Influenza vaccine preparation

Abstract

To provide an influenza HA vaccine preparation with enhanced stability.SOLUTION: An influenza HA vaccine composition comprising a hemagglutinin fraction of an influenza virus as an antigen, and further comprising two or more additives selected from sucrose, trehalose, N-acetyl-L-cysteine, glycine, glutamic acid, and aspartic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to influenza vaccine formulations with improved stability. [Background technology]

[0002] Influenza, which is prevalent in winter, is an acute respiratory infection caused by the influenza virus and is transmitted through droplets from sneezing and other sources, as well as through contact with objects carrying these droplets. During peak influenza seasons, the number of deaths from influenza and related diseases increases, particularly among the elderly. This phenomenon is common in developed countries, and is a serious social problem in Japan, where the proportion of elderly people is rapidly increasing.

[0003] One of the most effective methods for preventing seasonal influenza is vaccination. The influenza vaccine currently used in Japan is a split vaccine, called the influenza HA vaccine, which uses the hemagglutinin (HA) fraction of the virion split into virus particles as an antigen. Influenza HA vaccines use an HA antigen that is different from the natural form present on the surface of the virus. Therefore, the manufacturing process involves adding formalin or surfactants (e.g., polysorbate 80) to stabilize the antigen. However, this results in a decrease in potency during long-term storage stability tests, which is a problem.

[0004] It is generally known that the stability of vaccine preparations is improved by freeze-drying, and sugars are commonly added to prevent antigen denaturation during freeze-drying. For example, it has been reported that the stability of a recombinant influenza virus vector is improved by adding trehalose to the vector to prepare a dried formulation (Non-Patent Document 1). However, freeze-dried formulations require a long production cycle and large-scale capital investment.

[0005] In addition, it has been reported that the stability of antigens in liquid formulations of live attenuated vaccines is improved when sucrose, sodium glutamate, arginine, and albumin are used as additives (Non-patent Documents 2 and 3). However, no additives have been found that are particularly effective in improving the stability of influenza HA vaccine formulations. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Int J Pharm. 2019 Apr 20:561:66-73 [Non-patent document 2] Vaccine. 2016 Jul 12;34(32):3676-83 [Non-patent document 3] J Pharm Sci. 2019 Jul;108(7):2315-2322 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing influenza HA vaccine formulations with improved stability. [Means for solving the problem]

[0008] The present inventors have investigated the stability of influenza HA vaccines and found that the thermal stability in solution is significantly improved by using a combination of multiple specific sugars and amino acids.

[0009] That is, the present invention relates to the following 1) to 10). 1) An influenza HA vaccine composition containing an influenza virus hemagglutinin fraction as an antigen and two or more additives selected from sucrose, trehalose, N-acetyl-L-cysteine, glycine, glutamic acid, and aspartic acid. 2) The composition of 1), which is liquid. 3) The composition of 1) or 2), wherein the additive comprises sucrose and / or trehalose. 4) The composition of 1) or 2), wherein the additive comprises sucrose and trehalose, and also comprises N-acetyl-L-cysteine ​​and / or glycine. 5) The composition of 4), wherein the antigen comprises hemagglutinin fractions of influenza A and B viruses. 6) Any of the compositions 3) to 5), wherein the concentration of sucrose or trehalose in the composition solution is 60 to 200 mM. 7) The composition of 4) or 5), wherein the concentration of N-acetyl-L-cysteine ​​in the composition solution is 0.5 to 4 mM and the concentration of glycine is 20 to 60 mM. 8) The composition of 7), wherein the concentrations of sucrose and trehalose in the composition solution are each 60 to 200 mM. 9) An influenza HA vaccine composition containing hemagglutinin fractions of influenza A and B viruses as antigens, together with additives consisting of sucrose, trehalose, N-acetyl-L-cysteine, and glycine. 10) The composition of 9), wherein the concentrations of sucrose and trehalose in the composition solution are each 60 to 200 mM, the concentration of N-acetyl-L-cysteine ​​is 0.5 to 4 mM, and the concentration of glycine is 20 to 60 mM. [Effects of the Invention]

[0010] The vaccine composition of the present invention can improve the stability of influenza HA antigens, thereby increasing the robustness of immune induction in vaccinated recipients and enabling the extension of the temperature deviation tolerance time (stability budget) during long-distance transportation, such as overseas. [Brief explanation of the drawings]

[0011] [Figure 1-1] Heat stability evaluation using two types of additives (HA antigen: type B). [Figure 1-2] Heat stability evaluation using two types of additives (HA antigen: type B). [Figure 2-1] Heat stability evaluation using two types of additives (HA antigen: type A). [Figure 2-2] Heat stability evaluation using two types of additives (HA antigen: type A). [Figure 3] Heat stability evaluation using three types of additives (HA antigen: type B). [Figure 4] Heat stability evaluation using three types of additives (HA antigen: type A). [Figure 5] Relationship between N-acetyl-L-cysteine ​​concentration and the effect of improving thermal stability (HA antigen: type B). [Figure 6] Relationship between N-acetyl-L-cysteine ​​concentration and the effect of improving thermal stability (HA antigen: type A). [Figure 7] Relationship between glycine concentration and the effect of improving thermal stability (HA antigen: type B). [Figure 8] Relationship between glycine concentration and the effect of improving thermal stability (HA antigen: type A). [Figure 9] Relationship between aspartic acid concentration and the effect of improving thermal stability (HA antigen: type B). [Figure 10] Relationship between aspartic acid concentration and the effect of improving thermal stability (HA antigen: type A). DETAILED DESCRIPTION OF THE INVENTION

[0012] The influenza HA vaccine composition of the present invention (hereinafter also referred to as "vaccine composition") contains an influenza virus hemagglutinin (abbreviated as "HA") fraction as an antigen, and also contains two or more additives selected from sucrose, trehalose, N-acetyl-L-cysteine, glycine, glutamic acid, and aspartic acid. The influenza virus HA fraction used as an antigen may be either an HA derived from influenza A virus or an HA derived from influenza B virus, but preferably contains both. It may also contain HA derived from multiple subtype strains of influenza A or B virus. Subtypes include all currently known subtypes as well as subtypes that will be isolated and identified in the future. Suitable influenza virus HAs for use as antigens in the present invention include, for example, HA derived from two type A strains, the A / H1N1 subtype and the A / H3N2 subtype, and HA derived from one or two type B strains selected from the B / Victoria lineage and the B / Yamagata lineage.

[0013] The HA fraction of influenza virus can be obtained, for example, by growing a virus strain isolated from an influenza-infected animal or an influenza patient using the embryonated chicken egg method or cell culture method, and treating the virus liquid containing virus particles with a surfactant, ether, etc. to decompose or inactivate it. Specifically, an influenza virus strain is inoculated into embryonated chicken eggs or cultured cells and cultured at 30-37°C for approximately 1-7 days, preferably at 33-35°C for approximately 2 days. After completion of the culture, the virus suspension (infected allantoic fluid or infected cell culture supernatant) is collected and centrifuged or filtered for clarification. Subsequently, a barium salt adsorption elution reaction or ultrafiltration is performed for concentration. Virus purification can be performed using ultracentrifugation such as sucrose density gradient centrifugation or liquid chromatography. The purified virus solution is treated with a surfactant such as Triton X, sodium deoxycholate, or CTAB, or with diethyl ether, to promote cleavage of virus particles and inactivation of infectivity. 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 cells modified to highly express the receptor used by the virus for entry.

[0014] The content of influenza virus HA in the vaccine composition of the present invention is 15 μg or more per virus strain, ie, 15 μg HA / strain or more, preferably 15 to 60 μg HA / strain, and more preferably 15 to 21 μg HA / strain. The HA content is a value obtained by measurement using a test method established by WHO or national standards, such as a single radial immunodiffusion test.

[0015] In the vaccine composition of the present invention, two or more additives selected from sucrose, trehalose, N-acetyl-L-cysteine, glycine, glutamic acid and aspartic acid are used, preferably three, and more preferably four or five. By incorporating two or more of these additives, the thermal stability of the composition is improved even when the composition is in a liquid state, and the stability budget can be extended. Thermal stability can be evaluated, for example, using a protein higher-order structure evaluation device, Nano Temper's Tycho NT.6, by heating the composition solution in the range of 35 to 95°C at a rate of 30°C / min, measuring the fluorescence (330 nm and 350 nm) detected during this period, and evaluating the thermal denaturation of the protein based on the ratio of the obtained fluorescence intensities at 350 nm / 330 nm.

[0016] In terms of improving thermal stability, the following combinations of additives are preferred for influenza B HA: 1) sucrose and trehalose, 2) (sucrose or trehalose) and N-acetyl-L-cysteine, 3) glycine and (glutamic acid or aspartic acid), 4) glutamic acid and aspartic acid, 5) sucrose, trehalose and N-acetyl-L-cysteine, and 6) sucrose, trehalose, N-acetyl-L-cysteine ​​and glycine. Furthermore, for influenza A HA, preferred combinations include: 1) sucrose and trehalose, 2) (sucrose or trehalose) and (glycine, glutamic acid, or aspartic acid), 3) glycine and N-acetyl-L-cysteine, 4) glutamic acid and aspartic acid, 5) (sucrose or trehalose) and glycine and aspartic acid, 5) sucrose and trehalose and (glycine or aspartic acid), 7) sucrose and trehalose and N-acetyl-L-cysteine, 8) sucrose and trehalose and N-acetyl-L-cysteine ​​and glycine, and 9) sucrose and trehalose and N-acetyl-L-cysteine ​​and glycine and aspartic acid.

[0017] Influenza HA vaccines containing influenza A and B HA preferably contain saccharides such as sucrose or trehalose, or sucrose and trehalose, more preferably in combination with one or more selected from N-acetyl-L-cysteine ​​and glycine, and even more preferably in combination with three saccharides: sucrose, trehalose, and N-acetyl-L-cysteine, or four saccharides: sucrose, trehalose, N-acetyl-L-cysteine, and glycine.

[0018] The content of the additive in the vaccine composition is not limited as long as it is an amount that exerts a stabilizing effect, but the final concentration of sucrose or trehalose in the composition solution is preferably 60 mM or more, more preferably 60 to 200 mM, and even more preferably 60 mM. Glycine is preferably 20 mM or more, more preferably 20 to 250 mM, and even more preferably 20 mM. Glutamic acid is preferably 150 to 300 mM, and more preferably 200 to 250 mM. Aspartic acid is preferably 20 to 250 mM, and in the case of type A, more preferably 40 to 100 mM. The concentration of N-acetyl-L-cysteine ​​is preferably 0.5 mM or more, more preferably 0.5 to 4 mM, and even more preferably 0.5 to 2.0 mM.

[0019] When sucrose, trehalose, and N-acetyl-L-cysteine ​​are used in combination, the concentrations are preferably 60 to 200 mM sucrose, 60 to 200 mM trehalose, and 0.5 to 4 mM N-acetyl-L-cysteine. When sucrose, trehalose, N-acetyl-L-cysteine, and glycine are used in combination, the concentrations are preferably 60 to 200 mM sucrose, 60 to 200 mM trehalose, 0.5 to 4 mM N-acetyl-L-cysteine, and 20 to 100 mM glycine.

[0020] In an influenza HA vaccine containing influenza A and B HA, when sucrose, trehalose, and N-acetyl-L-cysteine ​​are used in combination, the concentrations are preferably 60 to 200 mM sucrose, 60 to 200 mM trehalose, and 0.5 to 4 mM N-acetyl-L-cysteine; and when sucrose, trehalose, N-acetyl-L-cysteine, and glycine are used in combination, the concentrations are preferably 60 to 200 mM sucrose, 60 to 200 mM trehalose, 0.5 to 4 mM N-acetyl-L-cysteine, and 20 to 60 mM glycine.

[0021] The vaccine composition of the present invention may be in the form of a liquid, powder (lyophilized powder, dry powder), capsule, or the like, but is preferably in the form of a liquid.

[0022] The vaccine composition of the present invention may further contain a pharmaceutically acceptable carrier. Examples of such carriers include those commonly used in vaccine production, such as saline, buffered saline, dextrose, glycerol, buffers, emulsifiers, preservatives (e.g., thimerosal), isotonicity agents, pH adjusters, inactivators (e.g., formalin), adjuvants, or immunostimulants. Adjuvants are substances that enhance the immune response to an antigen when administered together with the antigen. Examples of such adjuvants include aluminum salts (e.g., aluminum hydroxide gel), which are precipitating adjuvants; squalene, which is an oil-based adjuvant; MPL, which is a modified form of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacterial cell walls; nucleic acids derived from CpG or Poly I:C; and bacterial components that activate Toll-like receptors (TLRs).

[0023] The administration routes of the vaccine composition of the present invention include, for example, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, nasal administration, and oral administration. The administration method includes, for example, administration by syringe, microneedle, nebulizer, etc., with subcutaneous administration by syringe being preferred.

[0024] The single dose of the vaccine composition of the present invention may be 15 μg or more of HA per virus strain, and can be increased appropriately taking into consideration the age, sex, weight, etc. of the subject, but preferably 15 to 60 μg of HA / strain, more preferably 15 to 21 μg of HA / strain, and even more preferably 15 μg of HA / strain, is administered once or twice or more times. Multiple administrations are preferred, and in this case, administration is preferably performed at intervals of 1 to 4 weeks. Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this. [Example]

[0025] Example 1: Evaluation of thermal stability using two types of additives The contribution of two additives selected from sugars (sucrose, trehalose), amino acids (glycine (Gly), glutamic acid (Glu), aspartic acid (Asp)), and antioxidants (N-acetyl-L-cysteine: NAC) to the thermal stability of influenza HA vaccines was evaluated.

[0026] The approved influenza HA vaccine was used in a single stock solution to prepare a 50 μg / mL HA protein solution containing the additives listed above, either individually or in combination. The additives were added to a final concentration of 200 mM for sucrose, trehalose, glycine, glutamic acid, and aspartic acid, and a final concentration of 0.5 mM for N-acetyl-L-cysteine. The thermal stability of the HA antigen was evaluated using a Tycho NT.6 (NanoTemper) (Figures 1-1 and 1-2: Type B, Figures 2-1 and 2-2: Type A). The single stock solution of influenza HA vaccine used was manufactured using vaccine strains for the 2022 / 2023 season, with the A / Victoria / 1 / 2020 (IVR-217) strain for A / H1N1, the A / Darwin / 9 / 2021 (SAN-010) strain for A / H3N2, the B / Yamagata lineage being the B / Phuket / 3073 / 2013 strain, and the B / Vixctoria lineage being the B / Austria / 1359417 / 2021 (BVR-26) strain.

[0027] In Tycho NT.6, the prepared HA solution was heated in the range of 35 to 95°C at a rate of 30°C / min, the inflection point temperature (Ti value) of the thermal denaturation curve was measured, and the thermal stability was evaluated from the ΔTi value, which is the difference from the value when no additives were added (HA antigen only).

[0028] As shown in Figures 1-1, 1-2, 2-1, and 2-2, the ΔTi value was significantly higher when two additives were mixed than when only one additive was used for both Type B and Type A. Of these, the combination of two additives derived from sucrose, trehalose, and N-acetyl-L-cysteine, and two additives selected from glycine, glutamic acid, and aspartic acid for Type B, and the combination of two additives selected from sucrose, trehalose, glycine, glutamic acid, and aspartic acid for Type A were considered to be mixed formulations with higher thermal stability improvement.

[0029] Example 2: Evaluation of thermal stability using three types of additives Three additives were mixed to examine their thermal stability: sucrose, trehalose, glycine, and aspartic acid, which were confirmed to have an effect on the thermal stability of HA antigen (type A) in Example 1; and sucrose, trehalose, and N-acetyl-L-cysteine, which were confirmed to have an effect on the thermal stability of HA antigen (type B). Two or three of the additives listed above were mixed to prepare a 50 μg / mL HA protein content. The additives, sucrose, trehalose, glycine, and aspartic acid, were added to a final concentration of 60 mM, respectively, and N-acetyl-L-cysteine ​​to a final concentration of 0.5 mM. The thermal stability of the HA antigen was evaluated using a Tycho NT.6 (NanoTemper). (Figure 3: Type B; Figure 4: Type A) As shown in Figure 3, for type B, the ΔTi value was significantly higher for the formulation combining sucrose, trehalose, and N-acetyl-L-cysteine ​​than for the formulation combining only two of these, and it was thought that N-acetyl-L-cysteine ​​contributed most to improving thermal stability. Furthermore, Figure 4 shows that for type A, the formulation mixing three types selected from sucrose, trehalose, glycine, and aspartic acid had a higher ΔTi value than the formulation mixing two types, and it was thought that glycine and aspartic acid made a large contribution to improving thermal stability.

[0030] Based on the above, it was determined that the addition of sucrose and trehalose was important for improving thermal stability in both types A and B. We also determined the concentrations of N-acetyl-L-cysteine, glycine, and aspartic acid, which were thought to contribute significantly to improving thermal stability, and investigated the optimal formulation.

[0031] Example 3 Relationship between N-acetyl-L-cysteine ​​concentration and thermal stability improvement effect N-acetyl-L-cysteine ​​contributes significantly to thermal stability, and we investigated the relationship between N-acetyl-L-cysteine ​​concentration and its effect on improving thermal stability. HA antigen was prepared by adding sucrose, trehalose, and N-acetyl-L-cysteine ​​to the antigen to achieve a 50 μg / mL HA protein content. Sucrose and trehalose were added to final concentrations of 300 mM, and N-acetyl-L-cysteine ​​was added to final concentrations of 0.5, 1, 2, and 4 mM. The thermal stability of the HA antigen was evaluated using a Tycho NT.6 (NanoTemper) (Figure 5: Type B, Figure 6: Type A).

[0032] 5 and 6, there was no concentration-dependent change in the ΔTi values ​​within the set N-acetyl-L-cysteine ​​concentration range, and they were similar. Therefore, it was found that both type B and type A had similar thermal stabilization effects at concentrations of 0.5 to 4 mM N-acetyl-L-cysteine. Therefore, the concentration of N-acetyl-L-cysteine ​​was set to 0.5 mM.

[0033] Example 4 Relationship between glycine concentration and thermal stability improvement effect Glycine contributes significantly to thermal stability, and we investigated the relationship between glycine concentration and its effect on improving thermal stability. HA antigen was prepared by adding sucrose, trehalose, N-acetyl-L-cysteine, and glycine to a final concentration of 50 μg / mL. Sucrose and trehalose were added to a final concentration of 60 mM, N-acetyl-L-cysteine ​​to a final concentration of 0.5 mM, and glycine to final concentrations of 20, 40, 60, 80, and 100 mM. The thermal stability of the HA antigen was evaluated using a Tycho NT.6 (NanoTemper) (Figure 7: Type B, Figure 8: Type A).

[0034] In Figure 7, the ΔTi value decreased in a concentration-dependent manner within the set glycine concentration range, but in Figure 8, the ΔTi value increased in a concentration-dependent manner. However, in both types B and A, the ΔTi value increased when a glycine concentration of 20 mM was added compared to a mixture of sucrose, trehalose, and N-acetyl-L-cysteine, indicating improved thermal stability. Therefore, the glycine concentration was set to 20 mM.

[0035] Example 5 Relationship between aspartic acid concentration and thermal stability improvement effect Aspartic acid contributes significantly to thermal stability, we investigated the relationship between aspartic acid concentration and its effect on improving thermal stability. HA antigen was supplemented with sucrose, trehalose, N-acetyl-L-cysteine, glycine, and aspartic acid to prepare a solution containing 50 μg / mL of HA protein. The final concentrations of sucrose and trehalose were 60 mM, N-acetyl-L-cysteine ​​0.5 mM, glycine 20 mM, and aspartic acid 20, 40, 60, 80, and 100 mM, respectively. The thermal stability of the HA antigen was evaluated using a Tycho NT.6 (NanoTemper) (Figure 9: Type B, Figure 10: Type A). As shown in Figure 9, the addition of aspartic acid reduced the ΔTi value compared to a four-component mixture of sucrose, trehalose, N-acetyl-L-cysteine, and glycine. On the other hand, as shown in Figure 10, the addition of 40 mM or more of aspartic acid increased the ΔTi value compared to a four-component mixture of sucrose, trehalose, N-acetyl-L-cysteine, and glycine, indicating improved thermal stability. Therefore, the effect of adding aspartic acid to a four-component mixture of sucrose, trehalose, N-acetyl-L-cysteine, and glycine was thought to be effective only for type A.

[0036] Based on the above results, it was suggested that for influenza HA vaccines containing HA derived from types A and B, a composition of 60 mM sucrose, 60 mM trehalose, 0.5 mM N-acetyl-L-cysteine, and 20 mM glycine is a formulation that is highly effective in improving thermal stability.

Claims

1. An influenza HA vaccine composition containing an influenza virus hemagglutinin fraction as an antigen and two or more additives selected from sucrose, trehalose, N-acetyl-L-cysteine, glycine, glutamic acid and aspartic acid.

2. The composition of claim 1 which is a liquid.

3. The composition of claim 2 , wherein the additive comprises sucrose and / or trehalose.

4. The composition of claim 2, wherein the additive comprises sucrose and trehalose, and N-acetyl-L-cysteine ​​and / or glycine.

5. 5. The composition of claim 4, wherein the antigen comprises the hemagglutinin fraction of influenza A and B viruses.

6. The composition according to any one of claims 3 to 5, wherein the concentration of sucrose or trehalose in the composition solution is 60 to 200 mM.

7. 6. The composition according to claim 4, wherein the concentration of N-acetyl-L-cysteine ​​in the composition solution is 0.5 to 4 mM and the concentration of glycine is 20 to 60 mM.

8. 8. The composition according to claim 7, wherein the concentrations of sucrose and trehalose in the composition solution are each 60 to 200 mM.

9. An influenza HA vaccine composition containing hemagglutinin fractions of influenza A and B viruses as antigens, and also containing additives consisting of sucrose, trehalose, N-acetyl-L-cysteine, and glycine.

10. 10. The composition according to claim 9, wherein the sucrose and trehalose concentrations in the composition solution are each 60 to 200 mM, the N-acetyl-L-cysteine ​​concentration is 0.5 to 4 mM, and the glycine concentration is 20 to 60 mM.

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