Ii-keyT cell peptide of novel coronavirus Omicron series variants and its application

Abstract

The present invention belongs to the field of immunotherapy, and in particular to Ii-key T cell polypeptides and applications thereof of novel coronavirus Omicron series variants. The present invention provides a kind of Ii-key T cell polypeptide composition, wherein the antigenic peptide in the Ii-key T cell polypeptide includes SEQ ID NO.1-SEQ ID NO.6, and the antigenic peptide N-terminus is directly or amino acid deleted and connected to Ii-key by aminovaleric acid (ava), so that the polypeptide presents the response efficiency of CD4+ cells. The Ii-key T cell polypeptide of the present invention contains HLA-II and HLA-I epitopes, which can stimulate CD4+ and CD8+ T cell immune responses at the same time, and is well conserved in various variants of the new crown, reflecting the effect of anti-mutation. The Ii-key peptide mixture of the present invention can protect the lethal mouse model from complete survival when only one immunization is strengthened.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy, and specifically relates to Ii-key T cell polypeptides of novel coronavirus Omicron series variants and their applications. Background Art

[0002] The novel coronavirus infection (COVID-19) caused by the coronavirus SARS-CoV-2 is highly contagious. The COVID-19 vaccine (referred to as the novel coronavirus vaccine) mainly relies on humoral immunity, that is, the production of neutralizing antibodies to prevent viral infection, but neutralizing antibodies usually only last for 3-6 months; in addition, due to the continuous mutation of the virus, especially the amino acid mutation of the spike protein (S protein), which increases the virus's transmission speed and immune escape ability, the currently used novel coronavirus vaccine is difficult to prevent the infection and spread of the Omicron series of variants. Studies have shown that cellular immunity associated with the novel coronavirus vaccine plays a key role in clearing the virus and reducing severe mortality. Cellular immunity can target multiple proteins of SARS-CoV-2 and can widely cover various SARS-CoV-2 variants. It is almost unaffected by viral mutations, and its immune memory lasts for a long time, which can provide long-term protection. Therefore, the research and development of a new generation of vaccines should focus on vaccines that enhance cellular immune responses, or develop vaccines that only induce T cell immunity and use them in combination with other existing vaccines.

[0003] Peptide vaccines are a good path to solve the above problems. Since T cell recognition depends on short peptides presented by antigen-presenting cells (APC) and bound to the major histocompatibility complex (MHC), these T cell peptides with good functions can be screened out from the conserved region sequences of different SARS-CoV-2 proteins to prepare vaccines, thereby combating viral mutations and providing long-term immune protection. In addition, B cell peptides that only produce effective neutralizing antibodies can be screened to avoid the negative effects of non-neutralizing antibodies. Peptide vaccines have the characteristics of small molecular weight and simple structure, so they have high safety; the preparation process is simple, the production is rapid, and the production cost, storage and transportation conditions are low.

[0004] Peptide vaccines have disadvantages such as weak immunogenicity and easy degradation. In research, peptides are usually combined with carrier proteins, or the screened epitopes are embedded in other viral vectors to enhance their immunogenicity, but this has low safety and high cost. Therefore, the immunogenicity of peptides can be improved by selecting appropriate adjuvants, immunization pathways or chemical modifications such as Ii-key. The ideal new crown peptide vaccine should have good immunogenicity while retaining the above-mentioned advantages of synthetic peptides, mainly to produce anti-mutation and long-lasting T cell immunity.

[0005] In the prior art, Victoria K. Baxter et al. published a study on bioRxiv preprint in February 2022 using a mixture of 16 chemically synthesized T and B cell peptides from SARS-CoV-2 sequences and TLR adjuvants to boost mice and conduct a challenge experiment, but the mixed peptides did not provide any protection. The possible reasons are that the SARS-CoV-2 strain used for the challenge is a mouse-adapted strain, and the T cell response does not work on it; second, the T cell peptides are selected based on human HLA predictions and have poor binding to the MHC molecules of BALB / C mice; third, the B cell peptides do not produce any protective antibodies, and T cell immunity alone cannot provide effective protection in the absence of neutralizing antibodies.

[0006] Patent CN202110927606 discloses a series of epitope peptides of the novel coronavirus that are screened and recognized by human CD8+T cells, including conserved epitopes in coronaviruses, which can mediate cellular immunity against SARS-CoV-2 and contribute to the development of the next generation of vaccines to address immune escape caused by SARS-CoV-2 mutations. Patent CN112028978A specifically discloses some novel coronavirus-specific CD8+T cell epitope peptides. The novel coronavirus-specific CD8+T cell epitope peptides provided by this invention can produce a strong cellular immune response and secrete high levels of IFN-γ, which has important scientific significance and application prospects for the prevention of novel coronavirus infection, clinical treatment, and vaccine development.

[0007] However, the epitope peptides in the above inventions can only trigger CD8+T cell responses, and T cell epitopes have not been predicted and designed for Omicron variants. In addition, based on the fact that "Ii-key" can better activate cellular immune responses, it can be applied to the development of new coronavirus vaccines. Summary of the invention

[0008] In order to solve the above problems, the present invention provides the present invention provides a kind of Ii-key T cell polypeptide composition, the antigenic peptide in the Ii-key T cell polypeptide includes SEQ ID NO.1-SEQ ID NO.6, and the antigenic peptide N-terminus is directly or amino acid deleted and connected to Ii-key by aminovaleric acid (ava), so that the response efficiency of polypeptide presenting CD4+ cells is enhanced; the Ii-key T cell polypeptide of the present invention contains HLA-II class and HLA-I class epitopes, which can stimulate CD4+, CD8+T cell immune response at the same time, and has good conservatism in various variant strains of the new crown, reflecting the effect of anti-variation. The Ii-key peptide mixture of the present invention can protect the lethal mouse model from complete survival when only one immunization is strengthened.

[0009] On the one hand, the present invention provides an Ii-key T cell polypeptide composition, wherein the Ii-key T cell polypeptide refers to an antigen peptide with Ii-key connected to the N-terminus; the antigen peptide includes one or more amino acid sequences in SEQ ID NO.1-SEQ ID NO.6.

[0010] Specifically, the Ii-key T cell polypeptide contains HLA-I and HLA-II epitopes.

[0011] Specifically, the Ii-key and the antigen peptide are connected via 5-aminovaleric acid.

[0012] Specifically, the N-terminus of the antigen peptide is connected to Ii-key directly or after amino acid deletion.

[0013] More specifically, 1-5 amino acids are deleted from the N-terminus of the antigenic peptide.

[0014] Specifically, the sequence of the Ii-key is SEQ ID NO.7.

[0015] On the other hand, the present invention provides the use of the aforementioned Ii-key T cell polypeptide composition in the preparation of a new coronavirus vaccine.

[0016] Specifically, the vaccine also contains an adjuvant.

[0017] More specifically, the adjuvant may be one or more of aluminum salt, Titermax, emulsion, liposome, alum, and cytokine.

[0018] Preferably, the adjuvant is an aluminum salt.

[0019] More preferably, the adjuvant is aluminum phosphate and aluminum hydroxide.

[0020] On the other hand, the present invention provides a vaccine for preventing and / or treating novel coronavirus infection, wherein the vaccine comprises the aforementioned Ii-key T cell polypeptide composition.

[0021] Specifically, the new coronavirus is the Omicron variant.

[0022] More specifically, the variant may be alpha, beta, gamma, delta or omega.

[0023] The Omicron can be BA.1, BA.2, BA.5, BQ.1, XBB.1, CH.1.1, BF.7 or BA.5.2.

[0024] Specifically, the vaccine also contains an adjuvant.

[0025] More specifically, the adjuvant can be aluminum salt, Titermax, emulsion, liposome, alum, or cytokine.

[0026] Preferably, the adjuvant is an aluminum salt.

[0027] More preferably, the adjuvant is aluminum phosphate and aluminum hydroxide.

[0028] Specifically, the vaccine also includes pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze-drying stabilizers, wetting or emulsifiers, pH buffers, gel or viscosity-enhancing additives and preservatives.

[0029] Examples of the pharmaceutically acceptable carrier, excipient or diluent include, but are not limited to, demineralized or distilled water, saline solution, vegetable oil, silicone oil, volatile silicone, mineral oil, lower polyalkylene glycol or lower alkyl glycol, fatty acid ester, polyvinyl pyrrolidone, agar, carrageenan, gum arabic, and vaseline.

[0030] More specifically, the vegetable oil includes, but is not limited to, peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil or coconut oil.

[0031] More specifically, the silicone oil includes but is not limited to: polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methyl phenyl polysiloxane.

[0032] More specifically, the mineral oil includes but is not limited to: p-hydroxysiloxane.

[0033] More specifically, the lower polyalkylene glycol or lower alkyl glycol includes, but is not limited to, polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerol.

[0034] More specifically, the fatty acid ester includes, but is not limited to, isopropyl palmitate, isopropyl myristate or ethyl oleate.

[0035] In yet another aspect, the present invention provides a medicine comprising the aforementioned Ii-key T cell polypeptide composition.

[0036] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0037] More specifically, the pharmaceutically acceptable excipients include, but are not limited to, one or more of mannitol, sucrose, sodium chloride, magnesium chloride, polysorbate 80, glycerol and hydroxyethylpiperazine ethanesulfonic acid.

[0038] Specifically, the dosage form of the drug is an injection.

[0039] More specifically, the injection is one of a water injection, an oil injection or a powder injection.

[0040] The technical effects achieved by the present invention are as follows: the Ii-key T cell polypeptide of the present invention enhances the response efficiency of polypeptide-presenting CD4+ cells; the Ii-key T cell polypeptide of the present invention contains HLA-II and HLA-I epitopes, which can simultaneously stimulate CD4+ and CD8+ T cell immune responses, and is well conserved in various variants of the new coronavirus, reflecting the anti-mutation effect. The Ii-key peptide mixture of the present invention can protect the lethal mouse model from complete survival with only one booster immunization. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the coverage rate of six epitopes against HLA-II alleles; A is the coverage rate against HLA-II alleles in the Chinese population; B is the coverage rate against HLA-II alleles in the world population.

[0042] Figure 2 is the coverage rate of six epitopes against HLA-I class alleles; A is the coverage rate against HLA-I class alleles in the Chinese population; B is the coverage rate against HLA-I class alleles in the world population.

[0043] Figure 3 It is the coverage rate of six epitopes for HLA-I and HLA-II alleles; A is the coverage rate for HLA-I and HLA-II alleles in the Chinese population; B is the coverage rate for HLA- and HLA-III alleles in the world population.

[0044] Figure 4Schematic diagram of the Ii-key new coronavirus vaccine's rapid activation of CD4+T cell immunity, the MHC-II class molecule-mediated CD4+T cell immunity pathway; the blue arrow represents the normal pathway, and the red arrow represents the Ii-key-mediated pathway.

[0045] Figure 5 It is the technical roadmap for the Ii-key series peptide presentation efficiency test.

[0046] Figure 6 Identification of the presentation efficiency of P1 Ii-key series peptides on CD4+T cells.

[0047] Figure 7 Identification of the presentation efficiency of P5 Ii-key series peptides on CD4+T cells.

[0048] Figure 8 Identification of the presentation efficiency of P7 Ii-key series peptides on CD4+T cells.

[0049] Fig. 9 Identification of the presentation efficiency of P14 Ii-key series peptides on CD4+T cells.

[0050] Fig.10 To identify the presentation efficiency of P15 Ii-key series peptides on CD4+T cells.

[0051] Fig.11 To identify the presentation efficiency of P17 Ii-key series peptides on CD4+T cells.

[0052] Fig.12 Identification of the presentation efficiency of the original peptides P1, P5, P7, P14, P15 and P17 to CD8+T cells.

[0053] Fig.13 The results are shown in the flow cytometry analysis of intracellular factors IFN-γ+, IL-2+, TNF-α+, and IL-4+ in the spleen CD4+T cells of mice in different groups; the one on the far right is the PMA positive control group.

[0054] Fig.14 The intracellular factors IFN-γ+, IL-2+, TNF-α+, and IL-4+ of spleen CD4+T cells in different groups of mice were different (*, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001).

[0055] Fig.15 Immunization and virus challenge procedures for HLA-DR4 / H11-K18-ACE2 double transgenic mice.

[0056] Fig.16 The survival rate and weight change of HLA-DR4 / H11-K18-ACE2 double transgenic mice after virus challenge; A is the survival rate after virus challenge; B is the weight change after virus challenge.

[0057] Fig.17 The figure is the detection of viral load in lung and brain tissues of HLA-DR4 / H11-K18-ACE2 double transgenic mice after infection; A is the detection of viral nucleic acid copy number in lung tissue; B is the detection of viral nucleic acid copy number in brain tissue. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0059] Example 1 Screening of polypeptides

[0060] 1.1 The screening strategy is divided into the following steps:

[0061] (1) All sequences of all published MHC-II class molecule restricted peptides in IEDB (before November 2021) were summarized, and a total of 1933 results that met the criteria were obtained, removing redundancies; at the same time, the amino acid sequences of the original strain structural proteins S, M, N, and E were used for prediction.

[0062] (2) Select MHC-II class molecule alleles with high coverage in Asians and Chinese, use NetMHCIIpan4.1 and MIXMHC2pred for data analysis and epitope prediction. Each algorithm uses the high-frequency genotypes of MHC molecules in the Chinese population for calculation. When selecting prediction results, sequences that cover more HLA genotypes are given priority, followed by sequences with higher scores. For example, if a sequence has a very high score for binding ability with two genotypes but a low score for other genotypes, and another sequence has an upper-middle or medium score for binding ability with five genotypes, we will choose the latter sequence as a higher priority. When using NetMHCIIpan4.1 for prediction, the sequence length is limited to 10, 11, 12, 13, 14, and 15. Results with a rank above 10% are excluded, those with a rank within 5% are considered to have medium to high affinity, and those between 5% and 10% are considered to have low affinity. The rank% score of MIXMHC2pred prediction results is between 0% and 100%. The smaller the rank% value, the higher the affinity. Therefore, results above 10% are excluded, those with a rank within 5% are considered to have medium to high affinity, and those between 5% and 10% are considered to have possible affinity.

[0063] (3) Through the previous step, combined with software prediction and manual analysis, peptides that can bind to at least three HLA-DRB1 alleles are retained. Subsequently, HLA-A class molecule alleles with high coverage of Asians and Chinese are selected using the NetMHCpan4.1 prediction platform to predict embedded CD8+T cell epitope peptides. The strategy is the same as the previous step. The smaller the rank% value, the higher the affinity. Results with scores above 2% are excluded. Rank% within 1% is considered to have medium-high affinity, and rank% between 1% and 2% is considered to have low affinity. Peptides with poor MHC-I affinity are deleted. The length of the peptide segment of each prediction tool is set to 9, and the others are all default values. Peptides that can bind to at least three HLA-A alleles are retained.

[0064] (4) Search the original literature corresponding to the peptides from the literature, and based on the experimental results, delete the peptides with poor experimental results, or retain the peptides with average scores but good experimental results.

[0065] (5) The final peptide sequence was analyzed for sequence conservation. Based on the variant data from outbreak.info, especially various VOC variants, the peptide sequences were compared to see if they contained mutations specific to the variants. Peptide sequences with a large number of amino acid mutations were eliminated, and those with conservative amino acids were retained. Peptide sequences with 1-2 amino acid mutations were deleted based on the HLA affinity score results, whether they were enriched in different genotypes, including whether they came from structural proteins with strong immunogenicity, and finally 19 candidate T cell epitope peptides were retained for in vitro testing.

[0066] 1.2 In vitro ELISpot (enzyme-linked immunosorbent spot) assay

[0067] The PBMCs (peripheral blood mononuclear cells) of 8 patients with breakthrough infection of the new coronavirus and 14 recipients of the inactivated new coronavirus vaccine (full vaccination or booster immunization) were used to conduct in vitro ELISpot tests on 23 candidate peptides to evaluate whether these peptides can combine with polypeptides from different individuals to effectively stimulate the production of IFN-γ.

[0068] Each individual's PBMC was stimulated in vitro with two replicate wells for each peptide, and two negative control wells and one PHA positive control well were set up. Each well was stimulated with 1.5 × 10 5cells; considering the limited number of PBMC samples, some test wells are not set up with duplicate wells when the number of PBMCs is insufficient. If the number of PBMC samples of an individual is not enough to detect each polypeptide, the next individual will start from the last undetected polypeptide to ensure that each polypeptide is basically detected against the same number of individual PBMCs. The result judgment criteria are: when the results of the PHA control wells are normal, the positive cut-off value is the average number of SFU in the negative control wells plus two standard deviations, and the spot size and background of each well are observed by naked eye to exclude false positives and false negatives. The six antigen peptides with the highest binding rate of more than 30% were preliminarily screened through the experiment: 1, 5, 7, 14, 15 and 17 (see Table 1).

[0069] Table 1

[0070] Antigenic peptide SEQ ID NO sequence P1 SEQ ID NO.1 YNYLYRLFRKSNLKP P5 SEQ ID NO.2 AALALLLLDRLNQLESKMS P7 SEQ ID NO.3 KHFYWFFSNYLKRRV P14 SEQ ID NO.4 ALQIPFAMQMAYRFNGIGV P15 SEQ ID NO.5 QTLLALHRSYLTPGDSS P17 SEQ ID NO.6 FYVYSRVKNLNSSRV

[0071] 1.3 Prediction of genotype coverage

[0072] The genotype coverage of the Chinese population and the world population was predicted for the six antigen peptides 1, 5, 7, 14, 15 and 17 with the highest binding rate of more than 30%. The coverage of the screened HLA-II class molecules for the Chinese population was 85.35%, of which more than 50% of the population could bind to the four alleles; and the HLA-II class coverage in the world population also exceeded 80% (80.71%) ( Figure 1 ) reflects the high coverage of the screened genotypes for HLA-II of different ethnic groups. Similarly, the HLA-I class alleles of the Chinese population and the world population were predicted, with coverage of 93.67% and 90.26% respectively ( Figure 2 ), reflecting the high coverage of HLA-I class epitopes embedded in the six HLA-DR epitope peptides; in addition, combined with HLA-I, the coverage of HLA-II class epitopes for the Chinese and world populations is as high as 99.07% and 98.13% ( Figure 3 ), indicating that the six peptides screened contain the universality of different HLA alleles for different ethnic groups and can induce T cell immune response for most of the world's population.

[0073] 1.4 Conservative analysis

[0074] The conservation of six antigenic peptides was analyzed for the mutation sites of the five variants of concern (Alpha, Beta, Gamma, Delta, Omicron), and the conservation of the main subtypes was analyzed after the sub-branch of Omicron became the main dominant epidemic strain (Table 2). It can be seen that for the five VOCs, only peptide 1 produced an amino acid site "L452R" mutation for the Delta variant, but this amino acid site is not in the core binding region, and its mutation will not have a significant effect on the MHC molecule binding function. In addition, peptide 1 is in the RBD region of the main receptor binding region of the S protein, and the bioinformatics prediction and experimental results are good, so this peptide was selected to be retained.

[0075] Table 2

[0076] Antigenic peptide Protein Source alpha Beta Gamma Delta Omicron P1 S(RBD)-449-463 \ \ \ L452R \ P5 N-217-235 \ \ \ \ \ P7 ORF1ab-3151-3165 \ \ \ \ \ P14 S(S2)-895-911 \ \ \ \ \ P15 S(S1)-239-255 \ \ \ \ \ P17 E-56-70 \ \ \ \ \

[0077] In addition, the important offspring branch variants of Omicron (as of March 2023), BA.1, BA.2, BA.5, BQ.1, XBB.1, CH.1.1, BF.7 and BA.5.2 (see Table 3) were analyzed; except for XBB.1, BQ.1, and CH.1.1, which produced N460K mutations in the core binding region of peptide 1, only BA.1 produced N856K mutations in the non-core region for peptide 14, BA.5, BQ.1, CH.1.1, BF.7 and BA.5.2 produced L452R mutations in the non-core region for peptide 1, and peptide 15 produced G252V mutations in the non-core region for XBB.1. The remaining epitopes are conserved in all subtypes, indicating that these six T cell epitope peptides are well conserved in the variants. In addition, of these six T cell epitope peptides, three are located in the key antigen protein S protein, two of which are located in S1 (one is located in RBD), and the other is located in the S2 conserved region; although the S protein can produce the best immunogenicity, it belongs to a highly variable protein region, but these three epitopes are well conserved in various mutant strains, and the mutation sites are mostly in non-core regions; one is located in ORF1ab, one is located in N protein, and one is located in E protein, which reflects that while the S protein is the relatively main choice, other structural proteins and non-structural proteins are also selected, which is universal.

[0078] Table 3

[0079]

[0080] Example 2 Enhancement of CD4+ T cell presentation efficiency of HLA-DR epitopes based on Ii-key key peptide technology

[0081] When Ii-key is connected to the target peptide, it can bind to the MHC-II class molecule on the APC, thereby presenting the target peptide to the T cell; its essence is to directly form an "antigen peptide-MHC complex", which can induce immune effects in the human body without the need for internalization and presentation by antigen presenting cells, and without the need for antigen digestion and processing by antigen presenting cells in the body. This method also avoids the off-target and toxic side effects caused by "antigen digestion and processing by antigen presenting cells" ( Figure 4 ). In addition, the link between Ii-key and the target antigen peptide only requires a carbon chain of 5-6 carbon atoms, which can be easily achieved through chemical synthesis, without the need for time-consuming and labor-intensive artificial preparation processes such as expression and purification.

[0082] 5-aminovaleric acid (ava) is used as a carbon chain to link Ii-key and antigen peptide. The structure of MHC-Ⅰ and MHC-Ⅱ class molecules that accept antigen peptides is the antigen binding groove located at the far end of the molecule. The difference is that the groove of class I molecules is closed at both ends, and the length of the antigen peptide accepted is limited to 8-10 amino acid residues; the groove of class II molecules is open at both ends, and the length of the antigen peptide entering the groove varies greatly, ranging from 13-17 amino acid residues, or even more. Specifically, the peptide binding groove of MHC-II molecules can bind to peptides with a length of 9-10 amino acids, but the binding groove of MHC-II molecules is open at both ends, and can accommodate more antigenic peptides. The two sides of the peptide segment can extend outward or bend in the groove. Studies have shown that the extended part of the MHC-II epitope (called the flanking sequence PFRs) has a greater effect on the stability of the MHC, antigen epitope and TCR three-molecule complex; and Ii-key is at the amino end of the antigen peptide, which can be directly anchored in the antigen binding region of the MHC-II molecule. The target antigen peptide binds to the binding groove, so the number and nature of amino acids at the amino end of the flanking sequence are particularly important. Generally, several amino acids of the amino terminal flanking sequence are selected for serial deletion, and the amino acid at the amino terminal is retained in different forms, and the carboxyl end does not need to be changed. According to this strategy, each of the selected antigen peptide sequences is subjected to a serial synthesis of amino terminal amino acid deletion of the antigen peptide for Ii-key.

[0083] The peptide synthesis adopts the mature solid phase synthesis technology route, and the purification adopts high performance liquid chromatography. The final purified product is a lyophilized powder. The synthesis steps are as follows:

[0084] (1) "SPPS" is a sequential attachment process for amino acid synthesis, including: deprotection → neutralization and washing → condensation → washing. The cycle is as follows:

[0085] Deprotection (removal of the N-terminal Fmoc protecting group), and verification of the removal of excess piperidine after Fmoc deprotection by pH testing;

[0086] The completion of the coupling reaction during the protected peptide-resin precursor was monitored using ninhydrin and chloroaniline methods with dimethylformamide (DMF) washing;

[0087] Next coupling (condensation) of an amino acid derivative;

[0088] Wash with DMF.

[0089] The peptide synthesis repeats this cycle from the C-terminus to the N-terminus until each peptide is synthesized. All amino acid derivatives used in this process are preferably pharmaceutical excipients or food-grade raw materials.

[0090] (2) The protected peptide on the resin was treated with a cleavage mixture (trifluoroacetic acid [TFA]), water and dithiothreitol (DTT) to separate the crude peptide from the resin. The purity of the crude peptide was determined by high performance liquid chromatography (HPLC), and the homology of the crude peptide was determined by LC-MS (liquid chromatography-mass spectrometry).

[0091] (3) The peptide was purified using a reverse HPLC column with TFA (trifluoroacetic acid) and tested by RP-HPLC. The purity should be ≥ 90%.

[0092] (4) The peptide was purified by reverse HPLC column using HOAc (glacial acetic acid). The purity should be ≥95% by RP-HPLC detection, and no single impurity should be ≥3% unless it has been identified.

[0093] (5) Lyophilization: Filter through a 0.2 μm sterilizing filter and then lyophilize to obtain the acetate salt of the raw material drug.

[0094] The synthesized Ii-key series peptides are shown in Table 4.

[0095] Table 4

[0096]

[0097]

[0098] 2.1 Magnetic bead separation of CD4+T, CD8+T and CD14+ monocytes

[0099] In this study, magnetic cell sorting (MACS) was used to stimulate the CD4+T cells and CD8+T cells with peptides in vitro to determine the presentation efficiency of Ii-key HLA-II class molecule peptides and whether they contain class I cell epitopes.

[0100] In order to determine the effect of the screened HLA-DR peptides, the presentation efficiency comparison between the Ii-key series peptides and the original peptides, and whether the HLA-DR peptides contain HLA-I epitopes, the CD4+ and CD8+ T cells of the individual PBMCs that were positive for a certain peptide were separated and stimulated with the peptides for in vitro cell culture. The isolated CD14+ monocytes have two uses, one is to culture them in vitro with the isolated T cells as autologous APCs, and the other is to culture them into mature DCs as APCs for downstream ELISpot experiments. Sorting reference stem cell related cell sorting EasySep TM Instructions: First, divide PBMC into two parts according to the concentration required for calculation and culture, one part for CD14+ monocytes and one part for sorting CD4+ and CD8+ T cells. The general steps of sorting are as follows:

[0101] (1) Sorting of CD14+ monocytes: by immunomagnetic negative selection. Prepare samples with an indicated PBMC concentration in a volume range (3-5×10 7 cells / mL, 0.5-2mL), add the sample to a polystyrene round-bottom test tube, add the separation mixture, mix and incubate at room temperature for 3-5 minutes, vortex the specific antibody magnetic beads for 30 seconds during this period, and then add them to the sample at a concentration of 50μL / mL sample, mix and incubate at room temperature for 3-5 minutes, and insert the test tube into the magnetic pole for incubation for 2.5 minutes (do not cover the lid), then aspirate the cell suspension in the test tube or directly introduce it into a new test tube for use (mainly do not take the test tube out of the magnetic pole during this period), and obtain successfully sorted CD14+ monocytes for counting and subsequent use.

[0102] (2) Sorting of CD8+ T cells and CD4+ T cells: CD8+ T cells were positively selected and CD4+ T cells were negatively selected. Samples with indicated PBMC concentrations were prepared within a volume range (1×10 8 Cells / mL, 0.1-2.5mL), add the sample to a polystyrene round-bottom test tube, add the separation mixture, mix and incubate at room temperature for 3-5 minutes, vortex the specific antibody magnetic beads for 30 seconds during this period, and then add them to the sample at a concentration of 50μL / mL sample, mix and incubate at room temperature for 3-5 minutes, add culture medium to 2.5mL and insert the test tube into the magnetic pole for incubation for 2.5 minutes (do not cover the lid), then aspirate the magnetic bead suspension in the test tube or directly introduce it into a new test tube, and the suspension containing magnetic beads is subsequently used for CD4+T cell sorting, repeat the magnetic pole incubation step 2-3 times, and the successfully sorted CD8+ monocytes in the tube are counted and used later. The poured magnetic bead suspension continues to be negatively selected for CD4+T cells, and the steps are the same as those of the kit. The last step is to pour out the harvested CD4+T cells, which are counted and used later.

[0103] 2.2 In vitro peptide stimulation culture of CD4+T and CD8+T cells and culture of autologous DC (dendritic cells)

[0104] (1) In vitro peptide stimulation culture of CD4+T and CD8+T cells: ImmunoCult TM -XF T cell expansion medium, add human recombinant IL-2 and mix thoroughly to prepare fresh complete ImmunoCult TM -XF T Cell Expansion Medium (Note: Complete Expansion Medium must be prepared fresh each day of use and can be stored at 4°C for up to 2 weeks) in ImmunoCult TM -XF T cell expansion medium at 1×10 6 Human CD4+T cells were inoculated at 10 cells / mL, and 0.1ug / mL of the costimulatory molecule CD28 and 1 / 10 of the number of CD4+T cells were added. The cells were divided into the number of portions that needed to be evaluated for different peptides, and each Ii-key peptide and the original peptide were added at a final concentration of 50μg / mL (refer to generex literature Ii-Key / HER-2 / neu(776–790) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2 / neu+tumors). The same operation was performed on CD8+T cells, and only the original peptide was used for stimulation culture to evaluate whether MHC-I class molecule epitopes were embedded. The cells were incubated at 37°C and 5% CO2; T cells were counted after three and five days of culture, and the amplified T cells were counted at 1×10 6 Cells / mL were diluted with complete medium and incubated at 37°C and 5% CO2. The cultured T cells were harvested on the seventh day of culture, counted separately, and prepared for downstream FluoroSpot assay.

[0105] (2) Cultivation of autologous DCs: Simultaneous use of ImmunoCult TM The Dendritic Cell Culture Kit is used to culture CD14+ monocytes for DC differentiation. Prepare ImmunoCult using sterile techniques according to the instructions. TM DC differentiation medium (ImmunoCult TM -ACF Dendritic Cell Medium + ImmunoCult TM-ACF Dendritic Cell Differentiation Supplement), thaw the differentiation supplement at room temperature or 37°C until just thawed, and use the aliquots immediately after thawing. Do not refreeze, add 100 μL of differentiation supplement to 9.9 mL of dendritic cell culture medium, mix thoroughly; incubate the cell suspension at 37°C in a six-well cell culture plate for 3 days; on the third day, remove the culture medium, add it to a 15 mL centrifuge tube, and quickly add 5 mL of fresh ImmunoCult TM DC differentiation medium (this step is for treating suspension cells and adherent cells), centrifuge the tube at 300×g for 10 min, and discard the supernatant. Use a small amount of fresh ImmunoCult TM Resuspend cells in DC Differentiation Medium (i.e., 50 μL or no more than 10% of the original volume) and return to the culture flask (this step can preserve non-adherent cells or loosely adherent cells); incubate at 37°C for 2 days; on day 5, thaw ImmunoCult TM Dendritic cell maturation supplement, mix thoroughly, add maturation supplement directly to the culture flask at a dilution of 1:100, vortex gently to mix, no need to change the medium; on day 7, gently pipette up and down to harvest fully mature DCs, ensuring that all cells are in suspension, then transfer to an appropriate test tube, count, and prepare for downstream FluoroSpot assay

[0106] 2.3 In vitro FluoroSpot assay of peptide-loaded DCs for CD4+ and CD8+ T cells

[0107] Mature DCs were harvested, counted in 1 mL, and estimated to be 1 / 10 of the total number of CD4 / 8 T+ cells required after elispot plating. They were mixed with 25 ug / mL of the original peptide for loading and incubated at 37°C for 4 hours. The peptide-loaded individual DCs were mixed with individual T cells at a concentration of 1:10. The total number of cells mixed with individual CD4+, CD8+ T cells and DC cells was adjusted accordingly based on the actual final count (1-3×10 5), ensure that the number of cells of a single individual is the same in each well, and spread the FluoroSpot pre-coated plate at a concentration, and follow the operating steps of the Mabtech reagent manual. The general steps are: wash the pre-coated plate with PBS (200uL / well) three times, add 200μL serum-free medium (Serum-free medium for ELISPOT) to each well, and remove it after standing at room temperature for 30 minutes. The counted cell suspension is added to each experimental well, positive control well, and negative control well at 100μL / target cell number / well, and serum-free medium is added to the background control well; add a peptide stimulus with a final concentration of 10μg / mL to the experimental well, and add a positive stimulator 5μg / mL PHA to the positive control well; cover the plate and place it in a 37°C, 5% CO2 incubator for 18 to 24 hours. On the second day, pour out the cells and culture medium in the wells, add ice-cold deionized water, 200 μL / well, and place in a 4°C refrigerator for 10 minutes to hypotonic lyse the cells; discard the liquid in the wells, use PBS or washing buffer, 200-260 μL / well, wash the plate 5-6 times, stay for one minute each time, and dry it on absorbent paper each time; add the diluted biotinylated antibody (biotinylated Add 100μL / well of the working solution of ELISpot (antibody) to each experimental well, incubate at 37℃ for 1 hour or 2 hours; repeat the plate washing 5-6 times; add 100μL / well of the diluted enzyme-labeled avidin (streptavidin-HRP or ALP) (FluoroSpot is a fluorophore conjugate reagent) working solution to each experimental well, incubate at 37℃ or room temperature for 1 hour; repeat the plate washing 5-6 times, add 50μL / well of the fluorescence enhancer (FluoroSpot) to each experimental well; let it stand at room temperature and away from light for 5-30 minutes, and ELISpot will choose the termination time of color development according to the spot generation. Pour out the liquid in the well and uncover the base of the plate; place the plate in a cool place at room temperature, and pay attention to avoid light throughout the process. Use Mabtech IRIS plate reader to interpret the fluorescent spots, and the result judgment standard is: when the result of the PHA control well is normal, the positive cut-off value is the average number of SFU in the negative control well plus two standard deviations. The overall experimental steps are summarized as a technical roadmap ( Figure 5 ).

[0108] VAC are all PBMCs of inactivated vaccine recipients that can produce positive reactions to this peptide.

[0109] (1) The Ii-key series peptides of peptide P1 were used to test the CD4+ T cells isolated and amplified from the PBMC samples of VAC-4. Finally, the number of cells was 2×10 5 / wells were tested for IFN-γFluoroSpot, and the positive PHA wells were all normal. The results showed that both the Ii-key series peptides and the original peptides could produce a good CD4+T cell response, proving that this peptide contained an epitope that could bind to the HLA-II class of VAC-4 individuals, and the immune response results of the Ii-key series peptides were better, so P1-KEY1-KEY4 was selected as the Ii-key peptide of this application ( Figure 6 ).

[0110] The Ii-key series peptides of peptide P5 were used to test the CD4+T cells isolated and amplified from the PBMC samples of VAC-6. Finally, the number of cells was 1.5×10 5 / wells were tested for IFN-γFluoroSpot, and the positive PHA wells were all normal. The results showed that this peptide contained an epitope that could bind to the HLA-II class of VAC-6 individuals, but compared with the original peptide P5-O, the peptides produced by culturing CD4+T cells with P5-KEY1, P5-KEY2, and P5-KEY3 could not produce a better response from CD4+T cells, proving that these three Ii-key peptides could not produce a presentation efficiency superior to that of the original peptides, while P5-KEY4 could produce a response slightly superior to that of P5-O, stimulating the cultured CD4+T cells to secrete IFN-γ with a slightly higher number of SFUs than the original peptides. Therefore, the Ii-key peptide of P5-KEY4 was selected as the final form of this study ( Figure 7 ).

[0111] The Ii-key series of peptides P7 were used to test the CD4+T cells isolated and amplified from the PBMC samples of VAC-7. Finally, the number of cells was 2.5×10 5 / wells were tested for IFN-γFluoroSpot, and all positive PHA wells were normal. The results showed that the original peptide P5-O may not contain epitopes with high affinity for the HLA-II genotype of the VAC-7 individual. However, compared with the original peptide P7-O, the CD4+T cells cultured with P7-KEY3 produced a positive IFN-γSFU response. It is speculated that the core region of P7 binding to the HLA-II molecules of the VAC-7 sample individuals may not be the same as predicted, and the anchoring form of P-7KEY3 can just enable the core region to bind to the HLA-II antigen binding groove of VAC-7. It is speculated that the initial positive reaction of the P7 peptide to VAC-7 may be based on the function of its embedded HLA-I epitopes. The Ii-key peptide of P7-KEY3 was selected as the final form of this study ( Figure 8 ).

[0112] The Ii-key series peptides of peptide P14 were used to test the CD4+T cells isolated and amplified from the PBMC samples of VAC-1. Finally, the number of cells was 2.5×10 5 / wells were tested for IFN-γFluoroSpot, and all positive PHA wells were normal. The results showed that the original peptide P14-O contained an epitope that could bind to the HLA-II class genotype of VAC-1 individuals with a high affinity. Compared with the original peptide P7-O, all Ii-key series peptides exerted better CD4+ presentation function and response level, and the stimulated cultured samples all produced high CD4+IFNγSFU. Finally, all Ii-key peptide sequences with better effects than the original peptides were selected as the declared sequences. See Fig. 9 .

[0113] The Ii-key series of peptides P15 were used to test the CD4+T cells isolated and amplified from the PBMC samples of VAC-2. Finally, the number of cells was 3×10 5 / wells were tested for IFN-γFluoroSpot, and all positive PHA wells were normal. The results showed that the original peptide P5-O contained an epitope that could bind to the HLA-II class genotype of VAC-2 individuals with a high affinity. Compared with the original peptide P15-O, all Ii-key series peptides exerted better CD4+ presentation function and response level, and the stimulated cultured samples all produced high CD4+IFNγSFU. Finally, all Ii-key series peptides were selected for application. See Fig.10 .

[0114] The Ii-key series of peptides P17 were used to test the CD4+T cells isolated and amplified from the PBMC samples of VAC-13. Finally, the number of cells was 2.5×10 5 / wells were tested for IFN-γFluoroSpot, and all positive PHA wells were normal. The results showed that the original peptide P17-O contained an epitope that could bind to the HLA-II class genotype of VAC-13 individuals with a high affinity. Compared with the original peptide P17-O, all Ii-key series peptides exerted better CD4+ presentation function and response level, and the stimulated cultured samples all produced high CD4+IFNγSFU. Finally, all Ii-key peptides were selected as the final form of this study. See Fig.11 .

[0115] In summary, the Ii-key T cell epitope peptides finally selected in this study are shown in Table 5:

[0116] Table 5

[0117]

[0118]

[0119] (2) The original peptides of P1, P5, P7, P14, P15 and P17 were used to test the CD8+T cells isolated and amplified from individual PBMC samples. It was found that each peptide could stimulate the production of a large amount of IFN-γ by the CD8+T cells of individual PBMC, indicating that these HLA-DR peptides have epitopes that can bind to HLA-I molecules and stimulate the production of CD8+T cell responses. Fig.12 .

[0120] 2.5 In vivo validation of Ii-key peptide in HLA-DR4 transgenic mice

[0121] The Ii-key linked polypeptide with the best immune effect among the six polypeptides was selected and combined with aluminum phosphate and aluminum hydroxide adjuvants to immunize HLA-DR4 humanized transgenic mice, and T cell epitope peptides for C57BL / 6J mice were selected as polypeptide controls; 14 9-11 week old male HLA-DR4 transgenic mice were randomly divided into 3 groups with 5 mice in each group (4 in the adjuvant control group), one group was immunized with 6 Ii-key T cell peptides, one group was immunized with 3 mouse T cell peptides, and one group was used as adjuvant control; subcutaneous immunization was performed at the base of the tail on days 0 and 21, respectively (100 μL / mouse), with 30 μg / mouse for each T cell polypeptide.

[0122] Table 6 Immunization schemes for mice in different groups

[0123]

[0124] Note: n: quantity.

[0125] On the 35th day, HLA-DR4 mice were humanely killed, and the spleen was removed to isolate lymphocytes. The DR4-6key-pep group mice and the adjuvant control group mice were stimulated with 6 Ii-key peptides, and the DR4-3M-pep group mice were stimulated with 3 mouse peptides. The intracellular factors IFN-γ, IL-2, TNF-α, and IL-4 of splenic CD4+T cells were detected by flow cytometry. The detection was performed on a BD FACSCantoII flow cytometer, and the data were analyzed using Flowjo analysis software ( Fig.13 The results showed that the CD4+IFNγ+ cells, CD4+IL-2+ cells and CD4+TNF-α+ cells produced by DR4-6key-pep group HLA-DR4 mice stimulated by 6 Ii-key peptides were higher than those of DR4-3M-pep group mice and adjuvant control group mice ( Fig.14), and there was no significant difference in the results between the DR4-3M-pep group and the adjuvant control group, and there was no significant difference in CD4+IL-4+, indicating that the seven Ii-key peptides can stimulate HLA-DR4 mice to produce a good CD4-biased Th1 cellular immune response.

[0126] In summary, through in vitro and in vivo experiments, peptides that can enhance presentation efficiency and have a broad spectrum of HLA I-II class alleles in the Chinese population and the world population were screened. After linking to Ii-key, they produced stronger CD4+T cell responses than the original peptides, and could also produce CD8+T cell responses.

[0127] Example 3: Challenge experiment of mixed polypeptide double transgenic mice

[0128] In this study, HLA-DR4 / H11-K18-ACE2 double transgenic humanized mice were used to evaluate the protective effect of subcutaneous and intranasal immunization of HP vaccine against the virus. The mice were divided into three groups: HP-sc group (n=9), HP-in group (n=9) and control group Adju-Control-sc group (n=8). On the 29th day, the mice were transferred to the BSL-3 laboratory and challenged on the 30th day (0dpc). In order to widen the difference in lung viral load between the experimental group and the control group mice and verify the protectiveness of the HP vaccine, a higher dose of 2,0000TCID50 BA.5.2 / 50μL / mouse was used for the challenge, and the mice were infected by intranasal route. At 4dpc, 3 mice in the experimental group and 4 mice in the control group were killed. At the time point when the mice in the control group died or were dying, 4 mice in each group were killed. Assuming that the mice in the experimental group continue to survive, the last two surviving mice were selected and killed at 10 dpc, and the viral load and pathological conditions of mice in different groups at each time point were analyzed. Fig.15 .

[0129] The results showed that on day 8, all mice in the control group were dying (quadriplegia, unable to walk) or died, with a weight loss of about 25%, while all mice in the HP-sc group survived ( Fig.16 ), and their weight increased after the challenge, and they were in good condition. The weight of the mice in the HP-in group was slightly better than that of the control group at 3-4 dpc, but they began to lose weight at 5 dpc, and the decline was similar to that of the control group ( Fig.16 In terms of performance, the mice did not die on the 8th day, but they became weak and trembling, but could still walk, which was better than the control group mice. On the 10th day, one mouse died and the other survived ( Fig.16 A).

[0130] The lungs and brains of the control group mice were tested for SARS-CoV-2N gene copy number (e.g. Fig.17 After 4 days (i.e. 4 dpc), high levels of viral load were detected in the lungs, with the number of copies / μL being around 10 5 Up to 10 6 between( Fig.17 However, both the HP vaccine subcutaneous immunization group and the intranasal immunization group showed extremely low lung viral load at 4 dpc (10 1 This indicates that HP vaccine can produce good antiviral effects at 4dpc after subcutaneous and intranasal immunization of HLA-DR4 mice. At 8dpc, the lung viral load of the control group mice decreased by an exponential level, while the lung viral load of the subcutaneous immunization group mice was still very low (10 1 Viral nucleic acid copies / μL). Two mice in the intranasal immunization group had relatively low numbers (10 2 -10 3 The number of viral nucleic acid copies / μL) was slightly increased in one mouse in the subcutaneous immunization group at 10 dpc, but two of them were the same as the control group. This corresponds to the change in body weight, indicating that the intranasal immunization group mice could not prevent the progression of viral infection at 4-8 dpc. At 10 dpc, one mouse in the subcutaneous immunization group had a slightly increased lung viral load (10 2 Viral nucleic acid copies / μL), while the other remained low (10 1 The number of viral nucleic acid copies / μL). This indicates that mice in the subcutaneous immunization group almost avoided viral infection in the lungs. However, mice in the intranasal immunization group also showed a decrease in lung viral load at 10dpc, which corresponded to the decreasing trend of lung viral load in the control group at 4-8dpc.

[0131] The peptides screened in this application are the first to prove that a synthetic peptide mixture can protect a lethal mouse model from complete survival with only one booster immunization. The experimental design and results of this study strongly refute the discussion of Victoria K. Baxter et al. that 16 chemically synthesized mixed peptides did not provide any protection in a mouse challenge experiment.

Claims

1. A Ii-key T cell polypeptide composition, characterized in that: Ii-key is connected to the N-terminus of the antigen peptide, and the polypeptide composition consists of the following polypeptides: The antigenic peptide shown in SEQ ID NO.1; 4 amino acids are deleted from the N-terminus of the antigenic peptide shown in SEQ ID NO.2; 2 amino acids are deleted from the N-terminus of the antigenic peptide shown in SEQ ID NO.3; 2 amino acids are deleted from the N-terminus of the antigenic peptide shown in SEQ ID NO.4; 1 amino acid is deleted from the N-terminus of the antigenic peptide shown in SEQ ID NO.5 and the antigenic peptide shown in SEQ ID NO.6, and the Ii-key and the antigenic peptide are connected by 5-aminovaleric acid.

2. Use of the Ii-key T cell polypeptide composition according to claim 1 in the preparation of a new coronavirus vaccine, wherein the new coronavirus is a variant of Omicron.

3. The use according to claim 2, characterized in that: The vaccine also contains an adjuvant.

4. The use according to claim 3, characterized in that: The adjuvant is one or more of aluminum salt, emulsion, liposome and cytokine.

5. The use according to claim 4, characterized in that: The aluminum salt includes: alum; the emulsion includes: Titermax.

6. A vaccine for preventing infection with the new coronavirus, characterized in that: The vaccine includes the Ii-key T cell polypeptide composition described in claim 1, and the new coronavirus is the Omicron variant.

7. The vaccine according to claim 5 or 6, characterized in that The vaccine also contains an adjuvant.

8. The vaccine according to claim 7, characterized in that The adjuvant is one or more of aluminum salt, emulsion, liposome and cytokine.

9. The vaccine according to claim 8, characterized in that The aluminum salt includes: alum; the emulsion includes: Titermax.

10. A drug, characterized in that The medicine includes the Ii-key T cell polypeptide composition according to claim 1.

11. The drug according to claim 10, characterized in that The medicine also includes pharmaceutically acceptable excipients.

12. The drug according to claim 11, characterized in that The pharmaceutically acceptable excipients are one or more of mannitol, sucrose, sodium chloride, magnesium chloride, polysorbate 80, glycerol and hydroxyethylpiperazine ethanesulfonic acid.

13. The drug according to any one of claims 10 to 12, characterized in that The dosage form of the drug is injection.

14. The drug according to claim 13, characterized in that The injection is one of water injection, oil injection or powder injection.

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