TWI493038B - Complex for preparing influenza vaccine, novel influenza vaccine, and method for manufacturing the same - Google Patents

Description

Complex for preparing influenza vaccine, novel influenza vaccine and preparation method thereof

The invention relates to a compound for preparing influenza vaccine, a novel influenza vaccine and a preparation method thereof, in particular to a compound suitable for influenza vaccine with metal particles, a novel influenza vaccine and a preparation method thereof.

The flu virus is a ribonucleic acid (RNA) virus that has caused more than 30 pandemics since the 1580s and early 20th centuries. Every day, people catch a cold because of the flu virus. When the season changes, the flu virus often causes a pandemic, which plagues people's lives, and may be infected regardless of age. The severity of the flu varies from person to person. Most people will get better in a few days, but if you have respiratory, heart and kidney related diseases, diabetes or immunodeficiency, especially young children and the elderly, it is more prone to disease progression or pneumonia. The possibility of complications such as severe deaths, in the United States, about 20,000 people die each year from the flu.

Influenza viruses are easy to genetically mutate, making people responsibly diseased. Currently, they are used to fight the flu virus: using drugs or vaccinating drugs, but the effects of both are easy to fail. Among them, the current anti-influenza drugs are M2 blocker and neuraminidase inhibitor. In addition, most vaccines currently use inactivated vaccines, including whole virion vaccines and split-particle vaccines, combined with adjuvants to help the immune response or prolong the release of viral genes. Or use a subunit vaccine as a protection. Although the vaccines currently developed can reduce the severity of influenza and its complications, for vaccines or new influenza viruses, the protection of vaccines is poor and not long-lasting, and vaccines need to be used to maintain protection.

In view of this, there is an urgent need to develop a complex of influenza vaccines to make a new influenza vaccine. In addition to effective protection against multiple influenza viruses, it is possible to cause longer and better immunity without the need for adjuvants. reaction.

The main object of the present invention is to provide a metal particle-protein complex for preparing an influenza vaccine, which can form a strong affinity with a virus and can cause a good immune response in a living body.

Another object of the present invention is to provide an influenza vaccine which can attenuate viral toxicity and form a more effective protective force against a mutant virus.

Still another object of the present invention is to provide a method for preparing an influenza vaccine which can easily prepare attenuated vaccine containing metal particles.

The metal particle-protein complex of the present invention for preparing an influenza vaccine comprises: a metal nanoparticle; and a carbohydrate binding protein formed on the surface of the metal nanoparticle.

In the metal particle-protein complex of the present invention, the metal nanoparticles may have a particle diameter of 5 to 20 nm, preferably 7 to 18 nm, more preferably 10 to 15 nm. The material of the metal nanoparticle may be gold, platinum, or an alloy thereof, preferably gold, that is, the metal nanoparticle is preferably a gold nanoparticle. The gold nanoparticles have special chemical and physical properties, such as high stability, high biocompatibility, and good affinity with biomolecules, and are preferably suitable for protein or drug delivery. In addition, the size of the gold nanoparticles is preferably 13 nm, which achieves good efficacy in drug delivery and is not biologically toxic to organisms even at high concentrations. More specifically, the gold nanoparticles have a high affinity for thiols and amines on the surface of the protein, can form a strong gold-sulfur (Au-S) bond, and are not easily separated from the protein; Different subtypes of influenza virus, Jinnai particles have a good combination of effects. Further, after the preparation of the metal particle-protein complex of the present invention, it can be stored at 4 ° C for more than half a year and still has efficacy in use.

In the metal particle-protein complex of the present invention, the surface of the metal nanoparticle may form 20 to 70 of the carbohydrate binding protein, preferably 30 to 60 of the carbohydrate binding protein, more preferably 40- 55 of these carbohydrate-binding proteins. The saccharide-binding protein is mannose-binding protein (MBL), surfactant protein A, surfactant protein D, Conglutinin, CL- 43. Or galectin-1; preferably, galectin-1.

Among them, galectin-1 has different distribution and expression in normal tissues and pathogenic tissues, and various forms of cells produce galectin-1 in the case of viral infection or inflammation, which has sugars that recognize the surface of the pathogen. The ability to participate in a variety of biological reactions such as: intercellular adhesion, inflammation, cancer development, and host-pathogen interaction. Moreover, galectin-1 can regulate a variety of innate immunity and acquired immune responses, such as activation of immune cells.

Furthermore, the influenza vaccine of the present invention comprises: an influenza virus; and a metal particle-protein complex bound to the influenza protein, and the metal particle-protein complex comprises: a metal nanoparticle; and a sugar A class-binding protein is formed on the surface of the metal nanoparticle. That is, the influenza vaccine includes the above metal particle-protein complex to attenuate viral toxicity and form a more effective protective effect against a novel or mutant virus; and the influenza vaccine can provide an adjuvant function: attracting dendritic cells (DCs) and stimulating The immune response increases the phagocytosis of the virus by the antigen presenting cells.

In addition, the present invention further provides a method for preparing an influenza vaccine, comprising the steps of: (A) providing an influenza virus, and a metal particle-protein complex, wherein the metal particle-protein complex comprises: a metal nanoparticle, And a saccharide-binding protein, and the saccharide-binding protein is formed on the surface of the metal nanoparticle; and (B) mixing the influenza virus and the metal particle-protein complex; Flu vaccine.

Wherein the metal particle-protein complex binds to a set of membrane proteins of the influenza protein. Furthermore, the influenza virus of one virus titer unit may be combined with 10 ^-1 -10 ⁷ metal particle-protein complexes; preferably, it is combined with 10 ⁴ -10 ⁷ metal particle-protein complexes; more preferably It is combined with 10 ⁶ -10 ⁷ metal particle-protein complexes. Herein, a virus titer unit of influenza virus can be combined with "10 ^-1 " metal particle-protein complex, which means that a metal particle-protein complex can be combined with 10 influenza viruses. Furthermore, although a metal particle-protein complex can bind up to 10 viruses, in the examples of the present invention, the maximum amount of virus is not used, which is the purpose of the present invention to utilize metal particle-protein complexes. It is completely encapsulated or inhibits viral infectivity, so in terms of ratio, it is preferred to use a metal particle-protein complex in an amount greater than the amount of influenza virus.

Therefore, the simple-produced influenza vaccine provided by the present invention uses metal nanoparticles as a carrier of a carbohydrate-binding protein to enhance the affinity between the carbohydrate-binding protein and the virus. In addition, the metal particle-protein complex forms a strong affinity with the virus, thereby inhibiting or attenuating the virus's ability to infect, causing the virus to exhibit an attenuated state, and the attenuated virus forms a more effective protection against the mutant virus, except for various In addition to the effective protection of influenza viruses, the use of adjuvants can also result in longer and better immune responses.

[Preparation Example 1]

First, 108 ml of 0.6 mM HAuCl _{4 was} mixed with 2 ml of 195 mM sodium citrate and boiled under vigorous stirring for 10 minutes. The resulting burgundy suspension is then completely cooled, filtered under sterile conditions into a glass vial and stored at room temperature or 4 °C. Then, it was confirmed by a transmission electron microscope (TEM) that the spherical gold nanoparticles (AuNP) were uniformly dispersed in the liquid, and the AuNP diameter was about 13 ± 1.2 nm. The particle concentration of AuNP was calculated to be 9.2 nM (235 μg/ml) via the absorbance at 450 nm or the initial concentration of Au ³⁺ ions. Finally, 1 ml of AuNP (about 5-10x10 ¹² AuNPs) was added to 20-40 μg of galectin protein-1 and mixed uniformly. After gentle shaking and centrifugation, the gold nanoparticles-galectin-protein-1 complex was obtained. (AuNP/Gal-1) and suspended in a 10 nM sodium phosphate buffer solution at pH 7. The concentration or amount of AuNP/Gal-1 used in the following experimental examples was expressed by changing the Gal-1 content of the binding to AuNP.

[Experimental Example 1]

40 μg/ml of AuNP/Gal-1 was added to 512 HA of WSN/33 (H1N1) virus and mixed uniformly for 1 hour at room temperature, followed by centrifugation to quantify the vaccine composition, and then used 1% of pH 6.5 (w /v) phosphotungstic acid was applied to the composition for 3 minutes to fix and negatively stain the composition. The binding of AuNP/Gal-1 and WSN/33 (H1N1) viruses was observed by TEM at an acceleration voltage of 80 Kv, and the results are shown in Fig. 1.

Please refer to Figure 1. The photo on the left is WSN/33 (H1N1) virus, the middle and right photo is 40ug/ml of AuNP/Gal-1+10 ⁵ WSN/33 (H1N1) virus, TEM in two different fields of view. As a result, the smaller dark spherical particles were AuNP/Gal-1 complexes. The results showed that the AuNP/Gal-1 complex can be accurately bound to the surface of the influenza virus and wrapped in different forms and proportions outside the influenza virus envelope, which can cause viruses with different toxicities.

[Experimental Example 2]

The affinity of the AuNP/Gal-1 complex for influenza virus was measured by ELISA. First, 50 μl of 10 μg/ml Gal-1 was diluted with 0.1 M carbonate/bicarbonate buffer (pH 9.6), and then applied to a 96-well plate (Nunc-immuno plate, MaxiSorp surface, Nunc, Roskilde, Denmark), placed overnight at room temperature. Subsequently, it was treated with 1% (w/v) BSA for 2 hours, and after washing, 50 μl of 4HA different subtypes of influenza virus were added and cultured at 4 ° C for 24 hours. Thereafter, biotinylated goat anti-galectin-1 antibody (10 ng, R&D, Minneapolis, MN) was added for 2 hours at room temperature, followed by HRP-conjugated streptavidin (HRP-conjugated streptavidin, 1:200, R&D) was allowed to act at room temperature for 20 minutes. Finally, develop with 3,3',5,5'-tetramethylbenzidine (KPL), stop the enzyme reaction with 2N H ₂ SO ₄ after 5-10 minutes at room temperature, and detect it at a wavelength of 450 nm. The absorbance values are shown in Figure 2; the dissociation constant (Kd) is calculated as 1/2 Vmax by nonlinear regression analysis of Prism 5.0 kit software (GraphPad Software, San Diego, CA).

Please refer to Figure 2, from top to bottom, WSN/33 (H1N1) virus, Taiwan/N2723/06 (H3N2) virus, England /12/64 (H2N2) virus, and Philippines/2/62 (H3N2) virus, The solid group was added with AuNP/Gal-1, and the hollow group was added with Gal-1. The results showed that the Kd from top to bottom were 28 nM, 8 nM, 2.4 nM, 3.14 nM, 1.07 μM, 1.18 μM, 0.48 μM, and 0.89 μM, respectively, compared with the Gal-1 group. The Gal-1 complex has a higher affinity for different subtypes of influenza virus. Therefore, in this experimental example, the use of the gold nanoparticles as a carrier can enhance the affinity between the carbohydrate-binding protein and the virus.

[Experimental Example 3]

The AuNP/Gal-1 complex or Gal-1 stored at 4 ° C was observed for stability under different storage times. The method steps were the same as in Experimental Example 2 except that the virus was uniformly replaced with the 2HA WSN/33 (H1N1) virus, and the results are shown in FIG.

The results in Figure 3 show that the biological activity of the AuNP/Gal-1 complex can be maintained for at least six months, while the biological activity of Gal-1 is reduced by half after one month, and decreased to the original activity after two to six months. 20%. Thereby, the gold nanoparticles used in this experimental example can enhance the stability of biomolecules.

[Experimental Example 4]

Viral titer was detected by the Plaque assay. The MDCK cell suspension was mixed in a U-shaped 96-well plate (5×10 4 cells/well), infected with 0.1HA of WSN/33 (H1N1) virus, and then added with 1 μg/ml of AuNP/Gal-1, Gal- 1. AuNP/BSA, BSA, AuNP/PEG, or a buffer solution as a control group, which was allowed to act at room temperature for 2 days, and the results are shown in Fig. 4, and the plaque (PFU)/ml expressed in log ₁₀ units was expressed. And using t-test to analyze differences between groups.

Referring to Figure 4, the results show that the AuNP/Gal-1 complex exhibits potent antiviral activity, but if the AuNP/Gal-1 of this example, in which Gal-1 is substituted with BSA or PEG, the complex The excellent attenuation will disappear; in addition, the p-values of the AuNP/Gal-1 group and the Gal-1, AuNP/BSA, BSA, and AuNP/PEG groups were 0.033, 0.019, 0.014, and 0.0249, respectively, by t-test analysis. And 0.0249, there are statistically significant differences.

[Experimental Example 5]

The AuNP/Gal-1 complex obtained in Preparation Example 1 was combined with 1024HA virus to form an influenza vaccine. Among them, different proportions of AuNP/Gal-1 complexes (10μg, 20μg and 50μg in each group) were used to encapsulate influenza virus, and the viral infectivity was detected by a five-week-old female C57BL/6 mouse model. Only mice were tested. Here, the influenza vaccine was infected from the nasal cavity of the mouse, and the body weight and survival of the mice were observed daily, and the Kaplan-Meier survival curve and the log-rank test were used for survival analysis, and the t-test analysis was used. The mouse body weight difference, the results are shown in Figure 5, Figure 6.

Figure 5 is a mouse survival curve. All three groups of mice encapsulating the AuNP/Gal-1 complex survived and were significantly different (p = 0.03) compared to the influenza virus group (0 μg) alone. In addition, the influenza virus group alone caused about 70% of the mortality, which confirmed that the AuNP/Gal-1 complex effectively attenuated the infectivity of the influenza virus; and with reference to Figure 6, the mouse body weight change map, only the influenza virus group was injected alone. The mice were severely reduced in body weight and were significantly different from the other three groups (p < 0.001). Furthermore, AuNP/Gal-1 can inhibit the infection of viruses more stably than Gal-1 alone, and does not cause biological toxicity to normal cells. Thereby, the influenza vaccine of the present embodiment is attenuated vaccine with effective protection.

[Experimental Example 6]

Balb/c mice of six to eight weeks old were used to test the ability of influenza vaccines to be used for antibody production, and five mice per group were used for experiments. On the 0th and 14th day of the experiment, two doses of the AuNP/Gal-1/influenza virus influenza vaccine were administered intramuscularly, and there were three different ratio groups of 512HA, 256HA and 128HA and saline as the control group. Next, serum was collected on the 28th day, and analysis of influenza virus antibodies IgM, IgG, and IgA was performed, and the results are shown in Fig. 7 .

Figure 7 shows that three proportions of encapsulated influenza vaccines can promote influenza antibody production compared to the non-influenza virus group, while the 256HA ratio of influenza vaccine achieves optimal antibody production.

Based on the above results, galectin-1 inhibits the ability of viral infection by binding to influenza virus surface proteins, while the use of gold nanoparticles enhances galectin-1 function and provides strong multivalent linkages. , thereby increasing the affinity with the virus. Therefore, the AuNP/Gal-1 complex can be effectively developed into a novel vaccine against influenza virus infection.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

Fig. 1 is a TEM result chart of Experimental Example 1 of the present invention.

Fig. 2 is a graph showing the results of affinity analysis of Experimental Example 2 of the present invention.

Fig. 3 is a graph showing the results of the stability test of Experimental Example 3 of the present invention.

Fig. 4 is a graph showing the results of the plaque counting test of Experimental Example 4 of the present invention.

Fig. 5 is a graph showing the survival curve of the mouse of Experimental Example 5 of the present invention.

Fig. 6 is a graph showing changes in body weight of mice of Experimental Example 5 of the present invention.

Figure 7 is a graph showing the results of the production capacity of the antibody of Experimental Example 6 of the present invention.

Claims (16)

The use of a metal particle-protein complex for preparing an influenza vaccine comprises: a metal nanoparticle; and a carbohydrate binding protein formed on a surface of the metal nanoparticle; wherein the metal nanoparticle is The diameter system is 10-15 nm, and the surface of the metal nanoparticle is formed with 30 to 60 such carbohydrate-binding proteins. The metal particle-protein complex according to claim 1, wherein the material of the metal nanoparticle is gold, platinum, or an alloy thereof. The metal particle-protein complex of claim 1, wherein the metal nanoparticle is a gold nanoparticle. The metal particle-protein complex according to claim 1, wherein the carbohydrate-binding protein is mannose-binding protein (MBL), surfactant protein A, and surface activity. Protein D (Development Protein D), or Galectin-1. The metal particle-protein complex of claim 4, wherein the carbohydrate-binding protein is galectin-1. An influenza vaccine comprising: an influenza virus, which is 256HA or 512HA; and a metal particle-protein complex, which is combined with the influenza virus, and the metal particle-protein complex comprises: a metal nanoparticle; and a a saccharide-binding protein formed on the surface of the metal nanoparticle, wherein the metal nanoparticle has a particle diameter of 10-15 nm, and the surface of the metal nanoparticle is formed with 30-60 of the saccharide A binding protein; one of the viral titer units of the influenza virus system binds to 10 ^{-1 to} 10 ⁷ metal particle-protein complexes. The influenza vaccine of claim 6, wherein the metal particle-protein complex binds to a set of membrane proteins of the influenza virus. The influenza vaccine according to claim 6, wherein the material of the metal nanoparticle is gold, platinum, or an alloy thereof. The influenza vaccine of claim 6, wherein the metal nanoparticle is a gold nanoparticle. The influenza vaccine according to claim 6, wherein the carbohydrate-binding protein is mannose-binding protein, surfactant protein A, surfactant protein D, or galectin-1. The influenza vaccine according to claim 10, wherein the carbohydrate-binding protein is galectin-1. A method for preparing an influenza vaccine, comprising the steps of: (A) providing an influenza virus, and a metal particle-protein complex, wherein the influenza virus is 256HA or 512HA, and the metal particle-protein complex comprises: a metal naphthalene a rice particle, and a saccharide-binding protein, and the saccharide-binding protein is formed on the surface of the metal nanoparticle; wherein the metal nanoparticle has a particle diameter of 10-15 nm, and the metal nanoparticle The surface system is formed with 30-60 such glycoconjugate proteins; and (B) mixing the influenza virus and the metal particle-protein complex; wherein in step (B), a virus titer unit of influenza virus is used Mix with 10 ^-1 -10 ⁷ metal particle-protein complexes. The preparation method according to claim 12, wherein the material of the metal nanoparticle is gold, platinum, or an alloy thereof. The preparation method according to claim 12, wherein the metal nanoparticle is a gold nanoparticle. The preparation method according to claim 12, wherein the saccharide-binding protein is mannose-binding protein, surface active protein A, surface active protein D, or galectin-1. The preparation method according to claim 15, wherein the saccharide-binding protein is galectin-1.