Vaccine protective agent as well as preparation method and application thereof

Abstract

The invention provides a vaccine protective agent applied to an influenza attenuated live vaccine as well as a preparation method and application of the vaccine protective agent. The vaccine protective agent comprises recombinant human albumin, gelatin, cane sugar, sodium glutamate and arginine. The recombinant human albumin is adopted to replace human serum albumin, so that the potential safety hazard of plasma-derived viruses existing in a traditional human serum albumin protective agent can be avoided, and the occurrence rate of adverse reactions after inoculation is reduced. The influenza attenuated live vaccine prepared from the vaccine protective agent does not need freeze-drying and can be stably stored for a long time, and a solution is provided for preparation process optimization and safety upgrading of the influenza attenuated live vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biological products, and in particular to a vaccine protective agent, a preparation method and application thereof. Background Art

[0002] Influenza is an acute respiratory infection that poses a serious threat to human health. The World Health Organization (WHO) estimates that during an epidemic, it can spread rapidly worldwide, affecting 10% to 20% of the population. Even in non-epidemic years, influenza causes 3 to 5 million severe cases and 250,000 to 500,000 deaths annually. Influenza not only causes high morbidity and mortality, but also places a significant burden on public health systems and the economy. The elderly, children, pregnant women, and those with chronic illnesses or weakened immune systems are at high risk of influenza infection, prone to severe illness and even death. It is estimated that influenza causes billions of dollars in economic losses annually.

[0003] Influenza is caused by infection with influenza viruses, which belong to the Orthomyxoviridae family and are mainly divided into four types: type A (A), type B (B), type C (C), and type D (D). Influenza A and type B viruses are the main pathogens that cause seasonal influenza in humans. Influenza A viruses can be further divided into multiple subtypes (such as H1N1, H3N2, etc.) based on their surface antigens hemagglutinin (HA) and neuraminidase (NA). Influenza viruses are highly variable, especially influenza A viruses, whose antigenic drift and antigenic shift mechanisms lead to frequent viral mutations.

[0004] The World Health Organization (WHO) believes that the most effective way to prevent influenza and its severe consequences is vaccination. Safe and effective vaccines have been available and used for over 70 years. Influenza vaccines provide protection for healthy adults even when circulating viruses are not identical to the vaccine viruses. Vaccination is particularly important for people at high risk of severe influenza complications and for those who live with or care for people at high risk.

[0005] Currently, the influenza vaccines available on the market internationally include inactivated influenza vaccine (IIV), live attenuated influenza vaccine (LAIV), adjuvanted influenza vaccine and recombinant influenza vaccine, all of which are trivalent or quadrivalent vaccines.

[0006] The live attenuated influenza vaccine originated in 1957 at the Russian Academy of Medical Sciences Research, Institute of Experimental Medicine. It is a cold-adapted, attenuated influenza vaccine. In 2003, the FDA approved the live attenuated influenza vaccine produced by MedImmune. Immunogenicity data from clinical studies of the live attenuated influenza vaccine have shown that it is safe and highly effective. Immunization with LAIV can also reduce the incidence of pneumonia, bronchitis, and otitis media. This vaccine is recommended by the World Health Organization for the prevention of pandemic and seasonal influenza, and has been transferred to developing countries such as Thailand, India, and China.

[0007] LAIV vaccines have become an important option for preventing influenza. The live attenuated influenza vaccine currently available in the United States and Europe is produced by MedImmune. This vaccine is intended for people aged 2 to 49 years. Children aged 2 to 8 years should receive two doses of the vaccine at the first immunization, separated by at least four weeks. People aged 9 to 49 years should receive one dose each year before an influenza outbreak.

[0008] The production process for live attenuated influenza vaccines primarily involves inoculating the live attenuated influenza vaccine virus into chicken embryos, where the virus multiplies. The allantoic fluid from the chicken embryos is then harvested, purified through ultrafiltration and other purification processes, and a protective agent is added. To ensure vaccine stability and effectiveness, human albumin is often added as a protective agent. Although human albumin is a human-derived component and a batch-approved product, it carries safety concerns related to plasma-derived viruses.

[0009] Therefore, developing a safe, effective, stable and low-side-effect influenza live attenuated vaccine protective agent is one of the urgent issues to be addressed in this field. Summary of the Invention

[0010] In view of this, in order to address the potential safety risks of plasma-derived viruses in traditional human serum albumin protective agents in live attenuated influenza vaccines, the present invention provides a vaccine protective agent using recombinant human albumin instead of human serum albumin, as well as its preparation method and application. The vaccine protective agent provided by the present invention not only eliminates the potential risk of viral contamination of blood products, but also significantly improves the stability of the vaccine. The live attenuated influenza vaccine prepared using the vaccine protective agent of the present invention can achieve long-term stable storage without freeze-drying, providing an effective solution for optimizing vaccine preparation technology and improving safety. The specific amino acid sequence of the recombinant human albumin used in the present invention is detailed in: GenBank: CAA01491.

[0011] In a first aspect, the present invention provides a vaccine protective agent comprising the following components:

[0012] Recombinant human albumin: 0.1-5 wt.%, for example, 0.1, 0.2, 0.5, 1, 1.5, 2, 3, 4, 5 wt.%;

[0013] Gelatin: 0-4 wt.%, for example, 0, 0.2, 0.5, 1, 1.5, 2, 3, 4 wt.%;

[0014] Sucrose: 1-15 wt.%, for example, 1, 2, 3, 4, 5, 6, 6.84, 7, 8, 9, 10, 11, 12, 13, 14, 15 wt.%;

[0015] Sodium glutamate: 0.01-5 wt.%, for example, 0.01, 0.02, 0.05, 0.094, 0.2, 0.5, 1, 1.5, 2, 3, 4, 5 wt.%;

[0016] Arginine: 0.1-5 wt.%, for example, 0.1, 0.2, 0.5, 1, 1.21, 1.5, 2, 3, 4, 5 wt.%.

[0017] The gelatin content of 0 wt.% indicates that the vaccine protective agent of the present application does not contain gelatin.

[0018] In a preferred embodiment, the vaccine protective agent comprises the following components:

[0019] Recombinant human albumin: 0.2-4wt.%;

[0020] Gelatin: 0.2-3 wt.%;

[0021] Sucrose: 2-12 wt.%;

[0022] Sodium glutamate: 0.02-4wt.%;

[0023] Arginine: 0.2~4wt.%.

[0024] In a preferred embodiment, the vaccine protective agent comprises the following components:

[0025] Recombinant human albumin: 0.5-2wt.%;

[0026] Gelatin: 0.5-1.5wt.%;

[0027] Sucrose: 3-9 wt.%;

[0028] Sodium glutamate: 0.05-2wt.%;

[0029] Arginine: 0.5-2wt.%.

[0030] In a preferred embodiment, the vaccine protective agent further comprises a phosphate buffer.

[0031] In the second aspect, the present invention provides a method for preparing the vaccine protective agent described in the first aspect, which comprises the following steps: weighing each component according to its content, fully dissolving it in an appropriate amount of phosphate buffer, fixing the volume and adjusting the pH, filtering and sterilizing to obtain; or weighing each component in turn according to its content, separately dissolving each component fully in an appropriate amount of phosphate buffer, fixing the volume and adjusting the pH after mixing, filtering and sterilizing to obtain.

[0032] In a preferred embodiment, the prepared vaccine protective agent comprises the following components:

[0033] Recombinant human albumin: 0.1-5 g / ml, for example, 0.1, 0.2, 0.5, 1, 1.5, 2, 3, 4, 5 g / ml;

[0034] Gelatin: 0-4 g / ml, for example, 0, 0.2, 0.5, 1, 1.5, 2, 3, 4 g / ml;

[0035] Sucrose: 1-15 g / ml, for example, 1, 2, 3, 4, 5, 6, 6.84, 7, 8, 9, 10, 11, 12, 13, 14, 15 g / ml;

[0036] Sodium glutamate: 0.01-5 g / ml, for example, 0.01, 0.02, 0.05, 0.094, 0.2, 0.5, 1, 1.5, 2, 3, 4, 5 g / ml;

[0037] Arginine: 0.1-5 g / ml, for example, 0.1, 0.2, 0.5, 1, 1.21, 1.5, 2, 3, 4, 5 g / ml.

[0038] In a preferred embodiment, the prepared vaccine protective agent comprises the following components:

[0039] Recombinant human albumin: 0.2-4 g / ml;

[0040] Gelatin: 0.2-3 g / ml;

[0041] Sucrose: 2-12 g / ml;

[0042] Sodium glutamate: 0.02-4g / ml;

[0043] Arginine: 0.2~4g / ml.

[0044] In a preferred embodiment, the prepared vaccine protective agent comprises the following components:

[0045] Recombinant human albumin: 0.5-2 g / ml;

[0046] Gelatin: 0.5-1.5 g / ml;

[0047] Sucrose: 3-9 g / ml;

[0048] Sodium glutamate: 0.05-2g / ml;

[0049] Arginine: 0.5-2g / ml.

[0050] In a third aspect, the present invention provides use of the vaccine protective agent described in the first aspect in the preparation of a live attenuated influenza vaccine.

[0051] In a fourth aspect, the present invention provides a live attenuated influenza vaccine comprising a vaccine concentrate and the vaccine protective agent described in the first aspect.

[0052] In a preferred embodiment, the live attenuated influenza vaccine comprises influenza A virus and / or influenza B virus, wherein the titer of influenza A virus is ≥6.9 lg EID 50 / dose, influenza B virus titer ≥ 6.4lg EID 50 / dose.

[0053] In a fifth aspect, the present invention provides a method for preparing the live attenuated influenza vaccine described in the fourth aspect, comprising the following steps: mixing the qualified vaccine stock solution with the vaccine protective agent described in the first aspect, and packaging the mixture.

[0054] In a preferred embodiment, the vaccine stock solution and the 2X vaccine protectant are mixed in a 1:1 ratio.

[0055] The beneficial effects of the present invention are:

[0056] The present invention provides a vaccine protective agent containing recombinant human albumin and its use in live attenuated influenza vaccines. The vaccine protective agent of the present invention completely replaces traditional human serum albumin with recombinant human albumin as the main stabilizing component and does not contain dextran macromolecules. The live attenuated influenza vaccine preparation prepared using the vaccine protective agent of the present invention ensures the long-term stability of the liquid dosage form and eliminates the freeze-drying step required in the traditional process. The results of long-term stability experiments show that the stability of the live attenuated influenza vaccine preparation is significantly improved after recombinant human albumin is added to the formula, effectively solving the technical bottlenecks of existing vaccine protective agents, such as poor thermal stability, complex freeze-drying process and risk of sensitization by macromolecules. DETAILED DESCRIPTION

[0057] The advantages and technical effects of the present invention are described below with reference to specific examples. The scope of protection of the present invention shall be subject to the definition of the claims. The examples are merely illustrative of the concepts and principles of the present invention. It should be understood that these specific descriptions of the embodiments should not constitute a limitation on the scope of the present invention.

[0058] The present invention is described in detail through the following examples so that those skilled in the art can better understand the present invention. It should be understood that, unless otherwise stated, the instruments and equipment used in the following examples are conventional equipment in the art; the culture media used are all commercially available conventional culture media, the components and amounts of which are well known to those skilled in the art.

[0059] Example 1: Preparation of protective agents of different formulations of attenuated live influenza vaccines

[0060] The vaccine protectant for the live attenuated influenza vaccine was prepared by mixing the mixture with phosphate buffer at twice the concentration of each group of formulas in Table 1, fixing the volume and adjusting the pH to 6.0-8.0, and filtering and sterilizing. The vaccine protectant is a 2X vaccine protectant (the concentration is twice that in the formula, and the concentration after 2-fold dilution when used is the concentration in the following formula).

[0061] Table 1 Components and content information of vaccine protective agents (weight percentage)

[0062]

[0063]

[0064] Example 2: Preparation of live attenuated influenza vaccine

[0065] The vaccine is prepared by inoculating 9- to 11-day-old chicken embryos with attenuated strains of influenza A and B viruses recommended by the World Health Organization (WHO). After culture, harvesting, and purification of the virus fluid, the mixture is mixed in proportion. After adding the vaccine protective agent of Example 1, the vaccine is packaged according to the specification of 0.3 ml per vial to prepare a live attenuated influenza vaccine.

[0066] (1) Production virus: A reassortant attenuated influenza virus strain prepared by a WHO collaborating laboratory, H1N1: A / 17 / Victoria / 2022 / 28 (H1N1) pdm09; H3N2:

[0067] A / 17 / California / 2022 / 1410(H3N2); B: B / 60 / Austria / 2021 / 711.

[0068] (2) Virus inoculation and culture: Dilute the working seed batch virus with PBS, then inoculate the virus seed solution into the allantoic cavity of chicken embryos, 0.2 ml / piece, and incubate at 32.0°C for 2 to 3 days.

[0069] (3) Virus harvesting: Select chicken embryos with intact eggshells, clear blood vessels, and activity, place the embryos in a 4°C cold overnight, and then harvest the allantoic fluid, which is the single harvest fluid.

[0070] (4) Preparation of monovalent stock solution: The harvested single harvest solution is concentrated by ultrafiltration, and after concentration, it is purified by column chromatography or sucrose density gradient centrifugation. The purified virus solution is then sterilized and filtered through a 0.2 μm microporous filter membrane to obtain the monovalent stock solution.

[0071] (5) Preparation of live attenuated influenza vaccine: Mix the monovalent stock solutions of the three (sub)types (H1N1, H3N2, and B) and add 2X vaccine protective agent at a ratio of 1:1 to obtain the semi-finished product. Then, the product is packaged according to the specification of 0.3 ml / vial to obtain the live attenuated influenza vaccine preparation.

[0072] Example 3: Long-term stability study of live attenuated influenza vaccine

[0073] The influenza live attenuated vaccines of each formulation in Example 2 above were placed at 4°C for different periods of time, and samples were taken for appearance, pH, and virus titer determination to investigate the long-term stability of the product. The quality standards were in accordance with the 2020 edition of the Chinese Pharmacopoeia.

[0074] The experimental results are shown in Table 2:

[0075] Table 2 Virus titer results of 4℃ long-term storage experiment of influenza live attenuated vaccine (log EID 50 / dose)

[0076]

[0077]

[0078]

[0079] These long-term stability results demonstrate that the addition of recombinant human albumin to the live attenuated influenza vaccine formulation significantly improves stability. Furthermore, the live attenuated influenza vaccine prepared with recombinant human albumin as a protective agent exhibited minimal viral titer decline over the six-month observation period, significantly outperforming the vaccine prepared with human albumin as a protective agent. The present invention utilizes recombinant human albumin as a key component of the vaccine protective agent, significantly enhancing the long-term stability of the live attenuated influenza vaccine formulation.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A vaccine protective agent comprising the following components: Recombinant human albumin: 0.1-5wt.%; Gelatin: 0-4wt.%; Sucrose: 1-15wt.%; Sodium glutamate: 0.01-5wt.%; Arginine: 0.1~5wt.%.

2. The vaccine protective agent according to claim 1, comprising the following components: Recombinant human albumin: 0.2-4wt.%; Gelatin: 0.2-3 wt.%; Sucrose: 2-12 wt.%; Sodium glutamate: 0.02-4wt.%; Arginine: 0.2~4wt.%.

3. The vaccine protective agent according to claim 2, comprising the following components: Recombinant human albumin: 0.5-2wt.%; Gelatin: 0.5-1.5wt.%; Sucrose: 3-9 wt.%; Sodium glutamate: 0.05-2wt.%; Arginine: 0.5-2wt.%. The vaccine protectant according to any one of claims 1 to 3, further comprising a phosphate buffer.

5. A method for preparing the vaccine protective agent according to any one of claims 1 to 4, comprising the following steps: The components are weighed according to their contents, fully dissolved in an appropriate amount of phosphate buffer, fixed to volume and adjusted to pH, and then sterilized by filtration to obtain the product; or the components are weighed in sequence according to their contents, each component is fully dissolved in an appropriate amount of phosphate buffer, mixed, fixed to volume and adjusted to pH, and then sterilized by filtration to obtain the product.

6. The method according to claim 5, wherein: The prepared vaccine protective agent comprises the following components: Recombinant human albumin: 0.1-5 g / ml; Gelatin: 0-4g / ml; Sucrose: 1-15 g / ml; Sodium glutamate: 0.01~5g / ml; Arginine: 0.1~5g / ml.

7. Use of the vaccine protective agent according to any one of claims 1 to 4 in the preparation of a live attenuated influenza vaccine.

8. A live attenuated influenza vaccine comprising a vaccine stock solution and the vaccine protective agent according to any one of claims 1 to 4.

9. A method for preparing the live attenuated influenza vaccine according to claim 8, comprising the following steps: The vaccine concentrate that has passed the test is mixed with the vaccine protective agent according to any one of claims 1 to 4, and the mixture is divided into packages to obtain the vaccine.

10. The method according to claim 9, wherein the vaccine stock solution and the 2X vaccine protective agent are mixed in a ratio of 1:1.

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