Mixed vaccine for preventing influenza and coronavirus infections

Abstract

The invention discloses a mixed vaccine for preventing influenza and novel coronavirus infections. As a mixed vaccine for vaccination against influenza and novel coronavirus, a whole virus inactivated vaccine obtained by inactivating virus particles of influenza virus and novel coronavirus is used, whereby a neutralizing antibody against each virus is induced without affecting the effect of each vaccine. And a defense effect is obtained for attack of each virus. In addition, the mixed vaccine based on the combination shows a good neutralizing antibody induction and virus attack defense effect even if an adjuvant is not added.

Description

Technical Field

[0001] This invention relates to a combined vaccine for the prevention of influenza and novel coronavirus infection. Background Technology

[0002] In viral classification, influenza viruses belong to the Orthomyxoviridae family, which are classified as "enveloped, negative-sense, single-stranded RNA viruses." Types A, B, C, and D are known. The differences between types are based on the antigenicity of the M1 protein and nucleoprotein (NP), which are structural proteins within the viral particle. In addition to these differences, there are also variations in pathology, morphology, and genetics, particularly between types A and B and types C and D. Types A and B circulate repeatedly each year and are the primary cause of influenza in humans in most cases.

[0003] Influenza virus was isolated from pigs in 1931, and human influenza virus was isolated in 1933. Around the same time, it was discovered that influenza virus could replicate in chicken embryos, leading to the development of inactivated whole virus particle vaccines. These vaccines were produced by inoculating chicken embryos with the virus, culturing them, collecting and purifying the virus, and then inactivating it with formaldehyde. Inactivated whole virus particle vaccines are made by chemically inactivating purified virus particles with formaldehyde; the viral genes or proteins are chemically modified to lose only infectivity, thus the virus retains its particle form and becomes the vaccine antigen. However, early inactivated whole virus vaccines had strong side effects such as fever, leading to consideration of methods to remove lipid membrane components, which were considered the cause of these side effects. The following vaccine, which became the prototype of the current split vaccine, has high safety. It uses hemagglutinin (HA), the influenza antigen, as its main component. After zonal ultracentrifugation purification, it is separated with surfactants, and lipid membrane components that would cause fever are removed using ether. Split vaccines are vaccines containing purified HA (and NA) as the main antigen, and have been manufactured since 1964 and are still in use today (Non-Patent Literature 1).

[0004] Furthermore, in recent years, compared to split vaccines composed of a portion of viral components, whole-virus inactivated vaccines containing all viral particles have been considered to elicit a good immune response, especially in naive individuals without immunological memory, and are therefore considered to have high immunogenicity. New research and development are underway (Patent Document 1, Non-Patent Document 2). Patent Document 1 describes a precipitated inactivated influenza vaccine made by adding aluminum hydroxide gel to a whole-virus influenza vaccine.

[0005] On the other hand, the World Health Organization (WHO) officially named the novel coronavirus infection that occurred in 2019 COVID-19, caused by a virus known as SARS-CoV-2. SARS-CoV-2 is a single-stranded positive-sense RNA virus with a diameter of 50-200 nm, belonging to the Orthocoronavirus subfamily. Most people infected with COVID-19 experience mild symptoms, such as being asymptomatic or having flu-like symptoms, and recover naturally. However, in severe cases, it can lead to acute respiratory distress syndrome, sepsis, and / or multiple organ failure.

[0006] Vaccination is one method of preventing COVID-19. Regarding vaccines against SARS-CoV-2, various vaccines, including mRNA vaccines, viral vector vaccines, and recombinant protein vaccines, have been developed and approved, some of which are already on the market (Non-Patent Literature 3). In Japan, many people aged 5 and older have been vaccinated with mRNA and viral vector vaccines, with the aim of reducing severe cases and deaths through infection prevention and control of the disease. On the other hand, side effects following vaccination have been reported, including injection site pain, headache, fatigue, muscle pain, allergic reactions, thrombosis, pericarditis, and myocarditis. Additionally, undiagnosable adverse effects are also known.

[0007] Inactivated vaccines are known as vaccines other than those mentioned above. Inactivated vaccines are used, for example, as seasonal influenza vaccines, Japanese encephalitis vaccines, and quadrivalent vaccines. Inactivated vaccines are manufactured using pathogens, viruses, or components of those pathogens or parts of viruses that have lost their infectivity; they are known to generally have few side effects and high safety profiles. Therefore, it is hoped that inactivated SARS-CoV-2 vaccines can also be administered to children under 5 years of age. All inactivated vaccines against SARS-CoV-2 are whole-virus inactivated vaccines. Overseas, VALNEVA (COVID-19 vaccine (inactivated, adjuvant) Valneva) and SINOVAC (Sinovac COVID-19 vaccine, CoronaVac / PiCoVacc) have already received manufacturing and sales approval (Non-Patent Literature 4, 5). The VALNEVA vaccine adjuvant contains aluminum hydroxide and CpG1018 (cytosine-phosphate-guanine), while the SINOVAC vaccine adjuvant contains aluminum hydroxide. In Japan, KM Biologics is developing an inactivated (whole virus) COVID-19 vaccine, "KD-414," which is currently undergoing Phase III trials. "KD-414" incorporates aluminum hydroxide as an adjuvant (Non-Patent Literature 6).

[0008] Before the World Health Organization (WHO) declared a pandemic in 2019, more than half of respiratory infections were caused by seasonal influenza viruses, including influenza A and B. Following the COVID-19 pandemic, the morbidity and mortality rates caused by seasonal influenza viruses declined sharply due to restrictions on international travel and social distancing. However, with the lifting of these strict restrictions, the reopening of international travel, and the resumption of economic activities, the number of seasonal influenza cases is projected to increase. Given the anticipated number of COVID-19 cases, both diseases pose significant public health threats, particularly the risk of severe infection, which could become a burden on clinical care. SARS-CoV-2 and seasonal influenza share several similarities, including transmission routes, susceptibility, clinical symptoms, and the ongoing threat of new variants / subtypes, thus requiring preventative strategies such as annual vaccination against both viruses. Therefore, the WHO currently recommends the simultaneous administration of approved COVID-19 vaccines and seasonal influenza vaccines (Non-Patent Literature 7). Immunogenicity and safety have been reported when approved COVID-19 vaccines and seasonal influenza vaccines are administered simultaneously (Non-Patent Literature 8).

[0009] Against this backdrop, there is a growing expectation for vaccines that can provide protection against two viruses with a single dose. Recently, a mouse model was reported to have successfully protected against influenza and COVID-19 using an mRNA vaccine encoding the SARS-CoV-2 S protein and influenza hemagglutinin (HA) (Non-Patent Literature 9). However, as mentioned earlier, due to the potential for undiagnosable adverse effects, particularly with mRNA vaccines, there are arguments that annual vaccination should be avoided.

[0010] Vaccines containing antigens targeting multiple infectious diseases (viruses), i.e., mixed vaccines, have the advantage of providing protection against multiple viruses with a single dose. However, on the other hand, there are challenges that need to be overcome, making them impossible to achieve simply through a combination of ingredients. The immunological and biochemical effects of vaccines (e.g., interactions such as antigen interference, competition at adjuvant adsorption sites, epitope-specific inhibition, etc.) are difficult to predict; for example, sometimes the immunogenicity is reduced due to mixing. Furthermore, with monoantigen vaccines developed for single-dose administration, which include components optimized for their affinity to the antigen, especially adjuvants, there are concerns about undesirable interactions and unpredictable adjuvant effects from these components.

[0011] Existing technical documents Patent documents Patent Document 1: WO2008 / 041710 Non-patent literature Non-patent literature 1: Pharmaceutical Journal 131(12), 1723-1731 (2011): https: / / www.jstage.jst.go.jp / article / yakushi / 131 / 12 / 131_12_1723 / _pdf / -char / ja Non-patent literature 2: Vaccines 2022, 10(5), 804, https: / / www.mdpi.com / 2076-393X / 10 / 5 / 804 Non-patent literature 3: Clinical Immunology 222 (2021), 108634main.pdf (nih.gov) Non-patent document 4: European Medicines Agency website COVID-19 Vaccine (inactivated, adjuvanted) Valneva | EuropeanMedicines Agency (europa.eu) Non-patent document 5: World Health Organization website The Sinovac-CoronaVac COVID-19 vaccine: What you need to know (who.int) Non-patent document 6: Hum Vaccin Immunother. 2023 Apr 13;2193074 Immunogenicity and safety of single booster dose of KD-414 inactivated COVID-19 vaccine inadults: An open-label, single-center, non-randomized, controlled study inJapan - PubMed (nih.gov) Non-patent document 7: SAGE Working Group on Influenza. Coadministration of seasonal inactivated influenza and COVID-19 vaccines: Interim guidance. WorldHealth Organization Coadministration of seasonal inactivated influenza and COVID-19 vaccines (who.int) Non-patent document 8: Lancet Respir Med. 2022;10(2):167-79. Safety, immunogenicity, and efficacy of a COVID-19 vaccine (NVX-CoV2373) co-administered with seasonal influenza vaccines: an exploratory substudy of arandomised, observer-blinded, placebo-controlled, phase 3 trial (nih.gov) Non-patent literature 9: NPJ Vaccines. 2022 Jul 26;7(1):84. Rational development of a combined mRNA vaccine against COVID-19 and influenza - PMC (nih.gov) Summary of the Invention

[0012] The problem that the invention aims to solve The inventors of this application have obtained the following insight: As a combined vaccine for the prevention of influenza and COVID-19, a whole-virus inactivated vaccine is prepared by inactivating both viruses, thereby inducing neutralizing antibodies against each virus without affecting the efficacy of the individual vaccines, thus achieving a defensive effect against viral attacks. Furthermore, it has been confirmed that the combined vaccine based on this combination still exhibits good neutralizing antibody induction and defensive effects against viral attacks, even without the addition of adjuvants. This invention is based on the above insights.

[0013] Therefore, the object of the present invention is to provide a combined vaccine for the prevention of influenza and coronavirus infections.

[0014] Methods for solving problems Furthermore, the hybrid vaccine of the present invention is characterized by comprising an inactivated whole influenza virus vaccine and an inactivated whole SARS-CoV-2 virus vaccine.

[0015] The effects of the invention According to the present invention, the combined vaccine can prevent influenza infection and SARS-CoV-2 infection with a single dose, reducing the number of vaccinations required, thereby reducing the pain, time, and effort of the recipient, as well as the burden on doctors. Attached Figure Description

[0016] [ Figure 1 The following figure illustrates the results: The dosage of the SARS-CoV-2 whole virus inactivated vaccine was varied, and the adjuvant was changed to 0, 100 μg, or 200 μg and administered intraperitoneally to BALB / c mice (8 mice / group) to test whether the neutralizing antibody titer increased.

[0017] [ Figure 2 To illustrate the use of the mixed vaccine of the present invention, namely a SARS-CoV-2 whole-virus inactivated vaccine and an influenza whole-virus inactivated vaccine, in 7-week-old female BALB / c mice ( Figure 2 A), a graph showing whether HI antibody titers or neutralizing antibody titers were detected in serum 19 days later. Controls were provided by SARS-CoV-2 whole-virus inactivated vaccine, influenza whole-virus inactivated vaccine, and PBS (presumably a PBS-based vaccine). Figure 2 (B to D).

[0018] [ Figure 3 The following figure shows the results: BALB / c mice were administered influenza virus intranasally with the mixed vaccine of the present invention, and with either a SARS-CoV-2 whole-virus inactivated vaccine or an influenza whole-virus inactivated vaccine, and with PBS administered separately, and changes in body weight were monitored. Figure 3 A) and viral titers in the lungs on days 3 and 5 post-infection (dpi). Figure 3 B and C) were measured.

[0019] [ Figure 4 The following figure shows the results: BALB / c mice were administered the original SARS-CoV-2 strain intranasally to mice vaccinated with the mixed vaccine of the present invention, and mice vaccinated with SARS-CoV-2 whole-virus inactivated vaccine, influenza whole-virus inactivated vaccine, and PBS, respectively, and changes in body weight were monitored. Figure 4 A) and viral titers in the lungs on days 3 and 5 post-infection (dpi). Figure 4 B and C) were measured.

[0020] [ Figure 5The graph shows the serum HI antibody titer or neutralizing antibody titer 22 days later as a mixed vaccine of the present invention, in which a SARS-CoV-2 whole virus inactivated vaccine and a quadrivalent influenza whole virus inactivated vaccine strain (A / Singapore, A / Hong Kong, B / Phuket, B / Texas) were mixedly vaccinated.

[0021] [ Figure 6 The figure illustrates the following results: Serum samples were obtained on day 36 from mice that had been given a mixed vaccine of SARS-CoV-2 whole-virus inactivated vaccine and quadrivalent influenza whole-virus inactivated vaccine as the mixed vaccine of the present invention, to evaluate neutralizing antibodies against variants of the vaccine strain (i.e., Alpha strain, Delta strain, and Omeprone strain).

[0022] [ Figure 7 The following figure shows the results: BALB / c mice were administered the original SARS-CoV-2 strain intranasally to a mixed vaccine of SARS-CoV-2 whole-virus inactivated vaccine and quadrivalent influenza whole-virus inactivated vaccine (as described in this invention), and mice were administered the original SARS-CoV-2 strain intranasally to BALB / c mice that were inoculated with SARS-CoV-2 whole-virus inactivated vaccine, quadrivalent influenza whole-virus inactivated vaccine, and PBS, respectively, and the changes in body weight were monitored. Figure 7 A) and viral titers in the lungs on days 3 and 5 post-infection (dpi). Figure 7 B and C) were measured. Detailed Implementation

[0023] definition Unless otherwise specified, the terms "influenza virus" and "seasonal influenza virus" are used in this instruction manual with the same meaning.

[0024] In addition, unless otherwise specified, the terms "SARS-CoV-2" and "novel coronavirus" are used in this invention with the same meaning.

[0025] The mixed vaccine of the present invention The hybrid vaccine of the present invention is based on an inactivated whole-virus influenza vaccine and an inactivated whole-virus SARS-CoV-2 vaccine, and is further characterized by being adjuvant-free.

[0026] As mentioned earlier, it was thought that mixed vaccines could not be achieved simply by combining different vaccines. However, by making both influenza and SARS-CoV-2 vaccines into whole-virus inactivated vaccines, a mixed vaccine was obtained that induces neutralizing antibodies against each virus and shows a defensive effect against each virus attack without affecting the efficacy of the individual vaccines. This fact should be considered an unexpected one by those skilled in the art.

[0027] Furthermore, the combined vaccine of the present invention is adjuvant-free. "Adjuvant-free" in the context of the combined vaccine of the present invention means that it substantially does not contain any substances that, when administered together with the antigen, enhance humoral and cellular immune function. Here, "substantially" means that an unavoidable amount is permitted, but not an amount effective in enhancing humoral and cellular immune function.

[0028] As mentioned earlier, all reported, developed, or manufactured inactivated whole-virus vaccines against SARS-CoV-2 have added adjuvants such as aluminum hydroxide, which is a result of the determination that adjuvants are necessary for immunogenicity. Thus, it has been generally believed that inactivated whole-virus vaccines against SARS-CoV-2 require adjuvants. However, in the case of the mixed vaccine of this invention, by combining it with an inactivated whole-virus influenza vaccine, a mixed vaccine is obtained that induces neutralizing antibodies against each virus without adding adjuvants, without affecting the efficacy of each vaccine, and shows a defensive effect against each viral attack. This fact should also be considered unexpected by those skilled in the art.

[0029] The combined vaccine according to the present invention can prevent influenza and COVID-19 with a single dose, reducing the number of vaccinations required and minimizing pain, time, and effort for recipients. Furthermore, it can reduce the burden on physicians and other personnel.

[0030] Furthermore, compared to split vaccines that consist of only a portion of viral components, whole-virus inactivated vaccines containing all viral particles are considered to elicit a good immune response, especially in non-immune individuals without immunological memory. From the perspective of high immunogenicity, the mixed vaccine utilizing whole-virus inactivated vaccines of the present invention can also be considered advantageous.

[0031] Inactivated whole-virus influenza vaccine The "inactivated whole influenza virus vaccine" included in the mixed vaccine of the present invention refers to virus particles that retain the morphology of influenza virus, and the virus particles have been inactivated.

[0032] In this manual, "influenza virus" refers to the currently known influenza A virus, influenza B virus, influenza C virus, influenza D virus, or all their subtypes, and also includes any viruses that will be classified as influenza viruses in the future.

[0033] In this invention, the "inactivated whole influenza virus vaccine" can be a vaccine containing inactivated whole virus particles of a single influenza virus, or a vaccine containing inactivated whole particles of multiple influenza viruses.

[0034] According to a preferred embodiment of the present invention, the inactivated whole-virus influenza vaccine is prepared by inactivating at least one of the whole virus particles of influenza A virus or influenza B virus, and more preferably, it may contain both. Alternatively, it may be prepared by inactivating whole virus particles of two or more subtypes of influenza A virus or influenza B virus. For example, a quadrivalent inactivated whole-virus influenza vaccine containing two strains of influenza A virus and two strains of influenza B virus can be used as the mixed vaccine of the present invention.

[0035] The "inactivated whole-virus influenza vaccine" contained in the mixed vaccine of the present invention can be manufactured using known methods, for example, it can be obtained according to the method described in Vaccine, 2022, 10(5), 804 (Non-Patent Document 2).

[0036] Specifically, the influenza virus is cultured. In the presence of multiple influenza viruses, it is preferable to culture them separately. Culture can be performed using known methods such as the "chicken embryo method" or "cell culture method," but the "chicken embryo method" is more common. Next, the allantoic fluid containing the multiplied virus is purified and concentrated using methods such as sucrose density gradient centrifugation to obtain virus particles.

[0037] The obtained virus particles are then inactivated. Inactivation can be carried out by contacting an inactivating agent with a viral fluid containing the virus particles. Examples of inactivating agents include formaldehyde, paraformaldehyde, glutaraldehyde, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and β-propiolactone. Formaldehyde is preferred as the inactivating agent.

[0038] Then, as needed, it is prepared by diluting with, for example, a phosphate-buffered sodium chloride solution to contain a specified amount of inactivated influenza whole virus particles.

[0039] SARS-CoV-2 whole virus inactivated vaccine The "SARS-CoV-2 whole virus inactivated vaccine" contained in the mixed vaccine of the present invention refers to virus particles that retain the morphology of SARS-CoV-2, and the virus particles have been inactivated.

[0040] In this invention, "SARS-CoV-2" refers to all currently known SARS-CoV-2 strains, for example, including the original strain as well as its variants such as the Alpha, Beta, Gamma, Delta, and Omecron strains, and also includes any future virus strains classified as SARS-CoV-2.

[0041] In this invention, the “SARS-CoV-2 whole virus inactivated vaccine” can be a vaccine that contains not only a vaccine made by inactivating a single SARS-CoV-2 whole virus particle, but also a vaccine that contains multiple inactivated SARS-CoV-2 whole particles.

[0042] The "SARS-CoV-2 whole virus inactivated vaccine" contained in the mixed vaccine of the present invention can be manufactured using known methods, for example, it can be obtained according to the method described in Hum Vaccin Immunother. 2023 Apr 13;2193074 (Non-Patent Document 6).

[0043] Specifically, SARS-CoV-2 can be produced using a "cell culture method," which involves inoculating a viral strain into cultured cells, culturing them, clarifying, concentrating, purifying, and inactivating the viral suspension to obtain a viral fluid containing viral particles. Cells used in the culture can be those with good viral proliferation and that produce highly immunogenic antigens; for example, non-human animals (e.g., chickens) or strained cells derived from animals (strained animal cells). Specific examples of cells include Calu-3 and A549 cells derived from human lung adenocarcinoma cells, and Vero cells, VeroE6 cells, and VeroE6 / TMPRSS2 cells derived from African green monkey kidneys. Vero cells are preferred. Furthermore, the purification of viral particles can be performed using methods such as ultracentrifugation (e.g., sucrose density gradient centrifugation), ultrafiltration or filtration, and liquid chromatography.

[0044] Next, the obtained virus particles are inactivated. Inactivation can be carried out in essentially the same way as the inactivation of the aforementioned influenza virus, and can be implemented by contacting the inactivating agent with the viral fluid containing the virus particles. Examples of inactivating agents include formaldehyde, paraformaldehyde, glutaraldehyde, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, β-propiolactone, etc. Formaldehyde is preferred as an inactivating agent.

[0045] Mixed vaccine formulations and their uses The mixed vaccine of the present invention is a combination of an inactivated whole-virus influenza vaccine and an inactivated whole-virus SARS-CoV-2 vaccine. In addition to comprising a vaccine formulation consisting of the two vaccines, the combination also includes a method of providing the two vaccines as vaccine formulations filled into different vials and mixing them before use to form a mixed vaccine. According to a preferred embodiment of the present invention, the two vaccines are mixed and provided as a single vaccine formulation.

[0046] The mixed vaccine of the present invention does not require an adjuvant, but may contain components other than an adjuvant. For example, it may contain carriers permitted in vaccine formulations, specifically including saline, buffered saline, sugars such as glucose, amino acids, water, glycerol, isotonic buffer solutions, surfactants, and combinations thereof. Additionally, it may contain preservatives (e.g., thimerosal), isotonic agents, pH adjusters, and inactivating agents (e.g., formalin).

[0047] In the manufacture of the mixed vaccine of the present invention, the purified whole virus inactivated vaccine can be replaced with an appropriate buffer solution by ultrafiltration or other methods as needed, and then sterile filtered with a membrane filter before being mixed with other components. Alternatively, it can be mixed with other components and then sterile filtered with a membrane filter.

[0048] The mixed vaccine of the present invention can be administered systemically or locally. For local administration, examples include intramuscular, subcutaneous, intradermal, intranasal, and pharyngeal administration. Administration methods include injection, spraying, and application.

[0049] The concentration of the inactivated whole-virus influenza vaccine in the mixed vaccine of the present invention is preferably about 0.3 to 3.0 μg / mL, more preferably about 0.7 to 2.0 μg / mL. Furthermore, the dosage of the inactivated whole-virus influenza vaccine based on the mixed vaccine of the present invention can be appropriately determined considering the age, sex, weight, etc. of the recipient; in terms of antigen, it is generally about 3 to 15 μg, preferably about 7 to 11 μg. Additionally, when the mixed vaccine of the present invention comprises inactivated whole-virus influenza vaccines against multiple virus strains, the dosage for each virus strain can be set to the above-mentioned range.

[0050] Furthermore, the concentration of the SARS-CoV-2 whole-virus inactivated vaccine in the mixed vaccine of the present invention is generally preferably about 0.3 to 3.0 μg / mL, more preferably about 0.7 to 2.0 μg / mL. Additionally, the dosage of the SARS-CoV-2 whole-virus inactivated vaccine based on the mixed vaccine of the present invention can be appropriately determined considering the age, sex, weight, etc. of the recipient; in terms of antigen, it is generally about 3 to 15 μg, preferably about 7 to 11 μg.

[0051] The mixed vaccine of the present invention can also be administered more than twice, with intervals between preventive vaccinations.

[0052] Example The present invention will now be described in detail by way of examples, but the present invention is not limited to these examples in any way.

[0053] It should be noted that, in the following embodiments, the following abbreviations will be used as appropriate.

[0054] Flu-WPV (Inactivated Whole Virus Influenza Vaccine) Quadrivalent Inactivated Whole Virus Vaccine: qFlu-WPV SARS-CoV-2 whole virus inactivated vaccine: Co-WPV A combined vaccine (bivalent) of SARS-CoV-2 whole-virus inactivated vaccine and influenza whole-virus inactivated vaccine: Co / Flu-WPV A combined vaccine of SARS-CoV-2 whole-virus inactivated vaccine and quadrivalent influenza whole-virus inactivated vaccine: Co / qFlu-WPV Reference Example 1: The Necessity of Aluminum Hydroxide Adjuvant in SARS-CoV-2 Whole Virus Inactivated Vaccines The SARS-CoV-2 whole-virus inactivated vaccine was prepared using a SARS-CoV-2 virus strain, as described in Hum Vaccin Immunother. 2023 Apr 13;2193074 (Non-Patent Document 6). Specifically, Vero cells were cultured in a medium containing microcarriers, and the viral fluid was harvested from the resulting viral suspension. After concentration and inactivation, the inactivated SARS-CoV-2 antigen was obtained (the SARS-CoV-2 whole-virus inactivated vaccine).

[0055] 500 μL of a solution containing the amounts of immunizing agents (antigen and aluminum hydroxide adjuvant) listed in Table 1 below was administered intraperitoneally twice to BALB / c mice (8 mice / group) at 2-week intervals. Blood was collected 2 weeks after the second administration. Serum was centrifuged from the collected blood and frozen. The serum was used for neutralizing antibody titer testing. Based on the obtained neutralizing antibody titers, the geometric mean and 95% confidence interval of the neutralizing antibody titers for each administration group were calculated using the GEOMEAN and CONFIDENCE.T functions in Excel. A graph of the geometric mean of the neutralizing antibody titers for each administration group was created using the medical statistical analysis software "GRAPHPAD Prism 9 (ver. 9.3.1)". A significance test (unpaired Mann-Whitney test) was performed between the same administered antigens. p-value 0.001~0.01 p<0.0001). The results are as follows: Figure 1 As shown, this confirms the tendency for neutralizing antibody titers to increase with increasing antigen dosage. A significance test was performed, and significant differences were confirmed between all administration groups. The addition of adjuvant significantly increased neutralizing antibody titers; therefore, it is determined that adjuvants are necessary in inactivated whole-virus vaccines against COVID-19.

[0056] Example 1: SARS-CoV-2 whole-virus inactivated vaccine (Co-WPV) and influenza whole-virus inactivated vaccine (Flu-WPV) and preparation of qFlu-WPV Co-WPV was prepared as follows: The original SARS-CoV-2 viral strain was inoculated into Vero cells to induce viral proliferation. After incubation at 37°C for 2 days, the virus was centrifuged at 3000 rpm for 30 minutes at 4°C. Next, 10% formaldehyde aqueous solution was added to the supernatant (final concentration 0.1%), and the mixture was incubated at 4°C for 1 week to inactivate the virus. After inactivation, the virus was concentrated by centrifugation at 19,000 rpm, 4°C for 1.5 hours, and then purified by sucrose density ultracentrifugation at 27,000 rpm, 4°C for 1.5 hours. Total protein was determined using a spectrophotometer.

[0057] According to Vaccine, 2022, 10(5), 804 (Non-Patent Document 2), Flu-WPV (9 μg of A / California) was prepared by A / California.

[0058] In addition, a quadrivalent inactivated whole-virus influenza vaccine (qFlu-WPV: 9 μg of each of the four influenza vaccine strains) containing virus strains from 2017-2018 (A / Singapore, A / Hong Kong, B / Phuket, B / Texas) was prepared (Non-Patent Literature 2).

[0059] Co-WPV, Flu-WPV, and qFlu-WPV were stored in their respective vials at 4°C.

[0060] Example 2: A mixed vaccine of SARS-CoV-2 whole-virus inactivated vaccine and influenza whole-virus inactivated vaccine (bivalent vaccine) Preparation of seedlings (Co / Flu-WPV) and HI antibody titer or serum antibody titer Co / Flu-WPV (9 μg Co-WPV, 9 μg Flu-WPV (A / California)) was prepared by mixing the Co-WPV and Flu-WPV described in Example 1. To induce antibodies against each virus in the same manner, the antigen amounts were set to be equal. No adjuvant was added.

[0061] The comparative controls were Co-WPV (SARS-CoV-2 9 μg), Flu-WPV (A / California 9 μg), and PBS, which were inoculated into 7-week-old female BALB / c mice. Figure 2 A). Serum was collected from the tail vein 19 days after vaccination to determine the titer of HI antibodies or neutralizing antibodies. Results are as follows: Figure 2 Figures B through D are shown. Points represent individual values, and the line represents the mean using SEM. Statistical analysis used one-way ANOVA based on Tuke's multiple comparison test. p<0.0001).

[0062] Compared with mice vaccinated with PBS, mice vaccinated with Flu-WPV or Co-WPV induced significantly higher levels of HA inhibition (HI) and neutralizing antibodies against A / California. Figure 2 (B and C). Regarding neutralizing antibody titers, the range was 160–640 in mice vaccinated with Flu-WPV or Co / Flu-WPV, with no statistically significant difference among mice vaccinated with either vaccine. No HI antibodies or neutralizing antibodies against influenza virus were detected in animals vaccinated with either PBS or Co-WPV.

[0063] Regarding the neutralizing antibody titer against SARS-CoV-2, the titer ranged from 80 to 320 in animals vaccinated with Co-WPV alone, and from 80 to 160 in animals vaccinated with Co / Flu-WPV, significantly higher than that in animals vaccinated with PBS. Figure 2 D). Based on these results, the bivalent vaccines of Flu-WPV and Co-WPV did not show significant interference effects in antibody induction against their respective antigens, and induced sufficient antibodies even without the addition of adjuvants.

[0064] Example 3: Influenza virus and SARS-CoV-2 challenge test For BALB / c mice vaccinated with any of the vaccines or PBS in Example 2, 3000 PFU of influenza A / California virus or 10 PFU of PBS were administered intranasally 22 days after vaccination. 5 The original SARS-CoV-2 strain from PFU was used, and body weight changes were monitored daily. Furthermore, viral titers in the lungs were measured on days 3 and 5 post-infection (dpi). Specifically, three days after viral challenge, a subset of mice were humanely euthanized, and lung tissue was collected. The left lobe was placed in formalin for tissue sectioning. The right lobe was divided into two parts for viral titer determination and assay of gene expression of inflammatory cytokines and chemokines. The amount of influenza virus in the lungs was determined using standard methods. The results of the influenza virus challenge experiment are as follows: Figure 3 As shown in A~C, the results of the SARS-CoV-2 challenge experiment are as follows: Figure 4 Figures A through C are shown. Points represent individual values, and the lines represent the average using SEM. Statistical analysis used one-way ANOVA based on Tukey's multiple comparison test. p<0.0001).

[0065] Results of influenza virus challenge experiments showed that mice inoculated with PBS and Co-WPV experienced a sharp 20% decrease in body weight from the initial pre-inoculation value, while mice inoculated with Flu-WPV and Co / Flu-WPV maintained a normal body weight. Figure 3 A). To evaluate the effectiveness of Co / Flu-WPV in inhibiting viral replication, viral titers in the lungs were measured on days 3 and 5 post-infection (dpi). The viral titers in the lung homogenates of the PBS and Co-WPV groups were approximately 10 at 3 dpi, respectively. 5 PFU / ml. At 5 dpi, each is approximately 10. 4 PFU / ml ( Figure 3 B and C). As a control, viral titers in the lungs of mice inoculated with Flu-WPV or Co / Flu-WPV were below the detection limit (B and C). Figure 3 (B and C). These results show that viral replication was effectively suppressed in the lungs of mice inoculated with Flu-WPV or Co / Flu-WPV.

[0066] Results of SARS-CoV-2 challenge experiments showed that animals vaccinated with Flu-WPV or PBS experienced a 15% reduction in body weight from their initial pre-vaccination measurements, while animals immunized with Co-WPV or Co / Flu-WPV did not experience a weight loss throughout the observation period. Figure 4 A). On days 3 and 5 post-infection (dpi), a subset of animals in each group were euthanized, and viral titers in the lungs were determined using a plaque assay. At 3 dpi, viral titers were approximately 100-fold lower in animals inoculated with Co-WPV or Co / Flu-WPV compared to those inoculated with PBS or Flu-WPV. Figure 4 B). Furthermore, at 5 dpi, approximately 10 [units of something] were detected in the PBS or Flu-WPV administration groups. 5 The viral titer was PFU / ml, but in the Co-WPV and Co / Flu-WPV groups, the viral titer was below the detection limit. Figure 4 (B and C).

[0067] Example 4: A combined vaccine of SARS-CoV-2 whole-virus inactivated vaccine and quadrivalent influenza whole-virus inactivated vaccine (Co / Preparation of qFlu-WPV and HI antibody titer or serum antibody titer Co / qFlu-WPV was prepared by mixing Co-WPV (9 μg of SARS-CoV-2) described in Example 1 with qFlu-WPV (9 μg of each of the four influenza vaccine strains).

[0068] Co / qFlu-WPV, Co-WPV (9 μg SARS-CoV-2) as a control, qFlu-WPV (9 μg each), and PBS were inoculated into 7-week-old female BALB / c mice. Serum was collected from the tail vein 22 days after vaccination to determine HI antibody titers or neutralizing antibody titers. Results are as follows: Figure 5 As shown. Points represent individual values, and the line represents the mean using SEM. Statistical analysis used one-way ANOVA based on Tukey's multiple comparison test. p<0.0001).

[0069] In mice inoculated with qFlu-WPV or Co / qFlu-WPV, neutralizing antibodies against each viral antigen were induced. No statistically significant differences were observed between mice administered qFlu-WPV alone or Co / qFlu-WPV. Figure 5 A).

[0070] In addition, neutralizing antibodies against SARS-CoV-2 were confirmed in mice vaccinated with Co-WPV or Co / qFlu-WPV in both groups within the range of 40–80, but the neutralizing antibody titer in the PBS group mice was below the detection limit. Figure 5 B). These results indicate that in Co / qFlu-WPV, each whole-virus inactivated vaccine induces humoral immunity against each virus in a manner that does not significantly interfere with each other.

[0071] To evaluate neutralizing antibodies against the original strain (A), Alpha strain (B.1.1.7), Delta strain (B.1.6.17.2), and Omicron strain (BA.5), which are variants of SARS-CoV-2, the aforementioned mice were boosted on day 22, and serum samples were collected from each mouse 36 days later. Results are as follows: Figure 6 As shown. Points represent individual values, and the line represents the mean using SEM. Statistical analysis used one-way ANOVA based on Tukey's multiple comparison test. p<0.0001).

[0072] Neutralization tests after two vaccinations showed that antibodies against the original strain and the alpha strain were induced in mice vaccinated with Co-WPV and Co / qFlu-WPV. The average antibody levels against the original strain were 2560 and 2463, respectively, and against the alpha strain, they were 2048 and 1808, respectively. Figure 6(A and B). Notably, no statistically significant differences were observed between antibodies against the original strain and antibodies against the alpha strain in mice vaccinated with Co-WPV or Co / qFlu-WPV. Furthermore, high levels of neutralizing antibodies against the delta strain were also detected in mice vaccinated with Co-WPV or Co / qFlu-WPV, with average levels of 304 and 256, respectively. Figure 6 (C) On the other hand, mice inoculated with PBS or qFlu-WPV showed neutralizing antibodies below the detection limit for all viruses tested. These results indicate that Co / qFlu-WPV can effectively induce cross-reactive neutralizing antibodies.

[0073] In addition, neutralizing antibodies against the Omeprone strain were also measured. Figure 6 D). Compared with the strong antibody induction against the alpha and delta strains in mice inoculated with Co-WPV and Co / qFlu-WPV, antibody levels against the omeprone strain were low, and in some mice, the levels were below the detection limit. This suggests that a two-dose regimen may not be sufficient to enhance immunity against the omeprone strain.

[0074] Example 5: SARS-CoV-2 challenge experiment with Co / qFlu-WPV Similar to Example 3, the defensive effect of Co / qFlu-WPV against SARS-CoV-2 infection was investigated. The results are as follows: Figure 7 As shown. Points represent individual values, and lines are displayed using SEM to show the average. Statistical analysis used one-way ANOVA based on Tukey's multiple comparison test. p<0.0001).

[0075] 10 5 Following intranasal inoculation of SARS-CoV-2 with PFU into vaccinated mice, both PBS control mice and mice inoculated with Flu-WPV showed a sharp decrease in body weight until 4 days post-inoculation (dpi). Figure 7 A). On the other hand, in mice inoculated with Co-WPV or Co / qFlu-WPV, no weight loss was observed after viral inoculation ( Figure 7 A).

[0076] The viral titer in the lungs was determined using a plaque assay. Results showed that in mice inoculated with PBS and qFlu-WPV, an average viral titer of 2 × 10⁻⁶ was confirmed at 3 days post-infection (dpi). 7 and 1.9×10 7The PFU levels were more than 100 times higher than those in mice inoculated with Co-WPV or Co / qFlu-WPV. At 5 days post-infection (dpi), an average of 5.5 × 10⁻⁶ PFU was detected in mice in both the PBS and qFlu-WPV groups. 5 and 6.2×10 5 PFU was detected, but no viral titer was confirmed in mice inoculated with Co-WPV or Co / qFlu-WPV. Figure 7 (B and C).

[0077] In summary, the hybrid vaccine of the present invention induces neutralizing antibodies against each virus without affecting the efficacy of the individual vaccines, demonstrating a defensive effect against viral attacks. Furthermore, no adjuvant is required. Additionally, regarding SARS-CoV-2, it induces cross-immunity against variants other than the vaccine strain (original strain).

Claims

1. A combination vaccine, which includes an inactivated whole-virus influenza vaccine and an inactivated whole-virus SARS-CoV-2 vaccine.

2. The mixed vaccine as described in claim 1, wherein it does not contain an adjuvant.

3. The combined vaccine as described in claim 1 or 2, for the prevention of influenza infection and COVID-19.

4. The combined vaccine according to any one of claims 1 to 3, wherein, The influenza whole virus inactivated vaccine comprises an influenza whole virus inactivated vaccine that targets two or more influenza virus strains.

5. The mixed vaccine as described in claim 4, wherein, The inactivated whole-virus influenza vaccine is a quadrivalent inactivated whole-virus influenza vaccine using two type A virus strains and two type B virus strains as vaccine strains.

6. The combined vaccine according to any one of claims 1 to 5, wherein, Inactivated influenza whole virus vaccines and / or inactivated SARS-CoV-2 whole virus vaccines are made by inactivating the whole influenza virus and / or the whole SARS-CoV-2 virus using formaldehyde.

7. SARS-CoV-2 whole-virus inactivated vaccine, which, together with influenza whole-virus inactivated vaccine, is used as a combined vaccine for the prevention of influenza infection and COVID-19.

8. Inactivated whole-virus influenza vaccine, which, together with inactivated whole-virus SARS-CoV-2 vaccine, is used as a combined vaccine for the prevention of influenza infection and COVID-19.

9. A composition comprising an inactivated whole-virus influenza vaccine and an inactivated whole-virus SARS-CoV-2 vaccine, wherein the inactivated whole-virus influenza vaccine and the inactivated whole-virus SARS-CoV-2 vaccine are mixed and administered to humans as a combined vaccine for the prevention of influenza infection and COVID-19.

10. The composition of claim 9, wherein, The inactivated whole-virus influenza vaccine and the inactivated whole-virus SARS-CoV-2 vaccine are provided in separate vials.

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