RESTRICTIVE EPITOPE PEPTIDE OF MAJOR HISTOCOMPATIBILITY COMPLEX B2 OF H9N2 SUBTYPE AVIAN INFLUENZA VIRUS AND APPLICATION THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese Patent Application No. 202210449829.4, filed on Apr. 27, 2022, the contents of which are hereby incorporated by reference.

INCORPORATION BY REFERENCE STATEMENT

This statement, made under Rules 77(b)(5)(ii) and any other applicable rule incorporates into the present specification of an XML file for a “Sequence Listing XML” (see Rule 831(a)), submitted via the USPTO patent electronic filing system or on one or more read-only optical discs (see Rule 1.52(e)(8)), identifying the names of each file, the date of creation of each file, and the size of each file in bytes as follows:

- File name: 347_004_2023_3574 sequence listing
- Creation date: Apr. 20, 2023
- Byte size: 427,819 bytes

TECHNICAL FIELD

The present application relates to the technical field of genetic engineering, and in particular to a restrictive epitope peptide of a major histocompatibility complex (MHC) B2 of an H9N2 subtype avian influenza virus (AIV) and an application thereof.

BACKGROUND

Avian influenza virus (AIV) is a kind of segmented ribonucleic acid (RNA) virus belonging to the genus Influenza virus A of the family Orthomyxoviriade, with hosts ranged from various avian species to mammals including humans. Based on the serological differences in Hemagglutinin (HA) and Neuraminidase (NA), AIV can be classified into 18 HA subtypes and 11 NA subtypes, with H9N2 subtype AIV being prevalent in poultry in China. Despite of being a low pathogenic avian influenza, H9N2 subtype AIV can still cause significant economic losses by reducing egg production or co-infecting chickens with other pathogens in poultry, making it essential to strengthen prevention, control and research on H9N2 subtype AIV.

Currently, inactivated vaccines are mainly used for preventing and controlling this virus; however, the virus is likely to mutate and evade from the recognition of antibodies as a result of prolonged immune selection pressure, which leads to insufficient protection across subtypes by the specific antibodies produced by the vaccine. Therefore, the development of vaccines with broader coverage and longer-lasting protection is important for preventing and controlling avian influenza.

Extensive studies have shown that influenza-specific CD8⁺ T cells not only participate in viral clearance but also provide cross-protection against other subtypes of influenza viruses, as demonstrated by Dai et al. who found that CD8⁺ T cell response plays an important role in fighting AIV infection by comparing key protective factors generated by H9N2 AIV infection and vaccine immunization-induced immune response in pathogen-free chickens; while Seo et al. found that chickens infected with H9N2 AIV showed higher survival rates in H5N1 AIV infection assays and the survival rate of chickens infected with H5N1 AIV was increased by subsequent injection with activated H9N2 AIV-specific CD8⁺ T cells. In summary, the development of a vaccine that can induce T-cell immune responses is of great importance for the prevention and control of H9N2 AIV.

Immunogenic epitopes are prerequisites for inducting immune effects in T cells. By March 2022, the Immune Epitope Database (IEDB) showed a total of 34 T-cell epitopes of AIV against chickens, of which 24 T-cell epitopes had been functionally validated for immunogenicity, including 22 CD8⁺ T-cell epitopes and 2 CD4⁺ T-cell epitopes on nucleoproteins, polymerase proteins, matrix protein 1 and haemagglutinin, covering three subtypes of H5N1, H5N8 and H7N1, but no epitopes have been reported for the H9N2 subtype of AIV. Therefore, a systematic screening for immunogenic AIV epitopes is required to address the current long-term prevalence of H9N2 subtype AIV.

Antigenic epitopes are recognized by T cell receptors (TCRs) through binding to major histocompatibility complex (MHC) class I molecules; however, MHC class I molecules are polymorphic and different MHC class I molecules can bind different antigenic epitopes even for the same pathogen, so it is important to clarify the restriction of the MHC while screening for antigenic epitopes. Currently, chickens can be classified into 29 haplotypes from B1 to B29 based on the gene sequence of the MHC B gene region, of which haplotype B2, a common haplotype, has been widely reported to show resistance to certain diseases and is an excellent material for experimental studies on AIV and vaccine development.

SUMMARY

The objectives of the present application are to provide a restrictive epitope peptide of a major histocompatibility complex (MHC) B2 of an H9N2 subtype avian influenza virus (AIV) and an application thereof, so as to solve the problems existing in the prior art. According to the present application, an animal model of H9N2 subtype AIV infection in B2 haplotype chickens is established to demonstrate the important role of cellular immune response in the resistance of B2 haplotype chickens to AIV infection, and potential epitopes in H9N2 subtype AIV viral proteins are systematically screened using the binding motif of B2 haplotype MHC class I molecular, and finally peptide epitopes with immunogenicity are identified through functional assays to facilitate the development of AIV epitope vaccines.

To achieve the above objectives, the present application provides technical schemes as follows:

- the present application provides a restrictive epitope peptide of an MHC B2 of an H9N2 subtype AIV, where the restrictive epitope peptide has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID No.4.

The present application also provides an application of the restrictive epitope peptide in preparing vaccine against H9N2 subtype AIV.

The present application also provides a vaccine against H9N2 subtype AIV, including the restrictive epitope peptide.

The present application achieves the following technical effects:

- according to the present application, firstly, H9N2 subtype AIV (A/Chicken/Hunan/HN/2015) strain is used to infect B2 haplotype (BW/G3) SPF chickens, and the success of the infection model is determined by examining cloacal virus shedding, oropharyngeal virus load, changes in T-cell subtypes in peripheral blood mononuclear cells (PBMC) and expression of immune-related genes in PBMC to act as a follow-up test material; then, candidate peptide epitopes that are potentially immunogenic against H9N2 subtype AIV are screened based on the motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-X-V/I/L) of B2 haplotype chicken MHC class I molecule identified in the National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, and the immunogenicity of the above peptides is finally verified by the ELISpot assay to identify valid T cell epitope.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to explain the embodiments of the present application or the technical schemes in the prior art more clearly, the drawings needed in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and other drawings are available according to these drawings without creative work for ordinary people in the field.

FIG. 1 shows viral load of oropharyngeal swabs of B2 haplotype chicken, with n=7.

FIG. 2 illustrates antibody levels in serum, with n=4.

FIG. 3 illustrates changes of CD8α⁺ T cells in PBMC of B2 haplotype chickens after infection, with n=4.

FIG. 4 illustrates changes of CD4⁺ T cells in PBMC of B2 haplotype chickens after infection, with n=4.

FIG. 5 illustrates changes of proportions of CD4⁺ and CD8α⁺ T cells in PBMC of B2 haplotype chickens after infection; n=4.

FIG. 6 shows changes of CD⁴⁺/CD8α+ T cells in PBMC of B2 haplotype chickens after infection, n=4.

FIG. 7 represents expressions of natural immune related genes in PBMC of B2 haplotype chickens after H9N2 avian influenza virus (AIV) infection.

FIG. 8 represents expressions of cytotoxic T cells (CTLs)-related genes in PBMC of B2 haplotype chickens after H9N2 AIV infection.

FIG. 9 represents expressions of Th2-related genes in PBMC of B2 haplotype chickens after H9N2 AIV infection.

FIG. 10A, FIG. 10B and FIG. 10C show expression levels of interferon gamma (IFN-γ) of the splenocytes which are stimulated with peptide pool, while FIG. 10A shows ELISpot results of pool_1-pool_28, FIG. 10B shows ELISpot results of pool_29-pool_56, and FIG. 10C shows ELISpot results of pool_57-pool_85, with n=3 except in positive control.

FIG. 11 shows representative images of IFN-γ ELISpot responses of #1 chicken.

FIG. 12 shows representative images of IFN-γ ELISpot responses of #2 chicken.

FIG. 13 shows representative images of IFN-γ ELISpot responses of #3 chicken.

FIG. 14 shows secretion levels of IFN-γ in spleen lymphocytes of the #1 chicken, with three technical replicates of each peptide, except for the positive control.

FIG. 15 shows secretion levels of IFN-γ in spleen lymphocytes of the #2 chicken, with three technical replicates of each peptide, except for the positive control.

FIG. 16 shows secretion levels of IFN-γ in spleen lymphocytes of the #3 chicken, with three technical replicates of each peptide, except for the positive control.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various exemplary embodiments of the present application are now described in detail and this detailed description should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

It should be understood that the terms described in the present application are intended to describe particular embodiments only and are not intended to limit the present application. Furthermore, with respect to the range of values in the present application, it is to be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within a stated range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded from the scope.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as is commonly understood by those of ordinary skill in the field described in the present application. Although the present application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present application. All literature referred to in this specification is incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with said literature. In the event of conflict with any incorporated literature, the contents of this specification shall prevail.

Without departing from the scope or spirit of the present application, various improvements and variations can be made to specific embodiments of the specification of the present application, as will be apparent to those skilled in the art. Other embodiments derived from the specification of the present application are obvious to the skilled person. The specification and embodiments of the invention are only exemplary.

As used herein, the words “comprising”, “including”, “having”, “containing”, etc., are open-ended terms, i.e. meaning including but not limited to.

Terminology Explanation:

AIV: avian influenza virus; MHC I: class I major histocompatibility complex; ELISpot: enzyme-linked immune-absorbent spot; PBMC: peripheral blood mononuclear cells; SPF chicken: specific pathogens free chicken; CTL: cytotoxic T lymphocytes; IFN-γ: interferon-gamma; DPI: days post infection; EID₅₀: 50% embryo infective dose of chicken; FBS: fetal bovine serum; PMA+Ionomycin: phorbol myristoyl acetate and ionomycin.

Embodiment 1
1. Experimental Materials
1.1 Experimental Animals and Viruses

The chickens used in this experiment are 4-week-old B2 haplotype SPF chickens (BW/G3) purchased from the National Poultry Laboratory Animal Resource Center; the H9N2 subtype AIV (A/Chicken/Hunan/HN/2015) strain is isolated and stored in the National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control.

1.2 Main Experimental Reagents

Total RNA extraction kit is purchased from Jianshi Biotechnology Company; chicken peripheral blood lymphocyte separation liquid kit; chicken organ tissue mononuclear cell separation kit and red blood cell lysis buffer are purchased from Tianjin Haoyang Biological Manufacture Co., Ltd; ChamQ SYRB qPCR Master Mix are purchased from Nanjing Vazyme Biotech Co., Ltd.; Chicken IFN-γ ELISpot BASIC kit is purchased from Mabtech Company; flow antibodies including Anti-chicken CD3 antibody, Anti-chicken CD4 antibody, Anti-chicken CD8α antibody are purchased from Southern Biotech Company; PMA+Ionomycin and 3,3′,5,5′-tetramethylbenzidine (TMB) ELISpot chromogenic substrates are purchased from Dakewe Biotech Co., Ltd. of China; RPMI-1640 medium and Fetal Bovine Serum (FBS) Australian fetal bovine serum are purchased from GIBCO Company of the United States.

1.3 Preparation of Main Solution

(1) 1640 complete medium: 45 milliliters (mL) RPMI-1640 medium, 5 mL inactivated FBS and 500 microliters (μL) streptomycin (100×) are added into a 50 mL centrifuge tube, and mixed evenly at 4° C.;

(2) flow buffer: 49 mL RPMI-1640 culture medium and 1 mL of inactivated FBS are added into a 50 mL centrifuge tube, and mixed evenly at 4° C.;

(3) cells cryopreservation solution: 45 mL inactivated FBS and 5 mL dimethylsulfoxide (DMSO) are added into a 50 mL centrifuge tube, and mixed evenly at 4° C.

2 Experimental Methods
2.1 Virus Propagation

H9N2 AIV is melted and diluted 1,000 times in sterile PBS to obtain diluted virus solution, 9-11-day SPF chicken embryos are sterilized and placed on the biosafety cabinets, with each chick embryo allantoic cavity inoculated with 100 μL diluted virus solution, followed by sealing and continuing culture of the chick embryo for 24 hours (h); the survival of the chicken embryos is observed 24 h after the inoculation and the dead embryos are discarded; the remaining live embryos are continued to be cultured for 72 h to collect the virus, followed by aspirating the allantoic liquid into a centrifuge tube with a pipette, centrifuging the liquid at 4 degrees Celsius (° C.) for 10 minutes (min) at 2,000 revolutions per minute (rpm) to obtain a supernatant, passing the supernatant through a 0.22 micrometers (μm) filter membrane and sub-packing, storing at −80° C. for later use.

2.2 Determination of Hemagglutination Titer

The titer is determined with reference to Chinese national standards of the latest edition (GB/T 18936-2020).

2.3 Determination of EID₅₀

The 50% embryo infective dose (EID₅₀) of chicken embryo is determined as follows:

- the virus solution after propagation is taken out and melted on ice, then diluted 10 times with PBS; the virus solution with a dilution of 10⁻⁴-10⁻⁹is used to inoculate chicken embryos according to the method of 2.1, with 5 chicken embryos at each dilution and followed by culture 72 h; then, 25 μL allantoic liquid is collected from each chick embryo, the hemagglutination titer is determined by the method of 2.2, and the EID₅₀is calculated by Spearman-Karber method.

2.4 Establishment of Animal Model of H9N2 Subtype AIV infection in B2 Haplotype Chickens

H9N2 AIV virus solution is diluted with sterile PBS to 10⁷EID₅₀/200 μL; the experimental animals are divided into two groups, including B2 haplotype chicken experimental group and B2 haplotype chicken control group, with 7 chickens in each group. The experimental group is challenged intranasally and intratracheally, where 200 μL of virus is injected in the eyes of each animal with one drop in the left and one drop in the right eye firstly, then the rest of the virus is injected through the nasal cavity of one side; the control group is inoculated with equal volume of PBS in the same way. Swabs from the oropharyngeal and cloaca of chickens, peripheral anticoagulation and non-anticoagulation are collected and detected on 3, 5, 7, 9 and 11 DPI.

2.4.1 Detection of H9N2 AIV Shedding of Infected Chickens

The swabs collected on the sampling days are stored at −80° C. for unified detection. After the swab is taken out, it is melted on the ice, fully vortexed, and then the impurities are removed by centrifugation at 4° C. and 12,000 rpm for 5 min; then the supernatant is filtered through a 0.22 μm filter, and the EID₅₀is determined according to 2.3, usually with a dilution of 10⁰-10⁻⁶, so as to evaluate the virus shedding.

2.4.2 Detection of Serum Antibody Levels

The collected non-anticoagulants are placed at room temperature until the serum is precipitated, and the serum is collected into a 1.5 mL centrifuge tube, followed by centrifugation at 4° C. and 2,000 rpm for 10 min to remove the red blood cells, and the remaining serum is used to detect the serum antibody level.

The antibody level is detected by hemagglutination inhibition (HI) test, with reference to the latest edition of national standard (GB/T 18936-2020).

2.4.3 Detection of T Cell Percentage in Chicken PBMC

PBMC is separated according to the instruction of the kit, and appropriate cells are taken for flow staining; the following descriptions are based on 10⁶cells per tube: cells are added in flow tubes, then 1 mL of flow Buffer is added, followed by centrifugation at 440 g for 6 min, and CD3, CD4 and CD8 antibodies are diluted in the dark according to the recommended concentrations in the instruction; the supernatant is discarded after centrifugation, with 100 μL of diluted antibody added to each tube for re-suspension, and incubated for 30 min at 4° C. in the dark; then, 1 mL Buffer is added, and centrifuged at 440 g for 6 min, with precipitate resuspended with 250 μL flow Buffer; the data are collected by an up-flow cytometry and analyzed by a FlowJo software.

2.4.4 Expressions of Immune-Related Genes in Chicken PBMC by Fluorescence Quantitative Polymerase Chain Reaction (PCR) Detection

The total RNA is extracted according to the total RNA extraction kit from Jianshi Biotechnology Company, briefly, taking an appropriate amount of cells, centrifuging at 440 g for 6 min, discarding the supernatant, adding 1 mL of TRIzol then whirling; then adding equal volume of anhydrous ethanol, mixing well, transferring the liquid to a No.2 column, centrifuging for 1 min, and removing the filtrate; adding 400 μL RNA washing solution 2 to the column, centrifuging for 1 min, and discarding the filtrate; add 80 μL of DNase I reaction solution to the column, reacting at room temperature for 15 min, then adding 400 μL of RNA washing solution 1, centrifuging for 1 min, and discarding the filtrate; placing for 2 min, transferring the No.2 column to a centrifugal tube without RNase, adding 50 μL RNase-free water preheated to 70° C. in advance, standing for 2 min, centrifuging for 1 min to elute RNA, and detecting the concentration with ultra-micro spectrophotometer, and storing at −80° C.

RNA is reverse-transcribed according to the system in Table 1 following a reverse transcription procedure as follows: performing reverse transcription PCR at 37° C. for 15 min, inactivating at 85° C. for 5 s, and storing at 4° C.

TABLE 1

Reverse Transcription System

Components
Dosage

5 × M-MLV-Mix
4 μL

RNA
10 μL (less than 1,000 ng)

Sterilized water
6 μL

Total volume
20 μL

The reverse-transcribed complementary deoxyribonucleic acid (cDNA) is amplified by fluorescence quantitative PCR to detect the changes of immune-related genes in PBMC; the target genes and primers detected are as shown in Table 2, and the system is shown in Table 3. Reaction procedure: pre-denaturation at 95° C. for 30 s; cyclic reaction at 95° C., 10 s, 60° C. and 30 s, a total of 40 cycles; dissolution curve analysis 95° C., 15 s, 60° C., 60 s, 95° C., 15 s. The results are analyzed by 2^−ΔΔCtmethod with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as internal reference.

TABLE 2

qPCR25 Primers for Immune Related Genes

Gene name
Primer sequence (5′-3′)
Sequence ID

GAPDH
F-Primer
GAACATCATCCCAGCGTCCA
SEQ ID NO. 5

R-Primer
CGGCAGGTCAGGTCAACAAC
SEQ ID NO. 6

Granzyme
F-Primer
CGGGAAGCAACTGTTGAAAT
SEQ ID NO. 7

K
R-Primer
GAGTCTCCCTTGCAAGCATC
SEQ ID NO. 8

Perforin
F-Primer
ATGGCGCAGGTGACAGTGA
SEQ ID NO. 9

R-Primer
TGGCCTGCACCGGTAATTC
SEQ ID NO. 10

IFN-γ
F-Primer
CCTCCAACACCTCTTCAACATG
SEQ ID NO. 11

R-Primer
TGGCGTGCGGTCAAT
SEQ ID NO. 12

TNF-α
F-Primer
GCTGTTCTATGACCGCCCAGTT
SEQ ID NO. 13

R-Primer
AACAACCAGCTATGCACCCCA
SEQ ID NO. 14

IL-1β
F-Primer
GGTCAACATCGCCACCTACA
SEQ ID NO. 15

R-Primer
CATACGAGATGGAAACCAGCAA
SEQ ID NO. 16

IL-2
F-Primer
GCTAATGACTACAGCTTATGGAGCA
SEQ ID NO. 17

R-Primer
TGGGTCTCAGTTGGTGTGTAGAG
SEQ ID NO. 18

NK lysin
F-Primer
GATGGTTCAGCTGCGTGGGATGC
SEQ ID NO. 19

R-Primer
CTGCCGGAGCTTCTTCAACA
SEQ ID NO. 20

HMG-2
F-Primer
AGAGCACAAGAAGAAGCAC
SEQ ID NO. 21

R-Primer
GTCTTTTAGGAGCGTTGGGGTC
SEQ ID NO. 22

MHC-I
F-Primer
AAGAAGGGGAAGGGCTACAA
SEQ ID NO. 23

R-Primer
AAGCAGTGCAGGCAAAGAAT
SEQ ID NO. 24

IFN-α
F-Primer
GACAGCCAACGCCAAAGC
SEQ ID NO. 25

R-Primer
GTCGCTGCTGTCCAAGCATT
SEQ ID NO. 26

IFN-β
F-Primer
GCCCACACACTCCAAAACACTG
SEQ ID NO. 27

R-Primer
TTGATGCTGAGGTGAGCGTTG
SEQ ID NO. 28

IL-6
F-Primer
AAATCCCTCCTCGCCAATCT
SEQ ID NO. 29

R-Primer
CCCTCACGGTCTTCTCCATAAA
SEQ ID NO. 30

IL-10
F-Primer
AGCAGATCAAGGAGACGTTC
SEQ ID NO. 31

R-Primer
ATCAGCAGGTACTCCTCGAT
SEQ ID NO. 32

TLR3
F-Primer
ACAATGGCAGATTGTAGTCACCT
SEQ ID NO. 33

R-Primer
GCACAATCCTGGTTTCAGTTTAG
SEQ ID NO. 34

TLR7
F-Primer
TCTGGACTTCTCTAACAACA
SEQ ID NO. 35

R-Primer
AATCTCATTCTCATTCATCATCA
SEQ ID NO. 36

MHC-I
F-Primer
AAGAAGGGGAAGGGCTACAA
SEQ ID NO. 37

R-Primer
AAGCAGTGCAGGCAAAGAAT
SEQ ID NO. 38

MHC-II
F-Primer
CTCGAGGTCATGATCAGCAA
SEQ ID NO. 39

R-Primer
TGTAAACGTCTCCCCTTTGG
SEQ ID NO. 40

IL-4
F-Primer
TCGAGGAGTGACGGGTG
SEQ ID NO. 41

R-Primer
ACTATCCGGATGCTCTCCATC
SEQ ID NO. 42

IL-5
F-Primer
GGAACGGCACTGTTGAAAAATAA
SEQ ID NO. 43

R-Primer
TTCTCCCTCTCCTGTCAGTTGTG
SEQ ID NO. 44

IL-13
F-Primer
CTGCCCTTGCTCTCCTCTGT
SEQ ID NO. 45

R-Primer
CCTGCACTCCTCTGTTGAGCTT
SEQ ID NO. 46

Granzyme
F-Primer
ACTCATGTCGAGGGGATTCA
SEQ ID NO. 47

A
R-Primer
TGTAGACACCAGGACCACCA
SEQ ID NO. 48

MDA5
F-Primer
GGACGACCACGATCTCTGTGT
SEQ ID NO. 49

R-Primer
CACCTGTCTGGTCTGCATGTTATC
SEQ ID NO. 50

CXCLi1
F-Primer
AACTCCGATGCCAGTG
SEQ ID NO. 51

R-Primer
TTGGTGTCTGCCTTGT
SEQ ID NO. 52

CXCLi2
F-Primer
CATCATGAAGCATTCCATCT
SEQ ID NO. 53

R-Primer
CTTCCAAGGGATCTTCATTT
SEQ ID NO. 54

TGF-β3
F-Primer
TCTTTACATTGACTTCCGAC
SEQ ID NO. 55

R-Primer
TCCTCCCAACATAGTACAAG
SEQ ID NO. 56

MX1
F-Primer
AAGCCTGAGCATGAGCAGAA
SEQ ID NO. 57

R-Primer
TCTCAGGCTGTCAACAAGATCAA
SEQ ID NO. 58

OASL
F-Primer
AGATGTTGAAGCCGAAGTACCC
SEQ ID NO. 59

R-Primer
CTGAAGTCCTCCCTGCCTGT
SEQ ID NO. 60

ISG12-2
F-Primer
TCAATGGGTGGCAAAGGAG
SEQ ID NO. 61

R-Primer
TACAGGGAGAGCAAAGAAGAGAAGA
SEQ ID NO. 62

IFIT5
F-Primer
CAGAATTTAATGCCGGCTATGC
SEQ ID NO. 63

R-Primer
TGCAAGTAAAGCCAAAAGATAAGTGT
SEQ ID NO. 64

USP18
F-Primer
CAACGTGGGAAGAGGAGAAA
SEQ ID NO. 65

R-Primer
ACTTCATGAGCGGAGAAGGA
SEQ ID NO. 66

IRF3/7
F-Primer
ACTGACCAGCCCAGGAACTCT
SEQ ID NO. 67

R-Primer
AAGGCTTTCCCAACCACAAA
SEQ ID NO. 68

SST
F-Primer
GGTCCACGGTTATGGTGAAAG
SEQ ID NO. 69

R-Primer
GGTCAGAAATCACAACTCAAGCA
SEQ ID NO. 70

KHSRP
F-Primer
CAGCGGGGAAATGATTAAGAAG
SEQ ID NO. 71

R-Primer
TTTGTGTGTGGGGATGGAGA
SEQ ID NO. 72

PARP
F-Primer
ATTGTGGAGGAGCTGGGAGGAA
SEQ ID NO. 73

R-Primer
AGGCTTGCTGCACTTCCCATC
SEQ ID NO. 74

TABLE 3

Fluorescence Quantitative System

Reagent
Volume

2 × ChamQ Universal
10
μL

SYBR qPCR Mix

F-Primer
0.4
μL

R-Primer
0.4
μL

cDNA
2
μL

ddH₂O
7.2
μL

2.5 Screening Polypeptide Epitopes with Immunogenicity

According to the binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) of MHC class I molecular in B2 haplotype chickens determined in the National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, candidate polypeptide epitopes against H9N2 subtype AIV are screened, then the peptides are synthesized by Shanghai Top-Peptide Biotechnology Co., Ltd., with a purity of 95%, and each peptide is synthesized for 5 mg.

2.6 Immunogenicity Detection of Candidate Polypeptides
2.6.1 ELISpot Experiment for Detecting Immunogenicity of Peptide Pool

The synthesized polypeptide is dissolved in 200 μL DMSO, and then stored at −80° C.; five peptides are mixed into a pool, named from pool_1 to pool_85 respectively, and the immunogenicity is detected by ELISpot experiment, with experimental operation referring to the instructions of Chicken IFN-γ ELISpot BASIC Kit, the details are as follows:

the first day:

- (1) the PVDF membrane in the cell wells is activated by 15 μL of 35% ethanol per well using a multichannel pipette for no more than 1 min; then each well is added with 200 μL sterile water for washing, and repeated for 4 times; the monoclonal antibody against chicken IFN-γ is diluted with sterile water at 1:33, and 100 μL is added to each well, and coated at 4° C. overnight;

the second day:

- (2) the coating solution is discarded, and each well is washed with 200 μL sterile PBS for 4 times;
- (3) each well is added with 200 μL 1640 complete culture medium and incubated at room temperature for 1-2 h;
- (3) the solution is discarded, 100 μL spleen lymphocyte suspension (containing 3.5×10⁵cells) is added to each well: at the same time, peptide library (final concentration of 10 micrograms per microliter (m/mL) for each peptide) is added to the experimental group, an equal volume of DMSO is added to the negative control group, and 10 μL of PMA+Ionomycin mixture from the Dakewe Biotech Co., Ltd. is added into the positive control group;
- (4) after all the samples are added, the cell plate is placed into a 37° C. cell incubator containing 5% CO₂for culture of at least 18 h;

the third day:

- (5) after the culture, the culture medium and cells are discarded, and each well is washed five times by 200 μL sterile PBS; each well is added with 100 μL PBS containing 0.5% FBS and 1 μg/mL biotin-labeled detection antibody, and incubated for 2 h at room temperature; then the liquid is shaken off and 200 μL sterile PBS is added to each well for washing for 5 times;
- (6) each well is added with 100 μL diluted streptavidin-labeled horseradish peroxidase (HRP) and incubated at room temperature for 1 h; then the liquid is discarded and each well is added with 200 μL sterile PBS for washing for 5 times;
- (7) each well is added with 100 μL TMB chromogenic solution, and the reaction is stopped by washing the cell plate with ultrapure water until obvious spots appear at the bottom, then the cell plate is dried and counted in an automatic plate reader, and the number of spots is statistically analyzed.

2.6.2 ELISpot Experiment for Detecting Immunogenicity of Individual Peptide

The peptide pools in 2.6.1 that can significantly stimulate cells to produce spots are selected and detected for individual peptide by ELISpot experiment, the methods are the same as in 2.6.1.

2.7 Data Analysis

All the experimental data are statistically analyzed by using the software GraphPad Prism 8, in which ns means P>0.05, where the difference is not significant. * means P<0.05, with significant difference; ** means P<0.01, where the difference is extremely significant; *** means P<0.001, indicating an extremely significant difference, and **** means P<0.0001, where the difference is extremely significant.

3 Results
3.1 Detection of Detoxification of Infected Chicken

After H9N2 AIV infection of B2 haplotype chickens, swabs are collected and the virus shedding is detected according to 2.4.1. As shown in FIG. 1, in the H9N2 AIV-infected group, the viral load of oropharyngeal swabs peaked at 3 DPI, had fallen off since SDPI (p<0.0/), and disappeared at 11 DPI. The cloacal swabs were found to be positive at 3 DPI and SDPI and negative at 7 DPI in table 4, while control groups are all tested negative (the data is not included).

TABLE 4

The virus shedding of cloacal swabs in B2 Haplotype Chickens

Groups
3DPI
5DPI
7DPI

B2 chicken
3.5, 4.3 (2/7)
1.5, 1.5 (2/7)
(0/7)

Note:

the figures outside brackets indicate the virus shedding of cloacal swabs of infected chickens, which is the value of log₁₀EID₅₀. The figures inside brackets/before brackets indicate the number of chickens that have been detoxified, and the figures after/indicate the total number of chickens.

3.2 Detection of Serum Antibody Level in Infected Chickens

According to the results as shown in FIG. 2, the antibody levels are negative at 3 DPI (all less than 2 wells), all chickens tested at 5 DPI turn positive, and the HI antibody levels continue to rise until 11 DPI, indicating that the humoral immunity is initiated on 5 DPI, and the decrease the virus shedding in cloacal swabs from 5 DPI is associated with an increase in antibody levels. The control groups are all tested negative for antibody (the data is not included).

3.3 Changes of T Cell Subtypes in Chicken PBMC after Infection

In order to detect the immune response of T cells in B2 haplotype chickens after challenge, peripheral blood of chickens is collected from jugular vein according to the experimental arrangement, and PBMC is isolated and stained with flow antibody. As can be seen from FIG. 3, the proportion of CD8⁺ T cells significantly increased in the H9N2 AIV-infected group compared to that in the control group at 5 DPI, 7 DPI and 9 DPI (P<0.001); the results show that significant CD8+ T cell proliferation is detectable in chicken PBMC from day 5 after H9N2 AIV infection of B2 haplotype chickens and persists until 9 DPI, suggesting that virus clearance from 5 DPI is associated not only with elevated antibody levels but also subjects to an important effect of CD8+ T cell immune response.

The changes of CD4⁺ T cell subtypes are illustrated in FIG. 4. After H9N2 AIV infection of B2 haplotype chickens, the proportion of CD4⁺ T cells decreases significantly (P<0.05) at 5 DPI, 7 DPI, 9 DPI and returns to a normal level at 11 DPI; the proportion changes of CD4⁺CD8α⁺ double positive T cells in PBMC from B2 haplotype chickens after challenge are shown in FIG. 5, with no statistical difference in the proportion of CD4⁺CD8α⁺ double positive T cells in the challenge group compared to that in the control group. Moreover, the changes of CD4⁺/CD8α⁺ T cells in B2 haplotype chicken PBMC after the challenge are shown in FIG. 6, and the CD4⁺/CD8α⁺ T cell ratios in the 5 DPI, 7 DPI and 9 DPI challenge groups are significantly lower than those in the control group, indicating that the organism is in an immunosuppressed state at this stage.

3.4 Expression of Immune-Related Genes in PBMC of B2 Haplotype Chickens after Infection

In order to further verify the influence of immune response in the process of H9N2 AIV infecting B2 haplotype chickens, the changes of mRNA expression of important immune genes in PBMC after 5 days of in vivo infection are detected by fluorescence quantitative PCR, which mainly includes three parts: natural immune-related genes, CTLs genes and Th2 genes.

In the innate immunity gene fraction (FIG. 7), the expressions of the antiviral genes interferon-stimulated gene 12-2 (ISG12-2), 2′,5′ -oligoadenylate synthetase-like (OASL), interferon-induced proteins with tetratricopeptide repeats 5 (IFIT5), (ubiquitin specific peptidase 18) USP18 and myxovirus resistance 1 (MX1) significantly increased in the 5 DPI infected group compared to those in the control groups (P<0.05). As can be seen from the CTLs gene fraction (FIG. 8), the expressions of Granzyme k, IFN-γ, NK lysin, Poly-(ADP-ribose) Polymerase (PARP) increased significantly (P<0.05); and there no obvious changes of expression as detected in the Th2 gene (FIG. 9). Taken together with the increased proportion of CD8α⁺ T cells as shown in 3.3, it is further suggested that AIV infection has successfully activated the cytotoxic T cell immune response in B2 haplotype chickens. In summary, these results suggest that the model of H9N2 subtype AIV infection inducing cellular immune response in B2 haplotype SPF chickens (BW/G3) is successfully established.

3.5 Screening of Candidate Polypeptide Epitopes with Immunogenicity

According to the binding motif (A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) of MHC class I molecular of B2 haplotype chicken, the candidate polypeptide epitopes for H9N2 subtype AIV are screened, and the screened peptides are shown in Table 5.

TABLE 5

Candidate Polypeptides with Immunogenicity

Screened According to the Motif

Pool

Pool

serial
Peptide
Peptide
Sequence
serial
Peptide
Peptide
Sequence

number
name
sequence
ID
number
name
sequence
ID

Pool_1
P1
SAKEAQDVI
75
Pool_2
P6
LIGQGDVVL
216

P2
SAVLRGFLI
76

P7
ELVRKTRFL
217

P3
PIDNVMGMI
77

P8
VLTGNLQTL
218

P4
SLIIAARNI
78

P9
ILRKATKRL
219

P5
VLVNTYQWI
79

P10
AVKGIGTMV
1

Pool_3
P11
DVSFQGRGV
2
Pool_4
P16
MVGRRATAI
80

P12
GVRVSKMGV
355

P17
KVLFQNWGI
81

P13
RVRDQRGNV
356

P18
LVNTYQWII
82

P14
NVLLSPEEV
357

P19
NVRGSGMRI
83

P15
DVLGTFDTV
358

P20
YPITADKRI
84

Pool_5
P21
RLNPMHQLL
220
Pool_6
P26
IVVRGNSPV
359

P22
VLGKDAGAL
221

P27
NVLIGQGDV
360

P23
SVYIEVLHL
222

P28
GPVHFRNQV
361

P24
SVKREEEVL
223

P29
DPDEGTAGV
362

P25
FVNRANQRL
224

P30
TSTVHYPKV
363

Pool_7
P31
FPNEVGARI
85
Pool_8
P36
NVMGMIGIL
225

P32
MSQSRTREI
86

P37
VVVSIDRFL
226

P33
VSGKDEQSI
87

P38
AVLRGFLIL
227

P34
YSSSMMWEI
88

P39
APLMVAYML
228

P35
DSQTATKRI
89

P40
GPALSINEL
229

Pool_9
P41
LSAKEAQDV
364
Pool_10
P46
AGGTSSVYI
90

P42
SSVKREEEV
365

P47
IGGVRMVDI
91

P43
QSIAEAIIV
366

P48
GTFDTVQI
92

P44
YSSTERVVV
367

P49
KGEKANVLI
93

P45
VSIDRFLRV
368

P50
DAGSDRVMV
94

Pool_11
P51
TSSVYIEVL
230
Pool_12
P56
GGEVRNDDV
369

P52
VSADPLASL
231

P57
EGYEEFTMV
370

P53
QSRMQFSSL
232

P58
WGIEPIDNV
371

P54
ESAVLRGFL
233

P59
RGSGMRIVV
372

P55
LSINELSNL
234

P60
IGQGDVVLV
373

Pool_13
P61
VGRRATAIL
95
Pool_14
P66
GGVRMVDIL
235

P62
KATKRLIQL
96

P67
NLQTLKLRV
236

P63
KATKRLTVL
97

P68
VLFQQMRDV
237

P64
TAGVESAVL
98

P69
VLIGQGDVV
238

P65
DINPGHADL
99

P70
RVMVSPLAV
239

Pool_15
P71
VAYMLEREL
374
Pool_16
P76
RILTSESQL
100

P72
TGNLQTLKL
375

P77
DICKAAMGL
101

P73
LAKGEKANV
376

P78
MIKAVRGDL
102

P74
RATVSADPL
377

P79
EINGPESVL
103

P75
SADPLASLL
378

P80
LIIAARNIV
104

Pool_17
P81
PVAGGTSSV
240
Pool_18
P86
LLRTAVGQV
379

P82
AVRGDLNFV
241

P87
ASICTHLEV
380

P83
EAQDVIMEV
242

P88
NSICNTTGV
381

P84
CLLQSLQQI
243

P89
VSRPMFLYV
382

P85
SLQQIESMI
244

P90
ESRKLLLIV
383

Pool_19
P91
MAWTVVNSI
105
Pool_20
P96
DLEGLYEAI
245

P92
AAMDDFQLI
106

P97
GVTRREVHI
246

P93
KIKSEKTHI
107

P98
QVLSELQDI
247

P94
RIKTRLFTI
108

P99
DPSHEGEGI
248

P95
IIKPHKKGI
109

P100
EPRSLSCWI
249

Pool_21
P101
MATKADYTL
384
Pool_22
P106
EIGEDVAPI
110

P102
CAAMDDFQL
385

P107
ALLKHRFEI
111

P103
LAKSVFNSL
386

P108
LLKHRFEII
112

P104
SAESRKLLL
387

P109
EIGVTRREV
113

P105
NASWFNSFL
388

P110
PIGESPKGV
114

Pool_23
P111
KSEKTHIHI
250
Pool_24
P116
EITGTVRRL
389

P112
QSERGEETI
251

P117
FIIKGRSHL
390

P113
MSKEVNARI
252

P118
SIGKVCRTL
391

P114
SSWVELDEI
253

P119
LINDPWVLL
392

P115
DGFEPNGCI
254

P120
SLPPNFSSL
393

Pool_25
P121
SLENFRAYV
115
Pool_26
P126
EGIPLYDAI
255

P122
KLSQMSKEV
116

P127
FGWKEPNII
256

P123
ELDEIGEDV
117

P128
FLLMDALKL
257

P124
HLRNDTDVV
118

P129
PGTFDLEGL
258

P125
WGMEMRRCL
119

P130
ESSVKEKDL
259

Pool_27
P131
FLKTTPRPL
394
Pool_28
P136
IGKVCRTLL
120

P132
NSLYASPQL
395

P137
CVLEIGDML
121

P133
FSAESRKLL
396

P138
IVQALRDNL
122

P134
TGVEKPKFL
397

P139
ESGDPNALL
123

P135
KGINPNYLL
398

P140
CSORSKFLL
124

Pool_29
P141
VLEIGDMLL
260
Pool_30
P146
KGVYINTAL
399

P142
KLLLIVQAL
261

P147
YLLTWKQVL
400

P143
NLEPGTFDL
262

P148
SITIERMVL
401

P144
CLINDPWVL
263

P149
VGIDPFRLL
402

P145
GVYINTALL
264

P150
GIGTMVMEL
403

Pool_31
P151
TAGLTHLMI
125
Pool_32
P156
LSDNEGRLI
265

P152
AAGAAVKGI
126

P157
TSDMRTEII
266

P153
VAYERMCNI
127

P158
EGRLIONSI
267

P154
LILYDKEEI
128

P159
DGKWVRELI
268

P155
LIRMIKRGI
129

P160
IGTMVMELI
269

Pool_33
P161
LIFLARSAL
404
Pool_34
P166
RLIQNSITI
130

P162
KLSDNEGRL
405

P167
QLSTRGVQI
131

P163
HLMIWHSNL
406

P168
ELRSRYWAI
132

P164
FLARSALIL
407

P169
NLPFERATI
133

P165
CLPACVYGL
408

P170
SVGRMVSGI
134

Pool_35
P171
NATEIRASV
270
Pool_36
P176
LVGIDPFRL
409

P172
SALILRGSV
271

P177
CSLMQGSTL
410

P173
PACVYGLAV
272

P178
GSVAHKSCL
411

P174
SAAFEDLRV
273

P179
NGEDATAGL
412

P175
DPKKTGGPI
274

P180
PGNAEIEDL
413

Pool_37
P181
MVMELIRMI
135
Pool_38
P186
VSGIGRFYI
275

P182
RSGAAGAAV
136

P187
NPAHKSQLV
276

P183
ISVQPTFSV
137

P188
NSITIERMV
277

P184
RGQLSTRGV
138

P189
RASAGQISV
278

P185
DATAGLTHL
139

P190
IAIGSVSLI
279

Pool_39
P191
KADTRVLFI
414
Pool 40
P196
ASGKADTRV
140

P192
AIGSVSLII
415

P197
SSYVCSGLV
141

P193
AIICLLMQI
416

P198
RSGYETFRV
142

P194
SIGSWSKNI
417

P199
SGYETFRVV
143

P195
YINMADYSI
418

P200
SGYSGIFSV
144

Pool_41
P201
RVWWTSNSI
280
Pool_42
P206
HLGTKQVCI
419

P202
NPNQKIIAI
281

P207
VLFIREGKI
420

P203
APFSKDNSI
282

P208
SVSLIIAII
421

P204
GSVSLIIAI
283

P209
QVMPCEPII
422

P205
NSTIIEREI
284

P210
TVHLNSTII
423

Pool_43
P211
RGRPQEPRV
145
Pool_44
P216
GSNRPILYI
285

P212
FALGQGTTL
146

P217
KSQVNRQVI
286

P213
IIAIGSVSL
147

P218
FSVEGKKCI
287

P214
LIIAIICLL
148

P219
GSWPDGANI
288

P215
AILTTTMTL
149

P220
WAFDDGNDV
289

Pool_45
P221
ILERNTVHL
424
Pool_46
P226
SLIIAIICL
150

P222
IAIGSVSLI
425

P227
MATTTNPLI
151

P223
ASIIYDGML
426

P228
DLESLMEWI
152

P224
ESSYVCSGL
427

P229
ILSPLAKGI
153

P225
IGSWSKNIL
428

P230
ALASCMGLI
154

Pool_47
P231
GANINFMPV
290
Pool_48
P236
SGKADTRVL
429

P232
FSKDNSIRL
291

P237
PLAGSAQHV
430

P233
LVCATCEQI
292

P238
CSNPTNNQV
431

P234
PSGPLKAEI
293

P239
LSAGGDIWV
432

P235
LAKGILGFV
294

P240
PIILERNTV
433

Pool_49
P241
EVETYVLSI
155
Pool_50
P246
TAEGALGLV
295

P242
DPNNMDKAV
156

P247
VASQARQMV
296

P243
LGFVFTLTV
157

P248
LSIIPSGPL
297

P244
VIAANIIGI
158

P249
RGLORRRFV
298

P245
NIIGILHLI
159

P250
KAVKLYKKL
299

Pool_51
P251
CINGTCAVV
434
Pool_52
P256
GILHLILWI
160

P252
TLLMNELGV
435

P257
WIIRNWETV
4

P253
CLLMQIAIL
436

P258
PLVIAANII
161

P254
QAYQNRMGV
437

P259
DPLVIAANI
162

P255
EIAQRLEDV
438

P260
SGSSDPLVI
163

Pool_53
P261
GALASCMGL
300
Pool_54
P266
LIYNRMGTV
439

P262
GILGFVFTL
301

P267
LIRHENRMV
440

P263
ALSYSTGAL
302

P268
LLTEVETYV
441

P264
LSPLAKGIL
303

P269
SSTGLKDDL
442

P265
CSGSSDPLV
304

P270
LILWILDRL
443

Pool_55
P271
RIQIFPDTI
164
Pool_56
P276
IAANIIGIL
305

P272
RIKSNGNLI
165

P277
IIGILHLIL
306

P273
LIAPWYGHI
166

P278
YIVERPSAV
307

P274
KITSKVNNI
167

P279
GIKSLKLAV
308

P275
KIDDQIQDI
168

P280
AIDKITSKV
309

Pool_57
P281
ILHLIL WIL
444
Pool_58
P286
PLILDTCTI
169

P282
DGHFVNIEL
445

P287
RLNMINNKI
170

P283
TASLITILL
446

P288
LVNGLMGRI
171

P284
CATSLGHPL
447

P289
EVETRLNMI
172

P285
IAPWYGHIL
448

P290
VPSRSSRGI
173

Pool_59
P291
JIDHEFSEV
310
Pool_60
P296
IAMGFAAFL
449

P292
KILTIYSTV
311

P297
EINRTFKPL
450

P293
SLITILLVV
312

P298
RINYYWSVL
451

P294
TLTENNVPV
313

P299
YIGIKSLKL
452

P295
PLIGPRPLV
314

P300
FIEGGWSGL
453

Pool_61
P301
VSNADKICI
174
Pool_62
P306
DLKRGSCTV
315

P302
SSARSYQRI
175

P307
KLAVGLRNV
316

P303
LSGESHGRI
176

P308
TLDEHDANV
317

P304
SSRGIFGAI
177

P309
NVNNLYNKV
318

P305
ESKLERQKI
178

P310
ASLITILLV
319

Pool_63
P311
DIWAYNAEL
454
Pool_64
P316
KPLIGPRPL
179

P312
VLLENQKTL
455

P317
GPRPLVNGL
180

P313
PVTHAKELL
456

P318
NSTETVDTL
181

P314
SVLKPGQTL
457

P319
TSLGHPLIL
182

P315
VPVTHAKEL
458

P320
KSLKLAVGL
183

Pool_65
P321
QSTNSTETV
320
Pool_66
P326
GGREWSYIV
459

P322
YSTVASSLV
321

P327
NGLCYPGNV
460

P323
AATALANTI
322

P328
VPAEMLANI
461

P324
TANESGRLI
323

P329
LPSFGVSGI
462

P325
EAMVSRARI
324

P330
NPFVSHKEI
463

Pool_67
P331
ESEGTYKIL
184
Pool_68
P336
AIATPGMQI
325

P332
NGMLCATSL
185

P337
WIPKRNRSI
326

P333
YGNPSCDLL
186

P338
RIDFESGRI
327

P334
SGESHGRIL
187

P339
EIMKICSTI
328

P335
HGRILKTDL
188

P340
FLEESHPGI
329

Pool_69
P341
NSCLETMEI
464
Pool_70
P346
EGTYKILTI
189

P342
LSTVLGVSI
465

P347
WAYNAELLV
190

P343
QSSDDFALI
466

P348
GSCRCNICI
191

P344
SSYRRPVGI
467

P349
LGGREWSYI
192

P345
TGAPQLNPI
468

P350
TLLFLKVPV
193

Pool_71
P351
RLNKRSYLI
330
Pool_72
P356
RGKLKRRAI
469

P352
ILNTSQRGI
331

P357
DGGPNLYNI
470

P353
FVEALARSI
332

P358
TALANTIEV
471

P354
AVATTHSWI
333

P359
LIDFLKDVV
472

P355
NPRMFLAMI
334

P360
QIRGFVYFV
473

Pool_73
P361
RLIDFLKDV
194
Pool_74
P366
EIDSVNNAV
335

P362
KLEQSGLPV
195

P367
SSDDFALIV
336

P363
NPTLLFLKV
196

P368
ESADMSIGV
337

P364
RPVGISSMV
197

P369
VSHKEIDSV
338

P365
KSMKLRTQV
198

P370
PSSSYRRPV
339

Pool_75
P371
TIGKKKQRL
474
Pool_76
P376
QLNPIDGPL
199

P372
ISSMVEAMV
475

P377
LLIDGTASL
200

P373
MSRDWLMLI
3

P378
VLGVSILNL
201

P374
SGYAQTDCV
476

P379
NLHIPEVCL
202

P375
PGMQIRGFV
477

P380
DVNPTLLFL
203

Pool_77
P381
TATREGKHI
340
Pool_78
P386
LARSICEKL
478

P382
RAFTDEGAI
341

P387
VIFNRLEAL
479

P383
TLKANFSVI
342

P388
AIVGEISPL
480

P384
RSSTLGLDI
343

P389
RLRRDQKSL
481

P385
SIRLMDRCL
344

P390
SLRGRSSTL
482

Pool_79
P391
CVLEAMAFL
204
Pool_80
P396
CIRMDQAIV
345

P392
FVANFSMEL
205

P397
QVDCFLWHV
346

P393
LVSDGGPNL
206

P398
LPGHTDKDV
347

P394
QPEWFRNVL
207

P399
AIVDKNITL
348

P395
GPATAQMAL
208

P400
EGKHIVERI
349

Pool_81
P401
DVKNAIEVL
483
Pool_82
P406
IPKRNRSIL
209

P402
SSFQVDCFL
484

P407
ESGRLIDFL
210

P403
ASVPAPRYL
485

P408
KICSTIEEL
211

P404
RGRSSTLGL
486

P409
KIEKIRPLL
212

P405
QLSOKFEEI
487

P410
QLRSSSEDL
213

Pool_83
P411
SSEDLNGMI
350
Pool_84
P416
LLLEVEQEI
488

P412
QALQLLLEV
351

P417
TVSSFQDIL
489

P413
EIR WLIEEV
352

P418
EAAMRMGDL
490

P414
KLFSKQEWI
353

P419
LIEEVRHRL
491

P415
RVGMHKRIV
354

P420
EIRTFSFQL
492

Pool_85
P421
SLKLYRDSL
214

P422
RIVYWKQWL
215

3.6 Immunogenicity Detection of Candidate Peptides
3.6.1 ELISpot assay for Immunogenicity Detection of Peptide Pools

The synthesized peptides are mixed according to the requirement of five peptides as a pool, and the spleen lymphocytes of B2 haplotype chickens infected with H9N2 AIV for 28 days are stimulated respectively. As shown in FIGS. 10A-10C, it is found by statistical analysis that pool_2, pool_3, pool_52 and pool_75 significantly stimulate the production of IFN-γ spots by splenic lymphocytes, indicating the presence of immunogenic epitopes in the peptides that constitutes the above-mentioned peptide pools.

3.6.2 ELISpot Assay for Immunogenicity Detection of Polypeptide

FIGS. 11-13 suggest that a single peptide that is immunogenic may also stimulate IFN-γ production by splenic lymphocytes; according to the criteria provided in the reference (Identification of novel avian influenza virus derived CD8⁺ T-cell epitopes), a peptide that causes significant IFN-γ production in at least 2 of 3 chickens is considered immunogenic compared to a negative control, and four peptides, P10, P11, P373 and P257, satisfy this criteria and may be considered as B2 haplotype restrictive H9N2 AIV T-cell epitopes, as shown in FIGS. 14-16 (Shown in Table 6).

TABLE 6

Epitope Information for Four H9N2 Subtype AIV

T Cells Targeting Haplotype B2 Chickens

Protein in

which the

Polypeptide
Polypeptide
polypeptide
Protein

name
sequence
is located
sites

P10
AVKGIGTMV
NP
182-190

(SEQ ID NO. 1)

P11
DVSFQGRGV
NP
455-463

(SEQ ID NO. 2)

P373
MSRDWLMLI
NS1
98-106

(SEQ ID NO. 3)

P257
WIIRNWETV
PB2
552-560

(SEQ ID NO. 4)

The above-mentioned embodiments only describe the preferred mode of the present application, and do not limit the scope of the present application. Under the premise of not departing from the design spirit of the present application, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the present application shall fall within the protection scope determined by the claims of the present application.

RESTRICTIVE EPITOPE PEPTIDE OF MAJOR HISTOCOMPATIBILITY COMPLEX B2 OF H9N2 SUBTYPE AVIAN INFLUENZA VIRUS AND APPLICATION THEREOF

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