CN114685627A

CN114685627A - rAAV vector vaccine for preventing respiratory syncytial virus

Info

Publication number: CN114685627A
Application number: CN202111588672.5A
Authority: CN
Inventors: 不公告发明人
Original assignee: Guangzhou Update Biomedical Technology Co ltd
Current assignee: Guangzhou Update Biomedical Technology Co ltd
Priority date: 2020-12-28
Filing date: 2021-12-23
Publication date: 2022-07-01

Abstract

The invention discloses an rAAV vector vaccine for preventing respiratory syncytial virus. The inventor modifies F, G, SH protein and the like of RSV so that the corresponding nucleotide sequence can be effectively loaded, translated and expressed by rAAV vector, and partial examples have more stable structures, thereby effectively preventing RSV infection.

Description

rAAV vector vaccine for preventing respiratory syncytial virus

Technical Field

The invention relates to an rAAV vector vaccine for preventing respiratory syncytial virus RSV.

Background

Respiratory Syncytial Virus (RSV) was the first Respiratory Syncytial Virus in 1955Found (Morris et al, 1956) in Paramyxoviridae (Paramyxoviridae) Pneumovirinae (pneumovirinae)Pneumovirinae) Pneumovirus genus (A)Pneumovirus) The protein G is divided into two subtypes A and B according to the sequence of the protein G. RSV is widely prevalent around the world, is a viral pathogen causing Respiratory Tract Infection (RTI) worldwide, is the leading cause of Respiratory illness hospitalizations in children and elderly under 5 years of age, and causes lower Respiratory tract infection symptoms, with a significant proportion of patients with severe symptoms (e.g., bronchiolitis and pneumonia), requiring hospitalization, and having a high mortality rate. RSV can also infect adults, causing symptoms and hazards that are more severe than influenza. The risk of RSV infection is also greatly increased for people with congenital heart disease, chronic lung disease, and a history of specific disease. RSV is widely spread and prevalent worldwide and has become a worldwide public health problem, and the risk of RSV has raised high concerns and values for the World Health Organization (WHO) and for various levels of health regulatory agencies worldwide.

RSV is a non-segmented negative-strand RNA virus with a genome length of 15.2kb and 10 genes encoding 11 proteins in total, including the nonstructural proteins (NS 1, NS 2), the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the RNA-dependent RNA polymerase (L), the transcriptional elongation factor (M2-1), the regulatory factor (M2-2), and the 3 envelope glycoproteins (cohesin (G), fusion protein (F), and Small Hydrophobin (SH)). The virus is spherical particles of 100-350nm, but the virus particles which are usually dominant are filamentous structures with the diameter of 60-200 nm and the length of more than 10 μm. RSV can be transmitted by human-to-human contact, or inhalation by coughing or sneezing, and can acquire infections by exposure to contaminants, primarily infecting the epithelial cells of the nasal cavity and large and small airways of the lung, and possibly alveolar macrophages and other types of cells in the lung, which can cause the cells to fuse together to form syncytia.

The RSV envelope is provided with N, P, L, M and M2-1 protein below, and F, G and SH protein are expressed on the surface of the virus envelope, and because F protein is fusion protein, a unique cell fusion lesion is generated after a cell is infected; the G protein is adsorptive protein and can be combined with a receptor on the surface of a cell; the F protein is a typical paramyxovirus fusion glycoprotein and consists of two sections of proteins F1 and F2, and the middle parts of the proteins are connected by Furin enzyme cutting sites; the N end of F1 is peptide segment with fusion activity composed of continuous 19 hydrophobic amino acids, the C end is peptide segment composed of continuous 23 hydrophobic amino acids, which mediates the fusion of virus and cell membrane, the penetration of virus and the formation of syncytia; the G protein is involved in the mechanism of RSV infection and determines the diversity of RSV antigens.

RSV infection is mainly triggered by the protein F, G on the surface of the virus, and stimulates the organism to generate serum neutralizing antibodies and secretory IgA of respiratory mucosa; the F protein plays an important role in inducing immune protection and high-level serum neutralizing antibodies; meanwhile, RSV F and G protein are the main antigens for stimulating the body to generate protective antibodies, and the neutralizing antibodies induced by the G protein have type specificity; the F protein is highly conserved, the recognition sites of neutralizing antibodies are mainly distributed in the F1 fragment, and the induced neutralizing antibodies can simultaneously inhibit A, B virus infection of two subtypes. The Small Hydrophobin (SH) is another possible vaccine target, comprises a transmembrane domain and an extracellular domain, plays a role in replication and inflammation activation of viruses in vivo, and can induce antibody-dependent cell-mediated cytotoxicity (ADCC), so that the extracellular domain of the SH has potential application value in antigen research.

Vaccine research is the most concentrated field of RSV prevention and treatment research at present, and in the early 60 th 20 th century, a formalin inactivated RSV vaccine (FI-RSV) is developed for clinical research, which is the first RSV vaccine entering clinical trials, but the vaccine does not produce a protective effect on RSV diseases, and in subsequent natural infection, a vaccinated child has a serious disease Enhancement (ERD) phenomenon, the hospitalization rate is obviously increased, even death is caused, and therefore the vaccine cannot be clinically applied finally.

The research direction of the RSV vaccine in recent years is more extensive and deeper, and comprises attenuated live vaccines, inactivated vaccines, chimeric vector vaccines, subunit vaccines, virus-like particle vaccines, replication-defective virus vector vaccines, nucleic acid vaccines and the like as candidate vaccines, which are currently in the preclinical or clinical test stage, wherein the attenuated live vaccines, recombinant protein vaccines and chimeric vector vaccines are the hot directions of the RSV vaccine research in recent years; and the G protein and the F protein can induce good humoral immunity and cellular immunity reaction and are main target antigens of recombinant protein vaccine technology.

Subunit vaccines developed based on the F and G proteins of RSV, which are capable of inducing the production of neutralizing and protective antibodies, and the subunit vaccines prepared with them, including specific G proteins or purified F proteins, are more popular in vaccine application studies due to the considerable conservation of the F protein, whereas subunit vaccines based on both F and G proteins and live attenuated vaccines are most promising and have been applied in clinical research trials; the recombinant RSV nanoparticle vaccine is an oligomer particle consisting of F protein, can induce high-activity neutralizing antibody, can prevent lung infection, and can be used for mother-infant transfer and the prevention of RSV infection of old people; the adenovirus vector and the poxvirus vector vaccine expressing RSV F protein and G protein can induce good humoral immunity and cellular immunity reaction, has no ERD phenomenon, and is an ideal vaccine candidate vaccine variety; the goal of developing a nucleic acid vaccine encoding DNA or mRNA for RSV antigens is to be effective in protecting RSV infection in children and the elderly.

At present, the RSV vaccine is one of the most popular vaccine products in the market and is also a variety which is greatly supported and advocated by the WHO vaccine development program. In recent years, with the continuous improvement and development of technical levels of reverse genetics, vaccinology, molecular virology, genomics, immunology and the like, the development of vaccines for preventing RSV infection also makes a significant breakthrough, and a plurality of products show good immunogenicity and clinical application potential in the clinical research and test stage.

Adeno-associated virus (AAV) belongs to the genus parvovirus, the diameter of virus particles is about 22nm, and the AAV belongs to replication-defective icosahedral non-enveloped single-stranded DNA viruses, 12 AAV serotypes are found, and AAV causing diseases to human is not found at present. However, different AAV vectors have different capsid protein spatial structures, sequences, and tissue specificities. The recombinant AAV uses capsid as carrier, and transmits the virus packaged genome to nucleus for replication. AAV has a three-layer protein coat (VP 1, VP2, and VP 3) that surrounds and protects a single-stranded DNA genome of approximately 4.7kb in size.

The recombinant AAV for genetic engineering of wild AAV is an important vector for gene transmission and expression, has wide host range and tissue infection capacity, and can infect dividing cell and transduce exogenous gene into non-dividing cell to express exogenous gene for long term without depending on active division capacity of host cell.

The rAAV has the advantages of high safety, low immunogenicity, wide host range, stable expression and stable physical properties compared with other virus vectors. Currently, there are 3 AAV vector-based gene drugs approved abroad to be marketed and a number of AAV-based drugs in clinical research phase, and thus, AAV vector is considered by the FDA in the united states as one of the safest viral vectors for human gene therapy. According to the literature reports (Karen Nieto and Anna Salvetti), the vaccine based on AAV vector can stably express antigen in vivo and can induce body to generate durable and stable immune response.

The most important difficulties faced by RSV vaccines are currently considered to be the short duration of immunity and the imbalance of the induced Th1 and Th2 immune responses, while rAAV vector-based RSV vaccines are able to induce a sustained immune response and viral vector vaccines have significant advantages in altering the balance of Th1 and Th2 immune responses, which has become the most promising vaccine species for RSV. However, RSV has a large genome and is difficult to efficiently encapsidate with AAV vectors.

In the past, the preparation of rAAV has technical bottlenecks in the aspects of complex production process, multiple limiting factors and the like, and the difficulty of rAAV-based vaccines in both screening vaccine antigen sequences and large-scale production of rAAV is even more difficult.

Disclosure of Invention

The object of the present invention is to overcome at least one of the drawbacks of the prior art and to provide a vector vaccine for the prevention of respiratory syncytial virus.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

an RSV antigen having an amino acid sequence selected from at least one of the following amino acid sequences, or an amino acid sequence having at least 90%, 95%, 98%, 99% homology:

an RSV F antigen polypeptide sequence capable of inducing specific antibodies against the F protein, F1, F2 protein and neutralizing antibodies and immunoprotective effects against RSV, selected from the group consisting of:

using a connecting sequence GS to replace amino acid at the Furin site of the F protein to obtain fusion protein;

a connecting sequence GS is used for replacing the Furin site of the F protein and 10 amino acids connected with the rear fusion region of the F protein to obtain a fusion protein;

replacing amino acids at the Furin site of the F protein with a 2A connecting sequence to obtain an antigen polypeptide;

the 2A connecting sequence is used for replacing the F protein Furin site and the 10 amino acids connected with the post-fusion region of the F protein Furin site to obtain the antigen polypeptide;

an antigenic polypeptide obtained by adding a Foldon trimerization structural domain to the C-terminal residue of the F1 protein;

an IgE signal peptide sequence is added at the N-terminal of the F1 protein, and a Foldon trimerization structural domain is added at the C-terminal residue to obtain antigen polypeptide;

the F protein Furin B enzyme cutting site is mutated into KKQKQQ (SEQ ID NO. 1) to obtain antigen polypeptide;

an RSV G antigen polypeptide sequence capable of inducing specific antibodies against the G protein and neutralizing antibodies and immunoprotection effects against RSV selected from the group consisting of:

an antigenic polypeptide obtained by adding an IgE signal peptide sequence to the N-terminal of the G protein of RSV;

an RSV SH antigen polypeptide sequence capable of inducing specific antibodies against SH protein and neutralizing antibodies and immunoprotection effects against RSV selected from the group consisting of:

fusion proteins of the SH protein of RSV with the Fc region of an immunoglobulin IgG; preferably, the SH protein is coupled to the Fc region of an immunoglobulin IgG by a linker sequence; preferably, the linking sequence is selected from the group consisting of suitable sequences of 1 to 20 aa in length, immediately adjacent to the C-terminus of SH and the N-terminus of Fc; in particular, the amino acid sequence of the linker sequence is selected from GGGSGGGSGGGSGS (SEQ ID No.: 2), GGGSGGGS (SEQ ID No.: 3), GGGSGGGSGG (SEQ ID No.: 4), GGGSGGGSGGGS (SEQ ID No.: 5).

In some examples, the linker sequence GS is a GS linker, preferably, the amino acid sequence thereof is selected from GGGSGGGSGGGSGS, GGGSGGGS, GGGSGGGSGG, GGGSGGGSGGGS.

In some examples, the amino acid sequence of the 2A linker sequence is an amino acid sequence comprising D-X-E-X-N-P-G-P (SEQ ID NO: 6), preferably GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 7).

In a second aspect of the present invention, there is provided:

expressing the nucleotide sequence of an RSV antigen according to the first aspect of the invention.

In some examples, the nucleotide sequence further comprises at least one of an Inverted Terminal Repeat (ITR) of AAV, a signal peptide sequence.

In some examples, the Inverted Terminal Repeats (ITRs) of the AAV are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ/8 serotypes, preferably, the ITRs are of AAV2 serotypes.

In some examples, the signal peptide sequence is selected from a human interleukin 2 signal peptide and/or a human immunoglobulin e (ige) signal peptide; a preferred signal peptide is the IgE signal peptide, with the specific amino acid sequence being MDWTWILFLVAAATRVHS (SEQ ID No.: 8).

In some examples, the nucleotide sequence is codon optimized for human cells.

In some examples, the nucleotide sequence is as set forth in SEQ ID No.: 9 and SEQ ID No.: shown at 10.

In a third aspect of the present invention, there is provided:

a non-replicating recombinant adeno-associated virus vector vaccine for preventing respiratory syncytial virus, comprising at least an AAV capsid and RSV antigenic nucleotide sequences, wherein the nucleotide sequence according to the second aspect of the invention, or the nucleotide sequences encoding the F protein, F1 protein, F2 protein, G protein, and SH protein of RSV, is packaged in the AAV capsid.

In some examples, the AAV capsid protein sequences are derived from serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVDJ/8, and preferably AAV 5.

In some examples, the nucleic acid includes, but is not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof, preferably, the recombinant adeno-associated virus is ssAAV and scAAV.

In some examples, the expression cassette may be carried on any suitable vector for delivery to a packaging host cell; plasmids for use in the present invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells.

In some examples, at least one component of an acceptable viral diluent, buffer, protectant, stabilizer, excipient is also included.

In some examples, the non-replicating recombinant adeno-associated virus vector vaccine is formulated for intramuscular injection or intravenous use.

In some examples, the non-replicating recombinant adeno-associated virus vector vaccine is formulated for intranasal vaccination use.

In some examples, the non-replicating recombinant adeno-associated virus vector vaccine further comprises a container.

In some examples, the non-replicating recombinant adeno-associated virus vector vaccine further comprises instructions for use.

In a fourth aspect of the present invention, there is provided:

the third aspect of the present invention provides the use of a non-replicating recombinant adeno-associated virus vector vaccine in the preparation of a prophylactic RSV formulation.

In a fifth aspect of the present invention, there is provided:

a method of preventing RSV infection comprising vaccinating a population suitable for vaccination with a non-replicating recombinant adeno-associated virus vector vaccine according to the third aspect of the invention.

In some instances, requirements for an effective dosage to be used, and methods of use are included.

In some examples, the dose administered is about 10⁹～10¹²vg。

In some examples, the number of inoculations is 1-2.

In some examples, routes of vaccination include intranasal administration, intramuscular inoculation, and intravenous injection.

The invention has the beneficial effects that:

the antigen of some embodiments of the invention, whose encoding nucleic acid can be efficiently encapsulated within the AAV capsid, while having a more stable structure.

The antigen of some embodiments of the invention can be cut/autogenously cut into 2 segments after in vivo synthesis, then linked by disulfide bonds to form Post-F, and then polymerized to form a stable trimer structure.

Antigens according to some embodiments of the invention may form stable protein polypeptides/fusion proteins.

(1) The polypeptide sequence/fusion protein optimized and mutated on the basis of the RSV F, F1 and F2 antigenic polypeptides is one of the following amino acids capable of inducing specific antibodies against the F protein, F1 protein, F2 protein and neutralizing antibodies and immunoprotection effects against RSV:

RSV-F2-GS-F1 is characterized in that GS connecting sequence (SEQ ID NO: 2) is used for replacing amino acid at a Furin site of F protein to form stable Pre-F fusion protein;

② RSV-F2-2A-F1 replaces F protein Furin site amino acid with 2A connecting sequence (SEQ ID NO.: 7), 2A sequence is cut into F1 and F2, then F1 and F2 are connected by disulfide bond, and then polymerized to form stable trimer antigen protein structure;

③ RSV-F1-Foldon is formed by adding a Foldon trimerization domain (SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 11) to the C-terminal residue of F1 to form a more stable antigenic protein structure;

RSV-IgE-SP F1-Foldon is the N-terminal addition IgE signal peptide sequence of F1 (SEQ ID No.: 8), the C-terminal residue is trimerized by the addition of Foldon domain (SEQ ID No.: 11);

fifth, RSV-F2-2A-F1-10 is antigen polypeptide which uses 2A connecting sequence (SEQ ID NO: 7) to replace F protein Furin site amino acid and deletes 10 amino acids (137-146) of fusion zone after F protein Furin site;

sixthly, the RSV-F2-GS-F1-10 is formed by replacing amino acid at the Furin site of the F protein with a GS connection sequence (SEQ ID NO. 15) and deleting 10 amino acids (137-146) in a fusion region to form a stable Pre-F fusion protein;

seventhly, RSV-F mutation mutates the enzyme cutting site of F protein Furin B into KKQKQQ;

(3) the RSV G antigen polypeptide sequence is one of the following amino acid sequences: firstly, RSV G protein; ② RSV-IgE SP G is characterized by that an IgE signal peptide sequence (SEQ ID No.: 8) is added in the N-terminal of G protein; capable of inducing specific antibodies against the G protein and neutralizing antibodies and immunoprotection effects against RSV:

(4) the RSV SH antigen polypeptide sequence is one of the following amino acid sequences: firstly, RSV SH protein; ② RSV-SH-Fc is a fusion protein in which the C-terminal end of SH is linked to an immunoglobulin IgG Fc region (SEQ ID No.: 12) via a linker sequence (SEQ ID No.: 3); preferably, the linker sequence is GGGSGGGS; can induce specific antibody against SH protein, neutralizing antibody against RSV and immunoprotection effect.

Drawings

FIG. 1 is a schematic map of pAAV-MCS-F vector;

FIG. 2 is a schematic map of the pAAV-MCS-G vector;

FIG. 3 is an AAV-F protein neutralizing antibody titer assay (plaque assay);

FIG. 4 shows the rAAV-G protein neutralizing antibody titer assay (plaque assay);

FIG. 5 is an immune serum specific IgG assay;

FIG. 6 is a lung tissue virus titer test (plaque method);

figure 7 is a pathological examination of lung tissue.

Detailed Description

The present invention will be described in further detail below with reference to embodiments and the accompanying drawings, but the embodiments of the invention are not limited thereto.

Unless otherwise explicitly defined herein, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. In describing the present invention, the following terminology will be used, and the brief description will be defined below.

"RSV F protein" refers to all forms of RSV F protein, including F1 polypeptides and F2 polypeptides, i.e., including F0 precursor polypeptides, F0 precursor polypeptide trimers, mature F protein trimers, and the like.

Unless otherwise indicated, the terms "protein", "protein" and polypeptide are used interchangeably, as used herein.

As used herein, the terms "nucleic acid", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably; "nucleic acid" refers to naturally occurring or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrids, single-or double-stranded, sense or antisense, capable of hybridizing to complementary nucleic acids by Watson-Crick base pairing. The nucleic acids of the invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., Peptide Nucleic Acids (PNAs) or thiodiester linkages). Specifically, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

As used herein, the term "adjuvant" refers to a substance, such as aluminum hydroxide, that is capable of enhancing, accelerating or prolonging the immune response of the body to an immunogen or immunogenic composition.

As used herein, "AAV vector" refers to an adeno-associated virus (AAV) vector; "rAAV vector" refers to a recombinant adeno-associated virus (rAAV) vector, refers to a recombinant non-replicating adeno-associated virus, including a serotype capsid, and carries a recombinant genome comprising functional 5 'and 3' Inverted Terminal Repeats (ITRs) and linked foreign nucleotide sequences replacing the Rep or Cap genes of wild-type AAV. The ITR sequence provides functional rescue, replication and packaging for rAAV. In some embodiments, the ITR sequence is from AAV 2. The foreign nucleotide sequence is usually composed of a series of expression regulatory elements and coding regions.

As used herein, a vector refers to a means or element for delivery of genetic material, such as a plasmid, virus, phage, liposome, or the like, and this term is referred to either as a cloning vector, expression vector, or backbone vector, as well as a viral vector or viral expression vector, depending on the context of use.

AAV serotype plasmids include two Open Reading Frames (ORFs), encoding Rep and Cap expression products. Cap refers to the capsid protein of AAV, encodes VP1, VP2, VP3 and functional protein such as AAP, and the capsid sequence of AAV protein of different serotypes is different.

The technology in the implementation of specific experimental methods, usually can be according to conventional conditions, such as molecular cloning handbook, cold spring harbor laboratory manual and manufacturer provided instructions for implementation;

the experimental materials used in the practice were all commercially available, unless otherwise noted.

Example 1: construction of plasmid expressing RSV antigen polypeptide

The RSV F, G, SH protein gene sequences were obtained by NCBI database (www.ncbi.nlm.nih.gov) (see GenBank: AY 911262.1).

Analyzing the obtained polypeptide sequence characteristics, and selectively splicing with a signal peptide sequence. Preferably, the signal peptide is a human interleukin 2 signal peptide and/or a human immunoglobulin E (IgE) signal peptide. Performing human source codon optimization on the sequence; the gene sequence is sent to the gene synthesis company for completion.

Inserting the gene sequence into AAV expression plasmid vectors pAAV-MCS-F and pAAV-MCS-G to insert antigen gene sequence into plasmid vector containing ITR sequence to form antigen expression recombinant plasmid;

transforming Escherichia coli DH5 alpha by plasmid, coating on culture medium dish containing ampicillin resistance, culturing in 37 deg.C incubator for 48h, picking 2 white spots, streaking on LB plate containing resistance, and culturing in 37 deg.C incubator for 48 h; then picking white spots and inoculating the white spots in 5ml of resistant LB culture medium for shaking the bacteria for 16 hours, and extracting plasmids. Meanwhile, the positive clone is taken for enzyme digestion detection and sequencing, and the composition of each key element of the pAAV-MCS-F and pAAV-MCS-G plasmids is shown in figure 1 and figure 2.

The DH5 alpha bacteria of transformation helper plasmids pHelper, pAAV-RC, pAAV-MCS-F and pAAV-MCS-G are cultured in a shake flask, the bacteria are cracked, and the purified plasmids are used for transfecting cells.

FIG. 1 is a map diagram of a constructed pAAV-MCS-F vector, in which the key elements are described as follows: ITRs refer to the inverted terminal repeat of AAV 2; CGA promoter refers to an artificial promoter, F refers to a nucleotide sequence corresponding to a protein after the Furin enzyme cutting site is mutated; WPRE refers to woodchuck hepatitis virus post-transcriptional regulatory element; SV40 polyA refers to monkey vacuolar virus 40 (SV 40) polyadenylation sequence; signal Peptide (SP) is a Signal Peptide (e.g., IgE Signal Peptide sequence, IgE SP).

FIG. 2 is a map of the constructed pAAV-MCS-G vector; in the schematic diagram G refers to the SEQ ID number 11 sequence; other key elements are the same as those in FIG. 1.

Example 2: preparation of rAAV

Taking a tube of frozen HEK293 cells, and recovering at 25cm²The culture bottle is placed in a carbon dioxide incubator for 48 hours; subculturing according to the ratio of 1: 3-1: 5 until 10 layers of cell factories are reached, and culturing for 3-4 days;

plasmid transfection: removing a culture solution from a cell factory, adding PBS (phosphate buffer solution) for cleaning once, adding a mixed solution containing 50-500 mu G of pAAV-MCS-F or pAAV-MCS-G recombinant plasmid, 40-400 mu G of pHelper plasmid, 20-200 mu G of pAAV-RC plasmid and 30-500 mu G of transfection reagent Polyethyleneimine (PEI) for co-transfecting HEK293 cells, incubating and transfecting for 72 hours, adopting a hypotonic method to lyse the cells, simultaneously adding nuclease and 0.1-1% sodium deoxycholate according to 1-10 IU/mL, incubating for 1 hour at 37 ℃ to help cell lysis, and collecting cell lysate;

and (3) centrifugal clarification: centrifuging at 4 deg.C at 10000 × g for 30min, and collecting supernatant as virus crude cracking solution;

the rAAV purification adopts a iodixanol density centrifugation method, 60%, 40%, 25% and 15% of iodixanol are sequentially added into an ultracentrifuge tube, crude lysate is added into the upper layer, 65000rmp centrifugation is carried out for 60min, and 40% -60% of components are collected as rAAV purification samples; adopting an ultrafiltration tube (100 kDa) as a virus preservation solution (20 mmol/L, PBS (pH7.4) containing 1% of sucrose and 0.05% of poloxamer 188), and preserving the sample at a temperature below-20 ℃; and (3) determining the rAAV virus titer by adopting a qPCR method.

Alternatively, the method for constructing the rAAV of the present invention comprises the following steps:

the skilled worker can use known techniques for the production of three plasmid systems based on HEK293 cells, but also insect cell (Sf 9, Sf21 and Hi-5) -based Baculovirus Expression Vector Systems (BEVS) and packaging systems using herpes virus helper or adenovirus helper viruses for the production of rAAV.

rAAV purification may be carried out by a variety of conventional purification methods including physical lysis (repeated freeze-thaw, high pressure disruption), chemical lysis (addition of Triton, NP40, etc.), clarification (centrifugation or filtration), tangential flow ultrafiltration, chromatographic purification (affinity chromatography, ion exchange, etc.), gradient centrifugation (including cesium chloride, sucrose, iodixanol, etc.). The preferred method of AAV X affinity chromatography, density gradient centrifugation and ion exchange chromatography can obtain high purity rAAV particles.

Example 3: rAAV vector vaccine immunogenicity analysis

(1) Immunizing Balb/c mice with the age of 4-6 weeks: diluting the purified rAAV-F and rAAV-G to 5X 10¹¹vg/mL (high dose group) and 1X 10¹¹vg/mL (low dose group), 200 μ l of each mouse was injected intramuscularly in the hind leg, 100 μ l of each side, and one needle was used for immunization; collecting blood at 28 days, 35 days and 42 days, centrifuging, collecting serum, and packaging at-80 deg.C.

(2) Preparation of neutralizing antibody titer test samples: taking serum samples of each group of mice, inactivating the samples at 56 ℃ for 30min, and diluting the samples with serum-free RPMI-1640 culture medium by 2 times; RSV A2 strain virus was diluted to 100 PFU/100. mu.l in serum-free RPMI-1640 medium. The diluted virus solution and the sample were taken 100. mu.l each, mixed well, and placed in a cell culture chamber at 37 ℃ for neutralization for 2 hours.

(3) Plaque assay for neutralizing antibodies: removing the supernatant of the Hep-2 cell culture by aspiration, and adding 200. mu.l of the mixed solution to the Hep-2 cells cultured in a 6-well plateNegative control was added and the cells were incubated at 37 ℃ for 2 h. 4 ml of 1% methylcellulose overlay medium was added to each well and placed in CO₂And (3) 5 days in an incubator, removing the culture medium by aspiration, carrying out crystal violet staining for 2h, counting the plaque condition, judging the serum neutralization antibody titer by the serum dilution which can reduce the plaque number by 50%, and calculating the neutralization antibody titer of the RSV A2 neutralized by each serum sample.

(4) IgG1/IgG2a antibody titer assay: coating RSV-F or RSV-G protein (1-2 mug/hole) on a 96-well plate, adding 200 mu l of confining liquid, and standing at 37 ℃ for 2h for confinement; adding the mixture in a mode of dilution by multiple ratio (1: 2)²-2²⁵) Diluting the serum of the mouse to be detected, taking 100 mu l/hole of the diluted serum of the mouse as a negative control, and incubating the diluted serum of the mouse at 37 ℃ for 1 h; then taking out the ELISA plate, washing the plate 3 times by PBST, adding Goat anti-mouse IgG1 (diluted by antibody diluent 1: 10000) or Goat anti-mouse IgG2a (diluted by antibody diluent 1: 10000), 100 mu l/hole, and incubating for 1h at 37 ℃; washing the plate with PBST for 6 times, adding 100 μ l/well of color developing solution, and developing at 37 deg.C in dark for 10-15 min; 2M H was added₂Shaking the ELISA plate to mix with 50 mul/hole of SO4 stop solution, and reading the light absorption value of OD450nm with an enzyme-linked immunosorbent assay; and (5) judging a result: if the OD value of the hole to be measured is>0.1 and 2.1 times (P/N) greater than the negative control wells>2.1, wherein P is the OD value of the serum to be detected measured at a certain dilution factor, N is the OD value of the negative control), the serum is judged to be positive, and the highest dilution factor of the serum judged to be positive is the serum IgG1 or IgG2a antibody titer.

FIG. 3 is example 3: the detection result of neutralizing antibody in the immunogenicity analysis of rAAV vector vaccine is that recombinant adeno-associated virus rAAV-F (high dose group titer is 5 × 10)¹¹vg/mL and Low dose titers 1X 10¹¹vg/mL), one needle immunization, and 200. mu.l intramuscular injection per mouse; detecting mouse serum neutralizing antibody by plaque reduction method; the results indicate that both dose groups were able to induce high levels of neutralizing antibody titers, that the neutralizing antibody titers in the rAAV-F high dose groups were higher than 1:10240, and that the antibodies in the rAAV-F low dose groups were around 1: 2560.

FIG. 4 is example 3: neutralizing antibody detection result in rAAV vector vaccine immunogenicity analysis, namely recombinant glandRelated virus rAAV-G (high dose group titre 5X 10)¹¹vg/mL and Low dose titers 1X 10¹¹vg/mL), one needle immunization, and 200. mu.l intramuscular injection per mouse; detecting mouse serum neutralizing antibody by plaque reduction method; the results show that both dose groups can induce higher levels of neutralizing antibody titer, and the antibody titer in both the rAAV-G high dose group and the rAAV-G low dose group is about 1: 2560.

Fig. 5 is example 3: IgG1/IgG2a antibody titer determination results in rAAV vector vaccine immunogenicity analysis, namely, a single injection of a rAAV-F high-dose group, a low-dose group and a rAAV-G high-dose combined low-dose group is used for immunization, and 200 mu l of antibody is injected into each mouse muscle; the ELISA method is used for detecting specific IgG of F protein and G protein in serum, and the experimental result shows that the vaccine can induce high-level IgG1 and IgG2a, and the immune response is mainly of Th1 type.

Example 4: immune protection effect of rAAV vector vaccine

(1) Challenge protection test: immunizing Balb/c mice with the age of 4-6 weeks: diluting the purified rAAV-F and rAAV-G to 5X 10¹¹vg/mL (high dose group) and 1X 10¹¹vg/mL (low dose group), 200 μ l per mouse injected intramuscularly in the hind leg, 100 μ l per side, one injection immunization; and challenge tests were performed on day 42. Each dose group is divided into three groups, each is administered by dripping into nose 2 × 10⁶PFU RSV a2 strain or PBS, and on day 4 post challenge, mice were sacrificed by cervical dislocation, left and right lungs were aseptically placed in sterile collection tubes and 1ml of pre-cooled RPMI-1640 medium (2% FBS, double antibody) was added and stored at-80 ℃; or fixed with paraformaldehyde for histopathological observation.

(2) Lung tissue virus titer detection

Taking out lung tissue from refrigerator at-80 deg.C, weighing a certain amount, homogenizing under aseptic condition to obtain lung tissue suspension, centrifuging at 4 deg.C and 10000 Xg for 5min, and collecting supernatant. The supernatant was assayed for viral titer by the plaque method and the amount of virus in lung tissue (PFU/g or PFU/ml) was calculated.

The lung tissue virus clearance rate calculation formula is as follows:

virus clearance = (PBS control group pulmonary virus titer-sample group pulmonary tissue virus titer)/PBS control group pulmonary virus titer × 100%.

(3) Pathological observation of lung tissue

The lung tissue is fixed in paraformaldehyde solution for 48 hours, and is subjected to dehydration, transparency, wax dipping, embedding, slicing, HE dyeing and other steps to prepare pathological sections, and the pathological sections are observed under an optical microscope and digital images are collected.

(4) Lung tissue viral load detection

A certain amount of lung tissue is taken, total RNA is extracted and is subjected to reverse transcription to form cDNA, RSV F protein gene PCR amplification primers (PH-F: CCGGAATATTAATAGATCATGGAGA (SEQ ID NO.: 13); PH-R: TCATTTTATGTTTCAGGTTCAGGGG (SEQ ID NO.: 14)) are designed, the F protein gene is subjected to PCR amplification according to kit instructions, and whether a PCR product has a strip or not and the size of the strip are checked through electrophoresis.

Fig. 6 is example 4: detecting the titer of the lung tissue virus in the immune protection effect of the rAAV vector vaccine, namely immunizing by using a single injection of a rAAV-F high-dose group and a low-dose group, and a rAAV-G high-dose group and a low-dose group, and injecting 200 mu l of vaccine intramuscular injection into each mouse; the toxic attacking protection effect is detected through a mouse toxic attacking protection experiment, and the toxic attacking experiment result shows that 2 multiplied by 10⁶After the PFU RSV A2 virus strain is infected through nose, no virus is detected in the lung of the vaccine group mice, which indicates that the vaccine group mice have good protective effect on the RSV A2 virus challenge.

FIG. 7 is example 4: the observation result of pathological change conditions of mouse lung tissues in the immunity protection effect of the rAAV vector vaccine shows that the vaccine immunity group can obviously protect tissue lesions caused by RSV infected lungs, and obvious lung lesions appear after the virus challenge of a control group (rAAV-GFP).

In conclusion, the RSV vaccine based on the rAAV vector obtained by the invention can induce high-level neutralizing antibody titer and immune protection effect in mice by combining the rAAV-F protein immune serum and the rAAV-G protein immune serum, and is an ideal RSV vaccine candidate variety.

The above examples are given for clarity of illustration only and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

SEQUENCE LISTING

<110> Guangzhou Biomedicine technology Limited

<120> rAAV vector vaccine for preventing respiratory syncytial virus

<130> rAAV-RSV

<150> CN202011586494.8

<151> 2020-12-28

<160> 14

<170> PatentIn version 3.5

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Lys Lys Gln Lys Gln Gln

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Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser

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Claims

1. An RSV antigen having an amino acid sequence selected from at least one of the following amino acid sequences, or an amino acid sequence having at least 90%, 95%, 98%, 99% homology:

replacing amino acid at the Furin site of the F protein with a connecting sequence GS to obtain a fusion protein;

the F protein Furin B enzyme cutting site is mutated into KKQKQQ to obtain antigen polypeptide;

a RSV SH antigen polypeptide sequence capable of inducing specific antibodies against SH protein and neutralizing antibodies and immunoprotective effects against RSV selected from the group consisting of:

fusion proteins of the SH protein of RSV with the Fc region of an immunoglobulin IgG; preferably, the SH protein is coupled to the Fc region of an immunoglobulin IgG by a linker sequence; preferably, the linking sequence is selected from the group consisting of suitable sequences of 1 to 20 aa in length, immediately adjacent to the C-terminus of SH and the N-terminus of Fc; in particular, the amino acid sequence of the linker sequence is selected from GGGSGGGSGGGSGS, GGGSGGGS, GGGSGGGSGG, GGGSGGGSGGGS.

2. The RSV antigen of claim 1, wherein: the linker sequence GS is a GS linker, preferably, the amino acid sequence thereof is selected from GGGSGGGSGGGSGS, GGGSGGGS, GGGSGGGSGG, GGGSGGGSGGGS;

the amino acid sequence of the 2A linker sequence is the amino acid sequence comprising D-X-E-X-N-P-G-P, preferably GSGATNFSLLKQAGDVEENPGP.

3. A nucleotide sequence that expresses the RSV antigen of claim 1 or 2.

4. The nucleotide sequence of claim 3, wherein: the nucleotide sequence also includes at least one of an Inverted Terminal Repeat (ITR) of AAV, a signal peptide sequence.

5. The nucleotide sequence of claim 3, wherein: the Inverted Terminal Repeat (ITR) of the AAV is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVDJ/8 serotype, preferably, the ITR is ITR of AAV2 serotype;

the signal peptide sequence is selected from a human interleukin 2 signal peptide and/or a human immunoglobulin E (IgE) signal peptide; preferred signal peptides are IgE signal peptides, the specific amino acid sequence of which is as set forth in SEQ ID No.: shown in fig. 8.

6. The nucleotide sequence of claim 3, wherein: the nucleotide sequence is codon optimized for human cells; preferably, the nucleotide sequence is as set forth in SEQ ID No.: 9 or SEQ ID No.: shown at 10.

7. A non-replicating recombinant adeno-associated virus vector vaccine for the prevention of respiratory syncytial virus having at least an AAV capsid and an RSV antigenic nucleotide sequence, wherein: the AAV capsid comprises the nucleotide sequence of any one of claims 3 to 6, or a nucleotide sequence encoding the F protein, F1 protein, F2 protein, G protein, SH protein of RSV.

8. The non-replicating recombinant adeno-associated virus vector vaccine according to claim 7 wherein: the AAV capsid protein sequence is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVDJ/8 serotype, preferably AAV5 serotype.

9. The non-replicating recombinant adeno-associated virus vector vaccine according to claim 7 wherein: further comprises at least one component selected from acceptable viral diluents, buffers, protective agents, stabilizers, excipients and/or adjuvants.

10. Use of the non-replicating recombinant adeno-associated virus vector vaccine according to claims 7 to 9 in the preparation of a RSV prophylactic formulation.