Virus vector and application thereof in infecting B cells

Abstract

The invention relates to a lentiviral vector or retroviral vector and application thereof in infection of B cells, establishment of acquired immunity in subjects and preparation of broad-spectrum neutralizing antibodies. A virus envelope of the lentiviral vector or the retroviral vector contains a targeting molecule targeting B cells and carries a broad-spectrum neutralizing antibody gene; the viral glycoprotein of the lentiviral vector or retroviral vector may further comprise a first mutation that attenuates its ability to bind to a receptor and a second mutation that enhances its ability to antagonize complement inactivation, the capacity of the lentiviral vector or the retroviral vector for targeted transduction of B cells in a subject and preparation of broad-spectrum neutralizing antibodies is further enhanced.

Description

A viral vector and its application in infecting B cells Technical Field

[0001] The present invention relates to the field of medical immunology, and in particular to a lentiviral vector or a retroviral vector and its use in infecting B cells, establishing acquired immunity in a subject, and preparing broad-spectrum neutralizing antibodies. Background Art

[0002] HIV-1 (Human Immunodeficiency Virus-1) remains a major global public health problem, having claimed 42.3 million lives to date and continuing to spread in all countries worldwide; some countries are reporting an increase in new infections following previous declines. By the end of 2023, there were an estimated 39.9 million people (65% of whom were in the WHO African Region).

[0003] Although the emergence of highly effective antiretroviral therapy (ART) has greatly helped reduce the incidence of HIV-1 infection and improve the quality of life of infected people, the lack of continuous ART use and difficulty in obtaining ART treatment in infected people are considered to be associated with the persistent disease burden (Kumar R, et al., Broadly neutralizing antibodies in HIV-1 treatment and prevention. Ther Adv Vaccines Immunother. 2018 Oct 12; 6(4): 61-68.).

[0004] Although ART is highly effective, it does not eradicate infection, requiring lifelong adherence to ART for HIV-1 infected individuals. This is because ART primarily targets circulating virus and has limited access to proviruses that remain dormant in the viral reservoir (Id.). Therefore, while current ART is crucial for reducing HIV incidence, ART alone is insufficient to fully reduce HIV incidence and achieve a functional cure (Id.).

[0005] Neutralizing antibodies are specialized antibodies that act on microorganisms, such as viruses, to help them establish specific sites of infection. In viral infections, antibodies primarily prevent viral spread in two ways: by blocking viral entry into uninfected target cells (by inhibiting virus-receptor interactions) and through antibody-dependent cell-mediated cytotoxicity (ADCC) (Id.).

[0006] While neutralizing antibodies are generally effective against type-specific microorganisms with limited or no genetic variation, for complex viruses such as HIV-1 and influenza, whose genotypes exhibit considerable variation, "very specific" antibodies that are effective against this breadth of genetic variation are crucial. These "special antibodies" are called broadly neutralizing antibodies (bnAbs) (Id.).

[0007] A series of bnAbs identified in patients with chronic infections were classified and found to be derived from B cell precursors with unusual antigen receptor features, such as a long third heavy chain and complementarity determining region, and to require extensive somatic hypermutation to promote broad neutralization (Burton, DR &

[0008] Hangartner, L. Broadly neutralizing antibodies to HIV and their role in vaccine design. Annu. Rev. Immunol. 34, 635-659 (2016).) (Mascola, JR & Haynes, BFHIV-1 neutralizing antibodies: understanding nature's pathways. Immunol. Rev. 254, 225-244 (2013).).

[0009] B cells, also known as B lymphocytes, are a type of white blood cell that is a subtype of lymphocytes (Murphy K. (2012). Janeway's Immunobiology (8 th ed.). New York: Garland Science), B cells play a role in the humoral immune component of the adaptive immune system (Id.). B cells produce antibody molecules, which can be secreted outside the cell (secreted type) or inserted into the plasma membrane (membrane expressed type), where they become part of the B cell receptor (Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). "B Cells and Antibodies" Molecular Biology of the Cell (4 th ed.).Garland Science.), When naive or memory B cells are activated by antigens, they proliferate and differentiate into antibody-secreting effector cells, plasma cells (Id.).

[0010] Mature HIV bnAbs or other desired antibody genes can be directly integrated into the activated primary B cell genome in vitro through genetic engineering to express functional BCRs (B Cell Receptors, "BCRs") using endogenous HC constant genes (Voss, JE et al. Reprogramming the antigen specificity of B cells using genome-editing technologies (2019))(Hartweger, H. et al. HIV-specific humoral immune responses by CRISPR / Cas9-edited B cells. J. Exp. Med. 216, 1301-1310 (2019).)(Moffett, HF et al. B cells engineered to express pathogen-specific antibodies protect against infection. Sci. Immunol.). In this way, engineered BCRs can undergo type transformation into plasma cells and ultimately secrete protective antibodies (Kumar R, Qureshi H, Deshpande S, Bhattacharya J. Broadly neutralizing antibodies in HIV-1 treatment and prevention. Ther Adv Vaccines Immunother. 2018 Oct 12; 6(4): 61-68.).After being transplanted into immunocompromised mice, B cells genetically engineered in this way have been shown to provide protective levels of pathogen-specific antibodies in vivo for several weeks (Moffett, HF et al. B cells engineered to express pathogen-specific antibodies protect against infection. Sci. Immunol.), or in immune-complete mice, they provide the protective levels of antibodies for several days (Hartweger, H. et al. HIV-specific humoral immune responses by CRISPR / Cas9-edited B cells. J. Exp. Med. 216, 1301-1310 (2019).) (Moffett, HF et al B cells engineered to express pathogen-specific antibodies protect against infection. Sci. Immunol.).

[0011] At present, based on the developed bnAbs, such as b12, VRC01, VRC07, etc., a series of strategies for preparing drugs for treating HIV infection and AIDs have been developed: (1) direct administration of bnAbs proteins, however, direct use of antibodies is difficult to produce long-lasting immune protection; (2) lentiviral vectors carrying nucleic acid sequences encoding bnAbs are used to infect hematopoietic stem cells, and then the infected hematopoietic stem cells differentiate into B cells and plasma cells to produce bnAbs; however, the operation of infecting hematopoietic stem cells and transplanting infected hematopoietic stem cells is extremely difficult. This increases the difficulty for HIV-1 infected patients to obtain this treatment; (3) Intramuscular injection of AAV8 (adeno-associated virus 8, "AAV8") carrying the nucleic acid sequence encoding bnAbs, however, many HIV-1 infected patients have been infected with AAV and carry AAV antibodies, and the expression of AAV is limited in duration and cannot produce long-term antibody protection; (4) By infecting B cells with AAV carrying CRISPR / Cas9 and the nucleic acid sequence encoding bnAbs, the nucleic acid sequence encoding bnAbs is site-specifically integrated into the genome of B cells, however, the efficiency of AAV infection of B cells is low, and the efficiency of site-specific integration is also low.

[0012] Therefore, there is an unmet need for a lentiviral vector or retroviral vector that can effectively transduce B cells in vivo and / or in vitro, stably integrate the nucleic acid sequence encoding bnAbs into the B cell genome, and enable long-term and stable expression and secretion of bnAbs in B cells and their effector plasma cells.

[0013] Summary of the Invention

[0014] In view of this, in order to solve at least one of the above technical problems, the present invention provides a lentiviral vector or retroviral vector in one aspect, wherein (a) the viral envelope of the lentiviral vector or retroviral vector contains one or more targeting molecules that specifically bind to B cell endocytosis receptors, and the targeting molecules are not part of the envelope glycoprotein of the lentiviral vector or retroviral vector, and the targeting molecules are antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to B cell endocytosis receptors; and (b) the lentiviral vector or retroviral vector carries a nucleic acid sequence encoding bnAbs.

[0015] In some embodiments of the present invention, the bnAbs include but are not limited to bnAbs disclosed in the following patent documents: U.S. Patent No. 8673307, 9493549, 9783594, 10239935, US2018371086, US2020223907, WO2014 / 063059, WO2012 / 158948, WO2015 / 117008; and PCT / US2015 / 41272, WO2017 / 0 96221; these include the following bnAbs: 12A12, 12A21, NIH45-46, bANC131, 8ANC134, IB2530, INC9, 8ANC195, 8ANC196, 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM;

[0016] Other bnAbs include, but are not limited to, those described in the following literature: Klein et al., Nature, 492(7427):118-22 (2012), Horwitz et al., Proc. Natl. Acad. Sci. USA, 110(41):16538-43 (2013); Scheid et al., Science, 333:1633-1637 (2011); Scheid et al., Nature, 458;636-640 (2009); Eroshkin et al., Nucleic Acids Res., 42 (Database issue):DI 133-9 (2014); Mascola et al., Immumol Rev., 254(1):225-44(2013); for example, 2F5, 4E10, M66.6, CAP206-CH12 and 10E81 (all of these bnAbs can bind to the MPER (Membrane-Proximal External Region, “MPER”) of gp41); PG9, PG16 and CH01-04 (all of these bnAbs can bind to VIV2-glycan); 2G12 binds to the external domain of glycan; b12, HJ16, CH103-106, VRC01-03, VRC-PGO4, 04b, VRC-CH30-34, 3BNC62, 3BNC89, 3BNC91, 3BNC95, 3BNC104, 3BNC176 and 8ANC131 (all of these bnAbs can bind to the binding site of CD4);

[0017] and bnAbs described in the following patent documents, including but not limited to: U.S. Patent Nos. 8673307, 9493549 and 9783594; and WO2012 / 154312, WO2012 / 158948, WO2013 / 086533, WO2013 / 142324, WO2014 / 063059, WO2014 / 089152, WO 2015 / 048462, WO2015 / 103549; WO2015 / 117008, WO2016 / 014484, WO2016 / 154003, WO2016 / 196975, WO 2016 / 149710; WO2017 / 096221, WO2017 / 133639 and WO2017 / 133640;

[0018] Also included but not limited to the following bnAbs: bavituximab, UB-421, BF520.1, BiIA-SG, CHO1, CH59, C2F5, C4E10, C2F5+C2G12+C4E10, CAP256V2LS, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6, D1D2, 10-1074-LS, Cl3hmAb, GS-9722 (elipovimab) , DH411-2, BG18, GS-9721, GS-9723, PGT145, PGT121, PGT-121.60, PGT-121.66, PGT122, PGT-123, PGT-124, PGT -125, PGT-126, PGT-151, PGT-130, PGT-133, PGT-134, PGT-135, PGT-128, PGT-136, PGT-137, PGT-138, PGT-139, MDX010(ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1, N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH 45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12, PGDM21, PCDN-33A, 2Dm2m, 4Dm2m, 6Dm2m, PGDM1400, MDX010 (ipilimumab), VRCO1, VRC-01-LS, A32, 7B2, 10E8, VRC-07-523, VRC07-523LS, VRC24, VRC41,01, 10E8VLS, 3810109, 10E8v4, IMC-HIV, iMabm36, eCD4-Ig, IOMA, CAP256-VRC26.2 5. DRVIA7, VRC-HIVMAB080-00-AB, VRCHIVMAB060-00-AB, P2G12, VRC07, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426 , VRC29.03, CAP256, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.25, PCT64-24E, VRC38.01, PGT-151, CAP248-2B, 35022, ACS202, VRC34, VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, and LN01;.

[0019] All patent documents, literature, and publications mentioned above are incorporated herein by reference in their entirety.

[0020] Endocytosis receptors refer to receptors that mediate endocytosis. Lymphocytes, such as NK cells, T cells, and B cells, express a variety of endocytosis receptors on their surfaces.

[0021] Endocytosis refers to the process by which substances enter cells. During endocytosis, the substance to be taken in is surrounded by an area of ​​the plasma membrane, which then buds inside the cell to form a vesicle containing the taken in substance.

[0022] Endocytosis can be divided into four categories: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis, clathrin-mediated endocytosis), caveolae, pinocytosis and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p. vii., 2001).

[0023] Receptor-mediated endocytosis, also known as clathrin-mediated endocytosis, is mediated by the production of small (approximately 100 nm in diameter) vesicles with a morphologically characteristic coat composed of the cytoplasmic protein clathrin (McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nature Reviews. Molecular Cell Biology. 2011).

[0024] Clathrin-coated vesicles (CCVs) are present in almost all cells and form plasma membrane domains known as "clathrin-coated pits." Clathrin-coated pits can concentrate extracellular macromolecules that bind to different receptors responsible for receptor-mediated endocytosis of ligands, such as low-density lipoprotein, transferrin, growth factors, antibodies, and many other substances (Marsh M, McMahon HT (July 1999). The structural era of endocytosis. Science. 285(5425):215-220.).

[0025] Therefore, one or more targeting molecules that can specifically bind to B cell endocytosis receptors, that is, antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to B cell endocytosis receptors, are constructed on the surface of any lentiviral vector or retroviral vector provided by the present invention. The lentiviral vector or retroviral vector can enter B cells through endocytosis, thereby improving the targeting of the lentiviral vector or retroviral vector in transducing B cells; at the same time, the nucleic acid sequence encoding bnAbs carried by the lentiviral vector or retroviral vector is delivered to the genome of the B cells, thereby achieving stable expression and secretion of the bnAbs in B cells and their subsequently differentiated plasma cells.

[0026] In some embodiments of the present invention, the B cell endocytosis receptor is selected from CD5, CD19, CD20 (MS4A1), CD21 (CR2), CD22, CD23 (FCER2), CD38, CD40, CD79A, CD79B, CD83, IL-7R and CXCR5; preferably, the B cell endocytosis receptor is selected from CD19, CD20 (MS4A1), CD21 (CR2), CD22, CD23 (FCER2), CD38, CD40, CD79A, CD79B and CD83 (Stanislaw Pulczynski, et al., Modulation and intracellular transport of CD20 and CD21 antigens induced by B1 and B2 monoclonal antibodies in RAJI and JOK-1 cells-An immunofluorescence and immunoelectron microscopy study, Leukemia Research, Volume 18, Issue 7, 1994, Pages 541-552,ISSN 0145-2126)(Mary K.,et al.,CD22 Is a Recycling Receptor That Can Shuttle Cargo between the Cell Surface and Endosomal Compartments of B Cells.J Immunol 1 February 2011;186(3):1554-1563.)(Tessier J,et al.,Internalization and molecular interactions of human CD21 receptor. Mol Immunol. 2007 Mar; 44(9):2415-25.) (Karagiannis SN, et al., Endocytosis and recycling of the complex between CD23 and HLA-DR in human B cells. Immunology. 2001Jul; 103 (3): 319-31) (Ada Funaro, et al., CD38 Functions Are Regulated Through an Internalization Step.J Immunol 1 March 1998 160(5):2238-2247.)(Chen J, et al., Cholesterol-dependent and-independent CD40 internalization and signaling activation in cardiovascular endothelial cells. Arterioscler Thromb Vasc Biol. 2007 Sep; 27(9):2005-13.)(Langguth P, et al., CD83 acts as immediate early response gene in activated macrophages and exhibits specific intracellular trafficking properties.Biochem Biophys Res Commun.2023Mar 5;647:37-46.)(Henriques CM,et al.,IL-7induces rapid clathrin-mediated internalization and JAK3-dependent degradation of IL-7Ralpha in T cells.Blood.2010Apr 22;115(16):3269-77.)(Bürkle A,et al., Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood. 2007 Nov 1; 110(9): 3316-25.); the above document is incorporated herein by reference in its entirety.

[0027] The expression of the B cell endocytosis receptors CD5, CD19, CD20 (MS4A1), CD21 (CR2), CD22, CD23 (FCER2), CD38, CD40, CD79A, CD79B, CD83, IL-7R and CXCR5 in B cells and other cells (violin diagrams and bar diagrams) are shown in Figures 12 to 18.

[0028] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to a B cell endocytic receptor is selected from the group consisting of a full-length antibody, a half antibody, Fab, Fab', F(ab')2, Fv, VHH (single-domain antibody) and scFv (Single-Chain Fragment Variable).

[0029] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the B cell endocytic receptor is selected from at least one of: an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab')2, an Fv fragment, a single-chain variable region fragment (scFv), a disulfide bond-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or a light chain variable region (VL), an Fd fragment consisting of a VH and a CH1 domain, a linear antibody, a heavy chain antibody, and a nanobody (VHH).

[0030] In some embodiments of the present invention, the envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a first mutation, so that the ability of the envelope glycoprotein to specifically bind to a receptor is weakened or lost relative to before the first mutation occurs.

[0031] In some embodiments of the present invention, the envelope glycoprotein is selected from the following envelope glycoproteins and variants thereof: envelope glycoproteins of vesicular stomatitis virus strains and variants thereof, envelope glycoproteins of baboon endogenous retrovirus BaEV and variants thereof, envelope glycoproteins of feline endogenous retrovirus RD114 and variants thereof, and envelope glycoproteins of gibbon ape leukemia virus GALV and variants thereof;

[0032] Preferably, the envelope glycoprotein of the vesicular stomatitis virus strain and its variants include the following envelope glycoproteins and their variants: envelope glycoprotein of the vesicular stomatitis virus Indiana strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Cocal strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Maraba strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Morreton strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Alagoas strain and its variants, envelope glycoprotein of the vesicular stomatitis virus New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

[0033] In some embodiments of the present invention, the envelope glycoprotein of the Maraba strain of the vesicular stomatitis virus or its variant, the envelope glycoprotein of the Morreton strain of the vesicular stomatitis virus or its variant, the envelope glycoprotein of the Carajas strain of the vesicular stomatitis virus or its variant, the envelope glycoprotein of the Alagoas strain of the vesicular stomatitis virus or its variant, and the envelope glycoprotein of the New Jersey strain of the vesicular stomatitis virus or its variant comprise the amino acid sequence as shown in SEQ ID NO: 18-22, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 18-22.

[0034] The envelope glycoprotein of Vesicular Stomatitis Virus strains, such as the Indiana strain (VSV-G) or the Cocal strain (Cocal-G), can specifically bind to the low-density lipoprotein receptor (LDL-R), which is widely present on the surface of various cells and can mediate membrane fusion, resulting in broad infectivity. Therefore, lentiviral vectors or retroviral vectors often use VSV-G or Cocal-G to construct their envelope glycoproteins to enhance their infectivity (VSV-G or Cocal-G lentiviral vectors or retroviral vectors).

[0035] By causing the first mutation to occur in the envelope glycoprotein of vesicular stomatitis virus strains such as VSV-G and Cocal-G, the ability of the envelope glycoprotein to specifically bind to LDL-R is weakened or lost; at the same time, one or more targeting molecules that specifically bind to B cell endocytosis receptors are constructed on the surface of the lentiviral vector or retroviral vector, thereby further improving the targeting of B cells transduced by the lentiviral vector or retroviral vector.

[0036] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof; the receptor is the low-density lipoprotein receptor (LDL-R); the extracellular domain of the envelope glycoprotein comprises SEQ ID NO: 16 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence of SEQ ID NO: 16;

[0037] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0038] (a) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 16; and

[0039] (b) After optimal global alignment with SEQ ID NO: 16, the position corresponding to SEQ ID NO:16: substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353;

[0040] More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0041] (a) substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of 1182, substitution of M184, substitution of Y209, substitution of 1347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 16; and

[0042] (b) substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, corresponding to SEQ ID NO: 16 after optimal global alignment with SEQ ID NO: 16;

[0043] More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0044] Substitution or deletion of K47, substitution of R354;

[0045] Still further preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0046] The 47th amino acid is replaced by lysine K to glutamine Q, or the 47th amino acid lysine K is deleted, and the 354th amino acid is replaced by arginine R to glutamine Q;

[0047] Most preferably, the first mutation comprises a mutation in the amino acid sequence comprising the following amino acid: deletion of K47.

[0048] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is the low-density lipoprotein receptor (LDL-R); the extracellular domain of the envelope glycoprotein comprises SEQ ID NO: 17 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence of SEQ ID NO: 17;

[0049] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0050] (a) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 17; and

[0051] (b) after optimal global alignment with SEQ ID NO: 17, substitution or deletion at positions corresponding to Q8, S9, Q10, K47, K50, A51, D183, A179, Substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353;

[0052] More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0053] (a) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 17; and

[0054] (b) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, corresponding to SEQ ID NO: 17 after optimal global alignment with SEQ ID NO: 17;

[0055] Still further preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution or deletion of K47, substitution of R354;

[0056] Still further preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of amino acid 47 from lysine K to glutamine Q or deletion of amino acid 47 from lysine, and substitution of amino acid 354 from arginine R to glutamine Q;

[0057] Most preferably, the first mutation comprises a mutation in the amino acid sequence comprising the following amino acid: deletion of K47.

[0058] In some embodiments of the present invention, the envelope glycoprotein that undergoes any of the aforementioned first mutations retains the ability to mediate membrane fusion.

[0059] In some embodiments of the present invention, the envelope glycoprotein that undergoes any of the above-mentioned first mutations also undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement relative to before the second mutation occurs or prevents the envelope glycoprotein from being inactivated by complement.

[0060] The complement system is composed of a series of proteins and is part of the innate immune system. Complement (C) is present in the serum, tissue fluid, and cell membrane surfaces of normal humans and animals. After activation, it has enzymatic activity and can undergo complex cascade reactions. The complement system is initiated through a series of enzymes (enzymes) that cut each other, ultimately forming a membrane attack complex that resembles a hole on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, and clear immune complexes through lysis, conditioning, phagocytosis, and mediating inflammatory responses, demonstrating corresponding biological functions. Complement is widely involved in the body's defense response against microbial infection and immune regulation, and also mediates immunopathological damage reactions. It is an effector system and effector method system with important biological functions in the body.

[0061] Regulatory complement components exist in soluble or membrane-bound forms, including properdin (P factor), C1 inhibitor (C1INH), factor I, factor H, C4 binding protein (C4BP), S protein, SP40 / 40, membrane cofactor protein (MCP), decay accelerating factor (DAF), homologous restriction factor (HRF), and membrane inhibitor of reactive lysis (MIRL).

[0062] Enhancing the ability of VSV-G or Cocal-G lentiviral vectors or retroviral vectors to antagonize complement inactivation can increase the efficiency of VSV-G or Cocal-G lentiviral vectors or retroviral vectors infecting or transducing B cells in the body or blood of a subject.

[0063] In some embodiments of the present invention, the envelope glycoprotein that further undergoes a second mutation is an envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof, and the extracellular domain of the envelope glycoprotein comprises SEQ ID NO: 16, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence of SEQ ID NO: 16;

[0064] Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0065] (a) amino acid position 214 of SEQ ID NO: 16;

[0066] (b) after optimal global alignment with SEQ ID NO: 16, is located at amino acid position 214 corresponding to SEQ ID NO: 16;

[0067] (c) amino acid position 352 of SEQ ID NO: 16;

[0068] (d) after optimal global alignment with SEQ ID NO: 16, is located at amino acid position 352 corresponding to SEQ ID NO: 16;

[0069] (e) amino acid position 50 of SEQ ID NO: 16;

[0070] (f) after optimal global alignment with SEQ ID NO: 16, is located at amino acid position equivalent to 50 of SEQ ID NO: 16;

[0071] (g) amino acid position 146 of SEQ ID NO: 16; and

[0072] (h) after optimal global alignment with SEQ ID NO: 16, is located at amino acid position 146 corresponding to SEQ ID NO: 16;

[0073] More preferably, the amino acid mutation includes deletion, insertion or substitution of the amino acid;

[0074] More preferably, the amino acid mutation is a substitution of the amino acid.

[0075] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 16, comprising one or more of the following site mutations: substitution of T214, substitution of T352, substitution of K50, substitution of S146;

[0076] Preferably, the second mutation includes one or more of the following site mutations in the amino acid sequence: amino acid position 214 is replaced by threonine T to asparagine N, amino acid position 352 is replaced by threonine T to alanine A, amino acid position 50 is replaced by lysine K to threonine T, and amino acid position 146 is replaced by serine S to threonine T;

[0077] Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0078] (a) replacement of T214 and T352; and

[0079] (b) Replacement of T214, T352, K50 and S146;

[0080] More preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0081] (a) amino acid 214 is substituted from threonine T to asparagine N and amino acid 352 is substituted from threonine T to alanine A; and

[0082] (b) The amino acid at position 214 was replaced by threonine T to asparagine N, the amino acid at position 352 was replaced by threonine T to alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T.

[0083] In some embodiments of the present invention, the VSV-G or variant thereof having any of the first mutations also has any of the second mutations.

[0084] In some embodiments of the present invention, the envelope glycoprotein that further undergoes a second mutation is an envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the envelope glycoprotein comprises SEQ ID NO: 17 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence of SEQ ID NO: 17;

[0085] Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0086] (a) amino acid position 214 of SEQ ID NO: 17;

[0087] (b) after optimal global alignment with SEQ ID NO: 17, is located at amino acid position 214 corresponding to SEQ ID NO: 17;

[0088] (c) amino acid position 352 of SEQ ID NO: 17;

[0089] (d) after optimal global alignment with SEQ ID NO: 17, is located at amino acid position 352 corresponding to SEQ ID NO: 17;

[0090] (e) amino acid position 50 of SEQ ID NO: 17;

[0091] (f) after optimal global alignment with SEQ ID NO: 17, is located at amino acid position equivalent to 50 of SEQ ID NO: 17;

[0092] (g) amino acid position 146 of SEQ ID NO: 17; and

[0093] (h) after optimal global alignment with SEQ ID NO: 17, is located at amino acid position 146 corresponding to SEQ ID NO: 17;

[0094] More preferably, the amino acid mutation includes deletion, insertion or substitution of the amino acid;

[0095] More preferably, the amino acid mutation is a substitution of the amino acid.

[0096] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 17, comprising one or more of the following site mutations: substitution of K214, substitution of T352, substitution of K50, substitution of S146;

[0097] Preferably, the second mutation includes one or more of the following site mutations in the amino acid sequence: amino acid position 214 is replaced by lysine K to asparagine N, amino acid position 352 is replaced by threonine T to alanine A, amino acid position 50 is replaced by lysine K to threonine T, and amino acid position 146 is replaced by serine S to threonine T;

[0098] Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0099] (a) replacement of K214 and T352; and

[0100] (b) Replacement of K214, T352, K50 and S146;

[0101] More preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0102] (a) amino acid position 214 is substituted from lysine K to asparagine N and amino acid position 352 is substituted from threonine T to alanine A; and

[0103] (b) The amino acid at position 214 was replaced by lysine K to asparagine N, the amino acid at position 352 was replaced by threonine T to alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T.

[0104] The amino acid sequence shown in SEQ ID NO: 17 is at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 16.

[0105] In some embodiments of the present invention, the Cocal-G or variant thereof having any of the first mutations also has any of the second mutations.

[0106] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 23; relative to SEQ ID NO: 16, SEQ ID NO: 23 comprises a K47 deletion, T214N and T352A.

[0107] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 24, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 24; relative to SEQ ID NO: 16, SEQ ID NO: 24 comprises a K47 deletion, T214N, T352A, K50T and S146T.

[0108] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 25; relative to SEQ ID NO: 16, SEQ ID NO: 25 comprises R354Q, T214N and T352A.

[0109] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 26 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 26; relative to SEQ ID NO: 16, SEQ ID NO: 26 comprises R354Q, T214N, T352A, K50T and S146T.

[0110] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as set forth in SEQ ID NO: 37; relative to SEQ ID NO: 17, SEQ ID NO: 37 comprises a K47 deletion.

[0111] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO: 38; relative to SEQ ID NO: 17, SEQ ID NO: 38 comprises a K47 deletion, K214N, and T352A.

[0112] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as set forth in SEQ ID NO: 39 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as set forth in SEQ ID NO: 39; relative to SEQ ID NO: 17, SEQ ID NO: 39 comprises R354Q.

[0113] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 40, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 40; relative to SEQ ID NO: 17, SEQ ID NO: 40 comprises R354Q, K214N and T352A.

[0114] In some embodiments of the present invention, any of the envelope glycoproteins that have any of the first mutations and any of the second mutations mentioned above retain the ability to mediate membrane fusion.

[0115] In some embodiments of the present invention, the targeting molecule is directly or indirectly connected to the transmembrane domain and exposed on the surface of the lentiviral vector or retroviral vector;

[0116] Preferably, the transmembrane domain is selected from the transmembrane regions of the following proteins:

[0117] CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD95(Fas), CD134(OX40), CD137(4-1BB), CD150 (SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272(BTLA), CD273(PD-L2), CD274(P D-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LR P, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70;

[0118] More preferably, the transmembrane domain includes the transmembrane region of CD8α.

[0119] In some embodiments of the present invention, the targeting molecule is indirectly connected to the transmembrane domain via a linker domain and is exposed on the surface of the lentiviral vector or retroviral vector;

[0120] Preferably, the linker domain is selected from:

[0121] (i) an immunoglobulin hinge region selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0122] (ii) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0123] (iii) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and

[0124] (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0125] More preferably, the connecting domain includes the hinge region of CD8α.

[0126] In some embodiments of the present invention, the targeting molecule is indirectly connected to the transmembrane region of CD8α through the hinge region of CD8α and is exposed on the surface of the lentiviral vector or retroviral vector.

[0127] In some embodiments of the present invention, the broadly neutralizing antibody is selected from bavituximab, UB-421, BF520.1, BiIA-SG, CHO1, CH59, C2F5, C4E10, C2F5+C2G12+C4E10, CAP256V2LS, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6, D1D2, 10-1074-LS, Cl3hmAb, GS-9722 (elipovimab), DH411-2, BG18, GS-9721, GS-9723, PGT145, PGT121, PGT-121.60, P GT-121.66, PGT122, PGT-123, PGT-124, PGT-125, PGT-126, PGT-151, PGT-130, PGT-133, PGT-134, PGT-135, PGT-128, PGT-136, PGT-137, PGT-138, PG T-139, MDX010 (ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1, N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH 45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12, PGDM21, PCDN-33A, 2Dm2m, 4Dm2m, 6Dm2m, PGDM1400, MDX010(ipilimum ab), VRCO1, VRC-01-LS, A32, 7B2, 10E8, VRC-07-523, VRC07-523LS, b12, VRC24, VRC41, 01, 10E8VLS, 3810109, 10E8v4, IMC-HIV, iMabm36, eC D4-Ig, IOMA, CAP256-VRC26.25, DRVIA7, VRC-HIVMAB080-00-AB, VRCHIVMAB060-00-AB, P2G12, VRC07, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, VRC29.03, CAP256, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.25, PCT64-24E, VRC38.01, PGT-151, CAP248-2B, 35022, ACS202, VRC34, VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, LN01, 12A12, 12A21, NIH45-46, bANC131, 8ANC134, IB2530, INC9, 8ANC195, 8ANC196, 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM. .

[0128] In some embodiments of the present invention, the broadly neutralizing antibody is selected from b12, VRC07 and VRC07-523LS.

[0129] In some embodiments of the present invention, the nucleic acid sequence encoding the broadly neutralizing antibody comprises a nucleic acid sequence encoding a signal peptide of a secreted protein;

[0130] Preferably, the secretory protein is selected from IFN-α, IFN-β, IFN-γ, IL-1, IL-6 and IL-10;

[0131] More preferably, the secreted protein is IL-10.

[0132] In some embodiments of the present invention, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody;

[0133] Preferably, the nucleic acid sequence encoding the recombinant broadly neutralizing antibody comprises, from the 5' end to the 3' end, nucleic acid sequences encoding the following polypeptides: a signal peptide of a secretory protein, a heavy chain variable region (VH region) of a broadly neutralizing antibody, a heavy chain constant region 1 (CH1 region) of an immunoglobulin, a 2A peptide, a signal peptide of a secretory protein, a light chain variable region (VL region) of a broadly neutralizing antibody, and a light chain constant region (CL region) of an immunoglobulin;

[0134] More preferably, the secretory protein is selected from IFN-α, IFN-β, IFN-γ, IL-1, IL-6 and IL-10;

[0135] More preferably, the CH1 region of the immunoglobulin is the CH1 region of human IgG1;

[0136] More preferably, the 2A peptide is a FT2A peptide;

[0137] More preferably, the immunoglobulin CL region is the CL region of a human immunoglobulin kappa chain (κ chain).

[0138] In some embodiments of the present invention, the nucleic acid sequence encoding the recombinant broadly neutralizing antibody comprises, from the 5' end to the 3' end, nucleic acid sequences encoding the following polypeptides: a signal peptide of IL-10, a VH region of a broadly neutralizing antibody, a CH1 region of human IgG1, an FT2A peptide, a signal peptide of IL-10, a VL region of a broadly neutralizing antibody, and a CL region of a human immunoglobulin κ chain;

[0139] Preferably, the broadly neutralizing antibody is selected from b12 and VRC07-523LS.

[0140] In some embodiments of the present invention, the CL region of the immunoglobulin may be the CL region of a human immunoglobulin lambda chain (λ chain).

[0141] In some embodiments of the present invention, any of the aforementioned nucleic acid sequences encoding broadly neutralizing antibodies is operably linked to a promoter that can initiate transcription of polypeptides and / or proteins in B cells;

[0142] Preferably, the B cells are human B cells.

[0143] In some embodiments of the present invention, the promoter includes but is not limited to EEK promoter, MH promoter, PGK promoter, CASI promoter, CMV promoter, EF1α promoter, SV40 promoter, IgH promoter, CD19 promoter, CD20 promoter, LMP2 promoter and MHCⅡ promoter.

[0144] In another aspect, the present invention provides a composition comprising any of the aforementioned lentiviral vectors or retroviral vectors provided by the present invention and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions, and other auxiliary components;

[0145] Preferably, the composition comprises any one of the lentiviral vectors or retroviral vectors having any one of the first mutations and any one of the second mutations in the envelope glycoprotein provided by the present invention and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives and other auxiliary components.

[0146] In another aspect, the present invention provides a method for infecting or transducing B cells, comprising contacting a B cell with any of the aforementioned lentiviral vectors or retroviral vectors or any of the aforementioned compositions provided by the present invention;

[0147] Preferably, the contacting occurs outside the body of a subject, wherein the subject is an individual to whom B cells infected or transduced by the method of infecting or transducing B cells are administered;

[0148] Preferably, the contacting occurs in a subject, wherein the subject is an individual to whom the lentiviral vector or retroviral vector or the composition is administered;

[0149] More preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion techniques.

[0150] In some embodiments of the present invention, nucleic acids encoding broadly neutralizing antibodies are stably integrated into the genome of the transduced B cells.

[0151] In some embodiments of the present invention, when the contact occurs in the subject, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations; the composition comprises any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0152] In some embodiments of the present invention, the B cells are autologous B cells of the subject.

[0153] In some embodiments of the invention, the B cells are allogeneic B cells of the subject.

[0154] In some embodiments of the present invention, the B cells are B cells sorted from the subject's autologous and / or allogeneic peripheral blood mononuclear cells (PBMCs).

[0155] In some embodiments of the present invention, the B cells are B cells in the subject's autologous and / or allogeneic non-activated PBMCs.

[0156] In some embodiments of the present invention, the B cells are B cells sorted from the subject's autologous and / or allogeneic non-activated PBMCs.

[0157] In some embodiments of the present invention, the B cells are activated B cells.

[0158] In some embodiments of the present invention, the B cells are non-activated B cells.

[0159] In some embodiments of the present invention, the non-activated B cells are not cultured with in vitro activation.

[0160] In some embodiments of the present invention, the B cells are B cells differentiated from the subject's autologous and / or allogeneic hematopoietic stem cells.

[0161] In some embodiments of the present invention, the B cells are B cells differentiated from hematopoietic stem cells in the subject's autologous and / or allogeneic umbilical cord blood.

[0162] In some embodiments of the present invention, the B cells are B cells differentiated from hematopoietic stem cells in the subject's autologous and / or allogeneic peripheral blood.

[0163] In some embodiments of the present invention, the B cells are B cells differentiated from autologous and / or allogeneic hematopoietic stem cells in the subject's mobilized (eg, G-CSF-mobilized) peripheral blood.

[0164] In another aspect, the present invention provides a B cell expressing a broadly neutralizing antibody, wherein the B cell expressing a broadly neutralizing antibody is prepared by contacting any of the aforementioned lentiviral vectors or retroviral vectors or any of the aforementioned compositions provided by the present invention with a B cell;

[0165] Preferably, the contacting occurs outside the body of a subject, wherein the subject is an individual to whom the B cells expressing broadly neutralizing antibodies are administered;

[0166] Preferably, the contacting occurs in a subject, wherein the subject is an individual to whom the lentiviral vector or retroviral vector or the composition is administered;

[0167] More preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion techniques.

[0168] In some embodiments of the present invention, when the contact occurs in the subject, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations; the composition comprises any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0169] In some embodiments of the present invention, the expression is cytoplasmic expression (Cytoplasmic Expression);

[0170] Preferably, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody.

[0171] In another aspect, the present invention provides a B cell comprising a nucleic acid encoding a broadly neutralizing antibody, wherein the B cell is prepared by any of the aforementioned methods for infecting or transducing B cells provided by the present invention;

[0172] Preferably, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody.

[0173] In some embodiments of the present invention, the nucleic acid encoding the broadly neutralizing antibody is stably integrated into the genome of the B cell.

[0174] In another aspect, the present invention provides a plasma cell comprising a nucleic acid encoding a broadly neutralizing antibody, wherein the plasma cell is differentiated from any of the aforementioned B cells comprising a nucleic acid encoding a broadly neutralizing antibody provided by the present invention.

[0175] In some embodiments of the present invention, the plasma cells secrete broadly neutralizing antibodies in vivo or in vitro in a subject;

[0176] Preferably, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody.

[0177] In some embodiments of the present invention, the nucleic acid encoding the broadly neutralizing antibody is stably integrated into the genome of the plasma cell.

[0178] In another aspect, the present invention provides a method for preparing a broadly neutralizing antibody in a subject, the method comprising administering to the subject any of the aforementioned lentiviral vectors or retroviral vectors or any of the aforementioned compositions provided by the present invention to infect or transduce B cells in the subject, or administering to the subject any of the aforementioned B cells expressing a broadly neutralizing antibody, any of the aforementioned B cells comprising a nucleic acid encoding a broadly neutralizing antibody, or any of the aforementioned plasma cells comprising a nucleic acid encoding a broadly neutralizing antibody;

[0179] Preferably, the administration is at least one of intravenous, intratumoral, subcutaneous, intramuscular, sternal and infusion techniques.

[0180] In some embodiments of the present invention, the infected or transduced B cells, the B cells expressing broadly neutralizing antibodies, or the B cells containing nucleic acids encoding broadly neutralizing antibodies differentiate into plasma cells and secrete broadly neutralizing antibodies in the subject, or the plasma cells containing nucleic acids encoding broadly neutralizing antibodies secrete broadly neutralizing antibodies in the subject.

[0181] In some embodiments of the present invention, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations; the composition comprises any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0182] In another aspect, the present invention provides a method for establishing acquired immunity in a subject, the method comprising administering to the subject any of the aforementioned lentiviral vectors or retroviral vectors or any of the aforementioned compositions provided by the present invention to infect or transduce B cells in the subject, or administering to the subject any of the aforementioned B cells expressing a broadly neutralizing antibody, any of the aforementioned B cells comprising a nucleic acid encoding a broadly neutralizing antibody, or any of the aforementioned plasma cells comprising a nucleic acid encoding a broadly neutralizing antibody provided by the present invention;

[0183] Preferably, the administration is at least one of intravenous, intratumoral, subcutaneous, intramuscular, sternal and infusion techniques.

[0184] In some embodiments of the present invention, the infected or transduced B cells, the B cells expressing broadly neutralizing antibodies, or the B cells containing nucleic acids encoding broadly neutralizing antibodies differentiate into plasma cells and secrete broadly neutralizing antibodies in the subject, or the plasma cells containing nucleic acids encoding broadly neutralizing antibodies secrete broadly neutralizing antibodies in the subject.

[0185] In some embodiments of the present invention, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations; the composition comprises any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0186] In another aspect, the present invention provides a broadly neutralizing antibody prepared in a subject, wherein the broadly neutralizing antibody is produced by infecting or transducing B cells in the subject with any of the aforementioned lentiviral vectors or retroviral vectors or any of the aforementioned compositions provided by the present invention, or by administering to the subject any of the aforementioned B cells expressing the broadly neutralizing antibody, any of the aforementioned B cells comprising a nucleic acid encoding the broadly neutralizing antibody, or any of the aforementioned plasma cells comprising a nucleic acid encoding the broadly neutralizing antibody provided by the present invention;

[0187] Preferably, the administration is at least one of intravenous, intratumoral, subcutaneous, intramuscular, sternal and infusion techniques.

[0188] In some embodiments of the present invention, the infected or transduced B cells, the B cells expressing broadly neutralizing antibodies, or the B cells containing nucleic acids encoding broadly neutralizing antibodies differentiate into plasma cells and secrete broadly neutralizing antibodies in the subject, or the plasma cells containing nucleic acids encoding broadly neutralizing antibodies secrete broadly neutralizing antibodies in the subject.

[0189] In some embodiments of the present invention, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors provided by the present invention, in which any of the first mutations and any of the second mutations occur in the envelope glycoprotein; the composition comprises any of the lentiviral vectors or retroviral vectors provided by the present invention, in which any of the first mutations and any of the second mutations occur in the envelope glycoprotein, and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components; the lentiviral vector or retroviral vector or the composition is administered to a subject to infect or transduce B cells in the subject, and the B cells differentiate into plasma cells and secrete broadly neutralizing antibodies.

[0190] In some embodiments of the present invention, the B cells expressing broadly neutralizing antibodies and the B cells containing nucleic acids encoding broadly neutralizing antibodies differentiate into plasma cells in the subject and secrete broadly neutralizing antibodies.

[0191] In some embodiments of the present invention, the plasma cells comprising the nucleic acid encoding the broadly neutralizing antibody secrete the broadly neutralizing antibody in the subject.

[0192] In another aspect, the present invention provides use of any of the aforementioned lentiviral vectors or retroviral vectors, any of the aforementioned compositions, any of the aforementioned B cells expressing broadly neutralizing antibodies, any of the aforementioned B cells expressing broadly neutralizing antibodies, any of the aforementioned B cells comprising a nucleic acid encoding a broadly neutralizing antibody, or any of the aforementioned plasma cells comprising a nucleic acid encoding a broadly neutralizing antibody, in the preparation of a medicament for treating HIV infection and / or AIDs.

[0193] In some embodiments of the present invention, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations; the composition comprises any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0194] In another aspect, the present invention provides a method for treating a subject infected with HIV and / or suffering from AIDs, the method comprising administering to the subject any of the aforementioned lentiviral vectors or retroviral vectors, any of the aforementioned compositions, any of the aforementioned B cells expressing broadly neutralizing antibodies, any of the aforementioned B cells comprising a nucleic acid encoding a broadly neutralizing antibody, or any of the aforementioned plasma cells comprising a nucleic acid encoding a broadly neutralizing antibody;

[0195] Preferably, the administration is at least one of intravenous, intratumoral, subcutaneous, intramuscular, sternal and infusion techniques.

[0196] In some embodiments of the present invention, the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations; the composition comprises any of the lentiviral vectors or retroviral vectors in which the envelope glycoprotein provided by the present invention undergoes any of the first mutations and any of the second mutations and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0197] In some embodiments of the present invention, a therapeutically effective amount of the lentiviral vector or retroviral vector, the composition, the B cells expressing broadly neutralizing antibodies, the B cells comprising nucleic acids encoding broadly neutralizing antibodies, or the plasma cells is administered to the subject.

[0198] The beneficial effects of the present invention include:

[0199] The lentiviral vector or retroviral vector provided by the present invention, which carries the nucleic acid sequence encoding bnAbs, specifically binds to B cell endocytosis receptors through the targeting molecules contained in its viral envelope, can effectively transduce B cells (especially non-activated B cells), and the targeting of transduction is significantly improved; any of the lentiviral vectors or retroviral vectors provided by the present invention, in which the envelope glycoprotein undergoes any of the first mutations and any of the second mutations, can effectively infect B cells in AIDS patients or HIV-infected persons, stably integrate the nucleic acid sequence encoding bnAbs into the genome of the B cells, and the B cells can efficiently secrete bnAbs after differentiating into plasma cells, thereby achieving long-term, stable and efficient expression and secretion of bnAbs in AIDS patients or HIV-infected persons, and further enabling AIDS patients or HIV-infected persons to establish lasting immunity to HIV.

[0200] In this article:

[0201] "B cell": B lymphocyte, a subtype of white blood cell (Murphy K (2012). Janeway's Immunobiology (8 th ed.), B cells play a role in the humoral immune component of the adaptive immune system (Id.). B cells produce antibody molecules, which can be secreted outside the cell (secreted type) or inserted into the plasma membrane (membrane expression type), where they become part of the B cell receptor (Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). "B Cells and Antibodies" Molecular Biology of the Cell (4 th ed.).Garland Science.), When naive or memory B cells are activated by antigens, they proliferate and differentiate into antibody-secreting effector cells, plasma cells (Id.).

[0202] Herein, the B cells include Pre-Pro B Cell, Pre-B Cell, Immature B Cell (immature B cells), Transitional B Cell (transitional B cells), Marginal B Cell (marginal zone B cells), Follicular B Cell (follicular B cells), Activated Germinal B Cell (activated reproductive center B cells) and Memory B Cell (memory B cells). Herein, the B cells include non-activated B cells. In some embodiments of the present invention, the non-activated B cells include but are not limited to resting B cells, dormant B cells, B cells with low expression of activation markers such as CD69, CD80 and CD86, B cells without somatic high-frequency mutation and affinity maturation, and B cells with lower immunogenicity.

[0203] In some embodiments of the present invention, the B cells include but are not limited to B cells from the following sources: autologous B cells of the subject, allogeneic B cells of the subject, B cells sorted from the subject's autologous and / or allogeneic PBMCs, B cells in the subject's autologous and / or allogeneic non-activated PBMCs, B cells sorted from the subject's autologous and / or allogeneic non-activated PBMCs, activated B cells, non-activated B cells, B cells differentiated from the subject's autologous and / or allogeneic hematopoietic stem cells, B cells differentiated from the subject's autologous and / or allogeneic umbilical cord blood, B cells differentiated from the subject's autologous and / or allogeneic hematopoietic stem cells in the subject's autologous and / or allogeneic peripheral blood, and B cells differentiated from the subject's autologous and / or allogeneic hematopoietic stem cells in mobilized peripheral blood (such as G-CSF).

[0204] “HIV / AIDs”: Human Immunodeficiency Virus Infection (human immunodeficiency virus infection) and Acquired Immunodeficiency Diseases (Acquired Immunodeficiency Syndrome, also known as “AIDS”). AIDs are a series of diseases caused by HIV infection (Sepkowitz KA (June 2001). "AIDS-the first 20 years". The New England Journal of Medicine. 344(23): 1764-72; A, Kretzschmar M, Krickeberg K (2010). Modern infections diseases epidemiology concepts, methods mathematical models, and public health (Online-Ausg.ed.). New York: Springer.p.88; Kirch W (2008). Encyclopedia of Public Health. New York: Springer.pp.676–77.), is a kind of retrovirus ("Retrovirus" Definition”AIDSinfo.Archived from the original on December 28,2019.). After the initial infection, people may not notice any symptoms or may experience a brief, flu-like illness (“HIV / AIDS Fact Sheet N°360,” World Health Organization. November 2015. Archived from the original on February 17, 2016. Retrieved February 11, 2016.) If the infection progresses, it can further interfere with the immune system, increasing the risk of common infections such as tuberculosis, other opportunistic infections, and cancers that are rare in immunocompetent people (“Retrovirus Definition,” AIDSinfo. Archived from the original on December 28, 2019.). These late symptoms are called acquired immunodeficiency syndrome (“HIV / AIDS Fact Sheet N°360,” World Health Organization. November 2015. Archived from the original on February 17, 2016. Retrieved February 11, 2016.).

[0205] "Weaken or lose": When referring to the ability of a viral glycoprotein to bind to its receptor, the term "loss" includes completely eliminating the binding of the viral glycoprotein to its receptor, and the term "weaken" includes significantly reducing the binding. In a specific embodiment, "significantly reduce" refers to the ability of a viral glycoprotein or a native glycoprotein to bind to its receptor relative to the ability of the viral glycoprotein or a native glycoprotein to bind to any of the first mutations; "significantly reduce" is selected from a reduction of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2% and at least 1%.

[0206] "Variant": A variant is a mutant that has at least 50% identity to the amino acid sequence of a non-mutant (wild type / native), and "at least 50% identity" means that the variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identity to the amino acid sequence of the non-mutant (wild type); or, a variant "At least 50% identity" refers to a mutant in which the nucleic acid sequence encoding the variant is at least 50% identical to the nucleic acid sequence encoding the non-mutant (wild-type / native). "At least 50% identity" means that the nucleic acid sequence encoding the variant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence encoding the non-mutant (wild-type).

[0207] "Flexible linkers": Flexible linkers are usually used when the connected domains need to move or interact to a certain extent (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69.). Flexible linkers are usually composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211: 943–958.). These small amino acids provide flexibility while also allowing the movement of the connected functional domains. For commonly used flexible linker peptides, see Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69, which is incorporated herein by reference in its entirety.

[0208] "Antibody" refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to specifically bind to an antigen. "Antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0209] The "antibody" described herein includes a typical "four-chain antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain from its N-terminus to its C-terminus; and, when the full-length antibody is of the IgE isotype, it optionally further includes a heavy chain constant region CH4 domain; the light chain refers to a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) from its N-terminus to its C-terminus; the heavy chains and the light chains are connected by disulfide bonds to form a "Y"-shaped structure.

[0210] The "antibodies" described herein also include antibodies that do not contain light chains, for example, heavy chain antibodies (HCAbs) produced by dromedary camels (Camelus Dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanacos (Lama Guanicoe) and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (Ig New Antigen Receptor, IgNAR) found in cartilaginous fish such as sharks.

[0211] Herein, the terms "VHH domain," "nanobody," and "single domain antibody" (sdAb) have the same meaning and are used interchangeably. They refer to the construction of a single domain antibody (sdAb) consisting solely of a single heavy chain variable region by cloning the variable region of a heavy chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting solely of a single heavy chain variable region.

[0212] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0213] Herein, "antigen-binding fragment" refers to a fragment that does not have the entire structure of an intact antibody and only contains a portion or a partial variant of the intact antibody, wherein the portion or partial variant has the ability to bind to an antigen.

[0214] Illustratively, herein, "antibody or antigen-binding fragment thereof" includes but is not limited to full-length antibody, half antibody, Fab, Fab', F(ab')2, Fv, VHH (single domain antibody) and scFv; the Fab' fragment and F(ab')2 fragment may include a partial Fc fragment; the heavy chain variable region (VH region) of the scFv is connected to the light chain variable region (VL region) via a connecting peptide.

[0215] In some embodiments of the present invention, the order of the VH region and the VL region from the N-terminus to the C-terminus of the scFv is not particularly limited, such as VH region-Linker-VL region or VL region-Linker-VH region from the N-terminus to the C-terminus; the connecting peptide can be selected from a flexible connecting peptide, such as (G4S) n , where n = 1 to 4.

[0216] "Ligand": In receptor-ligand binding, a ligand is generally a molecule that binds to a site on a receptor to generate a signal, such binding typically resulting in a conformational change in the complex structure, thereby inducing the relevant physiological activity.

[0217] "Receptor binding fragment" refers to a fragment that does not possess the entire structure of a complete ligand but only contains a portion or a partial variant of the complete ligand, wherein the portion or partial variant has the ability to bind to the receptor. Exemplarily, "receptor binding fragment" herein includes but is not limited to the extracellular domain of the ligand.

[0218] "Nucleic acid" refers to any compound and / or substance including a polymer containing nucleotides, such as a polynucleotide. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed from 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. "Nucleic acid" can be linear or circular. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Moreover, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0219] "Viral envelope" refers to the outermost layer of many viruses (Hurlbert et al., Fundamentals of Microbiology, 102. Chapter #11: Viruses). The viral envelope protects the viral genetic material during its life cycle as it navigates through host cells. Not all viruses have a viral envelope. Many human pathogenic viruses are encapsulated in a lipid bilayer, and they infect target cells by fusing the viral envelope with the cell membrane.

[0220] "Retrovirus" and "retroviral vector": Retrovirus and retroviral vector. A retrovirus is a virus that can integrate a DNA copy of its RNA genome into the DNA of a host cell it infects, thereby altering the host cell genome. An overview of available packaging systems is provided in J.M. Coffin, S.M. Hughes, et al., Cold Spring Harbor Laboratory Press, 1997, page 447, which is incorporated herein by reference in its entirety.

[0221] "Lentivirus": Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain additional genes with regulatory or structural functions. This increased complexity allows the virus to regulate its life cycle, as it does during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV).

[0222] "Lentiviral vector": also known as Lentiviral Vector, which is produced by multiple attenuation of HIV virulence genes through gene editing, genetic engineering and other technical means. For example, the genes env, vif, vpr, vpu and nef are deleted, making the lentiviral vector biosafe.

[0223] Lentiviral vectors can stably integrate target genes, such as shuttle genes, into the chromosomes of target cells, allowing them to express the delivered shuttle genes long-term, offering significant advantages for gene therapy. Furthermore, they do not transfer viral genes, thus avoiding the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Furthermore, they have a relatively large cloning capacity, sufficient for most anticipated clinical applications.

[0224] "Shuttle gene": also known as Transgene, which is the target gene / nucleic acid sequence carried by a lentiviral vector or a retroviral vector.

[0225] Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey, et al., (1998) J. Virol. 72:9873-9880; Dull, et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516, and U.S. Pat. No. 5,994,136, each of which is herein incorporated by reference in its entirety.

[0226] Lentiviral vectors are generally packaged in packaging cell lines using a lentiviral vector system. For example, the process and method of packaging lentiviral vectors are described in Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016, which is incorporated herein by reference in its entirety.

[0227] Commonly used lentiviral vector systems include so-called third-generation lentiviral vector systems. The third-generation lentiviral vector system includes four plasmids. "Transfer plasmid" (Transfer Vector, "TV"; in some embodiments of the present invention, also referred to as "master plasmid") contains a lentiviral vector backbone genome and a polynucleotide / nucleic acid sequence delivered to the target cell by the lentiviral vector, such as a target gene such as a shuttle gene. The transfer plasmid typically has one or more target gene (shuttle gene) sequences flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, the transfer plasmid is typically designed to render the resulting vector replication ineffective. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious lentiviral particles in host cells. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby rendering the virus "self-inactivating" (SIN). See Dull, et al., J. Virol. 72:8463-71 (1998); Miyoshi, et al., J. Virol. 72:8150-57 (1998).

[0228] Third-generation lentiviral vector systems typically also include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically carries the gene encoding VSV-G, which is operably linked to a promoter, typically the CMV promoter. Third-generation lentiviral vector systems use two packaging plasmids, one encoding the genes gag and pol, and the other encoding the gene rev as a further safety feature, an improvement over the single packaging plasmid of so-called second-generation systems. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0229] As used herein, the term "lentiviral vector" is intended to refer to a lentiviral particle that comprises a viral envelope, has one or more characteristics of a lentivirus, and is capable of invading target cells, and is not capable of self-replication; the term "retroviral vector" is intended to refer to a retroviral particle that comprises a viral envelope, has one or more characteristics of a retrovirus, and is capable of invading target cells, and is not capable of self-replication.

[0230] The use of lentiviral vector systems relies on a "packaging cell line". Generally speaking, a packaging cell line is a cell line whose cells are capable of producing lentiviral vectors that do not have the ability to self-replicate and can infect target cells when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cells. Exemplarily, a transfection method including a chemically mediated transfection method, a physically mediated transfection method, or a biologically mediated transfection method can be used to introduce the plasmid into the packaging cell line. For example, chemically mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran, or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physically mediated transfection methods include transfection methods such as electroporation.

[0231] "Packaging cell line": Generally speaking, a packaging cell line is a cell line that, when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cell line, produces a lentiviral or retroviral vector capable of infecting host cells. Various methods for introducing plasmids into cells can be used, including but not limited to chemical transfection using reagents such as calcium phosphate, DEAE-dextran, and PEI, or physical transduction methods such as electroporation.

[0232] The packaging cell line is genetically engineered to improve the immunological properties of the lentiviral and retroviral vectors disclosed herein and / or to facilitate infection / transduction of target cells by the lentiviral and retroviral vectors in other ways; such other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0233] In some embodiments of the present invention, the packaging cell line includes but is not limited to at least one of the following cell lines: NS0 cell line, Vero cell line, HeLa cell line, COS cell line, CHO cell line, HEK cell line, BHK cell line and MDCKⅡ cell line.

[0234] In some embodiments of the present invention, the packaging cell line is a HEK-293T cell line.

[0235] "2A peptide": The term "2A peptide" refers to a self-cleaving peptide configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide, and P2A peptide, among others. 2A peptides are 18 to 22 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" can refer to peptides having different amino acid sequences. In the present disclosure, it should be understood that when a lentiviral vector or retroviral vector comprises two or more 2A peptides, the 2A peptides can be identical or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204. In the literature, 2A peptides are often referred to as self-cleaving peptides, but mechanistic studies have shown that the observed "self-cleavage" is actually the result of the ribosome skipping the formation of a glycylprolyl peptide bond at the C-terminus of the 2A peptide. Donnelly et al. (2001) J Gen Virol. 82: 1027-41.

[0236] "Envelope glycoprotein" refers to the glycoprotein coated on the outer layer of the virus (the outer layer of the viral envelope), which plays an important role in the adsorption and penetration of the virus into the host cells, pathogenicity, downregulation of the expression of host surface proteins, and increase in viral packaging and budding.

[0237] "Subject": As used herein, "subject," "patient," or "individual" are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, such as domestic animals, agricultural animals, or wild animals, as well as birds and aquatic animals. A "patient" is a subject who is infected with HIV, suffers from AIDs, is at risk of developing AIDs, or is infected with HIV, or is otherwise in need of any of the lentiviral or retroviral vectors, compositions, methods, bnAbs, or uses provided herein.

[0238] "Administration" or "Administer(ed)" is synonymous with "give" or "administer." The lentiviral or retroviral vectors or compositions disclosed herein can be administered by any effective route, including intravenous, intratumoral, subcutaneous, intramuscular, intrasternal, and infusion techniques. In some embodiments of the present invention, administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal, intralesional, sustained-release, and implantable.

[0239] “And / or”: should be understood to mean one or two alternatives.

[0240] "About" / "approximately": As used herein, when "about" or "approximately" is used to describe a numerical value "X", "about X" includes the numerical value "X" itself or a variation within the range of 0%-15% above or below the numerical value "X"; for example, "about 50%" means that it includes 50% itself or a variation within the range of 0%-15% above or below 50%. In one embodiment, "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0241] "Comprising": As used herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0242] "Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," "another embodiment," or "other embodiments" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, the various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0243] "Prevention": As used herein, "prevention" and similar words, such as "prevent," refer to methods used to prevent, inhibit, or reduce the likelihood of developing or recurring a condition, such as HIV infection and / or AIDs, or various complications caused by HIV infection and / or AIDs. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to onset or recurrence.

[0244] "Treatment": As used herein, "treatment" includes any beneficial or desired effect associated with treatment. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

[0245] "Therapeutically effective amount": i.e., "Therapeutically Effective Amount", as used herein, "therapeutically effective amount" is the amount of the lentiviral vector or retroviral vector, the composition, the B cells expressing broad-spectrum neutralizing antibodies, the B cells or plasma cells comprising nucleic acids encoding broad-spectrum neutralizing antibodies, or their active substances, administered / administered to an individual to provide a beneficial effect or otherwise reduce harmful, non-beneficial events. "Therapeutically effective amount" herein means a dosage that produces one or more desired or expected (e.g., beneficial) effects due to its administration / administration, which is performed once or multiple times within a specified time period. The exact dosage will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0246] "Autologous": As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0247] "Allogeneic": As used herein, "allogeneic" refers to a transplant derived from a different individual of the same species.

[0248] "Stable integration": also known as "stable transfection" or "stable expression" (Stable Gene Expression), refers to the integration of exogenous nucleic acid sequences into the cell genome after introduction into the cell, and their long-term stable expression in the cell and its progeny cells or differentiated cells.

[0249] "Specific binding": As used herein, the term "specific binding" refers to the binding that occurs between paired molecular species (e.g., receptor and ligand and antibody and antigen). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times the background binding (nonspecific binding, about 5 times the background binding, about 10 times the background binding, about 20 times the background binding, about 50 times the background binding, about 100 times the background binding, or about 1000 times the background binding) or more.

[0250] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the exact correspondence of nucleotide to nucleotide or amino acid to amino acid of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". Whether it is a nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, the advanced BLAST computer program purchased from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Briefly, the BLAST program defines identity as the number of aligned symbols (usually nucleotides or amino acids) that are identical divided by the total number of shorter symbols in the two sequences. The program can be used to determine percent identity over the entire length of the compared proteins.

[0251] "Signal peptide": Signal peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97(6): 422-441. (2018)) (Blobel G, Dobberstein B, et al., Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67(3):835-51.(1975)).

[0252] The function of a signal peptide is to prompt cells to transfer proteins, usually to the cell membrane or to secrete them outside the cell.

[0253] "MOI": Multiplicity of Infection (MOI) refers to the number of viral particles added to each host or target cell during infection. For example, if one million viral particles are added to one million cells, MOI = 1.

[0254] "Operably": A nucleic acid sequence is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the nucleic acid sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0255] "Transduction": As used herein, the terms "infection," "transfection," "transformation," or "transduction" are used synonymously to refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell, target cell, or packaging cell. An "infected," "transfected," "transformed," or "transduced" cell is a cell that has been infected, transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0256] Methods for introducing vectors such as viral particles or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into cells by physical, chemical or biological methods.

[0257] Physical methods for introducing vectors or isolated nucleic acids into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.

[0258] Biological methods for introducing vectors or isolated nucleic acids into cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

[0259] Chemical methods for introducing vectors or isolated nucleic acids into cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome.

[0260] "Pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary ingredients" include, but are not limited to, at least one of diluents, solubilizers, emulsifiers, preservatives, preservatives, and adjuvants. Excipients are preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration employed. Such excipients include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, pharmaceutical compositions may contain substances for improving, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.

[0261] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety. In the event of a conflict, the present application (including any definitions herein) will prevail. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be construed as an admission or any form of recommendation.

[0262] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0263] Figure 1 is a schematic diagram of the structure of the B cell-targeting lentiviral vector or retroviral vector;

[0264] FIG2 is a flow cytometry result showing the expression of GFP molecules in human non-activated PBMCs transduced with the lentiviral vector mVG1A19-GFP;

[0265] FIG3 shows the expression of GFP in H929 cells, Jurkat cells, and Nalm6 cells transduced with the lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12, and wtVG-EEK-b12, respectively;

[0266] FIG4 shows the results of detecting the secretion of IgG antibodies in the supernatant of each culture system after H929 cells, Jurkat cells and Nalm6 cells were transduced with the lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12 and wtVG-EEK-b12, respectively;

[0267] FIG5 is a graph showing the results of detecting the expression of GFP molecules in H929 cells after transduction of the PGK virus, the MH virus, and the EEK virus;

[0268] FIG6 shows the results of ELISA testing of the cell supernatants of each group (samples diluted 40-fold) after H929 cells were transduced with the PGK virus, the MH virus, and the EEK virus. The results show the binding of recombinant VRC07-523LS to gp120.

[0269] FIG7 shows the secretion of recombinant VRC07-523LS by two groups of plasma cells differentiated from B cells transduced with the lentiviral vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07, respectively;

[0270] FIG8 is a graph showing the secretion of recombinant VRC07-523LS in the plasma of NKG mice injected with the lentiviral vector mVG3A19-EEK-mVRC07 via the tail vein;

[0271] FIG9 is a flow cytometry result showing the expression of GFP molecules after transducing non-activated human PBMCs with the lentiviral vectors mVG2A79B-EEK-mVRC07 and mCG1A79B-EEK-mVRC07, respectively;

[0272] Figure 10: is the plasmid map of the pGClenti-GFP plasmid;

[0273] Figure 11: is the plasmid map of the main plasmid b12 containing the promoter EEK;

[0274] Figure 12: Figures 12A-B are violin plots and bar graphs, respectively, showing the expression of CD5 on the surface of B cells and other cells; Figures 12C-D are violin plots and bar graphs, respectively, showing the expression of CD19 on the surface of B cells and other cells;

[0275] Figure 13: Figures 13A-B are violin diagrams and bar graphs, respectively, showing the expression of MS4A1 (CD20) on the surface of B cells and other cells; Figures 13C-D are violin diagrams and bar graphs, respectively, showing the expression of CR2 (CD21) on the surface of B cells and other cells;

[0276] Figure 14: Figures 14A-B are violin diagrams and bar graphs, respectively, showing the expression of CD22 on the surface of B cells and other cells; Figures 14C-D are violin diagrams and bar graphs, respectively, showing the expression of FCER2 (CD23) on the surface of B cells and other cells;

[0277] Figure 15: Figures 15A-B are violin diagrams and bar graphs, respectively, showing the expression of CD38 on the surface of B cells and other cells; Figures 15C-D are violin diagrams and bar graphs, respectively, showing the expression of CD40 on the surface of B cells and other cells;

[0278] Figure 16: Figures 16A-B are violin diagrams and bar graphs, respectively, showing the expression of CD79A on the surface of B cells and other cells; Figures 16C-D are violin diagrams and bar graphs, respectively, showing the expression of CD79B on the surface of B cells and other cells;

[0279] Figure 17: Figures 17A-B are violin diagrams and bar graphs, respectively, showing the expression of CD83 on the surface of B cells and other cells; Figures 17C-D are violin diagrams and bar graphs, respectively, showing the expression of IL7R on the surface of B cells and other cells;

[0280] Figure 18: Figures 18A-B are violin diagrams and bar graphs respectively showing the expression of CXCR5 on the cell surface of B cells and other cells. DETAILED DESCRIPTION

[0281] The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0282] In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

[0283] Example 1

[0284] Construct a B cell-targeted lentiviral vector or retroviral vector that carries a nucleic acid sequence encoding bnAbs and can effectively infect / transduce B cells in a subject.

[0285] In this example, a lentiviral vector or retroviral vector is constructed that carries a nucleic acid sequence encoding bnAbs and can effectively infect / transduce B cells in a subject;

[0286] (i) the viral envelope of the lentiviral vector or retroviral vector comprises one or more targeting molecules that specifically bind to B cell endocytic receptors (the targeting molecules are directly or indirectly linked to the transmembrane domain and exposed on the surface of the lentiviral vector or retroviral vector);

[0287] (ii) the envelope glycoprotein of the lentiviral vector or retroviral vector undergoes any one of the first mutations and any one of the second mutations; and

[0288] (iii) The lentiviral vector or retroviral vector carries the nucleic acid sequence encoding bnAbs.

[0289] 1. Targeting molecules that specifically bind to B cell endocytic receptors

[0290] The one or more targeting molecules specifically bind to B cell endocytosis receptors; in this embodiment, exemplarily, the B cell endocytosis receptor is at least one of CD5, CD19, CD20 (MS4A1), CD21 (CR2), CD22, CD23 (FCER2), CD38, CD40, CD79A, CD79B, CD83, IL-7R and CXCR5.

[0291] The targeting molecule may constitute a membrane-expressed antibody or an antigen-binding fragment thereof. Exemplarily, the structure of the membrane-expressed antibody from N-terminus to C-terminus is: a signal peptide, a heavy chain variable region (VH) of scFv, a connecting peptide, a light chain variable region (VL) of scFv, a hinge region, and a transmembrane region;

[0292] The scFv specifically binds to CD5, CD19, CD20 (MS4A1), CD21 (CR2), CD22, CD23 (FCER2), CD38, CD40, CD79A, CD79B, CD83, IL-7R or CXCR5;

[0293] The connecting peptide may be a flexible connecting peptide, for example, a (G4S)3 connecting peptide whose amino acid sequence is shown in SEQ ID NO: 13;

[0294] Exemplarily, the hinge region can be selected from the hinge regions of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0295] Exemplarily, the transmembrane region can be selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10 , DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0296] 2. Mutated envelope glycoprotein

[0297] The envelope glycoprotein of the lentiviral vector or retroviral vector undergoes any of the first mutations and any of the second mutations, thereby weakening or losing the ability of the lentiviral vector or retroviral vector to specifically bind to a receptor, and enhancing the ability to antagonize complement inactivation, or preventing complement inactivation, thereby improving the efficiency of the B cell-targeting lentiviral vector in transducing B cells in the body or blood of a subject;

[0298] Preferably, the envelope glycoprotein is VSV-G or Cocal-G; more preferably, the first mutation is a K47 deletion mutation; the second mutation is a T214N+T352A mutation or K214N+T352A; the extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:23 or SEQ ID NO:38; relative to SEQ ID NO:16, SEQ ID NO:23 comprises K47 deletion, T214N and T352A; relative to SEQ ID NO:17, SEQ ID NO:38 comprises K47 deletion, K214N and T352A.

[0299] 3. Nucleic acid sequences encoding bnAbs

[0300] The lentiviral vector or retroviral vector carries a nucleic acid sequence encoding recombinant bnAbs;

[0301] Exemplarily, the structure of the recombinant bnAbs from N-terminus to C-terminus is: secretory protein signal peptide-VH region (bnAbs)-CH1 region-2A peptide-secretory protein signal peptide-VL region (bnAbs)-CL region.

[0302] Illustratively, the structural schematic diagram of the B cell-targeting lentiviral vector or retroviral vector is shown in FIG1 .

[0303] Example 2

[0304] Lentiviral vector mVG1A19-GFP was used to infect B cells in non-activated human PBMCs.

[0305] 1. Packaging of the Lentiviral Vector mVG1A19-GFP for Transduction of B Cells

[0306] A. Prepare the following four plasmids: envelope plasmid 1 carrying a nucleic acid sequence encoding mutant VSV-G1 and a nucleic acid sequence encoding a membrane-expressing anti-CD19 antibody, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and a lentiviral GFP plasmid (pGClenti-GFP plasmid, the plasmid map is shown in FIG10 ) (Green Fluorescent Protein, "GFP"); envelope plasmid 1 is synthesized by conventional molecular cloning methods;

[0307] The extracellular domain of the mutant VSV-G1 comprises the amino acid sequence shown in SEQ ID NO: 27; relative to SEQ ID NO: 16, SEQ ID NO: 27 comprises R354Q;

[0308] In this embodiment, exemplarily, the membrane-expressed anti-CD19 antibody is a scFv (scFv-FMC63) comprising a heavy chain variable region (VH region) and a light chain variable region (VL region) derived from FMC-63. The structure of the membrane-expressed anti-CD19 antibody (scFv-FMC63) from N-terminus to C-terminus is: CD8α signal peptide, FMC-63 VH region, connecting peptide ((G4S)3 connecting peptide), FMC-63 VL region, CD8α hinge region, CD8α transmembrane region;

[0309] (a) the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 12;

[0310] (b) the amino acid sequence of the FMC63-VH region is shown in SEQ ID NO: 1;

[0311] (c) the amino acid sequence of the (G4S)3 connecting peptide is shown in SEQ ID NO: 13;

[0312] (d) the amino acid sequence of the FMC63-VL region is shown in SEQ ID NO: 2;

[0313] (e) the amino acid sequence of the hinge region of CD8α is shown in SEQ ID NO: 14;

[0314] (f) The amino acid sequence of the transmembrane region of CD8α is shown in SEQ ID NO: 15.

[0315] B. Packaging of lentiviral vector mVG1A19-GFP: Mix the four plasmids and transfect them into the packaging cell line HEK-293T cell line using PEI reagent. The specific steps are as follows:

[0316] 9ug of the lentiviral GFP plasmid, 4ug of pMDLg / pRRE packaging plasmid, 2ug of pRSV-REV packaging plasmid, and 2ug of the envelope plasmid 1 were added to 1 mL of Opti-MEM medium, shaken well, and 64uL of PEI reagent was added. After pipetting evenly, the mixture was allowed to stand for 10 minutes and then added to the culture medium of HEK-293T cells. The culture medium was renewed after 6 hours. The culture supernatant was collected 48 hours after transfection, filtered using a 0.45um filter membrane, centrifuged at 50000g for 2.5h, the supernatant was discarded, and the lentiviral vector mVG1A19-GFP was resuspended in 200uL of F12 medium and frozen at -80°C.

[0317] Opti-MEM alpha Reduced Serum Medium: Brand: GIBCO, Catalog Number: #SP0272;

[0318] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

[0319] F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

[0320] Syringe filter: Brand: SORFA, item number: #622120.

[0321] 2. Transduction of Non-activated Human PBMCs

[0322] On Day 0, human (healthy human) PBMCs were transduced using the lentiviral vector mVG1A19-GFP at an MOI of 1 and an MOI of 5, respectively. The cells were infected at room temperature and cultured in an incubator at 37°C and a CO2 concentration of 5%. The PBMCs culture system contained 1640 culture medium and 10% FBS. On Day 2, 10 mL of DPBS buffer was added and mixed, followed by centrifugation at 500 g for 3 minutes. The supernatant was discarded, and the expression of CD20 (a B cell positive marker) and GFP molecules in the PBMCs was detected. The results are shown in Figure 2.

[0323] Antibody used in flow cytometry: APC-CD20, brand: BIOLEGEND, catalog number: #559776.

[0324] 3. Control group lentiviral wtVG-GFP vector transduced B cells in PBMCs

[0325] Referring to the above-mentioned method for packaging the lentiviral vector mVG1A19-GFP, the control lentiviral vector wtVG-GFP was packaged; the specific method is as follows:

[0326] Using PEI reagent, the lentiviral GFP plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and pMD2.G envelope plasmid (containing nucleic acid encoding wild-type VSV-G) were transfected into the packaging cell line HEK-293T cell line to package the control group lentiviral vector wtVG-GFP;

[0327] The control group lentiviral vector wtVG-GFP carries the GFP gene, and its viral envelope contains wild-type VSV-G, but does not contain the membrane-expressing anti-CD19 antibody, scFv-FMC63;

[0328] The extracellular domain of the wild-type VSV-G comprises the amino acid sequence shown in SEQ ID NO: 16;

[0329] The full-length protein of the wild-type VSV-G (including the signal peptide) comprises the amino acid sequence shown in SEQ ID NO: 31:

[0330] Wherein, the amino acid sequence shown at positions 1 to 16 of SEQ ID NO: 31:

[0331] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of wild-type VSV-G.

[0332] Referring to the method for transducing B cells in human non-activated PBMCs with the lentiviral vector mVG1A19-GFP, the control group lentiviral vector wtVG-GFP was used to transduce B cells in human (healthy) non-activated PBMCs at MOI=1 and MOI=5, respectively; on Day 2, flow cytometry was used to detect the expression of CD20 and GFP molecules in the PBMCs, respectively. The results are shown in Figure 2.

[0333] As shown in Figure 2, since the non-activated B cells in non-activated human PBMCs lowly express LDL-R, the control group lentiviral vector wtVG-GFP, whose viral envelope only contains wild-type VSV-G and does not contain the membrane-expressing anti-CD19 antibody, is difficult to effectively transduce B cells in non-activated human PBMCs;

[0334] Compared with the control group lentiviral vector wtVG-GFP, the lentiviral vector mVG1A19-GFP can specifically bind to the endocytic receptor CD19 on the surface of B cells through the anti-CD19 antibody contained in its viral envelope, enter B cells through endocytosis, effectively transduce B cells and deliver GFP gene.

[0335] Example 3

[0336] Detect the secretion of bnAbs transcribed from different promoters in different cells.

[0337] 1. Packaging of lentiviral vector wtVSVG-PGK / MH / EEK-b12

[0338] A. Construction of a master plasmid: Construct a master plasmid carrying the nucleic acid sequence encoding the bnAbs and the nucleic acid sequence encoding the GFP molecule; the nucleic acid sequence encoding the bnAbs is operably linked to the promoter PGK, MH, or EEK;

[0339] In this embodiment, exemplarily, the bnAbs is recombinant b12, and the nucleic acid sequence encoding the recombinant b12 comprises, from the 5' end to the 3' end, nucleic acid sequences encoding the following polypeptides: IL-10 signal peptide, VH region (derived from the VH region of b12), CH1 region (derived from the CH1 region of human IgG1), FT2A peptide, IL-10 signal peptide, VL region (derived from the VL region of b12), and CL region (derived from the CL region of human Kappa chain); the nucleic acid sequence encoding the recombinant b12 is operably linked to the promoter PGK, MH or EEK; the nucleic acid sequence encoding the recombinant b12 is shown in SEQ ID NO:41;

[0340] The master plasmid carries the nucleic acid sequence encoding the recombinant b12 and the nucleic acid sequence encoding the GFP molecule (master plasmid b12); illustratively, when the master plasmid b12 contains the EEK promoter, the plasmid map is shown in FIG11 ;

[0341] (a) the amino acid sequence of the IL-10 signal peptide is shown in SEQ ID NO: 6;

[0342] (b) the amino acid sequence of the VH region (derived from the VH region of b12) is shown in SEQ ID NO: 7;

[0343] (c) the amino acid sequence of the CH1 region (derived from the CH1 region of human IgG1) is shown in SEQ ID NO: 9;

[0344] (d) the amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 11;

[0345] (e) the amino acid sequence of the VL region (derived from the VL region of b12) is shown in SEQ ID NO: 8;

[0346] (f) the amino acid sequence of the CL region (derived from the CL region of human kappa chain) is shown in SEQ ID NO: 10;

[0347] (g) the nucleic acid sequence of the promoter PGK is shown in SEQ ID NO: 3;

[0348] (h) the nucleic acid sequence of the promoter EEK is shown in SEQ ID NO: 4;

[0349] (i) The nucleic acid sequence of the promoter MH is shown in SEQ ID NO: 5.

[0350] B. Packaging lentiviral vectors: Mix the pMD2.G envelope plasmid, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and the master plasmid b12. Referring to the method for packaging the lentiviral vector mVG1A19-GFP in Example 2, transfect the aforementioned four plasmids into the packaging cell line HEK-293T cell line using a PEI reagent to package the lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12, and wtVG-EEK-b12 carrying the nucleic acid sequence encoding the recombinant b12 linked to different promoters;

[0351] The lentiviral vector wtVG-PGK-b12 comprises a nucleic acid sequence encoding the recombinant b12, and the nucleic acid sequence encoding the recombinant b12 is operably linked to a PGK promoter;

[0352] The lentiviral vector wtVG-MH-b12 comprises a nucleic acid sequence encoding the recombinant b12, and the nucleic acid sequence encoding the recombinant b12 is operably linked to the MH promoter;

[0353] The lentiviral vector wtVG-EEK-b12 comprises a nucleic acid sequence encoding the recombinant b12, and the nucleic acid sequence encoding the recombinant b12 is operably linked to an EEK promoter.

[0354] 2. Detection of IgG secretion

[0355] The lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12 and wtVG-EEK-b12 were used to transduce LDL-R + H929 cell (human multiple myeloma cell) culture system, LDL-R + Jurkat cell (human T lymphoid leukemia cell) culture system and LDL-R + After the Nalm6 cell (human B lymphoid leukemia cell) culture system was established, the expression of GFP molecules in each group of cells and the secretion of IgG antibodies in the supernatant of each group of cells were detected; the cell culture system of each group contained 1640 culture medium and 10% FBS.

[0356] Day 0, at an MOI of 1, the lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12, and wtVG-EEK-b12 were added to H929 cells, Jurkat cells, and Nalm6 cells, respectively (i.e., forming 9 groups of cells mixed with viruses);

[0357] The cells of the above 9 groups mixed with viruses were cultured at a rate of 5×10 5 10 cells / well were inoculated into 6-well culture plates for cell culture; on Day 2, flow cytometry was used to detect the expression of GFP molecules in each group of cells, and the results are shown in Figure 3; on Day 2, the supernatant from each well was collected, and the secretion of IgG antibodies in each well was detected using an IgG detection kit according to the operating steps in the kit manual, and the results are shown in Figure 4.

[0358] As shown in FIG3 , the expression of GFP molecules was detected in each group of cells, and the lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12 and wtVG-EEK-b12 can effectively transduce B cells in human non-activated PBMCs.

[0359] As can be seen from Figure 4, after the lentiviral vectors wtVG-PGK-b12, wtVG-MH-b12 and wtVG-EEK-b12 were transduced into each group of cells, IgG antibodies were detected in the supernatant of each group of cells; the promoters PGK, EEK and MH can all initiate the transcription of IgG antibodies in human B cells.

[0360] IgG detection kit: human total immunoglobulin G, brand: Xinbosheng, product number: #EHC124.96.

[0361] Example 4

[0362] The binding of bnAbs (VRC07-523LS) with different promoters to gp120 was detected.

[0363] 1. Transduction of H929 cells with lentiviral vector wtVG-PGK / MH / EEK-mVRC07

[0364] A. Packaging of lentiviral vectors carrying nucleic acid sequences encoding the broadly neutralizing antibody VRC07-523LS

[0365] (1) Construction of a master plasmid: Construct a master plasmid (master plasmid mVRC07) carrying the nucleic acid sequence encoding the recombinant VRC07-523LS and the nucleic acid sequence encoding the GFP molecule;

[0366] In this embodiment, the nucleic acid sequence encoding the recombinant VRC07-523LS comprises, from the 5' end to the 3' end, nucleic acid sequences encoding the following polypeptides: the IL-10 signal peptide, the VH region (derived from the VH region of VRC07), the mutated CH1 region (derived from the mutated CH1 region of human IgG1), the FT2A peptide, the IL-10 signal peptide, the VL region (derived from the VL region of VRC07), and the CL region (derived from the CL region of human Kappa chain); the nucleic acid sequence encoding the recombinant VRC07-523LS is shown in SEQ ID NO: 42; the nucleic acid sequence encoding the recombinant VRC07-523LS is operably linked to the promoter PGK, MH or EEK;

[0367] (a) the amino acid sequence of the VH region (derived from the VH region of VRC07) is shown in SEQ ID NO: 28;

[0368] (b) the amino acid sequence of the mCH1 region (mutated human IgG1 CH1 region) is shown in SEQ ID NO: 30;

[0369] (c) the amino acid sequence of the VL region (derived from the VL region of VRC07) is shown in SEQ ID NO: 29;

[0370] The CH1 region of the VRC07-523LS contains mutations to improve the half-life of the VRC07-523LS (see Rudicell RS, et al., Enhanced potency of a broadly neutralizing HIV-1 antibody in vitro improves protection against lentiviral infection in vivo. J Virol. 2014 Nov; 88(21): 12669-82).

[0371] The half-life-improving mutation comprises introducing two amino acid mutations into the CH1 region of the amino acid sequence set forth in SEQ ID NO:9, namely, replacing the methionine M at position 311 of the amino acid sequence set forth in SEQ ID NO:9 with a leucine L (equivalent to the M428L mutation), and replacing the asparagine N at position 317 of the amino acid sequence set forth in SEQ ID NO:9 with a serine S (equivalent to the N434S mutation), to obtain a muted-CH1 region (i.e., the mCH1 region). The amino acid sequence of the mCH1 region is set forth in SEQ ID NO:30; relative to SEQ ID NO:9 (the CH1 region, the CH1 region of unmutated human IgG1), SEQ ID NO:30 comprises M311L and N317S.

[0372] (2) Packaging lentiviral vectors: mixing pMD2.G envelope plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid mVRC07, referring to the method for packaging the lentiviral vector mVG1A19-GFP, transfecting the aforementioned four plasmids into the packaging cell line HEK-293T cell line using PEI reagent, and packaging lentiviral vectors wtVG-PGK-mVRC07, wtVG-MH-mVRC07 and wtVG-EEK-mVRC07 carrying the nucleic acid sequence encoding the recombinant VRC07-523LS linked to different promoters;

[0373] The lentiviral vector wtVG-PGK-mVRC07 comprises a nucleic acid sequence encoding the recombinant VRC07-523LS, and the nucleic acid sequence encoding the VRC07-523LS is operably linked to a PGK promoter;

[0374] The lentiviral vector wtVG-MH-mVRC07 comprises a nucleic acid sequence encoding the recombinant VRC07-523LS, and the nucleic acid sequence encoding the VRC07-523LS is operably linked to the MH promoter;

[0375] The lentiviral vector wtVG-EEK-mVRC07 comprises a nucleic acid sequence encoding the recombinant VRC07-523LS, and the nucleic acid sequence encoding the VRC07-523LS is operably linked to the EEK promoter.

[0376] (3) Transduction of H929 cells: Day 0, at an MOI of 1, H929 cells were transduced using the lentiviral vectors wtVG-PGK-mVRC07 (PGK virus), wtVG-MH-mVRC07 (MH virus), and wtVG-EEK-mVRC07 (EEK virus), respectively;

[0377] The three groups of mixed cells transduced with the PGK virus, the MH virus or the EEK virus, i.e., H929 cells + PGK virus mixed cells, H929 cells + MH virus mixed cells and H929 cells + EEK virus mixed cells, were cultured at a rate of 5×10 5 cells / well, which were seeded in 6-well culture plates for cell culture. On Day 2, the expression of GFP molecules in cells of each group was detected. The results are shown in FIG5 .

[0378] As shown in Figure 5, the expression of GFP molecules was detected in all groups of H929 cells transduced by viruses. The PGK virus, MH virus and EEK virus can effectively transduce LDL-R + of H929 cells.

[0379] On Day 2, the supernatant from each well was collected and the binding of the antibody to gp120 in the supernatant of each group was detected by ELISA.

[0380] 2. Detect the binding of antibodies to gp120 in the supernatant of each group

[0381] gp120 is a glycoprotein exposed on the surface of the HIV viral envelope. The broadly neutralizing antibodies VRC07 and VRC07-523LS can both bind to gp120.

[0382] (1) Coating

[0383] Dilute gp120 protein (Sino Biological, Cat. No. #11233-V08H) to 1.0 μg / mL using coating solution (0.75 g of Na2CO3 and 1.46 g of NaHCO3, and adjust the volume to 1000 mL, pH 9.6). Coat two columns of ELISA plates with blank coating solution and 1.0 μg / mL gp120 solution, respectively. Add 100 μL to each well, apply sealing film, and incubate at 2-8°C overnight (12-16 hours).

[0384] (2) Washing the plate

[0385] Carefully remove the sealing film, discard any liquid in the wells, and add 300 μL of wash solution (8.0 g NaCl, 1.14 g Na2HPO4, 0.2 g KCl, 0.24 g KH2PO4, 0.5 mL TW-20, dilute to 1000 mL with ultrapure / deionized water to make 1× wash solution) to each well. Soak for 1-2 minutes. Wash the plate three times. Gently pat dry on absorbent paper after each wash.

[0386] (3) Closed

[0387] 200 μL of blocking solution (2% BSA in PBST, pH 7.4) was added to each well, and the plate was incubated at room temperature for 2.0 hours and then washed.

[0388] (4) Add samples

[0389] The isolated cell supernatants (H929 cells + PGK group, H929 cells + EEK group, and H929 cells + MH group) were diluted 2-fold with diluent (blocking buffer diluted 10-fold), with a sample-to-diluent volume ratio of 1:1. The test samples were added to the reaction wells at a volume of 100 μL per well, and 100 μL of diluent was added to the blank control wells. The negative control wells were inoculated with the supernatant of the H929 cell culture system that had not been infected with the virus. The positive control was a commercial gp120 antibody (Sino Biological, Cat. No. 11233-R011).

[0390] (5) Incubation

[0391] Seal the plate with sealing film, incubate at room temperature for 1 hour, and then wash the plate.

[0392] (6) Add secondary antibody

[0393] Dilute Goat Anti-Human IgG-Fc Secondary Antibody (HRP) to 0.25 μg / mL in diluent and add 100 μL to the corresponding wells. Seal the plate with sealing film and incubate at room temperature for 1 hour, then wash.

[0394] (7) Color development

[0395] Add 100 μL of TMB colorimetric solution to each well, seal the plate with film, and incubate at room temperature in the dark for 20 minutes.

[0396] (8) Termination

[0397] Add 100 μL of stop solution (20 mL of 98% concentrated sulfuric acid added to 180 mL of purified water) to each well, and gently shake the ELISA plate until mixed evenly.

[0398] (9) Reading

[0399] The absorbance of each well was measured at 450 nm and 630 nm using a microplate reader and read within 5 minutes after termination. The binding status of the antibody to gp120 in the supernatant of each group is shown in Table 1 below.

[0400] Table 1

[0401] As shown in Table 1, antibodies that can bind to gp120 were detected in the supernatants of H929 cells in each group to which the lentiviral vectors wtVG-PGK-mVRC07, wtVG-MH-mVRC07, and wtVG-EEK-mVRC07 were added respectively;

[0402] Moreover, among the three groups of supernatants, the amount of antibodies that can bind to gp120 was detected in the supernatant of H929 cells added with the lentiviral vector wtVG-EEK-mVRC07, the second highest, and the least in the supernatant of H929 cells added with the lentiviral vector wtVG-MH-mVRC07.

[0403] It can be seen from this that the recombinant VRC07-523LS transcribed by the promoter EEK is secreted in the largest amount in H929 cells, the recombinant VRC07-523LS transcribed by the promoter MH is secreted second in H929 cells, and the recombinant VRC07-523LS transcribed by the promoter PGK is secreted in the least amount in H929 cells.

[0404] 3. Detect the binding of antibodies to gp120 in the supernatant of each group at different sample dilution multiples

[0405] Referring to the aforementioned method for detecting the binding of the recombinant VRC07-523LS with different promoters to gp120, the remaining steps remained unchanged, and the step (4) "adding sample" was changed from "using diluent to dilute 2 times, with the volume ratio of sample to diluent being 1:1" to using the diluent to dilute 40 times, with the volume ratio of sample to diluent being 1:39. The commercial gp120 antibody was used as a standard to detect and calculate the binding of the recombinant VRC07-523LS with different promoters to gp120; the results are shown in FIG6 ;

[0406] As shown in Figure 6, the recombinant VRC07-523LS transcribed by the promoter MH is secreted in the largest amount in H929 cells, the recombinant VRC07-523LS transcribed by the promoter EEK is secreted in the second largest amount in H929 cells, and the recombinant VRC07-523LS transcribed by the promoter PGK is secreted in the smallest amount in H929 cells.

[0407] Example 5

[0408] Lentiviral vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07 were constructed, which targeted CD19, had the first mutation in the envelope glycoprotein, and carried the nucleic acid sequence encoding the recombinant broadly neutralizing antibody.

[0409] 1. Packaging of Lentiviral Vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07

[0410] A. Packaging of lentiviral vector mVG2A19-EEK-mVRC07

[0411] Referring to the method for packaging the lentiviral vector mVG1A19-GFP, the lentiviral vector mVG2A19-EEK-mVRC07 was packaged:

[0412] The envelope plasmid 2, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid mVRC07 were transfected into the packaging cell line HEK-293T cell line by PEI reagent; the envelope plasmid 2 carried the nucleic acid sequence encoding the mutant VSV-G2 and the nucleic acid sequence encoding the membrane-expressing anti-CD19 antibody (scFv-FMC63), the master plasmid mVRC07 contained the EEK promoter, and the nucleic acid sequence encoding the recombinant VRC07-523LS was operably linked to the EEK promoter;

[0413] The extracellular domain of the mutant VSV-G2 comprises the amino acid sequence shown in SEQ ID NO:32; relative to SEQ ID NO:16, SEQ ID NO:32 comprises a K47 deletion;

[0414] The viral envelope of the lentiviral vector mVG2A19-EEK-mVRC07 contains the membrane-expressed anti-CD19 antibody and the mutated glycoprotein, i.e., the mutant VSV-G2. The mutant VSV-G2 contains a K47 deletion mutation that weakens its ability to bind to LDL-R, thereby improving the efficiency of the lentiviral vector mVG2A19-EEK-mVRC07 in targeting and transducing B cells, and delivering the nucleic acid sequence encoding the recombinant VRC07-523LS.

[0415] B. Packaging of lentiviral vector mCG1A19-EEK-mVRC07

[0416] Referring to the method for packaging the lentiviral vector mVG1A19-GFP, the lentiviral vector mCG1A19-EEK-mVRC07 was packaged:

[0417] The envelope plasmid 3, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid mVRC07 were transfected into the packaging cell line HEK-293T cell line using PEI reagent; the envelope plasmid 3 carried the nucleic acid sequence encoding the mutant Cocal-G1 and the nucleic acid sequence encoding the membrane-expressing anti-CD19 antibody, the master plasmid mVRC07 contained the EEK promoter, and the nucleic acid sequence encoding the recombinant VRC07-523LS was operably linked to the EEK promoter;

[0418] The extracellular domain of the mutant Cocal-G1 comprises the amino acid sequence shown in SEQ ID NO:33; relative to SEQ ID NO:17 (the extracellular domain of wild-type Cocal-G), SEQ ID NO:33 comprises a K47 deletion;

[0419] The sequence of the full-length protein of wild-type Cocal-G (including the signal peptide) is shown in SEQ ID NO: 34;

[0420] Wherein, the sequence shown in positions 1 to 17 of SEQ ID NO: 34:

[0421] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of wild-type Cocal-G.

[0422] The viral envelope of the lentiviral vector mCG1A19-EEK-mVRC07 contains the membrane-expressed anti-CD19 antibody and a mutated glycoprotein, namely, mutant Cocal-G1. The mutant Cocal-G1 contains a K47 deletion mutation that weakens its ability to bind to LDL-R, thereby improving the efficiency of the lentiviral vector mCG1A19-EEK-mVRC07 in targeted transduction of B cells and delivering the nucleic acid sequence encoding the recombinant VRC07-523LS.

[0423] Day 0 to Day 13: Activate and transduce B cells isolated from PBMCs and differentiate transduced B cells into plasma cells

[0424] A. Day 0: B cell sorting

[0425] CD19 magnetic beads (trade name: CD19 MicroBeads, human; brand: MILTENYI BIOTEC; catalog number: #130-050-301) were used to isolate CD19 from non-activated PBMCs of human (healthy subjects) according to the product instructions. + B cells, for specific operation methods, see:

[0426] https: / / www.miltenyibiotec.com / US-en / products / cd19-microbeads-human.html#130-050-301; and

[0427] https: / / static.miltenyibiotec.com / asset / 150655405641 / document_p6or426nrl5s960tge5dom5h5q?content-disposition=inline.

[0428] B. Day 0 to Day 7: Activation and transduction of sorted B cells

[0429] The sorted B cells were resuspended in B cell activation medium (B cell activation culture system), and the lentiviral vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07 were added to the two groups of B cell activation culture systems, respectively, to activate and transduce the sorted B cells (refer to Cheng RY, et al., Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo. Nat Commun. 2022 Oct 16; 13(1): 6110.); the specific method is:

[0430] On Day 0, the lentiviral vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07 were added to the two B cell activation culture systems at an MOI of 5, respectively. The cells were infected at room temperature and cultured in an incubator at 37°C and 5% CO2. The sorted B cells were activated until Day 7.

[0431] The B cell activation medium contains B cell basal medium, 100 ng / mL megaCD40L (brand: ENZO LIFE SCIENCE), 1 μg / mL CpG ODN2006 (brand: INVITROGEN), 50 ng / mL IL-2, 50 ng / mL IL-10 (brand: PEPROTECH) and 10 ng / mL IL-15 (brand: MILTENYI); the B cell basal medium contains IMDM (GIBCO, brand: Thermo Fisher), 55 μM 2-mercaptoethanol (2-mercaptoethanol) and 10% FBS.

[0432] C. Day 7 to Day 10: Activated B cells differentiate into plasmablasts

[0433] On Day 7, the B cell activation medium was removed, and plasmablast differentiation medium was added to culture the cells until Day 10 to differentiate the activated B cells into plasmablasts.

[0434] The plasmablast differentiation medium contains the B cell basal medium, 50 ng / mL IL-6 (brand: PEPROTECH), 50 ng / mL IL-2, 50 ng / mL IL-10, and 10 ng / mL IL-15.

[0435] D. Day 10 to Day 13: Plasmablasts differentiate into plasma cells

[0436] On Day 10, the plasmablast differentiation medium was removed, and plasma cell differentiation medium was added to culture the cells until Day 13 to differentiate the plasmablasts into plasma cells.

[0437] The plasma cell differentiation medium contains the B cell basal medium, 50 ng / mL IL-6, 10 ng / mL IL-15, and 15 ng / mL interferon-α2B (brand: SIGMA-ALDRICH).

[0438] Day 14: Detection of VRC07-523LS secretion by plasma cells

[0439] On Day 14, referring to the aforementioned ELISA assay, the secretion of VRC07-523LS from the supernatants of two groups of plasma cells differentiated from B cells transduced with the lentiviral vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07, respectively (diluted 40-fold using the diluent), was detected and calculated. The results are shown in FIG7 .

[0440] As shown in FIG7 , VRC07-523LS was successfully detected in the supernatants of two groups of plasma cells obtained by differentiating B cells transduced with the lentiviral vectors mVG2A19-EEK-mVRC07 and mCG1A19-EEK-mVRC07.

[0441] Example 6

[0442] A lentiviral vector mVG3A19-EEK-mVRC07 was constructed, which targeted CD19, had the first and second mutations in the envelope glycoprotein, and carried the nucleic acid sequence encoding the recombinant VRC07-523LS.

[0443] 1. Packaging of Lentiviral Vector mVG3A19-EEK-mVRC07

[0444] Referring to the method for packaging the lentiviral vector mVG1A19-GFP, the lentiviral vector mVG3A19-EEK-mVRC07 was packaged:

[0445] The envelope plasmid 4, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid mVRC07 were transfected into the packaging cell line HEK-293T cell line using PEI reagent; the envelope plasmid 4 carried the nucleic acid sequence encoding the mutant VSV-G3 and the nucleic acid sequence encoding the membrane-expressing anti-CD19 antibody, the master plasmid mVRC07 contained the EEK promoter, and the nucleic acid sequence encoding the recombinant VRC07-523LS was operably linked to the EEK promoter;

[0446] The extracellular domain of the mutant VSV-G3 comprises the amino acid sequence shown in SEQ ID NO: 23; relative to SEQ ID NO: 16, SEQ ID NO: 23 comprises a K47 deletion, T214N, and T352A;

[0447] The viral envelope of the lentiviral vector mVG3A19-EEK-mVRC07 comprises the membrane-expressing anti-CD19 antibody and a glycoprotein with the first mutation (K47 deletion) and the second mutation (T214N and T352A), i.e., a mutant VSV-G3. The mutant VSV-G3 comprises a K47 deletion mutation that weakens its ability to bind to LDL-R and T214N and T352A mutations that enhance its ability to antagonize complement inactivation, thereby improving the efficiency of the lentiviral vector mVG3A19-EEK-mVRC07 in targeting and transducing B cells in a subject and delivering the nucleic acid sequence encoding the recombinant VRC07-523LS.

[0448] 2. Transduction of B cells in mice using the lentiviral vector mVG3A19-EEK-mVRC07

[0449] Two groups of NKG mice were selected, with 5 mice in each group. After irradiation with 0.5 Gy (Gray), 1×10 5 CD34 isolated from human umbilical cord blood + Six weeks later, one group was transplanted with CD34 +The hematopoietic stem cell-bearing mice were injected with the lentiviral vector mVG3A19-EEK-mVRC07 at 1E7 TU per mouse via the tail vein. One week later, peripheral blood plasma was collected from the two groups of mice. The secretion of VRC07-523LS in the plasma of each group of mice was detected and calculated by referring to the aforementioned ELISA detection method. The results are shown in FIG8 .

[0450] As shown in Figure 8, VRC07-523LS can be detected in the plasma of mice injected with the lentiviral vector mVG3A19-EEK-mVRC07. The lentiviral vector mVG3A19-EEK-mVRC07 can effectively transduce B cells in mice, and the transduced B cells can effectively differentiate into plasma cells, and then secrete the broad-spectrum neutralizing antibody VRC07-523LS.

[0451] Example 7

[0452] Lentiviral vectors mVG2A79B-EEK-mVRC07 and mCG1A79B-EEK-mVRC07 were constructed, which targeted CD79B, had the first mutation in the envelope glycoprotein, and carried the nucleic acid sequence encoding the recombinant broad-spectrum neutralizing antibody.

[0453] 1. Packaging of Lentiviral Vector mVG2A79B-EEK-mVRC07

[0454] Referring to the method for packaging the lentiviral vector mVG1A19-GFP, the lentiviral vector mVG2A79B-EEK-mVRC07 was packaged:

[0455] The envelope plasmid 5, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the master plasmid mVRC07 were transfected into the packaging cell line HEK-293T cell line using PEI reagent; the envelope plasmid 5 carried the nucleic acid sequence encoding the mutant VSV-G2 and the nucleic acid sequence encoding the membrane-expressing anti-CD79B antibody; the master plasmid mVRC07 contained the EEK promoter, and the nucleic acid sequence encoding the recombinant VRC07-523LS was operably linked to the EEK promoter;

[0456] In this embodiment, illustratively, the membrane-type expression of the anti-CD79B antibody from N-terminus to C-terminus is as follows: the CD8α signal peptide, the VH region derived from SN-8, the connecting peptide ((G4S)3 connecting peptide), the VL region derived from SN-8, the hinge region of CD8α, and the transmembrane region of CD8α.

[0457] The amino acid sequence of the VH region derived from SN-8 is shown in SEQ ID NO: 35;

[0458] The amino acid sequence of the VL region derived from SN-8 is shown in SEQ ID NO: 36;

[0459] 2. Packaging of Lentiviral Vector mCG1A79B-EEK-mVRC07

[0460] Referring to the method for packaging the lentiviral vector mVG1A19-GFP, the lentiviral vector mCG1A79B-EEK-mVRC07 was packaged:

[0461] The envelope plasmid 6, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the main plasmid mVRC07 were transfected into the packaging cell line HEK-293T cell line using PEI reagent; the envelope plasmid 6 carried the nucleic acid sequence encoding the mutant Cocal-G1 and the nucleic acid sequence encoding the membrane-expressed anti-CD79B antibody; the main plasmid mVRC07 contained the EEK promoter, and the nucleic acid sequence encoding the recombinant VRC07-523LS was operably linked to the EEK promoter.

[0462] 3. Transduction of Non-activated Human PBMCs

[0463] Referring to the method for transducing non-activated human PBMCs with the lentiviral vector mVG1A19-GFP, at an MOI of 5, on Day 0, non-activated human PBMCs were transduced with the lentiviral vectors mVG2A79B-EEK-mVRC07 and mCG1A79B-EEK-mVRC07, respectively. On Day 2, flow cytometry was used to detect the expression of GFP molecules in each group of PBMCs. The results are shown in Figures 9B and 9C, respectively.

[0464] As shown in Figures 9B and 9C, the lentiviral vectors mVG2A79B-EEK-mVRC07 and mCG1A79B-EEK-mVRC07 can express anti-CD79B antibodies contained in their viral envelopes to bind to LDL-R. - CD79B + The endocytic receptor CD79B on the surface of non-activated B cells successfully enters and transduces non-activated B cells through endocytosis.

Claims

1. A lentiviral vector or a retroviral vector, characterized in that: (a) the viral envelope of the lentiviral vector or retroviral vector comprises one or more targeting molecules that specifically bind to B cell endocytosis receptors, wherein the targeting molecules are not part of the envelope glycoprotein of the lentiviral vector or retroviral vector, and the targeting molecules are antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to B cell endocytosis receptors; and (b) The lentiviral vector or retroviral vector carries a nucleic acid sequence encoding a broad-spectrum neutralizing antibody.

2. The lentiviral vector or retroviral vector according to claim 1, characterized in that The B cell endocytic receptor is selected from CD5, CD19, CD20, CD21, CD22, CD23, CD38, CD40, CD79A, CD79B, CD83, IL-7RA and CXCR5.

3. The lentiviral vector or retroviral vector according to claim 1 or 2, characterized in that The envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a first mutation, so that the ability of the envelope glycoprotein to specifically bind to a receptor is weakened or lost relative to before the first mutation occurs.

4. The lentiviral vector or retroviral vector according to any one of claims 1 to 3, characterized in that The envelope glycoprotein is selected from the following envelope glycoproteins and variants thereof: envelope glycoproteins of vesicular stomatitis virus strains and variants thereof, envelope glycoproteins of baboon endogenous retrovirus BaEV and variants thereof, envelope glycoproteins of feline endogenous retrovirus RD114 and variants thereof, and envelope glycoproteins of gibbon ape leukemia virus GALV and variants thereof; Preferably, the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants thereof of the vesicular stomatitis virus Indiana strain, envelope glycoprotein and variants thereof of the vesicular stomatitis virus Cocal strain, envelope glycoprotein and variants thereof of the vesicular stomatitis virus Maraba strain, envelope glycoprotein and variants thereof of the vesicular stomatitis virus Morreton strain, and envelope glycoprotein and variants thereof of the vesicular stomatitis virus Variants, envelope glycoprotein of Alagoas strain of vesicular stomatitis virus and variants thereof, New The envelope glycoproteins of Jersey strain and variants thereof, the envelope glycoproteins of Carajas strain and variants thereof, the envelope glycoproteins of Chandipura strain and variants thereof, the envelope glycoproteins of Eptesicus strain and variants thereof, the envelope glycoproteins of Isfahan strain and variants thereof, the envelope glycoproteins of Jurona strain and variants thereof, the envelope glycoproteins of Malpais strain and variants thereof, the envelope glycoproteins of Perinet strain and variants thereof, the envelope glycoproteins of Piry strain and variants thereof, the envelope glycoproteins of Radi strain and variants thereof, the envelope glycoproteins of Rhinolopus strain and variants thereof, and the envelope glycoproteins of Yug Bogdanovac strain and variants thereof.

5. The lentiviral vector or retroviral vector according to claim 4, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is a low-density lipoprotein receptor LDL-R; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 16 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 16; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution or deletion of R354, deletion of amino acids 1-18, Deletion of amino acids 19-36, 37-51, 314-384, 321-374, 331-364, 344-354, 345-353; and (b) After optimal global alignment with SEQ ID NO: 16, the position corresponding to SEQ ID NO:16: substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 16; and (b) After optimal global alignment with SEQ ID NO: 16, the following sequences are located at positions corresponding to substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: Substitution of K47 or deletion of K47, substitution of R354; Still further preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: The amino acid at position 47 is replaced by lysine K to glutamine Q or the amino acid lysine at position 47 is deleted, and the amino acid at position 354 is replaced by arginine R to glutamine Q; Most preferably, the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acid: a deletion of K47.

6. The lentiviral vector or retroviral vector according to claim 4, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the receptor is a low-density lipoprotein receptor LDL-R; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 17 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 17; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183 in SEQ ID NO:

17. , substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; and (b) After optimal global alignment with SEQ ID NO: 17, the position corresponding to SEQ ID NO:17: substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 17; and (b) after optimal global alignment with SEQ ID NO: 17, substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 17; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: substitution or deletion of K47, substitution of R354; Still more preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: the 47th amino acid is replaced by lysine K to glutamine Q or the 47th amino acid lysine is deleted, and the 354th amino acid is replaced by arginine R to glutamine Q; Most preferably, the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acid: a deletion of K47.

7. The lentiviral vector or retroviral vector according to any one of claims 3 to 6, characterized in that The envelope glycoprotein of the lentiviral vector or retroviral vector also undergoes a second mutation, so that the ability of the envelope glycoprotein to antagonize inactivation by complement is enhanced compared to before the second mutation occurs, or the envelope glycoprotein is not inactivated by complement.

8. The lentiviral vector or retroviral vector according to claim 7, characterized in that The envelope glycoprotein is an envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof, wherein the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 16 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 16; Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 16; (b) after optimal global alignment with SEQ ID NO: 16, is located at the 214th amino acid corresponding to SEQ ID NO: 16; (c) amino acid position 352 of SEQ ID NO: 16; (d) after optimal global alignment with SEQ ID NO: 16, is located at amino acid position 352 corresponding to SEQ ID NO: 16; (e) amino acid position 50 of SEQ ID NO: 16; (f) after optimal global alignment with SEQ ID NO: 16, is located at the 50th amino acid corresponding to SEQ ID NO: 16; (g) amino acid position 146 of SEQ ID NO: 16; and (h) after optimal global alignment with SEQ ID NO: 16, is located at the 146th amino acid corresponding to SEQ ID NO: 16; More preferably, the amino acid mutation includes deletion, insertion or substitution of the amino acid; More preferably, the amino acid mutation is a substitution of the amino acid.

9. The lentiviral vector or retroviral vector according to claim 8, characterized in that The second mutation includes that the amino acid sequence contains one or more of the following site mutations: substitution of T214, substitution of T352, substitution of K50, substitution of S146: Preferably, the second mutation includes one or more of the following mutations in the amino acid sequence: the 214th amino acid is replaced by threonine T to asparagine N, the 352nd amino acid is replaced by threonine T to alanine A, the 50th amino acid is replaced by lysine K to threonine T, the 146th amino acid is replaced by threonine T to threonine T, Amino acid S is replaced by threonine T; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) replacement of T214 and T352; and (b) Replacement of T214, T352, K50 and S146; More preferably, the amino acid sequence comprises a combination of any of the following site mutations: (a) amino acid position 214 is substituted from threonine T to asparagine N and amino acid position 352 is substituted from threonine T to alanine A; and (b) The amino acid at position 214 is replaced by threonine T to asparagine N, the amino acid at position 352 is replaced by threonine T to alanine A, the amino acid at position 50 is replaced by lysine K to threonine T, and the amino acid at position 146 is replaced by serine S to threonine T.

10. The lentiviral vector or retroviral vector according to claim 7, characterized in that: The envelope glycoprotein is an envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, wherein the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 17 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 17; Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 17; (b) after optimal global alignment with SEQ ID NO: 17, is located at the 214th amino acid corresponding to SEQ ID NO: 17; (c) amino acid position 352 of SEQ ID NO: 17; (d) after optimal global alignment with SEQ ID NO: 17, is located at amino acid position 352 corresponding to SEQ ID NO: 17; (e) amino acid position 50 of SEQ ID NO: 17; (f) after optimal global alignment with SEQ ID NO: 17, is located at the 50th amino acid corresponding to SEQ ID NO: 17; (g) amino acid position 146 of SEQ ID NO: 17; and (h) after optimal global alignment with SEQ ID NO: 17, is located at the 146th amino acid corresponding to SEQ ID NO: 17; More preferably, the amino acid mutation includes deletion, insertion or substitution of the amino acid; More preferably, the amino acid mutation is a substitution of the amino acid.

11. The lentiviral vector or retroviral vector according to claim 10, characterized in that: The second mutation includes that the amino acid sequence contains one or more of the following site mutations: substitution of K214, substitution of T352, substitution of K50, substitution of S146; Preferably, the second mutation includes one or more of the following site mutations in the amino acid sequence: the 214th amino acid is replaced by lysine K to asparagine N, the 352nd amino acid is replaced by threonine T to alanine A, the 50th amino acid is replaced by lysine K to threonine T, and the 146th amino acid is replaced by serine S to threonine T; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) replacement of K214 and T352; and (b) Replacement of K214, T352, K50 and S146; More preferably, the second mutation comprises a combination of any one of the following site mutations in the amino acid sequence: (a) amino acid position 214 is substituted from lysine K to asparagine N and amino acid position 352 is substituted from threonine T to alanine A; and (b) The amino acid at position 214 is replaced by lysine K to asparagine N, the amino acid at position 352 is replaced by threonine T to alanine A, the amino acid at position 50 is replaced by lysine K to threonine T, and the amino acid at position 146 is replaced by serine S to threonine T.

12. The lentiviral vector or retroviral vector according to any one of claims 1 to 11, characterized in that The targeting molecule is directly or indirectly connected to the transmembrane domain and exposed on the surface of the lentiviral vector or retroviral vector; Preferably, the transmembrane domain is selected from the transmembrane regions of the following proteins: CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70; More preferably, the transmembrane domain includes the transmembrane region of CD8α.

13. The lentiviral vector or retroviral vector according to claim 12, characterized in that: The targeting molecule is indirectly connected to the transmembrane domain via a connecting domain and is exposed on the surface of the lentiviral vector or retroviral vector; Preferably, the linking domain is selected from: (i) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild type or a modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (ii) a hinge region selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (iii) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; and (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the connecting domain includes the hinge region of CD8α.

14. The lentiviral vector or retroviral vector according to any one of claims 1 to 13, characterized in that The broadly neutralizing antibody is selected from bavituximab, UB-421, BF520.1, BiIA-SG, CH01, CH59, C2F5, C4E10, C2F5+C2G12+C4E10, CAP256V2LS, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6, D1D2, 10-1074-LS, Cl3hmAb, GS-9722 (elipovimab), DH411-2, BG18, GS-9721, GS-9723, PGT145, PGT121, PGT-121.60, PGT-121.

70. .66, PGT122, PGT-123, PGT-124, PGT-125, PGT-126, PGT-151, PGT-130, PGT-133, PGT-134, PGT-135, PGT-128, PGT-136, PGT-137, PGT-138, PGT-1 39. MDX010 (ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1, N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH 45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12, PGDM21, PCDN-33A, 2Dm2m, 4Dm 2m, 6Dm2m, PGDM1400, MDX010(ipilimumab), VRCO1, VRC-01-LS, A32, 7B2, 10E8, VRC-07-523, VRC07- 523LS, b12, VRC24, VRC41, 01, 10E8VLS, 3810109, 10E8v4, IMC-HIV, iMabm36, eCD4-Ig, IOMA, CAP256 -VRC26.25, DRVIA7, VRC-HIVMAB080-00-AB, VRCHIVMAB060-00-AB, P2G12, VRC07, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, VRC29.03, CAP256, CAP256-VRC26.08, CAP25 6-VRC26.09, CAP256-VRC26.25, PCT64-24E, VRC38.01, PGT-151, CAP248-2B, 35022, ACS202, VRC34, VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, LN01, 12A12, 12A21, NIH45-46, bANC131, 8ANC134, IB2530, INC9, 8ANC195, 8ANC196, 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM.

15. The lentiviral vector or retroviral vector according to any one of claims 1 to 14, characterized in that The nucleic acid sequence encoding the broad-spectrum neutralizing antibody comprises a nucleic acid sequence encoding a signal peptide of a secreted protein; Preferably, the secretory protein is selected from IFN-α, IFN-β, IFN-γ, IL-1, IL-6 and IL-10; More preferably, the secreted protein is IL-10.

16. The lentiviral vector or retroviral vector according to any one of claims 1 to 15, characterized in that The broad-spectrum neutralizing antibody is a recombinant broad-spectrum neutralizing antibody; Preferably, the nucleic acid sequence encoding the recombinant broad-spectrum neutralizing antibody comprises, from the 5' end to the 3' end, the nucleic acid sequence encoding the following polypeptides: a signal peptide of a secretory protein, a heavy chain variable region (VH region) of a broad-spectrum neutralizing antibody, a heavy chain constant region 1 (CH1 region) of an immunoglobulin, a 2A peptide, a signal peptide of a secretory protein, a light chain variable region (VL region) of a broad-spectrum neutralizing antibody, and a light chain constant region (CL region) of an immunoglobulin; More preferably, the secretory protein is selected from IFN-α, IFN-β, IFN-γ, IL-1, IL-6 and IL-10; More preferably, the CH1 region of the immunoglobulin is the CH1 region of human IgG1; More preferably, the 2A peptide is a FT2A peptide; More preferably, the CL region of the immunoglobulin is the CL region of a human immunoglobulin Kappa chain (κ chain).

17. The lentiviral vector or retroviral vector according to claim 16, characterized in that The nucleic acid sequence encoding the recombinant broad-spectrum neutralizing antibody comprises, from the 5' end to the 3' end, the nucleic acid sequence encoding the following polypeptides: a signal peptide of IL-10, a VH region of a broad-spectrum neutralizing antibody, a CH1 region of human IgG1, a FT2A peptide, a signal peptide of IL-10, a VL region of a broad-spectrum neutralizing antibody, and a CL region of a human immunoglobulin κ chain; Preferably, the broadly neutralizing antibody is selected from b12 and VRC07-523LS.

18. The lentiviral vector or retroviral vector according to any one of claims 1 to 17, characterized in that: The nucleic acid sequence encoding the broad-spectrum neutralizing antibody is operably linked to a promoter that can initiate transcription of polypeptides and / or proteins in B cells; Preferably, the B cells are human B cells.

19. A composition, characterized in that The composition comprises the lentiviral vector or retroviral vector of any one of claims 1 to 18 and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions and other auxiliary components; Preferably, the composition comprises the lentiviral vector or retroviral vector according to any one of claims 7 to 18 and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservative solutions and other auxiliary components.

20. A method for infecting or transducing B cells, characterized in that: Contacting B cells using the lentiviral vector or retroviral vector of any one of claims 1 to 18 or the composition of claim 19; Preferably, the contacting occurs outside the body of a subject, the subject being an individual to whom B cells infected or transduced by the method of infecting or transducing B cells are administered; Preferably, the contacting occurs in a subject, wherein the subject is an individual to whom the lentiviral vector or retroviral vector according to any one of claims 1 to 18 or the composition according to claim 19 is administered. body; More preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

21. A B cell expressing broadly neutralizing antibodies, characterized in that: The B cells expressing broadly neutralizing antibodies are prepared by contacting the lentiviral vector or retroviral vector according to any one of claims 1 to 18 or the composition according to claim 19 with B cells; Preferably, the contacting occurs outside the body of a subject, and the subject is an individual to whom the B cells expressing broadly neutralizing antibodies are administered; Preferably, the contacting occurs in a subject, wherein the subject is an individual to whom the lentiviral vector or retroviral vector according to any one of claims 1 to 18 or the composition according to claim 19 is administered; More preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

22. The B cell expressing broadly neutralizing antibodies according to claim 21, characterized in that The expression is cytoplasmic expression (Cytoplasmic Expression); Preferably, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody.

23. A B cell comprising a nucleic acid encoding a broadly neutralizing antibody, characterized in that The B cells are prepared by the method of claim 20; Preferably, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody.

24. A plasma cell comprising a nucleic acid encoding a broadly neutralizing antibody, characterized in that The plasma cells are differentiated from the B cells of claim 23.

25. The plasma cell according to claim 24, characterized in that The plasma cells secrete broadly neutralizing antibodies in vivo or in vitro in the subject; Preferably, the broadly neutralizing antibody is a recombinant broadly neutralizing antibody.

26. A method for preparing broad-spectrum neutralizing antibodies in a subject, characterized in that: The method comprises administering to a subject a lentiviral vector or a retroviral vector according to any one of claims 1 to 18 or a composition according to claim 19 to infect or transduce B cells in the subject, or administering to a subject a B cell expressing a broadly neutralizing antibody according to any one of claims 21 to 22, a B cell comprising a nucleic acid encoding a broadly neutralizing antibody according to claim 23, or a plasma cell according to any one of claims 24 to 25; Preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

27. The method according to claim 26, characterized in that The infected or transduced B cells, the B cells expressing broad-spectrum neutralizing antibodies, or the B cells containing nucleic acids encoding broad-spectrum neutralizing antibodies differentiate into plasma cells and secrete broad-spectrum neutralizing antibodies in the subject, or the plasma cells containing nucleic acids encoding broad-spectrum neutralizing antibodies secrete broad-spectrum neutralizing antibodies in the subject.

28. A method for establishing acquired immunity in a subject, characterized in that: The method comprises administering to the subject the lentiviral vector or retroviral vector of any one of claims 1 to 18 or the composition of claim 19 to infect or transduce B cells in the subject, or administering to the subject the B cells expressing the broadly neutralizing antibody of any one of claims 21 to 22, the B cells comprising the nucleic acid encoding the broadly neutralizing antibody of claim 23, or the plasma cells comprising the nucleic acid encoding the broadly neutralizing antibody of any one of claims 24 to 25; Preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

29. The method according to claim 28, characterized in that The infected or transduced B cells, the B cells expressing broad-spectrum neutralizing antibodies, or the B cells containing nucleic acids encoding broad-spectrum neutralizing antibodies differentiate into plasma cells and secrete broad-spectrum neutralizing antibodies in the subject, or the plasma cells containing nucleic acids encoding broad-spectrum neutralizing antibodies secrete broad-spectrum neutralizing antibodies in the subject.

30. A broadly neutralizing antibody prepared in a subject, characterized in that: The broadly neutralizing antibody is prepared by infecting or transducing B cells in a subject with the lentiviral vector or retroviral vector of any one of claims 1 to 18 or the composition of claim 19, or by administering to a subject a B cell expressing a broadly neutralizing antibody of any one of claims 21 to 22, a B cell comprising a nucleic acid encoding a broadly neutralizing antibody of claim 23, or a plasma cell of any one of claims 24 to 25; Preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

31. The broadly neutralizing antibody according to claim 30, characterized in that The infected or transduced B cells, the B cells expressing broad-spectrum neutralizing antibodies, or the B cells containing nucleic acids encoding broad-spectrum neutralizing antibodies differentiate into plasma cells and secrete broad-spectrum neutralizing antibodies in the subject, or the plasma cells containing nucleic acids encoding broad-spectrum neutralizing antibodies secrete broad-spectrum neutralizing antibodies in the subject.

32. Use of the lentiviral vector or retroviral vector according to any one of claims 1 to 18, the composition according to claim 19, the B cell expressing a broadly neutralizing antibody according to any one of claims 21 to 22, the B cell comprising a nucleic acid encoding a broadly neutralizing antibody according to claim 23, or the plasma cell according to any one of claims 24 to 25 in the preparation of a medicament for treating HIV infection and / or AIDs.

33. A method for treating a subject infected with HIV and / or suffering from AIDs, characterized in that: The method comprises administering to the subject a lentiviral vector or a retroviral vector according to any one of claims 1 to 18, a composition according to claim 19, a B cell expressing a broadly neutralizing antibody according to any one of claims 21 to 22, a B cell comprising a nucleic acid encoding a broadly neutralizing antibody according to claim 23, or a plasma cell according to any one of claims 24 to 25; Preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

34. The method according to claim 33, characterized in that A therapeutically effective amount of the lentiviral vector or retroviral vector, the composition, the B cell expressing the broadly neutralizing antibody, the B cell comprising a nucleic acid encoding the broadly neutralizing antibody, or the plasma cell is administered to the subject.

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