Multi-antigen component PEI lipid delivery system and anti-avian influenza virus multivalent vaccine comprising same

Abstract

The invention relates to a multi-antigen component PEI lipid delivery system and an anti-avian influenza virus multivalent vaccine comprising the same, belongs to the technical field of biological medicines, and provides a delivery system consisting of PEI, squalene, cholesterol and mPEG. The preparation method comprises the following steps of: constructing mRNA (messenger ribonucleic acid) molecules and a composite vaccine antigen system consisting of prokaryotic expression purified H7N9 avian influenza NP antigen, loading the composite vaccine antigen system into a delivery system to obtain the nanoparticles with a virus-like structure, and displaying the HA protein antigen and the NP antigen in the structure of the nanoparticles in a particle form by the nanoparticles, so that the nanoparticles can be used for preparing the H7N9 avian influenza NP / H7N9 avian influenza NP / H7N9 avian influenza NP / H7N9 avian influenza virus. And the mRNA molecule for coding the NA antigen, which is combined with the mRNA molecule, can be delivered into a cell in a manner of fusing lipid and a cell membrane, so that the mRNA molecule can be effectively expressed to induce a body to generate specific antibody reactions aiming at HA, NA and NP, and the body can also generate specific CTL reactions aiming at HA, NA and NP of different influenza virus strain subtypes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a multi-antigen component PEI lipid delivery system and a multivalent anti-avian influenza virus vaccine containing the delivery system. Background Technology

[0002] Influenza is a group of infectious respiratory diseases in humans caused by influenza viruses, and it remains a significant long-term disease threat to all age groups worldwide. This class of pathogens has multiple types (A, B, C, and D) and multiple combined subtypes (such as H1N1, H3N2, H5N1, and H7N9). These different subtypes exhibit corresponding infectious pathological characteristics in different species. H1N1 and H3N2 subtypes are commonly threatening to humans. However, based on the epidemiological evolution of this pathogen, the H5, H7, and N9 subtypes, which commonly cause pandemics in various birds, currently pose the greatest potential threat to humans. This is because the cross-border infection between birds and humans has gradually broken through the traditional bird-swine-human model, as evidenced by the few reported cases of human infection caused by avian influenza (including H5 and H7). Furthermore, recent observations have confirmed that avian influenza infection in mammalian livestock (such as cattle) has become a large-scale epidemic trend.

[0003] Studies have shown that different antigens of the influenza virus exhibit different immunological effects: HA (hemagglutinin antigen), as a major antigen, can induce the production of major antiviral neutralizing antibodies; NA (neuraminidase), as another viral surface antigen, has been confirmed by numerous recent studies to stimulate the body to produce antibodies that block viral transmission between cells, thus providing immune protection; while NP (nucleocapsid protein) is an antigen that can induce a CTL response with a specific cytotoxic effect in the body's immune system and can enhance immune memory against the influenza virus. Furthermore, the NP antigen exhibits high sequence conservation across different types and subtypes of the virus.

[0004] Although the widespread prevalence of avian influenza viruses in poultry and livestock has posed a significant public health threat to humans, significant progress has not been made in vaccine prevention measures against human infection. This is primarily due to the following technical challenges: First, existing human influenza vaccine technology remains a "tracking" model, and there is no predictable data or model regarding the prevalence of various avian influenza strains in different poultry and animals, or their potential breakthrough infection in humans. Second, current industrialized influenza vaccine production models are insufficient to provide vaccine preparation technologies for different antigenic forms. Third, and another unresolved technical challenge, is the lack of an ideal vaccine antigen delivery system that can simultaneously deliver multiple antigenic components to exert immunological effects. This presents significant difficulties in implementing proactive vaccine prevention strategies in the face of potential prevalence of multiple avian influenza subtypes. Summary of the Invention

[0005] To address the problems existing in the background technology, the present invention provides a PEI lipid delivery system and constructs a composite vaccine antigen system. By loading the composite vaccine antigen system into the PEI lipid delivery system, virus-like nanoparticles are obtained. These nanoparticles can not only display the HA protein antigen and NP antigen in their structure in particle form, but also deliver the NA antigen-encoding mRNA molecules bound to them into the cell through lipid-cell membrane fusion and enable them to be effectively expressed, thereby forming antigen signal stimulation for different pathways of the body's immune system.

[0006] Based on this, the first objective of the present invention is to provide a multi-antigen component PEI lipid delivery system, characterized in that the delivery system is composed of low molecular weight polyethyleneimine (PEI), squalene, cholesterol, and mPEG.

[0007] Furthermore, the mass ratio of PEI, squalene, cholesterol, and mPEG is 1.6:0.5:1:0.06.

[0008] A second objective of this invention is to improve a multivalent vaccine against avian influenza virus, the multivalent vaccine comprising the aforementioned delivery system and composite vaccine antigen system.

[0009] Furthermore, the composite vaccine antigen system includes the HA recombinant antigen of avian influenza virus H5N1, an mRNA molecule prepared from the NA antigen coding region of human influenza H3N2 subtype, and the NP antigen of H7N9 avian influenza expressed and purified by Escherichia coli.

[0010] Furthermore, the expression level of mRNA molecules prepared from the NA antigen coding region in 293-T cells was ≥800 ng / ml, the content of HA recombinant antigen was ≥4000 ng / ml, and the content of NP antigen was ≥2000 ng / ml.

[0011] A third objective of this invention is to provide a method for preparing a multivalent vaccine against avian influenza, comprising the following steps: (1) Prepare the above-mentioned composite vaccine antigen system; (2) Preparation of a PEI lipid delivery system; (3) Add the composite vaccine antigen system from step (1) to a buffer solution containing 20 mM NaAc, pH 5.0, and 0.01% trehalose; (4) In microfluidic operation, the mixture from step (3) and the delivery system from step (2) are mixed to form a nanoparticle microsphere system; (5) Use a buffer solution containing 20mM NaAc, pH 5.0 and 0.01% trehalose to remove anhydrous ethanol by ultrafiltration through a 100kDa filter to obtain a diluent; (6) The diluent from step (5) is mixed with the nanoparticle microsphere system from step (4) to obtain the vaccine formulation.

[0012] Furthermore, in step (2), PEI, squalene, cholesterol and mPEG are mixed in anhydrous ethanol, with mass concentrations of PEI, squalene, cholesterol and mPEG of 1.6%, 0.5%, 1% and 0.06%, respectively.

[0013] The beneficial effects of this invention are: This invention provides a delivery system based on small molecule polyethyleneimine (PEI) – the PEI lipid delivery system. The PEI lipid delivery system of this invention is composed of PEI, squalene, cholesterol, and mPEG, which enables multiple antigen components to exert immune effects simultaneously.

[0014] This invention simultaneously constructs a composite vaccine antigen system composed of mRNA molecules of avian influenza subtype strain HA antigen (H5N1), human influenza virus H3N2 NA antigen, and avian influenza H7N9 NP antigen. This composite vaccine antigen system is loaded into a PEI lipid delivery system, and through a microfluidic process, virus-like nanoparticles are obtained. These nanoparticles not only display the HA protein antigen and NP antigen in particulate form, but also deliver the bound NA antigen-encoding mRNA molecules into cells via lipid-cell membrane fusion, enabling their effective expression and thus forming antigen signal stimulation targeting different pathways of the body's immune system. Animal immunological analysis based on this antigen model shows that this vaccine model can induce specific antibody responses against HA, NA, and NP, including HAI, during a two-immunization program; simultaneously, the body can also generate specific CTL responses against HA, NA, and NP of different influenza strain subtypes. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the eukaryotic cell expression and preparation process of the H5 subtype viral hemagglutinin HA recombinant antigen in this invention; Figure 2 This is a schematic diagram illustrating the design and preparation of the H3N2 subtype NA antigen mRNA molecule in this invention; Figure 3 This is a schematic diagram of the prokaryotic expression preparation of the H7N9 subtype NP antigen in this invention; Figure 4 These are electron microscope images of the structures of three batches of the multivalent antigen vaccine products of this invention. Figure 5 This is a Western blot analysis of the NA antigen expression results after cell transfection of three batches of the multivalent antigen vaccine products of this invention. Figure 6 The results show the detection of three antibodies and cellular immune responses in mice after immunization with the multivalent antigen vaccine of this invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0017] Example 1: A composite vaccine antigen system capable of targeting different subtypes, including avian influenza virus subtypes. 1. Obtaining the hemagglutinin (HA) recombinant antigen of avian influenza virus H5N1 (hereinafter referred to as HA recombinant antigen) Design of the H5N1 subtype hemagglutinin (HA) recombinant antigen pattern: Based on the HA sequence of the avian influenza virus H5N1 strain, a eukaryotic expression vector containing the HA signal sequence, head, and stem sequences was constructed (construction process as follows). Figure 1 The amino acid sequence of the constructed HA recombinant antigen is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2.

[0018] The HA recombinant antigen was obtained by transfecting CHO cells with the recombinant plasmid pCHO-HA and then purifying it. The design and operation method of the HA recombinant antigen of this invention are the same as those in patent CN202511851881.

[0019] 2. Obtaining mRNA molecules prepared from the NA antigen coding region of human influenza H3N2 subtype (hereinafter referred to as mRNA molecules prepared from the NA antigen coding region). Based on the NA antigen coding region gene sequence of the human influenza virus H3N2 subtype strain, corresponding 5'-UTR and 3'-UTR regions were designed to construct mRNA molecules capable of expressing the NA antigen (construction process as follows). Figure 2 The nucleic acid sequence of the constructed NA antigen is shown in SEQ ID NO.3.

[0020] The specific steps are as follows: The synthesized NA antigen gene was amplified by PCR for 25 cycles at 98℃ for 3 min / 98℃ for 25 sec / 62℃ for 15 sec / 72℃ for 1 min using primers NA-5 (SEQ ID NO.4) and NA-3 (SEQ ID NO.5). After purification by column chromatography, the product was ligated into the pVAX-1 vector purified by digestion with HindIII and BamHI to obtain the pVAX-NA recombinant plasmid. After amplification, preparation and purification of the plasmid, it was linearized by digestion with BamHI. The mRNA molecule was prepared by IVT (standard method) and the mRNA molecule content was determined for further dilution required for vaccine formulation.

[0021] 3. Obtaining the NP antigen of H7N9 avian influenza expressed and purified from prokaryotic cells (Escherichia coli) (hereinafter referred to as NP antigen). Based on the NP antigen coding region gene of the H7N9 avian influenza virus subtype (amino acid sequence as shown in SEQ ID NO. 6, nucleic acid sequence as shown in SEQ ID NO. 7), a prokaryotic expression system loaded with the pGEX prokaryotic expression vector was constructed (construction process as follows). Figure 3The construction process is as follows: The synthesized NP antigen coding region gene was amplified by PCR using primers NP-5 (SEQ ID NO.8) and NP-3 (SEQ ID NO.9) for 25 cycles at 98℃ for 3 min / 98℃ for 25 sec / 62℃ for 15 sec / 72℃ for 1 min to obtain PCR products. The PCR products were extracted using a gel extraction kit to obtain the target fragment. The target fragment was ligated to the pGEX-6p-1 prokaryotic plasmid digested and purified by BamHI via homologous recombination to construct the pGEX-NP recombinant plasmid. Subsequently, the pGEX-NP recombinant plasmid was transformed into E. coli DH5α competent cells for amplification and purification. The purified pGEX-NP recombinant plasmid was transferred into competent cells of expression-type Escherichia coli BL21(DE3). The specific method is as follows: expression-type Escherichia coli BL21(DE3) was placed in LB medium at a ratio of 1:100 and cultured overnight at 37°C and 200 rpm. After proliferation, the cells were incubated in 100 mM CaCl2 at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were resuspended in 1 ml of 100 mM CaCl2. The constructed pGEX-NP recombinant plasmid was added, and the cells were incubated in an ice-water bath at 4°C for 30 minutes, followed by heat shock at 42°C for 45 seconds. 0.6 ml of LB solution was added, and the cells were shaken at 37°C and 200 rpm for 1 hour. A small amount of the shaken bacterial culture was plated onto a kanamycin-resistant LB agarose culture plate and cultured overnight at 37°C. The next day, clones were selected and identified by BamHI enzyme digestion. After confirming positive clones, they were cultured overnight in LB liquid medium at 37°C. Then, the target protein was expressed by heat induction at 42°C. The purified NA protein was obtained by nickel ion affinity chromatography, and the protein content was determined for use in vaccine formulation.

[0022] The above-mentioned HA antigen, mRNA molecule and NP antigen were mixed in a ratio of 5:20:5 to obtain a compound vaccine antigen system preparation, 5μg:20μg:5μg / dose.

[0023] Example 2

[0024] (1) Preparation of PEI lipid delivery system: Polyethyleneimine (PEI), a polymer composed of polyethyleneimine units, exhibits a high charge density due to its numerous amino groups. Based on the structural characteristics of PEI, we used PEI with a molecular weight of 2500, combined with squalene (which has lipid-supporting and adjuvant functions), cholesterol (a structural component), and mPEG (a lipid-stabilizing agent). All four components were dissolved in GR-grade anhydrous ethanol. The mass percentages of PEI, squalene, cholesterol, and mPEG were 1.6%, 0.5%, 1%, and 0.06%, respectively, forming the organic phase (total lipid mass: 31.6 mg / ml anhydrous ethanol).

[0025] (2) Prepare a buffer solution with 20mM NaAc, pH 5.0 and 0.01% trehalose, and mix the composite vaccine antigen system of Example 1 with the buffer solution to form an aqueous phase.

[0026] (3) The aqueous phase from step (2) and the organic phase from step (1) are mixed in a microfluidic operation at a volume ratio of 1:3, using a microfluidic device (model: NanoAssemblr® Ignite). Tm (Flow rate: 100 ml / min) to form a nanoparticle microsphere system.

[0027] (4) Use the buffer solution from step (2) to remove anhydrous ethanol by ultrafiltration and centrifugation through a 100kDa filter. Ultrafiltration conditions: 3000rpm, 20min × 3. After ultrafiltration, concentration can be selected according to the subsequent antigen concentration.

[0028] Based on the calculation of 5 μg HA recombinant antigen / 20 μg NA antigen coding region prepared mRNA molecules / 5 μg NP antigen protein / per dose, the ultrafiltered (or ultrafiltered and concentrated) buffer solution is added to the nanoparticle microsphere system in step (3) to obtain nanoparticle multivalent avian influenza vaccine.

[0029] Example 3: Analysis and Determination of Vaccine Dosage Form Characteristics Three batches of vaccine products were prepared based on the method in Example 2. All three batches of vaccine products were tested by electron microscopy, and the results are shown in the attached figure. Figure 4 The vaccines of this invention are all spherical in shape, with particle diameter of 130-170 nm, PDI ≤ 0.15, and Zeta potential of 20-30 mV (Table 1).

[0030] Table 1: Test Results of Vaccine Products

[0031] Quantitative detection of antigens in vaccine nanoparticles: The content of HA antigen carried by the assembled vaccine nanoparticles was detected using an ELISA kit (SB: Cat: CW19DE0901), and the content of NP antigen was detected using an ELISA kit (SB: Cat: CW19OC2103). The corresponding quality requirements were: HA antigen content ≥ 4000 ng / ml and NP antigen content ≥ 2000 ng / ml. The detection results are shown in Table 2.

[0032] Table 2: Detection results of HA antigen and NP antigen in vaccine products

[0033] Assessing the ability of vaccine nanoparticles to transfect cells and deliver their carried mRAN into the cells: After transfection of 293T cells with the vaccine formulation, Western blot analysis (WB) was performed using specific anti-NA antigen antibodies to detect the NA antigen expressed in mRAN (according to standard methods). The detection results for different batches of samples are shown in the table below. Figure 5 .

[0034] Expression and level of NA antigen after transfection of cells with vaccine formulation: Detection results: NA antigen expression should be positive, and the expression level of NA antigen in three batches should be ≥800ng / ml.

[0035] Table 3. Detection results of HA and NP antigens in vaccine products.

[0036] Example 4: Immunological analysis of the multivalent vaccine against avian influenza virus of the present invention Structural studies and antigenic property analysis of PEI-based nanostructured vaccines have confirmed their ability to present antigens against major avian influenza subtypes to the body's immune system. These antigenic components can exert immunostimulatory effects similar to those produced by different antigens during viral infection in various forms.

[0037] Select healthy mice and immunize them with one dose each on days 0 and 21 (5 μg HA antigen protein, 20 μg NA-encoded mRNA molecule, and 5 μg NP antigen protein per dose).

[0038] Serological testing results in mice (using the approved Acro Biosciences ELISA kit for different antigen-associated antibodies against influenza virus) Figure 6 The results showed a significant increase in HA-specific antibodies against the H5 subtype and anti-NP antibodies against the H7 subtype. Simultaneously, anti-NA-specific antibodies also increased. This clear result indicates that the vaccine can simultaneously induce immune responses against different antigens of different avian influenza subtypes. Furthermore, Elispot assays showed that immunized animals exhibited corresponding specific responses to different influenza subtype antigen proteins. Figure 6 ).

[0039] The basic principles, core features, and technical advantages of this invention have been fully disclosed above. Those skilled in the art will understand that the scope of protection of this invention is not limited to the described embodiments; the embodiments and the content contained in the specification are only for illustrative purposes. Any changes, optimizations, or improvements made to this invention without departing from its spirit and without exceeding the scope defined by its claims should be covered within the scope of protection of this invention. The scope of the claims of this invention is determined by the appended claims and their equivalents.

Claims

1. A multi-antigen component PEI lipid delivery system, characterized in that, The delivery system consists of low molecular weight polyethyleneimine (PEI), squalene, cholesterol, and mPEG.

2. The delivery system according to claim 1, characterized in that, The mass ratio of PEI, squalene, cholesterol, and mPEG was 1.6:0.5:1:0.

06.

3. A multivalent vaccine against avian influenza virus, characterized in that, This includes the delivery system and composite vaccine antigen system as described in claim 1 or 2.

4. The multivalent vaccine according to claim 3, characterized in that, The composite vaccine antigen system includes the HA recombinant antigen of avian influenza virus H5N1, an mRNA molecule prepared from the NA antigen coding region of human influenza H3N2 subtype, and the NP antigen of H7N9 avian influenza expressed and purified by Escherichia coli.

5. The multivalent vaccine according to claim 4, characterized in that, The mRNA molecules prepared from the NA antigen coding region were expressed at a level ≥800 ng / ml in 293-T cells, the HA recombinant antigen content was ≥4000 ng / ml, and the NP antigen content was ≥2000 ng / ml.

6. The method for preparing a multivalent vaccine against avian influenza as described in any one of claims 3 to 5, characterized in that, Includes the following steps, (1) Prepare the composite vaccine antigen system as described in claim 4 or 5; (2) Prepare the delivery system according to claim 1 or 2; (3) Add the composite vaccine antigen system from step (1) to a buffer solution containing 20 mM NaAc, pH 5.0, and 0.01% trehalose; (4) In microfluidic operation, the mixture from step (3) and the delivery system from step (2) are mixed to form a nanoparticle microsphere system; (5) Use a buffer solution containing 20mM NaAc, pH 5.0 and 0.01% trehalose to remove anhydrous ethanol by ultrafiltration through a 100kDa filter to obtain a diluent; (6) The diluent from step (5) is mixed with the nanoparticle microsphere system from step (4) to obtain the vaccine formulation.

7. The preparation method according to claim 6, characterized in that, In step (2), PEI, squalene, cholesterol and mPEG are mixed in anhydrous ethanol, with mass concentrations of PEI, squalene, cholesterol and mPEG of 1.6%, 0.5%, 1% and 0.06%, respectively.

Images