Influenza virus vaccine composition

Abstract

The invention belongs to the technical field of biology, particularly relates to a tetravalent influenza vaccine, and more particularly relates to a tetravalent influenza mRNA (messenger Ribonucleic Acid) vaccine. The invention provides an influenza vaccine composition. The influenza vaccine composition comprises an influenza H1N1 antigen construct, an influenza H3N2 antigen construct, an influenza H5N6 antigen construct and an influenza Victoria antigen construct. Through the design of the composition, the influenza vaccine composition disclosed by the invention is creatively found to have the capability of generating high-titer antibodies to H1N1, H3N2, Victoria and H5N6, and also have the capability of generating high-titer antibodies to H7N9, H9N2 and Yamagata, and particularly, the influenza vaccine composition disclosed by the invention can generate antibodies to H7N9.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, it relates to a quadrivalent influenza vaccine, and more specifically, to a quadrivalent influenza mRNA vaccine. Background Art

[0002] Influenza is an acute respiratory infection caused by the influenza virus (IV). According to the World Health Organization, the seasonal prevalence of the influenza virus results in approximately 305 million cases of severe illness globally, leading to approximately 500,000 deaths, and causing significant economic losses and social burdens. The frequent mutation of the influenza virus leads to a certain lag in vaccine development and drug resistance to existing antiviral drugs. The selection of vaccine strains faces many challenges every year. The mismatch between vaccine strains and circulating infecting viruses is the main reason for poor efficacy. Most of the antibodies induced by vaccines are against the highly variable head region of hemagglutinin (HA). HA has high variability and low sequence conservation among different strains. Currently, the drugs against influenza virus only have moderate effects in treating influenza virus infections. Therefore, vaccination is the main and only way to protect the population from influenza virus infection.

[0003] Currently, seasonal influenza vaccines are mainly divided into three types: inactivated vaccines, live attenuated vaccines, and recombinant hemagglutinin vaccines. There is no approved influenza mRNA vaccine on the market yet. These vaccines usually consist of three or four different types of influenza virus components and are updated annually by the World Health Organization to match the latest prevalent strains.

[0004] mRNA vaccine technology has developed rapidly during the COVID-19 pandemic. Compared with traditional influenza vaccines, mRNA vaccines have a series of advantages. They have high safety, highly controllable antigen production processes, simple and rapid production processes, require less time to respond to antigen updates, and do not require the use of chicken embryos or cells. mRNA vaccines can effectively stimulate humoral and cellular immune responses by expressing antigens in vivo. These advantages of mRNA vaccines are exactly the characteristics that vaccines for producing against continuously mutating influenza strains should possess. There are literature reports that researchers combined the advantages of mRNA vaccines and nanoparticle vaccines and developed a self-assembling envelope-like virus-like particle (eVLP) mRNA vaccine based on the EABR technology. By adding ESCRT-I and ALIX binding domains EABR to the cytoplasmic tail of the antigen membrane protein, cellular components such as ESCR-related proteins TSG101 and ALIX can be recruited to bud and self-assemble the antigen protein into eVLP. An endocytosis inhibitory sequence (EPM) is added to the C-terminus of the EABR fusion protein to extend the residence time of the EABR fusion protein on the cell membrane, enhance its interaction with host proteins TSG101 and ALIX, and at the same time enhance the immune response of stimulated cells. This form of vaccine can enhance the immunogenicity and protective effect of the vaccine.

[0005] Therefore, there is a need in the art for a vaccine that can provide maximum protection against various influenza strains. Summary of the Invention

[0006] In view of this, in a first aspect, the present invention provides an influenza vaccine composition, comprising:

[0007] Influenza H1N1 antigen construct, influenza H3N2 antigen construct, influenza H5N6 antigen construct, and influenza Victoria antigen construct.

[0008] Further, the C-terminus of the antigen is connected to the C-terminal residue of the trimeric protein fiber of bacteriophage T4, the transmembrane region (TM) of SARS-Cov2, the truncated cytoplasmic tail region (ΔCT) of the S protein, an endocytosis prevention motif, and a sequence that can induce the self-assembly and budding of envelope-like virus-like particles to form the construct of the antigen.

[0009] Further, the C-terminus of the antigen is connected to the amino acid sequence shown in SEQ ID NO.1 to form the construct of the antigen.

[0010] Through the design of the above composition, it has been creatively found that the influenza vaccine composition of the present application, in addition to having the ability to produce high-titer antibodies against H1N1, H3N2, Victoria, and H5N6 it inherently possesses, also has the ability to produce high-titer antibodies against H7N9, H9N2, and Yamagata. In particular, the influenza vaccine composition of the present application unexpectedly can produce antibodies against H7N9, while the vaccines prepared by ordinary techniques do not have the ability to produce H7N9 antibodies.

[0011] Further, the antigen is the extracellular domain of the HA protein of the corresponding strain. For example, the influenza H1N1 antigen is the extracellular domain of the HA protein of the influenza H1N1 subtype.

[0012] Further, the amino acid sequence of the influenza H1N1 antigen is as shown in SEQ ID NO.2; the amino acid sequence of the influenza H3N2 antigen is as shown in SEQ ID NO.3; the amino acid sequence of the influenza Victoria antigen is as shown in SEQ ID NO.4; or the amino acid sequence of the influenza H5N6 antigen is as shown in SEQ ID NO.5.

[0013] Further, the nucleotide sequence of the influenza H1N1 antigen is as shown in SEQ ID NO.6; the nucleotide sequence of the influenza H3N2 antigen is as shown in SEQ ID NO.7; the nucleotide sequence of the influenza Victoria antigen is as shown in SEQ ID NO.8; or the nucleotide sequence of the influenza H5N6 antigen is as shown in SEQ ID NO.9.

[0014] In some specific embodiments, the influenza H1N1 antigen construct is composed of the amino acid sequence shown in SEQ ID NO.2 linked to the C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0015] In some specific embodiments, the influenza H3N2 antigen construct is composed of the amino acid sequence shown in SEQ ID NO.3 linked to the C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0016] In some specific embodiments, the influenza H5N6 antigen construct is composed of the amino acid sequence shown in SEQ ID NO.4 linked to the C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0017] In some specific embodiments, the influenza Victoria antigen construct is composed of the amino acid sequence shown in SEQ ID NO.5 linked to the C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0018] Vaccine compositions of the present invention can be used to prevent influenza in children and adults. Influenza vaccines are currently recommended for pediatric and adult immunizations, starting at 6 months of age. Therefore, patients can be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferably, the patients receiving the vaccine are elderly (e.g., ≥50 years old, ≥60 years old, preferably ≥65 years old), young people (e.g., ≤5 years old), hospitalized patients, health care workers, military and military personnel, pregnant women, chronic diseases, immunodeficiency patients, patients who take antiviral compounds in the first 7 days of receiving the vaccine, and people going abroad. However, these vaccines are not only suitable for these groups, but also can be applied to a wider range of people. For prevalent strains, it is preferably given to all age groups.

[0019] The vaccine composition of the present invention is administered to a patient simultaneously with an antiviral drug, particularly an antiviral drug effective against influenza (e.g., oseltamivir and / or zanamivir). The antiviral drug includes a neuraminidase inhibitor, such as (3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid or 5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glyceryl-D-galactonon-2-enoic acid, including their esters (e.g., ethyl esters) and salts (e.g., phosphates). A preferred antiviral compound is (3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester phosphate (1:1), also known as oseltamivir phosphate (Tamiflu).

[0020] In a second aspect, the present invention provides use of the above vaccine composition in the preparation of a medicament for treating and / or preventing influenza virus.

[0021] Furthermore, the drug is an mRNA vaccine.

[0022] Furthermore, the vaccine is constructed by EABR technology.

[0023] Furthermore, the vaccine composition is administered twice.

[0024] Furthermore, the administration interval is 7 to 28 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the antigenic construct of the vaccine composition of the present invention;

[0026] Figure 2 This is a diagram of the morphology of eVLPs formed by the vaccine composition of the present invention;

[0027] Figure 3 Protein expression verification of the vaccine composition of the present invention;

[0028] Figure 4 a - g are the immunogenicity results of the vaccine composition of the present invention in mice;

[0029] Figure 5 a - e are the immunogenicity results of the vaccine composition of the present invention in non - human primates. Detailed implementation manners

[0030] The present invention will be specifically described below in combination with specific implementation schemes and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific implementation schemes and examples are used to illustrate the present invention, rather than limiting the present invention.

[0031] The sequences used in the present invention are all exemplary. As is known to those skilled in the art, there are many sequences that can meet the functions of the various components of the present invention. For example, different nucleotide sequences encoding the same amino acid sequence, such as different amino acid sequences with an identity greater than 70% and having biological functions, etc. These are all included in the scope of protection of the present invention.

[0032] Example 1. Sequences used in the present invention The sequences used in the present invention are shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] Example 2. Preparation of the influenza vaccine composition of the present invention

[0040] Exemplary influenza vaccine composition of the present invention is as Figure 1As shown. Specifically, the CDS regions encoding the HA of influenza H1N1, H3N2, H5N6, and B / Victoria subtypes were downloaded from the Global Initiative on Sharing All Influenza Data (GISAID) website, and the part encoding the extracellular domain of HA was retained. The C-terminal 30 amino acid residues (T4 Foldon) of the trimeric protein fiber of bacteriophage T4 were connected to the C-terminus of the extracellular domain of HA to stabilize the trimeric conformation of the HA protein. T4 Foldon was then connected to the transmembrane region (TM) of SARS-Cov2 and the truncated cytoplasmic tail region (ΔCT) of the S protein, and the endocytosis prevention motif (EPM) was tightly connected after ΔCT. After the C-terminus of the EPM motif, a sequence EABR that can induce the self-assembly of envelope-like virus-like particles (eVLP) and budding on the cell surface was connected, thus successfully designing HA-EABR-eVLP of each influenza subtype. The HA-EABR-eVLP of each influenza subtype was sent to GenScript Biotech Corporation for synthesis and separately ligated to the eukaryotic expression vector pcDNA3.4, and the eukaryotic expression plasmid pcDNA3.4-HA-EABR-eVLP was successfully obtained ( Figure 1 ).

[0041] One day before transfection, the suspension cells HEK293T were seeded at 1.2×10 6 per well in a 6-well cell culture plate and cultured overnight in a 37°C CO2 cell culture incubator. The next day, the plasmid pcDNA3.4-HA-EABR-eVLP expressing the HA of each influenza subtype was transfected into the cells at 2 μg each using the jetPRIME transfection reagent at a ratio of 2:1 (transfection reagent: plasmid). 48 h after transfection, the supernatant was collected, and the expression of HA-EABR of each subtype was detected by Western blot. The results showed that HA-EABR of each subtype was successfully expressed. To verify whether HA-EABR could self-assemble into HA-EABR-eVLP after expression, we randomly selected the HA-EABR-eVLP expression plasmid of one subtype (H3N2) and transfected it in large amounts into Expi293 cells. After 3 days of suspension culture, the cells and cell debris were removed by centrifugation, and the supernatant of the medium was filtered through a 0.45 μM sterile filter, and the eVLP in the supernatant of the medium was enriched by centrifugation using a Backman ultra-high-speed centrifuge. The expression of the target protein in the product obtained by ultra-high-speed centrifugation was detected by Westen Blot. The results showed that HA-EABR-eVLP was present in the centrifuged precipitate. The enriched eVLP was negatively stained and observed under a transmission electron microscope. Nanoparticle structures of about 100 nm were observed by negative staining electron microscopy, thus determining that the constructed plasmid pcDNA3.4-HA-EABR-eVLP could successfully express the target protein and self-assemble into eVLP ( Figure 2 ).

[0042] The open reading frames of HA-mRNA-eVLP genes of various subtypes involved in influenza in this research institute were cloned into the pIVT vector. 5'UTR and 3'UTR sequences were added to both ends of the open reading frame, and a PolyA tail was added to the end. The target fragment was cloned into the vector pIVT through the 5'HindIII and 3'SalI restriction enzyme digestion sites, and the HA-mRNA-eVLP-pIVT plasmid of HA-EABR-eVLP of various subtypes of influenza virus was successfully expressed. After linearizing the plasmid with the restriction enzyme XhoI, mRNA was generated through an in vitro transcription reaction mediated by T7 RNA polymerase. N1-methylpseudouridine was used to replace the traditional uracil to stabilize the mRNA and reduce its immunogenicity. The RNA purification was carried out by the lithium chloride precipitation method to obtain the mRNA product. After mixing the mRNA in equal mass according to the various subtypes contained in the traditional four-antigen component and the new antigen component vaccines, the mixed mRNA product was encapsulated into lipid nanoparticles (LNP) by the microfluidic mixing method, and the mRNA-LNP was concentrated using a 100KDa ultrafiltration tube. The RNA dye was used to measure the concentration and encapsulation rate of the mRNA-LNP, thereby obtaining the HA-mRNA-eVLP vaccine encapsulated by LNP( Figure 3 ).

[0043] Example 3: Immunogenicity study of the composition of the present invention in mice

[0044] In the experiment, 6- to 10-week-old female BALB / c mice were grouped according to the following table (Table 1). The mRNA-eVLP group was the HA-mRNA-eVLP vaccine expressing the new four-antigen components (HA of H1N1, H3N2, BV (Victoria), and H5N6), and the QIV group was the traditional four-antigen component subunit vaccine (ordinary inactivated vaccine). At the same time, PBS was set as the blank control, as shown in Table 2.

[0045] Table 2

[0046]

[0047] After one week of quarantine and adaptation to the experimental environment, the mice were immunized with the vaccine for the first time, with 10 mice in each group. For the mRNA vaccine, the mRNA-LNP was diluted to 80 μg / mL, and 50 μL was injected into the leg muscles of each hind limb of each mouse, with an immunization dose of 8 μg; for the subunit vaccine, the volume was adjusted with PBS, and one human dose was used to immunize 10 mice. 50 μL was injected into the leg muscles of each hind limb of each mouse, with an immunization dose of 3 μg; for the PBS group, 50 μL of PBS with the same volume as the experimental group was injected into the leg muscles of each hind limb of each mouse. 14 days after immunization, blood was collected from the orbital venous plexus of the mice, and the serum was obtained by centrifugation. It was used for the detection of antibody titer and cross-antibody reaction, and the total IgG level in the serum binding to H1N1, H3N2, BV, BY, and H5N6 HA proteins was detected. 28 days after the first immunization, the mice were boost-immunized in the same manner. 7 days after the boost immunization, blood was collected from the orbital venous plexus of the mice, and the serum was obtained by centrifugation. It was used for the detection of antibody titer and cross-antibody reaction, and the total IgG level in the serum binding to H1N1, H3N2, BV, BY, and H5N6 HA proteins was detected.

[0048] After the boost immunization, each component of the HA-mRNA-eVLP vaccine could induce the production of antibodies against the corresponding virus subtypes, and the induced antibody levels were all higher than those of the commercial split vaccine; the mRNA-eVLP vaccine could induce the production of antibodies targeting the HA of the H5 subtype, and the levels were all higher than those of the commercial split vaccine; although the mRNA-eVLP vaccine did not include the BY HA antigen, it could also induce the production of antibodies targeting the BY HA protein, and the induced antibody levels were higher than those of the commercial split vaccine, indicating that the HA-mRNA-eVLP vaccine has the potential to induce the production of cross-antibodies. We further detected the broad spectrum of the cross-antibodies induced by the HA-mRNA-eVLP vaccine, and detected the total IgG level in the serum binding to H7N9 and H9N2 HA proteins 7 days after the second immunization. It was found that the mRNA-eVLP vaccine could induce the production of antibodies targeting the H7N9 and H9N2 HA antigens, and the antibody levels were all higher than those of the commercial inactivated vaccine. In particular, it was unexpectedly found that the vaccine composition prepared by the present invention could provide protection against H7N9, but the commercial inactivated vaccine could not ( Figure 4 a~g).

[0049] Example 4. Immunogenicity study of the composition of the present invention in non-human primates

[0050] Cynomolgus monkeys aged 12 - 15 years were used in the experiment to study the immunogenicity of the new four - antigen component HA - mRNA - eVLP vaccine. The experimental group included a total of 5 cynomolgus monkeys, with an immunization dose of 30 μg per monkey and a total immunization volume of 1 ml, 500 μl in each triceps brachii of the left and right arms; 14 days after immunization, blood was collected from the antecubital vein for antibody titer and cross - antibody reaction detection, and the total IgG level binding to H1N1, H3N2, BV, BY, and H5N6 HA proteins in the serum was detected. 28 days after the first immunization, the above 5 cynomolgus monkeys were boost - immunized with the vaccine using the same method as the first immunization. 7 days after the boost immunization, blood was collected from the antecubital vein for antibody titer and cross - antibody reaction detection, and the total IgG level binding to H1N1, H3N2, BV, BY, and H5N6 HA proteins in the serum was detected. After one boost immunization, the new antigen - component mRNA - LNP vaccine could induce the production of antibodies against the HA proteins of the corresponding virus subtypes ( Figure 5 a - e).

Claims

1. An influenza vaccine composition, comprising: An influenza H1N1 antigen construct, an influenza H3N2 antigen construct, an influenza H5N6 antigen construct, and an influenza Victoria antigen construct.

2. The influenza vaccine composition according to claim 1, wherein The antigen construct is composed of an antigen with the C-terminal residues of the trimeric protein fiber of bacteriophage T4, the transmembrane region of SARS-Cov2, the truncated cytoplasmic tail region of the S protein, an endocytosis prevention motif, and a sequence capable of inducing the self-assembly and budding of envelope-like virus-like particles connected to its C-terminus.

3. The influenza vaccine composition according to claim 2, characterized in that, The antigen construct is composed of an antigen with the amino acid sequence shown in SEQ ID NO.1 connected to its C-terminus.

4. The influenza vaccine composition according to claim 1, wherein The amino acid sequence of the influenza H1N1 antigen is shown in SEQ ID NO.2; the amino acid sequence of the influenza H3N2 antigen is shown in SEQ ID NO.3; the amino acid sequence of the influenza Victoria antigen is shown in SEQ ID NO.4; or the amino acid sequence of the influenza H5N6 antigen is shown in SEQ ID NO.

5.

5. The influenza vaccine composition according to claim 1, characterized in that, The nucleotide sequence of the influenza H1N1 antigen is shown in SEQ ID NO.6; the nucleotide sequence of the influenza H3N2 antigen is shown in SEQ ID NO.7; the nucleotide sequence of the influenza Victoria antigen is shown in SEQ ID NO.8; or the nucleotide sequence of the influenza H5N6 antigen is shown in SEQ ID NO.

9.

6. The influenza vaccine composition according to claim 3, characterized in that, The influenza H1N1 antigen construct is composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.2 connected to the amino acid sequence shown in SEQ ID NO.1; the influenza H3N2 antigen construct is composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.3 connected to the amino acid sequence shown in SEQ ID NO.1; the influenza H5N6 antigen construct is composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.4 connected to the amino acid sequence shown in SEQ ID NO.1; or the influenza Victoria antigen construct is composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.5 connected to the amino acid sequence shown in SEQ ID NO.

1.

7. Use of the influenza vaccine composition according to any one of claims 1 to 6 in the preparation of a drug for treating and / or preventing influenza virus.

8. The use according to claim 7, wherein The drug is an mRNA vaccine.

9. The use according to claim 8, characterized in that, The vaccine is constructed by the EABR technology.

10. Use according to any one of claims 7 to 9, characterized in that, The vaccine composition is administered twice.

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