CN114057846A

CN114057846A - Polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof

Info

Publication number: CN114057846A
Application number: CN202010788949.8A
Authority: CN
Inventors: 刘刚; 于文俊; 张庶民; 高军; 周荔葆; 毛昱; 孙韦强; 孙述学; 车兴华
Original assignee: Liaoning Chengda Biotechnology Co ltd; Tsinghua University
Current assignee: Liaoning Chengda Biotechnology Co ltd; Tsinghua University
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2022-02-18
Anticipated expiration: 2040-08-07
Also published as: CN114057846B

Abstract

The invention relates to the fields of immunology technology, biotechnology and biomedicine, in particular to a polypeptide for preventing novel coronavirus pneumonia COVID-19, an immunogenic conjugate and application thereof. An immunogenic conjugate comprising a polypeptide having an amino acid sequence shown as SEQ ID No.1 for use in the preparation of a vaccine for the prevention of COVID-19. The immunogenic conjugate is a synthetic peptide vaccine prepared by synthesizing a target polypeptide based on a known or predicted certain antigen epitope amino acid sequence in a pathogen antigen gene, combining the synthesized target polypeptide with a carrier protein to prepare a polypeptide conjugate and adding an adjuvant. Compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, the vaccine can quickly cope with virus variation; the site requirement required by vaccine production is reduced, and large-scale batch production is facilitated; the research and development cost and the research and development period are reduced; safe, nontoxic and stable; due to the small and simple molecular structure, serious complications and iatrogenic infection problems are rarely caused.

Description

Polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof

Technical Field

The invention relates to the fields of immunology technology, biotechnology and biomedicine, in particular to a polypeptide for preventing novel coronavirus pneumonia COVID-19, an immunogenic conjugate and application thereof.

Background

The coronavirus is a forward enveloped virus with RNA, the genome size of the coronavirus is about 26-32 kb, and the coronavirus is an RNA virus with the largest known genome. The genomic RNA and phosphorylated nucleocapsid (N) proteins are buried in a phospholipid bilayer and covered by a spike glycoprotein trimer (S), with membrane (M) proteins (type III transmembrane glycoproteins) and envelope (E) proteins located between the S proteins of the viral envelope. Coronaviruses have a variety of hosts, including avian and mammalian species, particularly bats. Coronavirus is a virus widely existing in nature and can cause multi-system diseases including respiratory tract, digestive tract and nervous system, and highly pathogenic coronavirus infection has become a public health problem which is widely concerned for nearly 10 years. In 11 months 2002, the first Severe Acute Respiratory Syndrome (SARS) occurred in Foshan mountain, China. In 2012, the middle east respiratory syndrome coronavirus (MERS-CoV) was the second highly pathogenic coronavirus found in the 21 st century. The mortality rate of MERS-CoV is high, with as many as 40% of patients dying. 2019 the new coronavirus (SARS-CoV-2) was first discovered in 12 months in 2019, and the infection caused by the new coronavirus pneumonia (COVID-19) is affecting millions of patients all over the world.

Patients with COVID-19 may develop symptoms to varying degrees, ranging from fever or mild cough to pneumonia, with more severe cases even dying. At present, the lethality rate of COVID-19 is about 2% to 4%, although the mortality rate is lower than that of SARS and MERS, the novel coronavirus (SARS-CoV-2) has the characteristics of long latent period, strong infectivity and high severe rate, unlike SARS virus causing SARS, the latent period of some cases has infectivity, and other virus carriers do not show any obvious symptoms, thereby increasing the difficulty in controlling epidemic situations. Therefore, the rapid development of preventive vaccines capable of improving the immunity level of the population and blocking the spread of viruses has become the most urgent major global need.

Since the first use of the biological vaccinia vaccine by humans at the end of the 18 th century, vaccines have played an irreplaceable role in the elimination of a variety of infectious diseases. After the COVID-19 is confirmed, a plurality of organizations at home and abroad develop the research of the COVID-19 vaccine, at least 5 technical lines are developed synchronously at present, including nucleic acid vaccines, virus vector vaccines, inactivated vaccines, recombinant protein vaccines, attenuated influenza virus vector vaccines and the like, but in view of the self characteristics of the vaccines, the vaccines are strictly carried out according to the research and development production flow specified by the state, after the animal experiments pass, pilot-scale research, clinical application and clinical test research in stages I to III must be completed, and the vaccines can formally enter the production stage after the approval is obtained. SARS vaccine has been left to date because after phase II clinical trials the epidemic situation has been completely controlled and phase III trials can no longer be performed. The design of the later clinical practice of the current COVID-19 vaccine is not clear, and a multi-country participation scheme needs to be established so as to determine the safety and the effectiveness of a candidate vaccine. Therefore, all vaccines under investigation have not yet been licensed to market.

The Nucleocapsid of SARS-CoV-2 is formed by encapsidating a single-stranded positive-strand RNA with Nucleocapsid protein (N); it is externally enveloped and embedded with three glycoproteins: spike protein (S), envelope protein (E) and membrane protein (M). The S protein is positioned at the outermost layer of the virus, is regularly arranged into a corona structure on a membrane, is involved in the process that the virus is combined with a virus receptor on the surface of a host cell and mediates the virus to enter the cell through membrane fusion, and plays an important role in the process of inducing the host to generate neutralizing antibodies. At present, the development of COVID-19 vaccines at home and abroad takes S protein as a primary target antigen, but it is reported that the S protein of SARS-CoV of coronaviruses possibly induces antibody-dependent immune enhancement reaction (ADE), so that specific fragments (namely polypeptides) in the S protein are screened as antigens, thereby effectively preventing COVID-19 and avoiding potential ADE.

Protein epitope profiling was studied using combinatorial chemistry techniques (the "cross-overlapping" polypeptide compounds), the smallest epitope was rapidly discovered, and a profile was constructed. The method is characterized in that a certain number of amino acid residue peptides (such as 1 to 50 amino acid residues) are synthesized into a cross-overlapping fragment, a protein sequence is completely synthesized in a mode of dislocation one by one (or interval dislocation, including the total number of amino acids from 2 to the synthesized peptide fragment), then antigen-antibody reaction (or screening reaction of other biological purposes, such as screening and finding T-cell immune antigenic determinants, SARS-CoV receptor ligands and the like) is carried out, all shortest polypeptide antigenic determinants can be obtained at one time, and then an antigenic determinant spectrum is drawn. Then the active short peptides are properly prolonged or orderly and linearly connected to carry out anti-SARS-CoV human positive serum screening reaction, thereby confirming the B-cell polypeptide compound and the map thereof which can be used for preparing SARS-CoV diagnostic reagent, medicine and vaccine. SARS-CoV-2 has high homology with SARS-CoV. The method is an effective way to quickly discover new coronavirus antigen by screening the 'cross-overlapping' chemical library for constructing SARS-CoV to obtain antigenic determinant and replacing it with SARS-CoV-2 sequence in the same position.

The traditional vaccine is prepared by inactivating or attenuating pathogens, and has the problems of biohazard, genetic variation, loss of the efficacy of the original vaccine and the like. The epitope vaccine is a vaccine prepared by using antigen epitope, and is the direction for developing infectious disease vaccines at present. Compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, the synthetic peptide vaccine is safe, nontoxic and stable, and has few serious complications and iatrogenic infection problems due to the small and simple molecular structure. The synthetic peptide vaccine is a vaccine prepared by synthesizing short peptide with antigenicity by a chemical technology according to a known or predicted antigen epitope amino acid sequence in a pathogen antigen gene, combining the short peptide with a carrier and then adding an adjuvant, is an ideal and safe novel vaccine, and is also a novel vaccine which is researched at present and is used for preventing and controlling infectious diseases and treating malignant tumors.

Disclosure of Invention

In view of the problems of the prior art, the invention aims to provide a polypeptide for preventing novel coronavirus pneumonia COVID-19, an immunogenic conjugate and application thereof. The synthetic peptide vaccine is used for preventing the COVID-19 virus, and compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, the synthetic peptide vaccine provided by the invention can quickly cope with virus variation; the site requirement required by vaccine production is reduced, and large-scale batch production is facilitated; the research and development cost and the research and development period are reduced; safe, nontoxic and stable; due to the small and simple molecular structure, serious complications and iatrogenic infection problems are rarely caused.

In order to achieve the purpose, the invention adopts the following technical scheme.

A polypeptide for preventing the novel coronavirus pneumonia COVID-19, the amino acid sequence of said polypeptide is chosen from any one of the following.

1) The amino acid sequence is the polypeptide shown in SEQ ID NO. 1.

2) A polypeptide which has more than 85 percent of same amino acid sequence with the amino acid sequence shown in SEQ ID NO. 1.

3) The polypeptide with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 1.

An immunogenic composition comprising a polypeptide as described above and at least one pharmaceutical carrier or excipient.

An immunogenic conjugate, comprising the polypeptide, wherein the immunogenic conjugate is prepared by cross-linking the polypeptide and a carrier protein by a polypeptide coupling method.

Further, the carrier protein is any one of diphtheria nontoxic mutant CRM197, tetanus toxoid TT, diphtheria toxoid DT or meningococcal outer membrane protein M-OMP.

Further, the molar ratio of the polypeptide to the carrier protein is: 1: 1-1: 30.

further, the binding rate of the polypeptide to the carrier protein is 40% or more.

A vaccine for preventing novel coronavirus pneumonia COVID-19, wherein the active ingredient of the vaccine comprises the polypeptide, the immunogenic composition or any one of the immunogenic conjugates.

Further, the vaccine further comprises a pharmaceutically acceptable adjuvant.

Further, the adjuvant is one or more combinations of aluminum hydroxide (al (oh)3), aluminum phosphate (AlPO4), monophosphoryl lipid a (mpl), or oligonucleotides (CpG).

Further, for administration to humans, the adjuvant is present in an amount of 1 μ g to 1000 μ g per dose, preferably 10 μ g to 500 μ g per dose, more preferably 10 μ g to 200 μ g per dose, more preferably 10 μ g to 100 μ g per dose, and most preferably 10 μ g to 50 μ g per dose.

Further, the vaccine is in any pharmaceutically acceptable dosage form, including intramuscular injection, subcutaneous injection, intradermal injection and microneedle injection.

Further, the vaccine is in any pharmaceutically acceptable dose.

The preparation method of the vaccine specifically comprises the following steps.

Step 1, using a full-automatic microwave polypeptide synthesizer, adopting an Fmoc solid-phase synthesis method, adding a solid-phase carrier, different amino acids, a remover, a polypeptide condensation reagent and the like, and carrying out full-automatic synthesis on target polypeptide.

And 2, respectively conjugating the polypeptide synthesized in the step 1 with a pharmaceutically acceptable carrier protein (including any one of diphtheria non-toxic mutant CRM197, tetanus toxoid TT, diphtheria toxoid DT or meningococcal outer membrane protein) through a Linker to form the immunogenic conjugate.

And 3, mixing the immunogenic conjugate prepared in the step 2 with a pharmaceutically acceptable adjuvant to prepare the vaccine.

Further, the specific method in step 3 is to mix the immunogenic conjugate with adjuvant and PBS solution, shake the mixture for 1 hour at room temperature and 30RPM, and obtain a final sample with a final concentration of 100 mug/ml polypeptide-containing antigen and 0.5mg/ml adjuvant; or firstly adsorbing the immunogenic conjugate on an aluminum adjuvant, shaking for 1 hour at room temperature and 30RPM, then adding MPL or CpG adjuvant, shaking for 1 hour at room temperature and 30RPM, and obtaining the final sample with the final concentration of 100 mu g/ml polypeptide antigen, 0.5mg/ml aluminum adjuvant and 0.5mg/ml MPL or CpG content.

Use of a polypeptide as described above, an immunogenic composition as described above, or an immunogenic conjugate as described in any of the above, in the manufacture of a medicament for the prevention or treatment of a novel coronavirus pneumonia COVID-19.

The medicament is in any pharmaceutically acceptable dosage form.

The drug is in any pharmaceutically acceptable dose.

Compared with the prior art, the invention has the following beneficial effects.

The synthetic polypeptide provided by the invention is a sequence with specific antigenicity screened from a COVID-19 virus S protein sequence, and is synthesized into a target polypeptide, so that the site requirement required by vaccine production is reduced, and the large-scale batch production is easy to realize; the synthetic peptide vaccine is safe, nontoxic and stable compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, and has small and simple molecular structure, so that serious complications and iatrogenic infection are rarely caused.

The polypeptide provided by the invention is based on a sequence with specific antigenicity screened from a COVID-19 virus S protein sequence, and short peptides are synthesized in vitro, and a plurality of short peptides are combined, so that the risk of side effects can be reduced compared with the full sequence; the prior report shows that the COVID-19 virus has variation, and compared with the polypeptide sequence provided by the invention, the phenomenon that all sequences have variation simultaneously does not occur, so that the antigenicity loss caused by variation is effectively reduced, meanwhile, the sequence variation part can be rapidly screened, new short peptides are added, and a new vaccine is synthesized and prepared, so that the virus variation can be rapidly responded, and the research and development cost and the research and development period are reduced.

The vaccine for preventing COVID-19 provided by the invention adopts a technology of combining polypeptide and protein carrier, enhances the immune response of human body, amplifies the antigenicity of polypeptide, increases the generation of neutralizing antibody, and enhances the efficacy of the vaccine, and adopts different adjuvants and different administration modes, so that the vaccine has universal applicability and is suitable for people of multiple ages.

Drawings

FIG. 1 is a comparison of antibody levels after combining polypeptides with different carriers and different adjuvants.

Detailed Description

The present invention is further illustrated by the following examples and the accompanying drawings, wherein the following examples are only preferred embodiments of the present invention, and are not intended to limit the present invention, and various changes and modifications may be made therein by those skilled in the art without departing from the spirit and the principle of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and scope of the present invention should be considered as being within the scope of the present invention.

Example 1 Synthesis of a polypeptide of interest (the amino acid sequence of which is the polypeptide shown in SEQ ID NO. 1).

1. The polypeptide with the amino acid sequence shown as SEQ ID NO.1 is prepared by a conventional solid phase polypeptide synthesis method, and the specific steps are as follows.

(1) Removing Fmoc protection.

A commercially available Rink Amide AM resin was charged into a reaction tube having a filter and resistant to an organic solvent, and was closed with an organic solvent-resistant cap. After washing with DMF (dimethylformamide) for 1 min, an excess of 20% piperidine/DMF (by volume) solution was added, and after capping the reaction tube was shaken gently to mix it well and keep the Fmoc protection removed for 15 min. After draining, washing three times with DMF.

(2) Peptide bond condensation.

Adding the Fmoc-protected amino acid with 3 times of resin amino molar weight, an activator HBTU with 3 times of resin amino molar equivalent and DIPEA (N, N-diisopropylethylamine) with 6 times of resin amino molar equivalent into a reaction tube after preactivation (after preactivation for 2-3 minutes), using DMF as a solvent, ensuring that the above reagents are completely dissolved, and completely covering the resin. Shaking the mixture every 2-3 min to mix the resin uniformly for 20-30 min.

Then, a 50-fold molar excess of acetic anhydride in DMF was added at room temperature and the mixture was drained after 10 minutes, and then an excess of 20% piperidine/DMF (by volume) solution was added at room temperature and reacted for 30 minutes. The solution was filtered off and the resin was washed 5 times with DMF.

The above steps are repeated in cycles until all the Fmoc-protected amino acids fitted to the polypeptide are attached to the resin in a linear fashion.

(3) And (3) polypeptide cleavage.

The polypeptide lysate is treated with a strong acid, such as TFA (trifluoroacetic acid). The resin was treated with TFA, water, EDT dimercaptoethanol, phenol (vol.: 92.5: 2.5: 2.5: 2.5) for 2 hours at room temperature. Then collecting the lysate carefully into a glass collector, adding ether precooled with ice, collecting the precipitated polypeptide, and continuing to wash with cold ether for 5-6 times to obtain crude peptide.

(4) And (5) polypeptide purification.

The crude peptide is purified by HPLC (high performance liquid chromatography), collected, lyophilized, and finally checked for purity (214nm wavelength) greater than 85% by HPLC and for correct molecular weight by mass spectrometry.

2. And (5) identifying the result.

The synthetic peptide shown in SEQ ID NO.1 had a purity of 90% and a molecular weight of 2016.23. The polypeptide has strong antigenicity determined by specific antigenicity experiment, and can induce neutralizing antibody in human body.

Example 2 preparation of synthetic peptide immunogenic conjugates shown in SEQ ID No. 1.

The synthetic peptide prepared in example 1 was bound to CRM197, TT, DT, bacterial outer membrane proteins by Linker to form conjugate polypeptides, respectively.

TT tetanus toxin protein and tetanus toxin carrier protein TT can be obtained by fermenting, cracking, centrifuging and purifying through chromatography of tetanus bacillus.

DT diphtheria toxin protein and diphtheria toxin carrier protein DT can be obtained by fermentation, lysis, centrifugation and chromatographic purification of Corynebacterium diphtheriae.

CRM197 recombinant diphtheria toxin protein, CRM197 gene sequence reconstructed diphtheria bacillus, and through fermentation, cracking, centrifugation and chromatographic purification.

Meningococcal outer membrane protein: the meningococcus is obtained by fermentation, cracking, centrifugation and chromatographic purification.

1. Synthetic peptides were combined with CRM197 by SMCC Linker or DSAP Linker to conjugate polypeptides.

CRM197 protein was formulated with a 1mg/ml concentration in PBS containing 2mM EDTA. A desired amount of CRM197 solution was added to Sulfo-SMCC (10mg/ml) at a molar ratio of 1:2 to 1:40, and reacted at room temperature for 1 hour, and the reaction mixture was concentrated by centrifugation at 3500rpm and 4 ℃ using an ultrafiltration concentration tube. After the volume is concentrated to 1/10 of the reaction solution, PBS is added to the volume of the original reaction solution, centrifugal concentration is continued, and centrifugal concentration is continuously carried out for 5 times to remove free Sulfo-SMCC. The final stock concentration of CRM197-SMCC was 10 mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or 10% DMSO-containing PBS solution, CRM197-SMCC stock solution was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, the reaction was carried out at room temperature for 1 hour, the conjugation was detected by SDS-PAGE, the amount of protein loading was 2. mu.g, and the electrophorogram was detected by SDS-PAGE.

The desired amount of synthetic peptide (example 1) was weighed out, dissolved in DMSO, mixed with DSAP (10mg/ml DMSO solution) at a molar ratio of 1:2 to 1:30, added with triethylamine (30-fold molar ratio), reacted at room temperature overnight, added with 500. mu.l NaPi (pH7.4, 0.1M), extracted three times with 1ml chloroform, centrifuged and the aqueous phase added to CRM197(2mg/ml NaPi (pH7.4, 0.1M)) solution at a molar ratio of synthetic peptide to CRM197 protein of 10:1 to 1:1 for 24 hours at room temperature, and examined for conjugation by SDS-PAGE.

2. The synthetic peptide is combined with TT protein through SMCC Linker or DSAP Linker to form a conjugated polypeptide.

The TT protein was formulated in a 1mg/ml concentration in PBS containing 2mM EDTA. A required amount of TT solution is taken, Sulfo-SMCC (10mg/ml) is added according to the molar ratio of 1:2-1:40, the reaction solution is reacted for 1 hour at room temperature, and the reaction solution is centrifugally concentrated by an ultrafiltration concentration tube at 4 ℃ and 3500 rpm. After the volume is concentrated to 1/10 of the reaction solution, PBS is added to the volume of the original reaction solution, centrifugal concentration is continued, and centrifugal concentration is continuously carried out for 5 times to remove free Sulfo-SMCC. The final TT-SMCC stock solution concentration was 10 mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or 10% DMSO-containing PBS, TT-SMCC stock was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, the reaction was carried out at room temperature for 1 hour, and the conjugation was detected by SDS-PAGE.

The desired amount of synthetic peptide (example 1) was weighed out, dissolved in DMSO, mixed with DSAP (10mg/ml DMSO solution) at a molar ratio of 1:2 to 1:30, added with triethylamine (30-fold molar ratio), reacted at room temperature overnight, added with 500. mu.l NaPi (pH7.4, 0.1M), extracted three times with 1ml chloroform, centrifuged and the aqueous phase added to TT (2mg/ml NaPi (pH7.4, 0.1M)) solution at a molar ratio of synthetic peptide to TT protein of 10:1 to 1:1, reacted at room temperature for 24 hours, and run for conjugation by SDS-PAGE.

3. The synthetic peptide is combined with DT into conjugated polypeptide through SMCC Linker or DSAP Linker.

The DT protein was formulated in a 1mg/ml concentration in PBS containing 2mM EDTA. The required amount of DT solution was added with Sulfo-SMCC (10mg/ml) at a molar ratio of 1:2-1:40, reacted at room temperature for 1 hour, and the reaction solution was concentrated by centrifugation at 3500rpm at 4 ℃ using an ultrafiltration concentration tube. After the volume is concentrated to 1/10 of the reaction solution, PBS is added to the volume of the original reaction solution, centrifugal concentration is continued, and centrifugal concentration is continuously carried out for 5 times to remove free Sulfo-SMCC. The final DT-SMCC stock solution concentration was 10 mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or 10% DMSO-containing PBS, DT-SMCC stock was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume and allowed to react at room temperature for 1 hour, and the conjugation was detected by SDS-PAGE.

The desired amount of synthetic peptide (example 1) was weighed out, dissolved in DMSO, mixed with DSAP (10mg/ml DMSO solution) at a molar ratio of 1:2 to 1:30, added with triethylamine (30-fold molar ratio), reacted at room temperature overnight, added with 500. mu.l NaPi (pH7.4, 0.1M), extracted three times with 1ml chloroform, centrifuged and the aqueous phase added to DT (2mg/ml NaPi (pH7.4, 0.1M)) solution at a molar ratio of synthetic peptide to DT protein of 10:1 to 1:1, reacted at room temperature for 24 hours, and run for conjugation by SDS-PAGE.

4. The synthetic peptide is combined with M-OMP to form conjugated polypeptide through SMCC Linker or DSAP Linker.

M-OMP protein was formulated in a 1mg/ml concentration in PBS containing 2mM EDTA. Taking a required amount of M-OMP solution, adding Sulfo-SMCC (10mg/ml) according to a molar ratio of 1:2-1:40, reacting at room temperature for 1 hour, and centrifugally concentrating the reaction solution at 3500rpm by using an ultrafiltration concentration tube at 4 ℃. After the volume is concentrated to 1/10 of the reaction solution, PBS is added to the volume of the original reaction solution, centrifugal concentration is continued, and centrifugal concentration is continuously carried out for 5 times to remove free Sulfo-SMCC. The final stock concentration of M-OMP-SMCC was 10 mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or 10% DMSO-in-PBS, M-OMP-SMCC stock was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, the reaction was carried out at room temperature for 1 hour, and the conjugation was checked by SDS-PAGE.

The desired amount of synthetic peptide (example 1) was weighed out, dissolved in DMSO, mixed with DSAP (10mg/ml DMSO solution) at a molar ratio of 1:2 to 1:30, added with triethylamine (30-fold molar ratio), reacted at room temperature overnight, added with 500. mu.l NaPi (pH7.4, 0.1M), extracted three times with 1ml chloroform, centrifuged and the aqueous phase added to M-OMP (2mg/ml NaPi (pH7.4, 0.1M)) solution at a molar ratio of synthetic peptide to M-OMP protein of 10:1 to 1:1, reacted at room temperature for 24 hours, and examined for conjugation by SDS-PAGE.

Example 3 preparation of vaccine and immunization effect.

Firstly, preparing a vaccine.

1. A material.

Polypeptide protein: the amino acid sequence is the polypeptide shown in SEQ ID NO. 1.

Carrier protein: diphtheria toxin null mutant (CRM197), Tetanus Toxoid (TT), Diphtheria Toxoid (DT), meningococcal outer membrane protein (Meningococcus OMP).

Adjuvant: aluminum hydroxide (al (oh)3), aluminum phosphate (AlPO4), MPL, CpG.

2. A preparation method.

Mixing the immunogenic conjugate with adjuvant and PBS solution, shaking for 1 hr at room temperature and 30RPM to obtain final concentration containing polypeptide antigen 100 μ g/ml and adjuvant 0.5mg/ml, and getting final sample; or firstly adsorbing the immunogenic conjugate on an aluminum adjuvant, shaking for 1 hour at room temperature and 30RPM, then adding MPL or CpG adjuvant, shaking for 1 hour at room temperature and 30RPM, and obtaining the final sample with the final concentration of 100 mu g/ml polypeptide antigen, 0.5mg/ml aluminum adjuvant and 0.5mg/ml MPL or CpG content.

Table 1. polypeptide conjugates and adjuvant cross-combination protocol.

Note: combination number naming rules: the "carrier initials" + "adjuvant number".

And II, immune effect experiment.

The immunogenicity of the vaccine immunized mice in table 1 was studied, healthy BALB/c mice weighing 16-18g were randomly grouped, 8-10 mice per group; injecting the sample to be tested in the table 1 into hind limb of mouse, injecting into muscle for 1 time on 0 th, 14 th and 21 st days, each time the administration volume is 100 uL/mouse, collecting blood for 1 time on 0 th, 21 st and 28 th days, separating serum, diluting the serum 20 times, and measuring the serum antibody level on 0 th, 21 st and 28 th days by enzyme linked immunosorbent assay (ELISA), specifically.

(1) Diluting the polypeptide stock solution into working solution (DMSO content is not higher than 0.05%) with a final concentration of 5 μ g/ml by using coating solution (0.05M carbonate-bicarbonate buffer solution), fully mixing uniformly, adding an enzyme label plate according to 100 μ l/hole, attaching sealing paper, and coating overnight at 2-8 ℃.

(2) The supernatant was aspirated off, washed 5 times with PBST, and blocked for 1h at room temperature by adding 100ul of 1% BSA in PBS.

(3) PBST 5 times.

(4) Sample adding: 100ul of diluted serum samples were added, incubated at room temperature for 2h, and washed 5 times with PBST.

(5) Adding an enzyme-labeled antibody: adding 100ul of diluted goat anti-mouse IgG HRP enzyme-labeled secondary antibody, and incubating for 1h at room temperature.

(6) PBST 5 times.

(7) Adding a substrate solution for color development: 100ul of TMB was added for color development.

(8) And (3) terminating the reaction: the reaction was terminated by adding 50ul of 1N sulfuric acid.

(9) And (4) judging a result: measuring OD₄₅₀The values and results are shown in Table 2.

TABLE 2 comparison of antibody levels after combination of polypeptides with different carriers and different adjuvants.

As shown in table 2, the carrier protein can significantly increase the antibody titer of the polypeptide; after the polypeptide is combined with the carrier protein, the antibody titer can reach more than 0.8, and the antibody titer is increased along with the increase of the dosage. The antibody titer of the carrier-free group is not more than 0.2 after the adjuvant is added, and is obviously lower than that of the carrier-containing group, so that the expected immunogenicity cannot be generated by combining the polypeptide with the adjuvant; the same dose, the same carrier protein, and the addition of adjuvant significantly increased antibody titers, with no significant difference between different adjuvants.

Sequence listing

<110> university of Qinghua, Liaoning Dabie shares GmbH

<120> polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> PRT

<213> Artificial sequence

<400> 1

Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys

1 5 10 15

Claims

1. A polypeptide for preventing novel coronavirus pneumonia COVID-19, wherein the amino acid sequence of the polypeptide is selected from any one of the following:

1) the amino acid sequence is the polypeptide shown in SEQ ID NO. 1;

2) a polypeptide having more than 85% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO. 1;

2. An immunogenic composition comprising the polypeptide of claim 1 and at least one pharmaceutical carrier or excipient.

3. An immunogenic conjugate comprising the polypeptide of claim 1, wherein the immunogenic conjugate is prepared by cross-linking the polypeptide of claim 1 to a carrier protein by polypeptide conjugation.

4. The immunogenic conjugate of claim 3, wherein the carrier protein is any one of diphtheria non-toxic mutant CRM197, tetanus toxoid TT, diphtheria toxoid DT, or meningococcal outer membrane proteins.

5. The immunogenic conjugate of claim 3, wherein the molar ratio of the polypeptide to the carrier protein is: 1: 1-1: 30.

6. the immunogenic conjugate of claim 3, wherein the binding rate of the polypeptide to the carrier protein is 40% or greater.

7. A vaccine for preventing novel coronavirus pneumonia COVID-19, wherein the active ingredient comprises the polypeptide of claim 1, the immunogenic composition of claim 2, or the immunogenic conjugate of any one of claims 3-6.

8. The vaccine of claim 7, further comprising a pharmaceutically acceptable adjuvant.

9. The vaccine of claim 7, wherein the adjuvant is one or more combinations of aluminum hydroxide Al (OH)3, aluminum phosphate AlPO4, monophosphoryl lipid A MPL, or oligonucleotide CpG.

10. The vaccine of claim 7, wherein the adjuvant is administered to a human in an amount of 1 μ g to 1000 μ g per dose, preferably 10 μ g to 500 μ g per dose, more preferably 10 μ g to 200 μ g per dose, more preferably 10 μ g to 100 μ g per dose, and most preferably 10 μ g to 50 μ g per dose.

11. The vaccine of claim 7, wherein the vaccine is in any pharmaceutically acceptable dosage form, including intramuscular injection, subcutaneous injection, intradermal injection, or microneedle injection.

12. The vaccine of claim 7, wherein the vaccine is in any pharmaceutically acceptable dose.

13. A method for the preparation of a vaccine according to any one of claims 7 to 12, in particular comprising the steps of:

step 1, using a full-automatic microwave polypeptide synthesizer, adopting Fmoc solid phase synthesis method, adding a solid phase carrier, different amino acids, a remover and a polypeptide condensation reagent to perform full-automatic synthesis of target polypeptide,

step 2, respectively conjugating and combining the polypeptides synthesized in the step 1 with pharmaceutically acceptable carrier proteins through a Linker to form immunogenic conjugates;

14. The method for preparing the vaccine according to claim 13, wherein the specific method in step 3 is to mix the immunogenic conjugate with the adjuvant and the PBS solution, shake the mixture for 1 hour at room temperature and 30RPM, and obtain a final sample with a final concentration of 100 μ g/ml of the polypeptide-containing antigen and 0.5mg/ml of the adjuvant; or firstly adsorbing the immunogenic conjugate on an aluminum adjuvant, shaking for 1 hour at room temperature and 30RPM, then adding MPL or CpG adjuvant, shaking for 1 hour at room temperature and 30RPM, and obtaining the final sample with the final concentration of 100 mu g/ml polypeptide antigen, 0.5mg/ml aluminum adjuvant and 0.5mg/ml MPL or CpG content.

15. Use of a polypeptide according to claim 1, an immunogenic composition according to claim 2, or an immunogenic conjugate according to any one of claims 3 to 6 in the manufacture of a medicament for the prevention or treatment of novel coronavirus pneumonia COVID-19.

16. The medicament of claim 15, wherein the medicament is in any pharmaceutically acceptable dosage form.

17. The medicament of claim 15, wherein the medicament is in any pharmaceutically acceptable dose.