Respiratory tract infection self-limiting virus live vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and particularly provides a respiratory tract infection self-limiting virus live vaccine as well as a preparation method and application thereof. The invention aims to solve the problems that in the prior art, the research and development progress of vaccines for respiratory tract infection self-limiting viruses is slow, existing candidate vaccines are invalid in single-time immunity, lack of cellular immunity, insufficient in broad-spectrum protection, high in safety risk and the like, and the production process is lack of standardization and non-uniform in quality control standard. Therefore, the invention provides the live vaccine for the respiratory tract infection self-limiting virus, which comprises the respiratory tract infection self-limiting virus which is not attenuated. The respiratory tract infection self-limiting virus live vaccine breaks through the safety limitation of a traditional attenuated live vaccine, it is proved for the first time that the unattenuated respiratory tract infection self-limiting virus can achieve safe and efficient immune response through subcutaneous inoculation, and a brand new technical path is provided for development of the respiratory tract infection self-limiting virus vaccine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically providing a live vaccine for a self-limiting respiratory infection virus, its preparation method, and its application. Background Technology

[0002] In recent years, respiratory viral infections have become a major challenge in global public health. While traditional live attenuated vaccines and inactivated vaccines have played a key role in the prevention of respiratory viral infections, they have significant limitations: although live attenuated vaccines can induce a strong immune response, there is a risk of virulence reversion; inactivated vaccines have a relatively high safety profile, but usually require the addition of adjuvants to achieve the desired immunization effect, and it is difficult to induce a sufficient cellular immune response.

[0003] In the existing technological system, influenza vaccine development is relatively mature, while the development of vaccines for other common respiratory viruses such as respiratory syncytial virus (RSV), parainfluenza virus (PIV), and human metapneumovirus (hMPV) has been slow.

[0004] It is noteworthy that the unique biological characteristics of these self-limiting respiratory viruses provide important insights for the development of novel vaccines: first, they mostly manifest as self-limiting infections in healthy adults; second, they primarily target and infect respiratory mucosal epithelial cells; and third, natural infection can induce a certain mucosal immune response.

[0005] However, existing vaccine technologies still face significant bottlenecks: First, traditional attenuation methods may alter the natural antigenic epitopes of the virus, affecting immunogenicity; second, inactivated vaccines are difficult to mimic the natural infection process of the virus, resulting in a gap between their immunization effect and natural immunity; and third, most vaccines cannot effectively induce mucosal and cellular immune responses, making it difficult to form the first line of defense and provide effective immune protection.

[0006] Furthermore, the requirements for culture conditions and purification processes vary significantly among different self-limiting viruses causing respiratory infections, increasing the difficulty of developing universal preparation systems. At the quality control level, key indicators such as the titer and residual host proteins in live viral vaccines have not yet been standardized, hindering vaccine quality stability.

[0007] In summary, the development of novel live vaccines against self-limiting respiratory viruses and their supporting preparation methods has become an urgent task. Summary of the Invention

[0008] The present invention aims to solve the above-mentioned technical problems, namely, to solve the problems of slow progress in vaccine development for self-limiting respiratory viruses (such as influenza virus, rhinovirus, respiratory syncytial virus, and some subtypes of coronaviruses), existing candidate vaccines have problems such as ineffectiveness of single immunization, lack of cellular immunity, insufficient broad-spectrum protection and high safety risks, and lack of standardization in production processes and inconsistent quality control standards.

[0009] In a first aspect, the present invention provides a live vaccine against a self-limiting respiratory infection virus, wherein the vaccine comprises an unattenuated self-limiting respiratory infection virus.

[0010] In the preferred embodiment of the above-mentioned self-limiting live vaccine for respiratory infections, the self-limiting live vaccine for respiratory infections does not contain adjuvant.

[0011] In the preferred embodiment of the above-mentioned self-limiting live vaccine for respiratory infections, the formulation of the self-limiting live vaccine for respiratory infections is suitable for subcutaneous injection.

[0012] In the preferred embodiment of the above-mentioned live vaccine for self-limiting respiratory infections, the viral titer of the unattenuated self-limiting respiratory infection virus is 10. 1 ~10 8 TCID 50 / mL, and the residual amount of host cell DNA is ≤25ng / mL, and the residual amount of host cell protein is ≤100μg / mL.

[0013] In the preferred embodiment of the above-mentioned live vaccine for self-limiting respiratory infections, the unattenuated self-limiting respiratory infection virus includes at least one of rhinovirus, coronavirus, seasonal influenza virus, human adenovirus, novel coronavirus infection, respiratory syncytial virus, or human metapneumovirus.

[0014] In a second aspect, the present invention provides a method for preparing the aforementioned self-limiting live viral vaccine for respiratory infections, comprising the following steps: S1. Culture: The self-limiting respiratory infection virus is inoculated into the culture of chicken embryos, MDCK, Vero, HEK293, 293T, MRC-5, A549 and other cells, and cultured to obtain cell culture virus solution; S2. Purification: The cell culture virus solution is subjected to deep filtration, ultrafiltration concentration or high-speed centrifugation, nuclease treatment and affinity chromatography purification in sequence, and the collected sample is the original solution; S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation.

[0015] In the preferred embodiment of the above preparation method, in step S2, the chromatography purification method includes ion exchange, SEC purification, affinity chromatography, etc.

[0016] In a third aspect, the present invention provides the use of the described respiratory infection self-limiting viral live vaccine in the preparation of a medicament for the prevention of diseases caused by respiratory infections via subcutaneous injection.

[0017] In the preferred embodiment of the above application, the drug is administered via a single or multiple immunization procedure.

[0018] The self-limiting live viral vaccine for respiratory infections of the present invention has the following technical effects: 1. The respiratory infection self-limiting virus live vaccine of this invention overcomes the safety limitations of traditional attenuated live vaccines. It is the first time that unattenuated respiratory infection self-limiting viruses have been demonstrated to achieve a safe and efficient immune response through subcutaneous inoculation, providing a novel technical pathway for vaccine development. Viral replication is strictly confined to draining lymph nodes and the spleen, and is completely cleared approximately 7 days after inoculation. No viral load or pathological damage was detected in vital organs such as the heart, liver, lungs, kidneys, spleen, intestines, and brain tissue. A single immunization of mice induces high levels of neutralizing antibodies, and mouse challenge experiments show a 100% protection rate, indicating that this strategy has a significant immunoprotective effect. 2. The formulation of the self-limiting live virus vaccine for respiratory infections of the present invention is suitable for subcutaneous injection. By changing the route of vaccination from respiratory infection (traditional live attenuated influenza vaccine route) to subcutaneous injection, the spread of the virus to the whole body is effectively prevented, and the potential risk of systemic infection is eliminated. 3. The live vaccine against self-limiting respiratory viruses of the present invention utilizes the natural conformational advantage of the intact antigens of live viruses to successfully activate a more advanced immune response. Compared with subunit vaccines, mRNA vaccines, split vaccines, and VLP vaccines, this strategy can more completely preserve viral antigen components, exhibiting superior broad-spectrum protective potential and providing a new solution to address the rapid mutation of self-limiting respiratory viruses. Attached Figure Description

[0019] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which: Figure 1 This is a process flow diagram of the method for preparing a self-limiting live virus vaccine for respiratory infections according to the present invention; Figure 2 This is a process flow diagram of the culture process in the preparation method of the self-limiting live virus vaccine for respiratory tract infection of the present invention; Figure 3 This is a process flow diagram of purification in the preparation method of the self-limiting live virus vaccine for respiratory infection of the present invention; Figure 4This diagram illustrates the subcutaneous immunization of Balb / c mice with high, medium, and low doses of live influenza PR8 strain virus and the results of weight changes after immunization. Figure 5 The results of the challenge protection experiment after immunization with high, medium and low doses of influenza PR8 strain; Figure 6 The results are for detecting high-dose influenza PR8 strain live virus subcutaneously immunized Balb / c mice; Figure 7 Results of immune challenge experiments with live and inactivated influenza vaccines; Figure 8 Results of cross-protection and heterologous challenge experiments with live influenza vaccines; Figure 9 Immunization challenge experiments for live virus vaccines, recombinant protein vaccines, and VLP vaccines; Figure 10 The results of qRT-PCR detection of viral load in various tissues and organs of mice challenged after a single immunization with live viral vaccine, recombinant protein vaccine, and VLP vaccine; Figure 11 The results show the viral load in organs of mice challenged after a single immunization with live virus, recombinant protein, and VLP booster immunization. Detailed Implementation

[0020] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0021] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0022] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0023] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0024] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0025] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0026] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0028] As noted in the background section, the development of vaccines against self-limiting respiratory viruses (such as rhinovirus and some subtypes of coronaviruses) is progressing slowly. Existing candidate vaccines suffer from problems such as ineffective single-dose immunization, lack of cellular immunity, insufficient broad-spectrum protection, and high safety risks. Furthermore, their production processes lack standardization, and quality control standards are inconsistent. This invention provides a live vaccine against self-limiting respiratory viruses. This vaccine is not attenuated during preparation and is administered via subcutaneous injection, inducing highly effective protection with a single immunization.

[0029] Specifically, in a first aspect, the present invention provides a live vaccine against a self-limiting respiratory infection virus, comprising an unattenuated self-limiting respiratory infection virus and free of adjuvant.

[0030] The respiratory infection self-limiting virus live vaccine provided by the present invention contains unattenuated respiratory infection self-limiting virus. It is not attenuated during the preparation process and is administered via subcutaneous injection. A single immunization can induce highly effective protection.

[0031] Preferably, the self-limiting live vaccine for respiratory infections does not contain adjuvants.

[0032] More preferably, the formulation of the self-limiting viral vaccine for respiratory infections is suitable for subcutaneous injection.

[0033] Attenuated live influenza vaccines, such as AstraZeneca's FluMist and Changchun BCHT's live attenuated vaccines, are nasal spray formulations. However, this invention achieves a subcutaneous injection formulation, overcoming the technical bias that traditional live vaccines cannot be injected. Furthermore, subcutaneous injection can bring about a better immune response or protection rate.

[0034] More preferably, the viral titer of the unattenuated respiratory infection self-limiting virus is 10. 1 ~10 8 TCID 50 / mL, and the residual amount of host cell DNA is ≤25ng / mL, and the residual amount of host cell protein is ≤100μg / mL.

[0035] Current technology generally holds that live viral vaccines must undergo attenuation to ensure safety. However, this invention directly uses unattenuated wild-type strains. By precisely controlling the viral titer and subcutaneous injection route, it was unexpectedly discovered that these strains can elicit a strong immune response without causing clinical infection symptoms. This discovery breaks through the technical prejudice that "wild-type strains inevitably lead to infection" and overturns the traditional understanding that "live vaccines must be attenuated." Furthermore, experimental results demonstrate that viral replication is strictly confined to draining lymph nodes and the spleen, and is completely cleared within 7-15 days post-inoculation. No viral load or pathological damage was detected in vital organs such as the heart, brain, and kidneys. A single immunization can induce high levels of neutralizing antibodies, and challenge experiments showed a 100% survival rate, indicating significant immunoprotective effects.

[0036] More preferably, the unattenuated respiratory infection self-limiting virus includes at least one of rhinovirus, coronavirus, seasonal influenza virus, adenovirus, novel coronavirus infection, respiratory syncytial virus, or human metapneumovirus.

[0037] In this invention, the rhinovirus is rhinovirus type A; the coronavirus is human coronavirus OC43; and the seasonal influenza virus is influenza virus H1N1 pdm09 strain.

[0038] Furthermore, in a second aspect, the present invention provides a method for preparing the aforementioned self-limiting live vaccine for respiratory infections, such as... Figure 1As shown, the preparation method is carried out according to the following steps: S1. Culture: The self-limiting respiratory infection virus was inoculated into chicken embryo, MDCK, Vero, HEK293, 293T, MRC-5, A549 and other cell cultures and cultured to obtain virus harvest fluid; S2. Purification: The virus harvested solution is subjected to deep filtration, ultrafiltration concentration or high-speed centrifugation, nuclease treatment and chromatography purification in sequence, and the collected sample is the original solution; S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation.

[0039] Traditional inactivated vaccines (such as Fluzone) require the addition of adjuvants and multiple administrations. This invention, through a combination of serum-free MDCK cell culture and specific purification processes, allows the unattenuated virus to retain its complete antigenicity, inducing durable humoral and cellular immunity with a single subcutaneous injection. This differs from the conventional understanding that "live vaccines require adjuvants to enhance their efficacy," thus overcoming the biases of existing technologies and achieving a synergistic effect of "adjuvant-free single-dose immunization."

[0040] In some preferred embodiments, such as Figure 2 As shown, when the unattenuated respiratory infection self-limiting virus is a seasonal influenza virus, the culture in step S1 is performed as follows: S11. Wash the culture container with MDCK cells that have grown at 80-100% confluence with PBS; S12. Mix the self-limiting respiratory infection virus with DMEM-F12 medium without FBS to obtain the inoculum solution; S13. Add the inoculum to the cleaned culture container and culture. S14. Add DMEM-F12 medium without FBS and add TPCK trypsin; S15. Continue culturing to obtain virus harvest fluid.

[0041] In some specific embodiments, in step S12, the volume ratio of influenza virus to FBS-free DMEM-F12 culture medium is 1:10 to 1:10000, preferably 1:100.

[0042] In this invention, the culture container includes, but is not limited to, cell culture flasks, cell factories, bioreactors, etc.

[0043] In some specific embodiments, in step S13, the culture is carried out at 30~37℃ and 0~5% CO2 for 0.5~3 hours; preferably, it is carried out at 37℃ and 5% CO2 for 2 hours.

[0044] In some specific embodiments, in step S14, the TPCK trypsin is added to a final concentration of 0~10 μg / mL, preferably 1 μg / mL.

[0045] In some specific embodiments, in step S15, the continued culture time is 24 to 72 hours, preferably 48 hours.

[0046] In some preferred embodiments, such as Figure 3 As shown, the purification in step S2 includes: S21. The virus harvested fluid is subjected to deep filtration to obtain filtrate; S22. The filtrate is concentrated by ultrafiltration to obtain a concentrated solution; S23. Add nuclease and MgCl2 to the concentrated solution, incubate overnight or digest with enzymes to obtain the digestion solution; S24. The enzyme digestion solution is filtered and purified by chromatography. The collected chromatographic solutions are combined to obtain the original solution.

[0047] In some specific embodiments, in step S21, the deep filtration uses a 0.8+0.6μm deep filter.

[0048] In some specific embodiments, in step S22, the ultrafiltration concentration is performed using a hollow fiber column of 100KD~500KD, preferably 300KD.

[0049] In some specific embodiments, in step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 100-1000 U / mL, preferably 250 U / mL, and adding MgCl2 to a final concentration of 1-10 mM, preferably 2 mM; the incubation is carried out at 37°C.

[0050] In some specific embodiments, in step S24, the filtration is performed using a 0.22μm filter; the chromatographic purification uses Core700 packing material.

[0051] In some preferred embodiments, in step S24, the equilibration buffer used in the chromatographic purification is a solution containing 20-60 mM PBS, 0.5-1.5 mM MgCl2, 80-120 mM NaCl, and with a pH of 7.4-7.6.

[0052] Existing technologies suggest that wild-type influenza viruses are prone to mutation during in vitro culture. This invention amplifies influenza viruses using MDCK cell culture technology, enabling unattenuated influenza viruses (H1N1 and H3N2) to maintain genetic stability during continuous passages. This overcomes the technical obstacle of "difficulty in industrializing wild-type viruses" and solves the technical problem of "stability of wild-type viruses in cell culture."

[0053] In some specific embodiments, in step S3, the filtration is performed through a 0.22 μm filter.

[0054] In this invention, the preparation method does not include the steps of inactivation treatment or toxicity reduction treatment.

[0055] It should be noted that, in this invention, the attenuation treatment refers to the operation that does not reduce the virulence of the self-limiting virus causing respiratory infection through molecular biological means or other methods (such as reverse genetics, gene recombination, continuous passage or chemical modification).

[0056] In a third aspect, the present invention provides the use of the described respiratory infection self-limiting viral live vaccine in the preparation of a medicament for the prevention of diseases caused by respiratory infections via subcutaneous injection.

[0057] In the preferred embodiment of the above application, the drug is administered via a single or multiple immunization procedure.

[0058] The following detailed embodiments illustrate the respiratory infection self-limiting live virus vaccine, its preparation method, and its application.

[0059] Example 1 This example involves the cultivation, concentration, and purification of influenza virus.

[0060] S1, Cultivation S11. Wash the MDCK cells (T175 flask) that have grown to 80-100% confluence twice with 10mL PBS; S12. Take 100 μl of H1N1 pdm09 and mix it with 10 mL of 0% FBS / DMEM-F12 medium to obtain the inoculum solution; S13. Add the inoculum to the cleaned T175 bottle and incubate at 34°C and 5% CO2 for 2 hours. S14. Add 20 mL of 0% FBS / DMEM-F12 and 30 μl of 1 mg / mL TPCK trypsin (final concentration 1 μg / mL). S15, culture 7 flasks, culture at 34℃ and 5% CO2 for 48h until cytopathic effect reaches 90%, aspirate the supernatant into 50mL centrifuge tubes, centrifuge at 3000rpm and 4℃ for 10min, aliquot into T75 cell culture flasks (about 200mL), and freeze at -80℃.

[0061] S2, Purification S21, Deep Filtration: Deep filtration was performed using a 0.8+0.6μm filter, followed by a 200mL pre-rinse and a 40mL top wash, yielding approximately 250mL of clear liquid.

[0062] S22, Ultrafiltration Concentration: The clarified liquid was concentrated and replaced using a 300KD hollow fiber column.

[0063] S23, Nuclease treatment: Add 1 KU of nuclease to a final concentration of 250 U / ml; then add 47 μL of 2 M MgCl2 to a final concentration of 2 mM (1:1000 addition), and incubate overnight at 4°C. S24, Chromatography: 0.45µm filter filtration Core 700 tomography steps: Regeneration: Buffer C (1M NaOH + 30% isopropanol); Equilibration: buffer A (40mM PBS, 1mM MgCl2, 100mM NaCl, pH 7.5); Sample loading: 5 mL / min, volume approximately 45.08 mL; Collection: Approximately 90 mL of elution buffer.

[0064] S3, Concentrated S31, Ultrafiltration Concentration: Concentrate 90mL of Core700 eluent using a 300KD hollow fiber column. The concentrate is about 11mL and the top wash is about 11mL. S32. The top wash solution was concentrated to 1 mL using a 100 KD ultrafiltration tube, and the solutions were combined to approximately 12 mL. S33. Sterilization and dispensing: Filter with a 0.22um syringe filter; add diluent (PBS) according to the concentration and dispense: 200μL×20 vials, 500μl×16 vials, freeze at -80℃.

[0065] Example 2 Unlike Example 1, the virus strain used in step S12 is the H1N1 PR8 strain.

[0066] Example 3 Unlike Example 1, the virus strain used in step S12 is H3N2.

[0067] Example 4 Unlike Example 1, the virus strain used in step S12 is of the Victoria lineage.

[0068] Example 5 Unlike Example 1, the virus strain used in step S12 is the Yamagata lineage of type B virus.

[0069] Example 6 Unlike Example 1, in step S24, the equilibration buffer A is a solution containing 20 mM PBS, 0.5 mM MgCl2, and 50 mM NaCl with a pH of 6.5.

[0070] Example 7 Unlike Example 1, in step S24, the equilibration buffer A is a solution containing 60 mM PBS, 1.5 mM MgCl2, 150 mM NaCl, and pH 8.0.

[0071] Example 8 The difference from Example 1 is: In step S13, the volume ratio of the virus strain to DMEM-F12 medium without FBS is 1:1000. In step S14, the incubation is carried out at 34°C and 5% CO2 for 1.5 hours. In step S15, the TPCK trypsin is added to a final concentration of 5 μg / mL; In step S16, the continued culturing time is 72 hours; In step S22, the concentration and fluid exchange are performed using a 300KD hollow fiber column; In step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 500 U / mL and adding MgCl2 to a final concentration of 5 mM; the incubation is carried out at 37°C.

[0072] Example 9 The difference from Example 1 is: In step S13, the volume ratio of the virus strain to DMEM-F12 medium without FBS is 1:10000. In step S14, the incubation is carried out at 34°C and 5% CO2 for 3 hours; In step S15, the addition of TPCK trypsin is to be done to a final concentration of 10 μg / mL; In step S16, the continued culturing time is 72 hours; In step S22, the concentration and fluid exchange are performed using a 300KD hollow fiber column; In step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 1000 U / mL and adding MgCl2 to a final concentration of 10 mM; the incubation is carried out at 37°C.

[0073] Example 10 The difference from Example 1 is: In step S13, the volume ratio of the virus strain to DMEM-F12 medium without FBS is 1:10. In step S14, the incubation is performed at 34°C for 0.5 hours; In step S15, TPCK trypsin was not added; In step S16, the continued culturing time is 72 hours; In step S22, the concentration and fluid exchange are performed using a 300KD hollow fiber column; In step S23, the addition of nuclease and MgCl2 involves adding nuclease to a final concentration of 100 U / mL and adding MgCl2 to a final concentration of 1 mM; the incubation is carried out at 37°C.

[0074] Experimental Example 1 The quality of the live influenza virus vaccine stock solution prepared in each embodiment was tested.

[0075] Detection methods: The viral titer, residual host cell DNA, and residual host cell protein of the influenza virus live vaccine stock solution prepared in each example were detected according to the methods in the Chinese Pharmacopoeia.

[0076] The results are shown in Table 1: Table 1

[0077] Experimental Example 2 This trial involved a complete protection trial using a single immunization with a high- or medium-dose live vaccine.

[0078] (1) Experimental steps: Twenty-five 6-8 week old female BALB / c mice were divided into 5 groups of 5 each. Each group was subcutaneously injected with PR8 strain live vaccine (derived from Example 2) (high-dose group: 5 × 10⁻⁶). 4 TCID 50 / ani, medium-dose group 5×10 2 TCID 50 / only and low-dose group 5×TCID 50 / ani), PBS (nasal drops) control group and PBS (subcutaneous injection) control group; 21 days post-immunization, the low, medium, and high dose groups and the PBS (subcutaneous injection) control group received 100×LD50. 50 The process of PR8 strain virus challenge via nasal drops is as follows: Figure 4 As shown in Figure A; Monitor weight and survival rate; Protection against PR8 strain challenge was performed 28 days post-immunization; viral load in lung / brain / liver / intestinal tissues was detected by qRT-PCR on days 4, 6, 8, and 14 post-challenge.

[0079] (2) Experimental results: The changes in body weight of BALB / c mice immunized subcutaneously with high, medium, and low doses of live influenza PR8 virus vaccine, compared with those in the PBS (subcutaneous injection) and PBS (nasal drop) control groups, are shown below. Figure 4 As shown in Figure B.

[0080] from Figure 4 The results in Figure B show that: after subcutaneous injection of high, medium, and low doses of influenza PR8 strain live vaccine into BALB / c mice, and subsequent challenge protection experiments were conducted on mice in the PBS (subcutaneous injection) control group and the PBS (nasal drop) control group, the body weight changes in the high, medium, and low dose groups were <5%. Figure 4 (Figure B) No respiratory distress or abnormal activity was observed.

[0081] The results of the challenge protection experiment for the high, medium, and low dose groups, the PBS (subcutaneous injection) control group, and the PBS (nasal drops) control group are as follows: Figure 5 As shown. Among them, Figure 5 Figure A shows the results of weight changes after immune challenge; Figure 5 Figure B shows the survival rate results after immune challenge; Figure 5 Figures C through F show the results of viral load detection in various tissues and organs of mice after challenge by qRT-PCR. Figure C shows the viral load detection results in lung tissue, Figure D shows the viral load detection results in brain tissue, Figure E shows the viral load detection results in liver tissue, and Figure F shows the viral load detection results in intestinal tissue.

[0082] from Figure 5 It can be seen that the protection rate of low, medium and high dose immunization groups in the challenge protection experiment was 100%. Figure 5 (Figure B).

[0083] like Figure 5 As shown, high and medium doses of the live viral vaccine immunization group can effectively protect BALB / c mice from high lethal doses (100×LD50). 50 The virus was infected with the PR8 strain. The PBS (subcutaneous injection) control group died 5 days after challenge, and a large number of viral gene copies were detected in the lungs (small amounts of virus were also found in brain and liver tissues); the medium-dose group (5×10⁻⁶)... 2 TCID 50 The immunized group had normal body weight and 100% survival rate after challenge, and the viral load in lung tissue decreased to 10. 2 ~10 3copies / μL (PBS (subcutaneous injection) control group was 10 6 ~10 7 copies / μL) Figure 5 Figure C). High-dose group (5×10) 4 TCID 50 After a single immunization (per animal), the protection rate against challenge was 100%, and the viral load in organs such as lung tissue, brain tissue, liver, and intestines was below the detection limit (<10 copies / μL). Figure 5 (Figures C, D, E, and F).

[0084] Conclusion: A single high-dose subcutaneous immunization can achieve complete protection in mice, and the virus can be rapidly cleared, indicating that it can induce good immunogenicity and protective effect.

[0085] Experimental Example 3 This trial involved a biodistribution study of a live viral vaccine (viral replication limitation and histopathological study).

[0086] (1) Experimental steps: Female BALB / c mice aged 6-8 weeks were subcutaneously injected with PR8 strain live vaccine (derived from Example 2) (5×10 4 TCID 50 / ani (subcutaneous immunization group) and intranasal immunization with PR8 strain live virus vaccine (50×TCID) 50 Five animals were included in each of the following groups: (nose-in-nasal immunization group) and PBS control group (NC). On days 3 and 7 post-immunization, mice subcutaneously immunized with the PR8 strain live vaccine were dissected, and lung, liver, heart, injection site skin, intestinal tissue, spleen, and lymph nodes were collected. The viral load in each tissue and organ was detected by qRT-PCR. On day 3 post-immunization, tissues and organs from the subcutaneous immunization group, the intranasal immunization group, and the control group were collected to prepare pathological sections and perform HE staining for observation. On days 7, 14, and 21 post-immunization, tissues and organs from the subcutaneously immunized group were collected to prepare pathological sections and perform HE staining for observation.

[0087] (2) Experimental results: Experimental results are as follows Figure 6 As shown, Figures A-1 and A-2 are the results of qRT-PCR detection of viral load in various tissues and organs of mice on days 3 and 7 after subcutaneous immunization with PR8 strain live vaccine, respectively; Figure B is the HE staining result of pathological sections of lung tissue from immunized mice; Figure C is the HE staining result of pathological sections of heart tissue from immunized mice; Figure D is the HE staining result of pathological sections of kidney tissue from immunized mice; Figure E is the HE staining result of pathological sections of brain tissue from immunized mice; and Figure F is the HE staining result of pathological sections of intestinal tissue from immunized mice.

[0088] from Figure 6 The results show that: PR8 strain live vaccine (5×10 4 TCID 50 (each animal) was immunized for 3 and 7 days, and the viral load in various tissues and organs was measured. The test results are as follows: Figure 6 Figures A-1 and A-2 show that 3 days post-immunization, low levels of nucleic acid copy numbers (102) were detected by qRT-PCR in lymph nodes, spleen, and skin at the injection site. 2 ~10 3 copies / μL); completely cleared 7 days after immunization (<10 copies / μL); 1 (copies / μL).

[0089] In addition, the PR8 strain live vaccine was administered via nasal drops (100×LD). 50 ) Infection group and PR8 strain live vaccine subcutaneously (5×10) 4 TCID 50 The pathological changes in the lungs of immunized mice (number of mice) were examined, and the results are as follows: Figure 6 Figure B shows that after subcutaneous immunization with the PR8 strain live virus vaccine, no significant changes were observed in the pathological changes of mouse lung tissue; however, after intranasal infection of mice with the PR8 strain live virus vaccine... Figure 6 The mice in Figure B showed lung congestion, significant hemorrhage, alveolar thickening, and inflammatory cell infiltration, confirming that the live virus vaccine was confined to the injection site and posed no risk of systemic spread. Furthermore, no significant abnormalities were observed in the pathological examination of the heart, kidneys, brain tissue, and intestines of mice in all groups (e.g., [figure B]). Figure 6 (Figures C-F) showed no obvious inflammatory cell infiltration.

[0090] Conclusion: After vaccination with PR8 strain live virus, the virus is strictly confined to the injection site and immune organs, cleared within 7 days, and there is no risk of systemic spread.

[0091] Test Example 4 This experiment involved cross-protection of different strains (subcutaneous immunization with H1N1 pdm09 followed by challenge with H1N1-PR8 strain).

[0092] (1) Experimental steps: Twenty-five female Balb / c mice aged 6-8 weeks were subcutaneously injected with a live H1N1 pdm09 virus vaccine (derived from Example 1) (high-dose group 5 × 10⁻⁶). 5 TCID 50 / ani, medium-dose group 5×10 3 TCID 50 / only and low-dose group 50×TCID 50The study included three groups of animals: a control group (PBS(SC) group) receiving subcutaneous injection of PBS solution and a negative control group (PBS group) receiving nasal drops of PBS solution (without immunization or challenge). Each group consisted of 5 animals. On day 21 post-immunization, the low, medium, and high dose groups and the PBS (SC) group were administered PR8 strain virus (100×LD). 50 Nasal droplet infection challenge, recorded as pdm09 (50 TCID) 50 SC) / PR8 group, pdm09 (5×10 3 TCID 50 SC) / PR8 group, 5×10 5 TCID 50 The SC / PR8 group and the PBS(SC) / PR8 group were used for challenge with PBS solution and were designated as the PBS / PBS group.

[0093] The body weight and survival rate of all groups were monitored, and the PR8 strain virus (100×LD) was detected by qRT-PCR. 50 The viral load in the lungs of each group after nasal droplet infection challenge.

[0094] (2) Experimental results: Experimental results are as follows Figure 7 As shown, where, Figure 7 Figure A shows the changes in body weight of mice challenged after immunization with the H1N1-pdm09 live vaccine; Figure 7 Figure B shows the survival rate of mice challenged after immunization with the H1N1-pdm09 virus live vaccine; Figure 7 Figure C shows the viral load in mouse lung tissue after challenge, detected by qRT-PCR. Figure 7 The D-plot shows the viral load in the heart of mice after challenge, as detected by qRT-PCR.

[0095] The results showed that ( Figure 7 Balb / c mice subcutaneously immunized with the high-dose H1N1 (pdm09) group showed no abnormal weight changes; the medium-dose group experienced a decrease followed by a recovery in weight; and the low-dose group and the PBS (SC) / PR8 group showed a continuous decrease in weight within one week. Figure 7 (Figure A), H1N1pdm09 virus live vaccine (5×10) 5 TCID 50 The survival rate was 100% for each animal, with an 80% survival rate in the medium-dose group, while all animals in the low-dose group and the PBS(SC) / PR8 group died. Figure 7 Figure B); H1N1 pdm09 virus live vaccine (5×10 5 TCID 50 / animal) lung virus load was 10 3 copies / μL (PBS(SC) / PR8 control group 10) 7copies / μL, reduced by 10 4 times) ( Figure 7 (Figure C); the viral load in the lungs of the medium-dose group was 10 5 The number of copies / μL was approximately reduced by about 100-fold, and there was no difference between the low-dose group and the PBS(SC) / PR8 control group. Figure 7 (Figure C).

[0096] Conclusion: Live influenza vaccines provide potent cross-protection through the advantage of whole viral components.

[0097] Experimental Example 5 This trial case involves a protective defect test of inactivated vaccines.

[0098] (1) Experimental steps: Twenty-five female Balb / c mice aged 6-8 weeks were divided into groups of five and subcutaneously injected with β-propiolactone-inactivated PR8 strain whole virus vaccine (with a viral titer of 5 × 10⁻⁶ before inactivation). 5 TCID 50 (Ina-PR8(5×10)) 5 TCID 50 (SC group), subcutaneous injection of PR8 strain live vaccine (derived from Example 2) 5×10 5 TCID 50 / group (PR8(5×10)) 5 TCID 50 Group SC) and subcutaneous injection of PR8 strain live vaccine (from Example 2) 5×10 6 TCID 50 / group (PR8(5×10)) 6 TCID 50 The study included two groups: a positive control group (PBS(SC) group) and a negative control group (PBS group) receiving PBS subcutaneous injection (PBS(SC) group) and PBS solution (nasal drops) without immunization or challenge (PBS group).

[0099] On day 21 post-immunization, Ina-PR8 (5×10) 5 TCID 50 SC group, PR8 (5×10) 5 TCID 50 SC group, PR8 (5×10) 6 TCID 50 SC group and PBS (SC group) with PR8 strain virus (100LD) 50 Nasal drops for drug administration, recorded as Ina-PR8 (5×10) 5 TCID 50 SC) / PR8 group, PR8 (5×10 5 TCID 50SC) / PR8 group, PR8 (5×10 6 TCID 50 The SC / PR8 group and the PBS(SC) / PR8 group; the PBS group was challenged with PBS solution and was designated as the PBS / PBS group.

[0100] The body weight and survival rate of all groups were monitored, and the PR8 strain virus (100×LD) was detected by qRT-PCR. 50 The viral load in the lungs of each group after nasal droplet infection challenge.

[0101] (2) Experimental results: Experimental results are as follows Figure 8 As shown, where, Figure 8 Figure A shows the changes in body weight of challenged mice after immunization with PR8 strain whole virus inactivated vaccine and different dose groups of live virus vaccine; Figure 8 Figure B shows the survival rate of mice challenged after immunization with PR8 strain whole virus inactivated vaccine and different dose groups of live virus vaccine; Figure 8 Figure C shows the results of qRT-PCR detection of viral load in mouse lung tissue after challenge.

[0102] Experimental results show that ( Figure 8 ): No abnormalities were observed in the body weight of Balb / c mice subcutaneously immunized with the PR8 strain live vaccine at any dose group, and all mice survived after challenge. Figure 8 Figure A); After a single immunization with the PR8 strain whole virus inactivated vaccine, the patient's weight initially decreased and then recovered, with a survival rate of 60% after challenge. Figure 8 (Figure B), and the viral load in the lung tissue after challenge is >10. 7 copies / μL ( Figure 8 Figure C); All members of the positive control (PBS(SC) / PR8) group died ( Figure 8 (Figure B), viral load in lung tissue >10 8 Approximately copies / μL ( Figure 8 (Figure C).

[0103] Conclusion: The PR8 strain live vaccine provides better protection than the whole virus inactivated vaccine.

[0104] Experimental Example 6 This trial case involves a recombinant protein vaccine that requires booster immunization testing.

[0105] (1) Experimental steps: Fifty female Balb / c mice aged 6-8 weeks were used. Five G1 mice were immunized once, subcutaneously injected with PR8-HA trimer (4.5 μg) + adjuvant (CpG + aluminum hydroxide). Twenty-one days later, the mice were challenged with the virus (100×LD50). 50PR8 strain virus challenged via nasal drops); G2: 5 mice were boosted with subcutaneous injections of PR8-HA trimer (4.5 μg) + adjuvant (CpG + aluminum hydroxide) twice (days 0 and 21), and challenged with virus 100×LD for 42 days later. 50 PR8 strain virus challenged via intranasal instillation); G3: 5 mice were immunized once with H1N1 VLP (non-PR8 strain) vaccine (3μg) + adjuvant (CpG + aluminum hydroxide), and challenged 21 days later (100×LD). 50 PR8 strain virus challenged via nasal drops); G4: 5 mice were booster immunized twice with H1N1 VLP (non-PR8 strain) vaccine (3μg) + adjuvant (CpG + aluminum hydroxide) (days 0 and 21); 42 days later, 100×LD50 was administered. 50 PR8 strain virus challenge via nasal drops; additional subcutaneous injection of 5×10 5 PR8 virus live vaccine (derived from Example 2) group, PBS solution (subcutaneous injection) positive control group (PBS(SC) group) (100×LD) 50 Two groups (n=5 each) were used to challenge PR8 strain virus via nasal drops, a negative control group (PBS group) with PBS solution (nasal drops), and a challenge group (no immunization and no challenge) with PBS solution. The challenge was performed on days 21 and 42, respectively.

[0106] Monitor body weight and survival rate; detect viral load in lung tissue, heart, brain tissue, and kidneys using qRT-PCR. 42 days later, the booster immunization group was given PR8 strain virus (100×LD). 50 Nasal drops for detoxification; Monitor body weight and survival rate; detect viral load in lung tissue, heart, brain tissue, and kidneys using qRT-PCR.

[0107] (2) Experimental results: Immune challenge and experimental results as follows Figure 9 , Figure 10 and Figure 11 As shown, where, Figure 9 Figure A is a schematic diagram of immune challenge; Figure 9 Figures B-1 and B-2 show the changes in body weight and survival rate of challenged mice after a single immunization (21 days). Figure 9 Figures C-1 and C-2 show the changes in body weight and survival rate of mice after challenge following booster immunization (42 days). Figure 9 The D-plot shows the serum antibody levels 14 days after immunization using a micro-neutralization assay. Figure 9 Figure E shows the antibody levels in serum 21 days after immunization, detected using the micro-cell method. Figure 9 The F-plot shows the serum antibody levels detected by the micro-neutralization assay 28 days after immunization. Figure 9The G-plot shows the serum antibody levels detected by the micro-neutralization method 35 days after immunization.

[0108] Experimental results show that ( Figure 9 No anti-HA antibodies were detected after a single immunization with PR8-HA, the survival rate after challenge was 0%, and the neutralizing antibody against PR8 strain was below the detection limit (<1:20). Figure 9 (Figure D); After challenge with the virus, the survival rate of mice in both the single-immunized and booster-immunized VLP groups was 0%, and the viral load of influenza virus HA in lung, heart, brain, and kidney tissues was >10. 6 copies / μL ( Figure 10 For the results of a single immunization and Figure 11 (As a result of enhanced immunity). Among them, Figure 10 In the figures, A shows the viral load in the lung tissue of challenged mice after a single immunization; B shows the viral load in the heart of challenged mice after a single immunization; C shows the viral load in the brain tissue of challenged mice after a single immunization; and D shows the viral load in the kidneys of challenged mice after a single immunization. Figure 11 In the figures, A shows the viral load in the lung tissue of mice challenged after booster immunization; B shows the viral load in the heart of mice challenged after booster immunization; C shows the viral load in the brain tissue of mice challenged after booster immunization; and D shows the viral load in the kidneys of mice challenged after booster immunization.

[0109] Following two immunizations with the recombinant protein (Day 35), the anti-HA antibody titer was 1:200, while in the live viral vaccine group, the anti-HA antibody titer was 1:800 on day 35. Viral load in various tissues was measured 21 days post-immunization; the residual viral load in the lungs of the live viral vaccine immunization group was <10. 3 copies / μL, while there was no significant difference between the PR8-HA protein single immunization group, the VLP single immunization group and the positive control (non-immunized group challenged only) (PBS(SC) / PR8) group (10 6 (copies / μL); viral load was measured in tissue samples 42 days post-immunization. The viral load in lung tissue of the live virus vaccine immunization group and the PR8-HA protein booster immunization group was 10 copies / μL). 2 ~10 3 copies / μL, while there was no significant difference between the VLP booster immunization group and the positive control (non-immunized group challenged only) (PBS(SC) / PR8) group (10 6 (copies / μL).

[0110] Conclusion: Recombinant protein vaccines rely on booster immunization and adjuvants, and their immunoprotective effect is significantly lower than that of live viral vaccines.

[0111] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A self-limiting live viral vaccine for respiratory infections, characterized in that, It includes unattenuated, self-limiting respiratory viruses.

2. The self-limiting live viral vaccine for respiratory infections according to claim 1, characterized in that, The described self-limiting live viral vaccine for respiratory infections does not contain adjuvants.

3. The self-limiting live viral vaccine for respiratory infections according to claim 2, characterized in that, The formulation of the self-limiting live viral vaccine for respiratory infections is suitable for subcutaneous injection.

4. The self-limiting live viral vaccine for respiratory infections according to claim 1, characterized in that, The viral titer of the unattenuated respiratory infection self-limiting virus was 10. 1 ~10 8 TCID 50 / mL, and the residual amount of host cell DNA is ≤25ng / mL, and the residual amount of host cell protein is ≤100μg / mL.

5. The self-limiting live viral vaccine for respiratory infections according to any one of claims 1-4, characterized in that, The unattenuated respiratory infection self-limiting viruses include at least one of rhinovirus, coronavirus, seasonal influenza virus, human adenovirus, novel coronavirus infection, respiratory syncytial virus, and human metapneumovirus.

6. A method for preparing a self-limiting live viral vaccine for respiratory infections according to any one of claims 1-5, characterized in that, The preparation method is carried out according to the following steps: S1. Culture: The self-limiting respiratory infection virus was inoculated into the cultures of chicken embryos, MDCK, Vero, HEK293, 293T, MRC-5, and A549 cells and cultured to obtain the virus harvest fluid. S2. Purification: The virus harvested solution is subjected to deep filtration, ultrafiltration concentration or high-speed centrifugation, nuclease treatment and chromatography purification in sequence, and the collected sample is the original solution; S3. Formulation: The stock solution is sterilized and filtered, and a diluent is added to prepare a liquid formulation, or a preservative is added and then freeze-dried to prepare a freeze-dried formulation.

7. The preparation method according to claim 6, characterized in that, In step S2, the chromatographic purification method includes ion exchange, SEC purification, and affinity chromatography.

8. The use of the self-limiting live viral vaccine for respiratory infections according to any one of claims 1-5 in the preparation of a medicament for the prevention of diseases caused by respiratory infections via subcutaneous injection.

9. The application according to claim 8, characterized in that, The drug is administered via a single or multiple immunization schedule.

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