Method for producing inactivated influenza vaccine and vaccine composition thereof

Abstract

Provided are an inactivated influenza vaccine having high immunogenicity and a method for producing the same.　This method for producing an inactivated influenza vaccine, wherein an inactivation treatment is performed using formaldehyde, comprises a step for preliminarily treating a virus solution containing influenza virus, which is collected from a host, with β-propiolactone.

Description

[Technical Field]

[0001] The present invention relates to a highly immunogenic inactivated influenza vaccine and a method for producing the same. [Background technology]

[0002] Human influenza viruses are single-stranded RNA viruses of the Orthomyxovirus genus, and are classified into types A, B, and C based on the antigenicity of their internal antigens. Of these, types A and B cause major epidemics every year, mainly in the winter, and in Japan, it is known that an estimated 10 million people or more are infected annually. The most effective means of preventing influenza viruses that cause such major epidemics every year is a vaccine. The influenza vaccine introduced in Japan in 1957 was triggered by the Asian flu epidemic, and at the time was an inactivated whole particle vaccine made from purified influenza virus that had been inactivated. Subsequently, in an effort to reduce side effects such as fever, split vaccines in which viral particles were split using diethyl ether or surfactant treatment and lipid components were removed were approved, and split vaccines are now widely used worldwide. On the other hand, in the pre-pandemic vaccine against the A / H5N1 subtype, inactivated whole particles are still used as the antigen because there is no history of infection or vaccination.

[0003] Inactivated whole particle vaccines and split vaccines are so-called inactivated vaccines in which the infectivity of the pathogenic virus has been eliminated. Vaccine inactivation treatments include treatments with β-propiolactone, formaldehyde, and diethyl ether or surfactants used to cleave virus particles (Non-Patent Document 1), with formaldehyde treatment being the most widely used. The aforementioned inactivated whole particle vaccines and split vaccines are both vaccines inactivated with formaldehyde, and formaldehyde is used not only as an inactivating agent but also as a storage stabilizer in the vaccine production process and as one of the components of vaccine formulations.

[0004] On the other hand, inactivated whole particle vaccines have a higher basic immune effect (priming effect) than split vaccines. This effect is due to the uptake of viral nucleic acids into cells and the activation of innate immunity via Toll-Like Receptor 7 (TLR7) (Akira S. Phil.Trans.R.Soc.B 2011;366:2748-2755). The high priming effect of inactivated whole particle vaccines can be attributed to the high viral nucleic acid content of inactivated whole particle vaccines. Furthermore, although the content is lower than that of inactivated whole particle vaccines, split vaccines still contain viral nucleic acids, which contributes significantly to their immunogenicity.

[0005] Formaldehyde is known to react with primary amines of proteins, phenyl groups of tyrosine, etc., and primary amines of nucleic acid bases to form crosslinks. Therefore, excessive denaturation of viral nucleic acids by formaldehyde is a concern, as it may reduce the immunogenicity of both inactivated whole particle vaccines and split vaccines. Therefore, inactivation treatment with formaldehyde must be performed under appropriate conditions so as not to attenuate innate immune activity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Pawar SD, Murtadak VB, Kale SD, Shinde PV, Parkhi SS. Evaluation of different inactivation methods for high and low pathogenic avian influenza viruses in egg-fluids for antigen preparation. J Virol Methods. 2015 Sep 15;222:28-33. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a highly immunogenic inactivated influenza vaccine and a method for producing the same. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to inhibit the decline in innate immune activity of vaccine antigens when influenza viruses are inactivated with formaldehyde. As a result, they unexpectedly discovered that pretreating influenza viruses with β-propiolactone, which is used as a virus inactivating agent like formaldehyde, before treating them with formaldehyde can inhibit the formaldehyde-induced decline in the innate immune activation ability of influenza vaccine antigens.

[0009] That is, the present invention relates to the following 1) to 5). 1) A method for producing an inactivated influenza vaccine that is inactivated using formaldehyde, the method comprising a step of pretreating a virus liquid containing influenza viruses recovered from a host with β-propiolactone. 2) Method 1), in which the inactivation treatment using formaldehyde involves adding formalin to the virus solution to a final concentration of 0.005 to 0.015 vol%. 3) The method of 2), wherein the inactivation treatment using formaldehyde is carried out at 2 to 8°C for 3 to 14 days or at 20 to 30°C for 3 days. 4) Any of the methods 1) to 3), in which the β-propiolactone treatment comprises adding β-propiolactone to a virus solution containing the recovered influenza virus to a final concentration of 0.0125 to 0.1 vol%, and allowing the mixture to react at 2 to 8°C for 18 hours or longer. 5) An inactivated whole particle or split influenza virus vaccine produced by any of the methods 1) to 4). [Effects of the Invention]

[0010] According to the method of the present invention, the decrease in the innate immune activation ability of influenza vaccine antigens caused by formaldehyde treatment can be suppressed while maintaining the pyrogenicity-reducing effect caused by formaldehyde treatment. Thus, the present invention makes it possible to provide inactivated influenza vaccines that are highly immunogenic and have as little pyrogenicity as possible, thereby making a significant contribution to the pharmaceutical industry. [Brief explanation of the drawings]

[0011] [Figure 1] Assessment of the TLR7 activation ability of influenza virus particles. [Figure 2] Formaldehyde treatment reduces TLR activation. [Figure 3] Resistance to formaldehyde by β-propiolactone treatment. [Figure 4] (A): Formaldehyde treatment conditions and changes in TLR activity for B / Yamagata strain antigens (reaction temperature: 4°C), (B): Formaldehyde treatment conditions and changes in TLR activity for B / Yamagata strain antigens (reaction temperature: 25°C). [Figure 5] (A): Formaldehyde treatment conditions and changes in TLR activity for A / H3N2 subtype antigens (reaction temperature: 4°C). (B): Formaldehyde treatment conditions and changes in TLR activity for A / H3N2 subtype antigens (reaction temperature: 25°C). [Figure 6A] Evaluation of densification of nucleoproteins by formaldehyde treatment (no treatment). [Figure 6B] Evaluation of nucleoprotein densification by formaldehyde treatment (4°C, 0.02%, 3 days). [Figure 6C] Evaluation of nucleoprotein densification by formaldehyde treatment (4°C, 0.02%, 14 days). [Figure 6D] Evaluation of densification of nucleoproteins by formaldehyde treatment (25°C, 0.02%, 3 days). [Figure 6E] Evaluation of densification of nucleoproteins by formaldehyde treatment (25°C, 0.02%, 14 days). [Figure 7] Evaluation of viral genome density by formaldehyde treatment. [Figure 8A] Results of mouse immunogenicity test (A / H1N1) (HI antibody titer). [Figure 8B] Results of mouse immunogenicity test (A / H3N2) (HI antibody titer). [Figure 8C] Results of mouse immunogenicity test (B / Victoria) (HI antibody titer). [Figure 9A] Results of mouse immunogenicity test (A / H1N1) (antigen-specific IgG titer). [Figure 9B] Results of mouse immunogenicity test (A / H3N2) (antigen-specific IgG titer). [Figure 9C] Results of mouse immunogenicity test (B / Victoria) (antigen-specific IgG titer). [Figure 10] Pyrogenic activity assessment in primates. DETAILED DESCRIPTION OF THE INVENTION

[0012] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments. In the present invention, "influenza virus" refers to influenza A virus or influenza B virus, or both. Influenza virus also includes all currently known subtypes, as well as subtypes that may be isolated and identified in the future. In the present invention, the term "influenza vaccine" refers to a vaccine containing at least an antigen of either influenza A virus or influenza B virus. That is, the influenza vaccine of the present invention may be a monovalent vaccine containing only one of influenza A virus or influenza B virus, or a polyvalent vaccine containing both. Furthermore, the antigen may be an inactivated whole particle vaccine in which the infectivity has been eliminated while the morphology of the virus particle is maintained, or a split vaccine in which the virus particles are cleaved with a surfactant or an organic solvent such as diethyl ether. The influenza virus strain used to prepare the vaccine of the present invention may be a strain isolated from an infected animal or patient, or may be a recombinant virus established in cultured cells by genetic engineering.

[0013] The method for producing an inactivated influenza vaccine of the present invention is a method for inactivating the vaccine using formaldehyde, and is characterized by including a step of pretreating a virus liquid containing influenza viruses recovered from a host with β-propiolactone.

[0014] The virus solution to be treated with β-propiolactone is a virus solution containing influenza viruses that have been infected in a host, cultured, and then collected. The "host" used for propagating influenza viruses herein may be either cultured cells or embryonated chicken eggs. That is, in the method of the present invention, the influenza viruses to be treated may be those propagated by either the embryonated chicken egg method or the cell culture method.

[0015] The "embryonated egg method" is a method in which a virus strain is inoculated into an embryonated egg and cultured, and then the virus suspension is clarified, concentrated, purified, and inactivated to obtain a virus liquid containing virus particles. Here, incubation is carried out by inoculating the influenza virus into embryonated eggs and then at 30-37°C for approximately 1-7 days, preferably at 33-35°C for approximately 2 days. After incubation, the virus suspension (infected allantoic fluid) is collected and centrifuged or filtered for clarification. Ultrafiltration is then carried out for concentration. Virus purification can be carried out using ultracentrifugation such as sucrose density gradient centrifugation, liquid chromatography, or other means.

[0016] The "cell culture method" is a method in which a virus strain is inoculated into cultured cells, cultured, and then clarified, concentrated, purified, and inactivated in the same manner as the embryonated egg method to obtain a virus liquid containing virus particles. Here, the cultured cells are not particularly limited as long as they are cells in which influenza viruses can grow, and examples include MDCK (Madin-Darby Canine Kidney), Vero, Caco-2, PER.C6, EB66, and these cells modified to highly express the receptor used by the virus for entry.

[0017] Influenza virus suspensions are cultured using embryonated eggs or cell culture methods, and then recovered and treated with β-propiolactone at any stage during clarification, concentration, or purification. Generally, an influenza virus suspension is inactivated with formaldehyde at one of the stages of clarification, concentration, or purification. However, in the present invention, the virus is treated with β-propiolactone in advance of the formaldehyde treatment. That is, in the method of the present invention, the β-propiolactone treatment and subsequent formaldehyde treatment can be performed before or after the clarification, concentration, and purification steps, preferably before or after the purification step, more preferably after the purification step, or, in the case of a split vaccine, before or after the virus cleavage step.

[0018] β-Propiolactone is a monoalkylating agent that is widely used for virus inactivation in the preparation of many vaccines. In the present invention, β-propiolactone treatment includes adding β-propiolactone to a virus solution containing influenza viruses recovered after culture to a final concentration of 0.0125 to 0.1 vol%, preferably 0.025 to 0.075 vol%, more preferably 0.05 vol%, and allowing the mixture to react at 2 to 8°C for 18 hours or longer, preferably 20 hours or longer, more preferably 24 hours or longer, and 50 hours or shorter, preferably 30 hours or shorter. In a more preferred embodiment, treatment is carried out at 2 to 8°C and at 0.05% for 24 hours.

[0019] In the present invention, the formaldehyde treatment is performed on the β-propiolactone-treated virus solution. The treatment conditions include adding formalin to the virus solution to a final concentration of 0.015 vol% or less, preferably 0.005 to 0.015 vol%, more preferably 0.01 to 0.015 vol%, and more preferably 0.01 vol%, and performing a reaction at 2 to 8°C for no more than 14 days or at 20 to 30°C for no more than 3 days. In a more preferred embodiment, for example, formalin is added to a final concentration of 0.01 vol %, and the reaction is carried out at 4°C for 3 to 14 days or at 25°C for 3 days. Typically, formaldehyde treatment is carried out at a final formalin concentration of 0.02 to 0.1 vol% at 2 to 8°C for 14 days to achieve an inactivating effect, but in the present invention, it can be carried out under milder conditions as described above. In the present invention, "formalin" refers to an aqueous formaldehyde solution containing 35 to 41% formaldehyde.

[0020] The influenza vaccine produced in this manner does not lose its TLR activation ability and retains the effect of reducing pyrogenic activity due to formalin treatment, making it an inactivated vaccine that maintains high immunogenicity and reduces pyrogenic activity as much as possible. Here, "the TLR activation ability is not reduced" means that the TLR stimulating activity is not significantly reduced compared to an antigen that has only been treated with β-propiolactone or an influenza virus that has not been inactivated. The TLR-stimulating activity can be measured, for example, by exposing RAW264.7 cells incorporating the TLR7 gene and the secretory alkaline phosphatase (SEAP) gene to the inactivated whole influenza antigen of the present invention and measuring the SEAP activity in the culture supernatant, as described in the Examples below.

[0021] The inactivated influenza vaccine of the present invention contains a hemagglutinin amount of 7.5 μg or more per virus strain, i.e., 7.5 μg HA / strain or more, preferably 9 to 21 μg HA / strain, and more preferably 15 μg HA / strain. The hemagglutinin content is a value obtained by measurement using a test method established by WHO or national standards, such as a single radial immunodiffusion test. The amount of antigen contained in the vaccine may be appropriately changed depending on the type of virus or the recipient.

[0022] The inactivated influenza vaccine of the present invention may further contain a pharmaceutically acceptable carrier in addition to the influenza virus antigen. Examples of such a carrier include those commonly used in vaccine production, such as buffers, emulsifiers, preservatives, isotonicity agents, pH adjusters, and adjuvants.

[0023] The dosage form of the inactivated influenza vaccine of the present invention may be, for example, a liquid, a freeze-dried powder, a capsule, or a tablet.

[0024] The route of administration of the inactivated influenza vaccine of the present invention may be, for example, subcutaneous administration, intramuscular administration, intradermal administration, nasal administration, sublingual administration, or oral administration, and the method of administration may be, for example, administration by syringe, microneedle, syringe equipped with a microneedle, transdermal patch, or spray.

[0025] The inactivated influenza vaccine of the present invention can be administered to humans and non-human mammals, preferably humans, including mice, rats, guinea pigs, rabbits, pigs, cattle, horses, goats, sheep, dogs, cats, rhesus monkeys, cynomolgus monkeys, orangutans, and chimpanzees. [Example]

[0026] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0027] Reference Example 1: Evaluation of influenza virus TLR activation ability The B / Phuket / 3073 / 2013 virus was inoculated into the chorioallantoic cavity of 12-day-old embryonated chicken eggs and incubated for 3 days before harvesting the allantoic fluid. The collected allantoic fluid was clarified by filtration, adsorbed onto barium sulfate, and eluted with 12% sodium citrate solution to recover influenza virus. The recovered virus was further purified by ultrafiltration into 6.7 mM phosphate-buffered saline (pH 7.2) after buffer replacement, followed by sucrose density gradient centrifugation to recover the influenza virus-containing fraction. The resulting virus fluid is referred to as purified influenza virus fluid. The inactivating agent β-propiolactone was added to this purified influenza virus fluid to a final concentration of 0.05% and incubated at 4°C for 24 hours to inactivate influenza virus infectivity. After this inactivation reaction, the buffer was replaced by 6.7 mM phosphate-buffered saline (pH 7.2) containing 1 w / w% sucrose by ultrafiltration (MWCO: 100,000), and this was used as a β-propiolactone-treated inactivated whole particle vaccine. 1.5 × 10 of RAW264.7 (NBP2-26261) carrying the secretory alkaline phosphatase gene 6 The TLR7 / 8 / 9 antagonist ODN2088 (Miltenyi) was added to the cells at final concentrations of 0, 0.1, 1, and 10 μM. 5 μg of inactivated whole particle vaccine of the B / Phuket / 3073 / 2013 strain prepared as described above or 5 μg of imiquimod was added to the ODN2088-added cells and cultured for 24 hours at 37 °C and 5% CO2. After culture, the supernatant was collected and the alkaline phosphatase activity of the supernatant was measured using a SEAP Reporter Assay Kit (trade name, Cayman). The relative activity values ​​of the ODN2088 signal at each concentration were calculated relative to the signal without ODN2088 for the inactivated whole particle vaccine and imiquimod, respectively.

[0028] The relative activity values ​​obtained after pretreatment with various concentrations of ODN2088 are shown in Figure 1. The relative activity values ​​of both the inactivated whole particle vaccine and imiquimod tended to decrease in an ODN2088 concentration-dependent manner, suggesting that the TLR7 activation ability of the inactivated whole particle vaccine can be evaluated in an in vitro system using NBP2-26261.

[0029] Reference Example 2: Decrease in TLR activation ability due to formaldehyde treatment Purified influenza virus solution prepared by the method described in Reference Example 1 above was adjusted to a protein concentration of 200 μg / mL, and 10% neutral buffered formalin solution (Fujifilm Wako Pure Chemical Industries, Ltd., formaldehyde content: 3.8-4.1%) was added to the solution to a final formalin concentration of 0, 0.01, or 0.02%, and the mixture was incubated at 4°C for 3, 7, or 14 days. After the incubation, glycine was added to a final concentration of 10 mM to quench the formaldehyde reaction, and the mixture was centrifuged at 4°C and 1,000,000 × g for 4 hours. After centrifugation, the supernatant was discarded, and the resulting virus pellet was suspended in 6.7 mM phosphate buffered saline. This was used as formaldehyde-treated virus. 1.5×10 6 Ten micrograms of each formaldehyde-treated virus was added to NBP2-26261 cells in total protein form and cultured for 24 hours at 37°C and 5% CO2. After culture, the supernatant was collected and its alkaline phosphatase activity was measured using the SEAP Reporter Assay Kit (Cayman). From the measurement results, the relative activity value was calculated for each sample relative to the signal obtained without formalin (formalin concentration: 0).

[0030] As shown in Figure 2, the relative activity tended to decrease with increasing formaldehyde concentration and reaction time. Therefore, it was thought that strong formaldehyde treatment reduced TLR activation, thereby causing a decrease in innate immune activity and immunogenicity.

[0031] Example 1: Formaldehyde resistance by β-propiolactone treatment The purified influenza virus solution and β-propiolactone-treated inactivated whole particle vaccine prepared in Reference Example 1 above were each adjusted to a protein concentration of 200 μg / mL, and 10% neutral buffered formalin solution (Fujifilm Wako Pure Chemical Industries, Ltd., formaldehyde content: 3.8-4.1%) was added to the solution to a final formalin concentration of 0, 0.01, or 0.02%. After the addition of formalin, the solution was incubated at 4°C for 14 days. After the reaction, glycine was added to a final concentration of 10 mM to stop the formaldehyde reaction, and the solution was centrifuged at 4°C and 1,000,000 × g for 4 hours. After centrifugation, the supernatant was discarded, and the resulting virus pellet was suspended in 6.7 mM phosphate buffered saline to serve as the sample. 1.5×10 6 10 μg of the prepared sample was added to NBP2-26261 cells in a total protein amount, and the cells were cultured for 24 hours at 37°C and 5% CO2. After the culture, the supernatant was collected and the alkaline phosphatase activity of the supernatant was measured using the SEAP Reporter Assay Kit (product name, Cayman). From the measurement results, the relative activity value was calculated for each sample relative to the signal obtained in the absence of formalin (formalin concentration: 0), and this was used as the relative activity value.

[0032] As shown in Figure 3, we found that prior β-propiolactone treatment can suppress the reduction in TLR activation ability caused by formaldehyde. Therefore, it was thought that by performing formaldehyde treatment before the preparation of influenza vaccine antigens, it is possible to prepare influenza vaccine antigens that maintain their innate immune activation ability.

[0033] Example 2 Optimization of formaldehyde treatment conditions β-Propiolactone-treated inactivated whole particle vaccines of the B / Phuket / 3073 / 2013 strain (B / Yamagata lineage) and the A / Kansas / 14 / 2017 strain (A / H3N2 subtype) were prepared using a method similar to that described in Reference Example 1. Each strain of vaccine was supplemented with 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, formaldehyde content: 3.8-4.1%) to achieve a final formalin concentration of 0, 0.01, or 0.02%. After formalin addition, the vaccine was incubated at 4°C or 25°C for 3, 7, or 14 days. After the incubation, glycine was added to a final concentration of 10 mM to quench the formaldehyde reaction, and the resulting mixture was centrifuged at 1,000,000 × g for 4 hours at 4°C. After centrifugation, the supernatant was discarded, and the resulting virus pellet was suspended in 6.7 mM Phosphate Buffered Saline to prepare a sample. 1.5×10 6 10 μg of the prepared sample was added to NBP2-26261 cells in a total protein amount, and the cells were cultured for 24 hours at 37°C and 5% CO2. After the culture, the supernatant was collected and the alkaline phosphatase activity of the supernatant was measured using the SEAP Reporter Assay Kit (product name, Cayman). From the measurement results, the relative activity value was calculated for each sample relative to the signal obtained in the absence of formalin (formalin concentration: 0), and this was used as the relative activity value.

[0034] Figure 4 shows the effect of formaldehyde treatment on the β-propiolactone-treated inactivated whole particle vaccine of the B / Phuket / 3073 / 2013 strain (B / Yamagata lineage). Under 4°C incubation conditions, no clear decrease in TLR activation was observed at 0.01% formalin over 3-14 days (Figure 4A). However, under 25°C incubation conditions, a tendency for TLR activation to decrease with increasing formalin concentration was observed at all incubation days (Figure 4B). The average relative activity values ​​are summarized in Table 1 below. Because this evaluation system uses cells to quantitatively assess enzyme activity, a change of 30% or more was considered significant, taking into account variability in the evaluation method. Therefore, it is believed that a significant decrease in TLR activation was observed under the conditions underlined in the table.

[0035] On the other hand, the effect of formaldehyde treatment on the β-propiolactone-treated inactivated whole-particle vaccine of the A / Kansas / 14 / 2017 strain (subtype A / H3N2) was confirmed as shown in Figure 5. Even at 4°C, a clear decrease in TLR activation was observed at 0.02% formalin for all incubation days (Figure 5A). Furthermore, at 25°C, even at 0.01% formalin, a significant decrease in TLR activation was observed over 7 days (Figure 5B). Table 2 below shows the relative activity values ​​for the A / Kansas / 14 / 2017 strain. Unlike the B / Phuket / 3073 / 2013 strain, the only changes observed were within 30% for 3-14 days at 4°C with 0.01% formalin or for 3 days at 25°C with 0.01% formalin. A decrease of more than 30% in TLR activation was observed in all other incubations.

[0036] Therefore, while conventionally, virus inactivation treatment has been performed using a formalin concentration of 0.02% at 4°C for a long period of time, it has been suggested that some strains are highly sensitive to formaldehyde, resulting in a decrease in TLR activation ability. Therefore, for both type A and type B virus strains, TLR activation ability can be maintained by treating them with β-propiolactone and then treating them with formaldehyde at a low concentration of formalin, for example, at room temperature for several days. Furthermore, while conventional formaldehyde treatment conditions have been relatively high and long, in order to achieve virus inactivation, the present invention allows for sufficient inactivation to be achieved even under relatively mild formaldehyde treatment conditions by performing β-propiolactone treatment in advance.

[0037] [Table 1]

[0038] [Table 2]

[0039] Reference Example 3 Cross-linking of viral genome and nucleoprotein (NP) by formaldehyde treatment A β-propiolactone-treated inactivated whole particle vaccine of the B / Phuket / 3073 / 2013 strain (B / Yamagata lineage) was prepared using a method similar to that described in Reference Example 1 and subjected to formaldehyde treatment under the conditions listed in Table 3. After each reaction, glycine was added to a final concentration of 10 mM to quench the formaldehyde reaction, and the resulting mixture was centrifuged at 1,000,000 × g at 4°C for 4 hours. After centrifugation, the supernatant was discarded, and the resulting virus pellet was suspended in 6.7 mM phosphate-buffered saline. Sodium dodecyl sulfate was added to the suspension to a final concentration of 1%, and the resulting mixture was fractionated into 23 fractions using a sucrose density gradient centrifugation (4°C, 206,500 × g for 3 hours) with a fractionation density of 10-60 w / w%. Equal volumes of each fraction solution were mixed with SDS-PAGE sample buffer (8% SDS, 40% glycerol / 250 mM Tris-HCl Buffer pH 6.8) and incubated at 95°C for 5 minutes before Western blotting. Additionally, 12.5 μL of Proteinase K (Roche) was added to 100 μL of each fraction solution from conditions 1, 3, and 5, and incubated at 70°C for 15 minutes before being subjected to qPCR for viral genome quantification. Western blotting was performed according to the method described below. qPCR was performed by extracting the viral genome using the High Pure Viral RNA Kit (Roche). Type B virus genome quantification was performed using the method described in the Influenza Diagnostic Manual (4th edition), targeting the Type B NS gene.

[0040] For Western blotting, the fraction solution containing sample buffer was electrophoresed on a 12.5% ​​polyacrylamide gel (ePAGEL, ATTO Corporation) and transferred to a PVDF membrane using a semi-dry transfer apparatus (ATTO Corporation). After transfer, the PVDF membrane was immersed in 75 mL of blocking buffer (TBS containing 10% skim milk) for a masking reaction at room temperature for 4 hours. After the reaction, the PVDF membrane was washed three times with an appropriate amount of TBS and then immersed in an anti-NP antibody solution (LifeSpan BioScience) and reacted at 4°C for approximately 16 hours (primary antibody reaction). After the primary antibody reaction, the PVDF membrane was washed five times with TBS containing Tween 20 (trade name), and an HRP-labeled anti-mouse antibody (Jackson Immuno Research Laboratories) solution was added and reacted at room temperature for 60 minutes (secondary antibody reaction). After the secondary antibody reaction, the PVDF membrane was washed five times with Tween 20 (trade name)-containing TBS, and nucleoproteins were detected using Super Signal (trade name) Western Lightning-Plus ECL (trade name, manufactured by Perkin Elmer).

[0041] First, the results of Western blotting are shown in Figure 6. Nucleoproteins were detected only at low density in the absence of formaldehyde treatment (condition 1, Figure 6A) and in the 3-day incubation with 0.02% formalin at 4°C (condition 2, Figure 6B). Nucleoproteins were detected at both high and low density in the 14-day incubation with 0.02% formalin at 4°C (condition 3, Figure 6C) and the 3-day incubation with 0.02% formalin at 25°C (condition 4, Figure 6D). Nucleoproteins were detected only at high density in the 14-day incubation with 0.02% formalin at 25°C (condition 5, Figure 6E). These results confirmed that increasing the formaldehyde treatment shifted nucleoproteins toward higher density.

[0042] qPCR quantification of viral genomes was performed under conditions 1, 3, and 5. Similar to nucleoprotein detection, viral genomes were detected only at low densities under condition 1, whereas viral genomes were detected at both high and low densities under condition 3. Under condition 5, viral genomes were localized only at high densities. The decrease in viral genome quantification values ​​with increasing formaldehyde treatment is thought to be due to the inhibition of polymerase chain reaction by residual formaldehyde cross-linking. However, the fraction of detected viral genomes was almost identical to the fraction of nucleoprotein detected by Western blotting. Therefore, formaldehyde treatment appears to cross-link nucleoprotein and viral genomes, and excessive cross-linking may attenuate TLR activation.

[0043] [Table 3]

[0044] Example 3 Mouse immunogenicity study In the same manner as described in Example 2, the following were prepared for three strains (A / H1N1: A / Brisbane / 02 / 2018, A / H3N2: A / Kansas / 14 / 2017, B / Victoria lineage: B / Maryland / 15 / 2016): 1) inactivated whole particle antigen treated with β-propiolactone, 2) inactivated whole particle antigen treated with β-propiolactone followed by treatment with formalin at a final concentration of 0.01% at 4°C for 14 days, and 3) inactivated whole particle antigen treated with β-propiolactone followed by treatment with formalin at a final concentration of 0.02% at 25°C for 14 days. Each antigen was adjusted with 6.7 mM phosphate-buffered saline (pH 7.2) containing 1 w / w% sucrose so that the hemagglutinin of each strain was 15 μg HA per 0.2 mL, and used as an inactivated whole particle vaccine (WV) in a mouse immunogenicity test. Inactivated whole particle vaccine and influenza HA vaccine (split vaccine, SV) as a control were administered subcutaneously to the dorsal region of 5-week-old female BALB / c mice at a dose of 15 μg HA / administration, and 21 days after administration, the HI antibody titer and antigen-specific IgG titer of the serum obtained from each mouse were measured. The results of HI antibody titers are shown in Figures 8A–8C. For all strains, WV treated with β-propiolactone alone induced higher antibody levels than SV. WV treated with 0.01% formalin for 14 days at 4°C (WV-0.01), which does not reduce spontaneous immune activation, induced antibodies at a similar level to WV treated with β-propiolactone alone. Furthermore, WV treated with 0.02% formalin for 14 days at 25°C (WV-0.02), which significantly reduces spontaneous immune activation, induced significantly less antibody than WV-0.01 for all strains (Mann-Whitney test, p<0.05). The results of antigen-specific IgG titers are shown in Figures 9A-9C. These titers show a similar trend to the HI antibody titers. WV-0.01 induced antibodies to the same extent as WV treated with β-propiolactone alone, while WV-0.02 showed a significantly reduced antibody induction compared to WV-0.01 (Mann-Whitney test, p<0.01). Therefore, it has been shown that the ability to activate innate immunity and the ability to induce antibodies are correlated. In order to maintain high immunogenicity of influenza vaccines, it is important to maintain the original activity of the vaccine antigen without impairing its ability to activate innate immunity.

[0045] Example 4: Evaluation of pyrogenic activity in primates Twelve quarantined cynomolgus monkeys were given atropine sulfate hydrate intramuscularly as a preanesthetic medication, followed by intramuscular administration of ketamine hydrochloride to sedate them. After sedation, a telemetry transmitter (TL11M2-D70-PCT, Data Sciences International Inc.) was implanted into the abdominal cavity and secured to the abdominal wall under isoflurane inhalation anesthesia. Immediately before surgery, antibiotics were administered intramuscularly to prevent infection, and ketoprofen was administered intramuscularly to relieve pain. An observation period of approximately 3 weeks was allowed after the telemetry transmitter implantation surgery, and the animals were then subjected to the study after confirming that no abnormalities had occurred. Cynomolgus monkeys implanted with telemetry transmitters were administered 0.5 mL of saline (Otsuka Pharmaceutical Factory) to each individual (Day 0), and body temperature was measured 24 hours after administration (baseline measurement for each individual). Seven days after saline administration, each individual was administered 0.5 mL of various tetravalent inactivated whole particle vaccines (15 μg HA / strain / 0.5 mL, WV) and influenza HA vaccine (15 μg HA / strain / 0.5 mL, SV) prepared as in Example 3. Body temperature was measured 24 hours after administration, and the difference from the baseline measurement was calculated for each individual. The difference in body temperature was considered fever, and the results are summarized in Figure 10. As shown in Figure 10, the split influenza HA vaccine did not exhibit pyrogenic activity, while the inactivated whole particle vaccine treated only with β-propiolactone exhibited the highest pyrogenic activity. Compared to the inactivated whole particle vaccine treated only with β-propiolactone, both formalin-treated inactivated whole particle vaccines exhibited reduced pyrogenic activity. Comparing WV-0.01 and WV-0.02, WV-0.02, which has a reduced ability to activate innate immunity, exhibited lower pyrogenic activity, but the peak fever values ​​(WV-0.01: 0.7°C, WV-0.02: 0.3°C) differed by only 0.4°C. Therefore, pyrogenic activity is reduced by formalin treatment, and the reduction in pyrogenic activity is greater when treatment is performed under stronger conditions (high concentration, high temperature, long period of time). However, as shown in Example 3, WV-0.02 exhibits a significant reduction in immunogenicity. Therefore, a vaccine with excellent efficacy and safety is one that has as low pyrogenic activity as possible without impairing its ability to activate natural immunity, such as WV-0.01.

Claims

1. A method for producing an inactivated whole influenza vaccine by inactivating the virus using formaldehyde, the method comprising the step of pre-treating a virus liquid containing influenza viruses recovered from a host with β-propiolactone before the formaldehyde treatment, Inactivation treatment using formaldehyde involves adding formalin to the virus solution to a final concentration of 0.005 to 0.015 vol %, and incubating the solution at 2 to 8°C for 3 to 14 days or at 20 to 30°C for 3 days. The β-propiolactone treatment comprises adding β-propiolactone to a virus solution containing the recovered influenza virus to a final concentration of 0.05 vol %, and allowing the solution to react at 2 to 8°C for 24 to 30 hours.

2. An inactivated whole particle influenza virus vaccine produced by the method of claim 1.

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