Method for regulating production of b cell high-affinity antibody

Abstract

Provided in the present invention is a method for regulating the production of a B cell high-affinity antibody. According to an embodiment of the present invention, the method comprises: regulating the expression level of an HMCES protein; or regulating the uracil DNA glycosidase activity of the HMCES protein, wherein the HMCES protein is located in the B cell, and the B cell is located in a germinal center. The method according to an embodiment of the present invention can effectively regulate the production of B cell high-affinity antibodies.

Description

Methods to regulate the production of high-affinity antibodies from B cells Technical Field

[0001] This invention relates to the field of cellular immunity, specifically to a method for regulating the production of high-affinity antibodies from B cells. More specifically, this invention relates to a method for regulating high-frequency mutations in B cell somatic cells, a method for regulating AID-induced DNA mutations in B cells, a method for predicting the level of high-affinity antibodies on B cells, the use of reagents in the preparation of kits, and a method for regulating pathogen-mediated immune responses. Background Technology

[0002] The high variability of the human immunodeficiency virus (HIV) is one of the major obstacles to developing an effective vaccine. The proteins on the surface of the virus frequently mutate, making it difficult for the immune system to recognize and neutralize the virus. However, despite this variability, a small percentage of HIV-infected individuals produce broadly neutralizing antibodies (bNAbs), which can recognize and neutralize a variety of different HIV strains. Currently, however, there is still no potent vaccine capable of inducing broadly neutralizing antibodies, failing to meet the needs for HIV prevention and treatment.

[0003] Therefore, there is an urgent need to develop a method for generating broad-spectrum neutralizing antibodies against HIV to meet the growing demand. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for regulating the production of high-affinity antibodies from B cells.

[0005] This invention is based on the following discoveries of the inventors:

[0006] The high variability of human immunodeficiency virus (HIV) is one of the major obstacles to developing effective vaccines, but the development of vaccines that induce broad-spectrum neutralizing antibodies against HIV still faces bottlenecks. To overcome this problem, the inventors discovered that the HMCES protein plays a promoting role in high-affinity antibodies and broad-spectrum HIV neutralizing antibodies, thereby regulating the production of high-affinity antibodies on B cells.

[0007] In a first aspect, the present invention provides a method for regulating the production of high-affinity antibodies on B cells. According to embodiments of the invention, the method includes: regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in germinal centers. The method according to embodiments of the invention can effectively regulate the production of high-affinity antibodies on B cells.

[0008] In a second aspect, the present invention provides a method for regulating high-frequency somatic mutations in B cells. According to embodiments of the present invention, the method includes: regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in germinal centers. The method according to embodiments of the present invention can effectively regulate high-frequency somatic mutations in B cells.

[0009] In a third aspect, the present invention provides a method for regulating AID-induced DNA mutations in B cells. According to embodiments of the present invention, the method includes: regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in germinal centers. The method according to embodiments of the present invention can effectively regulate AID-induced DNA mutations in B cells.

[0010] In a fourth aspect, the present invention provides a method for predicting the level of high-affinity antibodies on B cells. According to embodiments of the invention, the method includes: measuring the expression level of HMCES protein or its uracil DNA glycosidase activity in B cells. The method according to embodiments of the invention can effectively predict the amount of high-affinity antibodies produced on B cells.

[0011] In a fifth aspect of the invention, the invention provides the use of a reagent in the preparation of a kit for regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein; the kit for regulating the production of high-affinity antibodies on B cells or regulating high-frequency mutations in B cell somatic cells or regulating AID-induced DNA mutations in B cells.

[0012] In a sixth aspect, the present invention provides a method for modulating the immune response to pathogens. According to embodiments of the invention, the method includes the step of regulating the expression level of HMCES protein; or the step of regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within B cells located in germinal centers. The method according to embodiments of the invention is capable of modulating the immune response to pathogens.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0014] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0015] Figure 1 shows the immunofluorescence staining results of mouse spleen sections after immunization with sheep red blood cells in Example 1.

[0016] Figure 2 shows the results of HMCES specific expression in germinal center dark zone B cells in Example 1.

[0017] Figure 3 is a graph and statistical diagram showing the results of HMCES specific expression in germinal center dark zone B cells in Example 1.

[0018] Figure 4 shows the results of gene editing in Example 1 that resulted in the disruption of the Hha1 cleavage site.

[0019] Figure 5 is a schematic diagram of HMCES protein expression after gene editing in Example 1.

[0020] Figure 6 is a schematic diagram of the amino acid sequence after a 2bp deletion of the HMCES CDS region in Example 1.

[0021] Figure 7 shows Hmces in Example 1. - / - Image showing the successful knockout of HMCES in mouse germinal center B cells.

[0022] Figure 8 shows the effect of NP-CGG on Hmces in Example 1. - / - A flowchart showing the antibody production in immune detection of wild-type mice.

[0023] Figure 9 shows Hmces in Example 1. - / - The result of severely impaired production of high-affinity antibodies in mice.

[0024] Figure 10 shows the effect of NP-CGG on Hmces in Example 1. - / - A flowchart for detecting dominant W33L mutations after immunization with wild-type mice.

[0025] Figure 11 shows the results of a significant reduction in the frequency of dominant W33L mutations in the case of HMCES deletion in Example 1.

[0026] Figure 12 shows the results of a significant reduction in the point mutation frequency in the 5'Sμ and 3'JH4 regions of the target sites in Example 1.

[0027] Figure 13 shows the result in Example 1 where the point mutation spectrum did not change significantly.

[0028] Figure 14 shows the insertion or deletion mutation frequencies in the 5'Sμ and 3'JH4 regions of Hmces in Example 1. - / - The results showed a significant increase in mice.

[0029] Figure 15 shows the results of HMCES expression levels in peripheral blood mononuclear cells of HIV patients who produced / did not produce broad-spectrum neutralizing antibodies in Example 2.

[0030] Figure 16 shows the effect of eOD-GT8 60mer immunogen on VRC01 in Example 2. KI / KI A flowchart of the immunization process in mice.

[0031] Figure 17 shows the HMCES knockout and control VRC01 in Example 2. KI / KI The results showed no significant difference between the total eOD-GT8 specific IgG level and the proportion of GC B cells in mice.

[0032] Figure 18 shows the results of a significant reduction in the proportion of VRC01 class bNAbs in Example 2 when HMCES is missing.

[0033] Figure 19 shows Hmces in Example 2. - / - VRC01 KI / KI The result shows a significant reduction in the overall point mutation frequency in mice.

[0034] Figure 20 shows the results of a significant reduction in various DNA mutation types under the HMCES deletion condition in Example 2.

[0035] Figure 21 shows the results of similar mutation type distribution in the two groups of mice in Example 2.

[0036] Figure 22 shows the results of a significant decrease in the mutation frequency of the CDR3 region in HMCES-deficient mice in Example 2.

[0037] Figure 23 is a graph showing the results of SRAP hydrolysis of the dU-containing substrate in Example 3.

[0038] Figure 24 is an LC-MS / MS analysis result of uracil, a dU-containing substrate, hydrolyzed by SRAP in Example 3.

[0039] Figure 25 shows the results of uracil as the target of the enzyme activity reaction in Example 3.

[0040] Figure 26 shows the enzyme activity results after the nucleophilic group Cys2 in the SRAP catalytic triplet in Example 3 was mutated.

[0041] Figure 27 shows the results of SRAP exhibiting specific uracil DNA glycosidase activity at a nanomolar concentration (10 nM) in Example 3.

[0042] Figure 28 shows the results of a significant decrease in enzyme activity in the full-length HMCES C2A and C2S mutants in Example 3.

[0043] Figure 29 shows the results of SRAP recognition and cleavage of the VRC01 sequence containing dU in Example 3.

[0044] Figure 30 shows the results of the AP endonuclease APE1 cutting the DNA product generated by SRAP in Example 3.

[0045] Figure 31 shows the results of high-resolution mass spectrometry analysis of the substrate before and after the enzyme-catalyzed reaction in Example 3.

[0046] Figure 32 is a schematic diagram of Example 4 using dCas9-hAIDx to simulate the function of HMCES in AID-induced mutations.

[0047] Figure 33 shows the results of overexpression of HMCES-wild type in Example 4, which significantly increased the frequency of point mutations in the dCas9-hAIDx target region.

[0048] Figure 34 shows the results in Example 4 where neither the catalytic site defective mutant (mut1) nor the DNA binding defective mutant (mut2) increased the frequency of point mutations. Detailed Implementation

[0049] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0050] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0051] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0052] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0053] In this invention, the term "germinal centers (GCs)" refers to dynamic lymphoid tissue that develops in response to antigen stimulation and microbial infection. These centers are the sites of B cell somatic hypermutation (SHM) and antibody affinity selection, processes crucial for the production of high-affinity antibodies. The germinal centers are divided into two distinct regions: a dark zone (DZ) and a light zone (LZ). The dark zone primarily hosts rapid B cell proliferation and SHM, while the light zone is the site of B cell affinity selection.

[0054] This invention proposes a method for regulating the production of high-affinity antibodies on B cells, a method for regulating high-frequency mutations in B cell somatic cells, a pharmaceutical composition, and related uses, which will be described in detail below.

[0055] Methods to regulate the production of high-affinity antibodies from B cells

[0056] In a first aspect, the present invention provides a method for regulating the production of high-affinity antibodies on B cells. According to embodiments of the invention, the method includes: regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in germinal centers. The method according to embodiments of the invention can effectively regulate the production of high-affinity antibodies on B cells.

[0057] According to an embodiment of the present invention, the uracil DNA glycosidase activity of the HMCES protein is achieved by the specific binding of the SRAP domain of the HMCES protein to a DNA substrate containing uracil.

[0058] According to embodiments of the present invention, the high-affinity antibody is an antigen-specific high-affinity antibody, including but not limited to at least one of HIV-1 broad-spectrum neutralizing antibody, COVID-19 broad-spectrum neutralizing antibody, HBV broad-spectrum neutralizing antibody, HCV broad-spectrum neutralizing antibody, HER2 broad-spectrum neutralizing antibody, EGFR broad-spectrum neutralizing antibody and PD-1 broad-spectrum neutralizing antibody.

[0059] Methods to regulate high-frequency mutations in B cells

[0060] In a second aspect, the present invention provides a method for regulating high-frequency somatic mutations in B cells. According to embodiments of the present invention, the method includes: regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in germinal centers. The method according to embodiments of the present invention can effectively regulate high-frequency somatic mutations in B cells.

[0061] According to an embodiment of the present invention, the uracil DNA glycosidase activity of the HMCES protein is achieved by the specific binding of the SRAP domain of the HMCES protein to a DNA substrate containing uracil.

[0062] Methods for regulating AID-induced DNA mutations in B cells

[0063] In a third aspect, the present invention provides a method for regulating AID-induced DNA mutations in B cells. According to embodiments of the present invention, the method includes: regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in germinal centers. The method according to embodiments of the present invention can effectively regulate AID-induced DNA mutations in B cells.

[0064] According to an embodiment of the present invention, the uracil DNA glycosidase activity of the HMCES protein is achieved by the specific binding of the SRAP domain of the HMCES protein to a DNA substrate containing uracil.

[0065] Methods for predicting B cell high-affinity antibody levels

[0066] In a fourth aspect, the present invention provides a method for predicting the level of high-affinity antibodies on B cells. According to embodiments of the invention, the method includes: measuring the expression level of HMCES protein or its uracil DNA glycosidase activity in B cells. The method according to embodiments of the invention can effectively predict the amount of high-affinity antibodies produced on B cells.

[0067] Use of reagents in the preparation of reagent kits

[0068] In a fifth aspect of the invention, the invention provides the use of a reagent in the preparation of a kit for regulating the expression level of HMCES protein; or regulating the uracil DNA glycosidase activity of HMCES protein; the kit for regulating the production of high-affinity antibodies on B cells or regulating high-frequency mutations in B cell somatic cells or regulating AID-induced DNA mutations in B cells.

[0069] According to embodiments of the present invention, the reagent is used to promote the expression level of HMCES protein; or to promote the uracil DNA glycosidase activity of HMCES protein; the kit is used to promote the production of high-affinity antibodies on B cells; or to promote high-frequency mutations in B cell somatic cells or to promote AID-induced DNA mutations in B cells.

[0070] According to embodiments of the present invention, the reagent is used to inhibit the expression level of HMCES protein; or to inhibit the uracil DNA glycosidase activity of HMCES protein; the kit is used to inhibit the production of high-affinity antibodies on B cells; or to inhibit high-frequency mutations in B cell somatic cells or to inhibit AID-induced DNA mutations in B cells.

[0071] According to an embodiment of the present invention, the reagent includes triptolide.

[0072] According to embodiments of the present invention, the reagent comprises siRNA or protein factor for inhibiting the expression of HMCES protein.

[0073] According to an embodiment of the present invention, the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1.

[0074] The sequence of SEQ ID NO:1 is as follows: ACTCATCTGAGCGTATCATTG.

[0075] Methods to modulate the immune response to pathogens

[0076] In a sixth aspect, the present invention provides a method for modulating the immune response to pathogens. According to embodiments of the invention, the method includes the step of regulating the expression level of HMCES protein; or the step of regulating the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within B cells located in germinal centers. The method according to embodiments of the invention is capable of modulating the immune response to pathogens.

[0077] According to an embodiment of the present invention, the method includes the step of promoting the expression level of HMCES protein; or the step of promoting the uracil DNA glycosidase activity of HMCES protein, wherein the HMCES protein is located within the B cells, and the B cells are located in the germinal center.

[0078] According to embodiments of the present invention, the promotion of HMCES protein activity is achieved through small molecule activators, protein modification, and targeted inhibition of protein degradation.

[0079] According to embodiments of the present invention, the promotion of HMCES protein expression is achieved through viral / non-viral vector-mediated gene / mRNA transfection, transposase or recombinase-mediated gene integration, and CRISPR / Cas system-based gene editing or transcriptional activation.

[0080] According to embodiments of the present invention, the promotion of HMCES protein expression is achieved through viral / non-viral vector-mediated gene / mRNA transfection, transposase or recombinase-mediated gene integration, and CRISPR / Cas system-based gene editing or transcriptional activation.

[0081] According to embodiments of the present invention, the protein modification includes at least one of phosphorylation and ubiquitination.

[0082] According to embodiments of the present invention, the gene / mRNA transfection technology is achieved by utilizing at least one of the following methods: liposome or LNP-mediated transfection, calcium phosphate-mediated transfection, cationic transfection reagent-mediated transfection, virus-like particle (VLP)-mediated transfection, electroporation transfection, gene delivery via lentiviral vector, gene delivery via adeno-associated virus (AAV) vector, gene delivery via adenovirus vector, and gene delivery via Sendai virus vector.

[0083] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0084] Example 1:

[0085] To investigate the expression pattern of HMCES in B cells of germinal centers (GCs), this study immunized mice with sheep red blood cells (SRBCs) to induce GC formation in the spleen, and analyzed the results on day 7. Immunostaining of spleen sections showed that HMCES was induced to express after immunization, and in B220 cells... hi GL7 hi HMCES was significantly highly expressed in germinal center B cells, exhibiting an aggregation pattern compared to unimmunized spleen sections (see Figure 1). Furthermore, immunostaining of immunized spleen sections using the follicular dendritic cell marker CD35 as a bright area revealed that HMCES was specifically expressed in germinal center dark zone B cells (see Figures 2 and 3). Therefore, HMCES can respond to antigen stimulation and is highly expressed in germinal center dark zone B cells after immunization.

[0086] Germinal centers are the site of clonal expansion and seromas (SHM) in B cells, which is crucial for antibody diversity. The polar expression of HMCES in B cells in the dark zone of germinal centers suggests that HMCES plays a role in SHM and / or clonal expansion, thereby promoting the production of high-affinity antibodies.

[0087] In this embodiment, CRISPR / Cas9 technology was used to construct HMCES-deficient mice. Gene editing resulted in the disruption of the Hha1 cleavage site (see Figures 4 and 5) and a 2bp deletion in the HMCES CDS region (see Figure 6). Immunofluorescence and immunoblotting results showed that, compared with Hmces... + / + Compared to germinal center B cells, Hmces - / - HMCES was not detected in B cells of the germinal center, indicating that the knockout was successful, as shown in Figure 7.

[0088] Subsequently, in this embodiment, the hapten 4-hydroxy-3-nitrophenylacetyl (NP) conjugated with chicken gamma-globulin (NP-CGG) was used to target Hmces. - / - Immunization was performed on wild-type mice, and antibody production was detected (see Figure 8 for details). Hmces - / - Serum levels of anti-NP high-affinity (NP30) IgG antibodies in mice were comparable to those in wild-type mice. However, levels of anti-NP high-affinity (NP3) IgG antibodies were significantly reduced in HMCES deficiency, indicating that HMCES deficiency was associated with this deficiency. - / - The production of high-affinity antibodies in mice was severely impaired, as shown in Figure 9. The W33L amino acid substitution in the mouse VH186.2 gene induced by SHM represents high affinity for NP binding. In this embodiment, germinal center B cells from immunized mice were sorted, the VH186.2 gene was amplified, and sequencing was used to assess the frequency of this mutation, as shown in Figure 10. In the presence of HMCES deletion, the frequency of the dominant W33L mutation was significantly reduced, as shown in Figure 11. These results indicate that HMCES deletion impairs the production of high-affinity antibodies by affecting SHM.

[0089] In HMCES deficiency, the frequency of point mutations in the 5'Sμ and 3'JH4 regions, the target sites for activation-induced cytidine deaminase (AID), was significantly reduced in germinal center B cells (see Figure 12), decreasing by 29% and 33%, respectively. The mutation spectrum did not change significantly (see Figure 13). However, the insertion or deletion mutation frequencies in the 5'Sμ and 3'JH4 regions remained relatively stable in HMCES deficiency.- / - The levels were significantly increased in mice (see Figure 14). These results collectively indicate that HMCES plays a regulatory role in SHM. This suggests that HMCES promotes the production of high-affinity antibodies by facilitating the accumulation of point mutations during humoral immune responses.

[0090] Example 2:

[0091] The hallmark of most broad-spectrum HIV neutralizing antibodies is an abnormally high level of SHM (shearing motility), which is directly related to their ability to neutralize multiple strains. Given the role of HMCES in the production of high-affinity antibodies and the SHM process, HMCES may contribute to the production of broad-spectrum neutralizing antibodies.

[0092] To verify this hypothesis, this embodiment compared the expression levels of HMCES between HIV-infected individuals who produced broad-spectrum neutralizing antibodies and those who did not. The expression of HMCES in patients who produced broad-spectrum neutralizing antibodies was significantly higher than that in patients who did not produce broad-spectrum neutralizing antibodies (see Figure 15). The expression of HMCES was positively correlated with the production of broad-spectrum neutralizing antibodies, suggesting that HMCES may play a role in the production of broad-spectrum neutralizing antibodies.

[0093] VRC01-type broad-spectrum neutralizing antibodies recognize the CD4 binding site (CD4bs) epitope of HIV envelope glycoproteins. The VRC01 germline heavy chain (gH) knock-in mouse is a humanized mouse model used to study VRC01-type bNAbs and HIV vaccine detection. To investigate the role of HMCES in HIV bNAb production, this example examined the immune response of HMCES-deficient humanized VRC01 gH knock-in mice. HMCES... - / - Mice and VRC01 KI / KI Mouse hybridization yields Hmces - / - VRC01 KI / KI and Hmces + / + VRC01 KI / KI Mice were immunized with the eOD-GT860mer immunogen (see Figure 16). Serum levels of antibodies binding to eOD-GT8 or eOD-GT8-KO (designed to block CD4bs binding) were measured on day 17 post-immunization. Results showed no significant difference in total eOD-GT8-specific IgG levels and GC B cell proportions between HMCES knockout mice and wild-type mice under the VRC01 gH knock-in background (see Figure 17). However, the proportion of VRC01-like bNAbs was significantly reduced in the absence of HMCES (see Figure 18). Therefore, these findings suggest that the production of broad-spectrum HIV neutralizing antibodies requires the participation of HMCES.

[0094] Next, to further investigate the role of HMCES in the generation of bNAbs, this embodiment isolates Hmces. - / - VRC01 KI / KI and Hmces + / + VRC01 KI / KI Mouse GC B cells were used, and the VRC01 region was amplified and sequenced, followed by mutation analysis. (Compared with Hmces) + / + VRC01 KI / KI Compared to mice, Hmces - / - VRC01 KI / KI The overall point mutation frequency in mice was significantly reduced (see Figure 19). Furthermore, all DNA mutation types were significantly reduced under HMCES deletion conditions (see Figure 20), although the mutation type distribution was similar in both groups of mice (see Figure 21). In addition, the mutation frequency in the CDR3 region of HMCES-deficient mice also showed a significant decrease (see Figure 22). The significant reduction in mutation frequency under HMCES deletion corresponds to the reduction in VRC01-like bNAbs, indicating that HMCES promotes bNAb production by enhancing SHM.

[0095] Example 3:

[0096] Next, this embodiment investigates the molecular mechanism by which HMCES promotes SHM. HMCES consists of an N-terminal SRAP domain and a C-terminal unstructured peptide. Comparative analysis of the structures of the SRAP domain (PDB: 5KO9, 1ZN6, 2BDV, 2AEG, 2F20) from different species showed that the SRAP domain is highly conserved across species. The SRAP domain contains a unique helix-hairpin-helix (HhH) motif, an important DNA-binding motif commonly found in DNA repair proteins. In the outer pocket near the HhH motif, there are two highly conserved arginine residues (R98 and R212), which may contribute to stabilizing DNA binding. Furthermore, the SRAP domain also contains a predicted catalytic triplet structure composed of Cys2, Glu127, and His210. Given that catalytic triplet is a key characteristic of hydrolases, HMCES may function in SHM as a DNA-associated hydrolases, such as DNA glycosidases.

[0097] First, in this embodiment, the glycosidase activity of the SRAP domain of human HMCES was detected using single-strand (ss) and double-strand (ds) DNA substrates containing different pyrimidine derivatives (mC or hmC), uracil (dU), or thymine (T). Enzyme activity assays were performed by incubating purified human SRAP domain proteins with various DNA substrates. The results showed that only dU-containing substrates were hydrolyzed by SRAP (see Figure 23). Further, LC-MS / MS analysis of the products after 60 minutes of enzyme activity revealed that uracil was the product of this hydrolysis reaction (see Figure 24). Using ss dU-DNA substrates of various lengths with uracil damage in the middle, products of corresponding lengths were observed, indicating that uracil is the target of the enzyme activity reaction (see Figure 25). Therefore, the SRAP domain of HMCES exhibits specific glycosidase activity towards dU-containing substrates.

[0098] Furthermore, compared to the wild-type protein, mutating the nucleophilic group Cys2 in the SRAP catalytic triplet to alanine (C2A) significantly reduced its enzyme activity, as shown in Figure 26. SRAP also exhibited specific uracil DNA glycosidase activity at nanomolar concentrations (10 nM), as shown in Figure 27. Full-length HMCES purified from HEK 293T cells also showed uracil glycosidase activity, while the C2A and C2S mutants showed significantly reduced enzyme activity, with a decrease exceeding 90%, as shown in Figure 28.

[0099] During the GC reaction, AID-induced uracil damage in B cells initiates both CSR and SHM processes, leading to immunoglobulin diversification. Given the role of HMCES in SHM and HIV bNAbs production, this study sequenced the VRC01 region of VRC01 gH mice immunized with eOD-GT8 60mer, selecting the two highest-ranking SHM sites mutated by dC. By altering dC to dU to mimic AID deamination products, dU-containing ssDNA substrates were synthesized to test whether the SRAP domain of human HMCES could catalyze these substrates. Enzyme activity results showed that the dU-containing VRC01 sequence was recognized and cleaved by SRAP (see Figure 29). This indicates that HMCES functions as a uracil DNA glycosidase in the Ig gene sequence and may participate in SHM through its uracil glycosidase activity.

[0100] A monofunctional DNA glycosidase attacks N-glycosidic bonds, removing damaged nucleobases and generating AP sites while maintaining the integrity of the sugar-phosphate backbone. The AP endonuclease APE1 cleaves the DNA product generated by SRAP (see Figure 30), indicating that HMCES is a monofunctional uracil DNA glycosidase. To identify the enzymatic reaction products in the glycosidic reaction of HMCES with ss dU DNA substrate, high-resolution mass spectrometry was used to analyze the substrate before and after the reaction (see Figure 31). Besides the dU DNA substrate with m / z 3731.64, a new DNA product with m / z 3537.62 was detected after 2 hours, consistent with the predicted theoretical product m / z 3537.62 after N-glycosidic bond hydrolysis (a loss of m / z 94.02 from m / z 3731.64). Enzymatic activity assays were then performed using 1 μM SRAP protein and various concentrations of ss dU-DNA substrate. Compared to the reaction treated with NaOH, the N-glycosidic bond remained intact in the reaction without NaOH treatment. These data indicate that the AP site DNA-HMCES complex is a subsequent step in the HMCES glycosidase reaction, consistent with the enzymatic processes of other DNA glycosidases. These results suggest that SRAP functions as a monofunctional uracil DNA glycosidase.

[0101] Example 4:

[0102] Given HMCES's function as a uracil DNA glycosidase, it is hypothesized that HMCES recognizes uracil damage induced by AID and participates in error-prone DNA repair processes to promote SHM. To verify this hypothesis, this example establishes an in vitro SHM experiment in HEK 293T cells, using dCas9-hAIDx to simulate the function of HMCES in AID-induced mutations, as shown in Figure 32. This experiment uses a fusion protein composed of catalytically inactivated Cas9 (dCas9) and human AID (dCas9-hAIDx). The dCas9-hAIDx system targets a specific site in the GFP reporter gene region via gRNA to perform targeted deamination from dC to dU, effectively simulating the AID-induced mutation process in B cells during SHM under natural conditions.

[0103] Compared to the wild-type control (without HMCES overexpression), overexpression of HMCES-wild-type significantly increased the frequency of point mutations in the dCas9-hAIDx target region (see Figure 33). To determine whether the enzymatic activity or DNA-binding capacity of HMCES plays a role in AID-induced mutations, this embodiment introduced a catalytic site-deficient mutant (mut1) and a DNA-binding-deficient mutant (mut2) into this in vitro system. No increase in point mutation frequency was observed in either case compared to the control (see Figure 34). Therefore, HMCES plays a role in AID-induced mutations through its catalytic and DNA-binding activities.

[0104] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0105] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for regulating the production of high-affinity antibodies from B cells, characterized in that, include: Regulate the expression level of HMCES protein; or Regulates the uracil DNA glycosidase activity of HMCES protein. The HMCES protein is located within the B cells, which are located in the germinal center.

2. A method for regulating high-frequency mutations in B cells, characterized in that, include: Regulate the expression level of HMCES protein; or Regulates the uracil DNA glycosidase activity of HMCES protein. The HMCES protein is located within the B cells, which are located in the germinal center.

3. A method for regulating AID-induced DNA mutations in B cells, characterized in that, include: Regulate the expression level of HMCES protein; or Regulates the uracil DNA glycosidase activity of HMCES protein. The HMCES protein is located within the B cells, which are located in the germinal center.

4. The method according to any one of claims 1 to 3, characterized in that, The uracil DNA glycosidase activity of the HMCES protein is achieved through the specific binding of the SRAP domain of the HMCES protein to DNA substrates containing uracil.

5. The method according to claim 1, characterized in that, The high-affinity antibody is an antigen-specific high-affinity antibody, including at least one of the following: HIV-1 broad-spectrum neutralizing antibody, COVID-19 broad-spectrum neutralizing antibody, HBV broad-spectrum neutralizing antibody, HCV broad-spectrum neutralizing antibody, HER2 broad-spectrum neutralizing antibody, EGFR broad-spectrum neutralizing antibody, and PD-1 broad-spectrum neutralizing antibody.

6. A method for predicting the level of high-affinity antibodies on B cells, characterized in that, This includes measuring the expression level of HMCES protein or its uracil DNA glycosidase activity in B cells.

7. Use of the reagent in the preparation of the kit, wherein the reagent is used to regulate HMCES protein expression levels; or The kit is used to regulate the uracil DNA glycosidase activity of HMCES protein; it is used to regulate the production of high-affinity antibodies on B cells or to regulate high-frequency mutations in B cell somatic cells or to regulate AID-induced DNA mutations in B cells.

8. The use according to claim 7, characterized in that, The reagent is used to promote HMCES protein expression; or to promote HMCES protein uracil DNA glycosidase activity; the kit is used to promote the production of high-affinity antibodies in B cells; or to promote high-frequency mutations in B cell somatic cells or to promote AID-induced DNA mutations in B cells.

9. The use according to claim 7, characterized in that, The reagent is used to inhibit the expression of HMCES protein; or to inhibit the uracil DNA glycosidase activity of HMCES protein; the kit is used to inhibit the production of high-affinity antibodies on B cells; or to inhibit high-frequency mutations in B cell somatic cells or to inhibit AID-induced DNA mutations in B cells.

10. The use according to claim 7, characterized in that, The reagents include triptolide.

11. The use according to claim 9, characterized in that, The reagents include siRNA or protein factors used to inhibit the expression of HMCES protein.

12. The use according to claim 11, characterized in that, The nucleotide sequence of the siRNA is shown in SEQ ID NO:

1.

13. A method for modulating the immune response to a pathogen, characterized in that, This includes steps that regulate the expression level of HMCES protein; or Steps to regulate the uracil DNA glycosidase activity of HMCES protein. The HMCES protein is located within the B cells, which are located in the germinal center.

14. The method according to claim 13, characterized in that, Including steps that promote HMCES protein expression; or Steps to promote the uracil DNA glycosidase activity of HMCES protein. The HMCES protein is located within the B cells, which are located in the germinal center.

15. The method according to claim 14, characterized in that, The promotion of HMCES protein activity is achieved in the following manner: Small molecule activators, protein modification, and targeted inhibition of protein degradation.

16. The method according to claim 14, characterized in that, The promotion of HMCES protein expression is achieved in the following manner: Viral / non-viral vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / Cas-based gene editing or transcriptional activation.

17. The method according to claim 15, characterized in that, The protein modification includes at least one of phosphorylation and ubiquitination.

18. The method according to claim 16, characterized in that, The gene / mRNA transfection technology is achieved by using at least one of the following methods: liposome or LNP-mediated transfection, calcium phosphate-mediated transfection, cationic transfection reagent-mediated transfection, virus-like particle (VLP)-mediated transfection, electroporation transfection, gene delivery via lentiviral vector, gene delivery via adeno-associated virus (AAV) vector, gene delivery via adenovirus vector, and gene delivery via Sendai virus vector.

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