CN113186173A

CN113186173A - Novel coronavirus pneumonia vaccine based on attenuated influenza virus vector

Info

Publication number: CN113186173A
Application number: CN202110407536.5A
Authority: CN
Inventors: 罗剑; 高飞霞; 熊斐斐; 张敏; 刘雪颖; 郑眉; 丁亚红; 范蒋锋; 周旭; 李秀玲
Original assignee: SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Current assignee: SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2021-07-30
Anticipated expiration: 2041-04-15
Also published as: CN113186173B

Abstract

The invention discloses a novel coronavirus pneumonia vaccine based on an attenuated influenza virus vector. Specifically, the invention discloses an attenuated influenza virus strain, wherein the genome of the virus strain comprises an exogenous antigen gene sequence which is embedded at the 5' end of an NA gene of the attenuated influenza virus strain, and the exogenous antigen gene sequence, the NA gene and packaging signal sequences at two ends form an antigen expression cassette; and the genome of the virus strain contains a truncated influenza virus NS1 protein gene sequence. The attenuated influenza virus strain can be used for preparing vaccines, and the vaccines can be inoculated by a nasal spray immunization mode, induce organisms to generate protective immunity (including humoral immunity and mucosal immunity) against SARS-CoV-2 and influenza virus, and have double immunity effects on novel coronavirus pneumonia and influenza.

Description

Novel coronavirus pneumonia vaccine based on attenuated influenza virus vector

Technical Field

The invention relates to the field of genetic engineering technology and biological medicine, in particular to an attenuated influenza virus vector vaccine capable of expressing SARS-CoV-2RBD protein, which can be inoculated in a nasal drip mode and can simultaneously induce protective immunity against SARS-CoV-2 and influenza virus.

Background

The novel coronavirus pneumonia (CoVID-19) is an acute respiratory infectious disease caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The global pandemic of COVID-19 brings great harm to the life and property safety of people of all countries and social development. The development of preventive COVID-19 vaccine is strengthened, and the method has important significance for epidemic prevention and control. By far, more than 200 COVID-19 candidate vaccines are under development worldwide, and nearly 50 candidate vaccines enter human clinical trial evaluation.

SARS-CoV-2 is the 7 th coronavirus which is known to infect human, and the beta coronavirus of SARS-CoV-2 and SARS-CoV belong to the same genus coronavirus family is enveloped single-strand positive-strand RNA virus, and the virus particle is circular or elliptical and has a diameter of 60-140 nm. The SARS-CoV-2 virus genome is 29903 bp in total length, and encodes at least 27 kinds of protein including 15 kinds of non-structural protein, 4 kinds of structural protein and 8 kinds of auxiliary protein. The 4 structural proteins are spike protein (S), matrix protein (M), envelope protein (E) and nucleocapsid protein (N), respectively, wherein S, E and M are 3 glycoproteins on the surface of the viral envelope. S exists as a homotrimer, the monomer of which can be recognized by proteases and cleaved into S1 and S2 subunits, S1 mainly comprises a Receptor Binding Domain (RBD) which recognizes and binds to a cell surface receptor, and the S2 subunit mediates fusion of viral envelope and cell membrane. It is shown that SARS-CoV-2 takes human angiotensin converting enzyme 2 (angiotensin-converting enzyme 2, ACE2) as receptor to enter cells. Blocking the binding of S or its RBD to ACE2 helps to block viral entry into cells, and therefore the S protein or its RBD is also the primary antigen for the induction of protective antibodies to SARS-CoV-2.

Live Attenuated Influenza Vaccines are used to prevent infection by Influenza Viruses, and recombinant NS truncated Live Attenuated Influenza Vaccines are widely studied, which have shown that NS truncated trivalent Attenuated Influenza Vaccines induce high levels of neutralizing antibodies after immunization (Pu Wang, Min Zheng, Siu-Ying Lau, Pin Chen, et al Generation of DelNS1 Influenza Viruses: a Strategy for Optimizing Live infected Vaccines [ J ]. mBio.201Sep9; 10(5): e02180-19), and NS truncated Influenza based Vaccines are currently in clinical trials.

Therefore, there is a strong need in the art to develop a prophylactic vaccine capable of targeting both SARS-CoV-2 and influenza viruses.

Disclosure of Invention

The purpose of the present invention is to provide a novel coronavirus pneumonia vaccine based on an attenuated influenza virus vector, which can simultaneously target SARS-CoV-2 and influenza virus.

In a first aspect of the present invention, an attenuated influenza virus strain is provided, wherein the genome of the virus strain comprises an exogenous antigen gene sequence, wherein the exogenous antigen gene sequence is chimeric at the 5' end of the NA gene of the attenuated influenza virus strain, and forms an antigen expression cassette together with the NA gene and packaging signal sequences (packaging sequences) at both ends thereof;

the genome of the virus strain contains a truncated influenza virus NS1 protein gene sequence, and the truncated influenza virus NS1 protein is a protein with 102 amino acid deletions at the C terminal of an influenza virus NS1 protein.

In another preferred embodiment, the genome of said strain further comprises an influenza virus NEP sequence located 3' to the truncated influenza virus NS1 protein gene sequence.

In another preferred embodiment, the antigen expression cassette has a structure represented by formula I from 5 'end to 3' end:

Z0-Z1-Z2-Z3-Z4-Z5 (I)

wherein,

each "-" is independently a chemical bond or a nucleotide linking sequence;

z0 is a non-coding region sequence at the 5' end of the NA gene sequence;

z1 is a packaging signal sequence of a 5' end coding region of the NA gene;

z2 is an exogenous antigen gene sequence;

z3 is a linker peptide sequence or none;

z4 is the full-length sequence of NA gene containing 5' end coding region package signal sequence;

and

z5 is the 3' end non-coding region sequence of the NA gene sequence.

In another preferred embodiment, the exogenous antigenic gene sequence is derived from an antigen selected from the group consisting of: SARS-CoV-2, middle east respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV), or a combination thereof.

In another preferred embodiment, the exogenous antigen gene sequence is derived from SARS-CoV-2.

In another preferred embodiment, the exogenous antigenic gene sequence is identical to the sequence set forth in SEQ ID NO: 1, the coding sequence of the Receptor Binding Domain (RBD) amino acid sequence of the SARS-CoV-2 spike protein gene has at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

In another preferred embodiment, the exogenous antigenic gene sequence encodes a polypeptide as set forth in SEQ ID NO: 1.

In another preferred embodiment, the exogenous antigen gene sequence is as shown in SEQ ID NO: 2, respectively.

In another preferred embodiment, Z3 is a linker peptide having the sequence as set forth in SEQ ID NO: 3.

In another preferred embodiment, the full-length coding sequence of the NA gene is as set forth in SEQ ID NO: 4, said sequence comprising a 5 'and a 3' coding region packaging signal sequence.

In another preferred embodiment, the 5' coding region packaging signal sequence of the NA gene is shown in SEQ ID NO: 5, respectively.

In another preferred embodiment, the amino acid sequence of said truncated influenza virus NS1 protein is as set forth in SEQ ID NO: and 6.

In another preferred embodiment, the truncated influenza virus NS1 protein gene sequence is as set forth in SEQ ID NO: shown at 7.

In another preferred embodiment, the influenza virus is an influenza a virus.

In another preferred embodiment, the influenza virus is an H1N1 influenza virus.

In another preferred embodiment, the attenuated influenza virus strain has a 80-100 fold reduction in toxicity compared to its corresponding pre-attenuated influenza virus strain.

In another preferred example, the attenuated influenza virus strain is an attenuated influenza virus vector new coronary pneumonia vaccine candidate strain with the preservation number of CCTCC NO: V202115.

In a second aspect of the present invention, there is provided an expression vector comprising:

(a) a first expression vector comprising a first expression cassette for expression of said exogenous antigen;

(b) a second expression vector comprising a second expression cassette for expression of a truncated influenza NS1 protein;

wherein the first expression vector and the second expression vector are the same vector or different vectors.

In another preferred embodiment, the expression vector is selected from the group consisting of: plasmids, viral vectors.

In another preferred embodiment, the first expression cassette has a structure represented by formula II from the 5 'end to the 3' end:

Z0’-Z1’-Z2’-Z3’-Z4’-Z5’ (II)

wherein,

each "-" is independently a chemical bond or a nucleotide linking sequence;

z0 'is the non-coding region sequence of the 5' end of the NA gene sequence;

z1 'is a packaging signal sequence of a 5' end coding region of the NA gene;

z2' is exogenous antigen gene sequence;

z3' is a linker peptide sequence or none;

z4 'is the full-length sequence of the NA gene containing a 5' end coding region packaging signal sequence;

and

z5 'is the 3' end non-coding region sequence of the NA gene sequence.

In another preferred embodiment, the second expression cassette has a structure represented by formula III from the 5 'end to the 3' end:

Z6-Z7-Z8-Z9-Z10 (III)

wherein,

each "-" is independently a chemical bond or a nucleotide linking sequence;

z6 is a non-coding region sequence at the 5' end of an influenza virus NS1 gene sequence;

z7 is a truncated influenza virus NS1 gene sequence;

z8 is a linker peptide sequence or none;

z9 is an influenza virus NEP gene sequence;

and

z10 is the 3' non-coding region sequence of NEP gene sequence.

In another preferred embodiment, the expression vector is an influenza virus vector, and the vector comprises the first expression cassette and the second expression cassette.

In a third aspect of the invention, there is provided a method of preparing an attenuated influenza virus strain according to the first aspect of the invention, comprising the steps of:

(i) constructing a recombinant expression plasmid V1 with an exogenous antigen gene cDNA sequence embedded at the 5' end of the influenza virus NA gene cDNA sequence and a recombinant expression plasmid V2 with the C-terminal 102 amino acids truncated of the influenza virus NS1 gene;

(ii) respectively obtaining six auxiliary plasmids containing influenza virus PA, PB1, PB2, HA, NP and M genes;

(iii) co-transfecting host cells with the recombinant expression plasmids V1 and V2 constructed in the step (i) and the six helper plasmids obtained in the step (ii), respectively, culturing for 48 hours, and collecting culture supernatants; and

(iv) and (3) inoculating the supernatant into SPF (specific pathogen free) chick embryos, and harvesting chick embryo allantoic fluid to obtain the attenuated influenza virus strain.

In another preferred example, the attenuated influenza virus strain can be prepared into an attenuated live vaccine after further purification.

In another preferred embodiment, the exogenous antigenic gene is selected from the group consisting of: SARS-CoV-2, middle east respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV), or a combination thereof.

In another preferred example, the 3' end of the cDNA sequence of the influenza virus NS1 gene in the recombinant expression plasmid V2 is also connected with a complete cDNA sequence of influenza virus NEP.

In another preferred embodiment, the host cell is selected from the group consisting of: 293T cells, Vero cells, or combinations thereof.

In a fourth aspect of the invention, there is provided the use of an attenuated influenza virus strain according to the first aspect of the invention or an expression vector according to the second aspect of the invention in the preparation of a vaccine composition for use in a viral infectious disease.

In another preferred embodiment, the viral infectious disease is selected from the group consisting of: influenza, novel coronavirus pneumonia, Middle East Respiratory Syndrome (MERS), respiratory infection by human parainfluenza virus (HPIV), and respiratory infection by Respiratory Syncytial Virus (RSV).

In another preferred embodiment, the influenza is influenza elicited by influenza a (a) virus.

In another preferred example, the influenza is influenza elicited by H1N1 influenza virus.

In a fifth aspect of the invention, there is provided a vaccine composition comprising:

(a) an attenuated influenza virus strain according to the first aspect of the invention; and

(b) a vaccine acceptable carrier.

In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutically acceptable carrier comprises a liquid, preferably water, saline or a buffer.

In another preferred embodiment, the carrier further comprises auxiliary substances, preferably fillers, protectants, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like.

In another preferred embodiment, the vaccine composition is a bivalent vaccine or a multiple vaccine.

In another preferred embodiment, the vaccine composition is a bivalent vaccine.

In another preferred embodiment, the vaccine composition is a combined vaccine for the prevention of influenza virus and new coronavirus.

In another preferred embodiment, the influenza virus is an influenza a (a) virus.

In another preferred embodiment, the vaccine composition may further comprise a vaccine component derived from one or more pathogens selected from the group consisting of: SARS-CoV-2, middle east respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV), or a combination thereof.

In another preferred embodiment, the vaccine components comprise inactivated strains, attenuated strains, or proteins, nucleic acids, and the like.

In another preferred embodiment, the vaccine composition further comprises an adjuvant.

In another preferred embodiment, the adjuvant comprises: particulate and non-particulate adjuvants.

In another preferred embodiment, the particulate adjuvant is selected from the group consisting of: an aluminum salt, a water-in-oil emulsion, an oil-in-water emulsion, a nanoparticle, a microparticle, a liposome, an immunostimulatory complex, or a combination thereof;

in another preferred embodiment, the non-particulate adjuvant is selected from the group consisting of: muramyl dipeptide and its derivatives, saponin, lipid A, cytokine, derivative polysaccharide, bacterial toxin, microorganism and its product such as mycobacteria (Mycobacterium tuberculosis, Bacillus Calmette-Guerin), Bacillus pumilus, Bordetella pertussis, propolis, or combinations thereof.

In another preferred embodiment, the vaccine composition has a virus of at least 105EID50 per dose.

In another preferred embodiment, the vaccine composition is in a nasal spray form or a subcutaneous injection form.

In another preferred embodiment, the vaccine composition is in the form of a nasal spray.

In a sixth aspect of the invention, there is provided a method of preparing a vaccine composition according to the fifth aspect of the invention, comprising the steps of: the vaccine composition is prepared by mixing the attenuated influenza virus strain of the first aspect of the invention with a vaccinally acceptable carrier.

In a seventh aspect of the present invention, there is provided a prophylactic vaccination method for preventing influenza and neocoronary pneumonia, comprising the steps of: a subject in need thereof is vaccinated with an attenuated influenza virus strain according to the first aspect of the invention, or a vaccine composition according to the fifth aspect of the invention.

In another preferred embodiment, the subject is susceptible to influenza and/or new coronary pneumonia.

In another preferred embodiment, the inoculation mode is nasal spray inoculation.

In another preferred embodiment, the dose of the inoculation is not less than 10⁵EID50。

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

Drawings

Figure 1 shows a schematic of recombinant attenuated influenza vector vaccine plasmid construction.

Figure 2 shows the expression and identification of RBD in recombinant attenuated influenza vector vaccine.

Figure 3 shows the growth characteristics of recombinant attenuated influenza vector vaccines in cells and chicken embryos.

Figure 4 shows the attenuation properties of recombinant attenuated influenza vector vaccines in a mouse model.

FIG. 5 shows the serum Ig and mucosal IgA antibody responses induced against SARS-CoV-2RBD specificity following nasal immunization of mice with recombinant attenuated influenza vector vaccine.

FIG. 6 shows the serum neutralizing antibody response against SARS-CoV-2 induced by recombinant attenuated influenza vector vaccine after nasal drip immunization of mice.

Figure 7 shows the immune response against influenza virus induced by recombinant attenuated influenza vector vaccines after nasal drip immunization of mice.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly developed a novel coronavirus pneumonia vaccine mediated by an attenuated influenza virus vector chimeric with an antigenic gene of SARS-CoV-2 selected from the group consisting of RBD of spike protein of SARS-CoV-2 pathogen. Experiments prove that the vaccine of the invention shows sufficient attenuation characteristics in a mouse model; and can effectively induce the generation of specific mucosal IgA and serum Ig aiming at SARS-CoV-2, can effectively neutralize virus in a SARS-CoV-2 pseudovirus model, and can also induce the generation of protective immunity aiming at influenza. The vaccine of the present invention can thus provide dual protection against both influenza virus and SARS-CoV-2.

On the basis of this, the present invention has been completed.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the terms "subject", "subject in need thereof" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

Influenza virus

Influenza viruses are short for influenza viruses, and can be divided into four types, namely A (A), B (B), C (C) and D (D), and the influenza A viruses can be divided into different subtypes. In the present invention, "influenza virus" mainly refers to viruses of different subtypes of influenza virus type (a). In a preferred embodiment of the present invention, the influenza virus is an H1N1 influenza virus.

NA gene

The Neuraminidase (NA) gene is the 6 th segment of the influenza genome, about 1362 nucleotides in length, has NA activity, recognizes specific sialic acid, cleaves sialic acid at the end of cell surface receptors, and facilitates release of virions from infected cells.

NS1 gene

The non-structural protein (NS) gene is the 8 th segment of the influenza genome, about 890 nucleotides in length, and is primarily responsible for encoding non-structural protein 1(NS1) and Nuclear Export Protein (NEP). The length of NS1 is about 202-237 amino acids, and NS1 is a multifunctional protein, interacts with various host factors, resists host immune defense effect, and participates in virus infection and replication process.

NEP gene

Nuclear Export Protein (NEP), encoded by segment 8 gene of influenza A virus, plays an important role in many links such as viral genome replication and transcription, viral vRNP complex Nuclear Export, virion assembly, budding, etc.

Receptor binding domain of SARS-CoV-2 spike protein gene (SARS-CoV-2RBD)

The SARS-CoV-2 spike protein (S protein) has two subunits: s1 and S2, the receptor binding site (RBD) is located in the S1 subunit, and it constitutes a spike on the outer membrane surface of the virion in the form of trimer, whose main function is to recognize host cell surface receptors and mediate fusion with host cells. The RBD sequence of the invention totally encodes 266 amino acids (317-.

Attenuated influenza virus strains of the invention

The invention provides an attenuated influenza virus strain, which is the attenuated influenza virus strain with the preservation number of CCTCC NO: V202115. The genome of the attenuated influenza virus strain incorporates a receptor binding domain derived from the spike protein gene of SARS-CoV-2 (SARS-CoV-2RBD) as set forth in SEQ ID NO: 2, wherein SEQ ID NO: 2 encodes the amino acid sequence shown in SEQ ID NO: 1, SARS-CoV-2RBD amino acid sequence shown in the specification. And the genome of the virus strain contains a truncated influenza virus NS1 protein gene sequence (shown as SEQ ID NO: 7), and the truncated influenza virus NS1 protein is a protein with 102 amino acids deleted at the C terminal of the influenza virus NS1 protein (shown as an amino acid sequence in SEQ ID NO: 6). In addition, the genome of the strain also comprises an influenza virus NEP sequence located at the 3' end of the truncated influenza virus NS1 protein gene sequence.

An antigen expression cassette as shown in formula I in the genome of the attenuated influenza virus strain:

Z0-Z1-Z2-Z3-Z4-Z5(I)

wherein,

each "-" is independently a chemical bond or a nucleotide linking sequence;

z0 is a non-coding region sequence at the 5' end of the NA gene sequence;

z1 is a packaging signal sequence of a 5' end coding region of the NA gene;

z2 is SARS-CoV-2RBD sequence;

z3 is a linker peptide sequence;

z4 is a NA gene sequence containing a 5' end coding region packaging signal sequence;

and

z5 is the 3' end non-coding region sequence of the NA gene sequence.

The attenuated influenza virus can stimulate the organism to generate immune reaction aiming at the influenza virus H1N1 and SARS-CoV-2 after entering the organism by simultaneously expressing the antigen protein of the influenza virus H1N1 and the RBD protein of the SARS-CoV-2. Specifically, the attenuated influenza virus of the present invention can simultaneously activate humoral immune response and mucosal immune response of the body, and produce specific serum Ig antibody and specific mucosal IgA antibody against SARS-CoV-2RBD, and serum Ig antibody against influenza virus H1N 1.

In addition, the attenuation property of the attenuated influenza virus strain is realized by deleting 102 amino acids at the C terminal of the NS1 protein of the truncated influenza virus, so that the virus does not cause diseases after entering the body, but can still trigger the immune response of the body.

Expression vector

The present invention also provides an expression vector for preparing the attenuated influenza virus strain of the present invention, comprising (a) a first expression vector comprising a first expression cassette for expression of an exogenous antigen; (b) a second expression vector comprising a second expression cassette for expression of a truncated influenza virus NS1 protein;

As used herein, the term "exogenous antigen" refers to an exogenous antigen relative to an influenza virus, i.e., an antigen of a pathogen of other origin that the influenza virus itself does not contain. The antigen may be derived from a pathogen including, but not limited to, selected from the group consisting of: SARS-CoV-2, middle east respiratory syndrome coronavirus (MERS), human parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV), and the like. In a preferred embodiment of the present invention, the exogenous antigen is derived from SARS-CoV-2 and is the full length of the RBD protein of SARS-CoV-2 or a fragment thereof.

With the sequence information provided, the skilled artisan can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

The vector may be a plasmid or a virus. Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids may be complexed with agents to facilitate uptake of the viral vector by the target cell. Such agents include polycationic agents. Alternatively, a delivery system such as a liposome-based delivery system may be used. The vector for use in the present invention is preferably suitable for use in vivo or in vitro.

The vector will preferably comprise one or more regulatory sequences to direct expression of the nucleic acid sequence in the target cell. Regulatory sequences may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to a nucleic acid sequence. The vector may also include a selectable marker, e.g., to determine expression of the vector in a growth system (e.g., a bacterial cell) or in a target cell.

By "operably linked" is meant that the nucleic acid sequences are functionally related to the sequences to which they are operably linked such that they are linked in a manner such that they affect the expression or function of each other. For example, a nucleic acid sequence operably linked to a promoter will have an expression pattern that is affected by the promoter.

In a preferred embodiment of the present invention, the expression vector is a plasmid comprising a first expression vector and a second expression vector. Wherein the first expression vector is a plasmid containing exogenous antigen gene cDNA sequence (such as SARS-CoV-2 RBD); the second expression vector is a plasmid containing a cDNA sequence truncated by 102 amino acids at the C terminal of the influenza virus NS1 gene. The first expression vector and the second expression vector may be used to prepare an attenuated influenza virus strain of the invention by simultaneous transduction into a host cell. The host cell is selected from the group consisting of: 293T cells and Vero cells.

In another preferred embodiment of the present invention, the expression vector may be an influenza virus comprising the second expression cassette and the first expression cassette. The influenza virus is used as a virus live vector for expressing an exogenous antigen gene in the first expression cassette.

Vaccine composition

In the invention, a vaccine composition and a preparation method thereof are also provided.

The vaccine composition of the present invention comprises: (a) an attenuated influenza virus strain according to the first aspect of the invention; and (b) a vaccinally acceptable carrier.

Preferably, the carrier is a pharmaceutically acceptable carrier. These include any vehicle that does not itself induce the production of antibodies harmful to the individual receiving the composition. In a preferred embodiment, the pharmaceutically acceptable carrier comprises a liquid, preferably water, saline or a buffer. The carrier may also contain auxiliary substances, preferably fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like.

Preferably, the immunological composition may further comprise an adjuvant comprising: particulate and non-particulate adjuvants. The particulate adjuvant is selected from the group consisting of: an aluminum salt, a water-in-oil emulsion, an oil-in-water emulsion, a nanoparticle, a microparticle, a liposome, an immunostimulatory complex, or a combination thereof; the non-particulate adjuvant is selected from the group consisting of: muramyl dipeptide and its derivatives, saponin, lipid A, cytokine, derivative polysaccharide, bacterial toxin, microorganism and its product such as mycobacteria (Mycobacterium tuberculosis, Bacillus Calmette-Guerin), Bacillus pumilus, Bordetella pertussis, propolis, or combinations thereof.

More specifically, the vaccine composition of the invention comprises an immunologically effective amount of an attenuated influenza virus strain according to the first aspect of the invention, together with the other required components described above. An "immunologically effective amount" refers to an amount that is effective for treatment or prevention in a single dose or in a continuous dose administered to an individual. The amount will depend on the physiological condition of the animal (e.g., human), the ability of the immune system to synthesize antibodies, the degree of protection desired, and other relevant factors.

The vaccine composition of the invention is a bivalent vaccine or a multiple vaccine. In another preferred example, the vaccine composition is a combination vaccine, and the vaccine composition is a combination vaccine for preventing influenza virus and new coronavirus.

In another preferred embodiment, the vaccine composition may further comprise a vaccine component derived from one or more pathogens selected from the group consisting of: other viruses, or combinations thereof. The vaccine components comprise inactivated strains, attenuated strains, or proteins, nucleic acids and the like.

In the present invention, the vaccine composition or immunogenic composition can be made into nasal spray/drops, or injectable, such as liquid solution or emulsion; it can also be made into solid form suitable for being mixed with solution or suspension, or liquid excipient before nasal spray/drip or injection. The formulation may also be emulsified or encapsulated in liposomes, enhancing the adjuvant effect in the pharmaceutically acceptable carrier described above.

The conventional method is to administer the vaccine composition of the present invention by nasal spray or nasal drops. Other formulations suitable for other modes of administration include parenteral (subcutaneous or intramuscular) by injection, oral, and the like. The therapeutic dose may be a single dose regimen or a multiple dose regimen. The vaccine compositions of the present invention may be administered in combination with other immunomodulators.

Viral strain preservation

The attenuated influenza virus strain (attenuated influenza virus vector new coronary pneumonia vaccine candidate strain) is preserved in China center for type culture Collection (CCTCC, China, Wuhan) at 03.02-03.2021, and the preservation number is CCTCC NO: V202115.

The main advantages of the invention are:

(1) the attenuated influenza virus strain provided by the invention can be prepared into a novel coronavirus pneumonia vaccine candidate strain based on attenuated influenza virus mediation, and the vaccine can simultaneously activate humoral immunity and mucosal immune response of experimental mice and simultaneously provide dual protection for SARS-CoV-2 and influenza virus.

(2) Compared with the vaccine in the traditional injection inoculation mode, the attenuated influenza virus vector vaccine provided by the invention is inoculated in a nasal spray mode, simulates the natural immune process of a human body, and plays a role in three modes of mucosal immunity, cellular immunity and humoral immunity, and has obvious advantages.

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

Materials, reagents, instruments and the like used in examples are commercially available unless otherwise specified.

Experimental materials and methods

Cell: human embryonic kidney cell 293T cell line (

CRL-3216^TM) And Canine Kidney cells (Madin-Darby Canine Kidney, MDCK,

CCL-34)。

plasmid: the pHW2000 bidirectional transcription expression vector is preserved by Shanghai biological products research institute, Limited liability company.

Influenza virus vaccine strains: year 2020 northern hemisphere seasonal influenza virus vaccine strain A/Guangdong-Maonan/SWLI536/2019(H1N1) CNZC-1909 issued by WHO was purchased from National Institute for Biological Standards and Control, NIBSC, and maintained by the National Institute of Biotechnology, Inc.

The main reagents are as follows: DMEM medium was purchased from Gibco, cat #: 41500; fetal bovine serum was purchased from Gibco, cat # s: 25200; the chick embryo and BALB/C mouse are provided by the experimental animal center of the Shanghai biological product research institute, Limited liability company; TPCK-trypsin was purchased from ThermoFisher, USA, under the trade name: 20233. lipofectamine^TM2000 transfection reagents were purchased from ThermoFisher corporation, cat #: 11668019.

1. plasmid construction and strain rescue

Viral cDNA plasmids were constructed. Genomic RNAs of seasonal influenza vaccine component strain A/Guangdong-Maonan/SWL1536/2019(H1N1) were extracted, and the NA gene of H1N1 strain was obtained by RT-PCR using primers (FIG. 1A). SARS-CoV-2RBD sequence (Bio-engineering Co., Ltd.) was cloned from pUC57-RBD plasmid, after 183 nucleotides of the non-coding region and the adjacent coding region at the 5 ' end of NA cDNA, the full-length sequence of RBD gene was added, the 3 ' end of RBD was ligated with 2A "autoclear" peptide fragment, followed by the complete coding region and 3 ' non-coding region of NA cDNA (FIG. 1B). The above sequence was cloned into pHW2000 vector to obtain pHW-CoVRBD-NA plasmid. At the same time, pHW-DelNS was constructed, primers were used to obtain the 5 ' non-coding region of NS cDNA of H1N1 strain and 384 base sequences of NS1 coding region, the entire NEP sequence and 3 ' non-coding region were ligated at the 3 ' end, and a 2A "autoclear" peptide fragment was inserted between NS1 and NEP (FIG. 1C). Six plasmids of the other PA/PB1/PB2/HA/NP/M genes of H1N1 were additionally constructed onto the pHW2000 vector. The 8 plasmids are co-transfected to 293T cells by utilizing a reverse genetics technology, supernatant is harvested after 48 hours, the supernatant is inoculated to 9-11 day old chick embryos, chick embryo allantoic fluid is harvested after 48-72 hours, and the correctness of a virus sequence is verified through a hemagglutination experiment, gene extraction and sequencing. The strain is a candidate attenuated influenza virus vector novel coronavirus vaccine strain and is named as RBD-NA-DelNS.

2. Expression and characterization of recombinant attenuated influenza vector vaccines

DMEM with 10% fetal bovine serum at 37 deg.C and 5% CO₂MDCK and 293T cells were cultured in an incubator.

And (3) inoculating the RBD-NA-DelNS strain and the H1N1 strain to MDCK cells for 48H, and identifying the expression of the strains by a Western Blot and immunofluorescence method. The Western Blot comprises the following specific operation methods: after 48h of infection of MDCK cells by the strains, the medium was aspirated, the cells were washed once with PBS, 100. mu.L of RIPA lysate containing protease inhibitors was added to each well, the cells were scraped off with a cell scraper and transferred to a 1.5mL centrifuge tube, and the cells were lysed on ice for half an hour. Centrifuging at 10000 Xg for 10min, collecting 15 μ L cell lysate, adding 5 μ L LDS loading buffer solution, mixing, heating at 70 deg.C for 10min, cooling to room temperature, and adding SDS-PAGE gel into the sample. 120V, 90 min. After electrophoresis, the membrane was rotated at a constant voltage of 25V for 5 min. Blocking with 5% FBS-PBS for 1h at room temperature, washing with PBST three times for 10min each. Primary antibody (RBD protein-immunized mouse serum) was incubated overnight at 4 ℃ and HRP-anti-mouse secondary antibody was incubated at room temperature for 1h, membrane washed three times with PBST, and developed 10min each time.

The immunofluorescence method comprises the following steps: after 48h infection, the supernatant was removed, fixed with 4% paraformaldehyde for 20min, treated with 0.1% TritonX-100 for 20min, blocked with 2% BSA for 1h, incubated with primary antibody (RBD protein-immunized mouse serum) for 1h, and then treated with Alexa Fluor^TM488-anti-mouse secondary antibody (Jackson ImmunoResearch) after 1h incubation at room temperature, and DAPI stained nuclei, observed under a fluorescent microscope and photographed.

3. Characterization of attenuation characteristics of recombinant attenuated influenza vector vaccines

The RBD-NA-DelNS and H1N1 strains were used to determine the amount of infection in chick Embryos (EID) by chick embryo₅₀) According to the same EID₅₀Inoculated into MDCK cells and chick embryos, supernatant and allantoic fluid were harvested 48h and 60h after inoculation, respectively, and hemagglutination titer was determined with 1% chick blood.

Different dosages (10)⁵，10⁶，10⁷EID₅₀) RBD-NA-DelNS strain and 10⁵EID₅₀The H1N1 strain was nasally infected with BALB/C mice at 20. mu.l each, 20 mice per group, and the observation was carried outBody weight changes and survival rates of mice were recorded 14 days post infection. And detecting the virus load of organs such as lung, liver, brain and the like by using an RT-qPCR method (NP is a virus target gene) on the 3 rd day and the 7 th day after infection.

4. Immune response following immunization of mice with recombinant attenuated influenza vector vaccine

Different dosages (10)⁵，10⁶，10⁷EID₅₀) The RBD-NA-DelNS strain was used to immunize BALB/C mice each at 20. mu.l nasal drops, 5 mice per group, on

day

14 and 28 of immunization, tail vein bleeds and lungs were removed and homogenized under sterile conditions, and the immune response and neutralizing antibodies in the immunized mice were determined by ELISA and pseudovirus neutralization.

The titer of Ig antibody in serum and IgA antibody in lung tissue homogenate were measured by ELISA:

antigen coating: the RBD protein-immunized mouse serum and the seasonal influenza virus vaccine strain A/Guangdong-Maonan/SWL1536/2019(H1N1) split vaccine stock solutions were diluted with coating buffer to a final concentration of 2. mu.g/mL, respectively, and coated on different ELISA plates. Add 100. mu.L antigen dilution to each well of 96-well ELISA plate, incubate overnight at 4 ℃;

and (3) sealing: PBST washing 3 times, each hole is added with 100 u L PBST blocking liquid containing 5% fetal bovine serum, 4 degrees C placed and closed overnight;

sample adding: sucking out the blocking solution, performing 2-fold gradient dilution on each group of serum (or lung homogenate after immunization) on a dilution plate by using an antibody diluent, adding 100 mu L of each group of serum to a 96-hole ELISA plate, and incubating overnight at 4 ℃;

adding a secondary antibody: after PBST was washed 3 times, diluted HRP-labeled goat anti-mouse Ig (southern biotech, L3225-ZD05) (HRP-labeled goat anti-mouse IgA (southern biotech, 1040-05) was used as a secondary antibody in detecting lung homogenate IgA antibody titer) was added; 100 mu L/hole, and incubating for 1h at 37 ℃;

color development: PBST was washed 3 times, 100. mu.L of TMB was added to each well, and color development was carried out at room temperature in the dark for 3-10 min. Add 50. mu.L of 2M H per well₂SO₄Stopping developing, and reading at an OD position of 450nm of an enzyme-labeling instrument;

the mean +2SD of the PBS control group was used as a positive result criterion, and the experimental results were expressed as the mean. + -. SD of 5 mice per group.

Neutralizing antibodies in serum were determined by a pseudovirus neutralization method. The specific operation method comprises the following steps:

SARS-CoV-2 pseudovirus is first prepared. Plasmids encoding the full-length S protein and pNL4-3.luc RE were co-transfected into 293T cells. The supernatant was collected 48 hours after transfection, diluted with DMEM with an equal volume of diluted serum, and then incubated at 37 ℃ for 1h to transfer the mixture to Hela cells stably expressing human ACE 2. The cells were incubated at 37 ℃ for 48h, lysed with lysis buffer and luciferase activity was detected (Promega USA). The neutralization rate was calculated by comparing the luciferase value of the antibody or serogroup with the luciferase value of the pure virus control group.

Example 1

Production of recombinant attenuated influenza vector vaccines

According to a plasmid construction schematic diagram (figure 1), extracting genome RNA of a seasonal influenza vaccine strain A/Guangdong-Maonan/SWL1536/2019(H1N1), firstly constructing six plasmids (PB2, PB1, PA, NP, HA, M) of an H1N1 strain and a DelNS plasmid on a pHW2000 vector, simultaneously embedding a SARS-CoV-2RBD sequence on an NA gene and constructing on the pHW2000 vector, utilizing a reverse genetics technology to carry out rescue on 293T cells on the eight plasmids to obtain an attenuated influenza vector novel coronavirus pneumonia vaccine virus which is named as RBD-NA-DelNS. And identifying the virus sequence to be correct through sequencing.

When MDCK cells were infected with the recombinant attenuated influenza vector vaccines RBD-NA-DelNS and H1N1 strains, it was found that, in comparison with MDCK cells infected with H1N1 strain, RBD protein was expressed on cells and showed green fluorescence after infection with RBD-NA-DelNS strain, whereas H1N1 strain did not express RBD protein (fig. 2B), and the Western Blot result also showed that RBD protein was expressed in MDCK cells after infection with RBD-NA-DelNS strain (fig. 2A).

The above results demonstrate the successful generation of recombinant attenuated influenza vector vaccines.

Example 2

Attenuation characteristics of recombinant attenuated influenza vector vaccines

Will contain the sameRBD-NA-DelNS and H1N1 strains of virus number are found to be incapable of effectively replicating on MDCK cells after being infected with MDCK cells for 48 hours, the blood coagulation titer is 0 after infection, and the titer of the unmodified H1N1 strain is 2^7.67This result is expected to be recognized by various pattern recognition receptors in cells after infection of cells by influenza virus, and the cells produce interferon to inhibit influenza virus replication. However, the influenza virus NS can inhibit the interferon-related antiviral response in the host cell, the NS lacks an element that antagonizes the production of host interferon after truncation, and the influenza virus cannot effectively antagonize the production of interferon, so the cell inhibits the further replication of the influenza virus, and therefore, the RBD-NA-DelNS cannot effectively replicate in MDCK cells to produce progeny virus (fig. 3).

Meanwhile, two strains with different doses were infected into mice separately, and low doses were found (10)⁵EID₅₀) The H1N1 strain group of mice showed significant weight loss 7 days after infection and all died with a survival rate of 0. And 3 doses (10)⁵，10⁶，10⁷EID₅₀) The RBD-NA-DelNS strain group of mice did not lose weight within 14 days of infection, the survival rate was 100% (FIG. 4A), and it was preliminarily shown that the constructed recombinant strain was attenuated by at least about 100-fold. In addition, the viral load in lung, brain, liver and other organs was significantly lower than in the H1N1 strain group at 3 days and 7 days after infection (fig. 4B). The results show that the RBD-NA-DelNS strain has obvious attenuation in animal models and can be used as a candidate vaccine strain to carry out the next experiment.

Example 3

Immune response induced following immunization of mice with recombinant attenuated influenza vector vaccine

By adding different dosages (10)⁵，10⁶，10⁷EID₅₀) The RBD-NA-DelNS vaccine of (1) is used for nasal drop immunization of mice.

After 14 and 28 days of vaccination, the immune response in the immunized mice was tested by ELISA and pseudovirus neutralization. As a result, the RBD-NA-DelNS vaccine immunized mice stimulated the production of serum Ig antibodies specific for SARS-CoV-2RBD in vivo after 14 days of immunization and was dose-dependent. The serum Ig antibodies of mice immunized with the RBD-NA-DelNS vaccine increased at 28 days after immunization and were dose-dependent as compared to 14 days after immunization (FIG. 5A). Meanwhile, the RBD-NA-DelNS vaccine can induce to generate good mucosal immune response by detecting the mouse lung tissue homogenate RBD specific IgA antibody after vaccine immunization (figure 5B).

Similarly, serum neutralizing antibody detection based on SARS-CoV-2 pseudovirus system showed that neutralizing antibodies against SARS-CoV-2 were produced in mice immunized with the RBD-NA-DelNS vaccine and that the neutralizing antibody titer was dose dependent (FIG. 6). The above results show that the RBD-NA-DelNS vaccine can induce good serum and mucosal antibody response specific to SARS-CoV-2RBD, and can effectively neutralize virus.

In addition, the antibody response to influenza virus in mice after immunization was detected by ELISA method, and the immunization of mice with RBD-NA-DelNS vaccine stimulated the production of humoral immune Ig antibody against influenza virus in mice in vivo, which was significantly higher than that of PBS control group, and was dose-dependent, and the antibody titer after 28 days of immunization was slightly higher than that after 14 days of immunization (FIG. 7).

In general, after the recombinant attenuated influenza vector vaccine RBD-NA-DelNS is used for nasal drip immunization of mice, the recombinant attenuated influenza vector vaccine can simultaneously generate antibodies aiming at SARS-CoV-2 and influenza virus, activate humoral immunity and mucosal immune response in the mice, and can also induce the generation of neutralizing antibodies aiming at SARS-CoV-2.

Sequence information of the invention

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

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Claims

1. An attenuated influenza virus strain, wherein the genome of said virus strain comprises an exogenous antigen gene sequence, wherein said exogenous antigen gene sequence is chimeric at the 5' end of the NA gene of said attenuated influenza virus strain, and forms an antigen expression cassette together with the NA gene and packaging signal sequences (packaging sequences) at both ends thereof.

2. The virus strain of claim 1, wherein the genome of the virus strain comprises a truncated influenza virus NS1 protein gene sequence, and the truncated influenza virus NS1 protein is an influenza virus NS1 protein with a deletion of the C-terminal 102 amino acids.

3. The virus strain of claim 1, wherein the antigen expression cassette has the structure of formula I from 5 'to 3':

Z0-Z1-Z2-Z3-Z4-Z5 (I)

wherein,

each "-" is independently a chemical bond or a nucleotide linking sequence;

z0 is a non-coding region sequence at the 5' end of the NA gene sequence;

z1 is a packaging signal sequence of a 5' end coding region of the NA gene;

z2 is an exogenous antigen gene sequence;

z3 is a linker peptide sequence or none;

and

z5 is the 3' end non-coding region sequence of the NA gene sequence.

4. The virus strain of claim 1, wherein the exogenous antigenic gene sequence is derived from an antigen selected from the group consisting of: SARS-CoV-2, middle east respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV), or a combination thereof.

5. The virus strain of claim 1, wherein the exogenous antigenic gene sequence is identical to the sequence set forth in SEQ ID NO: 1, the coding sequence of the Receptor Binding Domain (RBD) amino acid sequence of the SARS-CoV-2 spike protein gene has at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

6. An expression vector, comprising:

7. A method of making an attenuated influenza virus strain according to any one of claims 1 to 5 comprising the steps of:

8. Use of an attenuated influenza virus strain according to any one of claims 1 to 5 or an expression vector according to claim 6 in the preparation of a vaccine composition for use in a viral infectious disease.

9. A vaccine composition, comprising:

(a) the attenuated influenza virus strain of any one of claims 1-5; and

(b) a vaccine acceptable carrier.

10. A method of preparing the vaccine composition of claim 9, comprising the steps of: mixing the attenuated influenza virus strain of any one of claims 1-5 with a vaccinally acceptable carrier to produce the vaccine composition.