Nucleic acid molecule related to HIV-1 (human immunodeficiency virus-1), fusion protein as well as preparation method and application of nucleic acid molecule

Abstract

The invention provides a nucleic acid molecule related to HIV-1 (human immunodeficiency virus-1), a fusion protein and a preparation method and application thereof, and belongs to the technical field of biological medicine.The mRNA vaccine provided by the invention encodes the fusion protein, and the fusion protein is any one of a Ub-Gag fusion protein and a Ub-Pol fusion protein, the protein can efficiently induce HIV-1 specific cytotoxic T lymphocyte (CTL) response, so that the capability of eliminating latent viruses of an organism is enhanced, and the defects that an existing HIV-1 vaccine is insufficient in immunogenicity and immune escape is likely to occur are effectively overcome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an HIV-1 related nucleic acid molecule, fusion protein, its preparation method and application. Background Technology

[0002] The immune control of human immunodeficiency virus type 1 (HIV-1) primarily relies on the specific response of cytotoxic T lymphocytes (CTLs). Numerous studies have shown that CTLs targeting highly conserved epitopes in the HIV-1 genome can effectively recognize and eliminate infected cells, significantly inhibiting viral replication and delaying disease progression. In contrast, immune responses targeting hypervariable regions of the virus are highly susceptible to mutation and can fail, leading to "immune escape." Therefore, designing multi-epitope vaccines centered on CTL epitopes in conserved functional regions can theoretically limit viral escape space to the greatest extent, improving the broad-spectrum and durable nature of the vaccine. However, traditional vaccine platforms based on DNA or recombinant proteins generally face problems in clinical practice, such as insufficient immunogenicity, low antigen presentation efficiency, and weak and unsustainable T-cell responses, severely limiting their protective efficacy.

[0003] Although antiretroviral therapy (ART) can effectively suppress viral replication, significantly reduce viral load, and prolong patient survival, it cannot completely eliminate the latent viral reservoir in the body. Once medication is discontinued, the virus often rebounds rapidly, requiring lifelong medication and presenting challenges such as poor adherence, long-term toxicity accumulation, and the risk of drug resistance. This indicates that relying solely on drug intervention is insufficient to achieve a functional cure for HIV. Against this backdrop, reconstructing or enhancing the body's specific immune response to HIV-1 through therapeutic vaccines, particularly inducing potent and durable conserved epitope-specific CTL responses, has become a key strategy for promoting a functional cure.

[0004] In recent years, mRNA vaccines, with their powerful immune activation capabilities demonstrated in previous epidemics, have provided new opportunities for the development of therapeutic HIV-1 vaccines. However, mRNA technology in the HIV field is still in the exploratory stage. Most studies focus on full-length or large segments of antigen sequences, lacking systematic screening and rational design of conserved CTL epitopes, which can easily lead to a dispersed immune response or even induce non-protective or interfering reactions. At the same time, the sequence stability, translation efficiency, and in vivo targeted delivery systems of mRNA further limit the strength and duration of its immune effect. Therefore, developing a novel vaccine platform based on conserved HIV-1 CTL epitopes, optimizing the mRNA sequence, and matching it with an efficient delivery system has significant scientific importance and clinical application prospects. Summary of the Invention

[0005] The main objective of this invention is to provide an HIV-1 related nucleic acid molecule, fusion protein, its preparation method and application, in order to overcome the technical problems of insufficient immunogenicity and easy immune escape of existing HIV-1 vaccines, thereby effectively enhancing the body's ability to clear latent viruses.

[0006] To achieve the above objectives, the present invention provides an HIV-1 related nucleic acid molecule, characterized in that the HIV-1 related nucleic acid molecule includes a first nucleotide encoding a Ub-Gag protein and / or a second nucleotide encoding a Ub-Pol protein, wherein the sequence of the first nucleotide is as shown in SEQ ID NO:3; and the sequence of the second nucleotide is as shown in SEQ ID NO:4.

[0007] The present invention further provides a fusion protein, wherein the fusion protein is either the Ub-Gag protein or the Ub-Pol protein; wherein, The amino acid sequence of the Ub-Gag protein is shown in SEQ ID NO:1; The amino acid sequence of the Ub-Pol protein is shown in SEQ ID NO:2.

[0008] This invention provides a method for preparing the combination of fusion proteins as described above, wherein the fusion protein combination includes Ub-Gag fusion protein and Ub-Pol fusion protein, and the preparation steps are as follows: The Ub-Gag gene fragment and the Ub-Pol target gene fragment were obtained separately; The gene fragments of Ub-Gag and Ub-Pol were inserted into expression vectors by enzyme digestion or cloning techniques, and then sequenced and verified to obtain recombinant plasmids of Ub-Gag and Ub-Pol, respectively. The recombinant plasmids of Ub-Gag and Ub-Pol were transfected into cells, respectively. The transfected cells were collected, lysed, and the proteins were collected and purified to obtain Ub-Gag protein and Ub-Pol protein.

[0009] In some implementations, the steps of obtaining the gene fragments of Ub-Gag and Ub-Pol include: CTL epitopes of 8-11 amino acid lengths were screened from HIV-1 database sequences, and HLA-A02 and HLA-A11 were selected for restriction HLA. Full-length gag and pol protein amino acid sequences were downloaded from the HIV database. Gag CTL epitopes with greater than 50% conservation and pol CTL epitopes with greater than 90% conservation were screened to obtain multiple epitopes of Ub-Gag and Ub-Pol, respectively. Multiple epitopes of the Ub-Gag are linked to obtain the Ub-Gag gene fragment, and multiple epitopes of the Ub-Pol are linked to obtain the Ub-Pol gene fragment.

[0010] The present invention also provides an expression vector carrying an HIV-1-related nucleic acid molecule encoding either the Ub-Gag fusion protein or the Ub-Pol fusion protein.

[0011] In some embodiments, the present invention further provides the use of the HIV-1-related nucleic acid molecules as described above, the fusion proteins as described above, the fusion proteins obtained by the preparation methods of combinations of the fusion proteins as described above, or the expression vectors as described above in the preparation of medicaments for the treatment or prevention of HIV-1-related infectious diseases.

[0012] In some embodiments, the HIV-1-related disease includes HIV-1 infection and its associated complications, including lung infections, digestive system infections, or malignancies associated with HIV-1 infection.

[0013] In some embodiments, the present invention also provides a vaccine composition of multiple CTL epitope mRNAs. The vaccine composition includes: (a) HIV-1-related nucleic acid molecules as described above; and (b) Pharmaceutically acceptable carriers and / or delivery systems.

[0014] In some embodiments, the delivery system is a lipid nanoparticle, liposome, emulsion, or polymer nanoparticle.

[0015] Overall, the Ub-Gag and Ub-Pol fusion proteins and their encoded nucleic acids, expression vectors, and mRNA vaccines provided by this invention can efficiently induce HIV-1-specific CTL responses and significantly enhance the body's ability to clear latent HIV-1 virus, thereby largely overcoming the technical difficulties of insufficient immunogenicity and immune escape in existing HIV-1 vaccines, and have good application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1This is a schematic diagram of the specific reaction for detecting epitope peptides using IFN-γ ELISpot. Figure 2 A schematic diagram of a vaccine design characterized by ubiquitin protein linking multiple CTL epitopes; Figure 3 This is a schematic diagram showing the expression levels of the antigen protein under different treatment conditions. Figure 4 A schematic diagram of an experiment induced by Ub-Gag and Ub-Pol proteins to kill HIV-1 reservoir cells; Figure 5 This is a plasmid map of pcDNA3.1+.

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] To make the objectives, technical solutions, and beneficial effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In recent years, mRNA vaccines, with their powerful immune activation capabilities demonstrated in previous epidemics, have provided new opportunities for the development of therapeutic HIV-1 vaccines. However, mRNA technology in the HIV field is still in the exploratory stage. Most studies focus on full-length or large segments of antigen sequences, lacking systematic screening and rational design of conserved CTL epitopes, which can easily lead to a dispersed immune response or even induce non-protective or interfering reactions. At the same time, the sequence stability, translation efficiency, and in vivo targeted delivery systems of mRNA further limit the strength and duration of its immune effect. Therefore, developing a novel vaccine platform based on conserved HIV-1 CTL epitopes, optimizing the mRNA sequence, and matching it with an efficient delivery system has significant scientific importance and clinical application prospects.

[0021] In view of this, the present invention provides an HIV-1 related nucleic acid molecule, the HIV-1 related nucleic acid molecule comprising a first nucleotide encoding a Ub-Gag protein and / or a second nucleotide encoding a Ub-Pol protein, wherein the first nucleotide sequence is shown in SEQ ID NO:3; and the second nucleotide sequence is shown in SEQ ID NO:4.

[0022] The specific sequence of the first nucleotide of the Ub-Gag is as follows: TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCTCTAGAGCCACCATGCAAATCTTCGTGAAGACCCTGACCGGCAAGACAATCACACTCGAGGTGGAACCTAGCGATACCATCGAGAACGTGAAGGCCAAAATTCAGGACAAGGAGGGCATCCCTCCAGACCAGCAGAGGCTGATCTTTGCCGGCAAGCAGCTGGAGGACGGCAGAACCCTGTCCGACTACAATATCCAGAAAGAGAGCACACTGCACCTGGTGCTGAGACTGAGAGGCGCCGCTGAAGCCGCCGCCAAGGAGGCCGCCGCTAAGGCCAAGATCAGACTGAGACCTGGCGGCAAAAAGCGCGTCCGAAGATCTCCTCGGACCCTGAACGCCTGGGTGAAAGTGCGGGTCAGACGGGGCCACCAGGCCGCTATGCAAATGCTGAAGGAAAGAGTGCGGAGAATCTATAAGAGATGGATCATCCTGGGACTGAGGGTGCGGAGAAGCATCCTGGATATCCGGCAGGGACCTAAGAGAGTTAGACGGGAACCCTTCAGAGATTACGTGGACAGATTCTACAGAGTGCGGCGGGCCACCCTGGAGGAAATGATGACCGCCAGAGTGAGAAGAGCCTGCCAGGGCGTGGGCGGCCCCGGACACAAGCGGGTGAGACGCTTCCTGGGCAAGATCTGGCCCAGCGCCGAGGCCGCCGCTAAGGAGGCCGCTGCCAAGGCTCACCATCACCACCACCACCACCACTAACTTAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA, wherein, the first T7 promoter + the first 5' UTR + the first target antigen + the first 8×His + the stop codon TAA + the restriction enzyme cutting site AfiⅡ + the first 3' UTR, and the specific sequences are as follows: The specific sequence of the first T7 promoter is TAATACGACTCACTATAGGG; The specific sequence of the first 5'UTR is as follows: AATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCTCTAGAGCCACC; The specific sequence of the primary target antigen is as follows: ; The specific sequence of the first 8×His is: CACCATCACCACCACCACCACCACCAC; The specific sequence of the stop codon is TAA; The specific sequence of the restriction enzyme cleavage site AfiⅡ is: CTTAAG; The specific sequence of the first 3'UTR is as follows: TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA.

[0023] The nucleic acid of Ub-Pol, i.e., the second nucleotide sequence, is as follows: TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCTCTAGAGCCACCATGCAAATCTTCGTGAAGACCCTGACCGGCAAGACAATCACACTCGAGGTGGAACCTAGCGATACCATCGAGAACGTGAAGGCCAAAATTCAGGACAAGGAGGGCATCCCTCCAGACCAGCAGAGGCTGATCTTTGCCGGCAAGCAGCTGGAGGACGGCAGAACCCTGTCCGACTACAATATCCAGAAAGAGAGCACACTGCACCTGGTGCTGAGACTGAGAGGCGCCGCTGAAGCCGCCGCCAAGGAGGCCGCCGCTAAGGCCAAAATGATCGGCGGCATCGGCGGATTTATCAGAGTTAGAAGAAAGCTGGTGGACTTCCGGGAACTGAACAAGAGAGTGCGGAGAGTGCTGGATGTGGGAGATGCCTACTTCAGCGTGCGGGTCAGAAGATACCAGTACATGGACGACCTGTACGTGAGAGTGCGGAGATTCGTGAACACACCTCCTCTGGTGAAAAGAGTGAGGCGCCCCCTCGTCAAGCTGTGGTATCAGCTGAGAGTGAGACGGGCTGTGTTCATCCACAATTTCAAGCGGAGAGTGCGGAGACTGCTGTGGAAGGGCGAGGGCGCCGTGCGGGTGAGAAGACACCTGAAGACCGCCGTGCAGATGGCCGTGGCCGAGGCCGCCGCTAAGGAGGCCGCTGCCAAGGCTCACCATCACCACCACCACCACCACTAACTTAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA, where the second T7 promoter + the second 5' UTR + the second target antigen + the second 8×His + the second stop codon TAA + the second restriction enzyme site Afi II + the second 3' UTR, and the specific sequences are as follows: The specific sequence of the second T7 promoter is: TAATACGACTCACTATAGGG; The specific sequence of the second 5'UTR is as follows: AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC; The specific sequence of the second target antigen is as follows: ; The specific sequence of the second 8×His is: CACCATCACCACCACCACCACCACCAC; The specific sequence of the stop codon is TAA; The specific sequence of the restriction enzyme cleavage site Afi II is: CTTAAG; The specific sequence of the second 3'UTR is as follows: TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA.

[0024] The present invention also provides a fusion protein, wherein the fusion protein is either the Ub-Gag protein or the Ub-Pol protein; wherein, The amino acid sequence of the Ub-Gag protein is shown in SEQ ID NO:1; The amino acid sequence of the Ub-Pol protein is shown in SEQ ID NO:2.

[0025] Specifically, the amino acid sequence of the Ub-Gag is as follows: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGAAEAAAKEAAAKAKIRLRPGGKKRVRRSPRTLNAWVKVRVR RGHQAAMQMLKERVRRIYKRWIILGLRVRRSILDIRQGPKRVRREPFRDYVDRFYRVRRATLEEMMTARVRRACQGVGGPGHKRVRRFLGKIWPSAEAAAKEAAAKAHHHHHHHH.

[0026] The amino acid sequence of the Ub-Pol is as follows: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGAAEAAAKEAAAKAKMIGGIGGFIRVRRKLVDFRELNKR VRRVLDVGDAYFSVRVRRYQYMDDLYVRVRRFVNTPPLVKRVRRPLVKLWYQLRVRRAVFIHNFKRRVRRLLWKGEGAVRVRRHLKTAVQMAVAEAAAKEAAAKAHHHHHHHH.

[0027] In relevant embodiments, the Ub-Gag and / or Ub-Pol proteins can be expressed in host cells from mRNA obtained through in vitro transcription, constituting the core immunogenic components of two multi-CTL epitope mRNA vaccines. Through the combination and optimized design of conserved CTL epitopes in the Gag and Pol regions, the fusion protein of the present invention can deliver multiple highly conserved and immunogenic CTL epitopes within a limited antigen length, thereby significantly improving the breadth and intensity of the cellular immune response against HIV-1 while ensuring safety.

[0028] It should be noted that in this invention, "Ub-Gag protein" and "Ub-Pol protein" are artificial fusion proteins designed in HIV (human immunodeficiency virus) vaccines or immunization research. Their names are derived from two parts: Ub (ubiquitin) and Gag or Pol (HIV viral structural proteins or enzyme proteins). Gag protein is a precursor to the core structural protein of HIV, playing a crucial role in viral assembly and is one of the most common CTL recognition targets in HIV-infected individuals. Pol protein encodes enzymes required for viral replication, including reverse transcriptase, integrase, and protease. These enzymes are essential for the viral life cycle and are also important CTL targets. Ub can guide multi-epitope peptides derived from Gag or Pol proteins to enter the proteasome degradation pathway more quickly, producing polypeptide fragments, which are then presented to CD8 via MHC class I molecules. + T cells, thereby more strongly activating CTL responses, these fusion proteins are often used as candidate antigens to test their ability to kill HIV-1 infected cells in animal models or clinical trials.

[0029] In some embodiments of the present invention, the present invention provides a method for preparing a combination of fusion proteins as described above, wherein the combination of fusion proteins includes Ub-Gag fusion protein and Ub-Pol fusion protein, and the preparation steps are as follows: The Ub-Gag gene fragment and the Ub-Pol target gene fragment were obtained separately; The gene fragments of Ub-Gag and Ub-Pol were inserted into expression vectors by enzyme digestion or cloning techniques, and then sequenced and verified to obtain recombinant plasmids of Ub-Gag and Ub-Pol, respectively. The recombinant plasmids of Ub-Gag and Ub-Pol were transfected into cells, respectively. The transfected cells were collected, lysed, and the proteins were collected and purified to obtain Ub-Gag protein and Ub-Pol protein.

[0030] It should be noted that a Kozak sequence (specifically GCCACC) is inserted upstream of the start codon (ATG) of each fusion gene (Ub-Gag or Ub-Pol) to enhance translation initiation efficiency. The expression vector is preferably a pcDNA3.1(+) eukaryotic expression vector, which contains a strong CMV promoter, supporting high-level expression in mammalian cells. Using the above technical approach, by screening for highly conserved Gag and Pol CTL epitopes, immunogenicity can be improved while effectively reducing the risk of immune escape.

[0031] In some embodiments of the present invention, the steps of obtaining the gene fragments of Ub-Gag and Ub-Pol include: CTL epitopes of 8-11 amino acid lengths were screened from HIV-1 database sequences, and HLA-A02 and HLA-A11 were selected for restriction HLA.

[0032] Full-length gag and pol protein amino acid sequences were downloaded from the HIV database. Gag CTL epitopes with greater than 50% conservation and pol CTL epitopes with greater than 90% conservation were screened to obtain multiple epitopes of Ub-Gag and Ub-Pol, respectively. Multiple epitopes in the Ub-Gag are linked to obtain the Ub-Gag gene fragment, and multiple epitopes in the Ub-Pol are linked to obtain the Ub-Pol gene fragment.

[0033] It should be noted that, to ensure broad coverage of the applicable population for the vaccine, this invention focuses on the major HIV-1 subtypes (type B, type C, and CRF01_AE recombinant type) prevalent globally, particularly in Asia. Known HLA-I class-restricted CTL epitopes are systematically screened from authoritative international HIV databases. The screening criteria are as follows: 1) Download a large number of full-length amino acid sequences of HIV-1 Gag and Pol proteins (covering multiple subtypes) from the HIV database. Among them, the CTL epitopes in Gag protein have a conservation rate of >50%, and the CTL epitopes in Pol protein have a conservation rate of >90%. Finally, highly conserved CTL epitopes were selected.

[0034] 2) The epitope length is selected from classic MHC class I binding peptides with a length of 8–11 amino acids, which meets the basic requirements for CTL recognition; 3) HLA restriction: The focus is on epitopes presented by HLA-A*02 and HLA-A*11 molecules, because these two alleles have a high carrier frequency in East Asian and Chinese populations (covering more than 60% of the population in total), which helps to improve the applicability of the vaccine in the target population.

[0035] Furthermore, in the process of constructing and expressing multiple epitopes, ubiquitin tags are preferably introduced to promote proteasome processing; cleavable linkers (RVRR) are used to ensure the correct release of epitopes; and flexible linker peptides are used to optimize the spatial conformation. The above construction strategies can be applied to the expression framework of DNA vaccines and can also serve as the design basis for the coding sequence of mRNA vaccines, thereby providing a structural platform for the further transformation of therapeutic vaccines.

[0036] Specifically, the operation steps are as follows: Screen for CTL epitopes of 8–11 amino acids in length from the database sequences of common HIV-1 subtypes (B, C, and CRF01_AE recombinant type), select HLA-A02 and HLA-A11 for restriction HLA, download the full-length gag and pol protein amino acid sequences from the HIV database, screen for gag CTL epitopes with greater than 50% conservation and pol CTL epitopes with greater than 90% conservation, and tandem the 9 selected gag epitopes or 9 pol epitopes to form two independent multi-epitope sequences.

[0037] Furthermore, multi-epitope constructs were designed by linking selected CTL epitopes with cleavage peptide-sensitive linkers containing RVRR sequences. Each construct had its N-terminus fused with a ubiquitin protein (Ub) via an A(EAAAK)2A linker peptide (specifically, the sequence AEAAAAKEAAAKA) to promote epitope presentation and enhance immunogenicity. An 8×His tag was linked to the C-terminus via the A(EAAAK)2A linker peptide for protein detection, resulting in two final vaccine candidates, named Ub-Gag and Ub-Pol, respectively. To ensure efficient transcription, a Kozak sequence was inserted upstream of each Ub-Gag / Pol fusion construct, and these sequences were cloned into the pcDNA3.1 expression vector.

[0038] The present invention provides an expression vector carrying a nucleic acid such as the polypeptide, which can be used to express the fusion protein in a suitable host cell.

[0039] The present invention further provides the use of the fusion protein prepared by the preparation method of the HIV-1 related nucleic acid molecule as described above, the fusion protein as described above, the combination of the fusion proteins as described above, or the expression vector as described above in the preparation of a drug for the treatment or prevention of HIV-1 related infectious diseases.

[0040] The above-mentioned fusion proteins and expression vectors were used to prepare effective drugs that can effectively control HIV-1 replication and reduce the risk of opportunistic infections (such as lung and gastrointestinal infections) and HIV-related malignancies caused by immunodeficiency. This provides a safe, scalable and promising new immunomodulatory intervention strategy for the prevention and treatment of HIV-1 infection.

[0041] Furthermore, the HIV-1 related diseases include HIV-1 infection and its related complications, including lung infections, digestive system infections, or malignancies associated with HIV-1 infection.

[0042] This invention provides a vaccine composition for multiple CTL epitope mRNAs, the vaccine composition comprising: (a) HIV-1-related nucleic acid molecules as described above; and (b) Pharmaceutically acceptable carriers and / or delivery systems.

[0043] Furthermore, the delivery system is a lipid nanoparticle, liposome, emulsion, or polymer nanoparticle.

[0044] The delivery system is a lipid nanoparticle, liposome, emulsion or polymer nanoparticle. After being delivered to the body through a suitable delivery system (e.g. lipid nanoparticle), it can express the corresponding fusion protein in vivo and efficiently induce HIV-1 specific cytotoxic T lymphocyte (CTL) response, thereby constituting two multi-CTL epitope mRNA vaccines.

[0045] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0046] The IFN-γ ELISpot detection kit used in the following examples is model number (3420-2H) MABTECH, USA.

[0047] Example 1: Screening and Determination of CTL Epitopes First, CTL epitopes of 8-11 amino acids in length from common HIV-1 subtypes B and C and the CRF01_AE recombinant type were screened from the HIV database. The restrictive HLA molecules were HLA-A02 and HLA-A11.

[0048] Subsequently, samples containing the full-length amino acid sequences of gag and pol proteins were downloaded from the database, and the conservation of each candidate epitope was calculated. The screening criteria were that the conservation of gag protein epitopes was ≥50% and that of pol protein epitopes was ≥90%. Finally, nine epitope sequences of each were obtained, as shown in Table 1. Table 1 is as follows: Table 1. Conserved CTL epitope sequences in Gag and Pol proteins.

[0049] The results showed that the conservation rate of the pol protein epitope was 94.26%, which was significantly higher than that of the gag protein epitope (72.37%).

[0050] Example 2: Evaluation of epitope reactivity To verify the immunogenicity of the selected epitopes (epitaxes described in Table 1), this embodiment uses the IFN-γ ELISpot method to detect the specific response of peripheral blood T cells from acute-phase HIV-1 infected individuals to candidate epitope peptides.

[0051] First, approximately 10 ml of peripheral venous blood was collected from an HIV-1 infected individual in the acute phase. Peripheral blood mononuclear cells (PBMCs) were obtained by conventional density gradient centrifugation, counted, and adjusted to a concentration of 1 × 10⁶ cells / mL. 6 Cells / mL. Subsequently, candidate epitope peptide working solutions were prepared by dissolving in DMSO and diluting with PBS. PBMCs were seeded into ELISpot plate wells pre-coated with anti-human IFN-γ capture antibody, 200 μL per well. Different candidate epitope peptides were added as stimulation groups. Negative control wells containing only culture medium and positive control wells containing a final concentration of 5 μg / mL Con AT cell stimulant were also included. After incubation for 36 hours in a cell culture incubator at 37°C with 5% CO2, the wells were washed according to the kit instructions. HRP biotin-labeled anti-IFN-γ detection antibody, streptavidin, and substrate chromogenic solution were added sequentially. The reaction was terminated after spots appeared. The plates were washed, dried, and read and analyzed using an ELISpot automated plate reader. Final results were expressed as per million PBMCs (1010). 6 The number of IFN-γ spots formed in PBMCs indicates the intensity of the T cell response.

[0052] The results are as follows Figure 1 As shown: Figure 1 In the graph, A represents the corresponding IFN-γ level, the x-axis represents different epitope peptides, and the y-axis represents the number of IFN-γ positive spots generated per million PBMCs, reflecting the intensity of the T cell response. This demonstrates that multiple Gag epitopes can induce significant IFN-γ release in patients with acute-phase infection, indicating that these epitopes possess good immunogenicity. Figure 1 In the image, B represents patients with acute infection who exhibit a low level of IFN-γ-related inflammatory response. The black dots on the blue background are spots formed by IFN-γ-secreting cells. The more spots there are, the more IFN-γ-secreting cells there are, and the stronger the immune response. The differences in cellular response intensity under stimulation of various epitopes are shown through photographs of actual ELISpot membranes.

[0053] Example 3: mRNA vaccine construction and design In sequence design, the selected multiconserved CTL epitopes are linked by flexible or cleavable linker peptides, including the A(EAAAK)2A rigid spacer sequence and the RVRR proteasome cleavage sequence. A ubiquitin (Ub) protein sequence is added to the N-terminus of the multi-epitope tandem structure to promote proteasome processing of the antigen peptide and MHC-I pathway presentation, and a His tag is added to the C-terminus for protein expression detection. A schematic diagram of the vaccine construction design is shown below. Figure 2 As shown, the DNA sequences of the candidate vaccines, specifically Ub-Gag and Ub-Pol, were synthesized in a pcDNA3.1-based plasmid vector backbone and cloned between the sequences tgcttactggcttatcgaaat and gggcccgtttaaacccgctga, in a 5' to 3' direction, resulting in plasmids pcDNA3.1 MVUb-Gag and pcDNA3.1 MV Ub-Pol, respectively. The pcDNA3.1 plasmid was purchased from the plasmid vector library of Nanjing GenScript Biotech Co., Ltd. The downloaded image is shown below. Figure 5 The purchase address is as follows: The specific steps are as follows: [https: / / www.genscript.com.cn / vector / detail?vector_name=cGNETkEzLjEoJTJCKQ==](https: / / www.genscript.com.cn / vector / detail?vector_name=cGNETkEzLjEoJTJCKQ==). I. Obtaining linearized templates via PCR reaction The following primers were selected: plasmid pcDNA3.1 MV Ub-Gag and pcDNA3.1 MV Ub-Pol were used as templates. The extension time was 1 min. MV is an abbreviation for mRNA vccine. The specific primers are as follows: MV Ub Forward:TAATACGACTCACTATAGGGAAATA; MV Ub Reverse:TCAGACTTTATTCAAAGACCAC.

[0054] 2. Gel recovery of the target band from the PCR product. II. In vitro transcription of linear fragments The linearized template was transcribed in vitro using the NEB HiScribe™ T7 High Yield RNA Synthesis Kit (E2040S). The reaction system is shown in Table 1. Table 1

[0055] Reaction conditions: Incubate at 37°C for 2 hours. (Note: A water bath / metal bath / PCR instrument can be used).

[0056] DNase digestion: After reacting the above system at 37°C for two hours, add 70 μl of RNase-free-H2O, 10 μl of 10* DNase I buffer, and 3 μl of DNase I to a 20 μl system, mix well, centrifuge, and react at 37°C for 15 minutes.

[0057] LiCl purification: The RNA product was purified by LiCl precipitation at -20℃ for about 2 hours to obtain the purified product.

[0058] III. mRNA Capping 1. Place 14 μl (10 μg) of dissolved RNA, i.e. the purified product, at 65°C for 5 minutes to denature it. 2. Remove the heat-denatured RNA and place it on ice for 5 minutes; 3. Configure the following reaction systems as shown in Table 2.

[0059] Table 2

[0060] 4. Place the above reaction at 37°C for 60 minutes (for RNA shorter than 200 nt, the reaction time can be increased to 2 hours) (using a metal bath). 5. Purify the capped RNA product by LiCl precipitation at -20℃. The RNA was precipitated at -20℃ for about 2 hours to obtain the purified product.

[0061] IV. mRNA tailing 1. The linearized template obtained by PCR does not contain an adenine tail and must be tailed using E. coli poly(A) polymerase.

[0062] 2. Configure the following tailing reaction system.

[0063] 3. Place the above system at 37°C for 30 min to react; 4. Remove the RNA after the reaction is complete and purify the capped and tailed RNA product by LiCl precipitation at -20℃. The RNA is precipitated at -20℃ for about 2 hours to obtain mRNA encoding Ub-Gag and Ub-Pol fusion proteins, respectively. The 5' end contains a Cap 1 structure and the 3' end contains a poly(A) tail, which can be used for in vitro or in vivo expression of antigen proteins.

[0064] Example 4: In vitro expression and degradation verification This embodiment aims to verify the in vitro expression of the Ub-Gag and Ub-Pol fusion protein constructed in Example 2 and whether it is degraded via the ubiquitin-proteasome pathway. To this end, an mRNA vaccine containing a ubiquitin tag and multiple epitope sequences was delivered to 293T cells via mRNA transfection reagent. Western blot was used to detect the expression levels of His-tagged proteins and the internal control β-actin, and the effect of the proteasome inhibitor MG132 on its stability was observed.

[0065] In Example 4, the experimental groups included a Mock control group, a Ub-Gag group, a Ub-Gag+MG132 group, a Ub-Pol group, and a Ub-Pol+MG132 group. The Ub-Gag or Ub-Pol encoded mRNA obtained through in vitro transcription was mixed with transfection reagent and added to 500 μL of 293T cells in the corresponding group. In the MG132 treatment group, 0.5 μL of the proteasome inhibitor MG132 was added to the culture medium at a ratio of 1:1000 after transfection, while the other groups received an equal volume of 0.5 μL of solvent as a control. The culture medium was discarded 24 h after transfection, and the cells were treated with pre-cooled PB... Cells were washed with S, lysed on ice with lysis buffer containing protease inhibitors, and the supernatant was collected by centrifugation as total protein extract. Protein concentration was determined by BCA method, and after uniform loading, SDS-PAGE electrophoresis was performed for separation and membrane transfer. Anti-His tag primary antibody (Proteintech, 66005-1-IG), anti-β-actin primary antibody (Elabscience, E-AB-40338), and corresponding HRP-labeled secondary antibody (Elabscience, E-AB-1001) were added sequentially. After membrane washing, chemiluminescent substrate was added for color development, and Western blot images were acquired using an imaging system. The His band signal was quantitatively analyzed to evaluate the expression level of Ub-Gag and Ub-Pol fusion proteins under different treatment conditions and their proteasome-mediated degradation.

[0066] The results are as follows Figure 3 As shown, His-tagged protein (26 kDa) was detected in cells transfected with Ub-Gag and Ub-Pol mRNA, while no corresponding band was found in untransfected cells. This means that both Gag and Pol proteins can be expressed, and their stability is regulated by the proteasome. This suggests that these two proteins may be ubiquitinated and then enter the proteasome pathway for degradation.

[0067] Figure 3In the figure, B represents the relative expression level of the His-labeled protein normalized to β-actin, determined by Western blot. P < 0.001 was used in the paired t-test. The results showed that after normalizing His expression to β-actin, quantitative analysis revealed significant differences in protein levels under different conditions. Treatment with the proteasome inhibitor MG132 significantly increased the relative expression levels of Ub-Gag and Ub-Pol (p < 0.001), indicating that both undergo ubiquitin-mediated proteasome degradation. Compared to Ub-Gag, the Ub-Pol construct exhibited a higher basal expression level and a greater increase in expression after degradation inhibition, suggesting differences in degradation kinetics.

[0068] Example 5: In vitro efficacy evaluation In Example 5, 10 ml of EDTA-anticoagulated peripheral venous blood was collected from 10 HIV-infected patients who had received long-term antiretroviral therapy and were virologically suppressed. PBMCs were obtained by density gradient centrifugation, washed 1-2 times with PBS or serum-free culture medium, and counted. The PBMCs were then divided into approximately 1×10⁻⁶ cells / mL. 6 Cells / well were seeded in 96-well plates and pre-cultured for approximately 12 h at 37°C and 5% CO2 in complete medium containing 10% fetal bovine serum and IL-2 (final concentration 10 ng / mL); then, the following transfection mixture was added to each well: Control group: Only 2 μL of commercial mRNA transfection reagent (Yeasen, 40809ES03) was added. Ub-Gag group: 2 μL transfection reagent + 1 μg Ub-Gag mRNA; Ub-Pol group: 2μL transfection reagent + 1μg Ub-Pol mRNA; Ub-Gag / Pol group: 4 μL transfection reagent + 1 μg Ub-Gag mRNA + 1 μg Ub-Pol mRNA; Ub-Gag / Pol + anti-PD-1 group: 4 μL transfection reagent + 1 μg Ub-Gag mRNA + 1 μg Ub-Pol mRNA + 2.5 μg pembrolizumab (anti-PD-1 antibody) were added to the corresponding wells for transfection. The control group only added transfection reagent or blank complex without mRNA. About 2 h after transfection, PHA (final concentration 5 μg / mL) was added to each well to activate latent HIV reservoir cells and promote viral genome transcription. The cells were incubated under the same conditions for about 48 h. After incubation, the cells from each well were collected, centrifuged and washed, and then surface labeling and intracellular staining were performed according to the standard flow cytometry staining procedure. Specifically, CD3 and CD4 surface staining was performed first, followed by fixation / permeabilization treatment, and intracellular staining was performed using fluorescently labeled anti-HIV p24 antibody. After data acquisition by flow cytometry, the mean fluorescence intensity (MFI) of HIV p24 was calculated within the CD3+ T cell phylum. Fluorescently labeled antibodies were used to detect the expression of CD3, CD4, and HIV p24 antigens. Finally, data were acquired and analyzed using flow cytometry for CD4. + Changes in T cell proportion and p24-positive cell proportion were used to further analyze the effects of Ub-Gag, Ub-Pol, and their mixtures on T cell response and HIV reservoir cell clearance in PBMCs. The procedures are as follows: Figure 4 As shown in A in the diagram.

[0069] Test results are as follows Figure 4 As shown in B, the horizontal axis represents different stimulus conditions, and the vertical axis represents CD4. + The percentage reduction in T cells, compared with the control group, and CD4 count in each stimulation group + T cell reduction, Ub-Gag, Ub-Pol and their combined constructs can all induce CD4 reduction. + The significant reduction in T cell numbers suggests a cytotoxic effect, and Gag protein activates CD4 more effectively than Pol. + T cells; the combined use of both can produce a synergistic effect and enhance the immune response.

[0070] Further linear regression analysis showed a positive correlation between the reduction in CD4+ T cells and HIV p24 expression levels; the higher the p24 expression, the lower the CD4+ T cell count. + The more T cells decrease, the better. Figure 4 CF was used to analyze the correlation between the reduction of CD4+ T cells and HIV p24 expression levels under different stimulation conditions. The x-axis represents the HIV p24 MFI under different stimulation conditions, and the y-axis represents CD4+ T cell expression. +The percentage reduction in T cells (%) was measured, and the results showed that CD4 was observed in both the Ub-Gag stimulation group and the Ub-Gag / Pol combined with anti-PD-1 antibody stimulation group. + There is a significant positive correlation between the reduction in T cells and the expression level of HIV p24. Among them, Ub-Gag is more immunogenic, and its expression is more strongly correlated with T cell activation. Exogenous mRNA transfection can effectively induce antigen expression and drive specific T cell responses. The addition of immune checkpoint inhibitors (such as PD-1) can enhance the response and further enhance the cell-killing effect, suggesting that it may play a synergistic role through immune checkpoint regulation mechanisms. That is, the expression of HIV Gag and Pol proteins through mRNA transfection can effectively activate CD8+ T cells, supporting its potential as a candidate antigen for vaccines.

[0071] In summary, compared with existing technologies, this invention has the following beneficial effects: This invention systematically screens multiple highly conserved CTL epitopes from HIV-1 Gag and Pol proteins, and employs ubiquitin (Ub) fusion, appropriately long flexible spacer peptides, and cleavable linker sequences for engineered design. This significantly improves the antigen processing efficiency in the proteasome pathway and the antigen presentation efficiency via the MHC-I pathway, thereby enhancing the strength and persistence of HIV-1-specific CTL responses. The highly conserved combination of multiple epitopes significantly reduces the risk of immune escape due to viral mutations, and the engineered design reduces crosstalk between epitopes, making the response more focused and efficient. Compared with traditional DNA or protein vaccines, this invention utilizes an mRNA / LNP platform to avoid vector integration and anti-vector immunity issues, ensuring safety and controllability, while simplifying the process and reducing costs, offering advantages in rapid iteration and large-scale production. Furthermore, since the preparation process does not rely on large-scale virus or cell culture, it reduces biological pollution and environmental risks, meeting the needs of green production and clinical translation.

[0072] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above-described embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention, regardless of differences in biological species, materials used, injection time and frequency, operational details, etc. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule associated with HIV-1, characterized in that, The HIV-1-related nucleic acid molecule includes a first nucleotide encoding a Ub-Gag protein and / or a second nucleotide encoding a Ub-Pol protein, wherein the first nucleotide sequence is shown in SEQ ID NO:3; and the second nucleotide sequence is shown in SEQ ID NO:

4.

2. A fusion protein, characterized in that, The fusion protein is either the Ub-Gag protein or the Ub-Pol protein; wherein... The amino acid sequence of the Ub-Gag protein is shown in SEQ ID NO:1; The amino acid sequence of the Ub-Pol protein is shown in SEQ ID NO:

2.

3. A method for preparing a combination of fusion proteins as described in claim 2, characterized in that, The fusion protein combination includes Ub-Gag fusion protein and Ub-Pol fusion protein, and the preparation steps are as follows: The Ub-Gag gene fragment and the Ub-Pol target gene fragment were obtained separately; The gene fragments of Ub-Gag and Ub-Pol were inserted into expression vectors by enzyme digestion or cloning techniques, and then sequenced and verified to obtain recombinant plasmids of Ub-Gag and Ub-Pol, respectively. The recombinant plasmids of Ub-Gag and Ub-Pol were transfected into cells, respectively. The transfected cells were collected, lysed, and the proteins were collected and purified to obtain Ub-Gag protein and Ub-Pol protein.

4. The method for preparing the combination of fusion proteins as described in claim 3, characterized in that, The steps to obtain the gene fragments of Ub-Gag and Ub-Pol include: CTL epitopes of 8-11 amino acid lengths were screened from HIV-1 database sequences, and HLA-A02 and HLA-A11 were selected for restriction HLA. Full-length gag and pol protein amino acid sequences were downloaded from the HIV database. Gag CTL epitopes with greater than 50% conservation and pol CTL epitopes with greater than 90% conservation were screened to obtain multiple epitopes of Ub-Gag and Ub-Pol, respectively. Multiple epitopes of the Ub-Gag are linked to obtain the Ub-Gag gene fragment, and multiple epitopes of the Ub-Pol are linked to obtain the Ub-Pol gene fragment.

5. An expression carrier, characterized in that, The expression vector carries any of the HIV-1-related nucleic acid molecules as described in claim 1.

6. The use of a fusion protein prepared by the method of preparing a combination of the HIV-1-related nucleic acid molecule of claim 1, the fusion protein of claim 2, or the fusion protein of claim 3 or 4, or the expression vector of claim 5, in the preparation of a drug, characterized in that, The drug is used to treat or prevent HIV-1-related infectious diseases.

7. The use as described in claim 6, characterized in that, The HIV-1-related infectious diseases include HIV-1 infection and its complications, including lung infections, digestive system infections, or malignancies associated with HIV-1 infection.

8. A vaccine composition containing multiple CTL epitope mRNAs, characterized in that, The vaccine composition includes: (a) The HIV-1-related nucleic acid molecule as described in claim 1; and (b) Pharmaceutically acceptable carriers and / or delivery systems.

9. The multi-CTL epitope mRNA vaccine composition according to claim 8, characterized in that, The delivery system is a lipid nanoparticle, liposome, emulsion, or polymer nanoparticle.

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