., suppositories or microcapsules. Implantable or microencapsulated slow release matrices include polylactic acid (U.S. Pat. No. 3,773,919;EP A0058481), copolymers of L-glutamic acid and gamma-ethyl L-glutamic acid [43], poly (2-hydroxyethyl methacrylate). Liposomes comprising the polypeptide are prepared by well known methods: [175,176]; EP-A-0052522; EP-A-0036676; EP-A-0088046; EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. patent nos. 4,485,045 and 4,544,545. Typically, the liposomes are small (about 200-800 angstroms) unilamellar type liposomes having a lipid content greater than about 30 mole% cholesterol, the ratio selected being tailored to the optimal polypeptide leakage rate. The composition may be administered to a tumor site or other desired site in a local manner, or may be delivered in a manner that targets the tumor or other cell.

The polypeptides of the invention may be produced wholly or in part by chemical synthesis. The polypeptides can be readily prepared according to established standard liquid or preferably solid phase peptide synthesis methods, the general description of which is widely available (see, e.g., J.M. Stewart and J.D. Young (1984) [177], M.Bodanzsky and A.Bodanzsky (1984) [178], or they can be prepared in solution by liquid phase methods or by any combination of solid phase, liquid phase and solution chemistry, e.g., by first completing the corresponding peptide moiety and then, if desired and appropriate, after removal of any protecting groups present, introducing the residue X by reaction of the corresponding carbonic or sulphonic acid or reactive derivative thereof.

Another convenient method of producing a polypeptide according to the invention is by using a nucleic acid in an expression system to express the nucleic acid encoding it. The invention also provides isolated nucleic acids encoding the polypeptides of the invention. Nucleic acids include DNA and RNA. Those skilled in the art will be able to determine substitutions, deletions and/or additions of these nucleic acids, which will still provide the polypeptides of the invention.

The invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as described above. The invention also provides recombinant host cells comprising one or more of the above constructs. As mentioned above, nucleic acids encoding polypeptides of the invention form one aspect of the invention, as does the method of producing the polypeptides, which method comprises expression from the nucleic acids encoding the polypeptides. Expression may conveniently be achieved by culturing a recombinant host cell comprising the nucleic acid under suitable conditions. After production by expression, the polypeptide may be isolated and/or purified using any suitable technique and then used as appropriate. Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacterial, mammalian, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include chinese hamster ovary/HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and the like. One common preferred bacterial host is E.coli. Expression of polypeptides in prokaryotic cells such as E.coli is well established in the art. For reviews, see, e.g., [179]. Expression in eukaryotic cells in culture is also useful as an option for producing the polypeptides of the invention to those skilled in the art, see for example, recent reviews [180,181].

Suitable vectors may be selected or constructed which contain suitable regulatory sequences including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other suitable sequences. The vector may be a plasmid, a virus such as a phage or a phagemid, as appropriate. See, e.g., [157]. A number of known techniques and protocols for manipulating nucleic acids are described in detail in Ausubel et al (1992) [182], such as preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and analyzing proteins.

Thus, in a further aspect the invention provides a host cell comprising a nucleic acid according to the invention. In a further aspect, methods of introducing such nucleic acids into host cells are provided. The introduction may be by any available technique. Suitable techniques for eukaryotic cells may include calcium phosphate transfection, DEAE dextran, electroporation, liposome-mediated transfection, and transduction with retroviruses or other viruses (e.g., vaccinia virus), or transduction with baculovirus for insect cells. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection with phage. Following introduction, expression of the nucleic acid may be caused or allowed, for example, by culturing the host cell under conditions in which the gene is expressed. In one embodiment, the nucleic acids of the invention are integrated into the genome (e.g., chromosome) of the host cell. Integration may be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques. The invention also provides methods comprising using the constructs described above in an expression system to express the polypeptides described above.

The fragment crystallizable region (Fc region) is the antibody tail region that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG antibody isotypes, the Fc region consists of two identical protein fragments from the second and third constant domains of the two heavy chains of the antibody. The Fc region of IgG comprises highly conserved N-glycosylation sites.

The Fc region of IgG consists of a pair of antibody HC domains, each domain having CH2 fused to CH3, which forms a structure of about 50 kDa. The designation "fragment, crystallizable" (Fc) comes from the fact: after cleavage of serum-derived myeloma IgG fractions with papain, the only fragments that can be crystallized are the paired CH2-CH3 fragments.

Within Fc, two CH3 domains are tightly bound to each other, while two CH2 domains are not in direct protein-protein contact with each other. The oligosaccharide binds to asparagine 297 (N297) in each of the two CH2 domains, filling part of the space between the two CH2 domains. In some crystal structures, hydrogen bonding between two carbohydrate chains, both directly and through bridging water molecules, has been observed. Although antibodies appear to be highly segmented molecules, the structure of Fc has been shown to affect binding of antigen binding fragments (Fab) to targeted antigens, and similarly, the content of variable chains in Fab affects Fc binding to various receptors. Recent circular dichroism studies have demonstrated a large number of structural couplings between Fab arms and Fc of IgG. Thus, igG molecules are highly complex molecules in which the different domains interact significantly, even over long distances.

Avidity refers to the cumulative strength of multiple affinities of a single non-covalent binding interaction (e.g., interaction between a protein receptor and its ligand), and may also be referred to as "functional affinity". Thus, avidity differs from the inherent affinity, which describes the strength of individual interactions. However, because individual binding events increase the likelihood of other interactions occurring (i.e., increase the local concentration of each binding partner near the binding site), the avidity should not be considered as simply the sum of the constituent affinities, but rather the combined effect of all affinities involved in biomolecular interactions. The distinction between "intrinsic affinity" and "functional affinity" is used from the different emphasis referred to in each term. The former is most useful when examining the structural relationship between the antibody binding site and the complementary region of the ligand, or when studying the kinetic mechanism of specific interactions. On the other hand, the latter is particularly important when detecting quantitative measurements of affinity enhancement, as in the present invention.

"functional affinity" and "intrinsic affinity" refer to the following formally identical reversible processes:

Complex (monovalent)

Complex (multivalent)

Wherein F is ₁ Is a monovalent antibody fragment, L ₁ Ab is a monovalent ligand _n Is a multivalent antibody, and L _m Is a multivalent ligand having m groups. In each case, two power units are reversibly combined to form one unit, and each process can in principle be characterized by an association constant. Since the association constant is a measure of thermodynamic affinity, both processes can specify quantitative values of affinity.

The invention also extends to variants of any of the peptide sequences disclosed herein. As used herein, the term "variant" refers to proteins having similar amino acid sequences and/or retaining the same function. For example, the term "variant" encompasses a protein or polypeptide that includes one or more amino acid additions, deletions, substitutions, and the like. Examples of variants of the invention are proteins comprising a peptide as defined below, except that one or more amino acids are replaced with one or more other amino acids. For example, amino acid substitutions may be made to reduce or eliminate the burden in the amino acid sequence.

The skilled artisan recognizes that the various amino acids have similar properties. One or more such amino acids of a substance may typically be replaced by one or more other such amino acids without eliminating the desired activity of the substance.

Thus, the amino acids glycine, alanine, valine, leucine and isoleucine are typically interchangeable (amino acids with aliphatic side chains). Among these possible substitutions, glycine and alanine are preferably used for the substitution with each other (because they have relatively short side chains), and valine, leucine and isoleucine are preferably used for the substitution with each other (because they have large hydrophobic aliphatic side chains). Other amino acids that are often interchangeable include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

Naturally occurring amino acids using three-letter and one-letter codes can be referred to as follows: glycine (G or Gly), alanine (a or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or lie), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or HIS), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or gin), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and threonine (T or Thr). Where the residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where the residue may be glutamic acid or glutamine, the notation Glx or Z may be used. Unless the context indicates otherwise, references to aspartic acid include aspartate and references to glutamic acid include glutamate.

Amino acid deletions or insertions may also be made with respect to the amino acid sequence of the fusion proteins described below. Thus, for example, amino acids which do not substantially affect the activity of the polypeptide, or at least amino acids which do not eliminate such activity, may be deleted. Such deletions are advantageous because the overall length and molecular weight of the polypeptide can be reduced while still retaining activity. This may allow for a reduction in the amount of polypeptide required for a particular purpose, e.g., the level of usage may be reduced.

In some embodiments, the following amino acids may be interchanged for conservative amino acid substitutions:

category(s)	Interchangeable amino acids
		Aliphatic series	Glycine, alanine, valine, leutonAmino acid, isoleucine
Hydroxy or sulfur/selenium containing	Serine, cysteine, threonine, methionine
		Aromatic series	Phenylalanine, tyrosine, tryptophan
Alkaline	Histidine, lysine, arginine
		Acid and amide thereof	Aspartic acid, glutamic acid, asparagine, glutamine

Thus, reference to a "conservative" amino acid substitution refers to the replacement of one or more amino acids in an antibody sequence (e.g., CDR or VH or VL sequences) with another amino acid in the same class as described above. Conservative amino acid substitutions are preferred in the CDR regions to minimize adverse effects on antibody function. However, conservative amino acid substitutions may also occur in the framework regions.

Amino acid changes relative to the sequences given below may be made using any suitable technique, for example by using site-directed mutagenesis or solid phase synthesis.

It is to be understood that amino acid substitutions or insertions within the scope of the invention may be made using naturally occurring or non-naturally occurring amino acids, but naturally occurring amino acids may be preferred. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids be present.

The invention also provides

Nucleic acid vaccine, wherein the antigen is a tumor specific antigen or a tumor associated antigen.

Nucleic acid vaccine, wherein the antigen is a viral antigen or a bacterial antigen.

Nucleic acid vaccine, wherein the antigen is two viral proteins that produce a T cell response and a VNAb.

Nucleic acid vaccine, wherein the antigen is two viral proteins that produce CD 8T cells, CD 4T cells and vnabs.

Nucleic acid vaccine, wherein the antigen is a key receptor binding domain of viral nucleocapsid protein and spike protein that produces T cell response and VNAb.

Nucleic acid vaccine, wherein the antigen is a key receptor binding domain of viral nucleocapsid proteins and spike proteins that produce CD 8T cells, CD 4T cells and vnabs.

Nucleic acid vaccine, wherein the antigen is a key receptor binding domain of coronavirus nucleocapsid protein and spike protein that produces a T cell response and VNAb.

Nucleic acid vaccine, wherein the antigen is a key receptor binding domain of coronavirus nucleocapsid proteins and spike proteins that produce CD 8T cells, CD 4T cells and vnabs.

Nucleic acid vaccine, wherein the antigen is a critical receptor binding domain of SARS-CoV-2 coronavirus nucleocapsid protein and spike protein that produces a T cell response and VNAb.

Nucleic acid vaccine, wherein the antigen is the critical receptor binding domain of the SARS-CoV-2 coronavirus nucleocapsid protein and spike protein that produce CD 8T cells, CD 4T cells and vnabs.

Nucleic acid vaccine as described above, wherein a low dose is delivered to elicit high avidity CD 8T cells.

A nucleic acid vaccine as described above, wherein the receptor binding domain is trimerized with a secondary fibrin.

A nucleic acid vaccine as described above, wherein the receptor binding domain is trimerized with a minor fibrin linked with a flexible GS linker.

A nucleic acid vaccine as described above, wherein the receptor binding domain is stabilised with additional disulphide bonds.

Nucleic acid vaccine encoding the covd-19 spike receptor binding domain aa 319-541 with a human heavy chain leader sequence and the nucleocapsid protein with a human heavy chain leader sequence linked to the hinge and CH2 and CH3 domains of human IgG1 Fc.

Nucleic acid vaccine encoding the covd-19 spike receptor binding domain aa 319-541 with a human heavy chain leader sequence linked to a minor fibrin and the nucleocapsid protein with a human heavy chain leader sequence linked to the hinge and CH2 and CH3 domains of human IgG1 Fc.

Nucleic acid vaccine encoding the covd-19 spike receptor binding domain aa 319-541 with a human heavy chain leader sequence linked to the hinge and CH2 and CH3 domains of human IgG1 Fc and a nucleocapsid protein with a human heavy chain leader sequence.

Nucleic acid vaccine encoding the covd-19 spike receptor binding domain aa 319-541 with a human heavy chain leader sequence and a nucleocapsid protein with a human heavy chain leader sequence.

Nucleic acid vaccine encoding the covd-19 spike receptor binding domain aa 330-525 with a GS linker and a secondary fibrin with a human heavy chain leader sequence and a nucleocapsid protein with a human heavy chain leader sequence linked to the hinge and CH2 and CH3 domains of human IgG1 Fc.

Nucleic acid vaccine encoding the covd-19 spike receptor binding domain aa 330-525 with GS linker and other cysteine residues with a human heavy chain leader sequence and nucleocapsid proteins with a human heavy chain leader sequence linked to the hinge and CH2 and CH3 domains of human IgG1 Fc.

A modified IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein said one or more residues of the Fc region are modified to the corresponding residues of a mouse IgG3 antibody and said modified IgG1 antibody or antigen-binding fragment thereof has an enhanced functional affinity compared to a corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc region residues.

A nucleic acid vaccine comprising a modified IgG1 antibody or antigen-binding fragment thereof, said modified IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of said Fc region are modified to the corresponding residues of a mouse IgG3 antibody and said modified IgG1 antibody or antigen-binding fragment thereof has an enhanced functional affinity compared to the corresponding IgG1 antibody or antigen-binding fragment thereof comprising wild-type Fc region residues.

A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein said one or more residues of the Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein said one or more residues of the Fc region are selected from the group consisting of: q342, P343, E345, N361, Q362, P374, D376, optionally, wherein the one or more modified residues of the Fc region are selected from the group consisting of: Q342R, P343A, E345T, N361K, Q K, P374S, D a.

A nucleic acid vaccine comprising a modified IgG1 antibody or antigen-binding fragment thereof, said modified IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of said Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein one or more residues of said Fc region are selected from the group consisting of: q342, P343, E345, N361, Q362, P374, D376, optionally, wherein the one or more modified residues of the Fc region are selected from the group consisting of: Q342R, P343A, E345T, N361K, Q K, P374S, D a.

A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein said one or more residues of the Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein said one or more residues of the Fc region are selected from the group consisting of: n286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, a378, optionally, wherein the one or more modified residues of the Fc region are selected from the group consisting of: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q K, G371N, P S, S375E, D376A, A378S.

A nucleic acid vaccine comprising a modified IgG1 antibody or antigen-binding fragment thereof, said modified IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of said Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein one or more residues of said Fc region are selected from the group consisting of: n286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, a378, optionally, wherein the one or more modified residues of the Fc region are selected from the group consisting of: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q K, G371N, P S, S375E, D376A, A378S.

A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein said one or more residues of the Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein said one or more residues of the Fc region are selected from the group consisting of: n286, K288, K290, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378, optionally wherein the one or more modified residues of the Fc region are selected from the group consisting of: N286T, K288W, K Q, A339P, Q342R, P343A, R344 3834 345T, L I, S354P, D Q, A357Q, L358M, T359S, N361K, Q K, K370 371N, Y373F, P38395 375E, D376A, A378S.

A nucleic acid vaccine comprising a modified IgG1 antibody or antigen-binding fragment thereof, said modified IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of said Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein one or more residues of said Fc region are selected from the group consisting of: n286, K288, K290, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378, optionally wherein the one or more modified residues of the Fc region are selected from the group consisting of: N286T, K288W, K Q, A339P, Q342R, P343A, R344 3834 345T, L I, S354P, D Q, A357Q, L358M, T359S, N361K, Q K, K370 371N, Y373F, P38395 375E, D376A, A378S.

A modified IgG1 antibody or antigen-binding fragment thereof, comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein said one or more residues of the Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein said one or more residues of the Fc region are selected from the group consisting of: n286, K288, K290, E294, Y300, V305, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378, optionally, wherein the one or more modified residues of the Fc region are selected from the group consisting of: N286T, K288W, K290Q, E294A, Y300 305A, A339P, Q342 339P, Q339 3775 342 35Q, E T, L351I, S354E, E357 5483 357 63357 358 359S, N361K, Q K, K361-35T, G N, Y373F, P374S, S E, D376A, A S.

A nucleic acid vaccine comprising a modified IgG1 antibody or antigen-binding fragment thereof, said modified IgG1 antibody or antigen-binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of said Fc region are modified to the corresponding residues of a mouse IgG3 antibody, and wherein one or more residues of said Fc region are selected from the group consisting of: n286, K288, K290, E294, Y300, V305, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378, optionally, wherein the one or more modified residues of the Fc region are selected from the group consisting of: N286T, K288W, K290Q, E294A, Y300 305A, A339P, Q342 339P, Q339 3775 342 35Q, E T, L351I, S354E, E357 5483 357 63357 358 359S, N361K, Q K, K361-35T, G N, Y373F, P374S, S E, D376A, A S.

Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis. The prior art documents mentioned herein are incorporated to the maximum extent allowed by law.

In describing embodiments of the present invention, the terms are not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

Examples

The invention will now be further described with reference to the following examples and figures. Future data collection was from healthy volunteers immunized with a new pDNA vaccine engineered to induce VNAb and potent T cell immunity using electroporation, which was known as a direct result of the effects of the examples.

Fig. 1: map and cloning strategy for pVAXDCIB68

Cloning sites BamHI/XhoI and HindIII/PstI are depicted for excision of light and heavy chains and substitution with S and N segments in the first and second round of cloning, respectively.

Fig. 2: the sequence of pVAXDCSN 1; SN1

The S glycoprotein and N full-length nucleotide and amino acid sequences within the expression vector pVAXDC. S chain codes for RBD amino acids 319-541 and murine IgK leader sequence. The nucleoprotein chain encodes amino acids 2-419 fused in frame with human IgG1 hinge-CH 2-CH3 and murine IgK leader sequence. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 3: the sequence of pVAXDCSN 2; SN2

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain murine IgK leader sequences. The S chain encodes RBD amino acids 319-541 linked to the secondary fibrin trimer motif by glycine serine. The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH 3. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 4: the sequence of pVAXDCSN 3; SN3

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain murine IgK leader sequences. The S chain encodes RBD amino acids 319-541 fused in-frame to human IgG1 hinge-CH 2-CH 3. The N-chain encodes amino acids 2-419. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 5: the sequence of pVAXDCSN 4; SN4

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain murine IgK leader sequences. The S glycoprotein chain encodes RBD amino acids 319-541 and the N chain encodes amino acids 2-419. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 6: the sequence of pVAXDCSN 5; SN5

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. S chain codes for RBD amino acids 319-541 and human IgH leader sequence. The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH3 and the human IgH leader. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 7: the sequence of pVAXDCSN 6; SN6

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The S chain encodes RBD amino acids 319-541 linked to the secondary fibrin trimer motif by glycine serine. The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH 3. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 8: the sequence of pVAXDCSN 7; SN7

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The S chain encodes RBD amino acids 319-541 fused in-frame to human IgG1 hinge-CH 2-CH 3. The N-chain encodes amino acids 2-419. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 9: the sequence of pVAXDCSN 8; SN8

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The S glycoprotein chain encodes RBD amino acids 319-541 and the N chain encodes amino acids 2-419. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 10: the sequence of pVAXDCSN 9; SN9

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. S chain coding is long (GGGS) ₃ The GS glycine serine linker is linked to RBD amino acids 330-525 of the secondary fibrin trimer motif (GTGGGSG). The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH 3. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 11: the sequence of pVAXDCSN 10; SN10

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. S chain code pass (GGGS) ₃ Glycine serine linker is linked to RBD amino acids 330-525 of the disulfide bridge motif. The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH 3. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 12: the sequence of pVAXDCSN 11; SN11

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. S chain coding is long (GGGS) ₃ The GS glycine serine linker is linked to RBD amino acids 330-525 of the secondary fibrin trimer motif (GTGGGSG). The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH3 iV1, with the murine IgG3 23AA substitution highlighted in bold. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 13: the sequence of pVAXDCSN 12; SN12

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The S chain encodes RBD amino acids 319-541 linked to the secondary fibrin trimer motif by glycine serine. The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH3 IV1, with the murine IgG3 23AA substitution highlighted in bold. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 14: the sequence of pVAXDCSN 13; SN13

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The S chain encodes RBD amino acids 319-541 fused in-frame to human IgG1 hinge-CH 2-CH 3. The N chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH3 iV1, with the murine IgG3 23AA substitution highlighted in bold. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 15: the sequence of pVAXDCSN 14; SN14

S and N full long chain nucleotide and amino acid sequences within the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The S chain encodes RBD amino acids 319-541 and the N chain encodes amino acids 2-419, all fused to the human IgG1 hinge-CH 2-CH3 iV1 constant region, with murine IgG3 23AA substitutions highlighted in bold. To reduce homology between the two codon-optimized human IgG1 hinge-CH 2-CH3 iV1 constant regions in SN14, the nucleotide sequences were not identical. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 16: RBD and N secretion levels in conditioned media 6 days after transiently transfecting HEK293 with the respective SN constructs (by sandwich ELISA).

Fig. 17: HHDII mice were immunized with SN8, SN9, SN10 and SN11 (A) or SN8, SN10 and SN11 (B) pDNA by gene gun on days 1, 8 and 15. Spleen cells were analyzed for ifnγ responses against medium controls, S1, RBD and N proteins, N and RBD peptide libraries, and individual N and RBD peptides by ELISpot assay on day 21. Responses are shown as mean spots per million splenocytes. The response of open circles to peptides, the response of filled circles to proteins, the response of red to N antigen, the response of blue to S antigen, RBD, S1.

Fig. 18: HHDII/DR1 mice were immunized with SN8 (A), SN9 (B), SN10 (C) and SN11 (D) pDNA by gene gun on days 1, 8 and 15. Spleen cells were analyzed for ifnγ responses against medium controls, S1, RBD and N proteins, N and RBD peptide libraries, and individual N and RBD peptides by ELISpot assay on day 21. Responses are shown as mean spots per million splenocytes. The response of open circles to peptides, the response of filled circles to proteins, the response of red to N antigen, the response of blue to S antigen, RBD, S1.

Fig. 19: in ELISpot, mice immunized with SN8, SN9 or SN11 pDNA by gene gun were compared for ifnγ responses to N protein (a), N138-147 peptide (B), S1 protein (C) and RBD protein (D). Data were collected from two independent studies. Responses were normalized to background control. Responses are shown as mean spots per million splenocytes.

Fig. 20: HHDII/DP4 mice were immunized with SN2 (A), SN3 (B) and RSN4 (C) pDNA by gene gun on days 1, 15 and 29. Spleen cells were analyzed for ifnγ responses to medium controls, S1, RBD and N proteins, RBD peptide libraries, and individual N and RBD peptides by ELISpot assay on day 35. Responses are shown as mean spots per million splenocytes. The response of open circles to peptides, the response of filled circles to proteins, the response of red to N antigen, the response of blue to S antigen, RBD, S1.

Fig. 21: HHDII/DP4 mice were immunized with SN2, SN3, and SN4 pDNA by gene gun on days 1, 15, and 29. Spleen cells were analyzed for ifnγ response to quantitative S1 protein drop by ELISpot assay on day 35. The affinity was calculated as the concentration of protein that elicited a 50% maximum response. Titration curves are shown as spots per million splenocytes and response normalization is shown as% maximum response curve.

Fig. 22: HHDII mice were immunized with SN11 pDNA by gene gun on days 1, 8 and 15. Spleen cells were analyzed for ifnγ response to RBD 417-425 peptide titer by ELISpot assay on day 21. The affinity was calculated as the concentration of protein that elicited a 50% maximum response. Titration curves are shown as spots per million splenocytes.

Fig. 23: HHDII mice were immunized with SN10 or SN11 pDNA by gene gun on days 1, 8 and 15. Spleen cells were analyzed for ifnγ response to quantitative N138-146 peptide drops by ELISpot assay on day 21. The affinity was calculated as the concentration of protein that elicited a 50% maximum response. Titration curves are shown as spots per million splenocytes.

Fig. 24: HHDII/DP4 mice were immunized with SN5, SN6, SN9, SN10, and SN11 (A) SN2, SN3, and SN4 (B) pDNA by gene gun on days 1, 15, and 29 or with SN3, SN8, SN10, and SN11 (C) pDNA by gene gun on days 1, 8, and 15. On day 35 (A and B) or day 21 (C), the serum was analyzed for antibody responses to S1, N and RBD proteins by ELISA at dilutions 1/100, 1/1000 and 1/10000.

Fig. 25: serum was collected on day 35 from HHDII/DP4 mice immunized with SN5, SN6, SN9, SN10 and SN11 by gene gun on days 1, 15 and 29 for replacement neutralization assays (RBD binding inhibition assays). Serum from naive mice was used as a negative control, and murine S1 antibody (sino biological) was used as an additional positive control.

Fig. 26: pseudovirus neutralization assay. HHDII/DP4 mice were immunized with SN5, SN6, SN9, SN10, SN11, SN2, SN3, and SN4 pDNA by gene gun on days 1, 15, and 29. Serum collected on day 35 was tested for neutralization of SARS-CoV-2 (A) or with unrelated virus (VSV G) (B) at 1/100 dilution. Virus neutralization (C) was also analyzed at different serum dilutions. 50% neutralization titer (ID 50) (D).

Fig. 27: sequence of VAXDCSN 15; SN15

The nucleotide and amino acid sequences of the spike and nucleoprotein full-length chains in the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The spike chain encodes amino acids 319-541. The nucleoprotein chain encodes amino acids 2-419 fused in frame to human IgG1 hinge-CH 2-CH3 iV1, with the murine IgG3 23AA substitution highlighted in bold. Stop codons are indicated by asterisks. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 28: the sequence of pVAXDCIB68 (SCIB 1)

Nucleotide and amino acid sequences of antibody heavy and light chain variable regions cloned in frame with human IgG1 CH 1-hinge-CH 2-CH3 constant region and human kappa constant region, respectively, within expression vector pVAXDC. The amino acids in the boxes encode HLA-DR7, HLA-F-in H1 and L3DR53 and HLA-DQ6 restricted gp100 _173-190 CD4 epitope (GTGRAMLGTHTMEVTVYH), HLA-0201TRP2 in H2 _180-188 Epitope (SVYDFFVWL), HLA-DR4 restricted gp100 in H3 and L1 _44-59 CD4 epitope (WNRQLYPEWTEAQRLD). HindIII/AFE I and BamHI/BsiWI restriction sites for variable heavy and light chain region transfer are highlighted. In order to directly replace the human IgG1 constant region domain (CH 1-hinge-CH 2-CH 3) with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 iV1 constant region, afeI and EcoRI were used as indicated. Stop codons are indicated by asterisks.

Fig. 29: the sequence of pVAXDCIB68 iV1 (iSCIB 1)

Nucleotide and amino acid sequences of antibody heavy and light chain variable regions cloned in frame with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 iV1 constant region (with murine IgG 3AA substitution highlighted in bold) and human kappa constant region, respectively, within the expression vector pVAXDC. The amino acids within the boxes encode HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gp100 in H1 and L3 _173-190 CD4 epitope (GTGRAMLGTHTMEVTVYH), HLA-0201TRP2 in H2 _180-188 HLA-DR4 restricted gp100 in epitopes (SVYDFFVWL), H3 and L1 _44-59 CD4 epitope (WNRQLYPEWTEAQRLD). HindIII/AFE I and BamHI/BsiWI restriction sites for variable heavy and light chain region transfer are highlighted. The human IgG1 constant region domain (CH 1-hinge-CH 2-CH 3) was directly replaced with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 iV1 constant region, as shown using AfeI and EcoRI. Stop codons are indicated by asterisks.

Fig. 30: the sequence of pVAXDCIB238 (SCIB 1 plus)

Nucleotide and amino acid sequences of antibody heavy and light chain variable regions cloned in frame with human IgG1 CH 1-hinge-CH 2-CH3 constant region and human kappa constant region, respectively, within expression vector pVAXDC. The amino acids in the box represent HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gp100 in CDR H1 retained from pVAXDCIB68 _173-190 CD4 epitope (GTGRAMLGTHTMEVTVYH), HLA-0201TRP2 in H2 _180-188 Epitope (SVYDFFVWL), HLA-DR4 restricted gp100 in L1 _44-59 CD4 epitope (WNRQLYPEWTEAQRLD). Other epitopes include gp100 nested in the insertion H3 site _471-492 Sequence (VPLDCVLYRYGSFSVTLDIVQG)In this, it is HLA-A1, B35 and a predicted HLA-DP4 epitope. Will TRP2 _177-205 And TRP2 _60-91 Sequences were grafted to the L2 and L3 sites of the variable light chain region, respectively. Together they comprise HLA-A2, A3, A31, A33, B35, B44, HLA-DR3 and another potential HLA-DP4 epitope as described elsewhere. HindIII/Afe I and BamHI/BsiWI restriction sites for variable heavy and light chain region transfer are highlighted. To directly replace the human IgG1 constant region domain (CH 1-hinge-CH 2-CH 3) with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 IV1 constant region, afeI and EcoRI were used as indicated. Stop codons are indicated by asterisks.

Fig. 31: the sequence of pVAXDCIB238 iV1 (iSCIB 1 plus)

Nucleotide and amino acid sequences of antibody heavy and light chain variable regions cloned in frame with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 iV1 constant region (with murine IgG 3AA substitution highlighted in bold) and human kappa constant region, respectively, within the expression vector pVAXDC. The amino acids in the box represent HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gp100 in CDR H1 retained from pVAXDCIB68 _173-190 CD4 epitope (GTGRAMLGTHTMEVTVYH), HLA-0201TRP2 in H2 _180-188 Epitope (SVYDFFVWL), HLA-DR4 restricted gp100 in L1 _44-59 CD4 epitope (WNRQLYPEWTEAQRLD). Other epitopes include gp100 nested in the insertion H3 site _471-492 Within the sequence (VPLDCVLYRYGSFSVTLDIVQG), it is HLA-A1, B35 and a predicted HLA-DP4 epitope. Will TRP2 _177-205 And TRP2 _60-91 Sequences were grafted to the L2 and L3 sites of the variable light chain region, respectively. Together they comprise HLA-A2, A3, A31, A33, B35, B44, HLA-DR3 and another potential HLA-DP4 epitope. HindIII/Afe I and BamHI/BsiWI restriction sites for variable heavy and light chain region transfer are highlighted. The human IgG1 constant region domain (CH 1-hinge-CH 2-CH 3) was directly replaced with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 iV1 constant region, as shown using Afe I and EcoRI. Stop codons are indicated by asterisks.

Fig. 32: sequence of pVAXDCIB178 (SCIB 2)

Antibody heavy and light chains cloned in frame with human IgG1 CH 1-hinge-CH 2-CH3 constant region and human kappa constant region, respectively, within expression vector pVAXDCNucleotide and amino acid sequences of the chain variable region. The amino acids in the boxes represent NYESO-1 in CDRs H1 and H2 _158-166 HLA-A24 epitope (LLMWITQCF) and HLA-A 2-restricted NNYESO-1 _157-165 Epitope (SLLMWITQC). NY-ESO-1 grafted into CDR H3 and L1 sites _83-111 Amino acid sequence (PESRLLEFYLAMPFATPMEAELARRSLAQ) and NY-ESO-1 _119-143 (PGVLLKEFTVSGNILTIRLTAADHR) together comprise a plurality of nested further epitopes as described elsewhere (xue et al ONCOIMMUNOLOGY 2016, VOL.5, no.6, e 1169353). HindIII/Afe I and BamHI/BsiWI restriction sites for variable heavy and light chain region transfer are highlighted. In order to directly replace the human IgG1 constant region domain (CH 1-hinge-CH 2-CH 3) with the enhanced human IgG1 CH 1-hinge-CH 2-CH3iV1 constant regions AfeI and EcoRI, afeI and EcoRI were used as indicated. Stop codons are indicated by asterisks.

Fig. 33: sequence of pVAXDCIB178 iV1 (iSCIB 2)

Nucleotide and amino acid sequences of antibody heavy and light chain variable regions cloned in frame with the enhanced human IgG1 CH 1-hinge-CH 2-CH3iV1 constant region (with murine IgG 3AA substitution highlighted in bold) and human kappa constant region, respectively, within the expression vector pVAXDC. The amino acids in the boxes represent NYESO-1 in CDRs H1 and H2 _158–166 HLA-A24 epitope (LLMWITQCF) and HLA-A 2-restricted NYESO-1 _157–165 Epitope (SLLMWITQC). NY-ESO-1 grafted into CDR H3 and L1 sites _83-111 Amino acid sequence (PESRLLEFYLAMPFATPMEAELARRSLAQ) and NY-ESO-1 _119-143 (PGVLLKEFTVSGNILTIRLTAADHR) together comprise a plurality of nested further epitopes [183 ] as described elsewhere]. HindIII/Afe I and BamHI/BsiWI restriction sites for variable heavy and light chain region transfer are highlighted. In order to directly replace the human IgG1 constant region domain (CH 1-hinge-CH 2-CH 3) with the enhanced human IgG1 CH 1-hinge-CH 2-CH3 iV1 constant region, afeI and EcoRI were used as indicated. Stop codons are indicated by asterisks.

Fig. 34: c57BL/6 or HLA-DR4 mice were immunized with SCIB1 or iSCIB1pDNA by gene gun on days 1, 8 and 15. The spleen cells were assayed for IFNγ response to TRP2180-188 peptide (A-C) or gp100 44-59 peptide (D) by ELISPot assay on day 21. The frequency of TRP2180-188 responses in different mouse strains was compared (a). B and C, TRP2180-188 response affinities were calculated as the peptide concentration that elicited 50% of the maximal response. Titration curves are shown as% of maximum response. Response frequency to gp100 44-59 peptide in HLA-DR4 mice at 0.1 μg/ml.

Fig. 35: c57BL/6, HHDII/DP4 or HLA-DR4 mice were immunized with SCIB1plus or iSCIB1plus pDNA by gene gun on days 1, 8 and 15. The spleen cells were assayed for IFNγ response to TRP2180-188 peptide (A-C) or gp100 44-59 peptide (D) by ELISPot assay on day 21. Response frequencies (a) in different mouse strains were compared. B and C, TRP2180-188 response affinities were calculated as the peptide concentration that elicited 50% of the maximal response. Titration curves are shown as% of maximum response. Response frequency of HLA-DR4 mice to 1 μg/ml gp100 44-59 peptide.

Fig. 36: c57BL6 mice were implanted with B16F1 tumor cells on day 1, followed by immunization with pDNA SCIB1, iSCIB1, SCIB1plus or iSCIB1plus on days 4, 11 and 18. A, tumor growth curve. Tumor volume comparison on day 18. And C, overall survival rate.

Fig. 37: HHDII or HHDII/DR1 mice were immunized with SCIB2 or iSCIB2pDNA by gene gun on days 1, 8 and 15. The spleen cells were analyzed for IFNγ responses to either the Nyeso1 157-165 peptide (A-C) or the Nyeso1119-143 peptide (D and E) by ELISPot assay on day 21. The response frequencies of Nyeso1 157-165 in the different mouse strains were compared (a). B and C, nyeso1 157-165 response affinities were calculated as the peptide concentration that elicited 50% of the maximum response. Titration curves are shown as% of maximum response. Response frequency to 10. Mu.g/ml Nyoso 1119-143 peptide in D, HHDII/DR1 mice. The response avidity of Nyeso1119-143 in E, HHDII/DR1 mice was calculated as the concentration of peptide that elicited 50% of the maximum response. Titration curves are shown as% of maximum response.

Fig. 38: HHDII mice were implanted with B16 HHDII Nyoso tumor cells on day 1, followed by immunization with pDNA SCIB2 or iSCIB2 on days 4, 8 and 11 and monitoring tumor-free survival over time.

Fig. 39: HLA-A2 transgenic or Balb/c mice were immunized with SN13 or SN14pDNA by gene gun on days 1, 8 and 15. The spleen cells were analyzed for their ifnγ response frequency (a) to the RBD peptide pool by ELISpot assay or for their affinity to RBD 417-425 peptides by peptide titration on day 21. The affinity was calculated as the concentration of peptide that elicited 50% of the maximum response. Titration curves are shown as% of maximum response. The serum dilutions on day 21 were analyzed for S1 protein-specific antibody responses (C) in ELISA assays or for SARS-CoV-2 neutralizing antibodies (D) in pseudovirus neutralization assays.

Fig. 40: RBD (a) and N (B) secretion levels (by sandwich ELISA) in conditioned medium and cell lysates 6 days after transient transfection of HEK293 with the respective SN constructs.

Fig. 41: c57BL/6 (C) or Balb/C (A and B) mice were immunized with SN11 (small RBD trimer), SN12 (RBD trimer), SN13 (RBD-Fc), SN15 (RBD monomer) pDNA by gene gun on days 1, 8 and 15, all of which constructs also contained modified Fc-N or intact S. The serum dilutions on day 21 were analyzed for S1 protein specific antibody responses in ELISA assays (A) or for SARS-CoV-2 neutralizing antibodies in pseudovirus neutralization assays (B and C).

Fig. 42: HLA-A2 transgenic C57B1/6 or Balb/C mice were immunized with SN11 (small RBD trimer), SN12 (RBD trimer), SN13 (RBD-Fc), SN15 (RBD monomer) by gene gun on days 1, 8 and 15, all of which constructs also contained modified Fc. The frequency of ifnγ responses of spleen cells to RBD peptide pools (Ai and B) and to RBD 417-425 peptides (Aii) was analyzed by ELISpot assay on day 21.

Fig. 43: real-time binding curves (SPR, biaT 200) of RBD-FC and RBD-iFCv1 interactions at increasing CD64 levels captured on CM5 chips.

Fig. 44: A. healthy donor T cell proliferation response (group of 20 donors). With iTV1, Following incubation with KLH, PBMCs were sampled from the bulk culture and evaluated for proliferation on days 5, 6, 7 and 8. Proliferation response with SI > 1.90 was significant using unpaired double sample student t test (p<0.05, indicated by a red dotted line) was considered positive. B. Shows healthy donor T cell pairs iTV1, < >>And->Box plot of response. The figure shows the maximum proliferation of cd4+ T cells obtained over time. Bars represent 10-90 percentiles. Statistical analysis using repeated measures one-way ANOVA (Friedman test) for paired comparison after Dunn's test is shown. * P<0.01

Fig. 45: healthy donor T cell proliferation response in group of 50 donors. With iTV1,Following incubation with KLH, PBMCs were sampled from the bulk culture and evaluated for proliferation on days 5, 6, 7 and 8. Proliferation response with SI > 1.90 was significant using unpaired double sample student t test (p<0.05, indicated by a red dotted line) was considered positive. B. Shows healthy donor T cell pairs iTV1, < >>And->Box plot of response. The figure shows the maximum proliferation of cd4+ T cells obtained over time. Bars represent 10-90 percentiles. Statistical analysis using repeated measures one-way ANOVA (Friedman test) for paired comparison after Dunn's test is shown. * P <0.01，****p<0.0001

Fig. 46: day 21 the specific antibody responses to N protein by gene gun immunization of mice sera with NP, NPFC or NPFciV1 on days 1, 8 and 15 were analyzed in ELISA assays.

Fig. 47: the sequence of pVAXDCSN 16; SN16

The nucleotide and amino acid sequences of the spike and nucleoprotein full-length chains in the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The spike chain encodes amino acids 319-541 carrying the N501Y mutation of Kent variant/lineage B.1.1.7, UK-VOC 202012/01. The nucleoprotein chain encodes amino acids 2-419 including D3L and S235F mutations from the variant fused in frame with a human IgG1 hinge-CH 2-CH3 iV1, with the murine IgG3 23AA replaced in bold. Stop codons are indicated by asterisks. Mutations of Kent variant/lineage B.1.1.7, UK-VOC 202012/01 are highlighted in bold grey. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 48: the sequence of pVAXDCSN 17; SN17

The nucleotide and amino acid sequences of the spike and nucleoprotein full-length chains in the expression vector pVAXDC. Both strands contain the human IgH leader sequence. The spike chain encodes amino acids 319-541 carrying the K417N, E484K and N501Y mutations of the south Africa variant VOC 501Y.V2/B1.351. The nucleoprotein chain encodes amino acids 2-419 including the T205I mutation from the variant, fused in frame with a human IgG1 hinge-CH 2-CH3 iV1, with the murine IgG3 23AA replaced in bold. Stop codons are indicated by asterisks. Mutations in south Africa variant VOC 501Y.V2/B1.351 are highlighted in bold grey. BamHI/XhoI and HindIII/PstI restriction sites for transferring both strands are highlighted.

Fig. 49: balb/c mice were immunized with SN15 (RBD monomer and N linked to modified Fc (FciV 1)) by gene gun on days 1, 8 and 15. The sera from day 21 were analyzed for antibody reactivity to the lineage a (martial), b.1.351 and b.1.1.7s1 protein variants in ELISA as reciprocal serum dilutions. Display EC ₅₀ Values.

Fig. 50: balb/c mice were immunized on days 1, 8 and 15 with SN15 (RBD monomer and N linked to modified Fc (FcifV 1)), SN16 (RBD monomer and N linked to modified Fc (FcifV 1) -B.1.1.7 variant), SN17 (RBD monomer and N linked to modified Fc (FcifV 1) -B.1.351 variant), or whole S DNA by gene gun. On day 21, the antibody reactivity of SN16 and SN17 immunized mice (a) or SN15, SN17 and intact S DNA immunized mice (B) sera to lineage a (armed), b.1.351 and b.1.1.7s1 protein variants was analyzed in ELISA with reciprocal serum dilutions. Display EC ₅₀ Values. C. ACE2 binding inhibition of variant RBD (i) and S1 (ii) proteins from SN15, SN17, whole S DNA, naive mice and control NIBSC 20/136 was assessed at 1/100 serum dilutions using MSD technology. BSA-PBS was used as a negative control.

Fig. 51: balb/c mice were immunized with SN15 or SN17 DNA constructs on days 1, 8 and 15 and serum was collected on day 21, analyzed for either lineage A or B.1.351 pseudotype virus (A) in pseudotyped neutralization assays or lineage A virus (B) in live virus neutralization assays. Data are readings at different serum titers and represent multiple experiments.

Fig. 52: balb/c mice were immunized with SN15 or SN17 DNA constructs on days 1, 8 and 15, and spleen cells were harvested on day 21 and analyzed for T cell responses to RBD or N peptide pools by IFNgamma ELISPot assay. The symbols represent the average response of individual mice and the lines represent the average between mice. Data were collected from multiple independent studies.

Fig. 53: balb/c mice were immunized with SN15 (RBD monomer and N linked to modified Fc (FciV 1)) or PSN17 (BD monomer and N linked to modified Fc (FciV 1) -B.1.351 variant) on days 1 and 29, followed by booster immunization with SN17 DNA on day 85. The serum of immunized mice was assayed for antibody reactivity against lineage a (martial) and b.1.351s1 protein variant (a) or b.1.351 and b.1.617.2rbd protein variant (B) in ELISA at reciprocal serum dilutions on days 42, 82 or 98. Display EC ₅₀ Values.

Fig. 54: SN15 complete plasmid vector nucleotide sequence (SEQ ID NO: 23).

Fig. 55: SN17 complete plasmid vector sequence (SEQ ID NO: 24).

Fig. 56: SN17 is complete doggbyone (dbDNA) vector sequence (SEQ ID NO: 25).

Fig. 57: the iSCIB1plus complete plasmid vector sequence (SEQ ID NO: 20).

Fig. 58: the iSCIB1plus had the complete doggybone (dbDNA) vector sequence (SEQ ID NO: 21).

Fig. 59: the iSCIB2 complete plasmid vector sequence (SEQ ID NO: 22).

Method

Materials, animals, cells and antibodies

Peptides and proteins

Covid19 peptides were selected based on the binding predictions of the IEDB database (http:// www.iedb.org /) for HLA-A 0201, HLA-DR 0101, and HLA-DP 0401, and based on the binding predictions of SYFPEITHI (http:// www.syfpeithi.de) for HLA-A 0201. Cancer antigen peptides were selected based on published sequences, IEDB database (http:// www.iedb.org /) binding predictions, and SYFPEITHI (http:// www.syfpeithi.de) binding predictions. Peptides (table 2) were synthesized in >90% purity (Genscript), aliquoted into disposable vials, and lyophilized for storage at-80 ℃ for reconstitution in PBS on the day of use. Recombinant N, S and His-tagged RBD proteins were purchased from Genescript (USA). N peptides were purchased from Miltenyi Biotec (UK), and RBD peptides were purchased from JPT Peptide Technologies (Germany).

TABLE 2Covid 19T cell epitopes

* MHCI binding predictions were performed at 04/03/2020 using IEDB analysis resource NetMHCpan (version 4.0) tool [184 ].

^￥ Peptides published as known Covid epitopes

TABLE 3 cancer antigen T cell epitopes

Plasmid(s)

The backbone of all the covd-19 plasmids pvaxdn 1-SN14 (SN 1-15) was derived from the FDA-regulated plastic vector backbone for human pVAX1 (Invitrogen). All nucleotide segments used for insertion are codons optimized for human expression. SN1-SN4 contains a murine IgK leader and SN5-15 contains a human IgH leader. Synthesizing a codon optimized nucleotide segment encoding: the leader sequence, amino acids 319-541 or 330-525 of the S glycoprotein RBD domain alone (accession number YP_ 009724390), amino acids of the S glycoprotein RBD domain 319-541 or 330-525 fused in frame with the hinge-CH 2-CH3 domain of the HuIgG1 constant domain (accession number P01857) or the variant hinge-CH 2-CH3iV1, in which 23 amino acids have been substituted with murine IgG3 residues, or amino acids of the S glycoprotein RBD domain 319-541 or 330-525 linked to the minor fibrin trimer folding motif or disulfide bridge motif by glycine serine linkers, and the synthetic nucleotide segments are inserted at the 5 'and 3' ends into BamHI and XhoI sites, respectively. In the first round of cloning, these segments were inserted into the BamHI/XhoI site of the pVAXDCIB68 construct shown in FIG. 1, directly replacing the kappa light chain in the first expression cassette to form an intermediate plasmid.

In the second round of cloning, codon optimized nucleotide segments encoding: leader sequence, full length nucleoprotein amino acids 2-419 alone (accession number yp_ 009724397), or full length nucleoprotein amino acids 2-419 fused in frame with the hinge-CH 2-CH3 domain or variant hinge-CH 2-CH3iV1 of HuIgG1 constant domain, and flanked by HindIII/PstI. The heavy chain from the intermediate vector formed in the first round was excised using HindIII/PstI and replaced with the N segment in the second expression cassette alongside the appropriate S segment shown in FIG. 1.

To enhance the ImmunoBody vectors pVAXDCIB68 (SCIB 1), pVAXDCIB238 (SCIB 1 plus) and pVAXDCIB178 (SCIB 2), the huIgG1 constant region of the antibody heavy chain encoding the CH 1-hinge-CH 2-CH3 domain (accession number P01857 amino acids 1-330) was substituted at a specific site with the same segment encoding the substituted 23 murine IgG3 residues. This is achieved by synthesizing a stretch of nucleotides encoding CH 1-hinge-CH 2-CH3iV1 flanked by AfeI and EcoRI. These restriction sites were used to excise the HuIgG1 constant region from the vector and insert the segment in frame along with the heavy chain variable region.

The sequences of the two strands in each of the expression cassettes of the pVAXDC vector, pVAXDCIB68 iV1, pVAXDCIB238 iV1 and pVAXDCIB178 iV1 of SN1-15 were confirmed by the dideoxy chain termination method [185 ]. FIGS. 2 to 15, 27 and 29, 31 and 33 show the DNA nucleotide and translated protein sequences of the two strands encoded in the plasmids SN1-SN15 and the enhanced ImmunoBody vectors pVAXDCIB68 iV1, pVAXDCIB238 iV1 and pVAXDCIB178 iV1, respectively.

The plasmid pCMV 3-2019-nCoV-spike (S1+S2) -length, which encodes the full-length spike protein from SARS-COV2 amino acids 1-1273 (accession number YP_009724390/QHD 43416.1), was obtained from Sino Biological (catalog number VG 40589-UT). It contains codon-optimized cDNA for expressing proteins inserted into the KpnI/XbaI site of the mammalian expression vector pCMV3-untagged under the control of a high level expression mammalian human enhanced cytomegalovirus immediate early (CMV) promoter in mammalian cells.

To construct pVAXDCSN16-17 (SN 16-17), two consecutive rounds of cloning were required. Two codon-optimized nucleotide segments encoding a spike chain comprising a human IgH leader (MDWIWRILFLVGAATGAHS) and spike glycoprotein RBD domain 319-541 amino acids (accession No. yp_ 009724390) were synthesized, wherein the spike glycoprotein RBD domain 319-541 amino acids comprise the N501Y mutation from Kent variant/lineage b.1.1.7, UK-VOC 202012/01, or the spike glycoprotein RBD domain 319-541 amino acids comprise the K417N, E484K and N501Y mutations from south africa variant/lineage VOC 501y.v2/B1.351, with the nucleotide segments flanked at the 5 'and 3' ends by BamHI and XhoI sites, respectively, for SN 17. In the first round of cloning, the segment was inserted into the BamHI/XhoI site of the pVAXDCIB68 (SCIB 1) plasmid, directly replacing the SCIB1 kappa light chain in the first expression cassette, to form two intermediate plasmids.

In the second round of cloning, two codon-optimized nucleotide segments encoding a nucleoprotein chain comprising the human IgH leader sequence, full-length nucleoprotein amino acids 2-419 (accession number YP 009724397) fused in-frame with the modified variant hinge-CH 2-CH3 iV1 human IgG1 constant domain (in which 23 amino acids have been substituted with murine IgG3 residues) were synthesized, wherein for SN16, full-length nucleoprotein amino acids 2-419 comprise D3L and S235F mutations from Kent variant/lineage b.1.1.7, UK-VOC 202012/01, or for SN17, full-length nucleoprotein amino acids 2-419 comprise T205I mutations from south africa variant/lineage VOC y.v2/B1.351, and the nucleotide segments are flanked by HindIII/PstI. SCIB1 heavy chain huIgG1 was excised from the intermediate plasmid formed in the first round using HindIII/PstI and replaced with a nucleoprotein segment in the second expression cassette alongside the appropriate spike segment to give SN16 and SN17.

The sequences of the two strands within each expression cassette of the pVAXDC vectors of SN16 and SN17 were verified by the dideoxy chain termination method. The DNA nucleotide and translated protein sequences of the two strands encoded within SN16 and SN17 are shown in FIGS. 47 and 48.

TABLE 4COVID19 constructs

Animal and cell line

C57Bl/6J, balb/C (Charles River), HLA-DR4 mice (model #4149, tacouc), HHDII/HLA-DP4 mice (EM: 02221, european mouse mutant library (European Mouse Mutant Archive)), HHDII mice (Pasteur institute), or HHDII/HLA-DR1 mice (Pasteur institute) were used at ages of 8 to 16 weeks. All work is done under the project license approved by the Home Office (Home Office) with ethical approval. In all studies, mice were randomized into different groups and blinded to the study personnel.

Culturing B16 melanoma involving expression of related MHCI and II alleles in RPMI medium 1640 with L-glutamine (2 mmol/L) and 10% FCS and appropriate antibiotics (previously described in [140,183,186,187 ]]) Cells contained to maintain the plasmid. As previously described [188 ]]HEK293T human embryonic kidney cells (ATCC CRL 1573) were propagated. In a kit having 10% FBS (Sigma), 2mM glutamine, 20mM HEPES buffer, 100 units/ml penicillin, 100mg/ml streptomycin and 10 ^-5 Murine spleen cells were cultured in RPMI-1640 with M2-mercaptoethanol. The cell lines used were validated by the provider as mycoplasma free (STR typing) and used within ten generations.

HEK293 transient transfection

Using an Expifectamine ^TM 293 transfection kit (Gibco, life technologies) transient transfection of Expi293F ^TM The cells were then assessed for secretion levels of the pDNA construct. Briefly, HEK293 cell suspensions (100 ml,2X 10) were transfected with 100. Mu.g DNA ⁶ /ml) and conditioned medium was harvested on day 6 post-transfection. The conditioned supernatant was passed through a 0.22 μm vial top filter (Merck Millipore) The sodium azide was filtered and added to a final concentration of 0.2% (w/v). Cell pellets were lysed in an appropriate volume of RIPA buffer (Sigma Aldrich, R0278) according to the manufacturer's instructions and clarified by centrifugation prior to analysis.

Immunization protocol

Unless otherwise indicated, mice were immunized intradermally with 1 μg of DNA by gene gun on days 1, 8 and 15 or days 1, 5 and 29 and analyzed for response on days 21 or 35, respectively.

For tumor treatment studies, mice were subcutaneously implanted with 2.5x10 on day 1 ⁴ The B16F1 or B16 HHDII Nyeso1 tumor cells were then vaccinated on days 4, 11 and 18 or days 4, 8 and 11. Tumors were measured at 3-4 day intervals. Tumor growth in mice was analyzed by measuring tumor size (length and width) using calipers. The volume is estimated by:

volume= (pi/6) x (width x length ² )

Pseudovirus neutralization assay

SARS-CoV-2 spike protein plasmid was generated and cloned as per [189 ]]The method for hepatitis c virus forms pseudo particles. The pseudo particles formed in the absence of plasmid served as negative control. For infection and neutralization assays of SARS-CoV-2 pseudoparticles, HEK293T cells were seeded per well in white 96-well tissue culture plates (Corning) and incubated overnight at 37 ℃. The following day, SARS-CoV-2 pseudoparticles were mixed with the appropriate amount of antibody, incubated for 1 hour at 37℃and then added to the cells. After incubation at 37℃for 72 hours, 100. Mu.l Bright-Glo (Promega) was added to each well and incubated for 2 minutes, or the cells were lysed with cell lysis buffer (catalog number E1500; promega) and placed on a shaker for 15 minutes. Luciferase activity was measured in Relative Light Units (RLU) using a SpectraMax M3 microplate reader (Molecular Devices) and SoftMax Pro6 software (Bright-Glo protocol), or 50. Mu.l of luciferase substrate was injected and read in each well using a FLUOstar Omega microplate reader (BMG Labtech) and MARS software. Infection with SARS-CoV-2 sham particles was measured by detection in the presence of the same dilutions of anti-SARS-CoV-2 mAb, test animal serum, preimmune animal serum and non-specific IgG. Each sample was examined in duplicate or triplicate And (5) measuring. Neutralization activity was reported as 50% inhibition dilution (ID ₅₀ ) Values were calculated by nonlinear regression (GraphPad Prism version 7) using lower and upper limits (0% and 100% inhibition) as constraints to assist curve fitting.

Live virus neutralization assay

SARS-CoV-2 infectious virus (CVR-GLA-1) was obtained from the national center for AIDS reagent (the National Centre For AIDS Reagents, NIBSC, UK) of NIBSC, UK.

Using the method previously described [190 ]]Live virus neutralization assays were performed except that 790TCID was used ₅₀ Each serum dilution was added per ml of SARS-CoV-2 virus. Furthermore, for the same assay, serum was diluted up to 1:81920.

ACE2 binding inhibition assay

Vaccine-raised antibodies were studied for their ability to block ACE2 binding to RBD or intact S protein using V-PLEX covd-19 ACE2 neutralization kit from Meso Scale Diagnostics LLC. V-plex SARS-CoV-2 Panel 7 multi-spot plates containing S1 RBD and intact S proteins from lineage A (initially identified in Wuhan) and variants (B1.1.7, B1.351, P.1) SARS-CoV-2 strains were blocked according to manufacturer' S instructions and then incubated with 1:100 diluted serum and sulfonated tagged human ACE2 protein. Serum incubated samples were compared to wells with dilution (no inhibition) and the results were expressed as percent inhibition of ACE2 binding.

RBD binding inhibition assay (surrogate neutralization assay)

RBD binding inhibition was assessed using a kit purchased from Genescript (USA). Briefly, different dilutions of serum from immunized animals were mixed with recombinant HRP-tagged RBD protein. The mixture was added to plates pre-coated with ACE2 receptor and RBD binding was detected using TMB substrate. RBD binding inhibition was calculated as colorimetric signal loss at 0% for the negative control.

ELISpot assay

ELISPot assays were performed using murine IFNγ capture and detection reagents according to the manufacturer's instructions (Mabtech AB, nacka Strand, sweden). Briefly, wells of 96-well Immobilin-P plates were coated with anti-IFNγ antibodies, and quadruplicates of wells were used with 5X10, unless otherwise indicated ⁵ Individual spleen cells were inoculated and added with synthetic peptide at a final concentration of 10. Mu.g/ml, 1. Mu.g/ml recombinant protein or 1. Mu.g/ml peptide pool. The plates were incubated at 37℃with 5% CO ₂ Incubate in atmosphere for 40 hours. After incubation, captured ifnγ was detected by biotinylated anti-ifnγ antibody and developed with streptavidin alkaline phosphatase and chromogenic substrate. Spots were analyzed and counted using an automated microplate reader (Cellular Technologies Ltd Europe GmbH, aalen, germany). Using a plot of effector function versus peptide concentration, functional avidity was calculated as the concentration that caused 50% of the maximal effector function.

ELISA of anti-S and N antibodies

Commercial N, S and RBD proteins (GeneScript, USA) were diluted in PBS and coated with high protein binding 96-well plates at 0.5 μg/well overnight at 4 ℃. Plates were washed and blocked with casein blocking agent (Thermo Scientific Ref: 37528) at 200 μl/well for at least 1 hour at room temperature, followed by incubation with murine serum diluted (at different dilutions) in PBS 2% bsa for 1 hour at room temperature. Plates were washed and incubated with anti-mouse Ig HRP antibody (2-step ELISA) or anti-mouse Fc-biotin in PBS containing 2% BSA followed by streptavidin-HRPO (3-step ELISA) for 1 hour at room temperature. After washing the added TMB substrate, 1N H was used ₂ SO ₄ The reaction was terminated. Commercially available murine IgG N and S1 specific antibodies (Sino Biological) were used as controls (N+ve and S1+ve). Serum from naive mice was included as a negative control. Absorbance was read at a wavelength of 450 nm.

Sandwich ELISA detection of secreted RBD and N proteins

In both cases a commercial kit/antibody pair was used. NPs in conditioned medium and cell lysates (6 days post transfection) were detected using SARS-CoV-2 NP ELISA kit from Bioss (catalog number BSKV 0001) according to the instructions of the supplier. Quantification was performed according to the NP standard curve provided by the kit. RBD (secreted and in cell lysates) was detected using a sandwich ELISA consisting of a capture antibody SARS-CoV-2 spike-neutralizing mouse mAb (Sino Biological,40591-MM 43) in combination with an HRPO-labeled detection antibody (epigenetek a 73682) from a SARS-CoV-2S protein RBD antibody pair. The capture antibody was coated at 200 ng/well; the detection antibodies were used at a dilution of 1:1000.

Analysis of CD64 binding by Fc-fusion construct SPR

Analysis was performed with BiaT 200. His-tagged CD64 (Acrobiosystems, FCA-H52H 1) was captured on a CM5 chip that binds anti-His antibodies. The chip includes 4 flow cells, 3 of which are used to capture increased density CD64, and the fourth flow cell is the reference cell. The Fc-construct was titrated over a concentration range of 50.0nM to 0.78nM and association and dissociation was monitored at a flow rate of 30ul/' for 210s and 700s, respectively. Kinetic parameters were derived by fitting the data to a 1:1 monovalent binding model.

Immunogenicity analysis of modified Fc (iFcv 1) constructs

Analysis was performed by Abzena (Cambridge) Ltd according to HTA standards. PBMCs were isolated from healthy population donor buffy coats (approved from commercial suppliers). Separation of cells by Density centrifugation Using lymphocyte separation Medium (Corning, amsterdam, netherlands) and use of CD8+Rosetteep ^TM (StemCell Technologies Inc, london, UK) depletes CD8+ T cells. The donors were characterized by identifying HLA-DR and HLA-DQ haplotypes to 4-digit resolution by HISTO Spot SSO HLA typing (MC Diagnostics, st. Asaph, UK). T cell responses to the neoantigen KLH (Invitrogen, paisley, UK) were also determined. PBMCs were then frozen and stored in a nitrogen phase until needed. A cohort of up to 50 donors was selected, covering 77% of HLA alleles. PBMCs from each donor were thawed, counted and viability assessed using trypan blue (Merck Life Science UK Ltd, gillingham, UK) dye exclusion. For each donor, 1ml of proliferation cell stock was added to the appropriate wells of a 24-well plate to create a large batch of culture. 1ml of the test sample (iTv 1) was added to the PBMC to give a final sample concentration of 0.3. Mu.M. For each donor, there were also included a reproducible control well (cells incubated with 0.3. Mu.M KLH), a clinical baseline control well (5. Mu.M Incubated cells), low immunogenicity control (with 0.3. Mu.M +.>Incubated cells) and medium-only wells. Culture in37 ℃ and 5% CO ₂ Incubate for a total of 8 days. On days 5, 6, 7 and 8, the cells in each well were gently resuspended by mixing 5x using an electronic pipette and 3x100 μl aliquots were transferred into each well of a 96-well round bottom plate. Cultures were treated with 100. Mu.l +.>0.75. Mu. Ci [3H ] in culture]Thymidine (Perkin->Beaconsfield, UK) was pulsed and incubated for a further 18 hours, and then harvested to a filter pad (Perkin) using a TomTec Mach III cell harvesterBeaconsfield, UK). CPM per well was measured with 1450 Microbeta Wallac Trilux liquid scintillation counter (Perkin +.>Beaconsfield, UK) by means of a Meltilex ^TM (PerkinBeaconsfield, UK) scintillation counts were determined as paralux low background counts. An empirical threshold for SI equal to or greater than 1.9 (SI. Gtoreq.1.90) has been established previously, and samples that induce responses above this threshold are considered positive.

Statistical analysis

Responses to nucleic acid plasmids were statistically analyzed using GraphPad Prism 7 software. Comparison analysis of ELISpot results was performed using paired or unpaired ANOVA or student t-test as appropriate and p-values calculated accordingly. Affinity curve/survival comparisons were evaluated by applying either F-test or log rank test using GraphPad Prism software. P (P) <0.05 was considered statistically significant. Inter-group neutralization titers (ID) were determined using Kruskal-Wallis analysis of variance (ANOVA) and Dunn's multiple comparison assay ₅₀ Values) and comparing. The data analysis was not blind. As described above, p-value<At 0.05, the difference was considered statistically significantWell and statistical analysis was performed using GraphPad Prism 8 software.

Preclinical studies

Example 1 RBD protein and N protein secretion of HEK293 transiently transfected with pDNA

To assess transfection efficiency of the pDNA construct, HEK293 cells were transiently transfected with pDNA using the Expi293 system of Thermofisher and protein secretion of RBD proteins and nucleoprotein in the medium was assessed using sandwich ELISA (fig. 16). This analysis shows that constructs comprising RBD-Fc fusions (SN 3 and SN 7) produced the highest secretion for RBD proteins; followed by unmodified RBDs (SN 4, SN5, SN 8). Trimeric RBDs (SN 2, SN6, SN9, SN10, SN 11) produce the lowest secretion levels. The nucleoprotein secretion of the constructs (SN 3, SN4, SN7, SN 8) containing unmodified NPs was highest. Since the latter targets are more associated with T cell responses than neutralizing antibodies are induced; lower secretion levels (as observed for N Fc fusion proteins) lead to a stronger T cell response is desirable.

Example 2 delivery of pDNA T cell response to RBD and N Protein (NP) by gene gun to HHDII mice

T cell responses to pVAXDC spike rbd+np (SN 8), pVAXDC spike RBD v2 trimer+npfc (SN 9), pVAXDC spike RBD v3 trimer+npfc (SN 10) and pVAXDC spike RBD v2 trimer+npfc iV1 (SN 11) were measured after 3 weeks immunization of HHDII mice with pDNA by gene gun administration. Ifnγ ELISpot response frequencies to all RBD constructs were measured using predicted or previously identified T cell epitopes and intact S1 proteins, RBD recombinant proteins and RBD peptide libraries. All four constructs showed strong responses to the S1 protein as well as to the RBD peptide pool and RBD aa417-425 peptides, which reached a significant response in all constructs (FIG. 17A). The frequency of responses to N antigen was measured using overlapping peptide libraries, recombinant N protein and identified T cell epitopes. All constructs showed strong responses to recombinant N protein, but only SN10 and SN11 constructs showed significant responses to the N aa138-147 peptide (fig. 17A).

Another study compared responses to pVAXDC spike RBD+NP (SN 8), pVAXDC spike RBD v3 trimer+NPFC (SN 10), and pVAXDC spike RBD v2 trimer+NPFC iV1 (SN 11) after immunization of HHDII mice for 3 weeks by gene gun administration of pDNA. All three constructs showed a significant response to the S1 protein as well as to the RBD peptide pool, but SN 11-only immunized mice showed a significant response to the RBD aa417-425 peptide (fig. 17B). For the N protein specific response, all four constructs showed strong responses to the N protein, but mice immunized with SN11 only showed a significant response to the N138-146 peptide (fig. 17B).

Example 3 delivery of pDNA T cell response to RBD and N Protein (NP) by gene gun to HHDII/DR1 mice

T cell responses to pVAXDC spike rbd+np (SN 8), pVAXDC spike RBD v2 trimer+npfc (SN 9), pVAXDC spike RBD v3 trimer+npfc (SN 10) and pVAXDC spike RBD v2 trimer+npfc iV1 (SN 11) were measured 3 weeks after immunization of HHDII/DR1 mice via gene gun administration of pDNA. The ifnγ ELISpot response frequency was measured for all RBD constructs using the identified T cell epitopes (RBD aa 417-425), intact S1 protein, intact RBD protein and RBD peptide libraries. Constructs SN8, SN9 and SN10 showed a significant response to only the intact S1 protein, whereas the SN11 construct gave a response to S1 and RBD proteins, RBD peptide libraries and RBD aa417-425 peptides (FIG. 18). The frequency of responses to N antigen was measured using overlapping peptide libraries, N protein and identified T cell epitopes (N aa 138-147). Constructs SN10 and SN11 gave a significant response to the intact N protein, but construct SN11 alone gave a significant response to the N aa138-147 peptide (FIG. 18).

Example 4 constructs comprising NPFC iV1 produce a higher frequency response to N protein

To compare T cell responses comprising N antigen (SN 8), N-linked to FC (SN 9), or N-linked to modified iV1 FC (SN 11), data were combined and normalized to background control. Construct SN11 showed significantly enhanced responses to N protein and N138-147 peptide when compared to SN8 or SN9 (p <0.01 and p <0.05, respectively) (fig. 19A and 19B). Interestingly, construct SN11 also responds more frequently to S1 protein than SN8 (< 0.05 p); construct SN11 also responded more frequently to RBD protein than SN8 and SN9 (< p < 0.001) (fig. 19C and 19D).

Example 5 delivery of pDNA T cell response to RBD and N proteins by gene gun to HHDII/DP4 mice

T cell responses to pVAXDC spike RBD trimer+npfc (SN 2), pVAXDC spike RBD fc+np (SN 3) and pVAXDC spike rbd+np (SN 4) were measured after HHDII/DP4 mice were immunized three times per two weeks with pDNA by gene gun. Using predicted and identified T cell epitopes, intact S1 protein and RBD protein, ifnγ ELISpot response frequency was measured for all RBD protein constructs. Significant responses of construct SN2 to RBD aa417-425 peptides, RBD proteins and RBD peptide libraries were observed, and significant responses of construct SN4 to S1 and RBD proteins (fig. 20). The ELISpot response frequency to the N construct was measured using the N protein and predicted and identified T cell epitopes. Constructs SN2 and SN4 showed strong responses to N protein (< p < 0.001), but the response of SN3 did not reach significance (fig. 20).

Example 6RBD FC and RBD trimer constructs deliver pDNA with better T cell response avidity to S1 protein by gene gun

After three immunization of HHDII/DP4 mice with pDNA administered once every two weeks by gene gun, T cell responses to pVAXDC spike RBD trimer+npfc (SN 2), pVAXDC spike RBD fc+np (SN 3) and pVAXDC spike rbd+np (SN 4) were evaluated for affinity to S1 protein titration. The response in mice immunized with SN2 and SN3 showed significantly higher response avidity (p < 0.0001) compared to mice immunized with SN4 (fig. 21). Affinity was measured using normalized data as the concentration that elicited 50% of the maximum response. This corresponds to an SN2 of 0.0001. Mu.g/ml, an SN3 of 0.000008. Mu.g/ml, and an SN4 of 0.17. Mu.g/ml.

Example 7pDNA immunization produces a high affinity peptide-specific response to the RBD 417-425 epitope

T cell responses to pVAXDC spike RBD v2 trimer+NPFiV 1 (SN 11) after immunization of HHDII/DP4 mice three times per week with pDNA by gene gun were evaluated for affinity to RBD 417-425 peptides by peptide titration. The response in mice immunized with SN11 showed a high affinity response greater than 0.0001ug/ml (figure 22).

Example 8 delivery of the NPFC iV1 construct by Gene gun the frequency and avidity of the T cell response of pDNA to the N138-146 peptide was better

After immunization of HHDII/DP4 mice three times per week with pDNA by gene gun, the T cell responses to pVAXDC spike RBD v3 trimer+NPFC (SN 10), pVAXDC spike RBD v2 trimer+NPFC iV1 (SN 11) were evaluated for affinity to the N138-146 peptide by peptide titration. Responses in mice immunized with SN11 showed a higher frequency and slightly higher response avidity than mice immunized with SN10 (fig. 23). The NP FC iV1 construct (SN 11) was shown to induce T cells with higher frequency and avidity than the NPFC construct (SN 10).

Example 9 delivery of antibody response of pDNA to RBD and N proteins by Gene gun

After immunization of HHDII/DP4 mice three times per two weeks with pDNA by gene gun, antibody responses to pVAXDC spike rbd+npfc (SN 5), pVAXDC spike RBD trimer+npfc (SN 6), pVAXDC spike RBD v2 trimer+npfc (SN 9), pVAXDC spike RBD v3 trimer+npfc (SN 10), pVAXDC spike RBD v2 trimer+npfciv 1 (SN 11) or pVAXDC spike RBD trimer+npfc (SN 2), pVAXDC spike RBD fc+np (SN 3) and pVAXDC spike rbd+np (SN 4) were measured. Antibody (Ab) titers to S1, RBD and N proteins were compared with mouse serum immunized with monomeric RBD construct, dimeric RBD presented as Fc fusion protein, RBD construct as trimer presented as minor fibrin construct, and shorter RBD as trimer. Antibodies were evaluated in serum at dilutions of 1/100 to 1/10,000. Strong reactivity to N protein was observed in the sera of all immunized mice, even at 1/10,000 dilution (FIG. 24). Reactivity to S1 and RBD proteins was observed in serum at 1/100 and 1/1000 dilutions, and reactivity to S1 and RBD proteins was highest from constructs SN5, SN6 and SN10 containing longer RBD constructs as monomers (SN 5) or trimers (SN 6) and shorter RBD v3 trimers (SN 10) (fig. 24A). Similar antibody responses to the S1 and N proteins were detected in sera of SN2, SN3 and SN4 immunized mice (fig. 24B).

After immunization of HHDII/DP4 mice three times per week by administration of pDNA by gene gun, antibody responses to pVAXDC spike rbdfc+np (SN 3), pVAXDC spike rbd+np (SN 8), pVAXDC spike RBD v3 trimer+npfc (SN 10) and pVAXDC spike RBD v2 trimer+npfciv 1 (SN 11) were measured in ELISA assay. Antibodies were evaluated in serum at dilutions of 1/100 to 1/10,000. Strong reactivity to N protein was observed in all immunized mouse sera, even at 1/10,000 dilution. Reactivity to S1 protein was observed in serum at 1/100 and 1/1000 dilutions, and reactivity to S1 protein from construct SN10 comprising the shorter RBD v3 trimer (SN 10) was highest, but was also detectable in serum from construct SN8 comprising full-length RBD monomer at 1/1000 dilutions (fig. 24C).

Example 10 pDNA delivered by gene gun elicited a virus-neutralizing antibody response of similar titer to the total antibody measurement

Inhibition of RBD binding to ACE2 receptor binding plates by virus-neutralizing antibodies was evaluated in a surrogate neutralization assay. Mouse sera immunized with constructs SN5, SN6 and SN10 containing longer RBD monomers, longer RBD trimer or shorter RBD v3 trimer, respectively, showed >50% inhibition at 1/100 dilution, and lower titers were observed for constructs SN9 and SN11 (fig. 25). Serum samples were also tested for virus neutralization in a pseudovirus neutralization assay. Sera from SN2, SN3 and SN4 immunized mice were evaluated at day 21 after only two immunizations, while SN5, SN6, SN9, SN10 and SN11 sera were evaluated at the end (day 35). In this assay, sera of mice immunized with constructs SN3, SN5 and SN6 alone showed >50% virus neutralization at 1/100 dilution, with SN5 showing near 90% neutralization and SN3 near 80% neutralization at 1/100 dilution (fig. 26A). No virus neutralization was observed for the unrelated virus (fig. 26B). Strong antibody titers against the N protein were observed from constructs comprising NP, NPFC and NPFC iV 1. Constructs comprising RBD monomers or RBD dimers presented as Fc fusions showed highest antibody titers targeting S protein and highest virus neutralizing antibodies similar to total Ig antibody titers.

In the pseudovirus neutralization assay, virus neutralization at different serum dilutions was also analyzed (fig. 26C). Titration data shows that mouse serum immunized with construct SN5 shows 50% neutralization titer (ID 50) at a serum dilution of 1/3517. Titer of mice immunized with construct SN6 at 1/236 dilution and mice immunized with construct SN3 at 1/600 dilution (fig. 26D).

TABLE 5 summary of the COVID19 constructs

TABLE 6 summary of immune response to the COVID-19 constructs M monomer, fc-fusion, fcv1 Fc-fusion enhanced, T trimer, sm minimonomer, st minitrimer, cc-extra cysteine

All the covd-19 constructs contained the S protein RBD as a monomer, trimer or Fc fusion protein, and the N protein as a monomer, fc fusion protein or Fc fusion protein modified to allow non-covalent association of antigen-Fc fusion proteins at the cell surface (tables 5 and 6).

SN11 expresses an N protein fused to a modified Fc, SN11 produces a significantly better T cell response to the N protein and to HLA-A2 epitope N138-146 than to the N protein fused to an unmodified Fc or to the N protein alone. More interestingly, this construct produced a stronger response to RBD, S1 and peptide RBD 417-425 than a similar construct expressing the same RBD construct but without N-Fc. This suggests that the modified N-Fc acts like an adjuvant and activates APC while enhancing T cell responses to other antigens.

In contrast, RBD trimers (SN 2, SN6, SN9, SN11, SN 12) and RBD-Fc (SN 3, SN7, SN13, SN 14) stimulated the best VNAB for RBD monomers (SN 1, SN4, SN5, SN8, SN 15). The resulting constructs compare RBD trimer to RBD-Fc and RBD-enhanced Fc to Fc modified N protein combinations. Constructs comprising RBD monomers and N proteins fused to the enhanced Fc region (SN 15), respectively, or RBD and N proteins fused to the enhanced Fc region (SN 14), respectively, produce the strongest antibody and T cell responses.

The above examples show that vaccines that incorporate RBD of spike protein to stimulate neutralizing antibodies and T cell responses, and N protein to induce memory T cell responses, can provide protection not only against covd 19 but also against any emerging coronavirus, as the N protein is highly conserved and rarely mutated.

Example 11 pDNA encoding T cell epitope within CDRs of Fc modified HuIgG1 construct (iSCIB 1) produces a strong T cell response

The immune response was assessed by immunization of conventional C57Bl/6 or HLA transgenic mice (HLA-DR 4) with pDNA encoding SCIB1 (WO 2008/116937-FIG. 28) or iSCIB1 (FIG. 29) by a gene gun and by an IFNγ ELISPot assay, and the two were compared. FIG. 34A shows that both SCIB1 and iSCIB1DNA produced high frequency TRP2 180-188 responses in immunized C57Bl/6 and HLA-DR4 mice. Analysis of response avidity by peptide titration showed that iSCIB1DNA immunization resulted in a higher avidity TRP2 180-188 specific CD8 response in C57Bl/6 and HLA-DR4 mice than SCIB1DNA immunization (fig. 35B and 35C). The response frequency of HLA-DR4 mice to gp100 44-59 epitopes was also analyzed, indicating that mice immunized with iSCIB1DNA tended to respond more frequently (FIG. 34D).

Example 12 pDNA (iSCIB 1 plus) encoding T cell epitopes within the CDRs of Fc modified HuIgG1 constructs produces a strong T cell response

The HLA transgenic mice (HLA-DR 4, C57Bl/6 or HHDII/DP 4) were immunized by gene gun with pDNA encoding SCIB1plus (FIG. 30) or iSCIB1plus (FIG. 31) and the immune response was assessed by the IFNγ ELISpot assay, and the two were compared. FIG. 35A shows that SCIB1plus and iSCIB1plus DNA produced high frequency TRP2 180-188 responses in immunized C57Bl/6, HHDII/DP4 and HLA-DR4 mice. Response avidity analysis by peptide titration showed that iSCIB1plus DNA immunization resulted in a higher avidity TRP2 180-188 specific CD8 response in C57Bl/6 and HLA-DR4 mice than SCIB1plus DNA immunization (fig. 35B and 35C). Analysis of gp100 44-59 specific response frequency in HLA-DR4 mice showed that mice immunized with iSCIB1plus DNA tended to respond more frequently (FIG. 35D).

Example 13 pDNA encoding T cell epitopes within the CDRs of Fc modified HuIgG1 constructs (iSCIB 1 and iSCIB1 plus) mediated effective tumor treatment

Conventional C57Bl/6 or HLA transgenic mice (HHDII/DP 4) were implanted with B16 melanoma cells expressing the appropriate MHC allele, followed by immunization with pDNA encoding SCIB1 (fig. 28), iSCIB1 (fig. 29), SCIB1plus (fig. 30) or iSCIB1plus (fig. 31), respectively, and comparing SCIB1 (fig. 28) with iSCIB1 (fig. 29), or comparing SCIB1plus (fig. 30) with iSCIB1plus (fig. 31), respectively. Tumor growth and survival were monitored. Tumor treatment studies in C57Bl/6 mice are shown in FIG. 36. Mice immunized with iSCIB1 DNA showed slower tumor growth than mice immunized with SCIB1 DNA and significantly slower tumor growth than control mice (p=0.0012) (fig. 36A). Mice immunized with SCIB1plus DNA or iSCIB1plus DNA showed significantly slower tumor growth (p < 0.0001) than controls (fig. 36B). Tumor volume analysis on day 18 showed that mice immunized with iSCIB1plus DNA resulted in slower tumor growth compared to SCIB1plus DNA (fig. 36B). Mice immunized with iSCIB1, SCIB1plus, and iSCIB1plus DNA showed significantly improved overall survival compared to control mice (p=0.0143, and p <0.0001, respectively) (fig. 36C). Although tumor treatment was provided with SCIB1plus and iSCIB1plus immunization, iSCIB1plus DNA immunization showed significantly improved overall survival (p=0.0003) compared to SCIB1plus DNA (fig. 36C).

Example 14 pDNA encoding T cell epitope within CDRs of Fc modified HuIgG1 construct (iSCIB 2) produces a strong T cell response

HLA transgenic mice (HHDII or HHDII/DR 1) were immunized with pDNA encoding SCIB2 (fig. 32) or iSCIB2 (fig. 33) by gene gun and the immune response was assessed by ifnγ ELISpot assay, and the two were compared. FIG. 37A shows that both SCIB2 and iSCIB2 DNA produced high frequency Nyoso 1 157-165 responses in immunized HHDII and HHDII/DR1 mice. Analysis of response avidity by peptide titration showed that both SCIB2 and iSCIB2 DNA immunization in HHDII mice produced a high avidity nyso 1 157-165 specific CD8 response, but iSCIB2 produced a higher avidity than SCIB2 in HHDII/DR1 mice (fig. 37B and 37C). The Nyeso 119-143 sequence contains a known HLA-DR1 epitope, and thus the response to the Nyeso 119-143 epitope was analyzed in HHDII/DR1 mice. These showed a significant enhancement in response frequency from iSCIB2 compared to SCIB2 (p= 0.0338) (fig. 37D). Affinity analysis showed that Nyeso 119-143 response generated by iSCIB2 also had higher affinity (p=0.0363) than response generated by SCIB2 in HHDII/DR1 mice (fig. 37E).

Example 15 pDNA encoding T cell epitope within the CDRs of Fc modified HuIgG1 construct (iSCIB 2) mediates effective tumor treatment

HLA transgenic mice (HHDII) were implanted with B16 melanoma cells expressing the appropriate MHCl allele, and then immunized with pDNA encoding SCIB2 (fig. 32) or iSCIB2 (fig. 33) by a gene gun, and the two were compared. Tumor growth and survival were monitored. Mice immunized with SCIB2 and iSCIB2 both showed significantly improved tumor-free survival (p <0.0001 and p=0.0010, respectively) over control mice, while there was no significant difference between SCIB2 and iSCIB2 immunized mice (fig. 38).

Example 16 COVID-19 specific T cell and neutralizing antibody response from pDNA delivered by Gene gun RBD Fc iV1 construct was superior to RBD Fc construct

Balb/c and HLA-A2 transgenic mice were immunized with pDNA comprising N protein fused to modified Fc (NPFC iV 1) and RBD domain fused to Fc (RBD FC, SN13, FIG. 14) or RBD domain fused to modified Fc (RBD FC iV1, SN14, FIG. 15). T cell responses were assessed by ifnγ ELISpot assay, mice immunized with RBD fciv 1, SN14 showed significantly higher frequency responses to overlapping peptide pools from RBD proteins than RBD Fc, SN13 (fig. 39A). Analysis of the response avidity of HLA-A2 transgenic mice to HLA-A2 epitope from RBD (RBD 417-425) indicated that SN14 (RBD Fc IV 1) immunized mice produced a higher avidity response than SN13 (RBD Fc) immunized mice (fig. 39B).

Antibody responses of immunized Balb/C mice were assessed by ELISA, SN13 and SN14 immunized mice showed similar S1 protein-specific antibody titers (fig. 39C). When the neutralizing antibody response was analyzed in the pseudo-virus neutralization assay, the mouse serum immunized with SN14 (RBD FC iV 1) showed a higher neutralizing ID50 titer than the mouse serum immunized with SN13 (RBD FC) (fig. 39D). This data provides evidence for excellent T cell and neutralizing antibody responses from pDNA constructs comprising antigen fused to modified Fc.

EXAMPLE 17 RBD protein and N protein secretion of HEK293 transiently transfected with SN11, 12, 13, 14 and 15pDNA

To assess transfection efficiency of SN11, 12, 13, 14 and 15pDNA constructs compared to SN 5pDNA, HEK293 cells were transiently transfected with pDNA using a thermofiser's Expi293 system and protein secretion of RBD proteins and nucleoprotein in culture media and cell lysates was assessed using sandwich ELISA (fig. 40). This analysis shows that, for the RBD protein, the construct comprising the RBD-Fc fusion protein (SN 13) produced the highest secretion; followed by unmodified RBD monomers (SN 5 and SN 15) and RBD monomer (SN 14) linked to modified Fc (fig. 40A). In cell lysates, the unmodified RBD monomers as well as the nucleoprotein linked to the modified Fc (SN 15) showed the highest RBD protein levels (fig. 40A). For constructs (SN 11, 12, 13, 14 and 15), the nucleoprotein in the lysates was similar, but the secreted protein level was slightly higher in constructs comprising RBD trimer (SN 12) and RBD monomer (SN 15) (fig. 40B).

Example 18 the covd-19 specific neutralizing antibody response of SN15 pDNA delivered by gene gun was superior to the response from intact S pDNA

Balb/C and C57B1/6 mice were immunized with pDNA comprising N protein fused to modified Fc (NPFC iV 1) and RBD monomer (SN 15), RBD trimer (SN 12) or RBD monomer linked to Fc (SN 13) or with intact S pDNA. The antibody response of immunized Balb/c mice was assessed by ELISA, SN15 and SN13 showed higher titers of S1 protein-specific antibodies and total IgG compared to whole S DNA or SN11 immunized mice (fig. 41A). In analyzing neutralizing antibody responses in the pseudovirus neutralization assay, mice sera immunized with SN15 (RBD monomer and NPFCiV 1) showed higher neutralizing ID50 titers than mice sera immunized with SN12 (RBD trimer and NPFCiV 1) or SN13 (RBD FC and NPFCiV 1) (fig. 41B). The antibody responses of C57Bl/6 mice immunized with SN15 or intact S pDNA were compared in a pseudovirus neutralization assay, and mice immunized with SN15 pDNA showed higher neutralization titers than intact S pDNA (fig. 41C). This suggests that in both mouse models, a more optimal neutralizing antibody was obtained after immunization with SN15 pDNA.

Example 19 the covd-19 specific T cell response from SN15 pDNA delivered via gene gun was superior to the response from intact S pDNA

Balb/C, HLA-A2 transgenes and C57B1/6 mice were immunized with pDNA comprising N protein fused to modified Fc (NPFC iV 1) and RBD monomer (SN 15), RBD trimer (SN 12), RBD short trimer (SN 11) or RBD monomer linked to Fc (SN 13) or with intact S pDNA. T cell responses of immunized mice were assessed by ifnγ ELISpot assay, showing high frequency responses of mice immunized with SN15, SN13, SN12, and SN11 pDNA to overlapping peptide pools from RBD proteins (fig. 42 Ai). The response of SN15, SN11 and SN12 immunized mice was significantly higher than the response from SN13 immunized mice (p < 0.0001). In analyzing peptide-specific responses in HLA-A2 transgenic mice, a similar response pattern (p=0.0002) was observed (fig. 42 Aii). Comparison of Balb/C and C57B1/6 mice immunized with SN15 pDNA with mice immunized with intact S pDNA showed significantly enhanced response (p=0.0002) to overlapping peptide pools from RBD proteins with SN15 immunized mice (fig. 42B). This data provides evidence that the T cell response of the SN15 pDNA construct is superior to that of the construct encoding the complete S protein.

EXAMPLE 20Fc modified HuIgG1 constructs showed stronger CD64 binding than unmodified Fc

Targeting antigens to high affinity fcγr1 (CD 64) induces better humoral and T cell responses through a combination of enhanced antigen internalization and improved APC activation. The purified RBD construct with modified Fc showed long-term interaction with CD64, which was more pronounced at higher CD64 receptor densities, compared to the unmodified RBD-Fc construct, indicating increased avidity (fig. 43, real-time SPR binding curve). This is evident from the kinetic parameters in Table 7, where the modified Fc observed slower dissociation at higher CD64 densities (300 and 900 RU) compared to the unmodified Fc construct (kd to RBD-Fc is 10 ^-3 Compared with 1/s, the kd of RBD-iFcv1 is about 10 ^-4 1/s) and higher maximum binding (Rmax).

Table 7 kinetic constants of interaction of Fc-containing constructs (Bia T200)

Example 21 modified iFcv1 constructs in the group of 20 donors were non-immunogenic

Theoretically, the presence of residue changes in modified Fc (iFcv 1) has the ability to induce immunogenicity when clinically administered to human volunteers. Thus, this was evaluated in a study by Abzena (UK) using trastuzumabAs a comparison. To assess whether the iFcv1 fusion construct has the potential to induce a CD 4T cell response in humans (which is an important driver of immunogenicity), a trastuzumab construct "iTV1" was created comprising modified iFcv 1. In proliferation assays (3H-thymidine uptake), immunogenicity iTV1 was assessed using CD8 depleted Peripheral Blood Mononuclear Cells (PBMC) from a group of 20 donors (covering about 77% of HLA alleles) representing european and north american populations. In combination with iTV1 or wild type trastuzumab +.>And->T cell responses were assessed on days 5-8 after control incubation (fig. 44A). The modified Fc construct iTV1 produced slightly increased proliferation in the 3/20 donor; this is in contrast to the low immunogenicity of Abzena +. >The observed results (slightly increased proliferation was observed in the 2/20 donor) were comparable. Both of which have a stimulation index of 2 to2.5, far below the use of positive control KLH orThe stimulation index observed, the latter, is known to induce anti-drug antibodies in 45% of patients. Comparison of the maximal proliferative response of CD8 depleted PBMC shows iTv1 and +.>There was no significant difference between (fig. 44B), indicating that like SN15, fc modification in iTV1 is unlikely to stimulate an effective CD 4T cell response in humans.

Example 22 modified iFcv1 constructs in a group of 50 donors were non-immunogenic

In addition to studies in groups of 20 donors, replicates were performed to extend to groups of 50 donors across a wider range of HLA types. Proliferation of modified Fc construct iTV1 was slightly increased in the 3/50 donor; this is also in comparison with the low immunogenicity control using AbzenaThe observed results (slightly increased proliferation was observed in the 2/20 donor and greater increase in the 1/50 donor) were comparable (FIG. 45A). For iTV1, a stimulation index of 2 to 2.4 was observed, well below that with positive controls KLH or +.>The stimulation index observed, the latter, is known to induce anti-drug antibodies in 45% of patients. Comparison of the maximal proliferative response of CD8 depleted PBMC shows iTV1 and +. >There was no significant difference between (fig. 45B), indicating that similar to SN15, SN17, iSCIB1plus, and iSCIB2, fc modification in iTV1 is unlikely to stimulate an effective CD 4T cell response in humans.

Example 23 pDNA encoding NP, NP Fc, and NP FciV1 delivered by a gene gun elicited a COVID-19N protein-specific antibody response

Antibody reactivity to SARS-CoV-2N protein was analyzed by ELISA using mouse serum immunized with pDNA encoding NP, NP Fc, or NP FciV 1. Similar antibody responses and ECs were observed in all constructs, whether or not fused to Fc ₅₀ Values (fig. 45).

Example 24 vaccination by SN15 pDNA induces antibody responses cross-reactive with variants

Balb/c mice were immunized with pDNA comprising N protein fused to modified Fc (NPFC iV 1) and RBD monomer (SN 15) and serum antibody responses to variant S1 proteins from the Wohan (lineage A), B.1.351 and B.1.1.7 strains were assessed by ELISA. Higher titers of S1 protein-specific antibodies from lineage a, b.1.1.7 and b.1.351 variants were observed with a detectable response compared to background at serum dilutions as low as 1/100,000. No significant difference was observed in reactivity to lineage a and b.1.1.7 variant S1 proteins (fig. 49). EC was observed for the b.1.351 variant ₅₀ Reduced, but the data indicate induction of cross-reactive antibody responses to b.1.1.7 and b.1.351 variants.

Example 25 induction of antibody responses cross-reactive with variants while inhibiting ACE2 binding by SN15, SN16 and SN17 pDNA vaccination

Balb/c mice were immunized with pDNA comprising RBD monomer (SN 15), RBD monomer from the B.1.1.7 variant (SN 16, FIG. 47) or RBD monomer from the B.1.351 variant (SN 17, FIG. 48), both fused to the related variant N protein of modified Fc (NPFC iV 1). Serum was assessed for antibody responses to variant S1 proteins from the wuhan (lineage a), b.1.351 and b.1.1.7 strains by ELISA. High titers of antibodies were observed from SN17 and SN16 specific for S1 proteins from the variants of b.1.1.7 and b.1.351 of lineages a, b.1.7, with a detectable response compared to background at serum dilutions as low as 1/100,000. No significant difference in reactivity of the lineage a, b.1.351 and b.1.1.7 variant S1 proteins was observed from SN17 (fig. 50 Ai). A similar response to SN15 (fig. 49) was observed from SN16 (fig. 50 Aii). EC was observed for the b.1.351 variant ₅₀ Reduced (fig. 50 Aiii), but nonetheless, the data indicated that cross-reactive antibody responses to b.1.1.7 and b.1.351 variants were induced. From using pedigree Comparison of the serum from mice immunized with the a and b.1.351 variant vaccine constructs with the serum from mice immunized with intact S DNA showed a higher titer response to both lineage A S protein and b.1.351S1 protein (fig. 50B).

The ability of immunized mice serum to inhibit ACE2 receptor binding to variant RBD or intact S proteins was assessed using a MesoScale Discovery platform. Mice vaccinated with the original lineage a vaccine construct (SN 15) and the b.1.351 variant vaccine construct (SN 17) had a higher inhibition of RBD binding to ACE2 than the NIBSC 20/136 control (fig. 50 Ci). In contrast, serum from mice immunized with intact S DNA showed a lower ability to inhibit ACE2 binding, similar to the NIBSC 20/136 control. Mice immunized with the original lineage a vaccine construct (SN 15) inhibited 80-100% of ACE2 binding to the original lineage a RBD and b.1.1.7 variants, with 50-60% decrease for b.1.351 and p.1rbd variants. The opposite result was observed in the serum of mice immunized with the b.1.351 variant vaccine (SN 17). Although inhibition of ACE2 binding to b.1.351 and p.1rbd variants was reduced, the level of inhibition was still higher than that observed with whole S DNA immunized mouse serum. In the ACE2 receptor binding inhibition assay, a similar trend was observed with the variant intact S protein (figure 50 Cii).

EXAMPLE 26 SN15 and SN17 immunized mice serum showed virus neutralization in pseudotyped and live virus neutralization assays

Balb/c mouse sera immunized with either the original lineage a (SN 15) or b.1.351 (SN 17) variant vaccine were also evaluated in pseudotyped and live virus neutralization assays for the original lineage a and b.1.351 variants. Serum from original variant vaccine immunized mice was shown to effectively neutralize original lineage a pseudotyped, with reduced potency against the b.1.351 variant vaccine (ID ₅₀ Values 6232 and 2137, respectively) (fig. 51A). Serum from mice immunized with either vaccine showed neutralizing b.1.351 pseudotyped variants, but little difference was noted (ID ₅₀ Values 948 and 997 respectively). In live virus neutralization assays, the ID of the original lineage a virus was neutralized using mouse serum immunized with original lineage a (SN 15) or b.1.351 (SN 17) variant vaccine ₅₀ Values 4964 and 1334, respectively, and both were superior to NIBSC controlFig. 51B).

Example 27T cell response was not affected by variation of viral strains

To examine whether T cell responses were affected by mutations in different viral variants, mouse spleen cells immunized with either the original lineage a vaccine (SN 15) or the b.1.351 vaccine (SN 17) were stimulated ex vivo with RBD and N peptide libraries derived from the original sequences. T cell responses specific for RBD and N were detected with little difference between responses induced by different vaccine constructs (fig. 52). These results indicate that mutations in the b.1.351 variant have less effect on T cell responses.

Example 28 antibody response can be effectively enhanced by vaccines specific for variant and show cross-reactivity to variants B.1.351 and B.1.617.2

To test whether the antibody response against lineage a virus using the SN15 vaccine could be boosted with a vaccine targeting the b.1.351 variant (SN 17), balb/c mice were immunized with the SN15 vaccine on days 1 and 29, followed by boosting with the SN17 vaccine on day 85. Serum samples taken on days 42, 82 and 98 were tested for antibody responses to lineage a and b.1.351s1 proteins by ELISA. As a comparison, mice were immunized with SN17 vaccine alone. In both sets of sera, antibody responses were detectable at dilutions as low as 1/100,000 (fig. 53A). In both groups of mice, a decrease in response titer was observed on day 82 as compared to day 42, but for the lineage A S protein, EC ₅₀ Value retention>1/3500. In both groups of mice, boosting the needle with SN17 vaccine on day 85 effectively boosted the response to lineage A S1 protein on day 98, indicating that the SN17 vaccine effectively boosted the responses primed by SN15 and SN17 vaccines. Reduced reactivity to the b.1.351s1 protein compared to the lineage A S1 protein, with lower EC in both groups of mice ₅₀ Values, and this was more evident in mice boosted with sn15+sn17. Nevertheless, the booster needle with SN17 vaccine was able to raise the response in the SN15 vaccine priming group to similar levels observed in mice receiving SN17 priming.

The reactivity of mouse sera immunized with these prime boost regimens to RBD proteins from b.1.351 and b.1.617.2 variants was analyzed by ELISA (fig. 53B). Compared with the B.1.351S1 proteinSerum from both groups of mice showed higher antibody titers and EC against b.1.351rbd protein ₅₀ Values. Also, it was shown that on day 98, the response to b.1.351rbd protein by SN15 or SN17 primed mice was effectively boosted by SN17 boosting needles, as compared to day 82. Higher titers after boosting the needle vaccine were noted in mice primed with SN17 vaccine. On day 82, cross-reactivity analysis of RBD protein from the b.1.617.2 variant showed high titer responses using SN15 primed mouse EC ₅₀ Mouse EC with value 8355 primed with SN17 ₅₀ The value was 5738. This is more reactive than that observed for the b.1.351rbd protein in the same serum, indicating that both vaccines have good cross-reactivity to this variant. Boosting needles with SN17 vaccine resulted in titers and ECs in two groups of mice ₅₀ The values increased to similar levels, indicating that the SN17 boost needle can boost the b.1.617.2 variant cross-reactive RBD-specific response initially induced by SN15 and SN17 vaccines.

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Sequence listing

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1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

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Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

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Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

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Asp Gly Val Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln

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Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

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Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

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Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Pro Lys Gly Arg Ala

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Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser

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Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu

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Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

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Val His Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln

20 25 30

Pro Gly Gly Ser Leu Arg Met Ser Cys Gly Thr Gly Arg Ala Met Leu

35 40 45

Gly Thr His Thr Met Glu Val Thr Val Tyr His Trp Val Arg Gln Ala

50 55 60

Pro Gly Lys Gly Leu Glu Trp Ile Ala Tyr Ile Gly Ser Gly Gly Ser

65 70 75 80

Val Tyr Asp Phe Phe Val Trp Leu Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ser Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Val Pro Leu Asp Cys Val Leu Tyr

115 120 125

Arg Tyr Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Trp Gly

130 135 140

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

145 150 155 160

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

165 170 175

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

180 185 190

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

195 200 205

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

210 215 220

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

225 230 235 240

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

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Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

260 265 270

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

275 280 285

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

290 295 300

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

305 310 315 320

His Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

325 330 335

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

340 345 350

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

355 360 365

Glu Lys Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val

370 375 380

Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser

385 390 395 400

Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val Glu

405 410 415

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

420 425 430

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

435 440 445

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

450 455 460

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

465 470 475 480

Pro Gly Lys

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

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Val His Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln

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Pro Gly Gly Ser Leu Arg Met Ser Cys Leu Leu Met Trp Ile Thr Gln

35 40 45

Cys Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala

50 55 60

Tyr Ile Gly Ser Gly Gly Ser Leu Leu Met Trp Ile Thr Gln Cys Arg

65 70 75 80

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Leu

85 90 95

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Pro

100 105 110

Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro

115 120 125

Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Trp Gly Gln Gly

130 135 140

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

145 150 155 160

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

165 170 175

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

180 185 190

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

195 200 205

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

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Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

225 230 235 240

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

245 250 255

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Thr

305 310 315 320

Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr

370 375 380

Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu Thr

385 390 395 400

Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp Glu

405 410 415

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

Lys

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Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr Phe

1 5 10 15

Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser

20 25 30

Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr

35 40 45

Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys

50 55 60

Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp

65 70 75 80

Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly

85 90 95

Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu

100 105 110

Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly

115 120 125

Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His

130 135 140

Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu

145 150 155 160

Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg

165 170 175

Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser

180 185 190

Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg

195 200 205

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

210 215 220

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

225 230 235 240

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

245 250 255

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

260 265 270

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

275 280 285

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

290 295 300

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

305 310 315 320

Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile

325 330 335

Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu

340 345 350

Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys

355 360 365

Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg

370 375 380

Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp

385 390 395 400

Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr

405 410 415

Gln Ala

<210> 5

<211> 418

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Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr Phe

1 5 10 15

Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser

20 25 30

Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr

35 40 45

Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys

50 55 60

Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp

65 70 75 80

Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly

85 90 95

Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu

100 105 110

Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly

115 120 125

Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His

130 135 140

Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu

145 150 155 160

Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg

165 170 175

Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser

180 185 190

Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Ile Ser Pro Ala Arg

195 200 205

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

210 215 220

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

225 230 235 240

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

245 250 255

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

260 265 270

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

275 280 285

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

290 295 300

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

305 310 315 320

Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile

325 330 335

Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu

340 345 350

Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys

355 360 365

Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg

370 375 380

Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp

385 390 395 400

Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr

405 410 415

Gln Ala

<210> 6

<211> 669

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<400> 6

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg

20 25 30

Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly

35 40 45

Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro

50 55 60

Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu

65 70 75 80

Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser

85 90 95

Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile

100 105 110

Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe

115 120 125

Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn

130 135 140

Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro

145 150 155 160

Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val

165 170 175

Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu

180 185 190

Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser

195 200 205

Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser

210 215 220

Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu

225 230 235 240

Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly

245 250 255

Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala

260 265 270

Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val

275 280 285

Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe

290 295 300

Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro

305 310 315 320

Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser

325 330 335

Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr

340 345 350

Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val

355 360 365

Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr

370 375 380

Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu

385 390 395 400

Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala

405 410 415

Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala

420 425 430

Asp Ser Thr Gln Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

435 440 445

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

450 455 460

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

465 470 475 480

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

485 490 495

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln

500 505 510

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

515 520 525

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

530 535 540

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

545 550 555 560

Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro

565 570 575

Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr

580 585 590

Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln

595 600 605

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

610 615 620

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

625 630 635 640

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

645 650 655

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 7

<211> 669

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 7

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg

20 25 30

Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly

35 40 45

Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro

50 55 60

Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu

65 70 75 80

Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser

85 90 95

Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile

100 105 110

Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe

115 120 125

Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn

130 135 140

Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro

145 150 155 160

Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val

165 170 175

Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu

180 185 190

Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser

195 200 205

Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Ile Ser

210 215 220

Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu

225 230 235 240

Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly

245 250 255

Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala

260 265 270

Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val

275 280 285

Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe

290 295 300

Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro

305 310 315 320

Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser

325 330 335

Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr

340 345 350

Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val

355 360 365

Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr

370 375 380

Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu

385 390 395 400

Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala

405 410 415

Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala

420 425 430

Asp Ser Thr Gln Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

435 440 445

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

450 455 460

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

465 470 475 480

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

485 490 495

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln

500 505 510

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

515 520 525

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

530 535 540

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

545 550 555 560

Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro

565 570 575

Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr

580 585 590

Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln

595 600 605

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

610 615 620

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

625 630 635 640

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

645 650 655

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 8

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 8

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 9

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 9

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 10

<211> 283

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 10

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Trp Asn Arg Gln Leu Tyr

35 40 45

Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Ala Asn Cys Ser Val Tyr Asp

65 70 75 80

Phe Phe Val Trp Leu His Tyr Tyr Ser Val Arg Asp Thr Leu Leu Gly

85 90 95

Pro Gly Arg Pro Tyr Arg Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

100 105 110

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

115 120 125

Thr Gly Val Tyr Tyr Cys Gln Cys Thr Glu Val Arg Ala Asp Thr Arg

130 135 140

Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln Asp Asp Arg Glu Leu

145 150 155 160

Trp Pro Arg Lys Phe Phe Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

165 170 175

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

180 185 190

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

195 200 205

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

210 215 220

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

225 230 235 240

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

245 250 255

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

260 265 270

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

275 280

<210> 11

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 11

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Pro Gly Val Leu Leu Lys

35 40 45

Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala

50 55 60

Asp His Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu

65 70 75 80

Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser

85 90 95

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

100 105 110

Ala Glu Asp Thr Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro

115 120 125

Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

130 135 140

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

145 150 155 160

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

165 170 175

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

180 185 190

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

195 200 205

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

210 215 220

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

225 230 235 240

Ser Phe Asn Arg Gly Glu Cys

245

<210> 12

<211> 2454

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 12

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctggctagcg tttaaactta agcttaccat gggatggagc tgtatcatcc tcttcttggt 720

agcaacagct accggagtcc actcccaggt gcagctggtg gagactgggg gaggcttaat 780

ccagcctgga gggtccctga gaatgtcctg cgggacaggc agggcaatgc tgggcacaca 840

caccatggaa gtgactgtct accattgggt tcggcaggct ccggggaagg ggctggagtg 900

gatcgcatac attggtagtg gtggtagtgt ttatgatttt tttgtgtggc tccgattcac 960

catttccaga gacaatagca agaacaccct gtatttgcaa ttgaacagtc tgagggctga 1020

ggacacagcc gtgtattact gtgcccgagt ccccctggat tgtgttctgt atcgatatgg 1080

ttccttttcc gtcaccctgg acattgtcca gggttggggc caaggaacca cggtcaccgt 1140

ctccagcgct tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 1200

ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac 1260

ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 1320

gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 1380

ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt 1440

tgagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct 1500

ggggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg 1560

gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 1620

caactggtac gtggacggcg tggaggtgca tacagcctgg acacagccgc gggaggagca 1680

gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 1740

tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac 1800

catctccaaa cccaaaggga gagcccagac accacaggtg tacaccatac ccccacctcg 1860

ggaacaaatg tccaagaaga aggtcagcct gacctgcctg gtcaccaact tcttctctga 1920

agccatcagt gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc 1980

tcccgtgctg gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag 2040

caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca 2100

ctacacgcag aagagcctct ccctgtctcc gggtaaatga tctaaagggc gatatcgccc 2160

ttaagggcga attctgcaga ttcgagcatg catctagagg gcccgtttaa acccgctgat 2220

cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 2280

ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 2340

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 2400

gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgg 2454

<210> 13

<211> 1836

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 13

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctagcttggt accgagctcg gatccaccat gggatggagc tgtatcatcc tcttcttggt 720

agcaacagct accggagtcc actccgatgt gttgatgacc caatctccac tctccctgcc 780

tgtcactcct ggggagccag cctcgatctc ttgctggaac aggcagctgt atccagagtg 840

gacagaagcc cagagacttg actggtatct gcagaaacca ggccagtctc cacagctcct 900

gatctacgcc aactgcagtg tttatgattt ttttgtgtgg ctccattatt attctgttag 960

agatacatta ttaggaccag gacgccccta caggggggtc ccagacagat tcagtggcag 1020

tggatcaggg acagatttca cactcaagat cagcagagtg gaggctgagg ataccggagt 1080

gtattactgc cagtgcacag aggtgcgagc cgacacaagg ccctggagtg gtccctacat 1140

cctacgaaac caggatgacc gtgagctgtg gccaagaaaa ttcttcttcg gtggaggcac 1200

caaggtggaa atcaagcgta cggtagcggc cccatctgtc ttcatcttcc cgccatctga 1260

tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact tctatcccag 1320

agaggccaaa gtacagtgga aggtggataa cgccctccaa tcgggtaact cccaggagag 1380

tgtcacagag caggacagca aggacagcac ctacagcctc agcagcaccc tgacgctgag 1440

caaagcagac tacgagaaac acaaagtcta cgcctgcgaa gtcacccacc agggcctgag 1500

ctcgcccgtc acaaagagct tcaacagggg agagtgttga ctcgagcatg catctagagg 1560

gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt 1620

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 1680

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 1740

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 1800

atagcaggca tgctggggat gcggtgggct ctatgg 1836

<210> 14

<211> 2448

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 14

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctggctagcg tttaaactta agcttaccat gggatggagc tgtatcatcc tcttcttggt 720

agcaacagct accggagtcc actcccaggt gcagctggtg gagactgggg gaggcttaat 780

ccagcctgga gggtccctga gaatgtcctg cctgttgatg tggatcacgc agtgcttttg 840

ggttcggcag gctccgggga aggggctgga gtggatcgca tacattggta gtggtggttc 900

cctgttgatg tggatcacgc agtgccgatt caccatttcc agagacaata gcaagaacac 960

cctgtatttg caattgaaca gtctgagggc tgaggacaca gccgtgtatt actgtgcccg 1020

accggagagc cgcctgcttg agttctacct cgccatgcct ttcgcgacac ccatggaagc 1080

agagctggcc cgcaggagcc tggcccagtg gggccaagga accacggtca ccgtctccag 1140

cgcttccacc aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg 1200

gggcacagcg gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc 1260

gtggaactca ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc 1320

aggactctac tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac 1380

ctacatctgc aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc 1440

caaatcttgt gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg 1500

accgtcagtc ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc 1560

tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg 1620

gtacgtggac ggcgtggagg tgcatacagc ctggacacag ccgcgggagg agcagtacaa 1680

cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa 1740

ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc 1800

caaacccaaa gggagagccc agacaccaca ggtgtacacc atacccccac ctcgggaaca 1860

aatgtccaag aagaaggtca gcctgacctg cctggtcacc aacttcttct ctgaagccat 1920

cagtgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt 1980

gctggactcc gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg 2040

gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac 2100

gcagaagagc ctctccctgt ctccgggtaa atgatctaaa gggcgatatc gcccttaagg 2160

gcgaattctg cagattcgag catgcatcta gagggcccgt ttaaacccgc tgatcagcct 2220

cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 2280

ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 2340

gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 2400

attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 2448

<210> 15

<211> 1728

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 15

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctagcttggt accgagctcg gatccaccat gggatggagc tgtatcatcc tcttcttggt 720

agcaacagct accggagtcc actccgatgt gttgatgacc caatctccac tctccctgcc 780

tgtcactcct ggggagccag cctcgatctc ttgcccaggg gtgcttctga aggagttcac 840

tgtgtccggc aacatactga ctatccgact gactgctgca gaccaccgct ggtatctgca 900

gaaaccaggc cagtctccac agctcctgat ctacaaagtt tccaaccgat tttctggggt 960

cccagacaga ttcagtggca gtggatcagg gacagatttc acactcaaga tcagcagagt 1020

ggaggctgag gataccggag tgtattactg ctttcaaggt tcacatgttc cgtggacgtt 1080

cggtggaggc accaaggtgg aaatcaagcg tacggtagcg gccccatctg tcttcatctt 1140

cccgccatct gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa 1200

cttctatccc agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa 1260

ctcccaggag agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac 1320

cctgacgctg agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca 1380

ccagggcctg agctcgcccg tcacaaagag cttcaacagg ggagagtgtt gactcgagca 1440

tgcatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct 1500

cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 1560

ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 1620

gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 1680

attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 1728

<210> 16

<211> 3018

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 16

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctggctagcg tttaaactta agcttgccac catggactgg atttggagaa tcctcttcct 720

ggtgggagct gccacaggcg cacacagcag cgacaacggc ccccagaacc agagaaacgc 780

ccccagaatc accttcggcg gccccagcga cagcaccggc agcaaccaga acggcgagag 840

aagcggcgcc agaagcaagc agagaagacc ccagggcctg cccaacaaca ccgccagctg 900

gttcaccgcc ctgacccagc acggcaagga ggacctgaag ttccccagag gccagggcgt 960

gcccatcaac accaacagca gccccgacga ccagatcggc tactacagaa gagccaccag 1020

aagaatcaga ggcggcgacg gcaagatgaa ggacctgagc cccagatggt acttctacta 1080

cctgggcacc ggccccgagg ccggcctgcc ctacggcgcc aacaaggacg gcatcatctg 1140

ggtggccacc gagggcgccc tgaacacccc caaggaccac atcggcacca gaaaccccgc 1200

caacaacgcc gccatcgtgc tccagctgcc ccagggcacc accctgccca agggcttcta 1260

cgccgagggc agcagaggcg gcagccaggc ctcaagccgt tccagcagca gatcccgcaa 1320

cagctcccgc aacagcaccc ctggaagctc aaggggcacc tctcctgcgc ggatggctgg 1380

taacggcggg gacgctgccc tggccctgct gctgctggac agactgaacc agctggagag 1440

caagatgagc ggcaagggcc agcagcagca gggccagact gtgaccaaaa aaagcgccgc 1500

cgaggccagc aagaagccca gacagaagag aaccgccacc aaggcctaca acgtgaccca 1560

ggccttcggc agaagaggcc ccgagcagac ccagggcaac ttcggcgacc aggagctgat 1620

cagacagggc accgactaca agcactggcc ccagatcgcc cagttcgccc ccagcgccag 1680

cgccttcttc ggcatgagca gaatcggcat ggaggtgacc cccagcggca cctggctgac 1740

ctacaccggc gccatcaagc tggacgacaa ggaccccaac ttcaaggatc aggtgatcct 1800

gctgaacaag cacatcgacg cctacaagac cttccccccc accgagccca agaaggacaa 1860

gaagaagaag gccgacgaga cccaggccct gccccagaga cagaagaagc agcagacagt 1920

gacattgctg cccgccgccg acctggacga cttcagcaag cagctccagc agagcatgag 1980

cagcgccgac agcacccagg ccgagcccaa atcgtgcgat aaaacgcata cgtgcccccc 2040

ctgtccagcc ccagagttgc tgggtggacc gagcgtgttc ctgttccccc ccaagcccaa 2100

ggacaccctg atgatcagca gaacccccga ggtgacctgc gtggtggtgg acgtgagcca 2160

cgaggacccc gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acacagcctg 2220

gacccagccc agagaggagc agtacaacag cacctacaga gttgtgtccg tgctgaccgt 2280

gctgcaccag gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct 2340

gcccgccccc atcgagaaga ccatcagcaa gcccaagggc agagcccaga caccccaggt 2400

gtacaccata ccccccccta gagaacaaat gtccaagaag aaggtgagcc tgacctgcct 2460

ggtgaccaac ttcttctctg aagccatcag tgtggagtgg gagagcaacg gccagcccga 2520

gaacaactac aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctctacag 2580

caagctgacc gtggacaaga gcagatggca gcagggcaac gtgttctctt gcagcgtgat 2640

gcacgaggcc ctgcacaacc actacaccca gaagagcctg agcctgagcc ccggcaagtg 2700

atgatctaaa gggcgaattc gcccttaagg gctaattctg cagattcgag catgcatcta 2760

gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 2820

ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 2880

cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 2940

gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 3000

atgcggtggg ctctatgg 3018

<210> 17

<211> 1719

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 17

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctagcttggt accgagctcg gatccgccac catggactgg atttggagaa tcctcttcct 720

ggtgggagct gccacaggcg cacacagcag agtgcagccc accgagagca tcgtgagatt 780

ccccaacatc accaacctgt gccccttcgg cgaggtgttc aacgccacca gattcgccag 840

cgtgtacgcc tggaacagga agagaatcag caactgcgtg gccgactaca gcgtgctcta 900

caacagcgcc agcttcagca ccttcaagtg ctacggcgtg agccccacca agctgaacga 960

cctgtgcttc accaacgtgt acgccgacag cttcgtgatc agaggcgacg aggtgagaca 1020

gatcgccccc ggccagaccg gcaagatcgc cgactacaac tacaagctgc ccgacgactt 1080

caccggctgc gtgatcgcgt ggaacagcaa taacctggac agcaaggtgg gcggcaacta 1140

caactacctc tacagactgt tcagaaagag caacctgaag cccttcgaga gagacatcag 1200

caccgagatc taccaggccg gcagcacccc ctgcaacggc gtggagggct tcaactgcta 1260

cttccccctc cagagctacg gcttccagcc caccaacggc gtgggctacc agccctacag 1320

agtggtggtc ctgagcttcg agctgctgca cgcccccgcc accgtgtgcg gccccaagaa 1380

gagcaccaat ctggtgaaga acaagtgcgt gaacttctga tgactcgagc atgcatctag 1440

agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc 1500

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 1560

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 1620

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 1680

acaatagcag gcatgctggg gatgcggtgg gctctatgg 1719

<210> 18

<211> 3018

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 18

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctggctagcg tttaaactta agcttgccac catggactgg atttggagaa tcctcttcct 720

ggtgggagct gccacaggcg cacacagcag cgacaacggc ccccagaacc agagaaacgc 780

ccccagaatc accttcggcg gccccagcga cagcaccggc agcaaccaga acggcgagag 840

aagcggcgcc agaagcaagc agagaagacc ccagggcctg cccaacaaca ccgccagctg 900

gttcaccgcc ctgacccagc acggcaagga ggacctgaag ttccccagag gccagggcgt 960

gcccatcaac accaacagca gccccgacga ccagatcggc tactacagaa gagccaccag 1020

aagaatcaga ggcggcgacg gcaagatgaa ggacctgagc cccagatggt acttctacta 1080

cctgggcacc ggccccgagg ccggcctgcc ctacggcgcc aacaaggacg gcatcatctg 1140

ggtggccacc gagggcgccc tgaacacccc caaggaccac atcggcacca gaaaccccgc 1200

caacaacgcc gccatcgtgc tccagctgcc ccagggcacc accctgccca agggcttcta 1260

cgccgagggc agcagaggcg gcagccaggc ctcaagccgt tccagcagca gatcccgcaa 1320

cagctcccgc aacagcaccc ctggaagctc aaggggcatc tctcctgcgc ggatggctgg 1380

taacggcggg gacgctgccc tggccctgct gctgctggac agactgaacc agctggagag 1440

caagatgagc ggcaagggcc agcagcagca gggccagact gtgaccaaaa aaagcgccgc 1500

cgaggccagc aagaagccca gacagaagag aaccgccacc aaggcctaca acgtgaccca 1560

ggccttcggc agaagaggcc ccgagcagac ccagggcaac ttcggcgacc aggagctgat 1620

cagacagggc accgactaca agcactggcc ccagatcgcc cagttcgccc ccagcgccag 1680

cgccttcttc ggcatgagca gaatcggcat ggaggtgacc cccagcggca cctggctgac 1740

ctacaccggc gccatcaagc tggacgacaa ggaccccaac ttcaaggatc aggtgatcct 1800

gctgaacaag cacatcgacg cctacaagac cttccccccc accgagccca agaaggacaa 1860

gaagaagaag gccgacgaga cccaggccct gccccagaga cagaagaagc agcagacagt 1920

gacattgctg cccgccgccg acctggacga cttcagcaag cagctccagc agagcatgag 1980

cagcgccgac agcacccagg ccgagcccaa atcgtgcgat aaaacgcata cgtgcccccc 2040

ctgtccagcc ccagagttgc tgggtggacc gagcgtgttc ctgttccccc ccaagcccaa 2100

ggacaccctg atgatcagca gaacccccga ggtgacctgc gtggtggtgg acgtgagcca 2160

cgaggacccc gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acacagcctg 2220

gacccagccc agagaggagc agtacaacag cacctacaga gttgtgtccg tgctgaccgt 2280

gctgcaccag gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct 2340

gcccgccccc atcgagaaga ccatcagcaa gcccaagggc agagcccaga caccccaggt 2400

gtacaccata ccccccccta gagaacaaat gtccaagaag aaggtgagcc tgacctgcct 2460

ggtgaccaac ttcttctctg aagccatcag tgtggagtgg gagagcaacg gccagcccga 2520

gaacaactac aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctctacag 2580

caagctgacc gtggacaaga gcagatggca gcagggcaac gtgttctctt gcagcgtgat 2640

gcacgaggcc ctgcacaacc actacaccca gaagagcctg agcctgagcc ccggcaagtg 2700

atgatctaaa gggcgaattc gcccttaagg gctaattctg cagattcgag catgcatcta 2760

gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg 2820

ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt 2880

cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg 2940

gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg 3000

atgcggtggg ctctatgg 3018

<210> 19

<211> 1719

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 19

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600

gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag 660

ctagcttggt accgagctcg gatccgccac catggactgg atttggagaa tcctcttcct 720

ggtgggagct gccacaggcg cacacagcag agtgcagccc accgagagca tcgtgagatt 780

ccccaacatc accaacctgt gccccttcgg cgaggtgttc aacgccacca gattcgccag 840

cgtgtacgcc tggaacagga agagaatcag caactgcgtg gccgactaca gcgtgctcta 900

caacagcgcc agcttcagca ccttcaagtg ctacggcgtg agccccacca agctgaacga 960

cctgtgcttc accaacgtgt acgccgacag cttcgtgatc agaggcgacg aggtgagaca 1020

gatcgccccc ggccagaccg gcaatatcgc cgactacaac tacaagctgc ccgacgactt 1080

caccggctgc gtgatcgcgt ggaacagcaa taacctggac agcaaggtgg gcggcaacta 1140

caactacctc tacagactgt tcagaaagag caacctgaag cccttcgaga gagacatcag 1200

caccgagatc taccaggccg gcagcacccc ctgcaacggc gtgaagggct tcaactgcta 1260

cttccccctc cagagctacg gcttccagcc cacctacggc gtgggctacc agccctacag 1320

agtggtggtc ctgagcttcg agctgctgca cgcccccgcc accgtgtgcg gccccaagaa 1380

gagcaccaat ctggtgaaga acaagtgcgt gaacttctga tgactcgagc atgcatctag 1440

agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc 1500

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 1560

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 1620

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 1680

acaatagcag gcatgctggg gatgcggtgg gctctatgg 1719

<210> 20

<211> 6321

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 20

gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctagcttg gtaccgagct cggatccacc 720

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctaccggagt ccactccgat 780

gtgttgatga cccaatctcc actctccctg cctgtcactc ctggggagcc agcctcgatc 840

tcttgctgga acaggcagct gtatccagag tggacagaag cccagagact tgactggtat 900

ctgcagaaac caggccagtc tccacagctc ctgatctacg ccaactgcag tgtttatgat 960

ttttttgtgt ggctccatta ttattctgtt agagatacat tattaggacc aggacgcccc 1020

tacagggggg tcccagacag attcagtggc agtggatcag ggacagattt cacactcaag 1080

atcagcagag tggaggctga ggataccgga gtgtattact gccagtgcac agaggtgcga 1140

gccgacacaa ggccctggag tggtccctac atcctacgaa accaggatga ccgtgagctg 1200

tggccaagaa aattcttctt cggtggaggc accaaggtgg aaatcaagcg tacggtagcg 1260

gccccatctg tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct 1320

gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat 1380

aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc 1440

acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 1500

tacgcctgcg aagtcaccca ccagggcctg agctcgcccg tcacaaagag cttcaacagg 1560

ggagagtgtt gactcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc 1620

tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 1680

ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 1740

tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 1800

gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 1860

ctctatggct tctgaggcgg aaagaaccag gctagcgttc gatgtacggg ccagatatac 1920

gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 1980

tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 2040

gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 2100

agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 2160

acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 2220

cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 2280

cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 2340

atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 2400

gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 2460

gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac 2520

tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca 2580

agctggctag cgtttaaact taagcttacc atgggatgga gctgtatcat cctcttcttg 2640

gtagcaacag ctaccggagt ccactcccag gtgcagctgg tggagactgg gggaggctta 2700

atccagcctg gagggtccct gagaatgtcc tgcgggacag gcagggcaat gctgggcaca 2760

cacaccatgg aagtgactgt ctaccattgg gttcggcagg ctccggggaa ggggctggag 2820

tggatcgcat acattggtag tggtggtagt gtttatgatt tttttgtgtg gctccgattc 2880

accatttcca gagacaatag caagaacacc ctgtatttgc aattgaacag tctgagggct 2940

gaggacacag ccgtgtatta ctgtgcccga gtccccctgg attgtgttct gtatcgatat 3000

ggttcctttt ccgtcaccct ggacattgtc cagggttggg gccaaggaac cacggtcacc 3060

gtctccagcg cttccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 3120

acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 3180

acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 3240

cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 3300

acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 3360

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 3420

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 3480

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 3540

ttcaactggt acgtggacgg cgtggaggtg catacagcct ggacacagcc gcgggaggag 3600

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 3660

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 3720

accatctcca aacccaaagg gagagcccag acaccacagg tgtacaccat acccccacct 3780

cgggaacaaa tgtccaagaa gaaggtcagc ctgacctgcc tggtcaccaa cttcttctct 3840

gaagccatca gtgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 3900

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 3960

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 4020

cactacacgc agaagagcct ctccctgtct ccgggtaaat gatctaaagg gcgatatcgc 4080

ccttaagggc gaattctgca gattcgagca tgcatctaga gggcccgttt aaacccgctg 4140

atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 4200

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 4260

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 4320

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 4380

tactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt 4440

aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg 4500

cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa 4560

gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 4620

gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 4680

ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca 4740

gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 4800

actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 4860

tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 4920

acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 4980

cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 5040

ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc 5100

gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 5160

ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 5220

acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 5280

ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 5340

tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt 5400

atttcacacc gcatcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 5460

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5520

tcaataatag cacgtgctaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 5580

ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 5640

ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 5700

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 5760

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 5820

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 5880

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 5940

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 6000

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 6060

atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 6120

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 6180

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 6240

gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 6300

gccttttgct cacatgttct t 6321

<210> 21

<211> 5071

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 21

gcgtataatg gactattgtg tgctgatatg tacacctgag gacacaggta caggactcag 60

cttaaggcta catcttagct tacgtcacta gagggtccac gtttagtttt taagatccat 120

tgatctccta aacgctgcaa gattcgcaac ctggaatact tagcgctagg tcctagtgaa 180

gcgggacttt ttttctaaag tcgttgagag gaggagtcgt cagaccagat agctttgatg 240

tcctgatcgg aaggatcgtt ggccccgatg tacgggccag atatacgcgt tgacattgat 300

tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 360

agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 420

gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 480

gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 540

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 600

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 660

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 720

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 780

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 840

ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg 900

cttactggct tatcgaaatt aatacgactc actataggga gacccaagct agcttggtac 960

cgagctcgga tccaccatgg gatggagctg tatcatcctc ttcttggtag caacagctac 1020

cggagtccac tccgatgtgt tgatgaccca atctccactc tccctgcctg tcactcctgg 1080

ggagccagcc tcgatctctt gctggaacag gcagctgtat ccagagtgga cagaagccca 1140

gagacttgac tggtatctgc agaaaccagg ccagtctcca cagctcctga tctacgccaa 1200

ctgcagtgtt tatgattttt ttgtgtggct ccattattat tctgttagag atacattatt 1260

aggaccagga cgcccctaca ggggggtccc agacagattc agtggcagtg gatcagggac 1320

agatttcaca ctcaagatca gcagagtgga ggctgaggat accggagtgt attactgcca 1380

gtgcacagag gtgcgagccg acacaaggcc ctggagtggt ccctacatcc tacgaaacca 1440

ggatgaccgt gagctgtggc caagaaaatt cttcttcggt ggaggcacca aggtggaaat 1500

caagcgtacg gtagcggccc catctgtctt catcttcccg ccatctgatg agcagttgaa 1560

atctggaact gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt 1620

acagtggaag gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca 1680

ggacagcaag gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta 1740

cgagaaacac aaagtctacg cctgcgaagt cacccaccag ggcctgagct cgcccgtcac 1800

aaagagcttc aacaggggag agtgttgact cgagcatgca tcaagagggc cctattctat 1860

agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc 1920

catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 1980

tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2040

tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2100

ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccaggcta gcgttcgatg 2160

tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta 2220

cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 2280

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 2340

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 2400

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 2460

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 2520

cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 2580

acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 2640

acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 2700

actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 2760

gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc 2820

actataggga gacccaagct ggctagcgtt taaacttaag cttaccatgg gatggagctg 2880

tatcatcctc ttcttggtag caacagctac cggagtccac tcccaggtgc agctggtgga 2940

gactggggga ggcttaatcc agcctggagg gtccctgaga atgtcctgcg ggacaggcag 3000

ggcaatgctg ggcacacaca ccatggaagt gactgtctac cattgggttc ggcaggctcc 3060

ggggaagggg ctggagtgga tcgcatacat tggtagtggt ggtagtgttt atgatttttt 3120

tgtgtggctc cgattcacca tttccagaga caatagcaag aacaccctgt atttgcaatt 3180

gaacagtctg agggctgagg acacagccgt gtattactgt gcccgagtcc ccctggattg 3240

tgttctgtat cgatatggtt ccttttccgt caccctggac attgtccagg gttggggcca 3300

aggaaccacg gtcaccgtct ccagcgcttc caccaagggc ccatcggtct tccccctggc 3360

accctcctcc aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta 3420

cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac 3480

cttcccggct gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 3540

ctccagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac 3600

caaggtggac aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg 3660

cccagcacct gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga 3720

caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga 3780

agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata cagcctggac 3840

acagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct 3900

gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc 3960

agcccccatc gagaaaacca tctccaaacc caaagggaga gcccagacac cacaggtgta 4020

caccataccc ccacctcggg aacaaatgtc caagaagaag gtcagcctga cctgcctggt 4080

caccaacttc ttctctgaag ccatcagtgt ggagtgggag agcaatgggc agccggagaa 4140

caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa 4200

gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca 4260

tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatgatc 4320

taaagggcga tatcgccctt aagggcgaat tctgcagatt cgagcatgca tatagagggc 4380

ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 4440

gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 4500

aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 4560

tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 4620

tgggctctat ggcttctact gggcggtttt atggacagca agcgaaccgg aattgccagc 4680

tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 4740

gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gagccgaccc 4800

ttagactctg tactcagttc tataaacgag ccattggata cgagatccgt agattgataa 4860

gggacacgga atatccccgg acgcaataga cggacagctt ggtatcctga gcacagtcgc 4920

gcgtccgaat ctagctctac tttagaggcc ccggattctg atggtcgtag accgcagaac 4980

cgattggagg gatgctagca ctagtgaggt gagattgtgt tcggcatgcc tcacaggcag 5040

atctatcagc acacaattgc ccattatacg c 5071

<210> 22

<211> 6207

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 22

gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctagcttg gtaccgagct cggatccacc 720

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctaccggagt ccactccgat 780

gtgttgatga cccaatctcc actctccctg cctgtcactc ctggggagcc agcctcgatc 840

tcttgcccag gggtgcttct gaaggagttc actgtgtccg gcaacatact gactatccga 900

ctgactgctg cagaccaccg ctggtatctg cagaaaccag gccagtctcc acagctcctg 960

atctacaaag tttccaaccg attttctggg gtcccagaca gattcagtgg cagtggatca 1020

gggacagatt tcacactcaa gatcagcaga gtggaggctg aggataccgg agtgtattac 1080

tgctttcaag gttcacatgt tccgtggacg ttcggtggag gcaccaaggt ggaaatcaag 1140

cgtacggtag cggccccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 1200

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 1260

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 1320

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 1380

aaacacaaag tctacgcctg cgaagtcacc caccagggcc tgagctcgcc cgtcacaaag 1440

agcttcaaca ggggagagtg ttgactcgag catgcatcta gagggcccta ttctatagtg 1500

tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 1560

tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 1620

ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 1680

gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 1740

ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc aggctagcgt tcgatgtacg 1800

ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg 1860

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 1920

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 1980

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 2040

ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 2100

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 2160

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 2220

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 2280

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 2340

cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 2400

tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta 2460

tagggagacc caagctggct agcgtttaaa cttaagctta ccatgggatg gagctgtatc 2520

atcctcttct tggtagcaac agctaccgga gtccactccc aggtgcagct ggtggagact 2580

gggggaggct taatccagcc tggagggtcc ctgagaatgt cctgcctgtt gatgtggatc 2640

acgcagtgct tttgggttcg gcaggctccg gggaaggggc tggagtggat cgcatacatt 2700

ggtagtggtg gttccctgtt gatgtggatc acgcagtgcc gattcaccat ttccagagac 2760

aatagcaaga acaccctgta tttgcaattg aacagtctga gggctgagga cacagccgtg 2820

tattactgtg cccgaccgga gagccgcctg cttgagttct acctcgccat gcctttcgcg 2880

acacccatgg aagcagagct ggcccgcagg agcctggccc agtggggcca aggaaccacg 2940

gtcaccgtct ccagcgcttc caccaagggc ccatcggtct tccccctggc accctcctcc 3000

aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 3060

ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 3120

gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 3180

ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 3240

aagaaagttg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 3300

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 3360

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 3420

gtcaagttca actggtacgt ggacggcgtg gaggtgcata cagcctggac acagccgcgg 3480

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 3540

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 3600

gagaaaacca tctccaaacc caaagggaga gcccagacac cacaggtgta caccataccc 3660

ccacctcggg aacaaatgtc caagaagaag gtcagcctga cctgcctggt caccaacttc 3720

ttctctgaag ccatcagtgt ggagtgggag agcaatgggc agccggagaa caactacaag 3780

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 3840

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 3900

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatgatc taaagggcga 3960

tatcgccctt aagggcgaat tctgcagatt cgagcatgca tctagagggc ccgtttaaac 4020

ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 4080

cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 4140

aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 4200

cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 4260

ggcttctact gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc 4320

tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc 4380

tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt 4440

gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat 4500

gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag 4560

gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac 4620

gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 4680

gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 4740

ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 4800

ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag 4860

cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat 4920

caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag 4980

gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 5040

ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 5100

ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg 5160

ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 5220

ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg 5280

tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat 5340

ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 5400

aatgcttcaa taatagcacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa 5460

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 5520

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 5580

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 5640

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 5700

tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 5760

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 5820

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 5880

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 5940

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 6000

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 6060

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 6120

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 6180

ttgctggcct tttgctcaca tgttctt 6207

<210> 23

<211> 6768

<212> DNA

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 23

gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctagcttg gtaccgagct cggatccgcc 720

accatggact ggatttggag aatcctcttc ctggtgggag ctgccacagg cgcacacagc 780

agagtgcagc ccaccgagag catcgtgaga ttccccaaca tcaccaacct gtgccccttc 840

ggcgaggtgt tcaacgccac cagattcgcc agcgtgtacg cctggaacag gaagagaatc 900

agcaactgcg tggccgacta cagcgtgctc tacaacagcg ccagcttcag caccttcaag 960

tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 1020

agcttcgtga tcagaggcga cgaggtgaga cagatcgccc ccggccagac cggcaagatc 1080

gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc gtggaacagc 1140

aataacctgg acagcaaggt gggcggcaac tacaactacc tctacagact gttcagaaag 1200

agcaacctga agcccttcga gagagacatc agcaccgaga tctaccaggc cggcagcacc 1260

ccctgcaacg gcgtggaggg cttcaactgc tacttccccc tccagagcta cggcttccag 1320

cccaccaacg gcgtgggcta ccagccctac agagtggtgg tcctgagctt cgagctgctg 1380

cacgcccccg ccaccgtgtg cggccccaag aagagcacca atctggtgaa gaacaagtgc 1440

gtgaacttct gatgactcga gcatgcatct agagggccct attctatagt gtcacctaaa 1500

tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 1560

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 1620

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 1680

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 1740

gggctctatg gcttctgagg cggaaagaac caggctagcg ttcgatgtac gggccagata 1800

tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 1860

tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 1920

accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 1980

aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 2040

agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 2100

gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 2160

ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 2220

tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 2280

tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 2340

gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct 2400

aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac 2460

ccaagctggc tagcgtttaa acttaagctt gccaccatgg actggatttg gagaatcctc 2520

ttcctggtgg gagctgccac aggcgcacac agcagcgaca acggccccca gaaccagaga 2580

aacgccccca gaatcacctt cggcggcccc agcgacagca ccggcagcaa ccagaacggc 2640

gagagaagcg gcgccagaag caagcagaga agaccccagg gcctgcccaa caacaccgcc 2700

agctggttca ccgccctgac ccagcacggc aaggaggacc tgaagttccc cagaggccag 2760

ggcgtgccca tcaacaccaa cagcagcccc gacgaccaga tcggctacta cagaagagcc 2820

accagaagaa tcagaggcgg cgacggcaag atgaaggacc tgagccccag atggtacttc 2880

tactacctgg gcaccggccc cgaggccggc ctgccctacg gcgccaacaa ggacggcatc 2940

atctgggtgg ccaccgaggg cgccctgaac acccccaagg accacatcgg caccagaaac 3000

cccgccaaca acgccgccat cgtgctccag ctgccccagg gcaccaccct gcccaagggc 3060

ttctacgccg agggcagcag aggcggcagc caggcctcaa gccgttccag cagcagatcc 3120

cgcaacagct cccgcaacag cacccctgga agctcaaggg gcacctctcc tgcgcggatg 3180

gctggtaacg gcggggacgc tgccctggcc ctgctgctgc tggacagact gaaccagctg 3240

gagagcaaga tgagcggcaa gggccagcag cagcagggcc agactgtgac caaaaaaagc 3300

gccgccgagg ccagcaagaa gcccagacag aagagaaccg ccaccaaggc ctacaacgtg 3360

acccaggcct tcggcagaag aggccccgag cagacccagg gcaacttcgg cgaccaggag 3420

ctgatcagac agggcaccga ctacaagcac tggccccaga tcgcccagtt cgcccccagc 3480

gccagcgcct tcttcggcat gagcagaatc ggcatggagg tgacccccag cggcacctgg 3540

ctgacctaca ccggcgccat caagctggac gacaaggacc ccaacttcaa ggatcaggtg 3600

atcctgctga acaagcacat cgacgcctac aagaccttcc cccccaccga gcccaagaag 3660

gacaagaaga agaaggccga cgagacccag gccctgcccc agagacagaa gaagcagcag 3720

acagtgacat tgctgcccgc cgccgacctg gacgacttca gcaagcagct ccagcagagc 3780

atgagcagcg ccgacagcac ccaggccgag cccaaatcgt gcgataaaac gcatacgtgc 3840

cccccctgtc cagccccaga gttgctgggt ggaccgagcg tgttcctgtt cccccccaag 3900

cccaaggaca ccctgatgat cagcagaacc cccgaggtga cctgcgtggt ggtggacgtg 3960

agccacgagg accccgaggt gaagttcaac tggtacgtgg acggcgtgga ggtgcacaca 4020

gcctggaccc agcccagaga ggagcagtac aacagcacct acagagttgt gtccgtgctg 4080

accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gagcaacaag 4140

gccctgcccg cccccatcga gaagaccatc agcaagccca agggcagagc ccagacaccc 4200

caggtgtaca ccataccccc ccctagagaa caaatgtcca agaagaaggt gagcctgacc 4260

tgcctggtga ccaacttctt ctctgaagcc atcagtgtgg agtgggagag caacggccag 4320

cccgagaaca actacaagac cacccccccc gtgctggaca gcgacggcag cttcttcctc 4380

tacagcaagc tgaccgtgga caagagcaga tggcagcagg gcaacgtgtt ctcttgcagc 4440

gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct gagccccggc 4500

aagtgatgat ctaaagggcg aattcgccct taagggctaa ttctgcagat tcgagcatgc 4560

atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc 4620

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 4680

cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 4740

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 4800

tggggatgcg gtgggctcta tggcttctac tgggcggttt tatggacagc aagcgaaccg 4860

gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg 4920

gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca agagacagga 4980

tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg 5040

gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc 5100

gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt 5160

gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt 5220

ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc 5280

gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc 5340

atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac 5400

caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag 5460

gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag 5520

gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat 5580

atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg 5640

gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa 5700

tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc 5760

ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg 5820

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca cttttcgggg 5880

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 5940

catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac ttcattttta 6000

atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 6060

tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 6120

tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 6180

ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 6240

agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 6300

ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 6360

tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 6420

gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 6480

cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 6540

ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 6600

agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6660

tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6720

ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctt 6768

<210> 24

<211> 6768

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 24

gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctagcttg gtaccgagct cggatccgcc 720

accatggact ggatttggag aatcctcttc ctggtgggag ctgccacagg cgcacacagc 780

agagtgcagc ccaccgagag catcgtgaga ttccccaaca tcaccaacct gtgccccttc 840

ggcgaggtgt tcaacgccac cagattcgcc agcgtgtacg cctggaacag gaagagaatc 900

agcaactgcg tggccgacta cagcgtgctc tacaacagcg ccagcttcag caccttcaag 960

tgctacggcg tgagccccac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 1020

agcttcgtga tcagaggcga cgaggtgaga cagatcgccc ccggccagac cggcaatatc 1080

gccgactaca actacaagct gcccgacgac ttcaccggct gcgtgatcgc gtggaacagc 1140

aataacctgg acagcaaggt gggcggcaac tacaactacc tctacagact gttcagaaag 1200

agcaacctga agcccttcga gagagacatc agcaccgaga tctaccaggc cggcagcacc 1260

ccctgcaacg gcgtgaaggg cttcaactgc tacttccccc tccagagcta cggcttccag 1320

cccacctacg gcgtgggcta ccagccctac agagtggtgg tcctgagctt cgagctgctg 1380

cacgcccccg ccaccgtgtg cggccccaag aagagcacca atctggtgaa gaacaagtgc 1440

gtgaacttct gatgactcga gcatgcatct agagggccct attctatagt gtcacctaaa 1500

tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 1560

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 1620

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 1680

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 1740

gggctctatg gcttctgagg cggaaagaac caggctagcg ttcgatgtac gggccagata 1800

tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 1860

tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 1920

accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 1980

aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 2040

agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 2100

gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 2160

ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 2220

tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 2280

tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 2340

gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct 2400

aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac 2460

ccaagctggc tagcgtttaa acttaagctt gccaccatgg actggatttg gagaatcctc 2520

ttcctggtgg gagctgccac aggcgcacac agcagcgaca acggccccca gaaccagaga 2580

aacgccccca gaatcacctt cggcggcccc agcgacagca ccggcagcaa ccagaacggc 2640

gagagaagcg gcgccagaag caagcagaga agaccccagg gcctgcccaa caacaccgcc 2700

agctggttca ccgccctgac ccagcacggc aaggaggacc tgaagttccc cagaggccag 2760

ggcgtgccca tcaacaccaa cagcagcccc gacgaccaga tcggctacta cagaagagcc 2820

accagaagaa tcagaggcgg cgacggcaag atgaaggacc tgagccccag atggtacttc 2880

tactacctgg gcaccggccc cgaggccggc ctgccctacg gcgccaacaa ggacggcatc 2940

atctgggtgg ccaccgaggg cgccctgaac acccccaagg accacatcgg caccagaaac 3000

cccgccaaca acgccgccat cgtgctccag ctgccccagg gcaccaccct gcccaagggc 3060

ttctacgccg agggcagcag aggcggcagc caggcctcaa gccgttccag cagcagatcc 3120

cgcaacagct cccgcaacag cacccctgga agctcaaggg gcatctctcc tgcgcggatg 3180

gctggtaacg gcggggacgc tgccctggcc ctgctgctgc tggacagact gaaccagctg 3240

gagagcaaga tgagcggcaa gggccagcag cagcagggcc agactgtgac caaaaaaagc 3300

gccgccgagg ccagcaagaa gcccagacag aagagaaccg ccaccaaggc ctacaacgtg 3360

acccaggcct tcggcagaag aggccccgag cagacccagg gcaacttcgg cgaccaggag 3420

ctgatcagac agggcaccga ctacaagcac tggccccaga tcgcccagtt cgcccccagc 3480

gccagcgcct tcttcggcat gagcagaatc ggcatggagg tgacccccag cggcacctgg 3540

ctgacctaca ccggcgccat caagctggac gacaaggacc ccaacttcaa ggatcaggtg 3600

atcctgctga acaagcacat cgacgcctac aagaccttcc cccccaccga gcccaagaag 3660

gacaagaaga agaaggccga cgagacccag gccctgcccc agagacagaa gaagcagcag 3720

acagtgacat tgctgcccgc cgccgacctg gacgacttca gcaagcagct ccagcagagc 3780

atgagcagcg ccgacagcac ccaggccgag cccaaatcgt gcgataaaac gcatacgtgc 3840

cccccctgtc cagccccaga gttgctgggt ggaccgagcg tgttcctgtt cccccccaag 3900

cccaaggaca ccctgatgat cagcagaacc cccgaggtga cctgcgtggt ggtggacgtg 3960

agccacgagg accccgaggt gaagttcaac tggtacgtgg acggcgtgga ggtgcacaca 4020

gcctggaccc agcccagaga ggagcagtac aacagcacct acagagttgt gtccgtgctg 4080

accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gagcaacaag 4140

gccctgcccg cccccatcga gaagaccatc agcaagccca agggcagagc ccagacaccc 4200

caggtgtaca ccataccccc ccctagagaa caaatgtcca agaagaaggt gagcctgacc 4260

tgcctggtga ccaacttctt ctctgaagcc atcagtgtgg agtgggagag caacggccag 4320

cccgagaaca actacaagac cacccccccc gtgctggaca gcgacggcag cttcttcctc 4380

tacagcaagc tgaccgtgga caagagcaga tggcagcagg gcaacgtgtt ctcttgcagc 4440

gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct gagccccggc 4500

aagtgatgat ctaaagggcg aattcgccct taagggctaa ttctgcagat tcgagcatgc 4560

atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc 4620

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 4680

cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 4740

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 4800

tggggatgcg gtgggctcta tggcttctac tgggcggttt tatggacagc aagcgaaccg 4860

gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg 4920

gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca agagacagga 4980

tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg 5040

gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc 5100

gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt 5160

gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt 5220

ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc 5280

gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc 5340

atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac 5400

caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag 5460

gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag 5520

gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat 5580

atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg 5640

gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa 5700

tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc 5760

ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg 5820

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca cttttcgggg 5880

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 5940

catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac ttcattttta 6000

atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 6060

tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 6120

tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 6180

ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 6240

agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 6300

ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 6360

tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 6420

gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 6480

cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 6540

ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 6600

agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6660

tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6720

ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctt 6768

<210> 25

<211> 5518

<212> DNA

<213> artificial sequence

<220>

<223> synthetic construct

<400> 25

gcgtataatg gactattgtg tgctgatatg tacacctgag gacacaggta caggactcag 60

cttaaggcta catcttagct tacgtcacta gagggtccac gtttagtttt taagatccat 120

tgatctccta aacgctgcaa gattcgcaac ctggaatact tagcgctagg tcctagtgaa 180

gcgggacttt ttttctaaag tcgttgagag gaggagtcgt cagaccagat agctttgatg 240

tcctgatcgg aaggatcgtt ggccccgatg tacgggccag atatacgcgt tgacattgat 300

tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 360

agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 420

gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 480

gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 540

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 600

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 660

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 720

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 780

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 840

ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg 900

cttactggct tatcgaaatt aatacgactc actataggga gacccaagct agcttggtac 960

cgagctcgga tccgccacca tggactggat ttggagaatc ctcttcctgg tgggagctgc 1020

cacaggcgca cacagcagag tgcagcccac cgagagcatc gtgagattcc ccaacatcac 1080

caacctgtgc cccttcggcg aggtgttcaa cgccaccaga ttcgccagcg tgtacgcctg 1140

gaacaggaag agaatcagca actgcgtggc cgactacagc gtgctctaca acagcgccag 1200

cttcagcacc ttcaagtgct acggcgtgag ccccaccaag ctgaacgacc tgtgcttcac 1260

caacgtgtac gccgacagct tcgtgatcag aggcgacgag gtgagacaga tcgcccccgg 1320

ccagaccggc aatatcgccg actacaacta caagctgccc gacgacttca ccggctgcgt 1380

gatcgcgtgg aacagcaata acctggacag caaggtgggc ggcaactaca actacctcta 1440

cagactgttc agaaagagca acctgaagcc cttcgagaga gacatcagca ccgagatcta 1500

ccaggccggc agcaccccct gcaacggcgt gaagggcttc aactgctact tccccctcca 1560

gagctacggc ttccagccca cctacggcgt gggctaccag ccctacagag tggtggtcct 1620

gagcttcgag ctgctgcacg cccccgccac cgtgtgcggc cccaagaaga gcaccaatct 1680

ggtgaagaac aagtgcgtga acttctgatg actcgagcat gcatcaagag ggccctattc 1740

tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc 1800

agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 1860

ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 1920

ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 1980

atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagg ctagcgttcg 2040

atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa 2100

ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 2160

atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2220

ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2280

aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2340

tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2400

ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2460

agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2520

ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2580

acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2640

gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga 2700

ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttgcca ccatggactg 2760

gatttggaga atcctcttcc tggtgggagc tgccacaggc gcacacagca gcgacaacgg 2820

cccccagaac cagagaaacg cccccagaat caccttcggc ggccccagcg acagcaccgg 2880

cagcaaccag aacggcgaga gaagcggcgc cagaagcaag cagagaagac cccagggcct 2940

gcccaacaac accgccagct ggttcaccgc cctgacccag cacggcaagg aggacctgaa 3000

gttccccaga ggccagggcg tgcccatcaa caccaacagc agccccgacg accagatcgg 3060

ctactacaga agagccacca gaagaatcag aggcggcgac ggcaagatga aggacctgag 3120

ccccagatgg tacttctact acctgggcac cggccccgag gccggcctgc cctacggcgc 3180

caacaaggac ggcatcatct gggtggccac cgagggcgcc ctgaacaccc ccaaggacca 3240

catcggcacc agaaaccccg ccaacaacgc cgccatcgtg ctccagctgc cccagggcac 3300

caccctgccc aagggcttct acgccgaggg cagcagaggc ggcagccagg cctcaagccg 3360

ttccagcagc agatcccgca acagctcccg caacagcacc cctggaagct caaggggcat 3420

ctctcctgcg cggatggctg gtaacggcgg ggacgctgcc ctggccctgc tgctgctgga 3480

cagactgaac cagctggaga gcaagatgag cggcaagggc cagcagcagc agggccagac 3540

tgtgaccaaa aaaagcgccg ccgaggccag caagaagccc agacagaaga gaaccgccac 3600

caaggcctac aacgtgaccc aggccttcgg cagaagaggc cccgagcaga cccagggcaa 3660

cttcggcgac caggagctga tcagacaggg caccgactac aagcactggc cccagatcgc 3720

ccagttcgcc cccagcgcca gcgccttctt cggcatgagc agaatcggca tggaggtgac 3780

ccccagcggc acctggctga cctacaccgg cgccatcaag ctggacgaca aggaccccaa 3840

cttcaaggat caggtgatcc tgctgaacaa gcacatcgac gcctacaaga ccttcccccc 3900

caccgagccc aagaaggaca agaagaagaa ggccgacgag acccaggccc tgccccagag 3960

acagaagaag cagcagacag tgacattgct gcccgccgcc gacctggacg acttcagcaa 4020

gcagctccag cagagcatga gcagcgccga cagcacccag gccgagccca aatcgtgcga 4080

taaaacgcat acgtgccccc cctgtccagc cccagagttg ctgggtggac cgagcgtgtt 4140

cctgttcccc cccaagccca aggacaccct gatgatcagc agaacccccg aggtgacctg 4200

cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag ttcaactggt acgtggacgg 4260

cgtggaggtg cacacagcct ggacccagcc cagagaggag cagtacaaca gcacctacag 4320

agttgtgtcc gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg 4380

caaggtgagc aacaaggccc tgcccgcccc catcgagaag accatcagca agcccaaggg 4440

cagagcccag acaccccagg tgtacaccat acccccccct agagaacaaa tgtccaagaa 4500

gaaggtgagc ctgacctgcc tggtgaccaa cttcttctct gaagccatca gtgtggagtg 4560

ggagagcaac ggccagcccg agaacaacta caagaccacc ccccccgtgc tggacagcga 4620

cggcagcttc ttcctctaca gcaagctgac cgtggacaag agcagatggc agcagggcaa 4680

cgtgttctct tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct 4740

gagcctgagc cccggcaagt gatgatctaa agggcgaatt cgcccttaag ggctaattct 4800

gcagattcga gcatgcatat agagggcccg tttaaacccg ctgatcagcc tcgactgtgc 4860

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 4920

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 4980

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 5040

acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctactggg cggttttatg 5100

gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg 5160

caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc 5220

tgatcaagag acaggatgag ccgaccctta gactctgtac tcagttctat aaacgagcca 5280

ttggatacga gatccgtaga ttgataaggg acacggaata tccccggacg caatagacgg 5340

acagcttggt atcctgagca cagtcgcgcg tccgaatcta gctctacttt agaggccccg 5400

gattctgatg gtcgtagacc gcagaaccga ttggagggat gctagcacta gtgaggtgag 5460

attgtgttcg gcatgcctca caggcagatc tatcagcaca caattgccca ttatacgc 5518

<210> 26

<211> 418

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 26

Ser Leu Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr Phe

1 5 10 15

Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser

20 25 30

Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr

35 40 45

Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys

50 55 60

Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp

65 70 75 80

Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly

85 90 95

Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu

100 105 110

Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly

115 120 125

Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His

130 135 140

Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu

145 150 155 160

Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg

165 170 175

Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser

180 185 190

Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg

195 200 205

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

210 215 220

Arg Leu Asn Gln Leu Glu Ser Lys Met Phe Gly Lys Gly Gln Gln Gln

225 230 235 240

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

245 250 255

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

260 265 270

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

275 280 285

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

290 295 300

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

305 310 315 320

Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile

325 330 335

Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu

340 345 350

Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys

355 360 365

Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg

370 375 380

Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp

385 390 395 400

Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr

405 410 415

Gln Ala

<210> 27

<211> 669

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 27

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Ser Leu Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg

20 25 30

Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly

35 40 45

Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro

50 55 60

Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu

65 70 75 80

Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser

85 90 95

Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile

100 105 110

Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe

115 120 125

Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn

130 135 140

Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro

145 150 155 160

Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val

165 170 175

Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu

180 185 190

Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser

195 200 205

Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser

210 215 220

Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu

225 230 235 240

Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Phe Gly Lys Gly

245 250 255

Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala

260 265 270

Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val

275 280 285

Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe

290 295 300

Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro

305 310 315 320

Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser

325 330 335

Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr

340 345 350

Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val

355 360 365

Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr

370 375 380

Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu

385 390 395 400

Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala

405 410 415

Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala

420 425 430

Asp Ser Thr Gln Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

435 440 445

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

450 455 460

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

465 470 475 480

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

485 490 495

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln

500 505 510

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

515 520 525

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

530 535 540

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

545 550 555 560

Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro

565 570 575

Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr

580 585 590

Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln

595 600 605

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

610 615 620

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

625 630 635 640

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

645 650 655

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 28

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 28

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 29

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 29

Gly Thr Gly Arg Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val

1 5 10 15

Tyr His

<210> 30

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 30

Ser Val Tyr Asp Phe Phe Val Trp Leu

1 5

<210> 31

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 31

Trp Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp

1 5 10 15

<210> 32

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 32

Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val Thr

1 5 10 15

Leu Asp Ile Val Gln Gly

20

<210> 33

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 33

Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser

1 5 10 15

Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg

20 25

<210> 34

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 34

Gln Cys Thr Glu Val Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr

1 5 10 15

Ile Leu Arg Asn Gln Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe

20 25 30

<210> 35

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 35

Leu Leu Met Trp Ile Thr Gln Cys Phe

1 5

<210> 36

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 36

Ser Leu Leu Met Trp Ile Thr Gln Cys

1 5

<210> 37

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 37

Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr

1 5 10 15

Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln

20 25

<210> 38

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 38

Pro Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr

1 5 10 15

Ile Arg Leu Thr Ala Ala Asp His Arg

20 25

<210> 39

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 39

Pro Glu Ser Arg Leu Leu Glu Phe Tyr

1 5

<210> 40

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 40

Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro

1 5 10

<210> 41

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 41

Leu Glu Phe Tyr Leu Ala Met Pro Phe

1 5

<210> 42

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 42

Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met

1 5 10

<210> 43

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 43

Met Pro Phe Ala Thr Pro Met Glu Ala

1 5

<210> 44

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 44

Leu Ala Met Pro Phe Ala Thr Pro Met

1 5

<210> 45

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 45

Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met

1 5 10

<210> 46

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 46

Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala

1 5 10 15

Glu Leu Ala Arg Arg Ser Leu Ala Gln

20 25

<210> 47

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 47

Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg Leu

1 5 10 15

<210> 48

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 48

Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr

1 5 10

<210> 49

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 49

Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile

1 5 10

<210> 50

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 50

Thr Val Ser Gly Asn Ile Leu Thr Ile Arg

1 5 10

<210> 51

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 51

Thr Val Ser Gly Asn Ile Leu Thr Ile

1 5

<210> 52

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 52

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu

1 5

<210> 53

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 53

Lys Leu Pro Asp Asp Phe Thr Gly Cys Val

1 5 10

<210> 54

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 54

Glu Leu Leu His Ala Pro Ala Thr Val

1 5

<210> 55

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 55

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Thr

1 5 10 15

Ala Trp Asn Arg

20

<210> 56

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 56

Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala

1 5 10 15

Ser Phe Ser Thr

20

<210> 57

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 57

Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg

1 5 10 15

Asp Ile

<210> 58

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 58

Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala

1 5 10 15

Pro Ala Thr Val

20

<210> 59

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 59

Ala Leu Asn Thr Pro Lys Asp His Ile

1 5

<210> 60

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 60

Leu Gln Leu Pro Gln Gly Thr Thr Leu

1 5

<210> 61

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 61

Leu Leu Leu Asp Arg Leu Asn Gln Leu

1 5

<210> 62

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 62

Gly Met Ser Arg Ile Gly Met Glu Val

1 5

<210> 63

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 63

Ile Leu Leu Asn Lys His Ile Asp Ala

1 5

<210> 64

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 64

Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu

1 5 10 15

Asn Gln Leu Glu

20

<210> 65

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 65

Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe

1 5 10 15

Phe Gly Met Ser

20

<210> 66

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 66

Met Leu Gly Thr His Thr Met Glu Val

1 5

<210> 67

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 67

Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val Thr Leu Asp Ile Val

1 5 10 15

<210> 68

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 68

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 69

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 69

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 70

<211> 196

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 70

Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr

1 5 10 15

Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys

20 25 30

Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe

35 40 45

Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr

50 55 60

Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln

65 70 75 80

Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu

85 90 95

Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu

100 105 110

Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg

115 120 125

Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr

130 135 140

Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr

145 150 155 160

Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr

165 170 175

Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro

180 185 190

Ala Thr Val Cys

195

<210> 71

<211> 1273

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 71

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 72

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 72

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 73

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 73

Cys Cys Gly Gly Gly Ser Gly

1 5

<210> 74

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 74

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser

1 5 10

<210> 75

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 75

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 76

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 76

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 77

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 77

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10

<210> 78

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 78

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

1 5 10 15

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

20 25 30

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

35 40 45

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

50 55 60

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

65 70 75 80

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

85 90 95

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

100 105 110

Lys

<210> 79

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 79

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 80

<211> 330

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 80

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Pro Lys Gly

210 215 220

Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln

225 230 235 240

Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe

245 250 255

Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 81

<211> 243

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 81

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro

20 25 30

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

35 40 45

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

50 55 60

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

65 70 75 80

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

85 90 95

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

100 105 110

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

115 120 125

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

130 135 140

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

145 150 155 160

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

165 170 175

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

180 185 190

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

195 200 205

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

210 215 220

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

225 230 235 240

Val Asn Phe

<210> 82

<211> 670

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 82

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro

20 25 30

Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn

35 40 45

Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu

50 55 60

Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys

65 70 75 80

Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn

85 90 95

Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg

100 105 110

Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr

115 120 125

Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala

130 135 140

Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr

145 150 155 160

Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile

165 170 175

Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala

180 185 190

Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg

195 200 205

Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr

210 215 220

Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu

225 230 235 240

Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys

245 250 255

Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu

260 265 270

Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn

275 280 285

Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn

290 295 300

Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp

305 310 315 320

Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met

325 330 335

Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr

340 345 350

Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln

355 360 365

Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro

370 375 380

Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala

385 390 395 400

Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala

405 410 415

Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser

420 425 430

Ala Asp Ser Thr Gln Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr

435 440 445

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

450 455 460

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

465 470 475 480

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

485 490 495

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

500 505 510

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

515 520 525

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

530 535 540

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

545 550 555 560

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

565 570 575

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

580 585 590

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

595 600 605

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

610 615 620

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

625 630 635 640

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

645 650 655

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665 670

<210> 83

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 83

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro

20 25 30

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

35 40 45

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

50 55 60

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

65 70 75 80

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

85 90 95

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

100 105 110

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

115 120 125

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

130 135 140

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

145 150 155 160

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

165 170 175

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

180 185 190

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

195 200 205

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

210 215 220

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

225 230 235 240

Val Asn Phe Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

245 250 255

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

260 265 270

<210> 84

<211> 670

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 84

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro

20 25 30

Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn

35 40 45

Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu

50 55 60

Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys

65 70 75 80

Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn

85 90 95

Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg

100 105 110

Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr

115 120 125

Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala

130 135 140

Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr

145 150 155 160

Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile

165 170 175

Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala

180 185 190

Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg

195 200 205

Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr

210 215 220

Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu

225 230 235 240

Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys

245 250 255

Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu

260 265 270

Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn

275 280 285

Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn

290 295 300

Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp

305 310 315 320

Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met

325 330 335

Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr

340 345 350

Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln

355 360 365

Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro

370 375 380

Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala

385 390 395 400

Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala

405 410 415

Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser

420 425 430

Ala Asp Ser Thr Gln Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr

435 440 445

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

450 455 460

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

465 470 475 480

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

485 490 495

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

500 505 510

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

515 520 525

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

530 535 540

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

545 550 555 560

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

565 570 575

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

580 585 590

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

595 600 605

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

610 615 620

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

625 630 635 640

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

645 650 655

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665 670

<210> 85

<211> 475

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 85

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro

20 25 30

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

35 40 45

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

50 55 60

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

65 70 75 80

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

85 90 95

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

100 105 110

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

115 120 125

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

130 135 140

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

145 150 155 160

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

165 170 175

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

180 185 190

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

195 200 205

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

210 215 220

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

225 230 235 240

Val Asn Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

370 375 380

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 86

<211> 438

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 86

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro

20 25 30

Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn

35 40 45

Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu

50 55 60

Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys

65 70 75 80

Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn

85 90 95

Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg

100 105 110

Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr

115 120 125

Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala

130 135 140

Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr

145 150 155 160

Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile

165 170 175

Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala

180 185 190

Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg

195 200 205

Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr

210 215 220

Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu

225 230 235 240

Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys

245 250 255

Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu

260 265 270

Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn

275 280 285

Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn

290 295 300

Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp

305 310 315 320

Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met

325 330 335

Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr

340 345 350

Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln

355 360 365

Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro

370 375 380

Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala

385 390 395 400

Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala

405 410 415

Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser

420 425 430

Ala Asp Ser Thr Gln Ala

435

<210> 87

<211> 243

<212> PRT

<213> artificial sequence

<220>

<223> synthetic sequence

<400> 87

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro

20 25 30

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

35 40 45

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

50 55 60

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

65 70 75 80

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

85 90 95

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

100 105 110

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

115 120 125

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

130 135 140

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

145 150 155 160

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

165 170 175

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

180 185 190

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

195 200 205

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

210 215 220

Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

225 230 235 240

Val Asn Phe

<210> 88

<211> 438

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 88

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro

20 25 30

Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn

35 40 45

Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu

50 55 60

Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys

65 70 75 80

Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn

85 90 95

Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg

100 105 110

Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr

115 120 125

Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala

130 135 140

Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr

145 150 155 160

Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile

165 170 175

Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala

180 185 190

Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg

195 200 205

Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr

210 215 220

Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu

225 230 235 240

Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys

245 250 255

Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu

260 265 270

Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn

275 280 285

Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn

290 295 300

Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp

305 310 315 320

Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met

325 330 335

Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr

340 345 350

Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln

355 360 365

Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro

370 375 380

Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala

385 390 395 400

Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala

405 410 415

Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser

420 425 430

Ala Asp Ser Thr Gln Ala

435

<210> 89

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 89

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe

<210> 90

<211> 669

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 90

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg

20 25 30

Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly

35 40 45

Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro

50 55 60

Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu

65 70 75 80

Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser

85 90 95

Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile

100 105 110

Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe

115 120 125

Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn

130 135 140

Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro

145 150 155 160

Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val

165 170 175

Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu

180 185 190

Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser

195 200 205

Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser

210 215 220

Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu

225 230 235 240

Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly

245 250 255

Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala

260 265 270

Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val

275 280 285

Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe

290 295 300

Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro

305 310 315 320

Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser

325 330 335

Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr

340 345 350

Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val

355 360 365

Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr

370 375 380

Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu

385 390 395 400

Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala

405 410 415

Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala

420 425 430

Asp Ser Thr Gln Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

435 440 445

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

450 455 460

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

465 470 475 480

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

485 490 495

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

500 505 510

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

515 520 525

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

530 535 540

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

545 550 555 560

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

565 570 575

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

580 585 590

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

595 600 605

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

610 615 620

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

625 630 635 640

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

645 650 655

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665

<210> 91

<211> 271

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 91

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala

245 250 255

Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

260 265 270

<210> 92

<211> 474

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 92

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 93

<211> 437

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 93

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg

20 25 30

Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly

35 40 45

Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro

50 55 60

Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu

65 70 75 80

Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser

85 90 95

Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile

100 105 110

Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe

115 120 125

Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn

130 135 140

Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro

145 150 155 160

Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val

165 170 175

Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu

180 185 190

Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser

195 200 205

Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser

210 215 220

Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu

225 230 235 240

Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly

245 250 255

Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala

260 265 270

Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val

275 280 285

Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe

290 295 300

Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro

305 310 315 320

Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser

325 330 335

Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr

340 345 350

Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val

355 360 365

Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr

370 375 380

Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu

385 390 395 400

Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala

405 410 415

Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala

420 425 430

Asp Ser Thr Gln Ala

435

<210> 94

<211> 263

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 94

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

20 25 30

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

35 40 45

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

50 55 60

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

65 70 75 80

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

85 90 95

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn

100 105 110

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

115 120 125

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

130 135 140

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

145 150 155 160

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe

165 170 175

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly

180 185 190

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

195 200 205

His Ala Pro Ala Thr Val Cys Gly Gly Gly Ser Gly Gly Gly Ser Gly

210 215 220

Gly Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

225 230 235 240

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

245 250 255

Gly Thr Gly Gly Gly Ser Gly

260

<210> 95

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 95

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

20 25 30

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

35 40 45

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

50 55 60

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

65 70 75 80

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

85 90 95

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn

100 105 110

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

115 120 125

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

130 135 140

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

145 150 155 160

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe

165 170 175

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly

180 185 190

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

195 200 205

His Ala Pro Ala Thr Val Cys Gly Gly Gly Ser Gly Gly Gly Ser Gly

210 215 220

Gly Gly Ser Cys Cys Gly Gly Gly Ser Gly

225 230

<210> 96

<211> 263

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 96

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

20 25 30

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

35 40 45

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

50 55 60

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

65 70 75 80

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

85 90 95

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn

100 105 110

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

115 120 125

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

130 135 140

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

145 150 155 160

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe

165 170 175

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly

180 185 190

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

195 200 205

His Ala Pro Ala Thr Val Cys Gly Gly Gly Ser Gly Gly Gly Ser Gly

210 215 220

Gly Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

225 230 235 240

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

245 250 255

Gly Thr Gly Gly Gly Ser Gly

260

<210> 97

<211> 474

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 97

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Pro Lys Gly

355 360 365

Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln

370 375 380

Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr Asn Phe Phe

385 390 395 400

Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 98

<211> 477

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 98

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln

20 25 30

Pro Gly Gly Ser Leu Arg Met Ser Cys Gly Thr Gly Arg Ala Met Leu

35 40 45

Gly Thr His Thr Met Glu Val Thr Val Tyr His Trp Val Arg Gln Ala

50 55 60

Pro Gly Lys Gly Leu Glu Trp Ile Ala Tyr Ile Gly Ser Gly Gly Ser

65 70 75 80

Val Tyr Asp Phe Phe Val Trp Leu Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ser Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asn Arg Gln Leu Tyr Pro Glu

115 120 125

Trp Thr Glu Ala Gln Arg Leu Asp Trp Gly Gln Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

145 150 155 160

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

165 170 175

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

180 185 190

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

195 200 205

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

210 215 220

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

225 230 235 240

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

245 250 255

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

260 265 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

275 280 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

290 295 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

305 310 315 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

325 330 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

340 345 350

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

355 360 365

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

370 375 380

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

385 390 395 400

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

405 410 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

420 425 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

435 440 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

450 455 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 99

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 99

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Trp Asn Arg Gln Leu Tyr

35 40 45

Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Thr Gly Val Tyr Tyr Cys

100 105 110

Gly Thr Gly Arg Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val

115 120 125

Tyr His Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

130 135 140

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

145 150 155 160

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

165 170 175

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

180 185 190

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

195 200 205

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

210 215 220

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

225 230 235 240

Ser Phe Asn Arg Gly Glu Cys

245

<210> 100

<211> 477

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 100

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln

20 25 30

Pro Gly Gly Ser Leu Arg Met Ser Cys Gly Thr Gly Arg Ala Met Leu

35 40 45

Gly Thr His Thr Met Glu Val Thr Val Tyr His Trp Val Arg Gln Ala

50 55 60

Pro Gly Lys Gly Leu Glu Trp Ile Ala Tyr Ile Gly Ser Gly Gly Ser

65 70 75 80

Val Tyr Asp Phe Phe Val Trp Leu Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ser Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Trp Asn Arg Gln Leu Tyr Pro Glu

115 120 125

Trp Thr Glu Ala Gln Arg Leu Asp Trp Gly Gln Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

145 150 155 160

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

165 170 175

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

180 185 190

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

195 200 205

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

210 215 220

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

225 230 235 240

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

245 250 255

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

260 265 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

275 280 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

290 295 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Trp Thr Gln

305 310 315 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

325 330 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

340 345 350

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

355 360 365

Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro

370 375 380

Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu Thr Cys Leu Val Thr

385 390 395 400

Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp Glu Ser Asn Gly Gln

405 410 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

420 425 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

435 440 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

450 455 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470 475

<210> 101

<211> 483

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 101

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln

20 25 30

Pro Gly Gly Ser Leu Arg Met Ser Cys Gly Thr Gly Arg Ala Met Leu

35 40 45

Gly Thr His Thr Met Glu Val Thr Val Tyr His Trp Val Arg Gln Ala

50 55 60

Pro Gly Lys Gly Leu Glu Trp Ile Ala Tyr Ile Gly Ser Gly Gly Ser

65 70 75 80

Val Tyr Asp Phe Phe Val Trp Leu Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ser Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Val Pro Leu Asp Cys Val Leu Tyr

115 120 125

Arg Tyr Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Trp Gly

130 135 140

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

145 150 155 160

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

165 170 175

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

180 185 190

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

195 200 205

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

210 215 220

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

225 230 235 240

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

245 250 255

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

260 265 270

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

275 280 285

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

290 295 300

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

305 310 315 320

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

325 330 335

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

340 345 350

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

355 360 365

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

370 375 380

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

385 390 395 400

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

405 410 415

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

420 425 430

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

435 440 445

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

450 455 460

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

465 470 475 480

Pro Gly Lys

<210> 102

<211> 283

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 102

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Trp Asn Arg Gln Leu Tyr

35 40 45

Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Ala Asn Cys Ser Val Tyr Asp

65 70 75 80

Phe Phe Val Trp Leu His Tyr Tyr Ser Val Arg Asp Thr Leu Leu Gly

85 90 95

Pro Gly Arg Pro Tyr Arg Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

100 105 110

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

115 120 125

Thr Gly Val Tyr Tyr Cys Gln Cys Thr Glu Val Arg Ala Asp Thr Arg

130 135 140

Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln Asp Asp Arg Glu Leu

145 150 155 160

Trp Pro Arg Lys Phe Phe Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

165 170 175

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

180 185 190

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

195 200 205

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

210 215 220

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

225 230 235 240

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

245 250 255

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

260 265 270

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

275 280

<210> 103

<211> 481

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 103

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln

20 25 30

Pro Gly Gly Ser Leu Arg Met Ser Cys Leu Leu Met Trp Ile Thr Gln

35 40 45

Cys Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala

50 55 60

Tyr Ile Gly Ser Gly Gly Ser Leu Leu Met Trp Ile Thr Gln Cys Arg

65 70 75 80

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Leu

85 90 95

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Pro

100 105 110

Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro

115 120 125

Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Trp Gly Gln Gly

130 135 140

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

145 150 155 160

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

165 170 175

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

180 185 190

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

195 200 205

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

210 215 220

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

225 230 235 240

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

245 250 255

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

260 265 270

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

275 280 285

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

290 295 300

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

305 310 315 320

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

325 330 335

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

340 345 350

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

355 360 365

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

370 375 380

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

385 390 395 400

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

405 410 415

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

420 425 430

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

435 440 445

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

450 455 460

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470 475 480

Lys

<210> 104

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 104

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe

<210> 105

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 105

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe

35 40 45

Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala

50 55 60

Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys

65 70 75 80

Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val

85 90 95

Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala

100 105 110

Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp

115 120 125

Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser

130 135 140

Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser

145 150 155 160

Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala

165 170 175

Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro

180 185 190

Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro

195 200 205

Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr

210 215 220

Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val

225 230 235 240

Asn Phe

<210> 106

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 106

Gly Gly Gly Gly Ser

1 5

<210> 107

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 107

Gly Gly Gly Ser Gly

1 5

<210> 108

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 108

Gly Gly Ser Gly Gly

1 5

<210> 109

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 109

Gly Ser Gly Gly Gly

1 5

<210> 110

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 110

Gly Ser Gly Gly Gly Pro

1 5

<210> 111

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 111

Gly Gly Glu Pro Ser

1 5

<210> 112

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 112

Gly Gly Glu Gly Gly Gly Pro

1 5

<210> 113

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 113

Gly Gly Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

1 5 10

<210> 114

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 114

Gly Gly Gly Ser Gly Gly Gly Gly

1 5

<210> 115

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 115

Gly Gly Gly Ser

1

<210> 116

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 116

Thr Val Leu Arg Thr

1 5

<210> 117

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 117

Thr Val Ser Ser Ala Ser

1 5

<210> 118

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 118

Thr Val Leu Ser Ser Ala Ser

1 5

<210> 119

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 119

Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser

1 5 10 15

<210> 120

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> synthetic construct

<400> 120

Thr Gly Arg Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr

1 5 10 15

His

Claims

1. A nucleic acid encoding a polypeptide comprising:

(i) A modified Fc region of human IgG1, and

(ii) At least one of the antigens is a heterologous antigen,

wherein (a) the modified Fc region comprises at least an Fc portion capable of binding CD64 as compared to a corresponding wild-type Fc region, (b) at least one residue of the Fc region is modified to a corresponding residue from a mouse IgG3 antibody, and (c) the modified Fc region has enhanced affinity for an Fc-gamma receptor (fcγr).

2. A nucleic acid encoding a polypeptide comprising:

(i) A modified Fc region of human IgG1, and

(ii) At least one of the antigens is a heterologous antigen,

3. A nucleic acid encoding a polypeptide comprising:

(i) A modified Fc region of human IgG1, and

(ii) At least one of the antigens is a heterologous antigen,

4. The nucleic acid of any one of claims 1 to 3, wherein the affinity for Fc-gamma receptor (fcγr) is enhanced by at least about 10% compared to a corresponding wild-type human IgG1 Fc region.

5. The nucleic acid of any one of claims 1-4, wherein at least one residue of the Fc region is selected from the group consisting of:

n286, K288, K290, a339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, a378, optionally wherein the at least one modified residue is selected from the group consisting of: N286T, K288W, K Q, A339P, Q342R, P343A, R344 3834 345T, L I, S354P, D Q, A357Q, L358M, T359S, N361K, Q K, K370 371N, Y373F, P38395 375E, D376A, A378S.

6. The nucleic acid of claim 5, wherein the human IgG3 Fc region comprises all of the following modifications: N286T, K288W, K Q, A339P, Q342R, P343A, R344 3834 345T, L I, S354P, D Q, A357Q, L358M, T359S, N361K, Q K, K370 371N, Y373F, P38395 375E, D376A, A378S.

7. The nucleic acid of claim 6, wherein the modified Fc region comprises the amino acid sequence provided in SEQ ID No. 1 or an amino acid sequence having at least 90% identity to SEQ ID No. 1.

8. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen is directly linked to the N-terminus of the modified Fc region of human IgG1 or linked by a linker.

9. The nucleic acid of any one of claims 1 to 7, wherein the polypeptide further comprises an antibody variable region into which the at least one heterologous antigen is inserted or substituted, optionally wherein the at least one heterologous antigen is substituted into a CDR of an antibody variable region.

10. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises a T cell epitope and/or a B cell epitope.

11. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen is from cancer or an infectious disease.

12. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the epitopes listed in any one of figures 28 to 33.

13. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the list of table 2 or table 3.

14. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)GTGRAMLGTHTMEVTVYH(SEQ ID NO:29)；

(b) SVYDFFVWL (SEQ ID NO: 30); and

(c)WNRQLYPEWTEAQRLD(SEQ ID NO:31)。

15. the nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)GTGRAMLGTHTMEVTVYH(SEQ ID NO:29)；

(b)SVYDFFVWL(SEQ ID NO:30)；

(c) WNRQLYPEWTEAQRLD (SEQ ID NO: 31); and

(d)VPLDCVLYRYGSFSVTLDIVQG(SEQ ID NO:32)。

16. the nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)GTGRAMLGTHTMEVTVYH(SEQ ID NO:29)；

(b)SVYDFFVWL(SEQ ID NO:30)；

(c)WNRQLYPEWTEAQRLD(SEQ ID NO:31)；

(d)VPLDCVLYRYGSFSVTLDIVQG(SEQ ID NO:32)；

(e) ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 33); and

(f)QCTEVRADTRPWSGPYILRNQDDRELWPRKFF(SEQ ID NO:34)。

17. the nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)LLMWITQCF(SEQ ID NO:35)；

(b)SLLMWITQC(SEQ ID NO:36)；

(c) PESRLLEFYLAMPFATPMEAELARRSLAQ (SEQ ID NO: 37); and

(d)PGVLLKEFTVSGNILTIRLTAADHR(SEQ ID NO:38)。

18. The nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)LLMWITQCF(SEQ ID NO:35)；

(b)SLLMWITQC(SEQ ID NO:36)；

(c)PESRLLEFYLAMPFATPMEAELARRSLAQ(SEQ ID NO:37)；

(d)PESRLLEFY(SEQ ID NO:39)；

(e)RLLEFYLAMPFATP(SEQ ID NO:40)；

(f)LEFYLAMPF(SEQ ID NO:41)；

(g)EFYLAMPFATPM(SEQ ID NO:42)；

(h)MPFATPMEA(SEQ ID NO:43)；

(i)LAMPFATPM(SEQ ID NO:44)；

(j)LLEFYLAMPFATPM(SEQ ID NO:45)；

(k)LLEFYLAMPFATPMEAELARRSLAQ(SEQ ID NO:46)；

(l)PGVLLKEFTVSGNILTIRLTAADHR(SEQ ID NO:38)；

(m)LKEFTVSGNILTIRL(SEQ ID NO:47)；

(n)KEFTVSGNILT(SEQ ID NO:48)；

(o)KEFTVSGNILTI(SEQ ID NO:49)；

(p) TVSGNILTIR (SEQ ID NO: 50); and

(q)TVSGNILTI(SEQ ID NO:51)。

19. the nucleic acid of any one of the preceding claims, wherein the at least one heterologous antigen comprises one or more epitopes selected from the group consisting of:

(a)KIADYNYKL(SEQ ID NO:52)；

(b)KLPDDFTGCV(SEQ ID NO:53)；

(c)ELLHAPATV(SEQ ID NO:54)；

(d)CPFGEVFNATRFASVTAWNR(SEQ ID NO:55)；

(e)RISNCVADYSVLYNSASFST(SEQ ID NO:56)；

(f)YLYRLFRKSNLKPFERDI(SEQ ID NO:57)；

(g)YQPYRVVVLSFELLHAPATV(SEQ ID NO:58)；

(h)ALNTPKDHI(SEQ ID NO:59)；

(i)LQLPQGTTL(SEQ ID NO:60)；

(j)LLLDRLNQL(SEQ ID NO:61)；

(k)GMSRIGMEV(SEQ ID NO:62)；

(l)ILLNKHIDA(SEQ ID NO:63)；

(m) GNGGDAALALLLLDRLNQLE (SEQ ID NO: 64); and

(n)KHWPQIAQFAPSASAFFGMS(SEQ ID NO:65)。

20. the nucleic acid of claim 9, wherein the antibody variable region is a heavy chain variable region comprising the following heterologous antigens substituted into CDR1, CDR2, and CDR3, respectively:

21. The nucleic acid of claim 20, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 2 or SEQ ID No. 3.

22. The nucleic acid of claim 11, wherein the infectious disease is caused by a virus, optionally a coronavirus, optionally SARS-Cov-2.

23. The nucleic acid of claim 22, wherein the heterologous antigen is an N protein of a coronavirus or an immunogenic fragment thereof.

24. The nucleic acid of claim 22 or 23, wherein the N protein comprises the amino acid sequence provided by SEQ ID No. 4.

25. The nucleic acid of claim 22 or 23, wherein the N protein comprises the amino acid sequence provided by SEQ ID No. 5.

26. The nucleic acid of any one of claims 22 to 24, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 6.

27. The nucleic acid of any one of claims 22, 23 or 25, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 7.

28. The nucleic acid of any one of the preceding claims in combination with a second nucleic acid encoding at least one heterologous antigen.

29. The nucleic acid of claim 28 when dependent on claim 23, wherein the second nucleic acid encodes a receptor binding domain of a coronavirus or an immunogenic fragment thereof.

30. The nucleic acid of claim 28 when dependent on claim 23, wherein the second nucleic acid encodes the receptor binding domain of SARS-Cov-2.

31. The nucleic acid of claim 30, wherein the receptor binding domain comprises the amino acid sequence provided in SEQ ID No. 8.

32. The nucleic acid of claim 30, wherein the receptor binding domain comprises the amino acid sequence provided in SEQ ID No. 9.

33. The nucleic acid of claim 30, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 6, and wherein the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided by SEQ ID No. 8.

34. The nucleic acid of claim 30, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 7, and wherein the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided by SEQ ID No. 9.

35. The nucleic acid of claim 28, wherein the second nucleic acid encodes an antibody light chain, the at least one heterologous antigen being inserted into or substituted into the antibody light chain, optionally wherein the at least one heterologous antigen is substituted into a CDR of an antibody light chain.

36. The nucleic acid of claim 35, wherein the antibody light chain encoded by the second nucleic acid comprises the following heterologous antigens substituted into CDR1, CDR2, and CDR3, respectively: WNRQLYPEWTEAQRLD (SEQ ID NO: 31), ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 33) and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 34).

37. The nucleic acid of claim 35, wherein the antibody light chain encoded by the second nucleic acid comprises sequence PGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO: 38) substituted into CDR 2.

38. The nucleic acid of claim 35, wherein the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided by SEQ ID No. 10 or SEQ ID No. 11.

39. The nucleic acid of claim 35, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 2, and wherein the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided by SEQ ID No. 10.

40. The nucleic acid of claim 35, wherein the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided by SEQ ID No. 3, and wherein the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided by SEQ ID No. 11.

41. A vector comprising the nucleic acid of any one of the preceding claims.

42. The vector of claim 41 further comprising a second nucleic acid encoding at least one heterologous antigen.

43. The vector of claim 41 or 42, comprising the nucleotide sequence provided below:

(a) SEQ ID NO. 12 and SEQ ID NO. 13; or (b)

(b) SEQ ID NO. 14 and SEQ ID NO. 15.

44. The vector of claim 41 or 42, comprising the nucleotide sequences provided in SEQ ID NO. 16 and SEQ ID NO. 17.

45. The vector of claim 41 or 42, comprising the nucleotide sequences provided in SEQ ID NO. 18 and SEQ ID NO. 19.

46. The vector of claim 41 or 42, which comprises the nucleotide sequence provided in SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22.

47. The vector of claim 41 or 42, comprising the nucleotide sequence provided as SEQ ID NO. 23.

48. The vector of claim 41 or 42, which comprises the nucleotide sequence provided in SEQ ID NO. 24 or SEQ ID NO. 25.

49. The vector of any one of claims 41 to 48, which is a DNA plasmid or doggybone (dbDNA) vector.

50. A peptide encoded by the nucleic acid of any one of claims 1 to 40, or the vector of any one of claims 41 to 49.

51. A vaccine composition comprising the nucleic acid of any one of claims 1 to 40, or the vector of any one of claims 41 to 49, and/or the peptide of claim 50, optionally in combination with an adjuvant.

52. The nucleic acid of any one of claims 1 to 40, the vector of any one of claims 41 to 49, the peptide of claim 50, and/or the vaccine of claim 51 for use as a medicament.

53. The nucleic acid, vector, peptide and/or vaccine for use according to claim 52, for preventing or treating cancer in a subject, optionally wherein the cancer is melanoma.

54. The nucleic acid, vector, peptide and/or vaccine for use according to claim 52, for preventing or treating an infectious disease in a subject, optionally wherein the infectious disease is covd-19.

55. The nucleic acid, vector, peptide and/or vaccine for use according to claim 53 or 54, wherein two or more different nucleic acids according to any one of claims 1 to 40, vectors according to any one of claims 41 to 49, peptides according to claim 50 or vaccines according to claim 51 are administered to a subject.

56. The nucleic acid, vector, peptide and/or vaccine for use according to claim 55, when dependent on claim 54, wherein the nucleic acid according to claim 33 and the nucleic acid according to claim 34 are administered to a subject.

57. The nucleic acid, vector, peptide and/or vaccine for use according to any one of claims 52 to 56, wherein the nucleic acid, vector, peptide and/or vaccine is administered to a subject using needleless injection.