New bunyavirus neutralizing antibody screening method based on single cell BCR sequencing

Abstract

The invention provides a new bunyavirus neutralizing antibody screening method based on single cell BCR sequencing. The new bunyavirus neutralizing antibody screening method comprises the following steps: S1, collecting peripheral blood B cells of acute stage and recovery stage patients and healthy volunteers of new bunyavirus infected persons; s2, carrying out CD19 < + > CD38 < + > CD138 < + > magnetic bead separation to obtain a single-cell suspension; s3, constructing a single cell transcriptome and a BCR library, separating single cells by using a microfluidic system, and performing high-throughput sequencing; s4, analyzing a B cell subset, a BCR cloning amplification mode and a VJ gene pair enrichment characteristic through bioinformatics analysis; and S5, screening a specific BCR clone type related to the severity of the disease as a neutralizing antibody candidate target. The method provided by the invention provides powerful support for immune monitoring and vaccine design of new bunyavirus infection, and is expected to provide new strategies and methods for clinical treatment and prevention.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for screening neutralizing antibodies against novel bunyavirus based on single-cell BCR sequencing. Background Art

[0002] Infection with novel bunyavirus (SFTSV) can lead to severe immune system disorders and high case fatality rates. Currently, the research on the immune response against SFTSV mainly relies on flow cytometry (FACS) and ELISA to detect B cell populations and antibody levels. However, these methods can only provide population-level data and cannot analyze the BCR clonal expansion, affinity maturation, and somatic hypermutation (SHM) of individual B cells. Although existing high-throughput sequencing technologies (such as bulk RNA-seq) can obtain BCR sequences, they lack single-cell resolution and are difficult to distinguish the immune characteristics of different B cell subsets. Therefore, there is an urgent need for a BCR analysis method based on single-cell sequencing to accurately analyze the immune response characteristics of SFTSV-infected individuals and provide technical support for the discovery of neutralizing antibodies.

[0003] Based on this, the present invention provides a method for screening neutralizing antibodies against novel bunyavirus based on single-cell BCR sequencing. Summary of the Invention

[0004] The present invention aims to analyze the dynamic B cell immune response of SFTSV-infected individuals through single-cell sequencing technology combined with BCR clonal analysis, discover characteristic BCR clonal patterns and potential neutralizing antibody targets related to disease severity, and provide a basis for vaccine design, antibody drug development, and clinical monitoring.

[0005] On the one hand, the present invention provides a method for screening neutralizing antibodies against novel bunyavirus based on single-cell BCR sequencing, and the steps include sample collection, single-cell library construction, sequencing, bioinformatics analysis, and screening.

[0006] 1. Further, the steps include:

[0007] S1. Collect peripheral blood B cells from acute-phase and convalescent-phase patients infected with novel bunyavirus and healthy volunteers.

[0008] S2. Obtain a single-cell suspension through magnetic bead sorting of CD19+CD38+CD138+.

[0009] S3. Construct a single-cell transcriptome and BCR library, and use a microfluidic system to isolate single cells and perform high-throughput sequencing.

[0010] S4. Analyze B cell subsets, BCR clonal expansion patterns, and VJ gene pair enrichment characteristics through bioinformatics analysis.

[0011] S5. Screen specific BCR clonotypes related to disease severity as candidate neutralizing antibody targets.

[0012] Furthermore, the acute phase of the infected individuals is the period when the nucleic acid of the virus infection is positive; the convalescent patients are those whose nucleic acid has turned negative for more than one week; the healthy volunteers are those without a history of new bunyavirus infection and without other underlying diseases.

[0013] Furthermore, the sequencing uses the Illumina HiseqX / Nova sequencing platform, the sequencing strategy is PE150, and the sequencing data volume of each transcriptome library is not less than 90G.

[0014] Furthermore, the library construction method is as follows: having a Singleron library construction system, separating single cells based on the principle of microfluidic micropore technology and capturing and labeling mRNA with magnetic beads with Barcode and UMI; using in combination single-cell transcriptome library construction reagents and single-cell immune receptor kits to complete the construction of single-cell transcriptome libraries and BCR libraries.

[0015] Furthermore, during the library construction process, the samples are treated with tissue preservation solution and dissociation solution, the cell viability is ≥85%, and the concentration is ≥1×10^5 cells / mL.

[0016] Furthermore, the bioinformatics analysis content includes quality control, alignment, and quantification of the sequencing data to obtain a single-cell expression profile matrix and remove cells with substandard sequencing quality.

[0017] Furthermore, the bioinformatics analysis content also includes integrating the single-cell expression profile data of multiple samples across batches and removing batch differences caused by technical noise.

[0018] Furthermore, the bioinformatics analysis content also includes at least one of marker gene analysis, trajectory analysis, cell communication analysis, functional enrichment analysis, TF coding ability prediction, cell cycle analysis, and protein interaction analysis.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention realizes a high-precision analysis of the B cell immune response of SFTSV-infected individuals through single-cell sequencing technology, overcoming the limitation of the prior art that can only provide population-level data. This method can not only subdivide B cell subsets but also reveal the dynamic changes of B cells in the acute phase, convalescent phase, and healthy population, providing an important basis for understanding the disease process;

[0021] The present invention has made remarkable progress in BCR clonal analysis, capable of resolving clonal spectra at single-cell resolution and identifying BCR clonal amplification patterns related to disease severity. In particular, the enrichment of specific VJ gene pairs (IGLV2-14_IGLJ1, IGLV2-14_IGLJ2) in severe SFTSV patients has been discovered, providing candidate targets for the development of SFTSV-specific antibodies and greatly advancing the development process of antibody drugs.

[0022] Through cell differentiation trajectory analysis and the study of the dynamic changes of antiviral genes in B cells, the present invention further reveals the mechanism of action of B cells in antiviral immune responses. These findings not only have important scientific research value but also provide strong support for the immune monitoring and vaccine design of novel bunyaviruses, and are expected to provide new strategies and methods for clinical treatment and prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram of cell integration annotation results. Among them, (A) is a UMAP diagram colored by cell type for clustering; (B) is a UMAP diagram colored by sample for clustering; (C) is a UMAP diagram colored by sample group for clustering; (D) is a diagram showing the proportion of cells by sample; (E) is a diagram showing the proportion of cells by group;

[0024] Figure 2 It is a diagram of B cell proportion analysis. Among them, (A) is a diagram showing the proportion of B cell subsets by sample; (B) is a diagram showing the proportion of B cell subsets by sample group;

[0025] Figure 3 It is a diagram of differential gene analysis between B cell groups. Among them, (A) is a diagram of differential gene analysis between group A and group H; (B) is a diagram of differential gene analysis between group C and group H; (C) is a diagram of differential gene analysis between group A and group C;

[0026] Figure 4 It is a diagram of B cell BCR clonal amplification analysis;

[0027] Figure 5 It is a diagram of the usage frequency analysis of VDJ genes in B cells of severe and mild cases during the acute phase;

[0028] Figure 6 It is a diagram of B cell pseudotime differentiation trajectory analysis. DETAILED DESCRIPTION OF THE INVENTION

[0029] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example

[0031] In this example, a method for screening neutralizing antibodies against novel bunyavirus based on single-cell BCR sequencing is provided. The steps include:

[0032] S1. Collect peripheral blood B cells from patients in the acute and convalescent phases of novel bunyavirus infection and healthy volunteers;

[0033] Among them,

[0034] Group A in the acute phase: 6 patients with positive nucleic acid for virus infection and whose nucleic acid has not turned negative;

[0035] Group C in the convalescent phase: 3 patients whose nucleic acid has turned negative for more than one week;

[0036] Healthy control group H: 3 healthy people without a history of novel bunyavirus infection and without other underlying diseases;

[0037] S2. Each subject collects 20 mL of peripheral blood, and performs flow cytometry sorting through CD19+CD38+CD138+ magnetic beads to obtain more than 10^6 B cells per case;

[0038] Among them, the processing and quality requirements of single-cell samples are: provide tissue preservation solution to ensure the stability of the sample after leaving the body and use it for the subsequent preparation of single-cell suspension; use tissue dissociation solution to prepare single-cell suspension for the sample. The dissociation process does not exceed 90 minutes, and the dissociation activity requirement is ≥85%, and the concentration is ≥1×10^5 cells / mL; use a cell counter and a microscope to perform quality inspection on the sample, and use the fluorescence technology principle to simultaneously detect important indicators such as cell concentration, cell activity, aggregation rate, cell diameter distribution, and whether there is red blood cell contamination to ensure meeting the library construction requirements;

[0039] S3. Construct a single-cell transcriptome and BCR library, and use a microfluidic system to isolate single cells and perform high-throughput sequencing;

[0040] Among them, the library construction method is:

[0041] Equipped with Singleron library construction system, based on the principle of microfluidic micropore technology, complete the separation of single cells and capture and label mRNA through magnetic beads with Barcode and UMI; used in combination with single-cell transcriptome library construction reagents and Single-cell immune receptor (TCR) kit, which can complete the construction of single-cell transcriptome libraries and BCR libraries, provide library construction training, and meet the library construction requirements of different samples; use an Agilent nucleic acid fragment analyzer to detect the quality of the constructed libraries, perform subsequent on-machine sequencing on the libraries that meet the standards, and for samples that meet the qualified standards (activity ≥ 85%, total cell count ≥ 1 * 105 / cell / mL), the target captured cell count is not less than 6,000 cells / sample;

[0042] Among them, the sequencing method is: use the Illumina HiseqX / Nova sequencing platform for sequencing, and the sequencing strategy is PE150;

[0043] Sequencing quality requirements: Q20 > 85%, Q30 > 80%, and the base type distribution is uniform; adopt strict data control standards to ensure data quality, and the sequencing data volume of each transcriptome library is not less than 90G on average.

[0044] S4. Analyze B cell subsets, BCR clonal expansion patterns, and VJ gene pair enrichment characteristics through bioinformatics analysis;

[0045] Among them, the bioinformatics analysis content includes:

[0046] 1) Perform quality control, alignment, and quantification on the sequencing data to obtain a single-cell expression profile matrix, and remove cells with substandard sequencing quality;

[0047] 2) Integrate the single-cell expression profile data of multiple samples across batches and remove batch differences caused by technical noise;

[0048] 3) Cluster and dimensionality reduction of single-cell expression profile data: generate two-dimensional displays of various algorithms such as t-SNE and UMAP of the data;

[0049] 4) Marker gene analysis: display the marker genes of each cluster in different forms, such as ridge plots, heatmaps, violin plots, and bubble plots;

[0050] 5) Annotate the cell type and function of the clustering results, or perform supervised cell type identification;

[0051] 6) Perform differential expression analysis, characteristic gene identification, and enrichment analysis of characteristic gene sets on cell functional subsets;

[0052] 7) Trajectory analysis: construct a cell pseudotime trajectory, perform differential gene analysis based on the differentiation state, perform differential gene analysis based on the pseudotime change, and perform differential gene analysis based on the trajectory branches;

[0053] 8) Cell communication analysis: analyze the interaction between cells;

[0054] 9) Functional enrichment analysis: Perform GO and KEGG functional enrichment analysis on the differentially expressed genes between different cell populations of the same sample and the differentially expressed genes in the inter-group comparison;

[0055] 10) TF coding ability prediction: Identify the TF coding ability of differentially expressed genes;

[0056] 11) Cell cycle analysis: Infer the cell cycle; Determine the proportion and distribution characteristics of cells in different cell cycles;

[0057] 12) Protein-protein interaction analysis: Analyze the interaction relationships between the encoded proteins of differentially expressed genes;

[0058] 13) Based on the heavy and light chain pairing of single-cell BCR data, conduct immunome research, such as the diversity and divergence of BCR, and calculate various indicators;

[0059] 14) According to the matched single-cell transcriptome and BCR data provided by the researcher, display the proportion of each B cell subtype's clonotype, the gene frequencies of V, D, and J, and the clonotypes shared by different cell types in the sample;

[0060] S5. Screen specific BCR clonotypes related to disease severity as candidate neutralizing antibody targets.

[0061] Based on the above steps, the following results were obtained Figure 1-6 . Among them, Figure 1 The results showed that the composition of B cell subsets changed during the acute phase: the proportion of plasma cells increased significantly. Figure 2 The results showed that group A had fewer memory B cells and more Naive B cells compared to groups H and C. Group C had more memory B cells and fewer Naive B cells than group H. Figure 3 The results showed that group A upregulated genes such as CXCR4 and IFI44L compared to groups C and H, and upregulated the antiviral-related pathways. Figure 4 The results showed that the plasma cell BCR of group A (including severe A-S and mild A-M) during the acute phase showed obvious clonal expansion. Figure 5 The results showed that in group A_S compared to group A_M, the VJ gene pairs IGLV2-14_IGLJ1 and IGLV2-14_IGLJ2 were significantly more used, which might be the VJ pairs preferentially used in disease severity. Figure 6 The results showed that the antiviral gene ISG15 decreased in expression level along the PB-PC differentiation trajectory, suggesting that the PB subset might mainly play an antiviral function.

[0062] Therefore, the results showed changes in the composition of B cell subsets during the acute phase, a significant increase in the proportion of plasma cells, and upregulation of antiviral genes such as CXCR4 and IFI44L; a decrease in BCR clonal diversity, with significant clonal expansion in the acute phase (Group A), especially in severe patients (Group A_S); enrichment of specific BCR clonotypes, and it was found that IGLV2-14_IGLJ1 and IGLV2-14_IGLJ2 were highly enriched in severe patients; changes in antiviral gene expression, with higher expression levels of genes such as ISG15 in the PB (plasmablast) subset, suggesting that it may be the main antiviral effector cell.

[0063] Based on the above steps, this embodiment provides a method for screening neutralizing antibodies against novel bunyavirus based on single-cell BCR sequencing.

[0064] For the points not fully exhausted in the technical scope claimed by the present invention in this embodiment, as well as the new technical solutions formed by the equivalent replacement of single or multiple technical features in the technical solutions of the embodiment, they are also within the scope claimed by the present invention: at the same time, in all the examples listed or not listed in the present invention, each parameter in the same example only represents an example of its technical solution (i.e., a feasible solution), and there is no strict cooperation and limitation relationship between the parameters. Among them, each parameter can be replaced with each other without violating the axiom and the requirements of the present invention, unless otherwise specifically stated.

[0065] The technical means disclosed in the present invention are not limited to the technical means disclosed above, but also include the technical solutions formed by any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention should be covered by the scope of the claims of the present invention.

Claims

1. A new method for screening neutralizing antibodies against bunyavirus based on single-cell BCR sequencing, characterized in that: The steps include sample collection, single-cell library construction, sequencing, bioinformatics analysis and screening.

2. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, characterized in that the steps include: S1. Collect peripheral blood B cells from patients with new bunyavirus infection in the acute and recovery phases and healthy volunteers; S2, obtain single cell suspension by CD19+CD38+CD138+ magnetic bead sorting; S3, construct single-cell transcriptome and BCR library, separate single cells using microfluidics system and perform high-throughput sequencing; S4. Analyze B cell subsets, BCR clone expansion patterns and VJ gene pair enrichment characteristics through bioinformatics analysis; S5. Screen specific BCR clonotypes associated with disease severity as candidate targets for neutralizing antibodies.

3. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, characterized in that: The acute stage of the infected person is the period when the virus infection nucleic acid is positive; the recovery period patients are patients whose nucleic acid turns negative for more than one week; the healthy volunteers are volunteers with no history of new brexit virus infection and no other underlying diseases.

4. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, characterized in that: The sequencing was performed using the Illumina HiseqX / Nova sequencing platform, the sequencing strategy was PE150, and the sequencing data volume of each transcriptome library was not less than 90G.

5. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, characterized in that: The library construction method is: with Singleron The library construction system is based on the principle of microfluidic micropore technology to complete single cell separation and capture and label mRNA through magnetic beads with barcodes and UMIs; Use with Single cell transcriptome library reagents and Single cell immune receptor kit, complete the construction of single cell transcriptome library and BCR library.

6. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 5, characterized in that: During the library construction process, the For samples treated with tissue preservation solution and dissociation solution, cell activity should be ≥85% and concentration should be ≥1×10^5cells / mL.

7. According to a new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, the bioinformatics analysis includes quality control, comparison and quantification of sequencing data, obtaining a single-cell expression spectrum matrix, and removing cells with substandard sequencing quality.

8. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, characterized in that: The bioinformatics analysis also includes batch integration of single-cell expression profile data of multiple samples and removal of batch differences caused by technical noise.

9. A new bunyavirus neutralizing antibody screening method based on single-cell BCR sequencing according to claim 1, characterized in that: The bioinformatics analysis content also includes at least one of marker gene analysis, trajectory analysis, cell communication analysis, functional enrichment analysis, TF coding ability prediction, cell cycle analysis and protein interaction analysis.

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