Measurement and comparison of immune diversity by high-throughput sequencing

• US 9,909,180 B2
• Filed: 02/04/2014
• Issued: 03/06/2018
• Est. Priority Date: 02/04/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Quake, Stephen R., Weinstein, Joshua, Jiang, Ning, Fisher, Daniel S.
• Primary Examiner(s): Martinell, James
• Application Number:
  US14/172,642
• Publication Number:
  US 20140235478A1
• Time in Patent Office: 1,491 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

High-throughput long read sequencing is used to perform immunogenomic characterization of expressed antibody repertoires in the context of vaccination. Informatic analysis allows global characterizations of isotype distributions, determination of the lineage structure of the repertoire and measure age and antigen related mutational activity. Global analysis of the immune system'"'"'s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

Claims

1. A method of identifying an antigen-specific antibody lineage in a subject exposed to the antigen, comprising,

sequencing nucleic acid from a biological sample obtained from the subject after exposure to the antigen, via high throughput sequencing, to determine CDR3 nucleic acid sequences, representing at least a portion of the subject'"'"'s antibody repertoire;

performing a linkage analysis of the CDR3 nucleic acid sequences to establish linked CDR3 sequences, wherein two CDR3 sequences are in a linkage if they have the same V and J assignment and no more than one amino acid difference in the CDR3 region;

grouping CDR3 sequences into a plurality of lineages, wherein individual lineages of the plurality comprise (i) identical CDR3 sequences and (ii) CDR3 sequences that are associated with one another through one or more linkages; and

comparing the CDR3 sequences in the plurality of lineages with a CDR3 sequence of an antigen-specific antibody obtained from the subject to determine the presence of the antigen-specific antibody lineage.

2. The method of claim 1, wherein the antigen is a vaccine.

3. The method of claim 2, further comprising,

(a) carrying out the method using a first biological sample obtained from the subject at a first time point;

(b) carrying out the method using a second biological sample obtained from the subject at a second time point; and

(c) comparing the lineage structure determined in (a) with the lineage structure determined in (b),

wherein after the first time point and prior to the second time point the vaccine is administered to the subject.

4. The method of claim 1, further comprising quantitating the abundance of individual CDR3 amino acid sequences within individual lineages by counting the number of sequencing reads of the counterpart CDR3 nucleic acid sequences.

5. The method of claim 1, further comprising,

quantitating an average number of mutations within the individual lineages, wherein the average number of mutations is the average over a plurality of sequence reads of the CDR3 regions within the individual lineages.

6. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, lymph, sputum, and tissue.

7. The method of claim 6, wherein the biological sample is blood.

8. The method of claim 1, further comprising quantitating sizes of the individual lineages by determining the number of linked CDR3 sequences in the individual lineages.