Back to results
US 2025/0102520 A1Published application
Simultaneous detection of antigens and antigen specific antibodies in a guinea pig model system
AssigneeVanderbilt University
InventorsIvelin Georgiev, Matthew Vukovich
Life of this patent
Estimating the term…
Abstract
Disclosed herein are methods of determining broadly neutralizing antibodies, antigen specific cell sorting and developing vaccine compositions in a guinea pig model system using LIBRA-seq.
Claims20
A method of determining broadly neutralizing antibodies in a guinea pig model system, comprising:
- immunizing a guinea pig with a cocktail of two or more viral strain antigens from a virus;
- isolating antigen-positive IgG-expressing B-cells from the guinea pigs immunized to the viral strain antigens using IgGpositive/IgMnegative flow panel;
- labeling a plurality of viral strain antigens with unique antigen barcodes;
- providing a plurality of barcode-labeled viral strain antigens to a population of the isolated IgG-expressing B-cells to form a mixture;
- allowing the plurality of barcode-labeled viral strains to bind to the population of the isolated IgG-expressing B-cells;
- washing unbound viral strain antigens from the population of isolated IgG-expressing B-cells;
- separating the isolated IgG-expressing B-cells into single cell emulsions;
- introducing into each single cell emulsion a unique cell barcode-labeled bead;
- preparing a single cell cDNA library from the single cell emulsions;
- performing nested PCR for target enrichment on the cDNA library using an inner reverse primer SEQ ID NO:
- 13, 14, or 15, an outer reverse primer SEQ ID NO:9, 10, 11, or 12, and a forward primer SEQ ID NO:
- 16 or 17;
- isolating B-cell receptor (BCR) genes from the cDNA library in the single cell emulsions;
- sequencing paired heavy and light chains of the BCR;
- performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise:
- the cell barcode and a nucleic acid sequence that identifies a particular viral strain antigen, wherein each amplicon comprises a unique molecular identifier (UMI);
- sequencing the plurality of amplicons;
- removing a sequence lacking the cell barcode, the UMI, or the nucleic acid sequence that identifies the particular viral strain antigens;
- aligning the antibody sequence to a reference library of immunoglobulin V, D, J and C sequences;
- constructing a UMI count matrix comprising the cell barcode, the nucleic acid sequence that identifies the particular viral strain antigens, and the antibody sequence;
- determining a LIBRA-seq score, wherein a low score means no predicted binding to antigens and a high score means high predicted binding to antigens;
- determining the nucleic acid sequence that identifies the particular viral strain antigens;
- and
- determining broadly neutralizing antibodies binding more than two viral strain antigens.
21 more elements
A method of developing a vaccine composition in a guinea pig model system, comprising:
- immunizing a guinea pig with a cocktail of two or more viral strain antigens from a virus;
- isolating antigen-positive IgG-expressing B-cells from the guinea pigs immunized to the viral strain antigens using IgG positive/IgM negative flow panel;
- labeling a plurality of viral strain antigens with unique antigen barcodes;
- providing a plurality of barcode-labeled viral strain antigens to a population of the isolated IgG-expressing B-cells to form a mixture;
- allowing the plurality of barcode-labeled viral strains to bind to the population of the isolated IgG-expressing B-cells;
- washing unbound viral strain antigens from the population of isolated IgG-expressing B-cells;
- separating the isolated IgG-expressing B-cells into single cell emulsions;
- introducing into each single cell emulsion a unique cell barcode-labeled bead;
- preparing a single cell cDNA library from the single cell emulsions;
- performing nested PCR for target enrichment on the cDNA library using an inner reverse primer SEQ ID NO:
- 13, 14, or 15, an outer reverse primer SEQ ID NO:9, 10, 11, or 12, and a forward primer SEQ ID NO:
- 16 or 17;
- isolating B-cell receptor (BCR) genes from the cDNA library in the single cell emulsions;
- sequencing paired heavy and light chains of the BCR;
- performing PCR amplification reactions to produce a plurality of amplicons, wherein the amplicons comprise:
- the cell barcode and a nucleic acid sequence that identifies a particular viral strain antigen, wherein each amplicon comprises a unique molecular identifier (UMI);
- sequencing the plurality of amplicons;
- removing a sequence lacking the cell barcode, the UMI, or the nucleic acid sequence that identifies the particular viral strain antigens;
- aligning an antibody sequence to a reference library of immunoglobulin V, D, J and C sequences;
- constructing a UMI count matrix comprising the cell barcode, the nucleic acid sequence that identifies the particular viral strain antigens, and the antibody sequence;
- determining a LIBRA-seq score, wherein a low score means no predicted binding to antigens and a high score means high predicted binding to antigens;
- determining the nucleic acid sequence that identifies the particular viral strain antigens;
- determining broadly neutralizing antibodies binding more than two viral strain antigens;
- and
- formulating a vaccine composition comprising the particular viral strain antigens that specifically bind to broadly neutralizing antibodies, wherein the particular viral strain antigens are capable of eliciting an immune response in the guinea pig.
22 more elements
Dependencies are inferred from the claim text.