Dendritic cell-targeted viroid DVLP, and preparation method therefor and use thereof

Abstract

A dendritic cell-targeted viroid DVLP, and a preparation method therefor and use thereof. The viroid DVLP is composed of VLP coated with an optimized Sindbis virus glycoprotein having a sequence as shown in SEQ ID NO.1. The preparation method therefor comprises: optimizing amino acids of an envelope protein of a wild-type Sindbis virus to obtain an SV-G gene sequence; constructing an SV-G gene expression vector pCMV-SV-G; co-transfecting HEK293T cells with the pCMV-SV-G, pRSV-REV, an antigen expression vector, pMDlg / PRE-D64V and pMS2S-PH-gagpol-D64V plasmid, and performing transfection and purification. The viroid DVLP has a very high DC cell targeting and protein stability, and after injection, the mRNA carried thereby is mainly concentrated at an injection site and a lymph node, with a low risk of diffusing to a non-target organ, such as spleen, liver, and kidney. Moreover, the viroid DVLP can generate a high-titer neutralizing antibody to fight virus infection and protect a host, and provide a platform for the development of tumor vaccines.

Description

Dendritic cell-targeted virus-like body DVLP and its preparation method and application Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to dendritic cell-targeted virosome DVLP and its preparation method and application, and specifically relates to the application of dendritic cell-targeted virosome mRNA delivery technology in vaccines. Background Art

[0002] Dendritic cells (DCs) are important antigen-presenting cells (APCs) that play a crucial role in vaccine effectiveness. Their functions are primarily manifested in two key aspects: First, DCs process and present antigens to T cells, thereby inducing cellular immunity; second, DCs process and present antigens to B cells, thereby inducing humoral immune responses. In 2010, the US Food and Drug Administration approved PROVENGE, the world's first DC cell vaccine, for the treatment of prostate cancer. However, the PROVENGE production process is complex and cumbersome, involving the isolation and in vitro preparation of the patient's own cells, followed by in vivo infusion. As a result, the vaccine's production costs and sales price are high, making it difficult to widely adopt. Furthermore, the translation of antigen mRNA by non-professional APCs may make antigen-expressing cells targets for CD8+ T cell-mediated killing. This is related to the side effects experienced by some individuals after receiving the COVID-19 mRNA vaccine. Furthermore, antibody-dependent cellular cytotoxicity (ADCC) may also damage host cells where antigenic proteins are embedded or secreted.

[0003] Therefore, specific targeting of DC is the development direction of the next generation of mRNA vaccines, which can simplify the production process, reduce production costs, improve vaccine safety, and enhance the immune effect of the vaccine. DC cell ICAM-3 integrin (DC-SIGN) is a pattern recognition receptor and adhesion receptor of DC cells, and plays an important role in DC cell migration and adhesion, inflammatory response, activation of T cells, initiation of immune response, etc. The wild-type Sindbis virus glycoprotein has two cell binding sites, which can target DC-SING protein (a C-type lectin-like receptor that can quickly bind and engulf substances and is an ideal targeting receptor on dendritic cells) and cell surface heparin sulfate (expressed by multiple cell types). In the prior art, by deleting amino acids 61 to 64 in the E3 region of the SV-G protein, mutating 157KE158 in the E2 region to 157AA158, and inserting a 10-amino acid tag sequence (MYPYDVPDYA) between amino acids 71 ​​to 74 in the E2 region, it is prevented from binding to heparan sulfate without affecting the DC cell binding ability. Recombinant lentiviral vectors (LVs) using Sindbis viral glycoprotein (SV-G) as a DC-SIGN ligand have the potential to achieve better immune effects than non-specific LVs. However, risk factors such as integration and insertion mutations limit the clinical translation of LV vaccines. Wild-type Sindbis viral glycoproteins, after being engineered to remove heparan sulfate binding functions, are also prone to poor protein stability, resulting in low binding efficiency with receptors and reduced binding specificity, making them difficult to apply to vaccine preparation. Furthermore, unstable proteins are prone to accelerated degradation, exposing hidden antigenic epitopes and thus triggering more immune responses. Although LNPs can achieve DC cell-targeting specificity by coupling with specific antibodies or ligands, there is still a lack of evidence for the effectiveness of LNP-based DC cell-targeted mRNA vaccines.

[0004] Summary of the Invention

[0005] To address the above technical problems, the present invention aims to provide a dendritic cell-targeted virosome (DVLP), a preparation method, and an application thereof. The virosome (DVLP) is derived from an engineered lentiviral vector (LV) and is capable of carrying mRNA to specifically target DC cells. To increase the stability of the engineered SV-G protein and reduce interatomic steric clashes, the present invention mutates the amino acid tag MYPYDVPDYA inserted in the E2 region to AAAAYPYDVPDYA, thereby increasing protein stability without changing the protein structure and maintaining DC cell targeting. Furthermore, the amino acids in the SV-G protein are codon-optimized for expression in human cells.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a dendritic cell-targeted virosome DVLP, wherein the virosome DVLP comprises a virus-like particle (VLP) and Sindbis viral glycoprotein, wherein the Sindbis viral glycoprotein is coated on the surface of the VLP, and mRNA is coated inside the VLP.

[0008] As one embodiment of the present invention, the amino acid sequence of the Sindbis virus glycoprotein (SV-G) is shown in SEQ ID NO. 1. SEQ ID NO. 1 was optimized based on the wild-type Sindbis virus glycoprotein (NP_062890.1) by engineering the SV-G protein by deleting amino acids 61 to 64 in the E3 region, mutating position 159KE160 in the E2 region to 159AA160, and inserting a 13-amino acid tag sequence (AAAAYPYDVPDYA) between amino acids 68 to 73 in the E2 region.

[0009] As one embodiment of the present invention, the mRNA includes at least one of mRNAs of spike, viral antigen, tumor antigen, and bacterial antigen.

[0010] Furthermore, the mRNA is transcribed from genes of spike, viral antigen, tumor antigen, or bacterial antigen.

[0011] In some embodiments, the mRNA includes spike mRNA, gB1 and gD1 mRNA of the HSV-1 virus.

[0012] In a second aspect, the present invention provides a method for preparing a dendritic cell-targeted virus-like body DVLP, comprising the following steps:

[0013] S1. Optimize the amino acid sequence of the envelope protein of the wild-type Sindbis virus (reverse translate into a gene sequence and perform codon optimization suitable for expression in human cells) to obtain the SV-G gene;

[0014] S2. Construct an expression vector for the SV-G gene to obtain the pCMV-SV-G plasmid;

[0015] S3. HEK293T cells were co-transfected with pCMV-SV-G, pRSV-REV, antigen expression vector, pMDlg / PRRE-D64V and pMS2S-PH-gagpol-D64V plasmids. After transfection, the cell culture supernatant was collected and purified.

[0016] As one embodiment of the present invention, in step S1, the nucleotide sequence of the SV-G gene is shown in SEQ ID NO. 2. The SV-G gene sequence is a nucleotide sequence encoding Sindbis virus glycoprotein.

[0017] In the present invention, the envelope protein of the wild-type Sindbis virus (i.e., the wild-type Sindbis virus glycoprotein) has a tendency to target DC cells. After modification, the ability to target DC cells is improved without affecting the titer of the virus.

[0018] As one embodiment of the present invention, constructing the expression vector of the SV-G gene in step S2 includes the following steps:

[0019] S2.1, integrate homology arms upstream and downstream of the SV-G sequence to obtain an artificially synthesized sequence;

[0020] S2.2. Double-digest the artificially synthesized sequence and the pMD2.G plasmid, purify and recover;

[0021] S2.3. Use T4 DNA ligase to ligate the recovered products to form plasmid pCMV-SV-G.

[0022] As an embodiment of the present invention, in step S2.1, the artificially synthesized sequence is shown as SEQ ID NO.3.

[0023] As one embodiment of the present invention, in step S2, the nucleotide sequence of the pCMV-SV-G plasmid is shown as SEQ ID NO.4.

[0024] As one embodiment of the present invention, the antigen expression vector is pCMV-mRNA-mut-6×MS2, wherein the mRNA is selected from at least one of spike, viral antigen, tumor antigen, and bacterial antigen mRNA.

[0025] In some embodiments, the antigen expression vector includes pCMV-Spike-mut-6×MS2, pCMV-gB1-gD1-6×MS2.

[0026] In the present invention, the construction method of pRSV-REV, pCMV-Spike-mut-6×MS2, pMDlg / PRRE-D64V and pMS2S-PH-gagpol-D64V refers to patent CN112168958A; wherein, the nucleotide sequence of pRSV-REV is shown in SEQ ID NO.5; pCMV-Spike-mut-6×MS2 refers to pCMV-nCoV-S-6×MS2-mut-flag in the patent, and its nucleotide sequence is shown in SEQ ID NO.6; pMDlg / PRRE-D64V refers to the integrase-deficient type of pMDlg / PRRE in the patent, and its nucleotide sequence is shown in SEQ ID NO.7; pMS2S-PH-gagpol-D64V refers to the plasmid sequence expressing the lentiviral GagPol long-chain protein containing RNA-binding protein in the patent, and its nucleotide sequence is shown in SEQ ID NO.8.

[0027] Furthermore, the antigen expression vector was constructed by replacing the Spike in pCMV-Spike-mut-6×MS2 with other types of mRNA using enzyme digestion and ligation methods, which are conventional techniques in the art.

[0028] In a third aspect, the present invention provides a dendritic cell-targeted virus-like body DVLP for use in the preparation of an mRNA vaccine.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) The present invention optimizes the wild-type Sindbis viral glycoprotein (SV-G), removing the heparan sulfate binding ability while retaining the DC cell binding ability; and optimizes the side chain conformation of SV-G, reducing the spatial conflict between atoms, thereby improving protein stability and further enhancing the binding effect with DC cell surface receptors, thereby improving DC cell targeting.

[0031] 2) The present invention provides an mRNA vaccine that can specifically target DC cells in vivo.

[0032] 3) The present invention demonstrates through a series of experiments that the virosomes can produce obvious co-localization with DC cells in the lymph nodes, proving that the vaccine has extremely high DC cell targeting in vivo.

[0033] 4) The present invention has demonstrated through a series of experiments that after injection of this type of virosome, the mRNA it carries is mainly concentrated in the injection site and lymph nodes, and the risk of spreading to non-target organs such as the spleen, liver, and kidneys is relatively low.

[0034] 5) The present invention has demonstrated through a series of experiments that this type of virosome can produce high-titer neutralizing antibodies after immunizing mice, thereby combating viral infection and protecting the host; it also provides a platform for the development of tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0036] Figure 1 is an AlphaFold3 modeling diagram of the SV-G protein mutant structure 1 and mutant structure 2 in Example 1;

[0037] FIG2 is a comparison of the mutant structures of SV-G protein mutant structure 1 and mutant structure 2 in Example 1;

[0038] FIG3 is an ELISA analysis of the production of spike-specific IgG antibodies against virus-like particles (DVLP) specifically targeting DC cells and virus-like particles (VSV-G VLP) non-specifically targeting DC cells in Example 3;

[0039] FIG4 is an ELISA analysis of the production of spike-specific IgG antibodies against DC-specific virus-like particles (DVLP) and liposome particles (LNP) in Example 3;

[0040] FIG5 is an immunofluorescence image of the distribution of DC-specific virus-like particles (DVLP), non-specifically targeted virus-like particles (VSV-G VLP) and liposome particles (LNP) in mouse lymph nodes;

[0041] FIG6 is a statistical analysis of the biodistribution of spike mRNA of virus-like particles (DVLP) specifically targeting DC cells, non-specifically targeting virus-like particles (VSV-G VLP) and liposome particles (LNP) in different organs and injection sites (footpad) of mice;

[0042] FIG7 is a graph showing the neutralizing antibody titer detection in the serum of mice inoculated with virus-like particles (DVLPs) (carrying HSV-1 gB1 and gD1 mRNA) that specifically target DC cells;

[0043] Figure 8 shows the viral load detection in the skin and dorsal root ganglia of mice vaccinated with DC cell-specific virus-like particles (DVLPs) (carrying HSV-1 gB1 and gD1 mRNA) after infection with HSV-1. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, provide detailed implementation methods and specific operating procedures, and will help those skilled in the art to further understand the present invention. It should be pointed out that the scope of protection of the present invention is not limited to the following embodiments, and a number of adjustments and improvements made under the premise of the concept of the present invention all fall within the scope of protection of the present invention.

[0045] Example 1

[0046] Optimization process operation steps and mutation stability calculation

[0047] Based on the wild-type Sindbis virus glycoprotein SV-G (amino acid sequence such as SEQ ID NO.9), an optimized engineering modification was performed. Referring to the methods of the prior art, the SV-G protein of the wild-type protein NP_062890.1 sequence had amino acids 61 to 64 of the E3 region deleted, position 159KE160 of the E2 region mutated to 159AA160, and a 10-amino acid tag sequence (MYPYDVPDYA) was inserted between amino acids 68 to 73 of the E2 region, which was defined as mutant structure 1 (amino acid sequence such as SEQ ID NO.18). The optimized solution of the present invention is to delete amino acids 61 to 64 of the E3 region of the SV-G protein of the wild-type protein NP_062890.1 sequence, mutate position 159KE160 of the E2 region to 159AA160, and insert a 13-amino acid tag sequence (AAAAYPYDVPDYA) between amino acids 68 to 73 of the E2 region, which was defined as mutant structure 2. First, AlphaFold3 modeling analysis was performed, using the amino acid sequences of SV-G from Mutation Structure 1 and Mutation Structure 2, respectively. Mutation Structures 1 and 2 were modeled and compared. The structural conservation of the two structures was initially assessed based on overall conformation and RMSD (Root Mean Square Deviation). RMSD is a metric used to quantify the differences between two structures (usually proteins or other macromolecules). Smaller RMSD values ​​indicate greater similarity; larger RMSD values ​​indicate greater differences. It measures similarity or difference by calculating the distance difference between corresponding atomic positions in the structures. RMSD can be used to analyze whether a mutation disrupts the spatial structure of a protein. Rosetta (using the default parameters of socre_jd2) was then used to further assess the stability of the two structures from an energy perspective. The structures were scored using the Ref2015 energy function, and the energy contribution of the amino acid results was then analyzed.

[0048] result:

[0049] Modeling analysis of Sindbis virus glycoprotein mutant structures 1 (hereafter referred to as SV-GM) and 2 (hereafter referred to as SV-G-4A, in which amino acid position 129 is mutated from M to AAAA (M129AAAA)) revealed that the introduction of the M129AAAA mutation did not significantly alter the overall conformation of the protein (see Figure 1, left: SV-G protein mutant structure 1, right: mutant structure 2). The RMSD between the two structures is 0.391 Å (RMSD < 1 Å indicates close similarity and is generally considered nearly identical), indicating a high degree of confidence in the predicted SV-G-4A structure and that the mutation does not disrupt the protein's spatial structure. Alignment of the mutant structures revealed that the mutation is located in a loop between the top β-sheets of the G protein, resulting in a longer loop (Figure 2). The stability of the two structures was further assessed using Rosetta energy-based scoring. The structures were scored using the Ref2015 energy function. The results showed that SV-G-4A is significantly less energetically similar than SV-GM, indicating greater stability (Table 1). After a specific division of the energy contribution items, it was found that SV-G-4A performed better in terms of amino acid side chain conformation and repulsion between amino acid atoms, indicating that the M129AAAA mutation optimized the side chain conformation of SV-G, reduced the spatial conflict between atoms, and thus improved protein stability.

[0050] Table 1 Rosetta energy calculation results

[0051] Note: total_score: total energy of protein, which is the sum of all the following items. The lower the score, the better the protein stability.

[0052] fa_dun: amino acid side chain conformation penalty. The higher the fa_dun, the more unfavorable conformational amino acids there are in the protein.

[0053] fa_rep: Penalty for inter-amino acid atomic repulsion. A higher value indicates more atomic conflicts in the protein.

[0054] Therefore, compared to mutant structure 1 of the Sindbis virus glycoprotein, the optimized mutant structure 2 has significantly lower protein energy and improved stability. The main reasons are more optimized side chain conformation and less steric conflict.

[0055] Example 2

[0056] Preparation of dendritic virus-like particles (DVLPs) specifically targeting DC cells

[0057] By modifying the envelope protein of non-specifically targeted virus-like particles, DC-specific virus-like particles (DVLPs) are obtained. The specific preparation steps are as follows:

[0058] S1. The amino acid sequence of the envelope protein of the wild-type Sindbis virus was optimized according to the mutant structure 2 in Example 1 to obtain an optimized amino acid sequence of the Sindbis virus glycoprotein. The amino acid sequence was reverse translated into a DNA sequence to obtain a nucleotide sequence encoding the amino acids of the Sindbis virus glycoprotein, which was named SV-G gene sequence; the amino acid sequence of the envelope protein of the wild-type Sindbis virus is shown in SEQ ID NO.9.

[0059] S2. Integrate homology arms upstream and downstream of the SV-G gene sequence to obtain an artificially synthesized sequence; double-digest the artificially synthesized sequence (2 μg) and the pMD2.G plasmid (2 μg) using AccI (1 μL) and KpnI (1 μL) at 37°C for 2 h; then subject the digestion products to agarose gel electrophoresis and purification and recovery; ligate the recovered products using T4 DNA ligase to form a new plasmid (the SV-G gene sequence replaces the VSV-G gene of the known plasmid pMD.2G), namely pCMV-SV-G; the nucleotide sequence of pMD2.G is shown in SEQ ID NO. 10. Integrating the upstream and downstream homology arms of the SV-G sequence according to the pMD2.G plasmid DNA sequence is a conventional technique in the art.

[0060] S3. Add pCMV-SV-G (9.07 μg), pRSV-REV (7.26 μg), pCMV-Spike-mut-6×MS2 (31.46 μg), pMDlg / PRRE-D64V (15.73 μg), and pMS2S-PH-gagpol-D64V (15.73 μg) plasmids to a sterile 15 mL centrifuge tube in sequence; add ultrapure water to make up to 1089 μL; add 1210 μL 2×HEBS and gently blow evenly; add 121 μL CaCl2 and gently blow evenly; after standing for 3 min 20 s, gently add dropwise to the cell culture supernatant and co-transfect HEK293T cells. Collect the cell culture supernatant 36 h and 60 h after transfection, respectively, and perform subsequent purification.

[0061] S4. The collected supernatant was preliminarily centrifuged at 4°C, 3000 g for 10 min.

[0062] S5. Filter the supernatant through a filter with a pore size of 0.45 μm; add the filtered supernatant to an ultracentrifuge tube, with a volume of approximately 32 mL; then slowly add a 20% mass concentration of sucrose in PBS solution to the bottom of the ultracentrifuge tube, with a volume of approximately 4 mL;

[0063] S6. Purify the virus solution in S5 by ultracentrifugation at 4°C, 25,000 rpm for 2 h.

[0064] S7. After discarding the supernatant, add 50 μL of PBS to soak and dissolve the virus at the bottom of the tube. After 2 hours, collect the virus to obtain DC cell-specific virus-like particles (DVLPs) and store them in a -80°C refrigerator.

[0065] Example 3

[0066] DC targeting test of optimized Sindbis virus glycoprotein pseudotyped VLPs, VSV-G pseudotyped VLPs and liposome-based LNPs

[0067] C57BL / 6J mice were immunized with a single dose of VSV-G VLPs or DVLPs (SV-G VLPs, 1.5 μg) carrying spike mRNA via footpad injection. A negative control group (Placebo) was injected with an equal volume of PBS. Fourteen days after immunization, mice were euthanized and serum was obtained. Serum spike-specific IgG levels were measured using a mouse IgG ELISA kit (Bethyl, Montgomery, Texas; E99-131). For the ELISA, the substrate was replaced with recombinant spike protein (Novoprotein, DRA49) and the standard was replaced with an anti-spike antibody (GeneTex, GTX632604). The results in Figure 3 show that under the same immune dose conditions, the DVLP (SV-G VLP) envelope protein with dendritic cell-specific targeting ability can induce the body to produce more spike-specific IgG than the VSV-G envelope protein-modified VLP with non-specific targeting, that is, DVLP (SV-G VLP) has higher immune efficiency, which means that the VLP with SV-G pseudotype envelope has higher DC cell targeting.

[0068] Mice (C57BL / 6J) were immunized with a single dose of LNPs (10 μg) carrying spike mRNA or DVLPs (SV-G VLPs, 2 μg p24) (VLPs modified with the DC-targeting envelope protein SV-G are referred to as DVLPs) via footpad injection. A negative control group (placebo) received an equal volume of PBS. Fourteen days after immunization, mice were euthanized, and serum was obtained for the detection of spike-specific IgG.

[0069] The results in Figure 4 show that the level of spike-specific IgG induced by DVLP was significantly higher than that of traditional nanoliposome LNP. Therefore, DVLP has a higher immune efficiency due to its DC cell targeting and mRNA delivery capabilities.

[0070] Example 4

[0071] In vivo targeting experiment of dendritic cell-specific virus-like particles (DVLPs)

[0072] (1) Experimental materials:

[0073] 6-week-old male C57BL / 6J mice, DC-specific virus-like particles (DVLPs), non-specifically targeted virus-like particles (VSV-G VLPs), liposome particles (LNPs), anti-CD11c antibody (Servicebio, B11059), anti-spike antibody (Genetex, GTX632604), 488-conjugated donkey anti-rabbit secondary antibody (Jackson, 11-547-003), 555-conjugated goat anti-mouse secondary antibody (Huabio, HA1118), and DAPI dye (Beyotime, P0131).

[0074] (2) Experimental methods:

[0075] A. DVLP (2 μg), VSV-G VLP (2 μg), and LNP (10 μg) were injected into mice via footpad injection;

[0076] B. 12 hours after injection, mice were euthanized and lymph nodes were removed for fixation and subsequent frozen sectioning.

[0077] C. Permeabilize the sections with 0.4% Triton X-100 at room temperature for 20 minutes.

[0078] D. Block the sections with PBS containing 1% BSA and 2% goat serum at room temperature for 20 minutes.

[0079] E. Dilute anti-CD11c and anti-Spike antibodies in blocking buffer at a dilution ratio of 1:400 and incubate with primary antibodies at 4°C overnight.

[0080] F. Wash the sections with PBS three times, 5 minutes each time.

[0081] G. Dilute the 488-conjugated donkey anti-rabbit secondary antibody and the 555-conjugated goat anti-mouse secondary antibody at a dilution ratio of 1:800 in PBS containing 1% BSA. Incubate with the secondary antibodies at room temperature for 1 hour.

[0082] H. Wash the sections with PBS three times, 5 minutes each time.

[0083] J. Use DAPI to stain the cell nucleus and seal the slides.

[0084] (3) Experimental results

[0085] As shown in Figure 5, the antigen (spike) expressed by DVLP highly overlaps with the DC cell marker (CD11c) (indicated by the arrow), demonstrating its DC targeting in vivo. The results show that compared with VSV-G VLP and LNP, DVLP can deliver more target mRNA to lymph nodes and successfully express it. At the same time, DVLP and the DC cell marker CD11c have more co-localization, demonstrating that DVLP has good DC cell targeting function.

[0086] Example 5

[0087] In vivo distribution experiment of dendritic cell-specific virus-like particles (DVLPs)

[0088] (1) Experimental materials:

[0089] 6-week-old male C57BL / 6J mice, DC-specific virus-like particles (DVLP), non-specifically targeted virus-like particles (VSV-G VLP), liposome particles (LNP), RNA isolater (Vazyme, R401-01), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, R323-01), ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711).

[0090] (2) Experimental methods:

[0091] A. DVLP (2 μg), VSV-G VLP (2 μg), and LNP (10 μg) were injected into mice via footpad injection;

[0092] B. 12 hours after injection, mice were euthanized, and the footpads, lymph nodes, liver, spleen, and kidneys were removed;

[0093] C. Use RNA isolater to extract RNA from footpads, lymph nodes, and the remaining liver, spleen, and kidneys;

[0094] D. Reverse transcribe the RNA in D using HiScript III RT SuperMix for qPCR (+gDNA wiper);

[0095] E. Quantitative analysis of cDNA was performed using ChamQ Universal SYBR qPCR Master Mix. The detection primer sequences used included Spike-F as shown in SEQ ID NO. 11, Spike-R as shown in SEQ ID NO. 12, Mouse Gapdh-F as shown in SEQ ID NO. 13, and Mouse Gapdh-R as shown in SEQ ID NO. 14.

[0096] (3) Experimental results:

[0097] The results in Figure 6 show that compared to LNP, both DVLP and VSV-G VLP have the characteristics of low organ off-target, and the delivered mRNA is concentrated in the injection site (footpad) and lymph nodes. Although there is no significant difference in the distribution of DVLP and VSV-G VLP in the footpad, the content of DVLP in the lymph nodes is significantly higher than that of VSV-G VLP, which indicates that DVLP has better lymph node targeting than VSV-G VLP.

[0098] Example 6

[0099] Delivery of HSV-1 gB1 and gD1 mRNA via dendritic virus-like particles (DVLPs) specifically targeting DCs effectively protected mice against HSV-1 viral infection.

[0100] (1) Experimental materials:

[0101] 6-week-old male C57BL / 6J mice, dendritic virus-like particles (DVLPs) specifically targeting DC cells, HSV-1 virus, Vero cells, low-melting point agarose (Sangon, A600015), TaKaRa MiniBEST Viral RNA / DNA Extraction Kit (TaKaRa, 9766), and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711).

[0102] (2) Experimental methods:

[0103] A. On days 0 and 14, DVLPs (2 μg) (carrying HSV-1 gB1 and gD1 mRNA) were injected into mice via footpad injection as primary and booster immunizations, respectively. The method for constructing DVLPs carrying HSV-1 gB1 and gD1 mRNA was similar to that in Example 1, except that pCMV-Spike-mut-6×MS2 in step S3 was replaced with pCMV-gB1-gD1-6×MS2. The sequence of pCMV-gB1-gD1-6×MS2 is shown in SEQ ID NO. 15 in the sequence listing. Mice in the negative control group (Placebo / NC) were injected with an equal volume of PBS.

[0104] B. On days 7 and 21, collect serum from mice for serum neutralization testing. Dilute the serum at a 1:10 ratio for the first time, then dilute it two-fold each time until the final dilution reaches 1:1280. Thoroughly mix the diluted serum with HSV-1 virus at a titer of 50 pfu and incubate at 37°C for 1 hour. Add the mixture to Vero cells and incubate for 1 hour. Discard the supernatant, add 1% low-melting-point agarose, and continue incubation for 72 hours. Read the corresponding EC50 titer.

[0105] C. On the 28th day, the back of the mouse was depilated near the spine, and the skin surface was gently scraped 16 times with disposable sandpaper, and then 10 7 pfu of HSV-1 (10 μL) for infection.

[0106] D. On day 34, the infected skin and dorsal root ganglia of the mice were removed, and the virus was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit. Viral titer and viral genome were detected (quantitative analysis was performed using ChamQ Universal SYBR qPCR Master Mix). The detection primer sequences used included HSV-1 gD-F as shown in SEQ ID NO. 16, HSV-1 gD-R as shown in SEQ ID NO. 17, Mouse Gapdh-F as shown in SEQ ID NO. 13, and Mouse Gapdh-R as shown in SEQ ID NO. 14.

[0107] (3) Experimental results:

[0108] The results in Figure 7 show that the primary immunization significantly induced the production of neutralizing antibodies against HSV-1, and booster immunization further increased the neutralizing antibody titer. The results in Figure 8 show that after two immunizations, the viral load in the mouse skin tissue was significantly reduced, and the presence of virus was almost undetectable in the dorsal root ganglia, indicating that vaccination with DVLP gB1-gD1 mRNA has a strong neuroprotective effect.

[0109] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A dendritic cell-targeted virus-like body DVLP, characterized in that: The virus-like body DVLP comprises virus-like particles VLP and Sindbis virus glycoprotein, wherein the Sindbis virus glycoprotein is wrapped on the surface of the VLP, and mRNA is wrapped inside the VLP.

2. The virus-like body DVLP according to claim 1, characterized in that The amino acid sequence of the Sindbis virus glycoprotein is shown in SEQ ID NO.

1.

3. The virus-like body DVLP according to claim 1, characterized in that The mRNA includes at least one of mRNAs of spike, viral antigen, tumor antigen, and bacterial antigen.

4. A method for preparing a virus-like body DVLP according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Optimizing the amino acid sequence of the envelope protein of the wild-type Sindbis virus to obtain the SV-G gene; S2, constructing the expression vector of SV-G gene to obtain pCMV-SV-G plasmid; S3. HEK293T cells were co-transfected with pCMV-SV-G, pRSV-REV, antigen expression vector, pMDlg / PRRE-D64V and pMS2S-PH-gagpol-D64V plasmids. After transfection, the cell culture supernatant was collected and purified.

5. The preparation method according to claim 4, characterized in that: In step S1, the nucleotide sequence of the SV-G gene is shown as SEQ ID NO.

2.

6. The preparation method according to claim 4, characterized in that: The construction of the expression vector of the SV-G gene in step S2 comprises the following steps: S2.1, integrate homology arms upstream and downstream of the SV-G sequence to obtain an artificially synthesized sequence; S2.2, double-digest the artificially synthesized sequence and the pMD2.G plasmid, purify and recover; S2.

3. Use T4 DNA ligase to ligate the recovered products to form plasmid pCMV-SV-G.

7. The preparation method according to claim 6, characterized in that: In step S2.1, the artificially synthesized sequence is shown as SEQ ID NO.

3.

8. The preparation method according to claim 4, characterized in that: In step S2, the nucleotide sequence of the pCMV-SV-G is shown in SEQ ID NO.

4.

9. The preparation method according to claim 4, characterized in that: The antigen expression vector is pCMV-mRNA-mut-6×MS2.

10. Use of the virosome DVLP according to any one of claims 1 to 3 or the virosome DVLP obtained by the preparation method according to any one of claims 4 to 9 in the preparation of an mRNA vaccine.

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