EPITOPE MAPPING METHOD

• US 20190325985A1
• Filed: 04/18/2019
• Published: 10/24/2019
• Est. Priority Date: 04/19/2018
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Hangartner, Lars, Ward, Andrew, Bianchi, Matteo, Turner, Hannah
• Granted Patent: US 11,217,328 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/6878 in eptitope analysis
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: H01J 37/26 Electron or ion microscopes; Electron or ion diffraction tubes
• CPC Class Codes: H01J 49/00 Particle spectrometers or separator tubes
• CPC Class Codes: H01J 49/0027 Methods for using particle spectrometers

Abstract

Provided herein are methods for mapping immune response to an immunogen, comprising: immunizing a subject with an immunogen and obtaining sera from the immunized subject at multiple time intervals following immunization, wherein the sera comprises one or more immune complexes between the immunogen and serum antibodies; imaging, by electron microscopy, the sera obtained from the immunized subject in each of the time intervals, to obtain structural images of the one or more immune complexes formed between the immunogen and serum antibodies; mapping immune response to the immunogen by measuring differences in structural images obtained at different time intervals to simultaneously visualize diverse antibodies targeting distinct epitopes in the immunized subjects. Further provided herein are vaccine design processes, comprising: administering a proposed vaccine to a test subject; imaging the immune complex formed in the test subject upon administration of the proposed vaccine; processing and visualizing the image to determine the likely immunogenicity of the proposed vaccine, and determining that the proposed vaccine is immunogenic if it binds to an antibody, and determining that the proposed vaccine should be redesigned if it does not bind or binds weakly to the antibody.

Claims

1. A method of mapping immune response to an immunogen, comprising:

immunizing a subject with an immunogen and obtaining sera from the immunized subject at multiple time intervals following immunization, wherein the sera comprises one or more immune complexes between the immunogen and serum antibodies,

imaging, by electron microscopy, the sera obtained from the immunized subject in each of the time intervals, to obtain structural images of the one or more immune complexes formed between the immunogen and serum antibodies;

mapping immune response to the immunogen by measuring differences in structural images obtained at different time intervals to simultaneously visualize diverse antibodies targeting distinct epitopes in the immunized subjects.

2. The method of claim 1, wherein the serum antibodies comprise IgA (immunoglobin A), IgD (immunoglobin D), IgE (immunoglobin E), IgG (immunoglobin G), IgM (immunoglobin M), or combinations thereof.

3. The method of claim 1, further comprising designing epitope specific probes for B-cell sorting of targeted specificities based on the electron microscopy structural images.

4. The method of claim 1, wherein the immune complex is purified by a chromatographic method prior to imaging.

5. The method of claim 4, wherein the chromatographic method comprises size exclusion chromatography (SEC).

6. The method of claim 1, wherein the electron microscopy comprises cyro electron microscopy or negative stain electron microscopy.

7. The method of claim 1, wherein the immune complex is further analyzed by Mass Spectrometry, next generation sequencing (NGS), MS/MS and/or NGS-assisted MS/MS.

8. The method of claim 1, wherein the subject is a human, mouse, humanized mouse, rat, or humanized rat.

9. The method of claim 1, wherein the rapid analysis of the immune complex allows for characterization and quantification of antibody responses elicited by immunization or infection.

10. The method of claim 1, wherein the method provides real time analysis of ongoing immunization experiments.

11. The method of claim 10, wherein the ongoing immunization experiments comprises human vaccine trials.

12. The method of claim 10, wherein the method provides cues for the improvement of the immunogen in case of failure of the human vaccine trial.

13. The method of claim 1, wherein the immunogen is a recombinant immunogen.

14. The method of claim 1, wherein the immunogen is an epitope and/or an antigen.

15. The method of claim 1, wherein the method maps the epitopes recognized by the antibody.

16. A method of forming an immune complex comprising:

providing an immunoglobulin (Ig) antibody;

enzymatically digesting the Ig into fragment antigen binding (Fab) and complexing the Fab with a soluble pathogen and/or antigen;

quantifying the specific Ig content for immune complex formation; and

forming the immune complex by incubating the pathogen and/or antigen with an excess of Fab.

17. A vaccine design process, comprising:

administering a proposed vaccine to a test subject;

imaging the immune complex formed by an elicited antibody in the test subject upon administration of the proposed vaccine;

processing and visualizing the image by electron microscopy to determine the likely immunogenicity of the proposed vaccine; and

determining that the proposed vaccine is immunogenic if it binds to an antibody and determining that the proposed vaccine should be redesigned if it does not bind or binds weakly to the antibody.

18. The vaccine design process of claim 17, wherein the elicited antibody is a secreted antibody, and/or an antibody from the tissue or feces of the test subject.

19. The vaccine design process of claim 17, wherein the test subject is a mammal.

20. The vaccine design process of claim 17, wherein the test subject is a rabbit, mouse, rat, and/or monkey.