CN117778473A

CN117778473A - Antigenic polypeptides and uses thereof

Info

Publication number: CN117778473A
Application number: CN202310869607.2A
Authority: CN
Inventors: 刘洪恩; 李展如
Original assignee: Guangdong Keguanda Pharmaceutical Technology Co ltd
Current assignee: Guangdong Keguanda Pharmaceutical Technology Co ltd
Priority date: 2022-07-14
Filing date: 2023-07-14
Publication date: 2024-03-29

Abstract

The present disclosure provides a recombinant adeno-associated virus (rAAV) vector and compositions comprising the rAAV vector. The disclosure also provides methods of producing an rAAV using the rAAV vector, and uses of the rAAV vector or rAAV in inducing an immune response against SARS-CoV-2 or a variant thereof in a subject, and preventing and/or treating a disease caused by infection with SARS-CoV-2 or a variant thereof in a subject.

Description

Antigenic polypeptides and uses thereof

Technical Field

The present disclosure relates to biotechnology, and is especially SARS-CoV-2 or its variant antigenic polypeptide and its recombinant adeno-associated virus and their application in preparing vaccine.

Background

As with the SARS-CoV-2 novel coronavirus, the major structural proteins of variant Omicron include Spike (Spike) protein (S), envelope (Envelope) protein (E), matrix (Matrix) protein (M) and Nucleocapsid (nucleoapsid) protein (N); among them, the most mutation sites on spike protein (S protein) are also the most critical. Spike proteins invade the body by binding to the human angiotensin converting enzyme 2 (ACE 2) receptor, a cell in humans, and therefore become the target protein for most new coronaries to exert protective efficacy.

Omicron variants have 32 sites of aberrant mutation at 50% prevalence of spike protein, in contrast to only 9 sites of 50% prevalence of destructive Delta variants, so Omicron variants are more prone to escape neutralizing antibodies than Delta variants. Related in vitro studies (Vikram Thakur, radha Kanta Ratho. OMICRON (B.1.1.529): anewSARS-CoV-2 variant of concern mounting worldwide fear.J Med Virol.2022;94:1821-1824) showed a 11.4-fold and 20-fold decrease in the neutralization capacity of Omicron, respectively, from individuals vaccinated with BNT162b2 and mRNA 1273; no neutralizing effect was observed in the serum of ChAdOx1 vaccinated individuals. In addition, the neutralizing effect of serum induced by vaccine prepared against wild-type toxic RBD of SARS-CoV-2 on Omicron is reduced, and the preventive and therapeutic effects are significantly reduced (MedRxiv.2021. Doi: 10.1101/2021.12.07.21267432). Thus, there is an urgent need to design a novel vaccine capable of not only against wild-type SARS-CoV-2 novel coronavirus, but also against various variants of SARS-CoV-2 including Omacron.

Summary of The Invention

The present disclosure provides a recombinant adeno-associated virus (rAAV) vector and compositions comprising the rAAV vector. The disclosure also provides methods of producing such rAAV vectors. The present disclosure also provides vaccine compositions comprising the rAAV vectors, as well as methods of using the rAAV vectors to induce an immune response against SARS-CoV-2 or a variant thereof in a subject, and methods of preventing and/or treating a disease caused by infection with SARS-CoV-2 or a variant thereof in a subject.

The purpose of the present disclosure is to overcome the shortcomings of the prior art, and to provide a recombinant adeno-associated virus (rAAV) vector vaccine for SARS-CoV-2 or variants thereof, and a preparation method and application thereof, which can be effectively used for preventing and treating various SARS-CoV-2 viruses including Omicron variants.

Accordingly, in one aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a nucleotide sequence encoding an S protein of SARS-CoV-2 variant Omicron or an antigenic fragment thereof. In some embodiments, a rAAV vector may comprise a nucleotide sequence encoding a polypeptide or antigenic fragment thereof, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID nos. 2-3, 16-17, and 23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID nos. 2-3, 16-17, and 23, or one or more (e.g., 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5) amino acid residues.

In some embodiments of the rAAV vectors of the present disclosure, the polypeptide comprises one or more mutations selected from the group consisting of: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

In some embodiments, the polypeptide comprises the following mutations: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO.2-3, 16-17 or 23.

In some embodiments of the rAAV vectors of the present disclosure, the polypeptide further comprises an N-terminal signal peptide sequence. In some embodiments, the signal peptide is derived from a signal peptide of a protein selected from the group consisting of: tissue plasminogen activator (tPA), granulocyte-macrophage colony-stimulating factor (GM-CSF), prolactin precursors, growth hormone, and immunoglobulins (e.g., igE). In some embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO.4 or SEQ ID NO. 5.

In some embodiments of the rAAV vectors of the present disclosure, the adenovirus-associated Inverted Terminal Repeat (ITR) sequences are included at the 5 'and 3' ends of the nucleotide sequence encoding the polypeptide or antigenic fragment thereof. In some embodiments, the ITR sequence is derived from an ITR sequence of an adeno-associated virus serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the ITR sequence is derived from an ITR sequence of an AAV2 serotype. In some embodiments, the ITR sequence has the nucleotide sequence set forth in SEQ ID No. 6. In some embodiments, the ITR sequence has the nucleotide sequence set forth in SEQ ID No. 28.

In some embodiments of the rAAV vectors of the present disclosure, the rAAV vectors further comprise expression regulatory elements, such as one or more of a promoter sequence, an upstream regulatory region, a transcriptional regulatory element, and a terminator.

In some embodiments of the rAAV vectors of the present disclosure, the nucleotide sequence encoding the polypeptide or antigenic fragment thereof is codon optimized of human origin.

In some embodiments of the presently disclosed rAAV vectors, the nucleotide sequence encoding the polypeptide or antigenic fragment thereof has the nucleotide sequence set forth in SEQ ID nos. 7-9, 18-20, 24 or 25.

In some embodiments, the rAAV vector has the nucleotide sequence depicted in SEQ ID NO.10-11, 21-22, or 26.

In another aspect, the present disclosure provides a composition comprising a rAAV vector of the present disclosure and an AAV serotype plasmid. In some embodiments of the compositions of the present disclosure, the serotype plasmid comprises nucleotide sequences encoding the Rep protein and Cap protein of an AAV.

In some embodiments, the Rep protein is a Rep protein of an adeno-associated viral serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the Rep proteins are from AAV2 serotypes.

In some embodiments, the Cap protein is a Cap protein of an adeno-associated virus serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the Cap protein is from an AAV5 serotype.

In some embodiments of the compositions of the present disclosure, the serotype plasmid comprises the nucleotide sequence shown in SEQ ID No.12 or SEQ ID No.13 at the 5 'and/or 3' end of the nucleotide sequences encoding the Rep proteins and Cap proteins of AAV.

In some embodiments, the composition further comprises a helper plasmid.

In yet another aspect, the present disclosure provides a method of producing a recombinant adeno-associated virus (rAAV), comprising:

i) Co-transfecting a host cell with the composition of the present disclosure;

ii) culturing the host cell under conditions suitable for the production of rAAV, and

iii) Isolation of rAAV from host cells and/or culture supernatant.

In some embodiments of the methods of the present disclosure, in step i) and/or step ii), the host cell is in an adherent state.

In some embodiments of the methods of the present disclosure, in step i) and/or step ii), the host cell is in suspension.

In another aspect, the present disclosure provides rAAV produced by the methods of the present disclosure.

In another aspect, the present disclosure provides a polypeptide comprising an amino acid sequence having one or more mutations in the amino acid sequence set forth in SEQ ID No.2 or 16 selected from the group consisting of: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

In some embodiments, the polypeptide comprises an amino acid sequence that has been subjected to the following mutations in the amino acid sequence set forth in SEQ ID No.2 or 16: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

In another aspect, the present disclosure provides a polypeptide comprising the amino acid sequence set forth in SEQ ID No.3, 17 or 23, or an antigen binding fragment thereof.

In yet another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide of the present disclosure or an antigen binding fragment thereof.

In yet another aspect, the present disclosure provides a vaccine composition comprising a rAAV vector of the present disclosure, a composition of the present disclosure, a rAAV of the present disclosure, a polypeptide of the present disclosure or an antigenic fragment thereof, or a nucleic acid of the present disclosure, and optionally one or more adjuvants and/or excipients.

In yet another aspect, the disclosure provides a method of inducing an immune response in a subject against SARS-CoV-2 or a variant thereof, the method comprising administering to the subject a rAAV vector of the disclosure, a composition of the disclosure, a rAAV of the disclosure, a polypeptide of the disclosure, or an antigenic fragment thereof, a nucleic acid of the disclosure, or a vaccine composition of the disclosure.

In yet another aspect, the present disclosure provides a method of preventing and/or treating a disease caused by infection with SARS-CoV-2 or a variant thereof in a subject, the method comprising administering to the subject a rAAV vector of the disclosure, a composition of the disclosure, a rAAV of the disclosure, a polypeptide of the disclosure, or an antigenic fragment thereof, a nucleic acid of the disclosure, or a vaccine composition of the disclosure.

In some embodiments of both aspects, the SARS-CoV-2 variant strain is selected from Alpha, beta, gamma, delta, epsilon, zeta, eta, iota, kappa, lambda, mu, omicron and subtypes thereof.

Compared with the prior art, the invention has at least the following advantages and effects:

1. the Omicron novel coronavirus antigenic polypeptide sequence adopted by the invention has definite structure and high targeting effect. AAV 5-type recombinant adeno-associated virus (rAAV) which adopts tPA novel signal peptide or tPA signal peptide to deliver antigenic polypeptide has extremely low immunogenicity and genetic toxicity, can improve the immunogenicity of the antigenic polypeptide, and can enhance the immunocompetence of the vaccine.

2. AAV 5-type recombinant adeno-associated virus (rAAV) delivering antigenic polypeptides can be delivered by nasal or intramuscular injection, and in vivo delivery and expression can produce fusion antigen polypeptides, can induce the production of antibodies with neutralizing titers against various novel coronaviruses including Omicron variants, and can be used for immunization against novel coronavirus infections caused by various novel coronaviruses including Omicron.

3. The effective vaccine can be prepared by adopting different preparation processes, so that the preparation process can be modified and optimized according to the requirements, thereby being beneficial to industrialized mass production.

Drawings

FIG. 1 is a schematic diagram of a vector map of vector plasmid 1. The key elements in the schematic are described as follows: ITR refers to the inverted terminal repeat of AAV 2; tPA signal NEW refers to a tPA novel signal peptide having the sequence SEQ ID No. 4; omicron (B.1.1529) -Spike refers to the Omicron novel coronavirus S protein with the sequence of SEQ ID NO. 3; WPRE refers to a woodchuck hepatitis virus posttranscriptional regulatory element; SV40polyA refers to the monkey vacuolated virus 40 (SV 40) polyadenylation sequence.

FIG. 2 is a schematic diagram of a vector map of vector plasmid 2. The tPA signal in the schematic diagram refers to the tPA signal peptide with the sequence of SEQ ID NO. 5; other key elements are the same as in fig. 1.

FIG. 3 is a graph showing the effect of ELISA detection on IgG antibody expression in sera of mice at day 40, day 60 and day 100 after immunization of C57 mice with recombinant adeno-associated virus (rAAV) vaccines TB-1 and TB-2.

FIG. 4 is a graph showing the effect of ELISA detection on IgG antibody expression in sera of mice at day 50 and day 70 after immunization of C57 mice with recombinant adeno-associated virus (rAAV) vaccines XF-1 and XF-2.

FIG. 5 is the results of serum virus neutralization assays performed on day 40, day 60 and day 100 after immunization of C57 mice with recombinant adeno-associated virus (rAAV) vaccines TB-1 and TB-2.

FIG. 6 is the results of serum virus neutralization assays performed on day 50 and day 70 after immunization of C57 mice with recombinant adeno-associated virus (rAAV) vaccines XF-1 and XF-2.

FIG. 7 shows the results of the expression level of the target protein of recombinant adeno-associated virus (rAAV) vaccine TB-3.

FIGS. 8A-8H show exemplary photographs of lung histopathological examination of recombinant adeno-associated virus (rAAV) vaccine immunized challenge group mice. Wherein fig. 8A and 8B show exemplary H & E staining results of lung tissue of mice of the control group (group 1), fig. 8C and 8D show exemplary H & E staining results of lung tissue of mice of the challenge group 2 (2E 11 vg), fig. 8E and 8F show exemplary H & E staining results of lung tissue of mice of the challenge group 3 (1E 11 vg), and fig. 8G and 8H show exemplary H & E staining results of lung tissue of mice of the challenge group 4 (5E 10 vg). Scale bar = 200 μm.

Detailed Description

Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. Although the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All published patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference. Other features and advantages of the disclosure will be apparent from the following detailed description, including the embodiments and claims.

For easier understanding of the present disclosure, certain terms are first defined below. Additional definitions for the following and other terms are set forth throughout the specification.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a", "an", "one or more" and "at least one" are used interchangeably.

The terms "comprising," "including," "containing," and "having," when used herein and in the appended claims, do not exclude other elements and may be used interchangeably. For the purposes of the present invention, the term "consisting of … …" is considered to be a preferred embodiment of the term "comprising".

The present disclosure provides recombinant adeno-associated virus (rAAV) vectors comprising a nucleotide sequence encoding an S protein of SARS-CoV-2 variant Omicron or an antigenic fragment thereof. In some embodiments, a rAAV vector may comprise a nucleotide sequence encoding a polypeptide or antigenic fragment thereof, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID nos. 2-3, 16-17, and 23, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID nos. 2-3, 16-17, and 23, or one or more (e.g., 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5) amino acid residues.

"vector" refers to a delivery means or element of genetic material, common vectors include, for example, vectors derived from bacterial plasmids, viral vectors, vectors derived from yeast plasmids, vectors derived from phages, cosmids, phagemids and the like, which term is also often referred to as cloning vectors, expression vectors or backbone vectors, as well as viral vectors, depending on the type or context of application. The term "expression vector" is typically a vector that enables expression of a gene of interest in a cell, and is typically a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide or protein and operably linked to expression regulatory elements.

As used herein, "rAAV vector" refers to a recombinant non-replicating adeno-associated virus, the rAAV vector comprising a serotype protein capsid and encapsulating a recombinant genome comprising functional 5 'and 3' inverted terminal repeats (ITR sequences) flanked by exogenous nucleotide sequences that replace the rep or cap genes of wild-type AAV.

The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably herein to refer to a compound consisting of amino acid residues covalently linked by peptide bonds, and do not limit the minimum length of the product. Thus, the above terms include peptides, oligopeptides, polypeptides, dimers (heterologous and homologous), multimers (heterologous and homologous), and the like. "proteins" and "polypeptides" encompass full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. In addition, for the purposes of this disclosure, polypeptides also refer to variants obtained by modification of the amino acid sequence of a native protein or polypeptide.

Typically, amino acid sequence modifications include substitutions, deletions, additions and insertions. As used herein, "substitution" refers to the removal of at least one residue and the insertion of a different residue at that position. "deletions" are characterized by the removal of one or more amino acid residues from the sequence. "addition" refers to amino and/or carboxy-terminal fusions, while "insertion" refers to intrasequence insertion of single or multiple amino acid residues. Amino acid substitutions are typically single residues but may occur at many different positions simultaneously. Typically, no more than about 2-6 residues are deleted at any one site within the protein molecule. The insertion will typically be a smaller insertion, e.g., about 1-4 residues, than the addition.

The term "variant" with respect to an amino acid sequence includes any substitution, variation, modification, substitution, deletion, or addition from the sequence or to one (or more) amino acids of the sequence. Substitutions, deletions, additions, insertions, or any combination thereof may be included in a single variant, provided that the polypeptide variant is antigenic.

Amino acid substitutions as used herein may be conservative or non-conservative substitutions. In this regard, it is understood in the art that amino acids may be grouped based on their physical characteristics. Examples of such groupings include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Conservative amino acid substitutions may involve substitution of a natural amino acid residue with a non-natural residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and in particular, does not result in a reduction in antigenicity.

In this context, substitution with a conserved amino acid refers to interchangeability with residues having similar side chains. The art has defined families of amino acid residues with side chains. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

In some embodiments, the substitutions are not conservative. For example, substitutions may be made with amino acids that alter some property or aspect of the polypeptide. In some embodiments, non-conservative amino acid substitutions may be made, e.g., altering the structure of the polypeptide, altering the binding properties of the polypeptide (e.g., increasing or decreasing the binding affinity of the polypeptide, and/or increasing or decreasing the binding specificity of the polypeptide).

The terms "nucleic acid", "nucleotide sequence" are used interchangeably to refer to any length of oligomer and polymer consisting essentially of nucleotides (e.g., deoxyribonucleotides and/or ribonucleotides). Nucleic acids may comprise purine and/or pyrimidine bases and/or other natural (e.g., xanthine, inosine), chemical or biochemical modifications (e.g., methylation), non-natural or derivatized nucleotide bases. The backbone of the nucleic acid may comprise sugar and phosphate groups, and/or one or more modified or substituted sugar and/or one or more modified or substituted phosphate groups, typically present in RNA or DNA. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The "nucleic acid" may be, for example, double-stranded, partially double-stranded or single-stranded. When single stranded, the nucleic acid may be the sense strand or the antisense strand. The "nucleic acid" may be circular or linear. As used herein, the term "nucleic acid" encompasses DNA and RNA, including genomic, pre-mRNA, mRNA, cDNA, recombinant or synthetic nucleic acids comprising a vector. For the purposes of the uses described herein, it will be appreciated that the polynucleotides may be modified by any method available in the art.

Those of skill in the art will appreciate that many different polynucleotides and nucleic acids may encode the same polypeptide due to the degeneracy of the genetic code. In addition, it will be appreciated that the skilled artisan can make nucleotide substitutions using conventional techniques that do not affect the polypeptide sequences encoded by the polynucleotides described herein, to reflect codon usage of any particular host organism in which the polypeptides are to be expressed.

"antigenicity" also known as "immunogenicity" or "immunoreactivity" refers to the ability of a protein or polypeptide to produce an antibody or to induce an antibody response in a subject. An "antigenic fragment" refers to a polypeptide fragment capable of inducing an immune response. The length of the antigenic fragment is not particularly limited and may be, for example, 5 to 300 amino acids, 301 to 600 amino acids, 601 to 900 amino acids, 901 to 1200 amino acids, or any number of amino acids therebetween or more. Antigenic fragments can be produced by methods known to those skilled in the art.

As used herein, "sequence identity" refers to the degree of identity between any given query sequence and the subject sequence. One of skill in the art will readily understand how to determine the identity of two polypeptides (e.g., unmodified peptides and peptide variants). For example, identity may be calculated after aligning two sequences such that their identity reaches a maximum level, e.g., gaps may be introduced. Another method of calculating identity may be implemented by the disclosed algorithm. Non-limiting examples of such mathematical algorithms include Myers and Miller (1988) CABIOS 4:11-17, smith et al (1981) adv. Appl. Math.2:482, needleman and Wunsch (1970) j.mol. Biol.48:443-453 homology alignment algorithm, pearson and Lipman (1988) proc.Natl. Acad.Sci.85:2444-2448 methods for searching for homology, karlin and Altschul (1990) proc.Natl. Acad.Sci.USA 87: modification of the algorithm of 2264 is described in Karlin and Altschul (1993) proc.Natl. Acad.Sci.USA 90: 5873-5877. By using programs based on such mathematical algorithms, sequence comparisons (i.e., alignments) for determining sequence identity can be performed. The program may be appropriately executed by a computer. Examples of such programs include, but are not limited to, the CLUSTAL, ALIGN program (version 2.0) of the PC/Gene program and GAP, BESTFIT, BLAST, FASTA and TFASTA of the Wisconsin genetics software package. The alignment using these procedures may be performed, for example, by using initial parameters.

In some embodiments of the rAAV vectors of the present disclosure, the polypeptide further comprises an N-terminal signal peptide sequence.

As used herein, "signal peptide" refers to a short peptide chain, typically 5-30 amino acids in length, that directs the transfer of a newly synthesized protein to the secretory pathway. In some embodiments, the signal peptide is derived from a signal peptide of a protein selected from the group consisting of: tissue plasminogen activator (tPA), granulocyte-macrophage colony-stimulating factor (GM-CSF), prolactin precursors, growth hormone, and immunoglobulins (e.g., igE). In some preferred embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO.4 or SEQ ID NO. 5.

In some embodiments of the rAAV vectors of the present disclosure, the adenovirus-associated Inverted Terminal Repeat (ITR) sequences are included at the 5 'and 3' ends of the nucleotide sequence encoding the polypeptide or antigenic fragment thereof.

ITR is a cis-acting element of the adeno-associated virus (AAV) vector genome, plays an important role in the integration, rescue, replication and genome packaging of adeno-associated viruses, and is involved in the integration and escape process of the viral genome on the host genome. The ITR sequence is typically from serotype AAV2 and is placed upstream and downstream of the sequence of interest. The ITR has a reverse self-complementary sequence, and thus forms a T-shaped double-stranded structure by self-complementarity in the state of single-stranded DNA. In some embodiments, the ITR sequence is derived from an ITR sequence of AAV2 serotype, and preferably has the nucleotide sequence shown in SEQ ID No.6, and/or has the nucleotide sequence shown in SEQ ID No. 28.

In some embodiments, the rAAV vectors of the present disclosure further comprise expression regulatory elements, such as one or more of a promoter sequence, an upstream regulatory region, a transcriptional regulatory element, and a terminator.

Expression regulatory elements are typically a collection of promoter sequences, upstream regulatory regions, transcriptional regulatory elements, and terminators, which together effect replication, transcription, and translation of the coding region sequences in the recipient cell. A promoter is a DNA sequence that recognizes, binds to, and initiates transcription by RNA polymerase and contains conserved sequences required for specific binding and transcription initiation by RNA polymerase, mostly upstream of the transcription initiation point of a structural gene, while the promoter itself is not transcribed. In some cases, a CAG promoter may be selected as the promoter sequence. Suitable promoters also include promoters known to those of skill in the art, such as the human Cytomegalovirus (CMV) promoter, the ubiquitin C promoter (UbC), the EF1 alpha promoter, and the like. In some cases, SV40polyA may be selected as the transcription terminator. Suitable polyA sequences include, but are not limited to, SV40, BGH, synthetic polyA, and the like, as known in the art. In some cases, the expression regulatory elements may also include transcriptional enhancing regulatory elements, such as woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs) and translational efficiency enhancing sequences, such as Kozak sequences.

In some embodiments, the nucleotide sequence encoding the polypeptide or antigenic fragment thereof is codon optimized of human origin. The manner in which codon optimization is performed is well known to those skilled in the art, and appropriate codon optimization can be performed according to host codon preference.

The disclosure also provides compositions comprising the rAAV vectors and AAV serotype plasmids of the disclosure. In some embodiments of the compositions of the present disclosure, the serotype plasmid comprises nucleotide sequences encoding the Rep protein and Cap protein of an AAV.

AAV serotype plasmid pRepCap comprises two Open Reading Frames (ORFs) encoding the Rep and Cap expression products, respectively. Wherein the Rep gene encodes a plurality of proteins for viral replication, and the Cap gene encodes functional proteins such as subunits VP1, VP2, VP3, AAP, etc., of capsid proteins. The Rep proteins are responsible for replication of the AAV genome and assist in assembly of AAV genome particles, and Cap proteins refer to capsid proteins of AAV familiar to those skilled in the art, with different capsid protein sequences for AAV of different serotypes.

In some embodiments, the Rep protein and Cap protein are each a Rep protein of an adeno-associated viral serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some preferred embodiments, the Rep proteins are from AAV2 serotypes and the Cap proteins are from AAV5 serotypes. In some more preferred embodiments, the serotype plasmid comprises the nucleotide sequence shown in SEQ ID No.12 or SEQ ID No.13 at the 5 'and/or 3' end of the nucleotide sequences encoding the Rep proteins and Cap proteins of AAV.

In some embodiments, the compositions of the present disclosure further comprise a helper plasmid. Helper plasmids suitable for the disclosed vector compositions are well known to those skilled in the art. In some embodiments, the helper plasmid may be an AAV helper plasmid pHelper, which generally includes coding regions for adenovirus vara, E4 ORF6, E2A, E B, and the like, providing the functions necessary for AAV replication.

The present disclosure provides methods of producing a recombinant adeno-associated virus (rAAV), comprising:

i) Co-transfecting a host cell with the composition of the present disclosure;

iii) Isolation of rAAV from host cells and/or culture supernatant.

In some embodiments, the host cell is in an adherent state during step i) and/or step ii), i.e. during co-transfection and/or cultivation of the composition. In other embodiments, the host cells are in suspension during step i) and/or step ii), i.e. during co-transfection and/or cultivation of the composition.

The disclosure also provides rAAV produced by the above methods.

Nucleic acids, such as vectors or expression vectors, can be delivered into prokaryotic and eukaryotic cells by a variety of methods known in the art, including but not limited to various chemical, electrochemical and biological methods, such as heat shock transformation, electroporation, transfection, such as liposome-mediated transfection, DEAE-Dextran-mediated transfection or calcium phosphate transfection, and the like. As used herein, "transfection" refers to the process of artificially introducing nucleic acid (DNA or RNA) into a cell using means other than viral infection. The purpose of transfection is to produce recombinant proteins, or to specifically enhance or inhibit gene expression in transfected cells. The transfection method is not particularly limited, and a person skilled in the art may select an appropriate transfection method according to, for example, the host cell and the type of vector or expression vector used.

The term "host cell" means any cell type that is readily transformed, transfected, transduced, or the like with the rAAV vectors of the present disclosure. The term "host cell" encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication. The host cell may be an isolated cell or cell line grown in culture, or a cell present in living tissue or organism.

The present disclosure provides a polypeptide comprising an amino acid sequence having one or more mutations in the amino acid sequence set forth in SEQ ID No.2 or 16 selected from the group consisting of: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

The present disclosure provides polypeptides comprising the amino acid sequence shown in SEQ ID No.3, 17 or 23, or antigen binding fragments thereof.

The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a polypeptide of the disclosure or an antigen-binding fragment thereof.

The present disclosure provides vaccine compositions comprising a rAAV vector of the present disclosure, a composition of the present disclosure, a polypeptide of the present disclosure or an antigenic fragment thereof, or a nucleic acid of the present disclosure, and optionally one or more adjuvants.

A "vaccine composition" is a composition that, when administered to a subject (e.g., a mammal or a human), induces or enhances an immune response against an antigenic substance contained within the composition. The response may include induction of antibody production (e.g., by stimulation of B cells) or a T cell-based response (e.g., a cytolytic response). These reactions may or may not be protective or neutralizing. A protective or neutralizing immune response is an immune response that is detrimental to an infectious organism (e.g., an organism from which an antigen originates) that is consistent with an antigen but beneficial to a subject (e.g., by reducing or preventing infection).

An "adjuvant" generally refers to an agent that increases, stimulates, activates, boosts or modulates an immune response against an active ingredient of the composition at a cellular or humoral level. Adjuvants can play a role in both acquired and innate immunity and function in a variety of ways.

Many substances (natural or synthetic) have been shown to act as adjuvants. Examples of suitable adjuvants include, but are not limited to, aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide, etc.), alum, cholera toxin, salmonella toxin, IFA (incomplete freund's adjuvant), CFA (complete freund's adjuvant), ISCOMatrix, GM-CSF and other immunostimulatory cytokines, oligodeoxynucleotides containing CpG motifs (CpG 7909, etc.), oil-in-water emulsions, saponins or derivatives thereof (QS 21, etc.), lipopolysaccharides such AS lipid a or derivatives thereof (MPL, RC529, GLA, E6020, etc.), lipopeptides, lactoferrin, flagellin, double stranded RNA or derivatives thereof (poliic, etc.), bacterial DNA, imidazoquinolines (Imiquimod, R848, etc.), C-lectin ligands (trehalose-6, 6' -behenic acid/ester (TDB), CD1d ligands (alpha-galactoceramide, etc.), squalene emulsions (MF 59, AS03, AF03, etc.), PLGA, etc. The adjuvant and composition may be administered to the subject continuously or mixed together immediately prior to administration to the subject.

The present disclosure provides methods of inducing an immune response in a subject against SARS-CoV-2 or a variant thereof, comprising administering to the subject a rAAV vector of the disclosure, a composition of the disclosure, a polypeptide of the disclosure or an antigenic fragment thereof, a nucleic acid of the disclosure, or a vaccine composition of the disclosure.

An immune response, also known as an immune response, refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either of these cells or the liver, which results in selective targeting, binding, damage, destruction, and/or elimination of invasive pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells in vertebrates, or normal human cells or tissues in the case of autoimmune or pathological inflammation. When administered to a subject, the composition comprises an amount of a polypeptide of the present disclosure, or an antigenic fragment thereof, sufficient to elicit an immune response. Compositions for use as vaccines comprise the polypeptides of the present disclosure, or antigenic fragments thereof, and any other components desired.

The term "inducing an immune response" refers to the immune system first encountering at least one antigenic protein or antigenic fragment thereof and then inducing an antigen-specific immune response for a defined period of time. The period of time is, for example, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or at least 10 years prior to induction. In one embodiment, the meeting of the immune system of an individual or subject with an antigenic protein or antigenic fragment thereof that does not induce an antigen-specific immune response is not considered to be "inducing an immune response". For example, an encounter of an individual's immune system with an antigenic protein or an antigenic fragment thereof that does not induce persistent immunity is not considered to be an "induction of an immune response" in accordance with the present disclosure. In yet another embodiment, the induction of persistent immunity is mediated by the production of memory B cells and/or memory T cells. In the case of cancer, for example, a particular antigen may be expressed by a cancer cell without eliciting an immune response. The mere presence of this antigen is not an "induction of an immune response" against said antigen as understood in the present application. In one embodiment, the individual or subject has not been intentionally immunized with an antigenic protein or antigenic fragment thereof or a vector containing nucleic acid encoding such protein or fragment to treat or prevent disease for a period of time as set forth herein.

Administration may occur once or may occur multiple times. In one embodiment, one or more administrations may occur as part of a so-called "prime-boost" regimen. Other application systems may include delayed release or sustained release delivery systems.

As used herein, "administering" refers to introducing a composition or agent into a subject, and includes introducing the composition or agent concurrently or sequentially. "administration" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo and experimental procedures. "administration" also encompasses in vitro and ex vivo treatments. Examples of suitable methods for administering a polypeptide, antibody or composition of the present disclosure include, but are not limited to, oral, nasal drip, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal, and intravenous injection, as well as systemic or topical administration to the vicinity of a target site. Administration includes self-administration and other administration. Administration may be by any suitable route. Suitable routes of administration allow the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the vein of the subject.

In certain embodiments of the disclosure, the rAAV vectors, compositions, polypeptides, or antigenic fragments thereof, nucleic acids, or vaccine compositions of the disclosure can be packaged with or stored in a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, auto-injectors, syringe pumps and injection pens. Devices for aerosolizing or powder formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. In some embodiments, the vaccine composition may be formulated as an injection for nasal or intramuscular injection.

It is contemplated that the "subject" administered includes, but is not limited to, humans (i.e., men or women of any age group, such as pediatric subjects (e.g., infants, young children, teenagers) or adult subjects (e.g., young, middle aged, or elderly) and/or other non-human animals, such as mammals (e.g., primates).

In some embodiments, the vaccine composition further comprises a pharmaceutically acceptable diluent and/or excipient.

The term "pharmaceutically acceptable" refers to a molecule or composition that is not harmful to its recipient, or that has a benefit over any deleterious effect on its recipient, when administered to the recipient. Regarding diluents and/or excipients used to formulate vaccine compositions as disclosed herein, pharmaceutically acceptable diluents and/or excipients must be compatible with the other ingredients of the vaccine composition and not deleterious to the recipient thereof or benefit to the recipient thereof beyond any deleterious effect.

The present disclosure also provides methods of preventing and/or treating a disease caused by infection with SARS-CoV-2 or a variant thereof in a subject, the method comprising administering to the subject a rAAV vector of the disclosure, a composition of the disclosure, a polypeptide of the disclosure or an antigenic fragment thereof, a nucleic acid of the disclosure, or a vaccine composition of the disclosure.

As used herein, "preventing" means administering a therapeutically effective amount of a rAAV vector, composition, polypeptide, or antigenic fragment thereof, or vaccine composition of the present disclosure to a subject in order to protect the subject from developing a disease caused by infection with SARS-CoV-2 or a variant thereof. When the term "preventing" is used herein in connection with a given treatment for a given condition (e.g., preventing infection by SARS-CoV-2 or a variant thereof), a subject intended to express the treatment does not develop clinically observable levels of the condition at all, or develops more slowly and/or to a lesser extent than a subject in the absence of the treatment. These terms are not limited to situations in which the subject does not experience any aspects of the condition. For example, a treatment may be considered to be a prevented condition if the treatment is administered during exposure of the patient to a stimulus that is expected to produce the manifestation of the given condition, and the treatment results in the subject experiencing fewer and/or milder symptoms of the condition than would be expected without administration. By causing the subject to exhibit only mild and pronounced symptoms of infection, it is believed that the treatment may "prevent" the infection, which does not mean that there must be no cells that are permeable to the virus.

As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the progression of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, increasing or improving quality of life, weight gain, and/or prolonging survival. The methods of the present invention contemplate any one or more of these aspects of treatment.

In certain embodiments, the treatment may be administered after one or more symptoms have occurred. In other embodiments, the treatment may be administered without symptoms. For example, the treatment may be administered to the susceptible individual prior to the onset of symptoms, or may be treated with another damaging agent (e.g., according to a history of symptoms, according to genetic or other susceptibility factors, disease therapy, or any combination thereof). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

Similarly, a decrease as used herein in connection with a risk of infection with a given treatment (e.g., decreasing the risk of infection with SARS-CoV-2 or a variant thereof) refers to a subject developing an infection more slowly or to a lesser extent than the basal level at which the infection developed in the control or in the absence of the treatment (e.g., without administration of a polypeptide of the present disclosure). The reduced risk of infection may cause the subject to exhibit only mild and pronounced symptoms of infection or to delay symptoms of infection, which does not mean that there must be no cells that are permeable to the virus.

SARS-CoV-2 virus, severe acute respiratory syndrome coronavirus 2, is a enveloped positive strand single strand RNA virus belonging to the genus coronaviridae, genus Severe acute respiratory syndrome-related coronavirus species. Its gene sequence and SARS virus and MERS virus belong to the same lineage but different clades, and is the seventh known human-infectious coronavirus. The host of viruses include mammalian and avian animals. The virus can invade human body through human upper respiratory tract, and reach infection by using ACE2 expressed on various cell surfaces as receptor; the main infectious organs include lung, heart, kidney, etc. At present, the mainstream detection means for SARS-CoV-2 infection cases is RT-PCR. "SARS-CoV-2 virus" and "novel coronavirus" are used interchangeably herein. SARS-CoV-2 variants include, but are not limited to Alpha, beta, gamma, delta, epsilon, zeta, eta, iota, kappa, lambda, mu, omicron and subtypes thereof.

SARS-CoV-2 virus and its variants cause an infectious disease (new coronavirus infection), and its common clinical symptoms include fever, cough and shortness of breath, and symptoms such as sore throat, muscle weakness, and phlegm accumulation are partially seen. Pathological studies on new coronavirus infection have shown that infection with SARS-CoV-2 virus and its variants can lead to respiratory damage (e.g., acute lesions of the lung), cytokine release syndrome (e.g., cytokine storm and acute inflammatory response), circulatory damage (e.g., cardiovascular damage such as arrhythmia, vascular embolism, pulmonary vasoconstriction, etc.), digestive damage (e.g., pancreas, liver, etc.), genitourinary damage (e.g., acute renal failure), nervous damage (e.g., loss of smell, taste), ocular damage (e.g., conjunctival infection), etc. In addition, some patients with new coronavirus infection may still experience a series of effects after recovery (long-term syndrome of new coronavirus infection).

Examples

The present disclosure is further described below in conjunction with specific embodiments, and advantages and features of the present disclosure will become apparent as the description proceeds. These embodiments are merely exemplary and do not limit the scope of the disclosure in any way. It will be understood by those skilled in the art that various changes and substitutions may be made in the details and form of the technical solutions of the present disclosure without departing from the spirit and scope of the present disclosure, but these changes and substitutions fall within the scope of the present disclosure.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as molecular cloning, as described in Sambrook et al: conditions described in the laboratory Manual (New York: cold spring harbor laboratory Press, 1989) or in accordance with the instructions provided by the manufacturer.

The materials used in the examples are all commercially available unless otherwise specified.

Example 1 construction of AAV expression vector plasmids

The SARS-CoV-2 variant Omicron novel coronavirus S protein gene sequence (GenBank accession number OM287553.1, SEQ ID NO. 1) was obtained by NCBI database (https:// www.ncbi.nlm.nih.gov/GenBank). The amino acid sequence number of the S protein coded by the gene sequence is SEQ ID NO.2 after removing the natural N-terminal signal peptide and the 63 amino acids at the C terminal. The following mutations were then made in this sequence: R682S, R685G, K986P and V987P (numbering according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27)) give the amino acid sequence shown in SEQ ID NO. 3.

After addition of the tPA novel signal peptide (SEQ ID NO. 4) at the N-terminus of the obtained sequence, human codon optimization was performed by GenSmart Optimization (version Beta 1.0) and primers were designed using DNAWorks (v3.2.4) (https:// hpcwebapps. Cit. Nih. Gov/DNAWorks /), using HSDNA polymerase (Takarabio) amplified synthetic sequences using the endonuclease FastDiget ^TM EcoRI and FastDiget ^TM HindIII (ThermoFisher) the adeno-associated virus backbone vector pAAV-CAG-MCS-WPRE-SV40polyA was subjected to homologous recombination ligation using ClonExpress MultiS One Step Cloning Kit recombination kit (Nanjinouzan), transformed E.coli JM08 or DH5a, plated on kanamycin-resistant dishes, picked up for 16 hours for colony detection, and the positive clones were sent to sequencing company (Suzhou Jin Weizhi) for sequencing (SEQ ID NO. 10). The plasmid with the correct sequencing result was designated as vector plasmid 1, and the composition of each element is shown in FIG. 1.

In addition, the added signal peptide was replaced with tPA signal peptide (SEQ ID No. 5), and vector plasmid 2 was constructed using the same method steps as vector plasmid 1. The gene sequence of the vector plasmid 2 is SEQ ID NO.11, and the components are shown in FIG. 2.

Example 2 preparation of recombinant adeno-associated Virus (rAAV) vaccine 1 (TB-1) and 2 (TB-2) by an adherence process

AAV293 cells are derived from HEK293 cell lines, a HEK293 cell line optimized for AAV viral particle packaging in unassisted systems. AAV-293 is used to package AAV viruses and can produce higher viral titers (Wei, J.J., S.M., zhang, and J.Z.xu., "Curt") The ure of 293 cells for the package of adeno-associated viruses, "Journal of Clinical Rehabilitative Tissue Engineering Research (2007)). AAV293 cells or HEK293 cells were transfected at 1X 10 per dish 24 hours prior to transfection ⁷ The density of individual cells was inoculated in 150mm dishes and co-transfected with 12. Mu.g helper plasmid pHelper (SEQ ID NO. 15), 8. Mu.g serotype plasmid pRep2Cap5 (SEQ ID NO. 14), 5. Mu.g vector plasmid 1 or 2 and 10. Mu.g transfection reagent polyethylenimine (25 kD). Cells were harvested 72 hours after transfection by centrifugation at 500 Xg for 5 minutes at 4 ℃. Cells were resuspended in lysis buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl. Lysates were harvested and subjected to three freeze-thaw cycles in a dry ice/ethanol and 37 ℃ water bath. Then 1unit/mL nuclease and 0.5% sodium deoxycholate were added, after incubating the cell suspension at 37℃for 1 hour, the cell suspension was centrifuged at 5,000Xg for 20 minutes, and the rAAV supernatant crude lysate was collected at 4 ℃.

The crude lysate was diluted to a final volume of 10mL with 10mM Tris-HCl (pH 8.0) buffer, and iodixanol was then loaded in a 39mL ultracentrifuge tube at a mass to volume gradient of 15%, 25%, 40%, 60%. Centrifugation was performed at 350,000Xg for 1 hour at 18℃to collect 3mL of a 40% lower fraction and 0.5mL of a 60% upper fraction as a purified solution. Ultrafiltration displacement to virus stock was performed with a 100kDa cut-off ultrafiltration tube (Millipore). The recombinant virus preservation solution is PBS phosphate buffer solution (pH 7.4) and 0.05 percent of poloxamer 188. Purified rAAVs were labeled as either vaccine 1 (TB-1) or vaccine 2 (TB-2) (corresponding to vector plasmid 1 or 2, respectively) and viral titers were determined using SYBRGreenIqPCR. The assay showed that the virus titer of vaccine 1 (TB-1) was 8.24X10 ¹² GC/mL, virus titer of vaccine 2 (TB-2) was 8.30X10 ¹² GC/mL. The virus preservation solution is preserved in a refrigerator at-80 ℃ before use.

Example 3 preparation of recombinant adeno-associated Virus (rAAV) vaccine 3 (XF-1) and 4 (XF-2) by suspension Process

Sampling and counting 293 cells to obtain cell density; cell separation is carried out according to the requirement of the required cell density, and a proper amount of culture medium is added; after separating the bottles, the cells were placed in a set temperature of 37℃and 5% CO ₂ Culturing in a shaking incubator at 100 rpm. Aspiration of fine with pipettorCell culture solution (6 x 10) ⁵ Mu.g pHelper (SEQ ID NO. 15), 8. Mu.g pRep2Cap5 (SEQ ID NO. 14), 5. Mu.g vector plasmid 1 or 2 and 10. Mu.g transfection reagent polyethylenimine were added sequentially to a centrifuge tube, shaken well and placed in a 5% CO solution set at 37℃C ₂ The culture was carried out in a shaking incubator at 100rpm for about 72 hours. Lysing and clarifying cell venom; adding PEG8000, ice-bathing for 2 hours, centrifuging to remove supernatant, and re-suspending to precipitate; performing iodixanol ultracentrifugation; ultrafiltering the venom obtained after ultracentrifugation; obtaining the virus stock solution with high purity and high titer. Purified rAAVs were labeled as vaccine 3 (XF-1) or 4 (XF-2) (corresponding to vector plasmid 1 or 2, respectively) and viral titers were determined using SYBRGreenI qPCR. The measurement result shows that the virus titer of vaccine 3 (XF-1) is 1X 10 ¹³ GC/mL, virus titer of vaccine 4 (XF-2) 1X 10 ¹³ GC/mL. The virus preservation solution is preserved in a refrigerator at-80 ℃ before use.

EXAMPLE 4 immunization of C57 mice

C57 mice at 8 weeks of age were randomly divided into 5 groups of 5 mice each. Recombinant adeno-associated virus (rAAV) vaccines prepared in examples 2-3 were administered intramuscularly to mice in groups TB-1, TB-2, XF-1 and XF-2 (titres 2.5X10) ¹² GC/mL, injection volume 40. Mu.L); blank vector AAV-GFP (titre 2.5X10) of recombinant green fluorescent protein adeno-associated virus was injected into the negative control group at the same dose ¹² GC/mL, injection volume 40. Mu.L).

Injection was performed on day 0 using a one-shot immunization protocol. The vaccine groups TB-1 and TB-2 were bled on days 40, 60 and 100, respectively, and the vaccine groups XF-1 and XF-2 were bled on days 50 and 70, respectively, with 0.1mL-0.2mL of blood per mouse. The obtained blood sample was left at 4℃for 60 minutes, centrifuged at 4000rpm for 15 minutes, and the supernatant serum was used for ELISA immunoassay detection and neutralizing antibody experiments.

EXAMPLE 5 ELISA immunoassays

(1) ELISA detection method

Spike protein (ECD, his tag) of Omacron novel coronavirus was formulated at a concentration of 0.333 μg/mL with 0.1M carbonate buffer (pH 9.6) Is added into a 96-well plate in an amount of 100 mu L per well, and is placed at 4 ℃ for incubation overnight; the next day is put into a 37 ℃ incubator for incubation for 1 hour; plates were washed 3 times with PBST (pbs+0.1% tween 20) at 300 μl wash/well; after washing the plates, 2% skim milk powder was added at 250 μl/well and incubated at 37deg.C for 1 hour; plates were washed 3 times with PBST (pbs+0.1% tween 20). Antibody serum (dilution 1:300) produced by each group of mice obtained in example 4 was added at 100. Mu.L/well, and incubated in an incubator at 37℃for 1 hour; after washing the plates 3 times, HRP (horseradish peroxidase) -labeled IgG was added to the mice serogroup at 100 μl/well and incubated for 1 hour at room temperature; the plate was then washed 3 times. TMB solution was added at 100. Mu.L/well and developed for 15 minutes at room temperature in the dark. 0.5. 0.5M H was added at 100. Mu.L/well ₂ SO ₄ The solution was stopped from developing and absorbance was measured using a microplate reader, wherein the detection wavelength was 450nm and the background wavelength was 570nm.

(2) Analysis of absorbance results

a. Absorbance (OD) values were plotted against recombinant adeno-associated virus vaccines TB-1 and TB-2 prepared in example 2. As shown in fig. 3, the IgG antibody titer produced by mice induced with the recombinant adeno-associated virus vaccines TB-1 and TB-2 prepared in example 2 of the present disclosure was significantly higher than that of AAV-GFP blank, demonstrating that the vaccine prepared by the adherence process induced a strong immune response in mice.

b. Absorbance (OD) values were plotted against recombinant adeno-associated virus vaccines XF-1 and XF-2 in the preparation of example 3. As shown in FIG. 4, the IgG antibody titer produced by mice induced by the recombinant adeno-associated virus vaccines XF-1 and XF-2 prepared in example 3 of the present invention was significantly higher than that of AAV-GFP blank, demonstrating that the vaccine prepared by the cell suspension culture process induced a strong immune response in mice during transfection.

Example 6 neutralizing antibody experiments

Vero E6 cells (2X 10) ⁴ Individual/well) was inoculated into 96-well plates at 37 ℃ and 5% co ₂ Incubate overnight until a monolayer is formed. 100TCID of Omicron New coronavirus ₅₀ Mixing with serial 4-fold dilution of mouse antiserum, incubating at 37deg.C for 1 hr, and mixing with serum sample at 5Heated at 6℃for 30 minutes, then added to a 96-well plate and mixed with Vero E6 cells. In each assay, omacron-new coronavirus-infected cells were used as positive controls, while virus-free cells were used as negative controls. CPE (cytopathic effect) was recorded by counting the number of Vero E6 cells infected with Omacron new coronavirus by the Reed-Muench method on day 3 post infection. The maximal dilution of serum from mice that produced cytopathic effects in 50% of the cells after vaccination was the neutralizing antibody titer CNA of this vaccine).

(1) The higher the neutralizing antibody titer, the lower the level of viral replication, the greater the protection against viral infection. The Neutralizing Antibody (NA) values were plotted against the recombinant adeno-associated virus vaccines TB-1 and TB-2 prepared in example 2. The results are shown in FIG. 5, and the induced serum neutralizing antibody titers of the vaccine TB-1 and TB-2 groups are significantly higher than those of the AAV-GFP blank group, which indicates that the vaccine TB-1 and TB-2 groups can generate strong humoral immune responses, the induced antibody serum has stronger activity, and cells are protected from infection of various novel coronaviruses including Omicron.

(2) The Neutralizing Antibody (NA) values were plotted against the recombinant adeno-associated virus vaccines XF-1 and XF-2 prepared in example 3. The results are shown in FIG. 6, and the serum neutralizing antibody titer induced by the vaccine XF-1 and XF-2 groups is significantly higher than that of the AAV-GFP blank group, which indicates that the vaccine XF-1 and XF-2 groups can generate a strong humoral immune response, the induced antibody serum has stronger activity, and the cells are protected from infection of various novel coronaviruses including Omicron. The results show that the vaccine obtained by the cell suspension culture method in the transfection process can induce strong immune response, which indicates that the prevention effect of the vaccine is not affected by adopting different preparation processes.

EXAMPLE 7 experiments on the expression of the protein of interest of recombinant adeno-associated Virus (rAAV) vaccine 3 (TB-3) prepared by the adherence Process

rAAV vaccine 3 was prepared according to the methods of examples 1 and 2, wherein the amino acid sequence of the Omacron novel coronavirus S protein used was SEQ ID NO.17 and the signal peptide added was the tPA signal peptide (SEQ ID NO. 5). 293T cells were diluted with medium and seeded into 12-well plates at 1 ml/wellIncubated overnight at 37 ℃. Diluting the vaccine to 2E11vg/ml, 1E11vg/ml and 5E10vg/ml, sucking the cell supernatant from the 12-well plate, adding diluted vaccine sample at 1 ml/well, 37 ℃ and 5% CO ₂ Infection was incubated for 8-10 hours in the conditions. The supernatant from the 12-well plate was then aspirated, and 1 ml/well of pre-warmed complete medium was added at 37℃with 5% CO ₂ Culturing for 38-40 hr. The supernatant was collected into a 1.5ml centrifuge tube for subsequent ELISA detection.

ELISA assays were performed as in example 5. In short, the standard substance is diluted for 6 gradients, the ELISA plate is taken out, the sample to be detected, the diluted 6 gradient standard substances and the blank group are added into the ELISA plate for incubation for 2 hours at room temperature, the secondary antibody is added, the incubation is carried out for 1 hour at room temperature, the chromogenic solution is added for color development for 15 minutes after the incubation is completed, the OD value is detected, and the target protein content is calculated. The detection results are shown in FIG. 7. The results show that the in vitro target protein expression amounts of the three vaccine dose groups of 5E10vg, 1E11vg and 2E11vg have obvious dose-effect relationship, and the target protein expression level of the 5E10vg low vaccine dose group is close to 5000pg/ml and has higher expression level.

Example 8 recombinant adeno-associated Virus (rAAV) vaccine is effective in ameliorating pulmonary viral infections in mice

SPF-class hACE2 transgenic mice (body weight 19-27 g) of 8-10 weeks old were collected and the experimental mice were grouped, immunized and challenged according to Table 1 below. Specifically, mice were randomly divided into 7 groups, of which 4 groups were set as challenge groups, 3 groups were set as satellite groups, and 6 groups each. Wherein the same immunization procedure was performed on the challenged mice as the satellite mice, i.e., the mice were immunized once by intramuscular injection of recombinant adeno-associated virus (rAAV) vaccine TB-2 prepared in example 2; the challenge group 1 was a control group, and mice were injected with dilutions of rAAV vaccine (0.01 g/L poloxamer 188, 50g/L sucrose, 3.58g/L disodium hydrogen phosphate dodecahydrate, 0.27g/L potassium dihydrogen phosphate, 0.20g/L potassium chloride, 8.00g/L sodium chloride, 0.2033g/L magnesium chloride, and water for injection). After 70 days of immunization, the challenged mice were immunized with 10 ⁵ TCID ₅₀ An infectious dose of 50. Mu.L/dose of SARS-CoV-2 variant Omicron (BA.1) by nasal drip infection; satellite group mice were euthanized and blood was collectedClear to detect neutralizing antibody titers. Continuously observing the mice in the virus challenge group for 5 days after virus challenge and recording weight change; on day 5 post challenge (i.e., day 75 post immunization), mice were sacrificed and lung tissue viral load detection and pathology detection were performed. Quantitative data generated by this experiment were analyzed by variance using statistical processing software SPSS (version.17.0).

TABLE 1 grouping and detection of laboratory animals

Results

(1) Animal body weight

The results of the mice weight measurements are shown in Table 2 below, wherein the average percent weight loss in the control group (group 1) was 1.29% at 5 days post challenge. The average body weight of the challenge group 2 (2E 11 vg) was reduced by 0.87%, and there was no significant difference (P > 0.05) from the control group. The average body weight of the group 3 (1E 11 vg) with the challenge group was reduced by 3.56%, and the group was not significantly different from the control group (P > 0.05). The average weight of the challenge group 4 (5E 10 vg) was reduced by 2.94%, and there was no significant difference (P > 0.05) from the control group. The weight of each group of mice immunized by the rAAV vaccine in the challenge group is not obviously different from that of the control group, so that the rAAV vaccine has no obvious toxicity to animals and has good safety.

TABLE 2 weight changes in mice in the challenge group

(2) Viral load

Total RNA was extracted from lung tissue of challenged mice using the kit RNeasy kit (Qiagen) and reverse transcribed. RT-qPCR was performed using the following cycling protocol and primers: the cycle was 40 times at 50℃2min,95℃2min,95℃15s,60℃30s, after which the dissolution profile program was run at 95℃15s,60℃1min,95℃45s. The primers used to detect SARS-CoV-2 are as follows:

forward primer, 5'-TCGTTTCGGAAGAGACAGGT-3' (SEQ ID No. 29);

Reverse primer, 5'-GCGCAGTAAGGATGGCTAGT-3' (SEQ ID NO. 30).

And (5) accurately and quantitatively detecting by a machine to obtain the viral load value.

The average viral load of mouse lung tissue is shown in Table 3 below, wherein the average viral load of control (group 1) lung tissue is 10 at 5 days post challenge ^6.19 Each copy/g. Challenge group 2 (2E 11 vg) lung tissue average viral load of 10 ^3.66 The copy/g was decreased by 2.52 lg values compared to the control group, significantly lower than the control group (P < 0.01); challenge group 3 (1E 11 vg) lung tissue average viral load of 10 ^3.77 The copy/g was decreased by 2.42 and lg values compared to the control group, significantly lower than the control group (P < 0.01); challenge group 4 (5 e10 vg) lung tissue average viral load of 10 ^3.94 The number of copies/g was reduced by 2.25 and lg values compared to the control group, significantly lower than the control group (P < 0.01). The results show that the three vaccine dose groups of 5E10vg, 1E11vg and 2E11vg can obviously reduce the viral load of the SARS-CoV-2 variant Omicron.

TABLE 3 pulmonary tissue viral load (log) of challenged mice ₁₀ Copy number/g)

Note that: * P < 0.05 has significant differences, P < 0.01 has very significant differences

(3) Pathology detection

The lung tissue of the challenged mice was H & E stained to observe pathological changes, and the results of the measurements are shown in FIGS. 8A to 8H and Table 4, wherein the degree of change in the pathology of the lung tissue of each group of mice is shown in Table 4 for 5 days of challenge, and exemplary photographs of the pathology of the lung tissue of each group of mice are shown in FIGS. 8A to 8H.

TABLE 4 toxicity-counteracting group mice lung tissue pathological changes profile

/>

Note that: ++, mild lesions; ++, moderate lesions.

The results showed that 5 days after challenge, 1 (1/6) of the animal lung tissues in the control group showed a slight interstitial pneumonia change, and a slight widening of the alveolar space, inflammatory cell infiltration, and a small amount of inflammatory cell infiltration around the blood vessels were seen in fig. 8A. In 5 (5/6) animals, lung tissue showed a change in the form of moderate interstitial pneumonia, and a more pronounced widening of alveolar space, inflammatory cell infiltration, and a small amount of inflammatory cell infiltration around blood vessels and bronchi, as shown in fig. 8B.

In the case of 5 (5/6) animals in group 2 (2E 11 vg) with slightly interstitial pneumonia changes, slight broadening of alveolar septum and inflammatory cell infiltration was seen, see FIG. 8C.1 (1/6) animal lung tissue showed a change in the form of moderate interstitial pneumonia, and a more pronounced widening of alveolar space, inflammatory cell infiltration, and a small amount of inflammatory cell infiltration around blood vessels and bronchi, as shown in fig. 8D.

3 (3/6) animals in group 3 (1E 11 vg) showed slight interstitial pneumonia changes in lung tissue, and slight broadening of alveolar septum and inflammatory cell infiltration were seen, see FIG. 8E.3 (3/6) animals showed changes in lung tissue with moderate interstitial pneumonia, and showed more pronounced alveolar septum broadening, inflammatory cell infiltration, and small inflammatory cell infiltration around blood vessels and bronchioles, as shown in fig. 8F.

3 (3/6) animals in group 4 (5E 10 vg) had slightly interstitial pneumonia changes, and it was seen that the alveolar space slightly widened, and inflammatory cell infiltration was seen in FIG. 8G.3 (3/6) animals showed changes in lung tissue with moderate interstitial pneumonia, and showed more pronounced alveolar septum broadening, inflammatory cell infiltration, and small inflammatory cell infiltration around blood vessels and bronchioles, as shown in FIG. 8H.

The results show that the vaccine of the application can improve the pulmonary viral infection of mice by using three doses of 5E10vg, 1E11vg and 2E11vg, wherein the 2E11vg dose has a remarkable effect of improving the pulmonary viral infection of mice.

(4) Satellite group neutralizing antibodies

Satellite group mouse sera were collected and the neutralizing antibody titer was determined by observing cytopathic effects using the method described in example 6. Briefly, serum samples were inactivated at 56℃for 30 min, diluted 2-fold in a gradient, and combined with an equal volume of 100TCID ₅₀ Omicron new crown virus liquid at 37 ℃,5% CO ₂ After 1 hour of co-incubation, the mixture was transferred to a pre-plated monolayer of 96-well Vero E6 cell plates, and after 3 days cytopathic effect was observed and the neutralizing antibody titer was calculated. The results are shown in table 5 below, where satellite group 7 (2E11 vg) neutralizing antibody GMT was 186.29 (n=6), satellite group 8 (1E11 vg) neutralizing antibody GMT was 72.22 (n=6), and satellite group 9 (5E10 vg) neutralizing antibody GMT was 52.31 (n-6). The average titer of neutralizing antibodies in serum of immunized mice has a dependence on the vaccine immunization dose and is positively correlated with the immunization dose.

TABLE 5 satellite group mouse serum anti-SARS-CoV-2 neutralizing antibody titres

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Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising a nucleotide sequence encoding an S protein of SARS-CoV-2 variant Omicron or an antigenic fragment thereof.

2. The rAAV vector of claim 1, comprising a nucleotide sequence encoding a polypeptide or an antigenic fragment thereof, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID nos. 2-3, 16-17 and 23, or an amino acid sequence that replaces, lacks, adds and inserts one or more (e.g., 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5) amino acid residues in the amino acid sequence set forth in any one of SEQ ID nos. 2-3, 16-17 and 23, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID nos. 2-3, 16-17 and 23.

3. The rAAV vector of claim 1 or 2, wherein the polypeptide comprises one or more mutations selected from the group consisting of: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

4. The rAAV vector of any one of claims 1-3, wherein the polypeptide comprises the following mutations: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

5. The rAAV vector of claim 1 or 2, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID No.2-3, 16-17, or 23.

6. The rAAV vector of any one of claims 1-5, wherein the polypeptide further comprises an N-terminal signal peptide sequence.

7. The rAAV vector of claim 6, wherein the signal peptide is derived from a signal peptide of a protein selected from the group consisting of: tissue plasminogen activator (tPA), granulocyte-macrophage colony-stimulating factor (GM-CSF), prolactin precursors, growth hormone, and immunoglobulins (e.g., igE).

8. The rAAV vector of claim 6, wherein the signal peptide has an amino acid sequence set forth in SEQ ID No.4 or SEQ ID No. 5.

9. The rAAV vector of any one of claims 1-8, comprising adeno-associated virus Inverted Terminal Repeat (ITR) sequences at the 5 'and 3' ends of the nucleotide sequence encoding the polypeptide or antigenic fragment thereof.

10. The rAAV vector of claim 9, wherein the ITR sequence is derived from an ITR sequence of an adeno-associated viral serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

11. The rAAV vector of claim 10, wherein the ITR sequence is derived from an ITR sequence of an AAV2 serotype.

12. The rAAV vector of claim 11, wherein the ITR sequence has the nucleotide sequence set forth in SEQ ID No. 6.

13. The rAAV vector of claim 11, wherein the ITR sequence has the nucleotide sequence set forth in SEQ ID No. 28.

14. The rAAV vector of any one of claims 1-13, further comprising an expression regulatory element, such as one or more of a promoter sequence, an upstream regulatory region, a transcriptional regulatory element, and a terminator.

15. The rAAV vector of any one of claims 1-14, wherein the nucleotide sequence encoding the polypeptide or antigenic fragment thereof is codon optimized of human origin.

16. The rAAV vector of claim 15, wherein the nucleotide sequence encoding the polypeptide or antigenic fragment thereof has the nucleotide sequence set forth in SEQ ID No.7-9, 18-20, 24 or 25.

17. The rAAV vector of any one of claims 1-16, wherein the rAAV vector has the nucleotide sequence set forth in SEQ ID No.10-11, 21-22, or 26.

18. A composition comprising the rAAV vector of any one of claims 1-17 and an AAV serotype plasmid.

19. The composition of claim 18, wherein the serotype plasmid comprises nucleotide sequences encoding Rep proteins and Cap proteins of an AAV.

20. The composition of claim 19, wherein the Rep protein is a Rep protein of an adeno-associated virus serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

21. The composition of claim 20, wherein the Rep protein is from an AAV2 serotype.

22. The composition of any one of claims 19-21, wherein the Cap protein is a Cap protein of an adeno-associated virus serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

23. The composition of claim 22, wherein the Cap protein is from an AAV5 serotype.

24. The composition of any one of claims 19-23, wherein the serotype plasmid comprises the nucleotide sequence shown in SEQ ID No.12 or SEQ ID No.13 at the 5 'and/or 3' end of the nucleotide sequences encoding the Rep proteins and Cap proteins of AAV.

25. The composition of any one of claims 18-24, further comprising a helper plasmid.

26. A method of producing a recombinant adeno-associated virus (rAAV), comprising:

i) Co-transfecting a host cell with the composition of any one of claims 18-25;

ii) culturing the host cell under conditions suitable for production of the rAAV, and

iii) Isolating the rAAV from the host cells and/or culture supernatant.

27. The method of claim 26, wherein in step i) and/or step ii) the host cell is in an adherent state.

28. The method of claim 26, wherein in step i) and/or step ii) the host cell is in suspension.

29. A rAAV produced by the method of any one of claims 26-28.

30. A polypeptide comprising an amino acid sequence comprising one or more mutations in the amino acid sequence set forth in SEQ ID No.2 or 16 selected from the group consisting of: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

31. The polypeptide or antigen binding fragment thereof of claim 30, wherein the polypeptide comprises an amino acid sequence that has been mutated in the amino acid sequence set forth in SEQ ID No.2 or 16: R682S, R685G, K986P and V987P, wherein numbering is according to the amino acid sequence of the S protein of the novel coronavirus strain NC 045512 (SEQ ID NO. 27).

32. A polypeptide or antigen binding fragment thereof, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID No.3, 17 or 23.

33. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of claims 30-32 or an antigen-binding fragment thereof.

34. A vaccine composition comprising the rAAV vector of any one of claims 1-17, the composition of any one of claims 18-25, the rAAV of claim 29, the polypeptide of any one of claims 30-32 or an antigenic fragment thereof, or the nucleic acid of claim 33, and optionally one or more adjuvants and/or excipients.

35. A method of inducing an immune response against SARS-CoV-2 or a variant thereof in a subject, the method comprising administering to the subject the rAAV vector of any one of claims 1-17, the composition of any one of claims 18-25, the rAAV of claim 29, the polypeptide of any one of claims 30-32 or an antigenic fragment thereof, the nucleic acid of claim 33 or the vaccine composition of claim 34.

36. A method of preventing and/or treating a disease caused by infection with SARS-CoV-2 or a variant thereof in a subject, the method comprising administering to the subject the rAAV vector of any one of claims 1-17, the composition of any one of claims 18-25, the rAAV of claim 29, the polypeptide of any one of claims 30-32, or an antigenic fragment thereof, the nucleic acid of claim 33, or the vaccine composition of claim 34.

37. The method of claim 35 or 36, wherein the SARS-CoV-2 variant strain is selected from the group consisting of Alpha, beta, gamma, delta, epsilon, zeta, eta, iota, kappa, lambda, mu, omicron and subtypes thereof.