Novel pathogen attenuated live vaccine and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a novel pathogen attenuated live vaccine and application thereof. The invention discloses an attenuated live vaccine, which comprises at least one pathogenic microorganism and a neutralizing antibody capable of carrying out a neutralizing reaction with the pathogenic microorganism, and provides a virus-antibody compound formed by combining the at least one pathogenic microorganism with the neutralizing antibody having a neutralizing capability on the pathogenic microorganism, the attenuated live vaccine, namely an attenuated live vaccine (such as an influenza attenuated live vaccine), has relatively good safety, immunogenicity and protectiveness, and has a better prevention or improvement effect compared with a traditional inactivated vaccine. Meanwhile, the preparation method of the attenuated live vaccine is simple, and compared with the traditional attenuated live vaccine, the attenuated live vaccine has the characteristics of quicker speed (the preparation time is about 1 hour) and easiness in preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel attenuated live pathogen vaccine and its application. Background Technology

[0002] Vaccination, as a core strategy for infectious disease prevention and control, aims to reduce the risk of infection and the severity of disease by activating specific immune responses. Currently approved vaccines primarily utilize inactivated vaccines, live attenuated vaccines, and subunit vaccines. While inactivated vaccines can effectively induce neutralizing antibody production, their limitation to humoral immune responses results in deficiencies in their protective efficacy and duration. In contrast, live attenuated influenza vaccines (LAIV) mimic the natural infection route, simultaneously stimulating mucosal, cellular, and humoral immune responses. Furthermore, the nasal spray delivery system offers the advantages of non-invasive administration and ease of use, making LAIV a crucial direction for next-generation influenza vaccine development.

[0003] Traditional attenuated virus technologies mainly rely on strategies such as cold adaptation, genome rearrangement, site-directed gene mutation, and single-cycle infection virus construction. However, these technologies generally suffer from bottlenecks such as lengthy development cycles (typically requiring 6-8 months) and insufficient attenuation stability (e.g., the potential risk of independent reversion). Although modern technologies such as reverse genetics have accelerated the construction of attenuated live vaccine strains, achieving a precise balance between attenuation efficacy and immunogenicity remains a key challenge for technological breakthroughs.

[0004] From the perspective of immune mechanisms, current research on infectious diseases mainly focuses on the neutralizing effect mediated by antibodies (Fab). This involves blocking the binding of key surface antigens of pathogens such as viruses (e.g., influenza virus HA and NA proteins) and bacteria through their antigen-binding domains, thereby weakening their toxicity, infectivity, or causing them to lose function, thus controlling pathogen infection. Recent studies have also revealed the significant synergistic role of antibodies (Fc) in antiviral immunity: their mediated effector functions include antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent trogocytosis (ADCT), and complement-dependent cell lysis, all of which are innate immune activation mechanisms. It is noteworthy that the Fc effects performed by different immune cell subsets exhibit functional heterogeneity: for example, dendritic cells can promote their maturation and high expression of MHC molecules through ADCP, effectively activating T cell immune responses; macrophage phagocytosis leads to pathogen destruction and release of pathogen-associated molecular patterns; neutrophil phagocytosis leads to pathogen destruction and recruitment of immune cells. These Fc-mediated immune responses can promote antigen presentation or secretion of inflammatory factors, thereby significantly enhancing the intensity of subsequent adaptive immune responses.

[0005] This section focuses on influenza viruses, also known as flu viruses, which are single-sense negative-sense RNA viruses belonging to the Orthomyxoviridae family. Currently, the main influenza viruses circulating in humans are influenza A (H1N1), influenza A (H3N2), and influenza B.

[0006] Based on the above situation, there is an urgent need to develop a live attenuated influenza vaccine with good preventive effect and simple and rapid preparation method. The Fab-Fc dual-function synergistic strategy, which makes full use of the neutralizing effect of antibody Fab and the ADCP effect of antibody Fc, may provide an innovative solution to break through the current bottlenecks in influenza vaccine development. Summary of the Invention

[0007] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention provides a novel attenuated live pathogen vaccine technology.

[0008] The first objective of this invention is to provide a live attenuated vaccine.

[0009] The second objective of this invention is to provide a method for preparing the live attenuated vaccine of the first aspect of this invention.

[0010] The third aspect of this invention aims to provide the use of the attenuated live vaccine of the first aspect of this invention in the preparation of medicaments for the prevention or improvement of diseases.

[0011] The fourth aspect of this invention is to provide a drug.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] A first aspect of the present invention provides an attenuated live vaccine comprising at least one pathogenic microorganism and a neutralizing antibody capable of neutralizing the pathogenic microorganism.

[0014] In some embodiments of the present invention, the pathogenic microorganism includes a virus.

[0015] In some embodiments of the present invention, the virus includes at least one of influenza virus, coronavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, norovirus, herpesvirus, EB virus, and Coxsackie virus.

[0016] In some embodiments of the present invention, the influenza virus includes at least one of influenza A virus, influenza B virus, and influenza A and C virus.

[0017] In some embodiments of the present invention, the pathogenic microorganism is an influenza A virus (such as influenza A virus A / Puerto Rico / 8 / 34PR8 strain), and the neutralizing antibody includes at least one of 1G01 antibody, 8F11 antibody, and MEDI8852.

[0018] A second aspect of the present invention provides a method for preparing a live attenuated vaccine according to the first aspect of the present invention, comprising the following steps: mixing viral microorganisms with neutralizing antibodies and incubating them to obtain a live attenuated vaccine.

[0019] In some embodiments of the present invention, the incubation conditions are 30-40°C and 3%-6% CO2 incubation for 0.5-2 hours.

[0020] In some preferred embodiments of the present invention, the incubation conditions are 35-40°C and 4%-6% CO2 for 1-2 hours.

[0021] In some more preferred embodiments of the present invention, the incubation conditions are 37°C and 5% CO2 incubation for 1 hour.

[0022] In some embodiments of the present invention, the titer of the pathogenic microorganism in the mixed system is 10–50 TCID. 50 / μL, the effective concentration of the neutralizing antibody is 0.1~2μg / μL.

[0023] In some preferred embodiments of the present invention, the titer of the pathogenic microorganism in the mixed system is 10–40 TCID. 50 / μL, the effective concentration of the neutralizing antibody is 0.1~1μg / μL.

[0024] In some more preferred embodiments of the present invention, the titer of the pathogenic microorganism in the mixed system is 20–30 TCID. 50 / μL, the effective concentration of the neutralizing antibody is 0.4 to 0.6 μg / μL.

[0025] A third aspect of the invention provides the use of the attenuated live vaccine of the first aspect of the invention in the preparation of a medicament for the prevention or improvement of a disease, including diseases caused by infection with pathogenic microorganisms.

[0026] In some embodiments of the invention, the disease includes diseases caused by infection with influenza viruses (influenza A, influenza B, and influenza A and C viruses).

[0027] A fourth aspect of the present invention provides a medicine comprising the live attenuated vaccine of the first aspect of the present invention.

[0028] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0029] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0030] In some embodiments of the present invention, the medicament may further comprise a pharmaceutically acceptable adjuvant or immunomodulator, selected from poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, MontanideISA51, etc.

[0031] In some embodiments of the present invention, the dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0032] In some preferred embodiments of the present invention, the route of administration of the drug includes at least one of nasal administration, transdermal administration, intraspinal administration, rectal administration, intra-articular administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and sublingual administration; preferably nasal administration or intramuscular injection.

[0033] In some embodiments of the present invention, the drug may also contain other pharmacologically active ingredients, thereby achieving combined treatment by using it in combination with other drugs.

[0034] In some embodiments of the present invention, the drug may be administered via local administration at a pharmaceutically acceptable dose.

[0035] The beneficial effects of this invention are:

[0036] This invention provides a virus-antibody complex formed by the binding of at least one pathogenic microorganism with a neutralizing antibody capable of neutralizing it, i.e., a live attenuated vaccine (such as a live attenuated influenza vaccine). This live attenuated vaccine has good safety, immunogenicity, and protective efficacy, and provides better prevention or improvement than traditional inactivated vaccines. Furthermore, the preparation method of this live attenuated vaccine is simple and faster (preparation time approximately 1 hour) and easier than traditional live attenuated vaccines. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 To illustrate the cytopathic effects under a microscope in the micro-neutralization experiment, the microscope was magnified 40 times. Among them, A shows the cytopathic effects of the 1G01+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 1) group at 0 and 24 hours after MDCK cell infection; B shows the cytopathic effects of the 8F11+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 2) group at 0 and 24 hours after MDCK cell infection; C shows the cytopathic effects of the PR8 positive control group at 0 and 24 hours after MDCK cell infection; and D shows the cytopathic effects of the negative control group at 0 and 24 hours after MDCK cell infection.

[0039] Figure 2 The graph shows the results of hemagglutination unit determination after a micro-neutralization experiment. In the graph, **** represents P < 0.001.

[0040] Figure 3 This is a line graph plotted based on mouse weight monitoring 14 days after immunization.

[0041] Figure 4 Survival curves were plotted based on the survival status of mice within 14 days after immunization.

[0042] Figure 5 The images show HE-stained pathological sections of mouse lungs on day 3 post-immunization, magnified at 400x. A represents HE-stained pathological section of mouse lungs on day 3 post-immunization in the PBS group; B represents HE-stained pathological section of mouse lungs on day 3 post-immunization in the PR8 (nasal drop group); C represents HE-stained pathological section of mouse lungs on day 3 post-immunization in the 1G01+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 1) group; D represents HE-stained pathological section of mouse lungs on day 3 post-immunization in the 8F11+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 2) group; and E represents HE-stained pathological section of mouse lungs on day 3 post-immunization in the PR8 (intramuscular injection group).

[0043] Figure 6 The images show HE-stained pathological sections of mouse lungs on day 5 post-immunization, magnified at 400x. A represents HE-stained pathological section of mouse lungs on day 5 post-immunization in the PBS group; B represents HE-stained pathological section of mouse lungs on day 5 post-immunization in the PR8 (nasal drop group); C represents HE-stained pathological section of mouse lungs on day 5 post-immunization in the 1G01+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 1) group; D represents HE-stained pathological section of mouse lungs on day 5 post-immunization in the 8F11+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 2) group; and E represents HE-stained pathological section of mouse lungs on day 5 post-immunization in the PR8 (intramuscular injection group).

[0044] Figure 7 The images show HE-stained pathological sections of mouse lungs on day 7 post-immunization, magnified at 400x. A represents HE-stained pathological section of the lungs of mice in the PBS group on day 7 post-immunization; B represents HE-stained pathological section of the lungs of mice in the PR8 (nasal drop group) group on day 7 post-immunization; C represents HE-stained pathological section of the lungs of mice in the 1G01+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 1) group on day 7 post-immunization; D represents HE-stained pathological section of the lungs of mice in the 8F11+PR8 (i.e., the live attenuated influenza vaccine prepared in Example 2) group on day 7 post-immunization; and E represents HE-stained pathological section of the lungs of mice in the PR8 (intramuscular injection group) group on day 7 post-immunization.

[0045] Figure 8 The figures show the viral titers in the lungs of mice on days 3, 5, and 7 post-immunization. In the figure, ns represents no significant difference, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.005.

[0046] Figure 9The titer of neutralizing antibodies in mouse serum on day 21 post-immunization.

[0047] Figure 10 The figure shows the concentration of sIgA in mouse bronchoalveolar lavage fluid. In the figure, ns represents no significant difference, and *** represents P < 0.005.

[0048] Figure 11 To monitor the body weight of mice after challenge with the virus.

[0049] Figure 12 Survival curves of mice after challenge with the virus.

[0050] Figure 13 The images show pathological sections of lung tissue stained with hematoxylin and eosin (HE) on day 3 after challenge with the virus in mice. The microscope magnification was 200x. Among them, A is a pathological section of lung tissue stained with HE on day 3 after challenge with the virus in the PBS group; B is a pathological section of lung tissue stained with HE on day 3 after challenge with the virus in the 1G01+PR8 group (i.e., the live attenuated influenza vaccine prepared in Example 1); C is a pathological section of lung tissue stained with HE on day 3 after challenge with the virus in the 8F11+PR8 group (i.e., the live attenuated influenza vaccine prepared in Example 2); and D is a pathological section of lung tissue stained with HE on day 3 after challenge with the virus in the PR8 (intramuscular injection group).

[0051] Figure 14 The figure shows the viral titer in mouse lung tissue on day 3 after challenge. In the figure, ns represents no significant difference, and * represents P < 0.05. Detailed Implementation

[0052] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0053] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] Unless otherwise specified, "room temperature" in this invention means 25℃±5℃.

[0055] In this embodiment of the invention, the term "influenza virus" refers to influenza A virus strain A / PR / 8 / 34, which is a variant of influenza A virus, subtype H1N1. It is a commonly used standard strain in the laboratory and is widely used in basic virology research, reverse genetics system construction, and vaccine development due to its ease of handling and high replication capacity in the laboratory.

[0056] The term "monoclonal antibody" refers to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen.

[0057] The term "Fab" refers to an antibody fragment formed by an intact light chain (the variable region (VL) and the constant region (CL) of the light chain) and an Fd fragment (the variable region (VH) of the heavy chain and the first constant region (CH1) of the heavy chain) through interchain disulfide bonds.

[0058] The term "Fc" refers to one of the fragments obtained after an antibody is hydrolyzed by papain. It has no antigen-binding activity but can interact with the Fc receptor.

[0059] The term "neutralizing antibody" refers to an antibody that protects cells from infection by specifically binding to the surface structure of pathogens or infectious particles via its Fab, thereby blocking their biological effects and significantly reducing or even completely eliminating their infectivity and pathogenicity (Reference: Klasse P J. Neutralization of virus infectivity by antibodies: old problems in new perspectives[J]. Advances in biology,2014,2014(1):157895.). Subsequently, the Fc of the neutralizing antibody binds to the Fc receptor on the surface of macrophages, thereby promoting the clearance of antigen-antibody complexes. Furthermore, previous literature has demonstrated that the immune responses induced by antibody Fc-mediated ADCP vary among different phagocytes. For example, phagocytosis by dendritic cells can promote their maturation and high expression of MHC molecules, effectively activating T cell immune responses (Reference: Pincetic A, Bournazos S, DiLillo DJ, et al. Type I and type II Fcreceptors regulate innate and adaptive immunity[J]. Nature immunology, 2014, 15(8):707-716.); phagocytosis by macrophages leads to pathogen destruction and release of pathogen-related molecular patterns (Reference: Sutterwala FS, Noel GJ, Clynes R, et al. Selective suppression of interleukin-12 induction after macrophage receptor ligation[J]. The Journal of experimental medicine, 1997, 185(11):1977-1985.); phagocytosis by neutrophils leads to pathogen destruction and recruitment of immune cells (Reference: Mayadas TN, Tsokos GC, Tsuboi N. Mechanisms of Immune complex–mediated neutrophil recruitment and tissue injury[J].Circulation,2009,120(20):2012-2024.), these responses can promote antigen presentation or secretion of inflammatory factors, thereby stimulating downstream adaptive immune responses.

[0060] As can be seen from the above, neutralizing antibodies can effectively inhibit the binding of pathogens to host cells by specifically binding to antigenic epitopes on the surface of pathogens, thereby significantly reducing or even completely eliminating their infectivity and pathogenicity. They also enhance the immune response. Therefore, this invention establishes a method for preparing a novel live attenuated vaccine, and uses influenza virus as a model to verify that the live attenuated vaccine has good safety, immunogenicity, and protective efficacy.

[0061] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0062] Example 1

[0063] A method for preparing an attenuated live influenza vaccine includes the following steps:

[0064] S1. Dilute the influenza A virus A / Puerto Rico / 8 / 34 (PR8) strain to obtain a titer of 1000 TCID. 50 / 25μL of virus dilution;

[0065] S2. Dilute the monoclonal antibody 1G01 (heavy chain amino acid sequence as shown in SEQ ID NO:1, light chain amino acid sequence as shown in SEQ ID NO:2) (solvent is PBS) to obtain a neutralizing antibody dilution with a concentration of 1 μg / μL.

[0066] S3. Mix equal volumes of virus diluent and neutralizing antibody diluent, place in a cell culture incubator, and incubate for 1 hour at 37°C and 5% CO2 to obtain an attenuated live influenza vaccine.

[0067] The amino acid sequence of the heavy chain of monoclonal antibody 1G01 is: METDTLLLWVLLLWVPGSTGDEVQLVESGGRALRPGGSLRLSCAASGFKFDDYAMSWVRQVPGKGLEFVSGLNWNGDITAYTDSVKGRFTVSRDNAKNSLYLHINSPKPEDTALYYCARTSSWGDYTRGPEPKITWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1).

[0068] Amino acid sequence of the light chain of monoclonal antibody 1G01: METDTLLLWVLLLWVPGSTGDDIQLTQSPSFLSASVGDRITITCRASQGIDGYLAWYQQRPGKAPNLLIYAASLLQSGVPSRFSGSGYGTEFTLTISSLQPEDFATYYCQH LDSYPLFTFPGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS(SEQ ID NO:2).

[0069] Example 2

[0070] A method for preparing an attenuated live influenza vaccine includes the following steps:

[0071] S1. Dilute the influenza A virus A / Puerto Rico / 8 / 34 (PR8) strain to obtain a titer of 1000 TCID. 50 / 25μL of virus dilution;

[0072] S2. Dilute the monoclonal antibody 8F11 (heavy chain amino acid sequence as shown in SEQ ID NO:3, light chain amino acid sequence as shown in SEQ ID NO:4) using PBS as solvent to obtain a neutralizing antibody dilution with a concentration of 1 μg / μL.

[0073] S3. Mix equal volumes of virus diluent and neutralizing antibody diluent, place in a cell culture incubator, and incubate for 1 hour at 37°C and 5% CO2 to obtain an attenuated live influenza vaccine.

[0074] Heavy chain amino acid sequence of monoclonal antibody 8F11: METDTLLLWVLLLWVPGSTGDQIQLVQSGPELRKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYSGEPTYADDFKGRFVFSLETSASTAYLLINNLNNEDMATYFCASLRRDAMDHWGPGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3).

[0075] The light chain amino acid sequence of monoclonal antibody 8F11 is: METDTLLLWVLLLWVPGSTGDDIVLTQSPASLAVSLGQRATISCKASHSVDFDGYVYMNWYQQKPGQPPKLLIYAASNLESGIPARFTGGGSGTDFTLNIHPVEEEDAATYSCQQSREDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS (SEQ ID NO:4).

[0076] Example 3

[0077] This example is used to evaluate the safety of the live attenuated influenza vaccines prepared in Examples 1 and 2, as detailed below:

[0078] A micro-neutralization experiment was performed in vitro as follows: 25 μL of 1 μg / μL 8F11 or 1G01 was mixed with 25 μL of 1000 TCID50. 50 After mixing 25 μL of PR8, the mixture was incubated at 37°C for 1 hour to obtain a live attenuated influenza vaccine (Examples 1-2). 50 μL of the live attenuated influenza vaccine and 500 μL / well of virus maintenance medium (500 μL 20% BSA + 500 μL triple antibody (penicillin-streptomycin-amphoteric B (100X)) + 50 μL 2 mg / mL TPCK trypsin + 48950 μL DMEM medium) were added to a 12-well MDCK cell plate washed with PBS. The PR8 positive control group received 25 μL of 1000 TCID after adding 500 μL / well of virus maintenance medium. 50 / 25μL of PR8. Incubate at 37℃ for 1 hour, then add 500μL / well of virus maintenance medium. After 24 hours, observe the cytopathic effect under a Nikon ECLIPSE Ti2 inverted microscope at a magnification of 40x (e.g., ...). Figure 1 As shown), and HA was measured (50 μL of culture supernatant was placed in a U-shaped plate, 50 μL of 2% guinea pig blood was added, and the results were recorded after 45 min). The cytopathic effect was as follows. Figure 1 As shown, the HA detection results are as follows: Figure 2 As shown.

[0079] Based on microscopic observation of cytopathic effects (in micro-volume neutralization experiments) Figure 1 The display shows that, Figure 1 China A and Figure 1Figure B shows the cytopathic effects of the live attenuated influenza vaccine prepared in Example 1 and the live attenuated influenza vaccine prepared in Example 2, respectively. It can be seen that the range of cytopathic effects caused by the two vaccines is significantly different. Figure 1 The extent and severity of lesions in C (PR8 positive control group) cells were significantly smaller. Combined with the results of the hemagglutination assay ( Figure 2 The hemagglutination units of the live attenuated influenza vaccine prepared in Example 1 and the live attenuated influenza vaccine prepared in Example 2 were significantly lower, indicating that they significantly inhibited the replication ability and pathogenicity of PR8, that is, they showed certain safety in in vitro experiments.

[0080] To further evaluate the safety of this live attenuated influenza vaccine, the inventors also conducted in vivo testing in mice. Specifically, 6-8 week old female mice were anesthetized with 4%–5% isoflurane at an oxygen flow rate of 1 L / min. Immunization was performed by intranasal instillation of 50 μL of the virus-antibody mixture per mouse using a 200 μL pipette. For the intramuscular injection group, 50 μL of the virus-aluminum adjuvant mixture (25 μL 1000 TCID50) was injected into each mouse using a 1 mL syringe. 50 / 25μL PR8 with 25μL aluminum adjuvant, mimicking an inactivated vaccine).

[0081] The mice were weighed daily for 14 consecutive days (e.g., Figure 3 As shown), and the survival status of the mice was recorded (e.g. Figure 4 (As shown). Eyeballs were removed on days 3, 5, and 7 post-immunization to collect serum. Lung tissue was obtained from dissection, and some lung tissue was subjected to HE staining for pathological examination (as shown). Figure 5 , 6 (See Tables 7 and 1). The remaining lung tissue was homogenized and then subjected to TCID. 50 Methods for detecting infectious viral titers in tissues (e.g.) Figure 7(As shown). The specific steps for determining the infectious virus titer in tissues are as follows: Add 230 μL of influenza virus maintenance medium to 7 wells in each of 6 columns of a 96-well plate, with intervals between columns. Then, transfer 105 μL of lung tissue homogenate to the first well, ensuring the pipette tip does not touch the maintenance medium. Immediately replace the pipette tip and mix the liquid in the first well with the new tip. Then, transfer another 105 μL to the second well, again ensuring the pipette tip does not touch the maintenance medium. Immediately replace the pipette tip and mix the liquid in the second well with the new tip. Repeat this process for the third well, and so on, for a total of 14 dilutions. After dilution, add 150 μL / well of 1×PBS to a 96-well plate containing MDCK cells. Gently tap the plate and then aspirate the liquid. Next, add 35 μL of virus dilution to the washed plate. One lung tissue homogenate sample is tested using one 96-well MDCK cell plate, with 5 replicates. After incubating at 37°C for 1 hour, add 200 μL / well of virus maintenance medium. The HA results were measured 48 hours later (50 μL of culture supernatant was placed in a U-shaped plate, 50 μL of 2% guinea pig blood was added, and the results were recorded and TCID was calculated after 45 min). 50 value).

[0082] Based on the above-mentioned influenza live attenuated vaccine mouse weight monitoring over 14 consecutive days ( Figure 3 The results showed that, except for the PR8 (nasal drop group), no significant weight loss occurred in any of the other groups. Furthermore, the survival curves... Figure 4 As shown in the figure, except for PR8 (nasal drop group), where one mouse died on day 5 and two mice died on day 7, no mice died in the other groups, indicating that the novel live attenuated influenza vaccine has good safety.

[0083] Figure 5 The results showed that, compared with the PBS group on day 3 post-immunization, the PR8 (nasal drop group) exhibited increased inflammatory cell infiltration, a higher number of samples with thickened alveolar walls, and small-scale hemorrhage and minimal necrosis in all samples; the 1G01+PR8 group (i.e., the influenza live attenuated vaccine treatment group of Example 1) showed no significant differences except for an increased number of samples with thickened alveolar walls; the 8F11+PR8 group (i.e., the influenza live attenuated vaccine treatment group of Example 2) showed no significant differences; and the PR8 (intramuscular injection group) showed minor hemorrhage in a few samples, but no other significant differences. Compared with the PR8 (nasal drop group) on day 3 post-immunization, the 1G01+PR8 group showed reduced inflammatory cell infiltration, and no hemorrhage or necrosis was observed in any samples; the 8F11+PR8 group showed reduced inflammatory cell infiltration, and no hemorrhage or necrosis was observed in any samples; and the PR8 (intramuscular injection group) showed reduced inflammatory cell infiltration, a decreased number of hemorrhage samples, and no necrosis was observed in any samples.

[0084] Figure 6The results showed that, compared with the PBS group on day 5 post-immunization, the PR8 (nasal drop group) exhibited increased inflammatory cell infiltration, a higher number of hemorrhage samples, and extensive mild to moderate thickening and significant necrosis in all samples; in the 1G01+PR8 group, some samples showed extensive mild to moderate alveolar wall thickening; in the 8F11+PR8 group, most samples showed extensive mild alveolar wall thickening; and in the PR8 (intramuscular injection group), most samples showed extensive mild alveolar wall thickening, with no other significant differences. Compared with the PR8 (nasal drop group) on day 5 post-immunization, the 1G01+PR8 group showed reduced inflammatory cell infiltration, a lower number of hemorrhage samples, and no necrosis in all samples; the 8F11+PR8 group showed reduced inflammatory cell infiltration and alveolar wall thickening, a lower number of hemorrhage samples, and no necrosis in all samples; and the PR8 (intramuscular injection group) showed reduced inflammatory cell infiltration, a lower number of hemorrhage samples, and no necrosis in all samples, with no other significant differences.

[0085] Figure 7 The results showed that, compared with the PBS group on day 7 post-immunization, the PR8 (nasal drop group) showed increased inflammatory cell infiltration and a larger number of samples, with most samples exhibiting alveolar wall thickening and necrosis; the 1G01+PR8 group showed increased inflammatory cell infiltration and a larger number of samples, with some samples showing extensive mild to moderate alveolar wall thickening and a small amount of necrosis, but no significant hemorrhage was observed in any of the samples, and there were no other significant differences; the 8F11+PR8 group showed extensive mild alveolar wall thickening in all samples, but no significant hemorrhage was observed in any of the samples; and the PR8 (intramuscular injection group) showed no significant hemorrhage, and there were no other significant differences. Compared with the PR8 (nasal drop group) on day 7 after immunization, no obvious bleeding was observed in the 1G01+PR8 group samples, and there were no other significant differences; the 8F11+PR8 group samples all showed a reduction in inflammatory cell infiltration, and no bleeding or necrosis was observed in the samples; the PR8 (intramuscular injection group) samples all showed a reduction in inflammatory cell infiltration, and no alveolar wall thickening, bleeding, or necrosis was observed in the samples, and there were no other significant differences.

[0086] Combination Figure 5 , Figure 6 , Figure 7 According to the results in Table 1, the lung pathological changes in the 1G01+PR8 and 8F11+PR8 groups were similar to those in the PBS group, and the lung pathological changes were significantly reduced compared to the PR8 (nasal drop group), so their safety was relatively good.

[0087] Table 1. HE staining pathological scores of mouse lungs on days 3, 5, and 7 post-immunization.

[0088]

[0089]

[0090] Note that in the table, "in" stands for Intranasal administration group, i.e., nasal drops group; and "im" stands for Intramuscular injection group, i.e., intramuscular injection group.

[0091] The pathological scoring criteria in Table 1 are referenced from [US] Peter Mann et al., International Standard for Terminology and Diagnostic Criteria of Pathological Changes in Rats and Mice (INHAND) [M]. Translated by Yang Lifeng, Zhou Xiangmei, and Zhao Deming. Beijing: China Agriculture Press, 2019.

[0092] Example 4

[0093] This embodiment tests the immunogenicity of the live attenuated influenza vaccines prepared in Examples 1 and 2, as detailed below:

[0094] Mice immunized in Example 3 were randomly divided into 3 groups of 15 mice each on day 19 post-immunization. Blood was collected from the ophthalmic veins, and serum was separated. On day 21 post-immunization, 3 mice were randomly selected from the above groups, their eyeballs were removed, and they were euthanized. Blood was collected, serum was separated, and mixed with the serum collected on day 19 for neutralizing antibody titer determination. Additionally, bronchoalveolar lavage fluid was collected for mucosal immunoassay sIgA titer determination.

[0095] Neutralizing antibody titer assay: Add 80 μL of the test sample to the first well of a 96-well plate, and 40 μL / well of virus maintenance medium to the second through tenth wells. Serially dilute the sample two-fold, and remove the 40 μL sample from the last well. Dilute the influenza virus PR8 strain to 200 TCID using virus maintenance medium. 50 Add 35 μL of virus sample mixture to each well, then add 40 μL of virus diluent to each well. Gently tap the 96-well plate to mix, and incubate at 37°C for 1 hour. After approximately 1 hour, wash the plated 96-well MDCK cell culture plate with 1×PBS and aspirate any remaining liquid. Add 35 μL / well of the incubated virus sample mixture to each well of the washed 96-well MDCK cell culture plate. Add 200 μL / well of virus maintenance solution to column 11 as a negative control, and add 35 μL / well of serially diluted virus diluent as TCID in column 12. 50 After quality control, the sample was incubated at 37°C for 1 hour. After 1 hour, samples were added to the sample wells and TCID250. 50 For quality control, add 200 μL / well of virus maintenance medium and incubate at 37°C for 48 hours. After 48 hours, detect HA (take 50 μL of culture supernatant into a U-shaped plate, add 50 μL of 2% guinea pig blood, and record the result after 45 minutes). Calculate the neutralization titer of the sample using the Reed-Muench method; for example, a value of 64 represents a 1:64 dilution of the immune serum.

[0096] Determination of sIgA titer in bronchoalveolar lavage fluid: The mouse secretory immunoglobulin A (sIgA) ELISA kit (catalog number: JL11763-96T) purchased from Jianglai Biotechnology was used for the determination according to its instructions.

[0097] The results of neutralizing antibody titer determination in each group are as follows: Figures 8-9 As shown, the results indicated that the neutralizing antibody levels induced by the live attenuated influenza vaccines prepared in Examples 1 and 2 were higher than those in the PR8 (intramuscular injection group) and PBS group. The concentrations of sIgA in the bronchoalveolar lavage fluid of mice in each group are shown below. Figure 10 As shown, the results indicate that the sIgA induced by the live attenuated influenza vaccine prepared in Example 2 was significantly higher than that in other groups. These results demonstrate that the live attenuated influenza vaccines prepared in Examples 1 and 2 possess good immunogenicity.

[0098] Example 5

[0099] Twelve mice immunized in Example 3 were used on day 21 post-immunization at a dose of 5 × 10⁻⁶ mg / L. 5 TCID 50 Influenza virus PR8 strain was challenged intranasally with a titer of 50 μL. The challenge volume was 50 μL. Mice were weighed daily for 14 days post-challenge, and their survival was recorded to evaluate vaccine protective efficacy. Figure 11 and Figure 12 (As shown), if a mouse's weight drops below 75% of its initial weight, it is considered dead and euthanized. On the third day after the viral challenge, the mouse was euthanized by removing its eyeballs, and blood and serum were collected. Lung tissue was then dissected, and some lung tissue was subjected to HE staining for pathological examination (e.g., ...). Figure 13 The remaining lung tissue was homogenized, and then the viral titer in the lung tissue was detected by qRT-PCR (e.g., ...). Figure 14 (As shown).

[0100] qRT-PCR method for detecting viral titers in lung tissue:

[0101] RNA extraction: The lung tissue homogenate sample of mice on day 3 after challenge was extracted using the Novizan FastPure viral DNA / RNA Mini Kit Pro (catalog number: RC323-01). For specific steps, please refer to its instruction manual.

[0102] Reverse transcription: Add 4 μL of extracted RNA sample, 2 μL of 5×Evo M-MLVRT Master Mix and 4 μL of RNase free water to an 8-tube PCR reaction system, and then perform reverse transcription according to the PCR program in Table 2.

[0103] Table 2 Reverse Transcription PCR Reaction Procedure

[0104]

[0105] qRT-PCR: First, add 27 μL of RNase-free water to an 8-tube strip, then add 3 μL of influenza standard plasmid (Influenza A standard nucleic acid sequence: cccaggtcgaaacgtacgttctctctatcgtcccgtcaggccccctcaaagccgagatcgcacagagacttgaagatgtctttgctggg aagaacaccgatcttgaggctctcatggaatggctaaagacaagaccgatcctgtca, SEQ ID NO:5) for a 10-fold serial dilution. After preparing the qRT-PCR reaction system according to Table 3, perform qRT-PCR according to the qRT-PCR reaction procedure in Table 4. The primer and probe sequences required for qRT-PCR are shown in Table 5.

[0106] Table 3 qRT-PCR reaction system

[0107]

[0108] Table 4 qRT-PCR reaction procedure

[0109]

[0110] Table 5 Primer and probe sequences used in qRT-PCR

[0111]

[0112] Weight monitoring data of mice in each group for 14 consecutive days after viral infection ( Figure 11 Mice in the 1G01+PR8 and 8F11+PR8 groups showed weight loss in the early stages of infection (first 72 hours), but the magnitude of this weight loss was less than that in the PR8 (intramuscular injection) and PBS groups. Notably, both the 1G01+PR8 and 8F11+PR8 groups showed a trend of weight recovery as early as day 4 post-infection. Furthermore, the survival curves... Figure 12 As can be seen, no mice died in the 1G01+PR8 group, one mouse died in the 8F11+PR8 group on day 5 after challenge, all mice died in the PR8 (intramuscular injection group) on day 5 after challenge, and one mouse died in the PBS group on day 4 after challenge, with all remaining mice dying on day 5. These results demonstrate that the live attenuated influenza vaccine prepared in Examples 1-2 has good protective efficacy, protecting mice from weight loss and death caused by influenza virus infection.

[0113] HE staining pathological analysis of lung tissue in mice on day 3 after viral challenge ( Figure 13 The results showed that the groups exhibited different pathological characteristics: in the PBS control group, some samples showed hemorrhage, necrosis, and a small amount of inflammatory cell infiltration; compared with the PBS group, no significant pathological changes were observed in the 1G01+PR8 group; the number of samples with inflammatory cell infiltration decreased in the 8F11+PR8 group, but some samples showed alveolar inflammatory cell aggregation, and some samples showed a small amount of alveolar dilation; the number of hemorrhage and necrosis samples decreased in the PR8 (intramuscular injection group). Pathological evidence indicates that the live attenuated influenza vaccine prepared in Examples 1-2 can significantly alleviate lung tissue pathological damage caused by high-dose influenza virus infection, suggesting that it has a clear immunoprotective effect.

[0114] The results of virus titer detection in mouse lung tissue on day 3 after challenge are as follows: Figure 14 The results showed that the CT value of the 8F11+PR8 group was significantly different from that of the PBS control group, meaning that its viral titer was lower than that of the PBS control group. There was no significant difference between 1G01+PR8 and PR8 (intramuscular injection group), but its viral titer was also lower than that of the PBS group.

[0115] In summary, the novel attenuated live vaccine (taking influenza attenuated live vaccine as an example) provided by this invention has the characteristics of rapid and simple preparation, and it has good safety, immunogenicity and protective effect, making it a novel attenuated live vaccine with application advantages.

[0116] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A live attenuated vaccine comprising at least one pathogenic microorganism and a neutralizing antibody capable of neutralizing said pathogenic microorganism.

2. The live attenuated vaccine according to claim 1, characterized in that, The pathogenic microorganisms include viruses.

3. The live attenuated vaccine according to claim 2, characterized in that, The viruses include at least one of the following: influenza virus, coronavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, norovirus, herpesvirus, EB virus, and Coxsackie virus.

4. The live attenuated vaccine according to any one of claims 1 to 3, characterized in that, The pathogenic microorganism is influenza A virus, and the neutralizing antibody includes at least one of 1G01 antibody, 8F11 antibody, and MEDI8852.

5. A method for preparing a live attenuated vaccine according to any one of claims 1 to 4, comprising the following steps: By mixing viral microorganisms with neutralizing antibodies and incubating them, a live attenuated vaccine can be obtained.

6. The preparation method according to claim 5, characterized in that, The incubation conditions are 30–40°C and 3%–6% CO2 for 0.5–2 hours.

7. The preparation method according to claim 5, characterized in that, In the mixed system, the titer of the pathogenic microorganism is 10–50 TCID. 50 / μL, wherein the concentration of the neutralizing antibody is 0.1–2 μg / μL.

8. The use of the attenuated live vaccine according to any one of claims 1 to 4 in the preparation of a medicament for the prevention or improvement of a disease, wherein the disease includes a disease caused by infection with a pathogenic microorganism.

9. A drug comprising the live attenuated vaccine according to any one of claims 1 to 4.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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