CN112535730B

CN112535730B - Novel coronavirus polypeptide vaccine and application thereof

Info

Publication number: CN112535730B
Application number: CN202011364278.9A
Authority: CN
Inventors: 徐晓慧; 史馨怡; 逯文姝; 王健苏; 崔道成; 朱仁英; 姜石松
Original assignee: Changzhou Wensong Biotechnology Co ltd
Current assignee: Changzhou Wensong Biotechnology Co ltd
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2021-09-17
Anticipated expiration: 2040-11-27
Also published as: CN112535730A

Abstract

The invention discloses a polypeptide capable of being used for preventing or treating new coronavirus infection, and a vaccine composition or a pharmaceutical composition containing the polypeptide. The amino acid sequence of the polypeptide is shown as SEQ ID NO 1 or 2. As a vaccine composition for preventing or treating a new coronavirus infection, the polypeptide of the present invention can stimulate both production of binding antibodies to the S1 protein and production of binding antibodies to the S2 protein, and at the same time, can stimulate a T cell response.

Description

Novel coronavirus polypeptide vaccine and application thereof

Technical Field

The present invention relates to the field of vaccines. In particular, the present invention relates to polypeptide vaccines for coronaviruses, in particular novel coronaviruses, and uses thereof.

Background

Coronaviruses are a class of enveloped single-stranded positive-stranded RNA viruses. The international committee for classification of viruses classified coronaviruses into 4 major groups of α, β, γ, and δ in 2012. At present, 7 kinds of human-infectable coronaviruses are found, namely human coronavirus 229E (HCoV-229E), human coronavirus NL63(HCoV-NL63), human coronavirus OC43(HCoV-OC43), hong Kong type I human coronavirus (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERCoV), and novel coronavirus (2019-nCoV). Among them, SARS-CoV, MERS-CoV and 2019-nCoV are highly pathogenic coronaviruses found at present, and bring serious harm to human health.

2019 the novel coronavirus (2019-nCoV) is a new strain of coronavirus that has not been previously found in humans. It is a non-segmented single-stranded positive-strand RNA virus belonging to the same genus as SARS-coV, family Coronaviridae, order Neuroviridae. The novel coronavirus pneumonia (Corona virus disease 2019, COVID-19) caused by the novel coronavirus (2019-nCoV) has extremely high infectivity, causes serious epidemic situations on the global scale and endangers the life safety of millions of people. Human infection with the virus often presents respiratory symptoms such as fever, cough, shortness of breath, dyspnea, and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.

At present, clear and effective preventive and therapeutic drugs and measures for COVID-19 are not available, and the clinical application mainly supports treatment and symptomatic treatment. The establishment of population immunity to SARS-CoV-2 by vaccine is the final way to control and block COVID-19 epidemic. At present, various types of COVID-19 vaccines are in preclinical and clinical trials, including live attenuated vaccines, inactivated virus vaccines, recombinant virus vector vaccines, recombinant protein vaccines, DNA vaccines, RNA vaccines, polypeptide vaccines and the like.

However, vaccines currently developed have limited immunoprotection, such as low immunogenicity, safety risks, low human response rates, and difficulty in overcoming viral immune escape. Therefore, there is an urgent need in the art to develop a novel vaccine with high human response rate and high efficiency to stimulate the human body to generate an immune response against SARS-CoV-2, so as to generate a blocking type anti-SARS-CoV-2 antibody in the vaccinee, thereby providing potent immunoprotection. And according to research predictions, new coronavirus may coexist with human in the future for a long time.

Therefore, there is an urgent need in the art to develop vaccines for the treatment of new coronavirus.

Disclosure of Invention

The aim of the present invention is to provide a new vaccine against a new coronavirus, which is able to produce a powerful immunoprotection in the vaccinee.

In a first aspect, the present invention provides a vaccine composition or immunogenic composition or pharmaceutical composition comprising a PCM polypeptide and/or an RH2 polypeptide and optionally an immunologically or pharmaceutically acceptable excipient;

wherein the PCM polypeptide is:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 1;

(b) a derivative polypeptide formed by adding, substituting or deleting one or more amino acids to or from the amino acid sequence of the polypeptide in (a), wherein the derivative polypeptide has the same or basically the same function as the polypeptide shown in SEQ ID NO. 1;

the RH2 polypeptide is:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2;

(b) and (b) a derivative polypeptide formed by adding, substituting or deleting one or more amino acids to the amino acid sequence of the polypeptide in the step (a), wherein the derivative polypeptide has the same or basically the same function as the polypeptide shown in SEQ ID NO. 2.

In a preferred embodiment, the vaccine composition or immunogenic composition or pharmaceutical composition is a vaccine composition or pharmaceutical composition for the prevention or treatment of a coronavirus infection.

In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus.

In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

In a preferred embodiment, the "addition, substitution or deletion of one or more amino acids" recited in (b) is an addition, substitution or deletion of one or more amino acids at either end of the polypeptide in (a); the addition of one or more amino acids is preferred.

In a preferred embodiment, the "one or more amino acids" are 1 to 7 amino acids, preferably 1 to 6 amino acids.

In a specific embodiment, the vaccine composition or pharmaceutical composition comprises a PCM polypeptide and an RH2 polypeptide and optionally an immunologically or pharmaceutically acceptable excipient.

In a second aspect, the present invention provides a polypeptide which is:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 1;

(b) and (b) a derivative polypeptide formed by adding, substituting or deleting one or more amino acids to the amino acid sequence of the polypeptide in the step (a), wherein the derivative polypeptide has the same or basically the same function as the polypeptide shown in SEQ ID NO. 1.

In a third aspect, the present invention provides a polypeptide which is:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2;

In a fourth aspect, the invention provides the use of a polypeptide of the second or third aspect in the manufacture of a vaccine composition or immunogenic composition or pharmaceutical composition.

In a fifth aspect, the present invention provides a nucleic acid encoding the polypeptide of the first or second aspect.

In a sixth aspect, the present invention provides an expression vector comprising the encoding nucleic acid of the fifth aspect.

In a seventh aspect, the present invention provides a host cell comprising the expression vector of the sixth aspect or a host cell having integrated in its genome the encoding nucleic acid of the fifth aspect.

In an eighth aspect, the invention provides the use of a polypeptide according to the second aspect to improve the effect of a vaccine composition or an immunogenic composition or a pharmaceutical composition.

In a preferred embodiment, said use refers to the use of a polypeptide according to the second aspect as a potentiator for enhancing a vaccine or immunogenic or pharmaceutical composition.

In a preferred embodiment, the vaccine composition or pharmaceutical composition is a vaccine composition or immunogenic composition or pharmaceutical composition for the prevention or treatment of a coronavirus infection.

In a ninth aspect, the present invention provides a vaccine booster comprising a polypeptide which is:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 1;

In a tenth aspect, the present invention provides a vaccine composition or immunogenic composition or pharmaceutical composition comprising a PCM polypeptide and other polypeptides for the treatment or prevention of infection by coronaviruses, in particular neocoronaviruses, and optionally an immunologically or pharmaceutically acceptable excipient;

wherein the PCM polypeptide is:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 1;

In an eleventh aspect, the present invention provides a method of increasing the therapeutic efficacy of a vaccine, the method comprising the step of including in the vaccine whose therapeutic efficacy is to be increased:

(a) a polypeptide having an amino acid sequence shown in SEQ ID NO. 1;

In a twelfth aspect, the present invention provides a method for producing the polypeptide of the second or third aspect, comprising the steps of:

1) culturing the host cell of the seventh aspect, thereby producing the polypeptide of the second or third aspect;

2) obtaining the produced polypeptide from the culture system obtained in step 1).

In a thirteenth aspect, the present invention provides a method of preventing or treating a coronavirus infection, comprising the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a vaccine composition or an immunogenic composition or a pharmaceutical composition according to the first aspect or a polypeptide according to the second or third aspect.

In a preferred embodiment, the subject is a mammalian subject, preferably a human.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of RBD binding antibody titer detection. The serum antibody titers to RBD generated by 3 groups of immunized mice of 100ug RH2, 100ug PCM, 50ug RH2+50ug PCM were compared. Control: serum from unimmunized mice. The abscissa is serum dilution.

Fig. 2 shows the results of S2 protein binding antibody titer detection. The serum antibody titers against the S2 protein produced by 100ug RH2, 50ug RH2+50ug PCM immunized mice were compared. The abscissa is serum dilution.

FIG. 3 shows the specific T cell response of spleen cells from RH2 immunized mice and RH2+ PCM combined immunized mice under stimulation of 10ug RCO and 10ug RH 2. NC: negative Control (Negative Control); RCO: 10 ug/well; PC: positive Control (Positive Control).

Detailed Description

The inventors have conducted extensive and intensive studies and have unexpectedly found that two polypeptides, PCM and RH2, can be used to stimulate the production of both binding antibodies to the S1 protein and binding antibodies to the S2 protein. In particular, the combination further produces a synergistic effect compared to the use alone. The present invention has been completed based on this finding.

Coronavirus (coronavirus)

The term "coronavirus (Coronaviruses)" as used herein is a single-stranded positive-strand RNA virus belonging to the order Nidovirales (Nidovirales) Coronaviridae (Coronaviridae) orthocoronaviridae (orthocoronaviridae). The virus can infect various species such as human, bat, pig, mouse, cow, horse, goat, monkey, etc. There are known 7 kinds of human-infecting coronavirus (HCoV), including middle east respiratory syndrome-associated coronavirus (MERSR-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr-CoV).

In a specific embodiment, the coronavirus described herein is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus. In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

The most recently isolated coronavirus was a novel coronavirus of the genus β, WHO named 2019-nCoV, which is the 7 th coronavirus that infects humans. At present, no effective vaccine and therapeutic drug aiming at the new coronavirus exist, and virus diffusion is mainly controlled through precautionary measures, epidemic situation is closely monitored, and suspected cases are isolated and observed. At present, no specific treatment method for coronavirus exists, and symptomatic support treatment is mainly adopted.

The new coronavirus enters the cell by binding with ACE2 receptor on the surface of human cell through S protein on the surface. The S protein consists of a longer extracellular domain, a transmembrane domain and an intramembrane domain, and belongs to a first Class of virus membrane fusion proteins (Class I viral fusion proteins). The most significant difference between the S proteins of different coronaviruses is whether the viruses are cleaved by host proteases during assembly and release. The mature S protein is typically cleaved into two subunits by host proteases (cysteine proteases, trypsin, etc.): s1 and S2. The S1 subunit can be further divided into two relatively independent regions, the N-terminal region and the C-terminal region, respectively. S1 contains a Receptor Binding Domain (RBD), and most of the RBDs of the coronavirus S protein are located in the C-terminal region. The S2 subunit is anchored to the membrane via a transmembrane domain, which contains essential elements required for the membrane fusion process, including: an intrinsic membrane Fusion Peptide (FP), two 7-peptide repeats (HR), a transmembrane domain (JMD) and a transmembrane domain (TMD), and a Cytoplasmic Domain (CD) at the C-terminus (about 40 amino acids in length). The two HR, HR1 and HR2, which are also called HR-N and HR-C depending on their position, are separated by a middle helix structure of about 140 amino acids, and when the RBD binds to the receptor, the S2 subunit changes conformation by inserting the FP into the host cell membrane, HR1 and HR2 each form a triple helix structure, which are arranged antiparallel to form a six helix bundle (6HB), together forming a fusion core, eventually leading to fusion of the viral membrane with the cell membrane. Therefore, the blocking of the RBD recognition of the host cell and the blocking of the fusion of the S2 subunit and the cell membrane can effectively inhibit the invasion of the virus.

The S protein is an ideal antigen due to its functional importance. However, the novel coronavirus is an RNA virus, and a vaccine for the RNA virus often causes side effects, such as ADE (antibody dependent enhancement). These side effects are often caused by the fact that some components of the vaccine stimulate an immune response that is not protective.

Polypeptides of the invention

Coronaviruses such as new coronaviruses have appeared in many varieties, but the main invasion route is still that the RBD of the S Protein (Spike Protein) on the surface thereof recognizes and binds to the ACE2 Protein on human cells, thereby completing the invasion of host cells. Therefore, if the compound can effectively block the binding of the S protein and ACE2, the compound has great possibility of preventing the invasion of new coronavirus to human cells and preventing or treating a series of symptoms caused by the infection of new coronavirus.

To this end, the inventors designed two recombinant proteins, PCM and RH 2. Experiments prove that PCM or RH2 can be used as a new crown vaccine candidate, and the combined immune effect of PCM + RH2 is better than that of one protein used alone, so that the PCM and RH combined immune strategy can be used as an advantageous new crown immune strategy.

Herein, the "polypeptide of the present invention" or "protein of the present invention" have the same meaning and may be used interchangeably herein. These terms all refer to proteins or polypeptides that block either the RBD recognition of a host cell by a coronavirus, or the fusion of the S2 subunit with a cell membrane, or both the RBD recognition of a host cell and the S2 subunit with a cell membrane.

In a specific embodiment, the polypeptide of the invention has an amino acid sequence as set forth in SEQ ID NO: 1:

MHHHHHHQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ (SEQ ID NO: 1); wherein the N-terminal has His6 label and methionine (M) coded by initiation codon;

or the polypeptide with the amino acid sequence shown as the following SEQ ID NO: 2:

MHHHHHHRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE (SEQ ID NO: 2); wherein the N-terminal has His6 label and methionine (M) encoded by initiation codon.

In view of the teachings of the present invention and the prior art, those skilled in the art will appreciate that the proteins of the present invention should also include variants of the proteins that have the same or similar function as the "protein of the present invention" but differ in amino acid sequence by a small amount from the amino acid sequence shown in any of SEQ ID NO 1 or 2. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 30, preferably 1 to 10, more preferably 1 to 6, most preferably 1 to 3) amino acids, and addition of one or more (usually up to 20, preferably up to 10, more preferably up to 6 or 3) amino acids at the C-terminus and/or N-terminus. For example, it is well known to those skilled in the art that substitutions with amino acids of similar or analogous properties, e.g., isoleucine and leucine, do not alter the function of the resulting protein. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus, such as a tag added for ease of isolation, does not generally alter the function of the resulting protein. For example, for ease of detection and experimentation, the proteins in the examples are those with a biotin label at the N-terminus, which does not adversely affect the performance of the protein.

Variants of the protein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding a "protein of the invention". The invention also includes other proteins, such as fusion proteins comprising a "protein of the invention" or a fragment thereof. In addition to almost full-length proteins, the invention also encompasses active fragments of the "proteins of the invention".

The invention also provides analogs of the "protein". These analogs may differ from the proteins of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

The conservative variant of the protein of the present invention is a protein in which an amino acid residue present in an amino acid sequence shown in SEQ ID NO. 1 or 2 is replaced with an amino acid residue having a similar or analogous property, but the conservative variant protein still has the same or similar activity as the protein having an amino acid sequence shown in SEQ ID NO. 1 or 2.

Thus, in view of the teachings of the present invention and the prior art, one skilled in the art can generate conservatively variant mutants by making amino acid substitutions as shown, for example, in the following table.

Therefore, the protein of the present invention also includes a protein derived from the protein of the present invention by substitution, deletion or addition of one or several amino acid residues and having the function of the protein of the present invention.

In a preferred embodiment, the derivative protein is a derivative protein formed by the substitution, deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, and most preferably 1 to 3 amino acid residues of the protein of the present invention and having the function of the protein of the present invention.

In a preferred embodiment, the derived protein is a protein derived from the protein of the present invention by deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, and most preferably 1 to 3 amino acid residues, and having the function of the protein of the present invention.

In a preferred embodiment, the derivative protein is a derivative protein formed by adding or deleting 1 to 30, 1 to 10, 1 to 6, or 1 to 3 amino acid residues to the C-terminal and/or N-terminal of the protein of the present invention and having the function of the protein of the present invention.

In a specific embodiment, the protein of the invention is a protein with a 6His tag at the N-terminus and an amino acid residue M encoded by the start codon, such as the amino acid sequence shown in SEQ ID NO 1 or 2.

On the basis of the protein, the invention also provides a polynucleotide sequence for encoding the protein. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature protein may be identical to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 or a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a protein having the amino acid sequence shown in SEQ ID NO. 1 or 2, but differs from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 or 2.

The polynucleotide sequences encoding the proteins of the invention may be inserted into a recombinant expression vector or genome. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Those skilled in the art can use well-known methods for constructing expression vectors containing the DNA sequences encoding the proteins of the present invention and appropriate transcription/translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the protein.

The host cells described herein include host cells comprising the above-described expression vectors or nucleotide sequences encoding the genome integrated with the coding sequence of the protein of the invention, preferably the amino acid sequence shown in SEQ ID NO. 1 or 2. The host cell of the invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells. In particular embodiments, the strains include, but are not limited to: coli (e.coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Hafnia alvei (Hafnia alvei), Bacillus subtilis (Bacillus subtilis). In a preferred embodiment, the strain is E.coli.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. The recombinant protein in the above method may be constitutively expressed or conditionally expressed, for example, when the host cell is grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cell is cultured for an additional period of time.

The recombinant protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The vaccine composition or the pharmaceutical composition of the present invention

The present invention further provides a vaccine composition or an immunogenic composition or a pharmaceutical composition comprising PCM or RH2, or both, based on the two recombinant proteins of the invention, PCM and RH 2.

The vaccine composition or the immunogenic composition or the pharmaceutical composition can induce and generate neutralizing antibodies which can block RBD from recognizing host cells and block the fusion of S2 subunit and cell membranes.

In addition, the inventor unexpectedly finds that the PCM polypeptide can stimulate the generation of RBD binding antibody and can better stimulate T cells due to stronger immunogenicity. Thus, the PCM polypeptides of the invention may be used in combination with RH2, providing further significant advantages. Because the immunogenicity of the PCM protein is strong, the PCM protein can stimulate T cells well, and can be used together with other coronavirus antigens to generate better immune protection effect. Therefore, the PCM protein of the invention can be used as a vaccine of coronavirus and a synergist of other vaccines aiming at coronavirus, thereby playing a synergistic effect.

As used herein, the term "binding antibody" refers to all antibodies that bind to the corresponding antigen. As used herein, the term "neutralizing antibody" refers to an antibody that is capable of binding to a corresponding antigen and of blocking the binding of that antigen to its receptor.

The vaccine composition or immunogenic composition or pharmaceutical composition of the invention comprises a polypeptide or protein of the invention, and is typically combined with an immunologically or pharmaceutically acceptable carrier, including any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Examples of suitable carriers include, but are not limited to, proteins, lipid aggregates (e.g., oil droplets or liposomes), and the like. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants").

In addition, the vaccine composition or immunogenic composition of the invention may also contain additional adjuvants. Representative vaccine adjuvants include (but are not limited to) the following classes: inorganic adjuvants such as aluminum hydroxide, alum, etc.; synthetic adjuvants such as artificially synthesized double-stranded polynucleotides (double-stranded polyadenylic acid, uridylic acid), levamisole, isoprinosine, and the like; oil agents, such as Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil, etc. Typically, the vaccine composition or immunogenic composition can be prepared as an injectable formulation, such as a liquid solution or suspension; it can also be made into solid form suitable for preparing solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

The composition can be made into unit or multi-component dosage form. Each dosage form contains a predetermined amount of active material calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, oral, or topical administration.

When using the (vaccine) composition, a safe and effective amount of the vaccine polypeptide of the present invention is administered to a human. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

In addition to the proteins of the invention, the vaccine compositions or immunogenic compositions or pharmaceutical compositions of the invention may be combined with other antiviral agents to enhance therapeutic efficacy. The other antiviral drug may be selected from one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, lanimivir, peramivir and chloroquine (chloroquine phosphate); one or more of lopinavir, ritonavir, ribavirin, rituxivir, and chloroquine (chloroquine phosphate) are preferred.

The invention has the advantages that:

1. the protein or vaccine composition or immunogenic composition of the invention is capable of producing protective neutralizing antibodies and avoids the production of side effects;

2. the protein or vaccine composition or immunogenic composition of the invention stimulates both production of binding antibodies to the S1 protein and production of binding antibodies to the S2 protein, and simultaneously stimulates a T cell response.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Examples

Materials and methods

All materials or reagents according to the invention are routinely available in the art, e.g. commercially available.

1. Sequence of

PCM sequence (containing N-terminal His)₆Marker, SEQ ID NO: 1):

MHHHHHHQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ

RH2 (containing N-terminal His)₆Marker, SEQ ID NO: 2):

MHHHHHHRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE。

2. inducible expression

The expression strain was spread evenly on LB plate containing kanamycin sulfate and placed upside down in an incubator at 37 ℃ overnight. From the transformed plate, a single clone was selected, inoculated into 50ml of LB broth containing kanamycin sulfate (25 g/L of LB broth of an organism, 50ug/ml of kanamycin), and cultured overnight at 37 ℃ and 150 rpm. Subsequently, the cells were expanded at 1:100 into 2YT medium (10g/L peptone, 10g/L yeast extract, 5g/L sodium chloride, pH 7.4, kanamycin 50ug/ml) containing kanamycin. When the OD reached about 0.8, IPTG was added to the cells to give a final concentration of 0.2mM, the cells were induced at 37 ℃ and 200rpm for 4 hours, and the cells were collected by centrifugation.

3. Protein purification

The inclusion body is obtained after the thalli are crushed and washed by ultrasonic wave. After the inclusion bodies are washed by using a solution of 20mM Tris (pH8.0), 300mM NaCl, 1% Triton X-100, 2mM EDTA and 5mM DTT, the inclusion bodies are dissolved and denatured by using 20mM Tris (pH8.0), 300mM NaCl and 8M Urea buffer solution, a Ni-IDA column is balanced, finally, target protein is eluted by using the balancing buffer solution of imidazole with different concentrations, and each eluted component is collected for SDS-PAGE analysis and detection.

4. Renaturation of proteins

Purifying with Ni-IDA affinity chromatography, collecting high-purity progenitor fraction, adding into treated dialysis bag, dialyzing at 4 deg.C into buffer (1 XPBS, 4mM GSH, 0.4mM GSSG, 0.4M L-arginine, 1M Urea, 5% glycerol) for renaturation, and dialyzing into stock solution (1 XPBS, 5% glycerol) for about 6-8 h. After the dialysis renaturation is finished, the supernatant is filtered by a 0.22 mu m filter and subpackaged to obtain the required protein.

5. Immunization of mice

20 BALB/c mice (5-7 weeks, female, 18-20g) were prepared, grouped as follows:

the immunization mode is as follows: the first immunization was performed on day 0, the second immunization was performed two weeks later (i.e., day 14), the third immunization was performed one week later (i.e., day 21), and the mice were sacrificed one week later (day 28), drawn, and subjected to ELISA and ELISPOT experiments.

IFN-gamma ELISA speckle method for detecting mouse T cell

This was performed using an ELISA-dot assay kit from BD PharminGen. Spleen cells of immunized mice were added to IFN-. gamma.antibody-precoated plates and stimulated with 10 ug/well RCO and 10 ug/well RH2 protein, incubated for 12-16h, spleen cells were discarded, and labeled anti-IFN-. gamma.antibody was added for incubation for 1 hour and then developed, and after the color development was terminated, spot counting was performed using a spot counter. The RCO protein is a recombinant overlapping polypeptide designed aiming at a virus RBD sequence and used for detecting a specific T cell response caused by new corona infection, and the amino acid sequence of the RCO protein is shown as SEQ ID NO. 3 (the RCO protein is referred to Chinese patent application No. CN 202011063193.7).

MHHHHHHSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFLRMKKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADLRMKEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLRMKPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLLRMKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYLRMKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYLRMKAGSTPCNGVEGFNCYFPLQSYGFQPTNGVLRMKYFPLQSYGFQPTNGVGYQPYRVVVLSFELRMKDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGLRMKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWLRMKIDLQELGKYEQYIKWPWYIWLGFIAGLIAIV(SEQ ID NO:3)

7. Antibody titer detection

The antibody titer of the immunized mice against the RBD and S2 proteins was measured by ELISA.

7.1 enzyme label plate coating 2ug/ml RBD protein: RBD protein purchased from Genescript was diluted with CB solution of pH9.0, 100ul per well was added to the plate, and the plate was coated overnight at 4 ℃.

7.2 sealing: after washing the plates with PBST (0.05% Tween-20), blocking was performed by adding 200ul of 2.5% BSA solution per well and blocking for 1h at 37 ℃.

7.3 serum sample dilution: the serum of the immunized and control mice is taken, 10ul of PBS solution and 990ul of PBS solution are respectively taken for each sample and mixed evenly, and the mouse serum with the dilution ratio of 1:100 is obtained. Then 4 times of gradient dilution is carried out, 250ul of mouse serum with the ratio of 1:100 is mixed with 750ul of PBS solution and mixed evenly, and the mouse serum with the dilution ratio of 1:400 is obtained; 250ul of mouse serum at a ratio of 1:400 was mixed with 750ul of PBS solution and mixed well to obtain mouse serum at a dilution of 1:1600, and so on. The highest dilution used was 1:1,680,000.

7.4 sample adding: after washing the blocked ELISA plate with PBST, the diluted 7.3 serum sample was added thereto, and the plate was incubated at 37 ℃ for 1 hour at 100ul per well.

7.5 adding enzyme-labeled antibody: after PBST washing, goat anti-mouse-HRP antibody diluted with PBS at a ratio of 1:20000 was added to 50ul per well and incubated at room temperature for 30 min.

7.6 adding a color developing solution and a stop solution: after PBST plate washing, 100ul of TMB color developing solution is added into each hole, 50ul of 15% sulfuric acid is added into each hole after color development is carried out for 5-10min to stop reaction, and OD450 value is detected.

7.8 mouse serum antibody titer curves were plotted using four-parameter fitting

8. Neutralizing antibody detection

The ELISA plate was coated with 2ug/ml RBD protein and blocked with 2.5% BSA in PBS for 1h at 37 ℃. Serum samples were diluted and mixed with labeled human ACE2 protein and applied to RBD-coated microtiter plates and incubated for 1 h. ACE2 was mixed with PBS or nonimmune mouse serum as a control. Then, washing, developing and reading the plate.

Example 1 preparation of PCM Polypeptides

The PCM polypeptide of the present invention was constructed and prepared by the methods described in materials and methods, Ministry of Biotechnology, Inc. of Ministry of technology, Nanjing.

Example 2 preparation of RH2 polypeptide

The RH2 polypeptide of the present invention was constructed and prepared by the methods described in materials and methods, Nanjing Kinshire Biotech Ltd.

Example 3 detection of antibody Titers

The inventors tested the titers of the polypeptides PCM of the invention and RH2 as described in materials and methods under "7" and "8". The concrete results are as follows:

after 28 days of immunization, mice immunized with 100ug RH2, 100ug PCM or 50ug RH2+50ug PCM all produced higher titers of RBD binding antibody. Although the amount of each protein used in the combined immunization was only one-half of that used in the individual immunization, the combined immunization produced RBD-binding antibody titers comparable to those of higher doses of PCM or RH2 alone (as shown in FIG. 1). The titer of the S2-binding antibody produced by the combined immunization group was even significantly better than that of RH2 alone (as shown in fig. 2), whereas the S2 protein sequence was not contained in PCM, so mice immunized with PCM alone did not produce the binding antibody of S2.

Neutralizing antibody detection experiments showed that neutralizing antibodies capable of blocking RBD and ACE2 binding were produced in three groups of immunized mice.

Example 4 determination of specific T cell response

Spleen lymphocytes from the spleens of the immunized mice of each group were extracted, and then stimulated with RH2 or RCO, respectively, a protein containing RBD-overlapping polypeptide as a stimulator, and IFN-. gamma.released from the specifically stimulated lymphocytes was detected by the ELISPOT method. Experiments have shown that mice immunized with either 100ug RH2 or 50ug RH2+50ug PCM are stimulated to produce a specific T cell response to RH 2. When RCO was used as a stimulator, all 5 mice immunized with 50ug RH2+50ug PCM developed specific T cell responses, and only 3, i.e., 60%, of 5 mice immunized with 100ug RH2 developed specific immune responses (as shown in FIG. 3).

And (4) conclusion:

as can be seen from the antibody detection, the 50ug RH2+50ug PCM combined immunization group can produce higher titer binding antibody regardless of whether the RBD protein or the S2 protein is targeted. Mice in the 100ug PCM immunized group, because PCM does not contain the S2 subunit fraction, were immunized to produce higher titers of RBD-binding antibodies, but not antibodies against S2. The 100ug RH2 immunization group, in addition to producing RBD-binding antibodies, also stimulated production of binding antibodies for a portion of the S2 protein. However, in comparison, the titer of the produced S2-binding antibody was much lower than that of the RH2+ PCM combined immune group. From the immunogenic composition, the immunogenic composition against the S2 subunit is higher and the antibody titer that should be generated is higher when 100ug RH2 alone compared to RH2+ PCM combined immunization with only 50ug RH2, but this is not the case.

Without wishing to be bound by any particular theory, the inventors believe that it is possible that the antibody titres are increased because the PCM proteins are more immunogenic and stimulate better T cells, while the cytokines secreted by CD4+ T cells stimulate the differentiation of B cell clones directed against PCM and RH 2.

Further analysis of specific T cell responses may also support this idea. Figure 2 shows that the T cell response of RH2+ PCM combined immunization is superior to that of RH2 alone. Therefore, RH2 or PCM can be used as candidate vaccine of coronavirus, but RH2 has obvious advantages when combined with PCM.

In addition, because the PCM protein has stronger immunogenicity, the PCM protein can better stimulate T cells, and can be used together with other coronavirus antigens, thereby generating better immune protection effect; in other words, the PCM protein of the present invention may be used not only as a vaccine for coronavirus itself, but also as a synergist for other vaccines against coronavirus, thereby achieving a synergistic effect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Claims

1. A vaccine composition or immunogenic composition or pharmaceutical composition comprising a PCM polypeptide and/or an RH2 polypeptide and optionally an immunologically or pharmaceutically acceptable excipient;

wherein the PCM polypeptide is a polypeptide with an amino acid sequence shown as SEQ ID NO. 1;

the RH2 polypeptide is a polypeptide with an amino acid sequence shown as SEQ ID NO. 2.

2. The vaccine composition or immunogenic composition or pharmaceutical composition of claim 1, wherein the vaccine composition or immunogenic composition or pharmaceutical composition is a vaccine composition or pharmaceutical composition for the prevention or treatment of a coronavirus infection.

3. The vaccine composition or immunogenic composition or pharmaceutical composition of claim 2, wherein the coronavirus is severe acute respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus, or a novel coronavirus.

4. The vaccine composition or immunogenic composition or pharmaceutical composition of claim 3, wherein the coronavirus is a severe acute respiratory syndrome coronavirus or a novel coronavirus.

5. The vaccine composition or immunogenic composition or pharmaceutical composition of claim 4, wherein the coronavirus is a novel coronavirus.

6. The vaccine composition or immunogenic composition or pharmaceutical composition of any one of claims 1-5, wherein the vaccine composition or pharmaceutical composition comprises a PCM polypeptide and an RH2 polypeptide and optionally an immunologically or pharmaceutically acceptable excipient.

7. A polypeptide, the polypeptide has an amino acid sequence shown as SEQ ID NO. 1.

8. A polypeptide, the polypeptide has an amino acid sequence shown as SEQ ID NO. 2.

9. Use of a polypeptide according to claim 7 or 8 in the manufacture of a vaccine composition or an immunogenic composition or a pharmaceutical composition.

10. The use according to claim 9, wherein the vaccine composition or immunogenic composition or pharmaceutical composition is a vaccine composition or pharmaceutical composition for the prevention or treatment of a coronavirus infection.

11. The use of claim 10, wherein the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus.

12. The use of claim 11, wherein the coronavirus is a severe acute respiratory syndrome coronavirus or a novel coronavirus.

13. The use of claim 12, wherein the coronavirus is a novel coronavirus.

14. A nucleic acid encoding the polypeptide of claim 7 or 8.

15. An expression vector comprising the coding nucleic acid of claim 14.

16. A host cell comprising the expression vector of claim 15 or having integrated into its genome the encoding nucleic acid of claim 14.

17. Use of a polypeptide according to claim 7 in the manufacture of a formulation for enhancing the effect of a vaccine composition or an immunogenic composition or a pharmaceutical composition.

18. Use according to claim 17, wherein the use is of a polypeptide according to claim 7 as a potentiator for enhancing a vaccine or immunogenic composition or pharmaceutical composition.

19. The use of claim 17, wherein the vaccine composition or pharmaceutical composition is a vaccine composition or immunogenic composition or pharmaceutical composition for the prevention or treatment of a coronavirus infection.

20. The use of claim 19, wherein the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus.

21. The use of claim 20, wherein the coronavirus is a severe acute respiratory syndrome coronavirus or a novel coronavirus.

22. The use of claim 21, wherein the coronavirus is a novel coronavirus.

23. A vaccine synergist comprises a polypeptide, wherein the polypeptide has an amino acid sequence shown as SEQ ID NO. 1.