Compositions and peptides with immune function

Abstract

The present application provides a peptide for regulation of immune response consisting essentially of an amino acid sequence selected from SEQ ID NOs. 1 to 4, and an isolated nucleic acid comprising a nucleotide sequence encoding the peptide. The present application also provides a composition for regulation of immune response comprising the peptide, and a composition of treatment or prevention of virus infection comprising the peptide.

Description

COMPOSITIONS AND PEPTIDES WITH IMMUNE FUNCTIONBACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present application relates to compositions and peptides having immune function, and more particularly to compositions and peptides capable of regulating immune responses.

[0003] 2. Description of the Related Art

[0004] Cytokines are small proteins (approximately 5 to 25 kDa) important in cell signaling. Due to their size, cytokines cannot cross the lipid bilayer of cells to enter the cytoplasm and therefore typically exert their functions by interacting with specific cytokine receptors on the target cell surface. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signaling as immunomodulating agents.

[0005] Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine may be produced by more than one type of cell. They act through cell surface receptors and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways. Cytokines are important in health and disease, specifically in host immune responses to infection, inflammation, trauma, sepsis, cancer, and reproduction.

[0006] Interferon (IFN) is a cytokine made by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. IFNs allow for communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.

[0007] However, given the short serum half-life, limited potency, difficulties in purification and storage of cytokine, there is still a need in the art for development of a protein and / or a peptide having cytokine-like functions and improving the protein properties to addressing the above disadvantages.SUMMARY

[0008] The present application provides a composition of regulation of immune response comprising: a truncated type I IFN consisting essentially of at least one domain selected from helix A, helix B, helix C, helix D, helix E, and AB loop, and a buffer, a carrier and / or an excipient.

[0009] The present application also provides a composition for of treatment or prevention of virus infection comprising: a truncated type I IFN consisting essentially of at least one domain selected from helix A, helix B, helix C, helix D, helix E, and AB loop, and a buffer, a carrier and / or an excipient.

[0010] The present application provides a peptide for regulation of immune response consisting essentially of at least one amino acid sequence selected from:SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK;SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN;SEQ ID NO. 3:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSA AADETELDKFYTEEYQQENDLEAC * ; wherein C* means the amino acid is modified by adding disulfide bond;SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE; and an amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4.

[0011] The present application provides an isolated nucleic acid comprising a nucleotide sequence that encodes the above peptide.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows the conserved sequence among various IFN-a subtypes.

[0013] FIG. 2 shows analysis of critical binding sites of !FN-a2b and 1FNAR1- IFNAR2 complex.

[0014] FIG. 3 (A) shows the predicted structure of cytopeptide- 1 and (B) shows the comparison of the similarity with IFN-a2b.

[0015] FIG. 4(A) and (B) show the analysis results of characteristics of cytopeptide- 1.

[0016] FIG. 5 (A) shows the predicted structure of cytopeptide-2 and (B) shows the comparison of the similarity with IFN-a2b.

[0017] FIG. 6 (A) and (B) show the analysis results of characteristics of cytopeptide-2.

[0018] FIG. 7 (A) shows the predicted structure of cytopeptide-3 and (B) shows the comparison of the similarity with IFN-a2b.

[0019] FIG. 8 (A) and (B) show the analysis results of characteristics of cytopeptide-3.

[0020] FIG. 9 (A) shows the predicted structure of cytopeptide-4 and (B) shows the comparison of the similarity with IFN-a2b.

[0021] FIG. 10 (A) and (B) show the analysis results of characteristics of cytopeptide-4.

[0022] FIG. 11 shows the result of cytotoxicity test of the solvent.

[0023] FIG. 12 shows the result of cytotoxicity test of the cytopeptide-4.

[0024] FIG. 13 shows the result of immune function assay for 4-hour cytopeptide-4 treatment.

[0025] FIG. 14 shows the result of immune function assay for 8-hour cytopeptide-4 treatment.

[0026] FIG. 15 shows the result of immune function assay for 16-hour cytopeptide- 4 treatment.

[0027] FIG. 16 shows the result of immune function assay for 24-hour cytopeptide- 4 treatment.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present application provides a composition of regulation of immune response comprising: a truncated type I IFN consisting essentially of at least one domain selected from helix A, helix B, helix C, helix D, helix E, and AB loop, and a buffer, a carrier and / or an excipient.

[0029] The type I IFN includes, for example, IFN-a, IFN-|3, IFN-s, IFN-K, IFN-co, which can bind to a specific cell surface receptor complex known as the IFN-a / p receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. IFN-a2b) belongs to the family of type I IFN with 180 to 190 amino acids. The first 20 amino acids from the N-terminal constitute a signal peptide, which is cleaved during the secretion process, and the remaining 160 amino acids constitute a functional protein that can bind to IFNAR which is on the cell membrane. The structure of type I IFN consists of five alpha-helices, including helix A, helix B, helix C, helix D, and helix E. First, helix A, AB loop, and helix E of type 1 IFN bind relatively higher affinity to the 1FNAR2. Then, they recruited IFNAR 1, which binds relatively lower affinity to type I IFN to closely helix B, helix C, and helix D. Finally, the type I IFN, IFNAR1 and IFNAR2 form the ternary complex to stimulate specific gene expression via JAK-STAT signaling transduction pathway activation.

[0030] The term "a truncated type I IFN" means that, comparing with an amino acid sequence or a nucleotide sequence of a native type I IFN ("the original sequence"), the truncated type I IFN has a shorter amino acid sequence or a shorter nucleotide sequence, and retains at least one functional fragment or domain of the original sequence. Namely, one or plural fragments of the sequence are removed from the original sequence of the native type I IFN. In the present application, at least 30% of length of the original sequence are removed to generate truncated type I IFN. In one embodiment, at least 40%of length of the original sequence are removed to generate the truncated type 1 1FN. In one embodiment, at least 50% of length of the original sequence are removed to generate the truncated type I IFN.

[0031] In some embodiments, the truncated type I IFN can be merely one or plural specific domains of the native type I IFN, for example, helix A, helix B, helix C, helix D, helix E, AB loop, or any combination thereof. In preferred embodiments, the truncated type I IFN contains helix A, helix B, helix C, helix D, helix E, AB loop, or any combination thereof. In one preferred embodiment, the truncated type I IFN mainly contains a polypeptide of helix B and helix C. In one preferred embodiment, the truncated type I IFN mainly contains a polypeptide of helix A and AB loop. In one preferred embodiment, the truncated type I IFN mainly contains a polypeptide of helix A, helix B, helix C and AB loop. In one preferred embodiment, the truncated type I IFN mainly contains a polypeptide of helix D and helix E.

[0032] In the present application, the truncated type I IFN can be a peptide having an amino acid sequence shown as follows:SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK;SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN;SEQ ID NO. 3:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSA AADETLLDKFYTELYQQLNDLEAC*, wherein C* means the amino acid is modified by adding disulfide bond;SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE; and an amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4.

[0033] In the present application, the peptide is designed based on the amino acid sequence of type I INFs. In embodiments, the peptide is designed based on the amino acid sequence of INF- a.

[0034] In some embodiments, the truncated type I IFN can have the amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4. For example, the sequence can have at least 85%, at least 90%, at least 95%, at least 99% of identity with any of SEQ ID NOs. 1 to 4.

[0035] In embodiments, the peptide of the truncated type 1 1FN can contains additional amino acids in the sequence for protein properties such as structural stability of protein, signaling, protein delivery, biocompatibility and the like.

[0036] In some embodiments, the peptide of the truncated type I IFN can be further modified for improvement of protein properties, such as affinity with cell membrane, protein structure stability and the like. In some embodiments, the modification can include, but not be limited to, phosphorylation, methylation, glycosylation, ubiquitination, acetylation.

[0037] In some embodiments, the modification is lipid modification, such as palmitoylation, myristylation, or prenylation. In some embodiments, the lipid modification such as palmitoylation is able to improve lipophilicity, which enhances the attraction between the modified peptide and the cell membrane. Thereby, the chance that the modified peptide contacts to the receptor on the cell membrane can be increased. In some embodiments, the lipid modification allows the modified peptide to penetrate the cell membrane more efficiently and will not be restricted by the receptor combination. In one preferred embodiment, the modification is palmitoylation. It is well known that the original IFN-a2b protein has to bind to two receptors on one cell membrane to work synergistically, then the original IFN-a2b protein enters the cell to express protein activity. However, the palmitic acid modification for the peptides can overcome or reduce the requirements involving receptors of the original IFN-a2b protein, for example, palmitic acid modification can enhance the attraction between the peptide and the cell membrane, thereby increasing the chance of the peptide binding to the receptor or assisting the peptide in crossing the cell membrane.

[0038] The present application also provides an isolated nucleic acid comprising a nucleotide sequence that encodes a peptide consisting essentially of an amino acid sequence selected from:SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK;SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN;SEQ ID NO. 3:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSAAADETLLDKFYTELYQQLNDLEAC*, wherein C* means the amino acid is modified by adding disulfide bond;SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE; and an amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4.

[0039] In some embodiments, the nucleotide sequence encodes a peptide selected from SEQ ID NO. 1, 2, 3, 4 or any combination thereof. In some embodiments, the nucleotide sequence can be carried by a vector including, but not being limited to, plasmid, viral vector, cosmid, and artificial chromosomes.

[0040] In the present application, the composition can be used for regulation of immune response, for example, in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. In one embodiment, the composition can be applied to trigger the protective defenses of the immune system that eradicate pathogens or tumors. In one embodiment, the composition can be applied to treat and / or prevent virus infection, e.g. Coronavirus infection.

[0041] In the present application, the peptide of the truncated type I IFN can be contained in the composition of regulation of immune response with a therapeutically effective amount. In one embodiment, the composition can contain 1 to millions International Units (IU) of the truncated type I IFN. In one embodiment, the composition can contain 1 to 1 ,000 microgram of the truncated type I IFN.

[0042] The composition also comprises a buffer, a carrier and / or an excipient. The buffer, carrier and excipient are a pharmaceutically acceptable ingredient and known buffer, carrier and excipients may be used as long as it can function as a buffer, a carrier and an excipient in the composition and do not adversely affect the peptide. For example, water, acids, alcohols, ethers, sulfoxides, polysaccharides, glycols, stearates, glycerin, and silicon oxides can be selected and used in the composition.

[0043] In the present application, the design process for peptide can comprise: conducting the protein analysis, truncating the original type I IFN sequence according to analysis results, evaluating the protein properties of the truncated type I IFN sequence, and generating the truncated type I IFN. The protein analysis can include, but not be limited to, analysis of conserved domain, acid dissociation constant, isoelectric point (pl),protein characteristics of type I IFN, binding site with IFN receptor and the like. The detail of the design process is described as follows, but not for limitation. The peptide of the truncated type I IFN is also called "cytopeptide" hereafter.

[0044] First, utilizing HOMCOS protein binding prediction program to find out interacting site between type I IFN and receptor on the cell surface. Next, based on the analysis, the candidate cytopeptides can be designed by retaining the important region of type I IFN that is indicated to influence biological activity and protein structure. Then, the candidate cytopeptides can be further calculated by protein analyzing program, e.g. Prot pi, EnCor and PROTEIN CALCULATOR v3.4 to find out peptide characteristics, including type of amino acid, pl, total charge at pH= 7.40, and charge over different environment (pH=6 to 8). Furthermore, for the purpose to close the original type I IFN protein structure, the candidate cytopeptide sequence can be modified partial site based on prediction of secondary structure, solvent accessibility, and protein binding. Finally, the cytopeptide structures can be constructed by protein modeling program, e.g. SWISS- MODEL according to established human IFN / IFN receptor complex to confirm the final cytopeptide sequence.

[0045] In embodiments, the design process is conducted to design and select cytopeptides, and the cytopeptides are applied for the gene expression assay. For example, cells can be treated with different types and concentration of the designed cytopeptide, RNA purification and reverse-transcription for obtaining complementary DNA (cDNA) can be conducted to detect expression pattern of downstream IFN-related pathway, such as JAK-STAT signaling pathway. By comparing the gene expression pattern, the functions of the designed cytopeptide can be evaluated.

[0046] In the present application, the claimed peptides have amino acid sequences truncated based on the original IFN-a2b sequence and combined with different partial domains of IFN-a2b. The truncated sequences may be partially similar with the original sequence, however, the changes of sequence simultaneously alter the protein structure and even affect the protein function. Accordingly, from a biological perspective, the truncated sequences are not identical to the original sequence.

[0047] Examples

[0048] Example 1

[0049] An IFN-a2b-derivated peptide was designed with the following procedures.

[0050] Thirteen types of IFN-a were aligned through the ClustalW multiple alignment program of BioEdit software. FIG. 1 shows the conserved sequence among various IFN-a subtypes. The amino acid sequences were calculated for the proportion of conserved regions in each a-helix of IFN-a, including Helix A, Helix B, Helix C, Helix D, and Helix E. (43% of the Helix A, 50% of the Helix B, 48% of the Helix C, 60% of the Helix D, and 75% of the Helix E.)

[0051] Subsequently, the proportion of the critical binding sites were examined. According to published papers, the inventors listed the critical binding sites between the IFN-a2b and IFN a / f> receptor. FIG. 2 shows analysis of critical binding sites of IFN-a2b and IFNAR1-IFNAR2 complex. The IFNAR2 binding site is highlighted in yellow, while the IFN ARI binding site is marked in blue. Then, each a-helical region is calculated as the proportion of critical binding sites. 21% of the Helix A, 30% of the Helix B, 33% of the Helix C, 18% of the Helix D, and 35% of the Helix E.

[0052] Accordingly, the peptides of the present application were designed to target the conserved regions and / or the critical binding sites, e.g. the Helix A, Helix B, Helix C, Helix D, and Helix E. In the following examples, the designed peptides were called cytopeptide-1, cytopeptide-2, cytopeptide-3, and cytopeptide-4, respectively.

[0053] Example 2

[0054] The cytopeptide-1 has the amino acid sequence:HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK (SEQ ID NO. 1)

[0055] Prediction of cytopeptide-1 structure

[0056] Cytopeptide- 1 structure was predicted through SWISS-MODEL. Model was constructed in SWISS-MODEL using a template from the PDB database (FIG. 3(A)). Next, structure of cytopeptide-1 was superposed with IFN-a2 to compare the similarity of each other (FIG. 3(B)). The prediction result showed that cytopeptide-1 was composed of two a-helix structures, which was similar to helix B and helix C of IFN-a2b.

[0057] Characteristics of cytopeptide-1

[0058] Amino acid composition, pl value and charge characteristics of cytopeptide- 1 were analyzed by protein calculator program, Prot pi and PROTEIN CALCULATOR v3.4. The analysis results were shown in FIG. 4(A) and FIG. 4(B). According to amino acid composition, both ratio of hydrophobic / hydrophilic and basic / acidic amino acid in cytopeptide-1 were closed to IFN-a2b (FIG. 4(A)). On the other hand, charge characteristics of cytopeptide-1 was with slightly negative charge over pH 6.00 to 8.00 (from -0.3 to -2.5), which was also similar to IFN-a2b (FIG. 4(B)).

[0059] Example 3

[0060] The cytopeptide-2 has the amino acid sequence:RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN (SEQ ID NO. 2)

[0061] Prediction of cytopeptide-2 structure

[0062] Cytopeptide-2 structure was predicted through SWISS-MODEL. Model was constructed in SWISS-MODEL using a template from the PDB database (FIG. 5(A)). Next, structure of cytopeptide-2 was superposed with IFN-a2 to compare the similarity of each other (FIG. 5(B)). The prediction result showed that cytopeptide-2 was similar to helix A and AB loop structure of IFN-a2b.

[0063] Characteristics of cytopeptide-2

[0064] Amino acid composition, pl value and charge characteristics of cytopeptide-2 were analyzed by protein calculator program, Prot pi and PROTEIN CALCULATOR v3.4. The analysis results were shown in FIG. 6(A) and FIG. 6(B). According to amino acid composition, both ratio of hydrophobic / hydrophilic and basic / acidic amino acid in cytopeptide-2 were closed to !FN-a2b (FIG. 6(A)). On the other hand, charge characteristics of cytopeptide-2 was with slightly positive charge over pH 6.00 to 8.00 (from 1.8 to 0.3) (FIG. 6(B)).

[0065] Example 4

[0066] The cytopeptide-3 has the amino acid sequence:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSA AADETLLDKFYTELYQQLNDLEAC* (SEQ ID NO. 3),* means the amino acid in cytopeptide-3 was modified by adding disulfide bond to stabilize peptide structure.

[0067] Prediction of cytopeptide-3 structure

[0068] Cytopeptide-3 structure was predicted through SWISS-MODEL. Model was constructed in SWISS-MODEL using a template from the PDB database (FIG. 7(A)). Next, structure of cytopeptide-3 was superposed with IFN-a2 to compare the similarity of each other (FIG. 7(B)). The prediction result showed that cytopeptide-3 was composed of three a-helix structures and one long loop, which was similar to helix A, helix B, helix C and AB loop of IFN-a2b.

[0069] Characteristics of cytopeptide-3

[0070] Amino acid composition, pl value and charge characteristics of cytopeptide-3 were analyzed by protein calculator program, Prot pi and PROTEIN CALCULATOR v3.4. The analysis results were shown in FIG. 8(A) and FIG. 8(B). According to amino acid composition, both ratio of hydrophobic / hydrophilic and basic / acidic amino acid in cytopeptide-3 were closed to IFN-a2b (FIG. 8(A)). On the other hand, charge characteristics of cytopeptide-3 was with slightly positive to slightly negative charge over pH 6.00 to 8.00 (from 0.5 to -2.9) (FIG. 8(B)), which was similar to IFN-a2b.

[0071] Example 5

[0072] As shown in FIG. 1 and FIG. 2, The result revealed that Helix E of IFN-a2b is the highest conserved region of the IFN-a subtypes, and Helix E has of the most critical receptor-binding sites. As such, a cytopeptide-4 to target the Helix E was designed.

[0073] The amino acid sequence of the cytopeptide-4 and IFN-a2b was performed by a multiple sequence alignment program, Clustal Omega. This program was established by EMBL-EBI (European Molecular Biology Laboratory - European BioinformaticsInstitute). The cytopeptide-4 was truncated from the original type I IFN sequence and designed to target the Helix E region based on published paper.

[0074] The cytopeptide-4 has the amino acid sequence of SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE

[0075] Prediction of the cytopeptide-4 structure

[0076] The cytopeptide-4 structure was predicted through the protein modeling program SWISS-MODEL according to the established human IFN / IFN receptor complex. First, the model was constructed in SWISS-MODEL using a template from the PDB database(FIG. 9(A)). Second, the structure of the cytopeptide-4 was superposed with IFN- a2 to compare their similarity (FIG. 9(B)). Green markers indicate higher similarity, while red ones indicate the lower one. The prediction showed that the cytopeptide-4 was composed of two a-helix structures, similar to parts of Helix D and Helix E of IFN-a2b (FIG. 9(A) and (B)).

[0077] Characteristics of the cytopeptide

[0078] Firstly, the HOMCOS protein binding prediction program was utilized to find out the interacting site between type I IFN and receptor on the cell surface. Secondly, the cytopeptide was further checked by two protein analyzing programs, Port pi and EnCor, to find peptide characteristics, including type of amino acids, percentage of amino acids, and pl value. Those results demonstrate the physical properties between truncated peptide and IFN-ot2b. Thirdly, the cytopeptide was estimated through the online analyzing programs, PROTEIN CALCULATOR v3.4 and PredictProtein, to realize total charge at pH= 7.40, charge over different environments (pH=6.00 to 8.00), prediction of secondary structure, solvent accessibility, and protein binding situations.

[0079] The Protein analyzing program Port pi was used to provide the percentage of the amino acids. Then, we further calculated the hydrophobic, hydrophilic, basic, and acidic proportion of amino acids in the cytopeptide and IFN-a2b sequences. In addition, the online analyzing program PROTEIN CALCULATOR v3.4 helps to predict molecular weight, estimated pl, and charge.

[0080] Both hydrophobic / hydrophilic and basic / acidic amino acid ratios in the cytopeptide were higher than IFN-a2b. On the other hand, the charge characteristics of the cytopeptide were mostly positive charges over pH 6.00 to 8.00 (from 2.1 to 1.2), while IFN-a2b were negative charges over pH 6.00 to 8.00 (from 0.8 to -3.5). In summary, the characteristics of the cytopeptide were different from the entire length of IFN-a2b (FIG. 10(A) and (B)).

[0081] Example 6

[0082] The incomplete IFN-a structure can still stimulate downstream signaling pathways without IFNAR1 involvement. However, the effect of IFNAR1 -induced deficiency may decrease the expression of immune-related genes compared to IFN-a- IFNAR1-IFNAR2 ternary complex. To improve the cytopeptide efficacy, the inventors added palmitoylation to the peptide. Palmitoylation is a lipid modification of the N- terminus of the peptide sequence by chemical synthesis to replace the modification in cells. The palmitoylated peptide can easily tend to the cell membrane because of the characteristics of the hydrophobic group. The cytopeptide can bind to the extracellular domain of the IFNAR2 with as increased probability as possible. After that, it can trigger the intracellular domain of the IFNAR2 to promote the expression of downstream immune-related genes.

[0083] Example 7

[0084] The cytopeptide-4 was assessed the safety through the cytotoxicity assay.

[0085] Cytotoxicity test of the solvent

[0086] The cytopeptide-4 was produced by the chemical synthesis. After several solubility tests, dimethyl sulfoxide (DMSO), which has amphipathic properties, was selected as the solvent for subsequent experiments. HEK293t (Human Embryonic Kidney) cells were seeded in the 6- well plates with 8x l05cells per well, and cells were treated with six different concentrations of DMSO (10%, 5%, 3%, 1%, 0.5%, and 0.25% v / v). Then, the cells were cultured with 5% CO2 at 37°C for 8 hr. Finally, the cell morphology was observed through optical microscopy.

[0087] To compare normal and abnormal cell morphology, the negative control without DMSO treatment was regarded as the control. The treatment groups were 10%, 5%, 3%, 1%, 0.5%, and 0.25% v / v DMSO.

[0088] The results revealed that the 10% v / v and 5% v / v DMSO treatment groups can observe severe abnormal cell morphology, including shrinkage and the characteristic of apoptosis (FIG. 11). The 3% v / v DMSO treatment group had slight cell apoptosis (FIG. 11). There was normal cell morphology at the 1% v / v, 0.5% v / v, and 0.25% v / v DMSO treatment (FIG. 11). Therefore, less than 1% DMSO is the best concentration as the solvent.

[0089] Example 8: Cytotoxicity assessment of the cytopeptide-4

[0090] HEK293t cells were seeded in the 96-well plates with 2xl04cells per well and cultured with 5% CO2 at 37°C. After the cells were exposed to the cytopeptide-4 at four concentrations for 4 hours (13.7, 41.1, 68.5, and 95.9 pg / ml), respectively, the reagent of cell counting kit-8 (CCK-8) was added to the cells for detecting cell survival rate (3 independent experiments). Keep incubation for 2 hours. Finally, 450 nm absorbance value was used to measure with the microplate reader.

[0091] DMSO was selected as the solvent for the cytopeptide-4. Thus, four concentrations of DMSO only were regarded as the control groups (0.15%, 0.44%, 0.73%, and 1.03% v / v). Ethanol is used as the positive control. The treatment group has 13.7, 41.1, 68.5, and 95.9 pg / ml cytopeptide-4 with 0.15%, 0.44%, 0.73%, and 1.03% v / v DMSO, respectively.

[0092] The data showed that 3-fold, 5-fold, and 7-fold concentrations of the cytopeptide-4 did not cause abnormal cell morphology or reduce the cell survival rate compared to DMSO groups (FIG. 12).

[0093] Example 9: Immune function assay

[0094] HEK293t cells were seeded in the 6-well plates with 8xl05cells per well and cultured with 5% CO2 at 37°C. The cells in each well were treated with 0.14% v / v DMSO and 13.7 pg / ml cytopeptide-4. The cells were then incubated for 4 hours (6 independentexperiments). After checking the cell morphology, the cells were collected using the Buffer RLT from the RNeasy Mini Kit. Subsequently, total RNA was extracted, and mRNA was converted to cDNA through the reverse transcription reaction. Lastly, the Quantitative real-time Polymerase Chain Reaction (qRT-PCR) was performed to detect the IFN downstream signaling pathway.

[0095] The negative control is the cells without treatment. It is used to prove that DMSO treatment cannot induce the expression of the immune-related gene. The control group consisted of cells treated with only 0.14% v / v DMSO, as DMSO was the solvent for the cytopeptide. The target genes included JAK1, STAT1, STAT2, and ISG15. The internal control is GAPDH.

[0096] Significantly, the expression of STAT1 and ISG15 increased after cytopeptide-4 treatment, compared to the DMSO control (FIG. 13; up to 19% and 18%). These findings demonstrate the successful stimulation of the initial regulation gene, STAT1 , by the cytopeptide-4 and the subsequent delivery of the signal to the downstream immune-related gene, ISG15.

[0097] 8, 16, and 24-hours treatments

[0098] The same experimental steps were conducted except the cell incubation time for 8, 16 or 24 hours.

[0099] HEK293t cells were treated with 13.7 pg / ml cytopeptide-4 for 8 hours (FIG. 14), 16 hours (FIG. 15), and 24 hours (FIG. 16). Compared to the DMSO control, expression of STAT1 had still significantly increased after cytopeptide-4 treatment for 8 hours. The data showed that the immune effect of the cytopeptide-4 disappeared after 8 hours treatment. This indicated the immune effect of the cytopeptide-4 was not cause the long-term systematic immune response.

[0100] Conclusion

[0101] The cytopeptide-4 is not a natural extract, its peptide sequence is referenced from the E-helical region of human INF-a. The tests prove that the cytopeptide-4 is able to induce a transient regulatory function of cellular immunity-related genes thantraditional interferons. Cell tests have confirmed that the cytopeptide-4 has no cytotoxicity and is expected to significantly reduce the side effects that may occur after traditional interferon therapy.

[0102] According to the results of cell experiments, the action time is estimated to be within 8 to 16 hours. It will slowly degrade and decline after the estimated action time is exceeded. Therefore, the cytopeptide-4 will not accumulate in the body and cause safety risks.

[0103] The unique peptide palmitoylation modification design can help improve bioavailability and speed up cell absorption.

[0104] In the gene expression assay, cells treated with different times and concentration of the cytopeptides were collected, purified their RNA and reverse- transcribed complementary DNA (cDNA) to detect expression pattern of downstream IFN-related pathway, such as JAK-STAT signaling pathway. Relative gene expression pattern of JAK-STAT pathway in cytopeptide group was similar to IFN treatment, which was significantly induced compared with control group. The results indicate that cytopeptide has the IFN-like function by modulating immune-related gene expression.

[0105] The experiment data suggests that cytopeptides have not only interferon-like immune-related genes regulatory functions but also without cytotoxicity. Therefore, the cytopeptide holds promise for variety applications. It could be developed into active pharmaceutical ingredients, raw materials for immune regulation products, non- traditional food raw materials, health food raw materials, and skin care products. Notably, the cytopeptide has the potential to replace traditional IFNs as a new drug ingredient, opening the exciting potential for further research.

[0106] While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than in a limiting sense. It is contemplated that modifications and combinations will readily occur to those skilled in the art, which modifications and combinations will be within the spirit of the invention and the scope of the following claims and its equivalent systems and methods.

Claims

WHAT IS CLAIMED IS:

1. A composition of regulation of immune response comprising: a truncated type I interferon (IFN) consisting essentially of at least one domain selected from helix A, helix B, helix C, helix D, helix E, and AB loop, and a buffer, a carrier and / or an excipient.

2. The composition of claim 1, wherein the type I IFN is IFN-a2b.

3. The composition of claim 1 , wherein the truncated type I IFN consists essentially of at least one amino acid sequence selected from:SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK;SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN;SEQ ID NO. 3:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSA AADETLLDKFYTELYQQLNDLEAC*, wherein C* means the amino acid is modified by adding disulfide bond;SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE; and an amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4.

4. The composition of claim 1 , wherein the amino acid sequence is further modified via lipid modification.

5. The composition of claim 4, wherein the lipid modification is palmitoylation.

6. The composition of claim 1 , wherein the buffer, the carrier and / or the excipient is selected from water, acids, alcohols, ethers, sulfoxides, polysaccharides, glycols, stearates, glycerin, and silicon oxides.

7. A peptide for regulation of immune response consisting essentially of at least one amino acid sequence selected from:SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK;SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN;SEQ ID NO. 3:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSA AADETLLDKFYTELYQQLNDLEAC*, wherein C* means the amino acid is modified by adding disulfide bond;SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAE1MRSFSLSTNLQESLRSKE; and an amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4.

8. The peptide of claim 7, wherein the peptide is further modified via lipid modification.

9. The peptide of claim 7, wherein the lipid modification is palmitoylation.

10. An isolated nucleic acid comprising a nucleotide sequence that encodes a peptide of claim 7.

11. A composition of treatment or prevention of virus infection comprising: a truncated type I IFN consisting essentially of at least one domain selected from helix A, helix B, helix C, helix D, helix E, and AB loop, and a buffer, a carrier and / or an excipient.

12. The composition of claim 11, wherein the type I IFN is IFN-a2b.

13. The composition of claim 11, wherein the truncated type I IFN consists essentially of at least one amino acid sequence selected from:SEQ ID NO. 1: HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNHLEKKK;SEQ ID NO. 2: RTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGN;SEQ ID NO. 3:C*ALPQTHSLGSRRTLALLAQARRISLFSALKDRHDFGHEAIQQIFNLFSTKDSSA AADETLLDKFYTELYQQLNDLEAC*, wherein C* means the amino acid is modified by adding disulfide bond;SEQ ID NO. 4: LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE; and an amino acid sequence having at least 80% of identity with any of SEQ ID NOs. 1 to 4.

14. The composition of claim 13, wherein the amino acid sequence is further modified via lipid modification.

15. The composition of claim 14, wherein the lipid modification is palmitoylation.

16. The composition of claim 11 , wherein the buffer, the carrier and / or the excipient is selected from water, acids, alcohols, ethers, sulfoxides, polysaccharides, glycols, stearates, glycerin, and silicon oxides.

17. The composition of claim 11, further comprising interferon beta and / or interferon gamma.

18. The composition of claim 11, further comprising an antiviral agent and / or antiinflammatory agent.

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