CN117964720A - HLA-A 02:01 restrictive SARS-CoV-2T cell epitope peptide and its application - Google Patents
HLA-A 02:01 restrictive SARS-CoV-2T cell epitope peptide and its application Download PDFInfo
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Abstract
The invention relates to HLa-a 02:01 restrictive SARS-CoV-2T cell epitope peptide and its application. The invention utilizes an immunoinformatics method and uses an IFN-gamma ELISPOT method to obtain HLa-a 02:01 restricted CTL epitope peptide by in vitro screening, and simultaneously performs screening verification of candidate vaccine epitopes in a B-HLa-a2.1 mouse model. Finally, 12 epitope peptides N1-N4 and A1-A8 are screened out, DNA candidate vaccines pCMV-4-N1-N4 and pCMV-8-A1-A8 are successfully constructed, and immune and virus attack protection verification is carried out in a B-HLa-a2.1 mouse model; wherein the A1, A2, A3, A4 and N2 epitope peptide fragments show good immunogenicity and conservation in the B-HLa-a2.1 mouse model. The results related to the toxicity attack of the mice after the immunization of the DNA vaccine disclosed by the invention show that the epitope peptide fragment based on prediction and preliminary test has HLa-a 02:01 restrictive immunogenicity, can induce obvious protective immune response, and is expected to provide candidates for the development of novel COVID-19 vaccines.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to HLA-A 02:01 restrictive SARS-CoV-2T cell epitope peptide and application thereof, especially application in preparing SARS-CoV-2 candidate vaccine.
Background
The global COVID-19 pandemic caused by SARS-CoV-2, which first appeared at the end of 2019, entered epidemic period after three years. Vaccines produced either on classical vaccine platforms (such as inactivated viruses, recombinant viral vectors and recombinant subunit vaccines) or on emerging platforms (most notably synthetic mRNA vaccines) play a vital role in reducing the damage caused by pandemics. The development and evaluation of SARS-CoV-2 vaccine candidates has generally focused on the induction and enhancement of antibodies that neutralize spike protein (S), which is responsible for receptor binding and viral entry. However, both infection-induced and vaccine-induced antibody selection resulted in significant acceleration of S protein variation relative to other structural and non-structural proteins. Thus, currently licensed vaccines need to be updated periodically to provide adequate protection against the emerging major variants of SARS-CoV-2. On the other hand, antibody titers in vaccinators and infected patients have been shown to decrease faster, resulting in reduced protection against re-infection, which requires additional injections.
In addition to neutralizing antibodies against the S protein, cellular immune response is also another important component of protective immunity to SARS-CoV-2. Unlike neutralizing antibodies, cellular immune responses are not limited to spike proteins, and can be directed against epitopes in all viral-encoded structural and non-structural proteins. For this reason, T cell immunity against conserved epitopes common to SARS-CoV-2 sublines and variants can theoretically provide significant cross protection, even against future variants. While some currently licensed vaccines targeting the S protein have been demonstrated to induce T cell responses longer than antibody responses, vaccine platforms focused on inducing T cell responses are expected to perform better in this regard. Indeed, a number of SARS-CoV-2T cell vaccine candidates have been studied and are currently in various stages of development.
Immunoinformatics-based T cell epitope prediction has become an increasingly popular and important approach for epitope discovery for prophylactic, therapeutic and other related applications. Epitopes predicted in vitro can be validated by analysis of their T cell recognition by infected patients. Prediction based on immunoinformatics can significantly reduce the number of epitopes that need to be tested and potentially reduce costs in terms of time, resources and human samples compared to epitope screening using various forms of epitope libraries. The validated predicted epitopes will form the basis of T cell vaccine design, and the constructed vaccine candidates will be subjected to immunogenicity and protective efficacy tests in humanized animal models and human subjects. This T cell vaccine development strategy has also been applied to SARS-CoV-2, but the studies published so far have generally involved epitopes from a limited number of viral proteins, limited to in vitro assays, or failed to provide in vivo efficacy test results.
Thus, there is an urgent need to develop a high-efficiency vaccine that is widely covered and has complete and long-lasting immunogenicity.
Disclosure of Invention
In order to overcome at least one of the problems of the prior art, the T cell epitope prediction algorithm based on immunoinformatics, such as IEDB, allows in vitro prediction of specific length and MHC specific potential immunogenic epitopes, the present invention performed immunoinformatics-based HLA-A 02:01 restriction epitope prediction on the whole proteome encoded by SARS-CoV-2 (Wuhan-hu-1). The present invention uses peripheral blood mononuclear cells of convalescent period martial (Wuhan-hu-1) infected patients to evaluate the immunogenicity of the predicted epitope. In addition, predicted conserved epitopes are used to construct DNA vaccines that express multi-epitope peptides. Most importantly, both DNA vaccine constructs induced an epitope specific CD8 + T cell response in HLA-A 02:01 restricted mouse model and protected mice from Wuhan-hu-1 virus in the challenge test after hACE2 transduction. These data provide candidate T cell epitopes for the development of novel T cell vaccines against SARS-CoV-2 and demonstrate a strategy for rapid T cell vaccine candidate development that can be applied to other emerging pathogens.
According to the invention, through immunoinformatics analysis of all encoding proteins of a new coronavirus original strain, aiming at HLA-A 02:01 alleles which are dominant in Chinese population, full coverage analysis is carried out on SARS-CoV-2 encoding protein sequences by adopting an immunoinformatics method, HLA-A 02:01 restrictive epitopes in the encoding protein sequences are predicted, and an ELISPOT detection method aiming at corresponding epitope CTL reactions is established through chemical synthesis of epitope peptides; preliminary tests were carried out on the immunogenicity of part of the predicted epitopes during natural infection and during artificial immunization of wild-type and HLA-A 02:01 transgenic mice; the invention also constructs SARS-CoV-2 candidate DNA vaccine, and performs immunization and toxicity test verification of the DNA vaccine in a B-HLA-A2.1 mouse model only expressing human HLA-A 02:01 restriction.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect, the present invention provides an HLA-A 02:01 restricted SARS-CoV-2T cell epitope peptide, comprising the steps of: full coverage analysis is carried out on SARS-CoV-2 coding protein sequence by adopting an immunoinformatics method, and HLA-A 02:01 restriction epitope of 14-site amino acid is predicted; chemically synthesizing epitope peptide based on the prediction result, and screening out specific epitope peptide with immune response in vitro; the epitope peptide obtained by in vitro screening and/or the epitope peptide synthesized chemically are subjected to conservation analysis, and T cell epitope peptide with high conservation and strong immunogenicity in SARS-CoV-2 original and other epidemic strains is screened.
The number of amino acid positions of the epitope peptide may be adjusted according to the experimental requirements, and may be, for example, amino acid 13, amino acid 15, amino acid 16, etc.
Further, in the in vitro screening process, the ELISPOT detection method for the corresponding CTL epitope reaction was established by chemically synthesized epitope peptides.
Further, the use of the immunoinformatics method tool IEDB (http:// tools. IEDB. Org/mhci /) predicts that the MHC class I parameter of the whole protein of SARS-CoV-2 virus is HLA-A.02:01, the species is CTL epitope of amino acid 14 of human, all predicted epitopes PERCENTILE RANK.ltoreq.2, predictive screening and chemical synthesis of 524 polypeptides (as shown in Table 1 in the examples).
Further, the T cell epitope peptides are located in all virally encoded structural and non-structural proteins, predominantly distributed over the S protein, ORF1ab, M and E proteins.
Further, the amino acid sequence of the CTL epitope peptide comprises sequences shown in SEQ ID NO. 1-SEQ ID NO. 47.
Specifically, human Peripheral Blood Mononuclear Cells (PBMCs) are used for evaluating the reaction of human IFN-gamma ELISPOT PRO bands on 524 epitope peptides targeted by SARS-CoV-2 protein, and the antigen specific reaction is measured, so that 34 specific epitope peptides with immune reaction are screened out, and the amino acids of the specific epitope peptides are SEQ ID NO. 1-SEQ ID NO. 34. The 34 epitope peptides are ：M-9、N28274-29533-2、ORF1ab1-1400aa-22、ORF1ab1-1400aa-26、ORF1ab1-1400aa-38、ORF1ab1-1400aa-4、ORF1ab1-1400aa-47、ORF1ab1-1400aa-60、ORF1ab1-1400aa-72、ORF1ab1-1400aa-82、ORF1ab1-1400aa-84、ORF1ab2787-4200aa-10、ORF1ab2787-4200aa-11、ORF1ab2787-4200aa-120、ORF1ab2787-4200aa-125、ORF1ab2787-4200aa-130、ORF1ab2787-4200aa-25、ORF1ab2787-4200aa-27、ORF1ab2787-4200aa-28、ORF1ab2787-4200aa-29、ORF1ab2787-4200aa-31、ORF1ab2787-4200aa-38、ORF1ab2787-4200aa-4、ORF1ab2787-4200aa-42、ORF1ab2787-4200aa-44、ORF1ab2787-4200aa-56、ORF1ab2787-4200aa-62、ORF1ab2787-4200aa-89、ORF1ab2787-4200aa-90、ORF1ab2787-4200aa-94、ORF1ab4187-4405aa-3、ORF1ab4187-4405aa-6、ORF1ab4397-5796aa-36、ORF1ab2787-4200aa-73., and in particular, due to the difference of T cell responses caused by the same virus infection of patients with similar HLA-A genetic background, in order to expand the range of candidate vaccines, the 524 synthetic epitope peptides are subjected to conservative analysis poetry anthology to obtain 13 epitope peptides, the amino acids of which are SEQ ID NO. 35-SEQ ID NO.47, in particular ：ORF1ab1-1400aa-23、ORF1ab4379-5796aa-58、S-36、S-52、ORF1ab2787-4200aa-60、ORF1ab1-1400aa-80、ORF1ab1-1400aa-56、M-10、ORF1ab1387-2800aa-4、ORF1ab1387-2800aa-62、ORF7a-8、ORF1ab1387-2800aa-24、S-47.
Further, the amino acid sequences of the CTL epitope peptides are shown as SEQ ID NO. 1-SEQ ID NO.12 and SEQ ID NO. 35-SEQ ID NO. 47.
Further, the T cell epitope peptide comprises N1-N4 polypeptides and A1-A8 polypeptides, wherein the amino acid sequences of the N1-N4 polypeptides are respectively shown in SEQ ID NO. 1-SEQ ID NO.4, and the amino acid sequences of the A1-A8 polypeptides are respectively shown in SEQ ID NO. 35-SEQ ID NO. 42.
Further, the T cell epitope peptide comprises an N2 polypeptide and an A1-A4 polypeptide, wherein the amino acid sequence of the N2 polypeptide is shown as SEQ ID NO.2, and the amino acid sequences of the A1-A4 polypeptides are respectively shown as SEQ ID NO. 35-SEQ ID NO. 38.
Specifically, the 34 epitope peptides screened in vitro are subjected to conservative analysis with a main epidemic virus strain of SARS-CoV-2 by using NCBI BLAST tool to obtain 12 epitope peptides, the corresponding amino acid sequences are SEQ ID NO. 1-SEQ ID NO.12, wherein the percentile Rank is less than or equal to 1, and 4 epitope peptides are obtained and respectively named as: N1-N4, the corresponding amino acid sequence is SEQ ID NO. 1-SEQ ID NO.4. And performing conservation analysis poetry anthology on 524 synthesized epitope peptides to obtain 13 epitope peptides, wherein the percentile Rank is less than or equal to 1,8 epitope peptides are obtained, which are respectively named A1-A8, and the corresponding amino acid sequences are SEQ ID NO. 35-SEQ ID NO.42.
Further, in the above-described conservation analysis, the main epidemic strains include Gamma(P.1),Delta(B.1.617.2,AY.3.1,AY.3,AY.25.1,AY.25,AY.39.1,AY.39,AY.44,AY.47,AY.100,AY.103),Beta(B.1.617.2,AY.3.1,AY.3,AY.25,AY.103),Omicron(B.1.1.529,BA.2.3,BA.2.9,BA.2.12,BA.2.18,BA.2.37,BA.2,BA.4,BA.5).
Further, in the screening process, the method further comprises the steps of: the epitope peptide obtained by in vitro screening was compared with sequences of other viral genes excluding SARS-CoV-2, and cross-reactive sequences conserved in different coronaviruses were identified.
Further, the amino acid sequence of the cross-reactive sequence is selected from the sequences set forth in SEQ ID NO. 48-SEQ ID NO. 55.
Specifically, NCBI-Blast-Protein (https:// www.medicgo.org) is used for comparing sequences of 34T cell epitope peptides obtained by in vitro screening with other viral genes, the screening is used for removing epitope peptides with homology and cross property, matching degree and coverage rate of 100% in other coronaviruses, 8 epitope peptides are finally screened to have conserved sequences in different coronaviruses, the corresponding amino acid sequences are SEQ ID NO. 48-SEQ ID NO.55, wherein 1M Protein and other 7 targeted antigens of ORF1ab proteins are specifically ：M-9、ORF1ab1-1400aa-26、ORF1ab2787-4200aa-11、ORF1ab2787-4200aa-10、ORF1ab2787-4200aa-25、ORF1a2787-4200aa-38、ORF1a2787-4200aa-90.
It is understood that each of the above amino acid sequences encompasses amino acid sequences in which any of the above sequences is substituted, deleted, inserted and/or added with 1 or more amino acid sequences without affecting the functionality thereof.
In a second aspect, the present invention provides a biomaterial associated with the T cell epitope peptide of any one of the first aspects of the invention, selected from one of a) to B):
a) A nucleic acid molecule encoding said T cell epitope peptide;
b) Recombinant vector and recombinant cell line containing the nucleic acid molecule as described in A).
Further, the above biological materials also include various preparations which can be prepared from the above nucleic acid molecules, recombinant vectors, recombinant cell lines, such as peptide-MHC tetramers, pMHC-TCR complexes, vaccines, detection reagents/kits, and the like.
It will be appreciated that nucleic acid molecules encoding epitope peptides are important for the production of epitope peptides within a host using genetic recombination techniques, and for vaccines, which may be delivered as naked nucleic acids, or may be delivered using appropriate viral or bacterial vectors. The recombinant vector may be a vector conventionally used in the art, for example pcDNA, pTT, pBV, pJV, pBJ, pCMV or the like. The recombinant cell line may be a host cell conventionally used in the art, such as HEK293-T cells, CHO cells, E.coli, yeast cells, and the like. The recombinant vectors described above may be transformed, transduced or transfected into host cells using methods conventional in the art, such as shock transformation and the like.
A third aspect of the invention provides the use of a T cell epitope peptide according to any one of the first aspect of the invention, or a biomaterial according to any one of the second aspect of the invention, selected from at least one of the following: the application of the kit in preparing a novel coronavirus infection or a cell immune response detection kit after infection, the application in preparing a novel coronavirus infected person screening detection reagent, the application in preparing a novel coronavirus preventive and therapeutic vaccine, the application in preparing an active or passive immunotherapy agent aiming at the novel coronavirus infection, and the application of the cross reaction sequences in preparing different coronavirus broad-spectrum vaccines.
Furthermore, in the active immunotherapy, the T cell epitope peptide can be used as a peptide vaccine, namely, the vaccine prepared from the T cell epitope peptide is administered to a patient, so that the vaccine plays a role in preventing and treating the infection of the novel coronavirus, and the used epitope peptide can be 1 or more than 2 peptide combinations; in passive immunotherapy, it comprises novel coronavirus-specific T cells obtained by stimulating peripheral blood lymphocytes with the aforementioned epitope peptides or antigen presenting cells presenting the epitope peptides to HLA.
Furthermore, the T cell epitope peptide can detect the frequency and the functional state (IFN-gamma secretion) of CD8 + T cells in a patient infected by the novel coronavirus, so that the T cell epitope peptide can be used as a novel coronavirus infection and a detection means of cellular immune response after the infection, and can also be used as a novel vaccine development aiming at the cellular immune response.
In a fourth aspect the invention provides a vaccine prepared from a T cell epitope peptide according to any one of the first aspects of the invention, or a biomaterial according to any one of the second aspects of the invention.
Further, the nucleic acid encoding the T cell epitope peptide can be used for preparing DNA vaccines, recombinant virus vector vaccines and the like; preferably, the vaccine is a DNA vaccine comprising at least one T cell epitope peptide.
It is understood that the vaccine may be administered by parenteral administration, including intravenous injection, subcutaneous injection, nasal administration, intramuscular injection and the like, and by oral administration, including suppositories; the dosage form for oral administration may use excipients conventionally used in the art, such as starch, cellulose, mannitol, magnesium stearate, lactose, and the like.
Further, the construction steps of the DNA vaccine comprise: and connecting each T cell epitope peptide sequence with a binding sequence, fusing the N end of the tandem epitope with a Flag tag, and cloning the homologous recombination on a vector plasmid to obtain the tandem epitope polypeptide expression plasmid. Specifically, the number of T cell epitope peptide sequences may be 1, 2, 3 …, etc., and if 1 epitope peptide is used, no binding sequence is required.
Further, the amino acid sequence of the binding sequence is EAAAK, the amino acid sequence of the Flag tag is DYKDDDDK, and the Vector plasmid is pCMV-Vector.
Further, a non-natural broadly mitotic CD4 + T cell epitope (PADRE) is also fused to the C-terminus of the tandem epitope via GPGPG linker.
Further, the tandem epitope polypeptide expression plasmids comprise pCMV-4-N1-N4 and pCMV-8-A1-A8.
Further, the amino acid sequence of pCMV-4-N1-N4 is shown as SEQ ID NO.56, and the amino acid sequence of pCMV-8-A1-A8 is shown as SEQ ID NO. 57.
Furthermore, the expression plasmids pCMV-4-N1-N4 and pCMV-8-A1-A8 respectively transfect HEK293-T cells and have good intracellular expression efficiency, and can be used for preparing recombinant DNA vaccines.
Specifically, specific information of each sequence described above is as follows:
The invention discloses a method for constructing and developing high-efficiency and long-lasting immunogenicity novel vaccine, which combines the virus epidemiological characteristics of SARS-CoV-2 by using an immunoinformatics screening method, and carries out full coverage analysis on SARS-CoV-2 coding protein sequence aiming at high-frequency HLA-A 02:01 alleles in Chinese population. Analyzing all coding protein sequences of SARS-CoV-2 original strain by using an immunoinformatics method, predicting to obtain 604 HLA-A 02:01 restrictive epitope from virus structural protein and non-structural protein, obtaining 524 epitope peptides by third-party chemical synthesis, and detecting the reactivity of PBMC of healthy individuals with 2 cases of HLA-A 02:01 genotype without new crown exposure history and 20 cases of HLA-A 02:01 genotype new crown original strain infected recovered individuals to grouping mixed epitope peptides and/or single epitope peptides by using an IFN-gamma ELISPOT method: only 1 healthy individual PBMC had a significant response to 1 epitope peptide, while 15 healthy individuals PBMC had a significant response to a plurality of groups of mixed epitope peptides, and 5 of them had a significant response to a total of 33 epitope peptide monopeptides, indicating that the relevant predicted epitope had some immunogenicity during the course of natural infection. Most of the 34 epitopes described above are highly conserved among the major new crown-pandemic variants. In addition, the invention also screens out the highly conserved 13 epitopes which are not recognized by the PBMC of the patient or the healthy donor from 524 synthetic epitopes, which suggests that the highly conserved epitopes have wider targeting as T cell vaccine epitope candidates and provide a new theoretical basis for preventing infection of SARS-CoV-2 as comprehensively as possible.
Meanwhile, based on the screening result, the immunogenic CTL epitope of the common human coronavirus may have cross with the CTL epitope of SARS-CoV-2, which means that the memory T cell aiming at the coronavirus with lower risk can target out the immunogenic epitope when being infected by SARS-CoV-2 and can be combined with the targeting epitope of other known coronaviruses with low infectivity, thereby being used for jointly constructing a vaccine for preventing SARS-CoV-2 in a broad spectrum. Specifically, among 33 epitope peptides screened by the rehabilitation sample, 8 epitope peptides have conserved sequences in different coronaviruses, wherein 1M protein and other 7 targeted antigens of ORF1ab proteins. These epitopes can continue to be validated in subsequent additional studies as conserved immunogens for the production of a variety of coronavirus pathogens, developing broad-spectrum vaccine candidates against different coronaviruses. Meanwhile, epitope peptide of targeted ORF1ab protein in other SARS-CoV-2 epidemic strains has better conservation in screening, and can be used for developing subsequent vaccines for more complete and comprehensive immunogens together with S protein, so as to cope with continuously mutated viruses.
In order to further screen and verify T cell epitopes close to candidate vaccines in an in vivo animal model, 12 polypeptides which are strong in immunogenicity and are highly conserved are selected, N1-N4 and A1-A8 sequences are respectively named, DNA vaccines pCMV-4-N1-N4 and pCMV-8-A1-A8 after codon optimization are constructed and identified, and systematic immunological evaluation and toxicity attack protection research are carried out in B-HLA-A2.1 mice. Experimental results show that pCMV-4-N1-N4, pCMV-8-A1-A8 and mixed immunization of the two can induce a CD8 + T cell immune response aiming at part of epitopes, wherein the immunogenicity and the specificity of the A1, A2, A3, A4 and N2 epitopes are better. In addition, the DNA vaccine constructed by the invention has the protection in the B-HLA-A2.1 model, reduces the virus titer of lung tissues and reduces the inflammatory reaction of the lung, thereby resisting the attack of SARS-CoV-2 virus and having the basic characteristics as a novel coronavirus vaccine candidate.
Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:
The invention predicts and successfully synthesizes 524 epitope peptides aiming at SARS-CoV-2 original virus strain whole protein HLA-A 02:01 based on an immunoinformatics method, and uses an IFN-gamma ELISPOT method to carry out specific epitope screening on PBMC samples of healthy and recovered patients; the invention obtains HLA-A 02:01 restrictive T cell epitope peptide by using an immunoinformatics method and in-vitro screening, and simultaneously carries out screening verification of candidate vaccine epitopes in a B-HLA-A2.1 mouse model and virus attack protection verification in the B-HLA-A2.1 mouse model. Specifically, the invention successfully predicts and synthesizes 524 SARS-CoV-2 epitope peptides, further screens out 12 epitopes, respectively named as N1-N4 and A1-A8, successfully constructs DNA candidate vaccine pCMV-4-N1-N4 and pCMV-8-A1-A8, and performs immunization and toxicity attack protection verification in a B-HLA-A2.1 mouse model, wherein A1, A2, A3, A4 and N2 epitope peptide segments show good immunogenicity and conservation in the B-HLA-A2.1 mouse model. The results related to the toxicity attack of the mice after the immunization of the DNA vaccine disclosed by the invention show that the epitope peptide fragments based on prediction and preliminary test have HLA-A 02:01 restrictive immunogenicity, can induce obvious protective immune response, and are expected to provide candidates for the development of novel COVID-19 vaccines.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a schematic diagram of the general concept of the present invention for constructing a SARS-CoV-2 vaccine candidate;
FIG. 2 is a schematic representation of the results of the reactivity of a pool of 524T cell epitope mixed peptides in a sample of human PBMC in accordance with one embodiment of the present invention; wherein A-T is a PBMC sample of different rehabilitation patients, and 1-26 are different mixed peptide pools; the peptide pool No. 26 contains 24 short peptides, and peptide pools No. 1-25 each contain 20 short peptides; comparison with negative control: [ SFC (peptide or pool) -SFC (mock) ]/SFC (mock), was considered to be a PBMC sample showing positive recognition relative to SFC.gtoreq.3, and graphPadprism 9.5 software generated this figure.
FIG. 3 is a schematic representation of the results of a conservative analysis of T cell epitopes in other epidemic coronavirus strains according to one embodiment of the invention; wherein, the abscissa is different epidemic strains of SARS-CoV-2, the ordinate is selected T cell epitope peptide, and the quantity is mutant amino acid; the closer to 0, the higher the epitope conservation; the ORF1ab protein has stronger conservation in different strains, and then M protein and N protein; N1-N4 and A1-A8 are the conserved epitope sequences of the subsequent candidate DNA vaccine, and all satisfy the percentile Rank less than or equal to 1.
FIG. 4 is a schematic diagram showing the results of construction and identification of SARS-CoV-2DNA vaccine according to an embodiment of the present invention; wherein, part A: connecting N1-N4 and A1-A8 epitope peptide sequences with EAAAK binding sequences, fusing the N-end of the tandem epitope with Flag tags, and cloning homologous recombination on pCMV-Vector, which are named pCMV-4-N1-N4 and pCMV-8-A1-A8 respectively; part B: transfecting pCMV-4-N1-N4 and pCMV-8-A1-A8 expression plasmids into HEK293T cells, wherein blank control holes are HEK293T cells, and detecting the expression of epitope polypeptides carrying Flag tags by an immunofluorescence method; part C: the expression of Flag tag recombinant proteins in cell lysates transfected with pCMV-Flag-N1-N4 and pCMV-Flag-A1-A8 is detected by Western blot, the calculated molecular weights are respectively 9.76 and 17.6kDa, the molecular weights are basically consistent with the predicted molecular weights, and M is Marker.
FIG. 5 is a schematic diagram showing the results of detection of IFN-gamma, a cytokine of SARS-CoV-2 recombinant DNA vaccine according to an embodiment of the present invention; wherein, part A: stimulation of B-HLA-A2.1 mice spleen lymphocyte suspension with 10mg/mL SARS-CoV-2N1-N4 and A1-A8 peptide Chi Tiwai for 8 hours after immunization; part B: flow cytometry examined IFN-gamma + production in CD8 + T cells following stimulation by different immune groups A1-A8; part C: flow cytometry detects IFN-gamma + production in CD8 + T cells following N1-N4 stimulation of different immune groups; the data results are shown as mean + standard error, and statistical analysis is performed by using a 2WAY-ANOVA test; * P <0.0001, < p <0.001, < p <0.05, 4 samples per group.
FIG. 6 is a schematic diagram showing the results of a recombinant DNA vaccine of one embodiment of the present invention to protect B-HLA-A2.1 mice from SARS-CoV-2 infection; wherein, part A: vaccinated mice received 100. Mu.L of SARS-CoV-2 (1.5X10 5 TCID 50) challenge and protection after the second immunization; part B: monitoring daily weight change; part C: collecting lung tissues, and detecting viral load by RT-qPCR; part D: lung histochemical analysis after H & E staining; data results are expressed as mean + Standard Error (SEM) and were statistically analyzed using the unpaired t-test with p <0.0001, < p <0.001, < p <0.01, 4 to 6 samples per group.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.
In a specific embodiment, the invention adopts an immunoinformatics method to carry out full coverage analysis on SARS-CoV-2 coding protein sequence aiming at HLA-A 02:01 alleles, predicts HLA-A 02:01 restriction epitope of 14 amino acids, establishes an ELISPOT detection method aiming at corresponding T cell epitope reaction through chemical synthesis of epitope peptide, screens out specific epitope peptide with immune reaction in vitro, and carries out preliminary test on immunogenicity of part of the predicted epitope in natural infection process and artificial immunity wild type and HLa 02:01 transgenic mice on the basis; the epitope peptide obtained by in vitro screening and/or the epitope peptide synthesized chemically are subjected to conservation analysis, the epitope peptide with stronger immunogenicity which is highly conserved in SARS-CoV-2 original and other epidemic strains is screened out, so that SARS-CoV-2 candidate DNA vaccine is constructed, and immunization and toxicity attack experiments of the DNA vaccine are carried out in a B-HLA-A2.1 mouse model only expressing human HLA-A 02:01 restriction. The above steps are specifically shown in fig. 1.
In the following examples, samples of peripheral blood mononuclear cells from a rehabilitee: PBMC (labeled A-T) were prepared from peripheral blood samples of 20 cases of HLA-A 02:01 homotype SARS-CoV-2 (Wuhan-hu-1) infected rehabilitation patients hospitalized with Shanghai public health clinical center at the beginning of 2020 using Percoll isolation (Sigma, china) (see Table 3 below) and HLA-A sequencing was performed using PCR-SBT technique. Healthy blood sample: two PBMC samples collected from healthy blood donors prior to the end of 2019 were purchased from Shanghai Baite Biotech Inc. All PBMC samples were cultured in R10 medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin, 2mM L-glutamine, 20mM HEPES, all from Siemens technology, U.S.A.) at the Shanghai medical college laboratory animal center at the university of double denier.
Example 1-HLA-A.02:01 selection of restricted SARS-CoV-2T cell epitope peptide
This example screens specific HLA-A 02:01 restricted T cell epitope peptides with strong immunogenicity according to analysis of SARS-CoV-2 whole protein sequence, which specifically comprises the steps of:
(1) SARS-CoV-2 protein sequence search and sequence analysis
All protein amino acid sequences (GenBank accession NC-045512) encoded by SARS-CoV-2 (Wuhan-hu-1) for epitope prediction were searched. To analyze epitope conservation, sequences corresponding to major lineages and variants were searched for, including Beta(variant B.1.351/MZ433432.1),Gamma(variant P.1/MZ477859.1),Delta(variants B.1.617.2/OK091006.1,AY.3.1/ON834880.1,AY.3/OQ905725.1,AY.25.1/ON834811.1,AY.25/ON834805.1,AY.39.1/ON834816.1,AY.39/ON834809.1,AY.44/ON834818.1,AY.47/ON834815.1,AY.100/ON834822.1,AY.103/ON834807.1,Omicron(variants B.1.1.529/OM570283.1,BA.2.3/OR352440.1,BA.2.9/OR325311.1,BA.2.12/OP790345.1,BA.2.18/OR325322.1,BA.2.37/OR325193.1,BA.2/ON834972.1,BA.4/OR325403.1,BA.5/OR277772.1).
(2) Prediction and synthesis of T cell epitopes
All protein sequences encoded by SARS-CoV-2 (Wuhan-hu-1) were submitted to the Immune Epitope Database (IEDB) (http:// tools. IEDB. Org/mhci /) for predicting 14 amino acid epitopes limited by HLA-A.02:01, and further analysis was performed using the NETMHCPAN EL 4.1.1 algorithm (see Reynisson B,Alvarez B,Paul S,et al.NetMHCpan-4.1and NetMHCIIpan-4.0:improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data[J].Nucleic Acids Res,2020,48(W1):W449-W454). select epitopes with "percentile rank" values equal to or less than 2 (PERCENTILE RANK.ltoreq.2).
The method predicts HLA-A 02:01 restrictive T cell epitope in SARS-CoV-2 protein group based on immunoinformatics, specifically: to screen and verify the potential CD8 + T cell epitope of SARS-CoV-2, all ORFs encoded by the original Wuhan-hu-1 isolate were submitted to IEDB software, predicting the epitope presented by the 14 amino acid peptide fragment by HLA-A x 02:01 (one of the most common subtypes of HLA-A). A total of 604 epitopes were predicted (percentile rank.ltoreq.2, see table 1 below) and the epitope peptides were located in almost all structural and non-structural proteins and included about 30-90% of the length of the corresponding protein (see table 2 below). All 604 predicted epitopes were chemically synthesized (gold, china), but only 524 were successfully synthesized and purified to > 85%, so that 524 epitopes were used for subsequent research analysis. The predicted polypeptide sequences are shown in Table 1 below:
TABLE 1 HLA-A 02:01 restrictive CTL epitope peptide predicted by SARS-CoV-2 holoprotein
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Note that: each short peptide is 14 amino acids in length, and two short peptides overlap by 9 amino acids. According to the rank value of IEDB software NETMHCpanCL4.1, the sequence is considered to have better specificity with HLA-A 02:01. Pool number is the pool number of the mixed peptide when the samples of the convalescence patients are screened. A total of 524 short peptides from No.1 to No. 26 were studied. "none" means that the short peptide meets the predicted conditions, but is not successfully synthesized in the subsequent chemical synthesis, and therefore does not participate in the experimental study.
(3) Polypeptide stimulated PBMC detection Using IFN-gamma ELISpot assay
The polypeptides were divided into 26 groups, each containing 20 or 24 peptides (see peptide pool number in Table 1), and the recognition of PBMC from healthy and convalescent patients was initially examined. The final concentration of polypeptide per group was 5. Mu.g/mL, or the polypeptide pools each stimulated PBMC (2X 10 5 cells) at 5. Mu.g/mL. After 24 hours, IFN-gamma production was detected using the IFN-gamma ELISPot assay (human IFN-gamma (ALP) ELISPot Pro, mabtech). The negative control was unstimulated cells and the positive control was anti-CD 3 stimulated cells. Counts were used iSpot (AID, germany) and the results were expressed as relative dot forming cell (SFC) numbers compared to negative control: (polypeptide or pool of polypeptides SFC) - (negative control SFC)/(negative control SFC). Relative SFCs of 3 and above indicated that PBMC samples showed positive recognition and response to the corresponding polypeptides (pools), and the results are shown in table 2.
TABLE 2 IFN-. Gamma.ELISPot assay results for predicting T cell epitope peptides
1) Screening results of T cell epitope peptides of serum samples of rehabilitators
To verify whether the predicted epitope was immunogenic in natural infection, PBMC were isolated from peripheral blood of 20 patients (labeled A-T, see Table 3 below) infected with strain Wuhan-hu-1 at the beginning of 2020, with a recovery of the type HLA-A.02:01, and assayed for secretion of IFN-gamma following peptide stimulation. Due to the limited number of PBMCs, 524 epitope peptides were first divided into 26 groups of 20 or 24 peptides each (see peptide pool number in table 1). IFN-y production was detected using the IFN-y ELISPOT method with 5 μg/mL stimulation of PBMC from rehabilitation patients per peptide concentration.
Table 3-20 clinical information of rehabilitators
| Number of recovered person | Age of | Sex (sex) | Hospitalization time | Discharge time | Symptoms of |
| A | 42 | Male men | 2020.01.28 | 2020.02.08 | Mild symptoms |
| B | 30 | Male men | 2020.02.07 | 2020.02.14 | Severe cases of severe disease |
| C | 28 | Female woman | 2020.02.08 | 2020.02.18 | Severe cases of severe disease |
| D | 39 | Male men | 2020.02.07 | 2020.02.14 | Mild symptoms |
| E | 51 | Female woman | 2020.02.13 | 2020.02.19 | Mild symptoms |
| F | 30 | Male men | 2020.02.08 | 2020.02.16 | Mild symptoms |
| G | 56 | Male men | 2020.01.20 | 2020.01.27 | Severe cases of severe disease |
| H | 53 | Female woman | 2020.01.20 | 2020.01.27 | Severe cases of severe disease |
| I | 59 | Male men | 2020.02.08 | 2020.02.18 | Severe cases of severe disease |
| J | 66 | Male men | 2020.01.26 | 2020.02.20 | Severe cases of severe disease |
| K | 32 | Male men | 2020.01.21 | 2020.01.29 | Mild symptoms |
| L | 59 | Female woman | 2020.02.06 | 2020.02.15 | Severe cases of severe disease |
| M | 41 | Male men | 2020.02.06 | 2020.02.20 | Mild symptoms |
| N | 43 | Male men | 2020.01.20 | 2020.02.24 | Mild symptoms |
| O | 46 | Female woman | 2020.01.27 | 2020.02.18 | Mild symptoms |
| P | 29 | Male men | 2020.01.27 | 2020.02.08 | Severe cases of severe disease |
| Q | 41 | Female woman | 2020.01.28 | 2020.02.16 | Severe cases of severe disease |
| R | 56 | Male men | 2020.01.26 | 2020.02.21 | Severe cases of severe disease |
| S | 77 | Female woman | 2020.01.26 | 2020.02.01 | Severe cases of severe disease |
| T | 49 | Male men | 2020.01.30 | 2020.02.18 | Severe cases of severe disease |
As shown in FIG. 2, most PBMC samples showed IFN-gamma production following stimulation of the polypeptide pool, especially PBMC from patients A, D, M and R. Next, the member peptides in the pool of 1 or 3 (depending on the number of PBMCs) peptides shown in fig. 2 that stimulated IFN- γ highest were stimulated using patient PBMCs, and the IFN- γ ELISPOT method identified 33 epitope peptides that were located in M, N and multiple nonstructural proteins were recognized by patient PBMCs.
2) Results of screening T cell epitope peptides of healthy serum samples
All 524 epitope peptides were also used alone to stimulate two HLA-A 02:01 isotype PBMC samples purchased from healthy donors (collected prior to epidemic). Only one epitope peptide (ORF 1ab2787-4200aa-73: KLLGVGKPCIKVA) was positive for IFN-. Gamma.ELISPOT detection of one PBMC sample (not recognized in the 33 above).
From the above results, it was found that 34T cell epitope peptides (including 33 epitope peptides selected from PMBC of the rehabilitee and 1 epitope peptide selected from PMBC of the healthy person) were obtained by in vitro screening, and the amino acid sequences thereof were shown as SEQ ID NO.1 to SEQ ID NO.34. The specific T cell epitope peptides with immune response obtained by the in vitro screening are shown in the following table 4:
TABLE 4 epitope peptides for in vitro PMBC screening
(4) Homology analysis of selected T cell epitope peptides with other viruses
To identify peptides with potential cross-reactive sequences between SARS-CoV-2 and other coronaviruses, the screened CTL peptides were compared to the sequences of other viral genes using NCBI-Blast-Protein (https:// www.medicgo.org). Cross-reactive sequences were identified, excluding SARS-CoV-2.
The NCBI-Blast-Protein online assay was used to screen for epitope peptides that removed SARS-CoV-2 that had homology, cross-over, match and coverage in other coronaviruses by 100%. Among the 33 epitope peptides screened in the rehabilitation sample, 8 epitope peptides have conserved sequences among different coronaviruses (see table 5 below), and the amino acid sequences of the epitope peptides are numbered as SEQ ID NO. 48-SEQ ID NO.55, wherein 1M protein and other 7 targeted antigens of ORF1ab proteins. These epitopes can continue to be validated in subsequent additional studies as conserved immunogens for the production of a variety of coronavirus pathogens, developing broad-spectrum vaccine candidates against different coronaviruses.
TABLE 5 identity of T cell epitopes of other coronaviruses (Cross sequences)
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(5) Conservation analysis of T cell epitope peptide in different SARS-CoV-2 variants
The major epidemic strains of SARS-CoV-2 virus were searched for using NCBI BLAST (https:// www.ncbi.nlm.nih.gov/labs/viruses /), and the in vitro selection was analyzed for conservation of 34 peptide fragments. In addition, to further expand the scope of candidate vaccines, the synthesized 524 epitope peptide and the epidemic strain described above were conservatively analyzed using the same assay.
The 34 epitope peptides screened in vitro (as shown in Table 4 above) were analyzed for conservation in the major epidemic strain of the SARS-CoV-2 portion by IEDB software selection Epitope Conservancy Analysis on-line. 12 of the 34 epitopes screened in vitro are conserved among different epidemic strains, and the result is shown in the A part of figure 3, and the amino acid sequences of the 12 epitope peptides are numbered as SEQ ID NO. 1-SEQ ID NO.12.
Meanwhile, in order to further expand the range of candidate vaccines, based on immunoinformatics technology, the synthesized 524 epitope peptides were subjected to a conservative analysis on the main epidemic strain of SARS-CoV-2, and the result is shown in section B of FIG. 3. The result of the conservation analysis screened 13 epitopes (epitopes not recognized by patient or healthy donor PBMC) conserved in other SARS-CoV-2 epidemic strains, and the amino acid sequence numbers thereof were SEQ ID NO. 35-SEQ ID NO.47.
The conservation analysis is carried out to obtain 25 highly conserved T cell epitope peptides, namely ORF1ab2787-4200aa-42、ORF1ab2787-4200aa-94、ORF1ab2787-4200aa-4、ORF1ab4187-4405aa-6、N28274-29534-2、ORF1ab1-1400aa-22、ORF1ab1-1400aa-72、ORF1ab1-1400aa-82、ORF1ab1-1400aa-84、ORF1ab2787-4200aa-28、ORF1ab2787-4200aa-29、ORF1ab4187-4405aa-3、ORF1ab1-1400aa-23、ORF1ab4379-5796aa-58、S-36、S-52、ORF1ab2787-4200aa-60、ORF1ab1-1400aa-80、ORF1ab1-1400aa-56、M-10、ORF1ab1387-2800aa-4、ORF1ab1387-2800aa-62、ORF7a-8、ORF1ab1387-2800aa-24、S-47,, the sequence information of which is detailed in the invention content.
Example 2 design of T cell epitope peptide candidate DNA vaccine
In order to construct a candidate vaccine with better immunogenicity and broad spectrum, the method further comprises the steps of: since significant variation and evolution of the virus has occurred since the onset of the viral pandemic, this example analyzes the sequence conservation of the predicted epitope relative to all major SARS-CoV-2 lineages and variants. Because the binding force between MHC and epitope is represented by the percentile Rank, the smaller the number is, the better the immunogenicity is, the 34 epitopes screened in vitro have 12 conserved epitopes in other SARS-CoV-2 epidemic strains, the epitope with the percentile Rank less than or equal to 1 is selected, and the total number of 4 epitopes meets the condition and is named as N1-N4 (sequences shown as SEQ ID NO. 1-SEQ ID NO. 4); because patients with similar HLA-A genetic background have different T cell responses caused by the same virus infection, in order to expand the screening range of candidate vaccines, 13 conserved epitopes (epitopes not recognized by patients or healthy donor PBMC) in other SARS-CoV-2 epidemic strains are screened from 524 synthetic epitopes, and the epitopes with the percentile Rank less than or equal to 1 are selected, and total 8 epitopes meet the condition and are named as A1-A8 (sequences shown as SEQ ID NO. 35-SEQ ID NO. 42). In summary, the epitope peptides finally screened for vaccine design were N1-N4 and A1-A8.
Based on the above-mentioned screening of N1-N4 and A1-A8, the construction of DNA vaccine expressing multiple epitopes was further designed according to predicted and partially validated HLA-A.02:01 restriction SARS-CoV-2T cell epitopes, specifically: in order to further verify the immunogenicity of the candidate epitopes in an in vivo animal model, DNA candidate vaccines pCMV-4-N1-N4 and pCMV-8-A1-A8 (sequences shown as SEQ ID No.56 to SEQ ID No. 57) were constructed. Gene coding framework analysis was performed using Snapgene (version 3.1) software. The gene is synthesized by Beijing engine biotechnology Co., ltd, subcloned into HindIII and PstI sites of pCMV-HBV1.1 vector, and pCMV-4-N1-N4 and pCMV-8-A1-A8 expression plasmids are constructed. As shown in part A of FIG. 4, the N1-N4 and A1-A8 epitope peptide sequences were ligated to the EAAAK binding sequence, the N-terminus of the tandem epitope was fused to the Flag tag, a non-natural broadly mitogenic CD4 + T cell epitope (PADRE) was also fused to the C-terminus via GPGPG linker, the corresponding cDNA sequence was chemically synthesized, and a CMV promoter-driven mammalian expression Vector (homologous recombination cloned into pCMV-Vector) was inserted to prepare pCMV-4-N1-N4 and pCMV-8-A1-A8. Immunofluorescence and western blot analysis using anti-FLAG monoclonal antibodies demonstrated that these DNA vaccine plasmids were capable of expressing designed multi-epitope polypeptides.
And after the expression plasmids pCMV-4-N1-N4 and pCMV-8-A1-A8 are respectively transfected into HEK293-T cells, immunofluorescence and immunoblotting methods are used for detecting the expression of tandem epitope polypeptides carrying Flag tags. The expression of the Flag-tagged recombinant protein was evident in transfected cells (FIG. 4, part B), and the WB experiments detected Flag-tagged recombinant protein expression with apparent molecular weights of 9.76 and 17.6kDa in the transfected cell lysates, which was substantially consistent with the predicted molecular weight, as shown in FIG. 4, part C. Therefore, the tandem epitope polypeptide expression plasmid has good intracellular expression efficiency, and can be used for preparing recombinant DNA vaccine.
Example 3-DNA candidate vaccine induced CD8 + T cell response in HLA-A 02:01 transgenic mice
This example demonstrates the mouse immunity of pCMV-4-N1-N4 and pCMV-8-A1-A8 constructed in example 2, comprising the steps of:
To test a DNA vaccine based on predicted HLA-A.times.02:01 restricted SARS-CoV-2T cell epitope, a mouse model of human HLA-A.times.02:01 restriction (B-HLA-A 2.1) was used. Endogenous mouse B2m gene was replaced by human B2m gene in B-HLA-A2.1 mice, and the human HLA-A 02:01 epitope binding domain was fused to other domains of the mouse H2D gene. Only the expression and function of the transgenic HLA-A 02:01 was detected and observed in these mice, whereas endogenous mice H2D b were not.
The B-HLA-A2.1 mice were given intramuscular injections of 25 μg empty vector, pCMV-4-N1-N4, pCMV-8-A1-A8 or both constructs (12.5 μg each) and the same dose was given as booster injections after 3 weeks (FIG. 5, part A). Killing mice two weeks after the intensive injection, taking spleen tissue to prepare mouse spleen lymphocytes, grinding and separating single spleen cells by using a 200-mesh net, grinding the cells by using the tail end of a syringe, centrifuging a mixed suspension of the cells and a culture medium, adding 2mL of erythrocyte lysate, and stopping adding R10 culture medium at room temperature for 4 min; centrifugation at 1000rpm for 8min, discarding the waste liquid, re-suspending spleen cells with 2mL of complete 1640 medium, counting lymphocytes with a cytometer, 2X 10 6 cells per sample; stimulation was with N1-N4 and A1-A8 polypeptides at a final concentration of 5. Mu.g/mL. IFN-gamma + cells in the percentage of viable CD3 +CD8+ T lymphocytes in polypeptide-stimulated spleen cells were measured by intracellular IFN-gamma staining. As shown in parts B and C of FIG. 5, pCMV-4-N1-N4 immunity induction detected a CD8 + T cell response to N2, while pCMV-8-A1-A8 immunity induced a detectable response to the A1, A2, A3 and A4 epitopes. However, in mice injected with pCMV-4-N1-N4 and pCMV-8-A1-A8, only specific responses to the A5 epitope were detected. No specific response was detected in the empty vector immunized mice except that no specific stimulation (IFN- γ producing CD8 + T lymphocytes) was also present on the N1 epitope (parts B and C of fig. 5). These data indicate that constructing immunity using designed DNA vaccines can induce CD8 + T cell responses against at least a portion of the epitopes. In other words, these predicted HLA-A 02:01 restriction tables can be processed and presented in this HLA-A 02:01 restriction model.
EXAMPLE 4 protection of mice against SARS-CoV-2 infection with DNA candidate vaccine
This example demonstrates whether the immunization with the pCMV-4-N1-N4 and pCMV-8-A1-A8 DNA vaccines constructed in example 2 provides immunoprotection against SARS-CoV-2 infection, comprising the steps of: as shown in FIG. 6, part A, 8 days after boost injection of B-HLA-A2.1 mice, recombinant adenovirus was instilled to express human ACE2 (rAD 5-hACE 2) in the nasal cavity, rendering respiratory cells susceptible to SARS-CoV-2. The injection dose of the mice is calculated according to the weight of 0.2mL/10g, after the mice are completely comatose, 50 mu L of rAD5-hACE2 adenovirus 2.5X10 9 PFU is sucked by a liquid-transferring gun, the mice are carefully dripped into the noses of the anesthetized mice, after the mice are completely sucked into the lungs, the mice are horizontally placed and observed for 1h until the mice wake up after the anesthesia, and the rest experiment is continued. After 5 days, nasal cavity inoculation Wuhan-hu-1 virus (100. Mu.L SARS-CoV-2 (1.5X10 5 TCID 50)) was performed, and after 5 days the lung tissue was harvested by monitoring for sacrifice. The RT-qPCR method is used for detecting SARS-CoV-2 virus genome RNA and sgRNA. The protection effect of the vaccine was evaluated by comprehensive evaluation of lung tissue inflammation using HE staining (aka biosome company).
Quantification of lung tissue viral load also indicated that empty vector immunized mice contained significant amounts of viral RNA in their lungs, whereas viral RNA was undetectable in lung tissue of pCMV-4-N1-N4, pCMV-8-A1-A8, or both immunized mice (fig. 6, part C). DNA vaccine immunized mice reduced weight slightly after virus inoculation compared to empty vector injected mice (fig. 6, part B); also, histochemical analysis of lung tissue sections showed severe destruction of alveolar structure in empty vector immunized mice, whereas vaccine immunized mice did not (part D of fig. 6). The above results indicate that immunization induced by a DNA plasmid comprising a polypeptide expression predicted to limit the SARS-CoV-2 epitope by HLA-A 02:01 can combat viral infection in this mouse model.
As can be seen from the above examples, the present invention selects one of the most common HLA-A subtypes, HLA-A 02:01, the epitope length selects 14 amino acid residues for prediction, and these epitopes are tested in related human PBMC and corresponding B-HLA-A2.1 mouse models, which can be screened to identify SARS-CoV-2T cell epitopes with a broader HLA-A specificity. When PBMCs from HLA-A 02:01 homotypic recovery COVID-19 patients were stimulated using the same polypeptide pool panel and analyzed for IFN- γ production, 33 single epitope peptides capable of stimulating production of IFN- γ by convalescent PBMCs and 1 single epitope peptide capable of stimulating production of IFN- γ by healthy PBMCs were identified from these predicted polypeptide pools and 4 polypeptides N1-N4 were selected for subsequent validation by conservative analysis; and, to expand the candidate vaccine screening range, 8 polypeptides A1-A8 were selected for subsequent validation by performing a conservative analysis from 524 synthetic epitopes. The present invention constructs two DNA candidate vaccines comprising highly conserved predicted epitopes (pCMV-4-N1-N4 and pCMV-8-A1-A8) in major SARS-CoV-2 lineages and variants, and data obtained in B-HLA-A2.1 mouse validation indicate that at least some of the epitopes are immunogenic in this HLA-A 02:01 restriction model.
Recombinant adenovirus-mediated human ACE2 gene transduction to B-HLA-A2.1 mice respiratory tract used in the present invention allows testing for protection of DNA vaccine constructs without the need for cumbersome B-HLA-A2.1 mice to hybridize and homozygote selection with human ACE2 transgenic mice. Vaccine immunized mice lost less weight after virus inoculation than empty vector immunized mice, almost no virus was detected in lung tissue 5 days post infection, and alveolar structure destruction was much less, which could safely be considered to induce a similar epitope-specific CD8 + T cell response in these mice, likely to be effective against the observed challenge of SARS-CoV-2.
The present invention is based on immunoinformatics' T cell epitope prediction and screening of epitopes using PBMC of convalescent patients allows identification of HLA-A 02:01 restricted SARS-CoV-2T cell epitope peptides that are immunogenic in natural infection. Furthermore, immunogenicity and efficacy tests on DNA vaccines containing identified epitopes have shown that these epitopes can also induce immunogenicity in vaccination, and more importantly, epitope-specific CD8 + T cell responses and against SARS-CoV-2 virus challenges. The epitopes identified and tested in the present invention are complementary to other known T cell epitopes of SARS-CoV-2, which together support the future design and development of vaccines aimed at stimulating better T cell responses in humans. Meanwhile, the T cell vaccine development method demonstrated in the invention can also be used as a supplement to the initial response of other new pathogens possibly occurring in the future so as to supplement other classical and emerging rapid vaccine development methods.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (10)
- HlA-A 02:01 restricted SARS-CoV-2T cell epitope peptide, characterized in that the step of screening for said T cell epitope peptide comprises: full coverage analysis is carried out on SARS-CoV-2 coding protein sequence by adopting an immunoinformatics method, and HLA-A 02:01 restriction epitope of 14-site amino acid is predicted; chemically synthesizing epitope peptide based on the prediction result, and screening out specific epitope peptide with immune response in vitro; the epitope peptide obtained by in vitro screening and/or the epitope peptide synthesized chemically are subjected to conservation analysis, and T cell epitope peptide with high conservation and strong immunogenicity in SARS-CoV-2 original and other epidemic strains is screened.
- 2. The T cell epitope peptide of claim 1, wherein the amino acid sequence of said T cell epitope peptide comprises the sequence shown in SEQ ID No. 1-SEQ ID No. 47.
- 3. The T cell epitope peptide of claim 2, wherein said T cell epitope peptide comprises N1-N4 polypeptides and A1-A8 polypeptides, the amino acid sequences of said N1-N4 polypeptides are shown in SEQ ID No. 1-SEQ ID No.4, respectively, and the amino acid sequences of said A1-A8 polypeptides are shown in SEQ ID No. 35-SEQ ID No.42, respectively.
- 4. The T cell epitope peptide of claim 2, wherein said T cell epitope peptide comprises an N2 polypeptide and A1-A4 polypeptide, the amino acid sequence of said N2 polypeptide is shown in SEQ ID No.2, and the amino acid sequences of said A1-A4 polypeptides are shown in SEQ ID No. 35-SEQ ID No.38, respectively.
- 5. The T cell epitope peptide of claim 1, further comprising the step of, during the screening process: comparing the epitope peptide obtained by in vitro screening with sequences of other viral genes excluding SARS-CoV-2, and identifying cross-reactive sequences conserved in different coronaviruses; wherein the amino acid sequence of the cross reaction sequence is selected from the sequences shown in SEQ ID NO. 48-SEQ ID NO. 55.
- 6. A biomaterial associated with the T cell epitope peptide of any one of claims 1-5, wherein the biomaterial comprises one of a) to B):a) A nucleic acid molecule encoding said T cell epitope peptide;b) Recombinant vector and recombinant cell line containing the nucleic acid molecule as described in A).
- 7. Use of a T cell epitope peptide according to any one of claims 1-5, or a biomaterial according to claim 6, wherein said use is selected from at least one of the following: the application of the kit in preparing a novel coronavirus infection or a cell immune response detection kit after infection, the application in preparing a novel coronavirus infected person screening detection reagent, the application in preparing a novel coronavirus preventive and therapeutic vaccine, the application in preparing an active or passive immunotherapy agent aiming at the novel coronavirus infection, and the application of the cross reaction sequences in preparing different coronavirus broad-spectrum vaccines.
- 8. A vaccine prepared from the T cell epitope peptide of any one of claims 1 to 5, or the biological material of claim 6.
- 9. The vaccine of claim 8, wherein the vaccine is a DNA vaccine comprising at least one T cell epitope peptide; wherein, the construction steps of the DNA vaccine comprise: and connecting each T cell epitope peptide sequence with a binding sequence, fusing the N end of the tandem epitope with a Flag tag, and cloning the homologous recombination on a vector plasmid to obtain the tandem epitope polypeptide expression plasmid.
- 10. The vaccine of claim 9, wherein the tandem epitope polypeptide expression plasmid comprises pCMV-4-N1-N4, pCMV-8-A1-A8, wherein the pCMV-4-N1-N4 has an amino acid sequence as shown in SEQ ID No.56, and wherein the pCMV-8-A1-A8 has an amino acid sequence as shown in SEQ ID No. 57.
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