Assays and methods for assessing vaccine preparations

Abstract

Provided herein are assays and methods of assessing vaccine preparations, and engineered B cell receptors (BCR) and engineered B cell lines used in such assays and methods.

Description

Attorney Docket No.01217-0002-00PCT ASSAYS AND METHODS FOR ASSESSING VACCINE PREPARATIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No. 63 / 601,866, filed November 22, 2023, which is incorporated by reference herein in its entirety for any purpose. SEQUENCE LISTING

[0002] The present application contains a Sequence Listing, which has been submitted electronically in XML format. Said XML copy, created on November 17, 2024, is named “01217-0002-00PCT.xml” and is 26,256 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. FIELD OF INVENTION

[0003] Provided herein are assays and methods of assessing vaccine preparations, and engineered B cell receptors (BCR) and engineered B cell lines used in such assays and methods. BACKGROUND

[0004] Vaccines have been one of the most successful public health interventions in preventing disease and death due to infections. A vaccine is a biological product that can be used to safely induce an immune response that confers protection against infection and / or disease. To achieve this, the vaccine must contain antigens that are either derived from the pathogen or produced synthetically to represent components of the pathogen. Noteworthy, the antigens may be derived from a foreign (external) antigen, e.g. a virus or an allergen, or may be derived from a self- antigen, e.g. tumor antigen, cytokine or prion protein. Some current vaccines consist of live attenuated or non-virulent variant strains of microorganisms, or killed or otherwise inactivated organisms. Other vaccines utilize more or less purified components of pathogen lysates such as surface carbohydrates, recombinant pathogen-derived proteins that are sometimes fused to other molecules, or replicative viruses that produce an antigen from a pathogen. Both the innate and adaptive immune subsystems are necessary to provide an effective immune response to an immunization. Further, effective immunizations must induce long-term stimulation of both the humoral and cell-mediated arms of the adaptive system by the production of effector cells and memory cells.Attorney Docket No.01217-0002-00PCT

[0005] Predicting immune responses before vaccination is challenging because of the complexity of the governing parameters. There remains a need to provide effective methods for assessing and / or predicting immunogenicity and potency of vaccines. SUMMARY OF INVENTION

[0006] The present disclosure provides assays and methods of assessing a vaccine preparation, and engineered B cell receptors (BCR) and engineered B cell lines used in such assays and methods. Embodiment 1. A method of assessing a vaccine preparation, comprising: a) contacting an engineered B cell line with a vaccine preparation; and b) detecting (i) BCR-mediated cell survival and / or (ii) BCR-mediated activation of a reporter gene, wherein the engineered B cell line comprises an engineered B-cell receptor (BCR), wherein the engineered BCR comprises a heavy chain and a light chain, wherein: the heavy chain comprises: a heavy chain variable region (VH) of an antibody that binds a target antigen; and a heavy chain constant region of a membrane IgM (mIgM); and the light chain comprises: a light chain variable region (VL) of the antibody; and a light chain constant region. Embodiment 2. The method of embodiment 1, wherein the B cell does not express endogenous surface IgM. Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the VH comprises a mature complementarity determining region 3 (H-CDR3). Embodiment 4. The method of any one of embodiments 1-3, wherein the VH comprises a mature framework region 4 (H-FR4). Embodiment 5. The method of any one of embodiments 1-4, wherein the VL comprises a mature complementarity determining region 3 (L-CDR3). Embodiment 6. The method of any one of embodiments 1-5, wherein the VL comprises a mature framework region 4 (L-FR4). Embodiment 7. The method of any one of embodiments 1-6, wherein the VH comprises one or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3.Attorney Docket No.01217-0002-00PCT Embodiment 8. The method of any one of embodiments 1-6, wherein the VH comprises a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. Embodiment 9. The method of any one of embodiments 1-6, wherein the VH comprises one or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3. Embodiment 10. The method of any one of embodiments 1-9, wherein the VL comprises one or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. Embodiment 11. The method of any one of embodiments 1-9, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline- reverted L-CDR2, and a germline-reverted L-FR3. Embodiment 12. The method of any one of embodiments 1-9, wherein the VH comprises one or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3. Embodiment 13. The method of any one of embodiments 1-12, wherein the engineered B cell line comprises a reporter gene operably linked to a promoter that is responsive to activation of the engineered BCR. Embodiment 14. The method of embodiment 13, wherein the method comprises detecting BCR-mediated activation of the reporter gene. Embodiment 15. The method of embodiment 14, wherein detecting BCR-mediated activation of the reporter gene comprises detecting expression of the reporter gene operably linked to the promoter that is responsive to activation of the engineered BCR. Embodiment 16. The method of any one of embodiments 13-15, wherein the promoter comprises one or more enhancer elements. Embodiment 17. The method of any one of embodiments 13-16, wherein the promoter comprises one or more nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) responsive enhancer elements. Embodiment 18. The method of embodiment 17, wherein the promoter is a NF-κB / activator protein 1 (AP-1) inducible promoter. Embodiment 19. The method of any one of embodiments 13-16, wherein the promoter comprises one or more NFAT-response elements.Attorney Docket No.01217-0002-00PCT Embodiment 20. The method of any one of embodiments 1-19, wherein the reporter gene encodes an enzyme or a fluorescent protein. Embodiment 21. The method of any one of embodiments 1-20, wherein the reporter gene encodes a luciferase, an alkaline phosphatase, a β-lactamase, a β-galactosidase, β-glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase. Embodiment 22. The method of any one of embodiments 1-21, wherein the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP). Embodiment 23. The method of any one of embodiments 1-22, wherein BCR-mediated activation of the reporter gene is detected using an alkaline phosphatase activity assay. Embodiment 24. The method of any one of embodiments 1-23, wherein the method comprises detecting BCR-mediated cell survival. Embodiment 25. The method of any one of embodiments 1-24, wherein the BCR-mediated cell survival is assessed using a cell viability assay. Embodiment 26. The method of any one of embodiments 1-25, wherein the engineered B cell line is contacted with the vaccine preparation, and an inhibitor of cell proliferation and / or an inhibitor of actin polymerization element. Embodiment 27. The method of any one of embodiments 1-26, wherein the engineered B cell line is contacted with the vaccine preparation and Cytochalasin D. Embodiment 28. The method of any one of embodiments 1-27, wherein the engineered B cell line is primed with human Interleukin-4 (IL4) before being contacted with the vaccine preparation. Embodiment 29. The method of any one of embodiments 1-28, wherein the vaccine is a liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine or conjugate vaccine, preferably a liposomal vaccine. Embodiment 30. The method of any one of embodiments 1-29, wherein the vaccine is a liposomal vaccine. Embodiment 31. The method of any one of embodiments 1-30, wherein the target antigen is an antigenic peptide presented on the surface of a vaccine. Embodiment 32. The method of embodiment 31, wherein the VH comprises a mature H- CDR3 and a mature H-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11.Attorney Docket No.01217-0002-00PCT Embodiment 33. The method of embodiment 31 or embodiment 32, wherein the VL comprises a mature L-CDR3 and a mature L-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. Embodiment 34. The method of any one of embodiments 31-33, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21. Embodiment 35. The method of any one of embodiments 31-34, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22. Embodiment 36. The method of any one of embodiments 31-33 and 35, wherein the VH comprises a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H- FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10. Embodiment 37. The method of any one of embodiments 31-34, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. Embodiment 38. The method of any one of embodiments 31-37, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12. Embodiment 39. The method of any one of embodiments 31-38, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. Embodiment 40. The method of any one of embodiments 31-39, wherein the heavy chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. Embodiment 41. The method of any one of embodiments 31-40, wherein the light chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at leastAttorney Docket No.01217-0002-00PCT 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18. Embodiment 42. The method of any one of embodiments 31-41, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. Embodiment 43. The method of any one of embodiments 31-42, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14. Embodiment 44. The method of any one of embodiments 31-43, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7. Embodiment 45. The method of any one of embodiments 31-44, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8. Embodiment 46. The method of any one of embodiments 1-45, wherein the engineered B cell line comprises an engineered Ramos cell line, DT40 cell line, CH12 cell line, Burkitt’s lymphoma B cell line, primary B cell line, or stem cell-derived B cell line. Embodiment 47. The method of any one of embodiments 1-46, wherein the engineered B- cell line comprises an engineered Ramos cell line. Embodiment 48. The method of any one of embodiments 1-47, wherein the method comprises comparing BCR-mediated cell survival of the engineered B cell line contacted with the vaccine preparation to BCR-mediated cell survival of the same engineered B cell line contacted with a second vaccine preparation and / or a control vaccine preparation. Embodiment 49. The method of embodiment 48, wherein the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated cell survival of the engineered B cell line. Embodiment 50. The method of any one of embodiments 1-49, wherein the method comprises comparing BCR-mediated activation of the reporter gene of the engineered B cell line contacted with the vaccine preparation to BCR-mediated activation of the reporter gene of theAttorney Docket No.01217-0002-00PCT same engineered B cell line contacted with a second vaccine preparation and / or a control vaccine preparation. Embodiment 51. The method of embodiment 50, wherein the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated activation of the reporter gene of the engineered B cell line. Embodiment 52. The method of any one of embodiments 48-51, wherein the vaccine preparation and the second vaccine preparation comprise the same peptide antigen. Embodiment 53. The method of any one of embodiments 48-52, wherein the vaccine preparation and the second vaccine preparation are different preparations of the same vaccine. Embodiment 54. The method of any one of embodiments 1-53, wherein the vaccine preparation comprises an antigenic peptide derived from a self-antigen. Embodiment 55. The method of embodiment 54, wherein the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α-synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27. Embodiment 56. The method of embodiment 54 or embodiment 55, wherein the antigenic peptide is derived from β-amyloid (Aβ). Embodiment 57. The method of any one of embodiments 1-56, wherein the vaccine preparation is a liposomal vaccine preparation and comprises a β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome. Embodiment 58. The method of embodiment 56 or embodiment 57, wherein the antigenic peptide comprises amino acids 1-15 of β-amyloid (Aβ) (SEQ ID NO: 23). Embodiment 59. The method of any one of embodiments 1-58, wherein the method comprises assessing presentation of the peptide antigen on the vaccine surface. Embodiment 60. The method of any one of embodiments 1-59, wherein the method comprises assessing the antibody response to the vaccine preparation. Embodiment 61. The method of any one of embodiments 1-59, wherein the method comprises assessing potency of the vaccine preparation. Embodiment 62. The method of any one of embodiments 1-59, wherein the method comprises assessing the ability of an antigen to activate B-cell or to induce BCR signaling. Embodiment 63. The method of any one of embodiments 1-59, wherein the method comprises predicting in vivo immunogenicity of the vaccine preparation. Embodiment 64. The method of any one of embodiments 48-63, wherein the control vaccine preparation is a preparation of a vaccine that does not comprise a peptide antigenAttorney Docket No.01217-0002-00PCT recognized by the BCR or wherein the control vaccine preparation is a preparation of a vaccine that has previously been determined to have immunogenicity in vivo. Embodiment 65. The method of any one of embodiments 1-64, wherein the method comprises detecting BCR-mediated cell survival and BCR-mediated activation of the reporter gene. Embodiment 66. The method of embodiment 65, wherein the vaccine preparation is identified as having suitable predicted in vivo immunogenicity if it induces both BCR-mediated reporter gene activation and BCR-mediated cell survival. Embodiment 67. The method of any one of embodiments 1-66, wherein the method comprises comparing multiple batches of the vaccine preparation comprising the same antigen. Embodiment 68. An engineered B-cell line comprising: a) an engineered B-cell receptor (BCR), wherein the engineered BCR comprises a heavy chain and a light chain, wherein: the heavy chain comprises: a heavy chain variable region (VH) of an antibody that binds a target antigen; and a heavy chain constant region of a membrane IgM (mIgM); and the light chain comprises: a light chain variable region (VL) of the antibody; and a light chain constant region; and b) a reporter gene operably linked to a promoter that is responsive to activation of the BCR, wherein the promoter comprises one or more nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) responsive enhancer elements; and wherein the target antigen is a self-antigen. Embodiment 69. The engineered B-cell line of embodiment 68, wherein the B-cell does not express endogenous surface IgM. Embodiment 70. The engineered B-cell line of embodiment 68 or embodiment 69, wherein the VH comprises a mature complementarity determining region 3 (H-CDR3). Embodiment 71. The engineered B-cell line of any one of embodiments 68-70, wherein the VH comprises a mature framework region 4 (H-FR4). Embodiment 72. The engineered B-cell line of any one of embodiments 68-71, wherein the VL comprises a mature complementarity determining region 3 (L-CDR3). Embodiment 73. The engineered B-cell line of any one of embodiments 68-72, wherein the VL comprises a mature framework region 4 (L-FR4).Attorney Docket No.01217-0002-00PCT Embodiment 74. The engineered B-cell line of any one of embodiments 68-73, wherein the VH comprises one or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. Embodiment 75. The engineered B-cell line of any one of embodiments 68-73, wherein the VH comprises a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H- FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. Embodiment 76. The engineered B-cell line of any one of embodiments 68-73, wherein the VH comprises one or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3. Embodiment 77. The engineered B-cell line of any one of embodiments 68-76, wherein the VL comprises one or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. Embodiment 78. The engineered B-cell line of any one of embodiments 68-76, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L- FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. Embodiment 79. The engineered B-cell line of any one of embodiments 68-76, wherein the VH comprises one or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L- CDR2 and a mature L-FR3. Embodiment 80. The engineered B-cell line of any one of embodiments 68-79, wherein the promoter is a NF-κB / activator protein 1 (AP-1) inducible promoter. Embodiment 81. The engineered B-cell line of any one of embodiments 68-80, wherein the reporter gene encodes an enzyme or a fluorescent protein. Embodiment 82. The engineered B-cell line of any one of embodiments 68-81, wherein the reporter gene encodes a encodes a luciferase, an alkaline phosphatase, a β-lactamase, a β- galactosidase, β-glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase. Embodiment 83. The engineered B-cell line of any one of embodiments 68-82, wherein the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP). Embodiment 84. The engineered B-cell line of any one of embodiments 68-83, wherein the target antigen is an antigenic peptide presented on the surface of a liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine or conjugate vaccine, preferably a liposomal vaccine.Attorney Docket No.01217-0002-00PCT Embodiment 85. The engineered B-cell line of any one of embodiments 68-84, wherein the target antigen is an antigenic peptide presented on the surface of a liposomal vaccine. Embodiment 86. The engineered B-cell line of embodiment 84 or embodiment 85, wherein the VH comprises a mature H-CDR3 and a mature H-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11. Embodiment 87. The engineered B-cell line of any one of embodiments 84-86, wherein the VL comprises a mature L-CDR3 and a mature L-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. Embodiment 88. The engineered B-cell line of any one of embodiments 84-87, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21. Embodiment 89. The engineered B-cell line of any one of embodiments 84-88, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22. Embodiment 90. The engineered B-cell line of any one of embodiments 84-87 and 89, wherein the VH comprises a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10. Embodiment 91. The engineered B-cell line of any one of embodiments 84-88, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L- FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. Embodiment 92. The engineered B-cell line of any one of embodiments 84-91, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12.Attorney Docket No.01217-0002-00PCT Embodiment 93. The engineered B-cell line of any one of embodiments 84-92, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. Embodiment 94. The engineered B-cell line of any one of embodiments 84-93, wherein the heavy chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. Embodiment 95. The engineered B-cell line of any one of embodiments 84-94, wherein the light chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18. Embodiment 96. The engineered B-cell line of any one of embodiments 84-95, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. Embodiment 97. The engineered B-cell line of any one of embodiments 84-96, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14. Embodiment 98. The engineered B-cell line of any one of embodiments 84-97, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7. Embodiment 99. The engineered B-cell line of any one of embodiments 84-98, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8. Embodiment 100. The engineered B-cell line of any one of embodiments 68-99, wherein the engineered B cell line comprises an engineered Ramos cell line, DT40 cell line, CH12 cell line, Burkitt’s lymphoma B cell line, primary B cell line, or stem cell-derived B cell line. Embodiment 101. The engineered B-cell line of any one of embodiments 68-100, wherein the engineered B-cell line comprises an engineered Ramos cell line.Attorney Docket No.01217-0002-00PCT Embodiment 102. The engineered B-cell line of any one of embodiments 84-101, wherein the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α- synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27. Embodiment 103. The engineered B-cell line of any one of embodiments 84-102, wherein the antigenic peptide is derived from β-amyloid (Aβ). Embodiment 104. The engineered B-cell line of any one of embodiments 84-103, wherein the liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine or conjugate vaccine, preferably a liposomal vaccine, comprises a β-amyloid (Aβ)- derived peptide antigen displayed on its surface. Embodiment 105. The engineered B-cell line of any one of embodiments 84-104, wherein the liposomal vaccine comprises a β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome. Embodiment 106. The engineered B-cell line of any one of embodiments 84-105, wherein the antigenic peptide comprises amino acids 1-15 of β-amyloid (Aβ) (SEQ ID NO: 23). Embodiment 107. The engineered B-cell line of any one of embodiments 68-106, wherein the B-cell line is selected to present a high, medium or low expression of the engineered BCR. Embodiment 108. A kit comprising: a) the engineered B-cell line of any one of embodiments 68-107; and b) reagents for detecting BCR-mediated activation of the reporter gene and / or BCR- mediated cell survival. BRIEF DESCRIPTION OF FIGURES

[0007] FIG.1A-1E shows isolation of mIgM-surface negative Ramos-Blue cells by FACS. 1A shows staining of endogenous mIgM using anti lambda-PE and anti hIgM-APC antibodies, and sorting of the double-negative (DN; = IgM-surface negative) cell population. FIG.1B shows the gating strategy for the sorting of mIgM surface-negative Ramos-Blue cells using a FACS Aria II. FIG.1C shows staining control on unsorted Ramos-Blue cells with anti-lambda-PE only. FIG.1D shows staining control on unsorted Ramos-Blue cells with anti hIgM-APC only. FIG. 1E shows mIgM staining on isolated surface-negative Ramos-Blue cells with both anti lambda- PE and anti hIgM-APC antibodies. For FIG.1C, 1D, and 1E, flow cytometry analysis of mIgM expression was performed using an Attune NxT flow cytometer.

[0008] FIG.2A shows the gating strategy for the selection of low, medium, and high mIgM- expressing cells. FIG.2B shows engineered 6E10 mIgM BCR binding A-beta 1-15 ofAttorney Docket No.01217-0002-00PCT biotinylated ACI-24.012 liposomal vaccine by flow cytometry (Attune NxT). Streptavidin conjugated to a fluorophore (Strep-488) binds biotin on ACI-24 vaccine to allow detection by fluorescence.

[0009] FIG.3 shows 6E10.10.H2 reporter B cells stimulation by ACI-24.012 liposomal vaccine at decreasing concentration. Cells were stimulated over 48h. ACI-24.012 (left bars) is a liposomal vaccine displaying an amyloid-beta (A-beta) 1-15 peptide and ACI-24E.012 (right bars) is a negative control lacking A-beta 1-15.

[0010] FIG.4 shows 6E10.10.H2 reporter B cells stimulation with or without addition of co- stimulatory reagents. Cells were stimulated over 72h with or without priming of the cells 24h before stimulation with co-stimulatory reagents. Left bars show stimulation with anti-hIgM F(ab’)2 and right bars show stimulation with ACI-24.012.

[0011] FIG.5 shows 6E10.10.H2 reporter B cells stimulated at various cell densities. SEAP secretion increase from baseline (BCR-mediated NF-κB / AP-1 reporter gene activation) of 6E10.10.H2 B cells seeded at different densities and stimulated by different concentrations of ACI-24.012 liposomal vaccine (defined by A-beta 1-15 peptide antigen concentration) is shown.

[0012] FIG.6 shows 6E10.10.H2 reporter B cells stimulation with or without pre-incubation at +4°C. Cells were stimulated over 72h with liposomal vaccine ACI-24.012 or ACI-24.043 (defined by A-beta 1-15 peptide antigen concentration) with or without pre-incubation step at +4°C. ACI-24E.012 and ACI-EE.043 are negative controls lacking A-beta 1-15 (used at the same dilution as their counterpart vaccine).

[0013] FIG.7 shows 6E10.10.H2 reporter B cells stimulation with increasing Cytochalasin D (CD) concentration (No CD, 0.2, 1 and 5µM). Cells were stimulated over 72h with an ACI- 24.012 vaccine or negative controls ACI-EE.043 or ACI-24E.012 (at equivalent dilution), with exposure to different concentrations of Cytochalasin D.

[0014] FIG.8A-8B shows a comparison of two different readouts for B-cell activation. 6E10.10.H2 reporter B cells were stimulated with four different ACI-24 liposome samples over 48h, and B-cell activation was measured by SEAP secretion increase with the BCR-mediated NF-kB / AP-1-induced reporter gene activity (FIG.8A) or BCR-mediated cell survival increase using 2 different viability assays (FIG.8B). ACI-24 and ACI-24.012 are liposomal vaccine comprising an A-beta 1-15 peptide antigen. ACI-24.043 S vaccine batch has a suboptimal presentation of the B cell antigen on the vaccine surface. ACI-24E.012 is a negative control lacking A-beta 1-15 (used at equivalent dilution).Attorney Docket No.01217-0002-00PCT

[0015] FIG.9A-9D shows a comparison of two isolated B cell pools expressing high (H2) or low (L2) levels of the 6E10 mIgM engineered BCR for the ranking of ACI-24 vaccine samples by B-cell activation. FIG.9A-9B shows BCR-mediated reporter gene activity (SEAP secretion). FIG.9C-9D shows BCR-mediated cell survival readout. ACI-24.012 and ACI- 24.043 are liposomal vaccine comprising an A-beta 1-15 peptide antigen. ACI-24.043 S is a suboptimal vaccine batch. ACI-EE.043 is a negative control lacking A-beta 1-15 (used at equivalent dilution).

[0016] FIG.10A-10B shows 6E10.10.H2 reporter B cell line stimulation using different ACI- 24 liposomal vaccine batches comprising A-beta 1-15 peptide antigen (ACI-24.012, ACI-24.008 and ACI-24.043), A-beta protein and protein fragment (A-beta 1-15 and biotinylated A-beta 1- 42) and negative controls lacking A-beta 1-15 peptide antigen (ACI-24E.012 and ACI-EE.043). FIG.10A shows BCR-mediated reporter gene activity (SEAP secretion). FIG.10B shows BCR- mediated cell survival readout (Orangu assay). All antigens are expressed in molarity of A-beta content for the purpose of this comparison (except for the negative controls used at equivalent dilution). Analysis performed in duplicates in a single experiment.

[0017] FIG.11A-11B shows the comparison of B cells (6E10.10.H2) BCR-mediated survival assay versus in vivo titers post vaccination of mice using different batches of liposomes. Batch A with immunogenic peptide A-beta 1-15, and adjuvants; B is a suboptimal batch with immunogenic peptide A-beta 1-15 and adjuvants; C without target antigen (negative control).

[0018] FIG.11A shows area under the curve (AUC) ratio (normalization to a reference vaccine batch) of the BCR-mediated cell survival response. FIG.11B shows anti-A-beta antibody titer in mice at one-week post-immunizations. DETAILED DESCRIPTION

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the arts to which the invention belongs.

[0020] Before the present compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.Attorney Docket No.01217-0002-00PCT Definitions

[0021] The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0022] The term antibody includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0023] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.),Attorney Docket No.01217-0002-00PCT small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment”.

[0024] The term “antigen” refers to any agent (e.g., protein, peptide, lipid, polysaccharide, glycoprotein, glycolipid, nucleic acid or any combination of any of the foregoing) that, when introduced into a host, animal or human, having an immune system (directly or upon expression as in, e.g., DNA vaccines), is recognized by the immune system of the host and is capable of eliciting an immune response. The antigen-induced immune response can be humoral or cell- mediated, or both. An agent is termed “antigenic” when it is capable of or comprises a component capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor (TCR). “Surface antigens” are expressed naturally on the surface of a pathogen, or the surface of an infected cell, or the surface of a tumor cell. In various embodiments, an antigen is a peptide. In some such embodiments, the peptide is a portion of a natural protein.

[0025] The term “self-antigen”, as used herein, refers to any peptide derived from an antigen naturally produced by an individual. In general, the immune system of the individual is tolerant to self-antigen molecules and therefore no immune reaction occurs. In some cases, the immune system is not tolerant to the self-antigens and auto-immune diseases may occur. The use of a self-antigen allows the targeting of a molecule to which the immune system is tolerant and therefore may help to induce an immune response that otherwise would not occur.

[0026] The term “foreign antigen”, as used herein, refers to any molecule that is not naturally produced by an individual (human). In general, the immune system of the individual will recognise the foreign antigen and an immune response will be generated against the antigen. For examples, the foreign antigen may be a viral antigen, or an allergen. Examples of viral antigens include, but are not limited to, antigens from rhinoviruses, coronaviruses, enteroviruses, adenoviruses, parainfluenza viruses and respiratory syncytial viruses.

[0027] The term “epitope” refers to the part of an antigenic molecule to which an antibody is produced and to which the antibody will bind. The term “immunogenic epitope,” refers to the portion of a protein that elicits an antibody response in an animal or in human.

[0028] The term “antigenic epitope,” refers to the portion of a protein to which an antibody binds via its antigen binding region (i.e., domains containing the complementarity determining regions or antigen binding site). An antigenic epitope refers to a three dimensional molecular structure (linear, non-linear, and / or conformational) that is capable of immunoreactivity with a monoclonal antibody. Antigenic epitopes may comprise proteins, protein fragments, peptides,Attorney Docket No.01217-0002-00PCT carbohydrates, lipids, oligopeptide mimics (i e, organic compounds that mimic the antibody binding properties of the antigen), and other molecules, or combinations thereof. Antigenic epitopes preferably contain a sequence of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50 amino acids. Furthermore, epitope bearing polypeptides of the invention may be modified, for example, by the addition of amino acids to the polypeptides, for example, but not limited to, at the amino- and / or carboxy-termini of the peptide. Such modifications may be performed, for example, to alter the conformation of the epitope bearing polypeptide such that the epitope will have a conformation more closely related to the structure of the epitope in the native protein.

[0029] An “antigenic peptide” is understood to encompass any peptide capable, upon administration to a mammal, particularly a human, of inducing an immune response in said mammal. The antigenic peptide may be derived from a foreign (external) antigen, e.g. a virus or an allergen, or may be derived from a self-antigen, e.g. tumour antigen, cytokines such as, for example, IL-17, IL-27, or proteins such as Abeta, Tau, a-syn, involved in proteinopathies. A “vaccine” is an immunogenic composition that can be used to elicit protective immunity or a protective immune response in a recipient. The protective immunity may for example be towards pathogens such as viruses, fungi, parasites, yeast, bacteria, and protozoa, or allergens, or may for example be towards self-antigens such as tumour antigen, cytokines such as, for example, IL-17, IL-27, or pathological forms of proteins such as Abeta, Tau, a-syn, prion protein or huntingtin-involved in proteinopathies. A vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine. Said vaccine preparations may include prophylactic as well as therapeutic vaccine preparations. “Liposomal vaccines” contains antigen(s) (protein, peptide, lipid, lipopeptide etc.) from the agent of interest that is incorporated into or attached (covalently or non-covalently) to or within a liposome.

[0030] “Potency” refers to the biological activity of a vaccine preparation required in the treatment or prevention of diseases, disorders, or conditions.

[0031] “Immunogenicity” refers to a measure of the ability of an antigen to elicit an immune response (humoral or cellular) when administered to a recipient.

[0032] “Promoter” refers to a nucleic acid stretch typically located upstream a nucleic acid molecule encoding a functional polypeptide or protein. The promoter is functional in host cells and regulates the transcription of nucleic acid stretches found downstream to the promoter. The promoter comprises transcription factor binding sites as well as a transcription initiation region.Attorney Docket No.01217-0002-00PCT

[0033] “Operably linked” refers to the linking of nucleotide regions encoding specific genetic information such that the nucleotide regions are contiguous, the functionality of the region is preserved and will perform relative to the other regions as part of a functional unit.

[0034] A “reporter gene” is an exogenous coding region joined to a promoter sequence or element in an expression vector that is introduced into cells to provide the means for measuring the promoter activity and / or activity of upstream pathways.

[0035] The term “about” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems (e.g., when measuring an immune response), the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.

[0036] A “mature antibody” in the context of this patent refers to an immunoglobulin that has been selected and developed through in vivo processes as well as those selected and optimized through in vitro technologies, including but not limited to hybridoma, phage display, mammalian display, ribosome display and yeast display, as well as single-cell technologies, such as single-cell RNA sequencing and single-cell cloning. A "Mature antibody" has undergone in vivo somatic hypermutation and / or in vitro affinity selection and / or maturation, resulting in enhanced specificity and binding affinity to a target antigen. Overview

[0037] Provided herein are assays, methods, and kits for assessing a vaccine preparation. Also provided herein are engineered B cell receptors (BCRs) and engineered B cell lines used in such assays, methods, and kits.

[0038] In some embodiments, the vaccine preparation is an antigen-presenting vaccine preparation. In some embodiments, the antigen-presenting vaccine preparation is a liposomal vaccine preparation, DNA scaffold vaccine preparation, nanoparticle vaccine preparation, Virus- Like Particles (VLP) vaccine preparation or conjugate vaccine preparation, etc. In some embodiments, the vaccine preparation is a liposomal vaccine preparation.

[0039] In some embodiments, the method comprises assessing and / or predicting antibody response to a vaccine preparation. In some embodiments, the method comprises assessing and / or predicting immunogenicity, potency, or both, of the vaccine preparation. In some embodiments, the method comprises assessing the ability of an antigen to activate a B-cell or to induce BCR signaling.

[0040] In some embodiments, the method comprises contacting an engineered B cell line discussed herein with a vaccine preparation, wherein the engineered B cell line comprises anAttorney Docket No.01217-0002-00PCT engineered BCR discussed herein, and assessing BCR activation. In some embodiments, assessing the BCR activation comprises analyzing and / or detecting 1) alterations (such as downstream signaling) induced by BCR activation and / or 2) cell survival. In some embodiments, the method comprises analyzing and / or detecting alterations induced by BCR activation. In some embodiments, the method comprises analyzing and / or detecting BCR- mediated cell survival. In some embodiments, the method comprises analyzing and / or detecting alterations induced by BCR activation and BCR-mediated cell survival. In some embodiments, alterations induced by BCR activation comprises BCR-mediated activation of a reporter gene.

[0041] In some embodiment, the vaccine preparation is identified as having suitable potency if it mediates BCR activation. In some embodiments, the vaccine preparation is identified as having suitable potency if it mediates alterations induced by BCR activation and / or BCR- mediated cell survival. In some embodiment, the vaccine preparation is identified as having suitable potency if it mediates alterations induced by BCR activation. In some embodiments, alterations induced by BCR activation comprises BCR-mediated activation of a reporter gene, and the vaccine preparation is identified as having suitable potency by detecting the product generated by the reporter gene (i.e., luciferase or a fluorescent protein, or a metabolite or product from an enzyme, etc.). In some embodiments, the vaccine preparation is identified as having suitable potency if it mediates BCR-mediated cell survival. In some embodiment, the vaccine preparation is identified as having suitable potency if it mediates alterations induced by BCR activation and cell survival.

[0042] In some embodiments, the vaccine preparation is identified as having suitable immunogenicity if it mediates BCR activation. In some embodiments, the vaccine preparation is identified as having suitable immunogenicity if it mediates alterations induced by BCR activation and / or BCR-mediated cell survival. In some embodiment, the vaccine preparation is identified as having suitable immunogenicity if it mediates alterations induced by BCR activation. In some embodiments, alterations induced by BCR activation comprises BCR- mediated activation of a reporter gene, and the vaccine preparation is identified as having suitable potency by detecting the product generated by the reporter gene (i.e., luciferase or a fluorescent protein, or a metabolite or product from an enzyme, etc.). In some embodiments, the vaccine preparation is identified as having suitable immunogenicity if it mediates BCR-mediated cell survival. In some embodiment, the vaccine preparation is identified as having suitable immunogenicity if it mediates alterations induced by BCR activation and BCR-mediated cell survival.Attorney Docket No.01217-0002-00PCT Engineered B Cell Receptor (BCR)

[0043] B cell receptor (BCR) is an immunoglobulin complex that has the function of antigen binding and signaling when an antigen binds the receptor. When the B cell receptor binds to an antigen, it initiates a signal through the cytoplasmic tails of lg-alpha and lg-beta chains that are each associated with distinct sets of downstream signaling / effector molecules.

[0044] Provided herein is an engineered BCR which is capable of binding to a target antigen, optionally with low affinity. In some embodiments, the engineered BCR mimics the naïve BCR.

[0045] In some embodiments, the target antigen is an antigenic peptide presented on the surface of a vaccine. In some embodiments, the vaccine is an antigen-presenting vaccine. In some embodiments, the antigen-presenting vaccine is a liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine, conjugate vaccine, etc. In some embodiments, the vaccine is a liposomal vaccine. In some embodiments, the antigenic peptide is derived from a self-antigen. In some embodiments, the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α-synuclein, huntingtin, prion, an amylin protein, IL-17, or IL-27. In some embodiments, the antigenic peptide is derived from β-amyloid (Aβ). In some embodiments, the liposomal vaccine comprises a β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome. In some embodiments, the antigenic peptide comprises amino acids 1-15 of β-amyloid (Aβ) (SEQ ID NO: 23).

[0046] In some embodiments, the BCR is engineered by grafting variable domains of heavy and light chains from an antibody of interest (capable of binding to a target antigen) into an immunoglobulin molecule. In some embodiments, the variable domains of heavy and / or light chains from an antibody can be mature or germline-reverted. In some embodiments, the variable domains of heavy and / or light chains from an antibody can be mature. In some embodiments, the variable domains of heavy and / or light chains from an antibody can be germline-reverted. In some embodiments the membrane-bound immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY). In some embodiments, the membrane-bound immunoglobulin molecule is IgM.

[0047] In some embodiments, the engineered BCR is in a membrane IgM format (mIgM). In some embodiments, the BCR is engineered by grafting variable regions of heavy and light chains from an antibody of interest into an mIgM backbone. In some embodiments, the mIgM backbone is a human mIgM backbone.

[0048] The generation of the engineered BCR can be performed as described in Weaver et al., 2016, Nat. Protocols, 11(2): 193-213, wherein antibodies which bind to the peptide antigenAttorney Docket No.01217-0002-00PCT displaced on the surface on vaccine preparation are reformatted into low-affinity engineered membrane IgM (mIgM) format to best mimic the naïve BCR.

[0049] In some embodiments, the engineered BCR comprises a heavy chain and a light chain. In some embodiments, the heavy chain comprises: a heavy chain variable region (VH) of an antibody that binds a target antigen; and a heavy chain constant region of a membrane IgM (mIgM); and the light chain comprises: a light chain variable region (VL) of the antibody; and a light chain constant region.

[0050] Antibody heavy and light variable domains are encoded by a group of genes, called Variable (V), Diversity (D), and Joining (J). The recombination of these genes encodes for the two structural elements of the variable domain, the framework regions (FRs) and complementarity-determining regions (CDRs). In some embodiments, the VH comprises 3 complementary determining regions (H-CDR1, H-CDR2, and H-CDR3) and 4 framework regions (H-FR1, H-FR2, H-FR3, and H-FR4). In some embodiments, the VL comprises 3 complementary determining regions (L-CDR1, L-CDR2, and L-CDR3) and 4 framework regions (L-FR1, L-FR2, L-FR3, and L-FR4).

[0051] Immunoglobulin genes undergo various modifications during maturation of the immune response, including recombination between V, D and J gene segments, isotype switching, and hypermutation in the variable regions. Recombination and somatic hypermutation are the foundation for generation of antibody diversity and affinity maturation, but they can also generate sequence liabilities that may make commercial production of such immunoglobulins as therapeutic agents difficult or increase the immunogenicity risk of the antibody. In general, mutations in CDR regions are likely to contribute to improved affinity and function, while mutations in framework regions may increase the risk of immunogenicity.

[0052] In some embodiments, where a particular mature antibody differs from its germline sequence at the amino acid level, it may be desirable to mutate the mature antibody sequence back to the germline sequence.

[0053] In certain embodiments, a FR and / or a CDR may comprise one or more amino acid changes for the purposes of “germlining”, mutating back to the germline sequence. For example, the amino acid sequences of selected mature antibody heavy and light chains are compared to germline heavy and light chain amino acid sequences and where certain residues of the selected mature VL and / or VH chains differ from the germline configuration (e.g., as a result of somatic mutation of the immunoglobulin genes used to prepare the phage library), it may be desirable to “revert” the mature altered CDR and / or framework residues of the selected antibodies to theAttorney Docket No.01217-0002-00PCT germline configuration (i.e., change the CDR and / or framework amino acid sequences of the selected mature antibodies so that they are the same as the germline complementary determining and / or framework amino acid sequences). Such “back mutation” (or “reverting”) of mature CDR and / or framework residues can be accomplished by standard molecular biology methods for introducing specific mutations (e.g., site- directed mutagenesis; PCR-mediated mutagenesis, and the like).

[0054] In some embodiments, variable light and / or heavy chain CDRs and FRs can be any combination of mature or germline-reverted.

[0055] In some embodiments, the VH comprises a mature complementarity determining region 3 (H-CDR3). In some embodiments, the VH comprises a mature framework region 4 (H-FR4). In some embodiments, the VL comprises a mature complementarity determining region 3 (L- CDR3). In some embodiments, the VL comprises a mature framework region 4 (L-FR4).

[0056] In some embodiments, the VH comprises one or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. In some embodiments, the VH comprises two or more of: a germline- reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. In some embodiments, the VH comprises three or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. In some embodiments, the VH comprises four or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3. In some embodiments, the VH comprises a germline-reverted H-FR1, a germline-reverted H- CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H- FR3.

[0057] In some embodiments, the VH comprises one or more of: a mature H-FR1, a mature H- CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3. In some embodiments, the VH comprises two or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H- CDR2 and a mature H-FR3. In some embodiments, the VH comprises three or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3. In some embodiments, the VH comprises four or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3. In some embodiments, the VH comprises a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3.Attorney Docket No.01217-0002-00PCT

[0058] In some embodiments, the VL comprises one or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. In some embodiments, the VL comprises two or more of: a germline- reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. In some embodiments, the VL comprises three or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. In some embodiments, the VL comprises four or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3. In some embodiments, the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3.

[0059] In some embodiments, the VL comprises one or more of: a mature L-FR1, a mature L- CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3. In some embodiments, the VL comprises two or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L- CDR2 and a mature L-FR3. In some embodiments, the VL comprises three or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3. In some embodiments, the VL comprises four or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3. In some embodiments, the VL comprises a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3.

[0060] In some embodiments, the VH comprises a mature H-CDR3 and a mature H-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11.

[0061] In some embodiments, the VL comprises a mature L-CDR3 and a mature L-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17.

[0062] In some embodiments, the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21.

[0063] In some embodiments, the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22.Attorney Docket No.01217-0002-00PCT

[0064] In some embodiments, the VH comprises a germline-reverted H-FR1, a germline- reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline- reverted H-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

[0065] In some embodiments, the VL comprises a germline-reverted L-FR1, a germline- reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline- reverted L-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16.

[0066] In some embodiments, the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12.

[0067] In some embodiments, the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15.

[0068] In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13.

[0069] In some embodiments, the light chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18.

[0070] In some embodiments, the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

[0071] In some embodiments, the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14.

[0072] In some embodiments, the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7.Attorney Docket No.01217-0002-00PCT

[0073] In some embodiments, the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, for 100% identity to SEQ ID NO: 8.

[0074] In some embodiments, the engineered BCR comprises a reporter gene operably linked to a promoter that is responsive to activation of the BCR.

[0075] In some embodiments, the promoter comprises one or more enhancer elements. In some embodiments, the promoter comprises one or more nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) responsive enhancer elements. In some embodiments, the promoter comprises one or more NFAT-response elements.

[0076] In some embodiments, the promoter comprises an NFAT promoter, NF-kB promoter, AP-1 promoter, FOXO promoter, STATS promoter, or an IRF promoter.

[0077] In some embodiments, the promoter comprises one or more nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) responsive enhancer elements. In some embodiments, NF-κB activity is upregulated following BCR activation. In some embodiments, the promoter comprises a NF-κB / activator protein 1 (AP-1) inducible promoter.

[0078] In some embodiments, the reporter gene encodes an enzyme or a fluorescent protein. In some embodiments, the reporter gene encodes a luciferase, an alkaline phosphatase, a β- lactamase, a β-galactosidase, β-glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase. In some embodiments, the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP).

[0079] In some embodiments, the engineered BCR does not comprise a reporter gene operably linked to a promoter. Engineered B Cell Line

[0080] Provided herein is an engineered B cell line comprising an engineered B cell receptor (BCR) as discussed herein.

[0081] In some embodiments, the engineered B cells are capable of transmitting down-stream signals following BCR activation, or such signaling capacity is provided by co-expressing elements of the signaling molecules otherwise not present. In some embodiments, the engineered B cell line is selected to present a high, medium, or low expression of the engineered BCR.

[0082] In some embodiments, the B cell line does not express an endogenous immunoglobulin heavy chain or an endogenous immunoglobulin light chain. In some embodiments, expression of endogenous immunoglobulin in the B cell line is eliminated or reduced. In some embodiments,Attorney Docket No.01217-0002-00PCT the B cell line does not express endogenous surface IgM. A B cell line that does not express an endogenous surface IgM excludes interference from the endogenous surface IgM with the engineered B cell receptor and / or B cell receptor downstream activation signal.

[0083] Any cell that is capable, or that is made to be capable of transmitting down-stream signals following B cell receptor activation, whether expressed endogenously or in trans, can be used. These include, for example, but not limited to, mature B cells isolated from humans, vertebrates, mammals, birds, rodents, or other animals, transgenic animals, or immortalized cell lines. B cells can be immortalized by any method known in the art, preferably in such a way that does not interfere, or negatively affect the signal to noise ratio of the assays discussed herein (see, for example, Wiesner et al., 2008. Conditional immortalization of human B cells by CD40 ligation. PLoS ONE 3(l):el464; Kusam & Dent, 2007. Common Mechanisms for the Regulation of B Cell Differentiation and Transformation by the Transcriptional Repressor Protein Bcl-6. Immunol Res 37(3): 177-86).

[0084] A cell can be made to be capable of transmitting down-stream signals following BCR activation by expressing other proteins of the B cell receptor signaling complex, including, for example, but not limited to, immunoglobulin alpha and beta, and any other molecules required for downstream signaling, including, for example, but not limited to, CD19, CD2, CD40, CD45, PIR-B, FcγRIIBl, CRAC Channel (Ca +), Lyn, Syk, Btk, PI3K p85 & pi 10, Akt, PRK2, PKC, TAKl, MEKKs, MKK3 / 46, MKK4 / 7, p38, JNK, JNK1 / 2, c-Raf, MEK1 / 2, Erkl / 2, IKK, GSK-3, mTOR, p70 S6K,CaMK, IP3R, SHP1, SHP2, SHIP, PTEN, Calcineurin, Rho, Rac / cdc42, RhoA, Rap, Ras, Rheb, Gab, BCAP, She, Dok-3, ezrin, BAM-32, clathrin, Nek, BLNK, Cbl, GRB2, LAB, STIM1, TSC2, CARMAl , BcllO, CaM, ΙκΒ, RapL, Riam, PLCγ2, MALT1, Vav, SOS, RasGRP, RasGAP, NFκB, NFAT, CREB, ATF02, Jun, Bcl-6, Egr-1, Elk-1, Bfl-1, Oct-2, Ets-1, FoxO, and Bcl-xL.

[0085] In some embodiments, mature B cells from non-transgenic vertebrate animals, such as, for example, but not limited to, humans, mammals, birds, primates, or rodents are isolated by any means known to one of ordinary skill in the art and altered by any method known to one of ordinary skill in the art to suppress the expression of endogenous immunoglobulin. In some embodiments, mature primary B cells are isolated by any means known to one of ordinary skill in the art from transgenic vertebrate animals, such as, for example, but not limited to, transgenic mammals, birds, primates, or rodents, in which gene expression is altered by any method known to one of ordinary skill in the art to suppress the expression of endogenous immunoglobulin. In some embodiments, immortalized mature B cells from any vertebrates, such as, for example, butAttorney Docket No.01217-0002-00PCT not limited to, humans, mammals, birds, primates, or rodents are altered by any method known to one of ordinary skill in the art to suppress the expression of endogenous immunoglobulin. In some embodiments, the primary cells described above are immortalized by such methods as described above. In another preferred embodiment, commercially available, immortalized cell lines are used.

[0086] A non-limiting example of a cell line that may be used to practice the invention described herein is the chicken DT40 cell line. B-cell development in chickens and mammals are similar processes, including at the molecular level and in the regulatory networks. The chicken DT40 cell line is an avian leucosis virus-induced bursal B-cell lymphoma line that overexpresses c-myc and lacks p53 expression but otherwise has a stable pheno- and karyotype. The cell line appears to be arrested at the bursal stem cell stage of differentiation as it has on-going Ig diversification, and BCR ligation leads to apoptosis rather than proliferation. DT40 cells have several orders of magnitude higher homologous integration frequency than any other vertebrate cell lines described to date. This provides versatile options for deleting and replacing genes, which may prove very useful in refinement of an assay (Kohonen P et al.2007. Scand J Immunol 66:113-21).

[0087] DT40 cells express surface IgM, and have been used to study BCR signaling. Much of what is known about the mechanisms of activation, interactions and hierarchy relationships of the multiple components that make up the BCR signaling pathway has been elucidated in DT40 cells (Winding & Berchtold, 2001. J Immunol Methods 249: 1-16; Kurosaki, 2002. Nat Rev Immunol 2:354-63). The DT40 cell line has also played a central role in elucidating the role and mechanism of action of AID, and has on-going Ig diversification due to AID expression (Winding & Berchtold, 2001. J Immunol Methods 249: 1-16; Arakawa, Saribasak, & Buerstedde, 2004. PLoS Biol 2:el79). For the purpose of expressing characterized immunoglobulin on the surface of these cells, however, somatic mutation would obscure the understanding of antibody / epitope-specific BCR signaling. Therefore, an AID knock-out DT40 cell line is preferable (Arakawa, Hauschild, & Buerstedde, 2002. Science 295:1301-6).

[0088] Background would not be expected to be high due to cross-reactivity of DT40 endogenous surface immunoglobulin. However, heterologous co-expression of the antibodies selected for expression as BCR components may result in competition for other components of the BCR complex, such as the chicken Iga and IgP chains. Therefore, a DT40 cell line that, in addition to its AID- / -karyotype, lacks IgM heavy and light expression would be even more preferable (Arakawa, Hauschild, & Buerstedde, 2002. Science 295:1301-6).Attorney Docket No.01217-0002-00PCT

[0089] Other cell lines that may be used to practice the invention include, as non-limiting examples, Ramos and CH12 cells. The Ramos human Burkitt’s lymphoma cell line, like DT40 cells, is transformed by c-myc over-expression and does not possess the Epstein Barr Virus (EBV) genome. The cells have B lymphocyte characteristics, with surface associated μ and κ chains, and have been used extensively as model B lymphocytes for apoptosis studies. CH12 is a murine B- cell lymphoma-derived cell line that expresses both I-A and I-E class II molecules and μ / κ surface IgM with specificity for SRBC. CH12 cells can, in some embodiments, be stimulated with LPS.

[0090] In some embodiments, the engineered cell line is an engineered Ramos cell line, DT40 cell line, CH12 cell line, Burkitt’s lymphoma B cell line, primary B cell line, or stem cell- derived B cell line. In some embodiments, the engineered B-cell line is an engineered Ramos cell line.

[0091] In some embodiments, the engineered B cells comprise a reporter gene operably linked to a promoter that is responsive to activation of the BCR. In some embodiments, the engineered BCR does not comprise a reporter gene operably linked to a promoter.

[0092] In some embodiments, the promoter comprises one or more enhancer elements. In some embodiments, the promoter comprises one or more nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) responsive enhancer elements. In some embodiments, the promoter comprises one or more NFAT-response elements.

[0093] In some embodiments, the promoter comprises an NFAT promoter, NF-kB promoter, AP-1 promoter, FOXO promoter, STATS promoter, or an IRF promoter.

[0094] In some embodiments, the promoter comprises one or more NF-κB responsive enhancer elements. In some embodiments, NF-κB activity is upregulated following BCR activation. In some embodiments, the promoter comprises a NF-κB / activator protein 1 (AP-1) inducible promoter.

[0095] In some embodiments, the reporter gene encodes an enzyme or a fluorescent protein. In some embodiments, the reporter gene encodes a luciferase, an alkaline phosphatase, a β- lactamase, a β-galactosidase, β-glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase. In some embodiments, the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP). Methods

[0096] Provided herein is a method of assessing a vaccine preparation. In some embodiments, the method comprises assessing and / or predicting antibody response to a vaccine preparation. InAttorney Docket No.01217-0002-00PCT some embodiments, the method comprises assessing and / or predicting immunogenicity, potency, or both, of a vaccine preparation. In some embodiments, the method comprises assessing the ability of an antigen to activate B-cell or to induce BCR signaling.

[0097] In some embodiments, the vaccine preparations are antigen-presenting vaccine preparations. In some embodiments, the antigen-presenting vaccine preparation is a liposomal vaccine preparation, DNA scaffold vaccine preparation, nanoparticle vaccine preparation, Virus- Like Particles (VLP) vaccine preparation or conjugate vaccine preparation. In some embodiments, the vaccine preparation is a liposomal vaccine preparation. In some embodiments, the method comprises assessing presentation of the peptide antigen on the liposomal vaccine surface.

[0098] Provided herein are methods comprising of assessing and / or predicting antibody response to a vaccine preparation, comprising contacting an engineered B cell line disclosed herein with a vaccine preparation and optionally an inhibitor of cell proliferation and / or an inhibitor of actin polymerization element, wherein the engineered B cell line comprises an engineered BCR disclosed herein; and assessing BCR activation. Assessing BCR activation comprises assessing and / or detecting 1) alterations (such as downstream signaling) induced by BCR activation and / or 2) BCR-mediated cell survival.

[0099] In some embodiments, the inhibitor of cell proliferation and / or inhibitor of actin polymerization element is present. In some embodiments, the inhibitor of cell proliferation and / or inhibitor of actin polymerization element comprises Cytochalasin D. [000100] In some embodiments, the method comprises assessing and / or predicting antibody response to a vaccine preparation, comprising contacting an engineered B cell line disclosed herein with a vaccine preparation and optionally an inhibitor of cell proliferation and / or an inhibitor of actin polymerization element, wherein the engineered B cell line comprises an engineered BCR disclosed herein; and assessing and / or detecting BCR-mediated cell survival. [000101] In some embodiments, the engineered BCR comprises a reporter gene operably linked to a promoter that is responsive to activation of the BCR. In some embodiments, the engineered BCR does not comprise such a reporter gene. [000102] In some embodiments, the methods comprise detecting and comparing BCR-mediated cell survival of the engineered B cell line contacted with the vaccine preparation to BCR- mediated cell survival of the same engineered B cell line contacted with a second (or more) vaccine preparation(s) and / or a control vaccine preparation. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are different preparations of theAttorney Docket No.01217-0002-00PCT same vaccine. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising the same antigen. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising different antigens. In some embodiments, the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated cell survival of the cell line. In some embodiments, the methods can identify defective batches of vaccine preparations and / or define process parameters that significantly impact the B-cell epitope presentation. [000103] In some embodiments, the method comprises assessing and / or detecting antibody response to a vaccine preparation, comprising contacting an engineered B cell line as disclosed herein with a vaccine preparation and optionally an inhibitor of cell proliferation and / or an inhibitor of actin polymerization element, wherein the engineered B cell line comprises a BCR as disclosed herein; and assessing and / or detecting alterations (such as downstream signaling) induced by BCR activation. [000104] In some embodiments, the engineered BCR comprises a reporter gene operably linked to a promoter that is responsive to activation of the BCR. In some embodiments, assessing and / or detecting alterations induced by BCR activation comprises assessing and / or detecting BCR-mediated activation of the reporter gene. [000105] In some embodiments, assessing and / or detecting alterations induced by BCR activation comprises assessing and / or detecting expression of the reporter gene operably linked to the promoter that is responsive to activation of the BCR. In some embodiments, assessing and / or detecting expression of the reporter gene includes detecting the presence of a product generated by the reporter gene (i.e., photon from luciferase or a fluorescent protein, or a metabolite or product form an enzyme, etc.) and / or quantifying the mRNA or protein expression of the reporter gene. [000106] In some embodiments, the methods comprise detecting and comparing alterations induced by BCR activation of the engineered B cell line contacted with the vaccine preparation to alterations induced by BCR activation of the same engineered B cell line contacted with a second (or more) vaccine preparation(s) and / or a control vaccine preparation. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are different preparations of the same vaccine. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising the same antigen. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising different antigens. In some embodiments, the method comprises ranking the vaccineAttorney Docket No.01217-0002-00PCT preparations according to their ability to induce BCR-mediated alterations (such as downstream signaling). In some embodiments, the methods can identify defective batches of the vaccine preparation and / or define process parameters that significantly impact the B-cell epitope presentation. [000107] In some embodiments, the methods comprise detecting and comparing BCR-mediated activation of the reporter gene of the engineered B cell line contacted with the vaccine preparation to BCR-mediated activation of the reporter gene of the same engineered B cell line contacted with a second vaccine preparation and / or a control vaccine preparation. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising the same antigen. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising different antigens. In some embodiments, the method comprises ranking the vaccine preparations according to their ability to induce BCR- mediated activation of the reporter gene of the engineered B cell line. In some embodiments, the methods can identify defective batches of the vaccine preparation and / or define process parameters that significantly impact the B-cell epitope presentation. [000108] In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) comprise the same peptide antigen. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) comprise different peptide antigen. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are different preparations of the same vaccine. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are preparations of different vaccines. In some embodiments, the control vaccine preparation is a preparation of a vaccine that does not comprise a peptide antigen recognized by the BCR. In some embodiments, the control vaccine preparation is a preparation of a vaccine that has previously been determined to have immunogenicity in vivo. [000109] In some embodiments, the method comprises assessing and / or detecting alterations induced by BCR activation and BCR-mediated cell survival. In some embodiments, the method comprises assessing and / or detecting BCR-mediated activation of a reporter gene and BCR- mediated cell survival. [000110] In some embodiments, the engineered B cell line is contacted with the vaccine preparation(s) and the inhibitor of cell proliferation and / or inhibitor of actin polymerization element. In some embodiments, the engineered B cell line is contacted with the vaccine preparation(s) and cytochalasin D. In some embodiments, the engineered B cell line is contactedAttorney Docket No.01217-0002-00PCT with vaccine preparation(s) and about 0.1 - 10 µM, about 0.2-5 µM, or about 5 µM cytochalasin D. In some embodiments, the engineered B cell line is contacted with a vaccine preparation and about 5 µM cytochalasin D. [000111] In some embodiments, the engineered B cells as discussed herein are primed with a co-stimulatory reagent before being contacted with the vaccine preparation(s). In some embodiments, the engineered B cells are primed with a co-stimulatory reagent for at least about 24 hours before being contacted with the vaccine preparation(s). In some embodiments, the co- stimulatory reagent is CD40L, IFNα, or human Interleukin-4 (IL4). In some embodiments, the co-stimulatory reagent is human IL4. In some embodiments, the engineered B cells are primed with about 1-20 mg / mL, about 5-10 ng / mL, or about 10 ng / mL of IL4. In some embodiments, the engineered B cells are primed with about 10 ng / mL of IL4. 1) Detecting BCR activation and analyzing / detecting alterations induced by BCR activation [000112] Any method known to one of ordinary skill in the art may be used to assay biochemical, biophysical, any other alterations of downstream signaling, or changes in their subcellular localization before and after exposure of the engineered B cell line as discussed herein (comprising the engineered BCR as discussed herein) to an antigen or vaccine preparation comprising a peptide of the antigen. [000113] Alterations induced by BCR activation that may be assayed include, for example, but not limited to, elevated or diminished enzymatic activity (e.g. tyrosine or serine / threonine kinase and phosphatase activities), protein / substrate phosphorylation or dephosphorylation, or any other kind of post-translational modifications, and association with other molecules. As non-limiting examples, G-proteins may more prevalently be associated with GTP or GDP following B cell stimulation, adapter proteins may associate with, or dissociate from, cytoskeletal / structural or enzymatic proteins. Other methods for assaying signaling downstream of B cell receptor activation include second messenger analysis, such as, for example, but not limited to, measuring intracellular calcium flux. Non-limiting examples of methods for assaying signaling molecules are described in US Application 2009 / 0111710, which may be adapted by methods known to one of ordinary skill in the art to assaying for signaling molecules downstream of BCR activation. [000114] Increases or decreases in transcriptional activity may be monitored as a method of analyzing signaling downstream of BCR activation. Transcription rates of genes known or discovered to be regulated by BCR activation-induced signaling can be analyzed by any methodAttorney Docket No.01217-0002-00PCT known to one of ordinary skill in the art, including, for example, but not limited Northern blot analysis and RT PCR. [000115] One example include transcription factor responsive reporter gene assays, whereby DNA constructs, such as, for example, plasmids, cosmids, BACs, etc., comprising a transcription factor responsive promoter, such as, for example, a promoter comprising an NF kappa B-responsive element, that drives / regulates transcription of a reporter gene, such as, for example, a firefly or a Renilla luciferase, and any other components required for replication, selection, integration, etc., are transfected, and luciferase activity is assayed by luminescence or fluorescence before and after exposure of one or more cells expressing an antibody of interest to a target antigen. Cells may comprise one or more stably integrated copies of the DNA construct, stably maintained copies of the DNA construct, or the DNA construct may be available for the assay temporarily, such as, for example, but not limited to, following transient transfection. [000116] In some embodiments, alterations induced by BCR activation comprises a reporter gene operably linked to a promoter that is responsive to activation of the BCR. In some embodiments, assessing and / or detecting alterations induced by BCR activation comprises assessing and / or detecting BCR-mediated activation of the reporter gene. [000117] In some embodiments, assessing and / or detecting alterations induced by BCR activation comprises assessing and / or detecting expression of the reporter gene operably linked to the promoter that is responsive to activation of the BCR. In some embodiments, assessing and / or detecting expression of the reporter gene includes detecting the presence of a product generated by the reporter gene (i.e., photon from luciferase or a fluorescent protein, or a metabolite or product form an enzyme, etc.) and / or quantifying the mRNA or protein expression of the reporter. In some embodiments, expression of the reporter gene is detected by measuring transcription rates of a reporter gene that is under transcriptional regulation of a promoter that is responsive to a transcription factor that is itself up- or down-regulated in response to BCR activation. [000118] In some embodiments, the promoter comprises one or more enhancer elements, which can be nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) responsive enhancer elements or NFAT-responsive elements. In some embodiments, the reporter gene is under the control of promoters comprising one or more c-Rel NFκB- responsive elements, which provides highly sensitive assays. These assays are applied to assay for cRel / NFκB-mediated induction of transcription, which itself can serve as a marker for cellular responses and differentiation. In response to BCR activation and downstream signaling that leads to aAttorney Docket No.01217-0002-00PCT proliferative response, cRel binds to the canonical NFκB binding site, and up-regulates transcription of genes under the control of promoters that contain these binding sites. Thus, an assay that reports cRel-mediated up-regulation provides a functional read-out of BCR-induced signals that lead to proliferative responses. These assays can be carried out in cells that lead to a proliferative response, such as, for example, but not limited to, human peripheral mature naive B cells (Kohonen P et al.2007. Scand J Immunol 66:113-21). [000119] In some embodiments, the promoter comprises an NFAT promoter, NF-kB promoter, AP-1 promoter, FOXO promoter, STATS promoter, or an IRF promoter. [000120] In some embodiments, the promoter comprises a NF-κB / activator protein 1 (AP-1) inducible promoter. [000121] In some embodiment, the reporter gene encodes an enzyme or a fluorescent protein. In some embodiments, the reporter gene encodes a luciferase, an alkaline phosphatase, a β- lactamase, a β-galactosidase, β-glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase. In some embodiments, the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP). [000122] In some embodiments, BCR-mediated activation of the reporter gene is detected using an alkaline phosphatase activity assay. In some embodiments, BCR-mediated activation of the reporter gene is detected by measuring SEAP secretion. [000123] In some embodiments, the promoter is a NF-κB / activator protein 1 (AP-1) inducible promoter and the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP). Engagement and crosslinking of the engineered BCRs by the vaccine preparation triggers B-cell / BCR activation, which can be measured by a dose-dependent SEAP release upon upregulation of the reporter gene for the NF-kB / AP-1 pathway. [000124] In some embodiments, the BCR-mediated activation of the reporter gene of the engineered B cell line contacted with the vaccine preparation is compared to the BCR-mediated activation of the reporter gene of the same engineered B cell line contacted with a second vaccine preparation and / or a control vaccine preparation. In some embodiments, the method comprises comparing multiple batches of vaccine preparations comprising the same antigen. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising different antigens. In some embodiments, the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated activation of the reporter gene of the engineered B cell line.Attorney Docket No.01217-0002-00PCT [000125] In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) comprise the same peptide antigen. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) comprise different peptide antigens. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are different preparations of the same vaccine. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are preparations of different vaccines. In some embodiments, the control vaccine preparation is a preparation of a vaccine that does not comprise a peptide antigen recognized by the BCR. In some embodiments, the control vaccine preparation is a preparation of a vaccine that has previously been determined to have immunogenicity in vivo. 2) BCR-mediated Cell Survival [000126] In some embodiments, engagement and crosslinking of the engineered BCRs by the vaccine preparation triggers B-cell / BCR activation, which can be measured by a dose- dependent BCR-mediated cell survival. [000127] In some embodiments, the BCR-mediated cell survival is assessed using a cell-toxicity assay or cell viability assay, for example, such as those discussed in Weyermann et al., 2004, Int. J. Pharm., 288(2): 369-376. In some embodiments, the assay measures cytoplasmic enzyme activity released by damaged cells, such as lactate dehydrogenase (LDH), which is rapidly released into the cell culture supernatant upon damage of the plasma membrane. In some embodiments, the assay measures the reduction of a tetrazolium salt by oxydoreductase enzymes released by dead cells. In some embodiments, the assay measures the number of viable, uninjured cells in cultures, for example, by using neutral red (3-amino-m-dimethylamino-2- methyl-phenazine hydrochloride) and measuring the uptake and subsequent lysosomal accumulation of the supravital dye, neutral red. In some embodiments, the assay measures adenosine triphosphate (ATP) that is present in all metabolically active cells, which can be determined in a bioluminescent measurement. [000128] In some embodiments, the assay measures the reduction of a tetrazolium salt by oxydoreductase enzymes released by dead cells. In some embodiments, the cell viability indicator reagent is a tetrazole selected from the following: MTT (3-[4,5-dimethylthiazole-2-yl]- 2,5-diphenyltetrazolium bromide), XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- carboxanilide-2H-tetrazolium), MTS (5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3- (4-sulfophenyl)tetrazolium, inner salt) or WSTs (i.e., (4-[3-4-iodophenyl]-2-(4-nitrophenyl)-2H- 5-tetrazolio)-1,3-benzene disulfonate).Attorney Docket No.01217-0002-00PCT [000129] In some embodiments, the assay comprises measuring reduction of MTT, XTT, MTS, or WSTs, such as water-soluble tetrazolium 8 (WST-8). In some embodiments, the assay comprises MTT assay or OranguTMassay. [000130] In some embodiments, the BCR-mediated survival of the cell line contacted with the vaccine preparation is compared to the BCR-mediated survival of the same cell line contacted with a second vaccine preparation and / or a control vaccine preparation. In some embodiments, the method comprises comparing multiple batches of vaccine preparations comprising the same antigen. In some embodiments, the methods comprise comparing multiple batches of vaccine preparations comprising different antigens. In some embodiments, the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated cell survival of the engineered B cell line. [000131] In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are different preparations of the same vaccine. In some embodiments, the vaccine preparation and the second (or more) vaccine preparation(s) are preparations of different vaccines. In some embodiments, the control vaccine preparation is a preparation of a vaccine that does not comprise a peptide antigen recognized by the BCR. In some embodiments, the control vaccine preparation is a preparation of a vaccine that has previously been determined to have immunogenicity in vivo. Vaccine preparations [000132] In some embodiments, the vaccine preparation is an antigen-presenting vaccine preparation. In some embodiments, the antigen-presenting vaccine preparation is a liposomal vaccine preparation, DNA scaffold vaccine preparation, nanoparticle vaccine preparation, Virus- Like Particles (VLP) vaccine preparation, or conjugate vaccine preparation. In some embodiments the vaccine preparation is a liposomal vaccine preparation. In some embodiments, the vaccine preparation comprises a peptide antigen presented on the surface of the liposome. [000133] In some embodiments, the vaccine preparation comprises at least one adjuvant, for example, such as those discussed in Facciolà, Alessio, et al. "An overview of vaccine adjuvants: current evidence and future perspectives." Vaccines 10.5 (2022): 819. As used herein, the term “adjuvant” refers to any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens. In some embodiments, the at least one adjuvant is an aluminum salt (such as such as aluminum hydroxide, aluminum phosphate or aluminum sulfate), an oil-in-water emulsion (such as MF59, AS03, Freund’s adjuvants), a microparticle (such as virus-like particles, virosomes, polylacticAttorney Docket No.01217-0002-00PCT acid, polylactic acid-co-glycolic acid), a toll-like receptor (TLR) agonist, a mucosal adjuvant (such as Cholera toxin, heat-labile enterotoxin (LTK3 and LTR72), Chitosan) or a combined adjuvant (such as AS01, AS02, AS04). In some embodiments, the toll-like receptor (TLR) agonist is a TLR1 / 2 agonist (such as L-pampo, MALP-2, Pam2CSK4, Pam3CSK4), a TLR3 agonist (such as Poly(I:C) (polyinosinic:polycytidylic acid)), a TLR4 agonist (such as Monophosphoryl lipid A), a TLR5 agonist (such as Flagellin), a TLR7 / 8 agonist (such as Imiquimod (R837; 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) and resiquimod (R848, 4-amino-2-(etoximetil)-a,a-dimethyl-1H-imidazo [4,5-c]quinoline-1-ethanol)) or a TLR9 agonist (such as CpG). [000134] In some embodiments, the vaccine preparation comprises at least one pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier that may be administered to a patient, together with an antigen, and does not destroy the pharmacological activity thereof and is non-toxic when administered in doses sufficient to deliver a pharmaceutically effective amount of the compound. Such pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. The vaccine preparations may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen- free, phosphate-buffered physiologic saline is a typical carrier. [000135] In some embodiments, the liposomal vaccine comprises an antigenic peptide derived from a self-antigen. In some embodiments, the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α-synuclein, huntingtin, prion, an amylin protein, IL-17, or IL-27. In some embodiments, the antigenic peptide is derived from β-amyloid (Aβ). [000136] In some embodiments, the liposomal vaccine comprises a β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome. In some embodiments, the antigenic peptide comprises, consists essentially of or consists of amino acids 1-15 of β-amyloid (Aβ) (SEQ ID NO: 23). Such anti-Aβ vaccines are disclosed, for example, in WO2007 / 068411 and WO2019 / 197414. [000137] In some embodiments, the liposomal vaccine comprises a Tau-derived peptide antigen displayed on the surface of the liposome. Such anti-Tau vaccines are disclosed, for example, in WO2010 / 115843 and WO2019 / 084118. [000138] In some embodiments, the liposomal vaccine comprises a α-synuclein -derived peptide antigen displayed on the surface of the liposome. Such anti- α-synuclein vaccines are disclosed, for example, in WO2023 / 152260.Attorney Docket No.01217-0002-00PCT [000139] In some embodiments, the liposomal vaccine is anti-Aβ vaccine (ACI-24). [000140] In some embodiments, the vaccine preparation comprises an antigenic peptide derived from a self-antigen. In some embodiments, the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α-synuclein, huntingtin, prion, an amylin protein, IL-17, or IL-27. In some embodiments, the antigenic peptide is derived from β-amyloid (Aβ). [000141] In some embodiments, the vaccine preparation comprises a β-amyloid (Aβ)-derived peptide antigen conjugated to a carrier. Such anti- β-amyloid (Aβ) vaccines are disclosed, for example, in WO2014 / 143087. [000142] In some embodiments, the vaccine preparation comprises a Tau-derived peptide antigen conjugated to a carrier. Such anti-Tau vaccines are disclosed, for example, in WO2013 / 041962 and WO2019 / 084488. [000143] In some embodiments, the vaccine preparation comprises an α-synuclein-derived peptide antigen conjugated to a carrier. Such anti-α-synuclein vaccines are disclosed, for example, in WO2009 / 103105 and WO2022 / 029181. Certain Kits [000144] The present invention also provides kits. In some embodiments, the kits comprise one or more of the engineered B-cell line disclosed herein. In some embodiments, the kits comprise a vaccine preparation as discussed herein. [000145] In some embodiments, the kits may comprise reagents for detecting BCR activation. In some embodiments, the kits may further comprise reagents for detecting alterations induced by BCR activation. In some embodiments, the kits may further comprise reagents for detecting BCR-mediated cell survival. In some embodiments, the kits may further comprise reagents for detecting alterations induced by BCR activation and cell survival. [000146] In some embodiments, the kit may comprise reagents for detecting BCR-mediated activation of the reporter gene and / or BCR-mediated cell survival. In some embodiments, the kit may further comprise reagents for detecting BCR-mediated activation of the reporter gene. In some embodiments, the kit may further comprise reagents for detecting BCR-mediated cell survival. In some embodiments, the kit may further comprise reagents for detecting BCR- mediated activation of the reporter gene and BCR-mediated cell survival. [000147] In some embodiments, the kit can also contain instructions for detecting BCR activation.Attorney Docket No.01217-0002-00PCT EXAMPLES [000148] The following examples are included to further describe some embodiments of the present disclosure and should not be used to limit the scope of the disclosure. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. [000149] While aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the aspects of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. [000150] The following abbreviations are used in the Examples and elsewhere in the application. Name Abbreviation Amino acids AA Amyloid beta Abeta Alexa fluor AF Alkaline phosphatase AP Activator protein 1 AP-1 Allophycocyanin APC Area under the curve AUC B cell receptor BCR Biotinylated vaccine Biot-vaccine Cytochalasin D CD Complementarity-determining regions CDRs Chinese hamster ovary CHO Certificate of analysis CoA Diversity gene D gene Double-negative DN Deoxyribonucleic acid DNA Enzyme-linked immunosorbent assay ELISA Extracellular signal-regulated kinase ERK Fluorescence-activated cell sorting FACSAttorney Docket No.01217-0002-00PCT Framework region FR Heavy chain HC Heavy chain Complementarity-determining region HC_CDR Immunoglobulin G IgG Interleukine-4 IL-4 Joining gene J gene Light chain LC Light chain Complementarity-determining region LC_CDR Membrane Immunoglobulin M mIgM Milliliter mL (unit) Multiplicity of infection MOI Nuclear factor kappa B NF-kB Nanomolar nM Phosphate-buffered saline PBS Phycoerythrin PE Proof of concept PoC Secreted embryonic alkaline phosphatase SEAP Streptavidin Strep Transduction units Tu Variable gene V gene Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element WPRE Microgram μg (unit) Microliter μL Example 1. ACI-24 Cell-Based Potency Reporter Assay Development Overview [000151] Commercially available anti A-beta antibodies were screened for their ability to bind to ACI-24 vaccine B-cell epitope – palmitoylated peptides from the amino-acid sequence 1-15 of Amyloid beta (A-beta1-15; SEQ ID NO: 23) at the surface of ACI-24 liposomes.6E10 was able to bind to biotinylated ACI-24 in an ELISA format and selected (data not shown). [000152] 6E10 antibody was reformatted into a low-affinity engineered membrane IgM (mIgM) format (see Example 2) to best mimic the naïve BCR. After validating the integrity and binding properties of the IgM constructs, the Ramos-BlueTMreporter cell line (see Example 3) was transduced with engineered anti Abeta BCRs and stable mIgM-expressing cells were isolated by fluorescence-activated cell sorting (FACS) (see Example 4 and 5). These cells were then used to develop a B-cell activation assay by 1) BCR-mediated reporter gene activity for the NF-kB / AP- 1 pathway and 2) BCR-mediated cell survival (see Example 6). Example 2. Reformatting Anti-Abeta Antibodies as BCR IgMAttorney Docket No.01217-0002-00PCT [000153] The generation of engineered mIgM is performed as described in Weaver et al., 2016, Nat. Protocols, 11(2): 193-213. Briefly, antibody heavy and light variable domains are encoded by a group of genes, called Variable (V), Diversity (D), and Joining (J). The recombination of these genes encodes for the two structural elements of the variable domain, the framework regions (FRs) and complementarity-determining regions (CDRs). The germline precursors of the antibodies of interest are first inferred and then configured into a membrane IgM format. [000154] V gene–only reversions of heavy and light chains (FR1, CDR1, FR2, CDR2 and FR3) are engineered using the IMGT webtool (www.imgt.org / IMGT_vquest / vquest), while retaining the mature CDR3 and FR4 (remaining sequence downstream to the CDR3 but preceding the immunoglobulin constant region, the Joining segment). [000155] A leader sequence is added N-terminal of the V gene–reverted variable region. The V regions were reverted to Mus musculus germlines. The variable region (V gene–reverted FR1, CDR1, FR2, CDR2, FR3 + mature CDR3 and FR4) is synthesized and preceded by the leader sequence to direct translation into the lumen of the endoplasmic reticulum (ER). Leader sequences can be analyzed by the SignalP 4.1 server (www.cbs.dtu.dk / services / SignalP / ) to confirm the presence and location of signal peptide cleavage sites. [000156] The variable domain of the germline-reverted heavy chain is added to the constant region encoding mIgM available in GenBank (accession no. X17115). [000157] The final gene is subcloned into a pcDNA3.4-TOPO expression vector. [000158] For anti-Abeta antibody 6E10, the following DNA sequences were used to predict the germline gene segments (see US Patent No.8,614,298 B2): >VH_6E10_humanized_variant GAGGTTCAGTTGGTGCAGTCTGGGGCAGAGCTTAAGAAGCCAGGGGCCTCAGTCAAGGTGTCCTGTACAGCTTCTGGTTTCA ACATTAAAGACACCTATATACATTGGGTGAGGCAGGCCCCTGGACAGCGCCTGGAGTGGATTGGAAGGTTTGATCCTGTGAA TGTTAATACTAGATATGACTCGCGGTTCCGGGGCAGGGCCACTATAACATCAGACGCATCCACCAATACAGCCTACATGGAGC TCAGCAGCCTGAGATCTGAGGACACTGCCGTCTATTACTGTTCTAGGTCTTATTACAACGGTAGAAGACGCTTTACTTACTGGG GCCAAGGGACTCTGGTCACTGTCTCTTCA (SEQ ID NO: 1) >6E10_VL_humanized_variant GACATTGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCACTGGGAGAGAGGGCCACTATCAACTGCAAGGCCAGTCAGA GTCTGTTAAGCAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAACAGAAACCAGGGCAGCCTCCTAAATTGTTGATCTAC TGGGCATCTATTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACATTTTTCACTCTCACCATCAGTAGT CTGCAGGCTGAAGACGTGGCAGTGTATTACTGTCAGAATGATTATAATTATCCATTCACATTCGGCCAGGGGACAAAGTTGGA GATAAAA (SEQ ID NO: 2) [000159] The results are shown in Tables 1 and 2:Attorney Docket No.01217-0002-00PCT Table 1: 6E10 VH: IMGT V-QUEST Output Result summary: Productive IGH rearranged sequence (no stop codon and in-frame junction) Mus musculus IGHV14- identity = 88.89% (256 / 288 V-GENE and allele 3*02 F score = 1147 nt) J-GENE and allele Mus musculus IGHJ3*01 F score = 181 identity = 87.23% (41 / 47 nt) D-GENE and allele by IMGT / Junction Mus musculus IGHD1- D-REGION is in reading frame 3 Analysis 1*01 F FR-IMGT lengths, CDR-IMGT lengths and [25.17.38.11] [8.8.13] CSRSYYNGRRRFTYW AA JUNCTION (SEQ ID NO: 19) Table 2: 6E10 VL: IMGT V-QUEST Output Result summary: Productive IGK rearranged sequence (no stop codon and in-frame junction) V-GENE and allele Mus musculus score = 1273 identity = 92.26% (274 / 297 IGKV8-19*01 F nt) J-GENE and allele Mus musculus IGKJ4*01 F score = 149 identity = 89.19% (33 / 37 nt) FR-IMGT lengths, CDR- IMGT lengths and AA [26.17.36.10] [12.3.9] CQNDYNYPFTF (SEQ ID NO: 20) JUNCTION [000160] A human IgM leader (ATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGTCCAGTCC; SEQ ID NO: 3) is added to the heavy chain and a different leader (ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGATCCACTGGT; SEQ ID NO: 4) is added to the light chain. In addition, certain silent mutations are added to the human mIgM constant regions DNA to remove restriction sites (underlined in SEQ ID NO: 5). The human mIgM and mIgκ constant region DNA sequences used to assemble the IgM antibody DNA sequences are shown below. Human mIgM constant region DNA sequence: GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCG TGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAA CTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAG GTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCA ACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGT CCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTC AGTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTCGGGTCTGGCGTCACCACGGACC AGGTGCAGGCTGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGA GAGCGACTGGCTCAGCCAGAGCATGTTCACATGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATAttorney Docket No.01217-0002-00PCT GCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCA GCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGAC CATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAAT GCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGT GCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCT GCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATC ACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGT CCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAG CATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACATGCGTGGTGGCCCATGAGGCC CTGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGAGGGGGAGGTGAGCGCCGACGAGGAGG GCTTTGAGAACCTGTGGGCCACCGCCTCCACCTTCATCGTCCTCTTCCTCCTGAGCCTCTTCTACAGTAC CACCGTCACCTTGTTCAAGGTGAAA (SEQ ID NO: 5) Human mIgκ constant region DNA sequence: GCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAG TCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACG ACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACC CTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAA CTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (SEQ ID NO: 6) [000161] The final amino acid sequences of the mIgM heavy chain and mIgκ light chain based on the 6E10 antibody are as follows (signal peptides are in italics; V-gene germline-reverted variable region is underlined, and mature CDR3 and FR4 for heavy and light chains are underlined and in bold, SEQ ID NO: 11 and SEQ ID NO: 17, respectively): Reverted 6E10 mIgM HC: MDWTWRFLFVVAAATGVQSEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRI DPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSRSYYNGRRRFTYWGQGTLVTVSSG SASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQV LLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRSKSKLICQATGFS PRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNA SSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNA TFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATIT CLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEAL PNRVTERTVDKSTEGEVSADEEGFENLWATASTFIVLFLLSLFYSTTVTLFKVK (SEQ ID NO: 7) Reverted 6E10 mIgκ LC: METDTLLLWVLLLWVPGSTGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYNYPFTFGQGTKLEIKRADAAPT VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKD EYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 8) Example 3: mIgM-Surface Negative Single Cell Isolation and Expansion [000162] The following procedure was used to isolate B cells that do not express any endogenous mIgM on their surface in order to ectopically express the engineered mIgM afterAttorney Docket No.01217-0002-00PCT transduction. Endogenous mIgMs have a lambda light chain while the engineered BCR displays a kappa constant domain. [000163] A sub-population of the Ramos-BlueTMcells (a B lymphocyte cell line comprising a NFκB / AP-1-inducible gene reporter (SEAP); InvivoGen) that do not express endogenous mIgM BCR (lambda light chain) was isolated by FACS. Briefly, cells were stained with a violet dead cell stain (L34963; ThermoFisher) for viability; and with anti hIgM-APC antibody and anti human lambda chain-PE antibodies to label the endogenous BCR. Approximately 60% of the Ramos-BlueTMcells population was identified negative for endogenous mIgM expression (FIG. 1A). Single cells were sorted on a FACS ARIA II, gating for APC- / PE- live cells, into 96-well plates containing 100µL Ramos conditioned growth medium in each well and 10 separate cell pools were expanded gradually by transferring them into 300 μL, 500 μL, 1 mL, and 2 mL in 48-, 24-, 12- and 6-well formats, respectively. After the 6-well format, cell pools were transferred into T25 and T75 flasks with normal growth medium (not conditioned). After expansion, the sorted Ramos-BlueTMcells were confirmed to be free of endogenous IgM by FACS. Sorted Ramos-BlueTMcells showed no signal from anti Lambda-PE nor anti hIgM-APC (FIG.1E) compared to unsorted Ramos-BlueTMcells (FIG.1C and 1D). After validation, the isolated cells are expanded further to generate aliquots for transduction (5-10 vials at 3-5x106cells). Example 4: Surface-negative Ramos-Blue Reporter Cells Transduction [000164] Isolated surface-negative Ramos-BlueTMcell were transduced with lentiviral particles coding for 6E10 mIgM constructs. [000165] Briefly, lentivirus vector encoding the 6E10-derived mIgM constructs included a puromycin resistance gene and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to enhance expression. The Spleen focus-forming virus (SFFV) promoter was selected for optimal expression in B-cells. See Winiarska et al., 2017, Mol Med Rep, 16(3): 3041-3048. Lentiviruses were then packaged for transduction using a self-inactivating 3rd generation system. The viral titers were about 1-5x108transduction units (Tu) / mL. [000166] 24-well plates were coated with RetroNectin (transduction enhancer) and B cells were transduced overnight at 37°C following the manufacturer’s recommendations for suspension cells. Cells were cultured for 48h, and then washed with phosphate buffer saline (PBS) to remove residual viruses in the medium.Attorney Docket No.01217-0002-00PCT Example 5: Engineered mIgM-expressing B Cells Isolation and Expansion [000167] Ramos-BlueTMcells expressing the 6E10 mIgM were sorted on a FACS ARIA II using violet dead cell stain, APC–anti IgM antibody as well as PE–anti kappa chain antibody (double staining of transduced engineered mIgM). [000168] Three different gates were defined to specifically select for low; medium; and high mIgM BCR expression, as depicted in FIG.2A. Cells were sorted for high, medium and low mIgM expression in 96-well plates, with either 1, 10, or 500 cells / well, in conditioned medium. Individual cell pools were expanded gradually by transferring them into 300 μL, 500 μL, 1 mL, and 2 mL in 48-, 24-, 12- and 6-well formats, respectively. The cells were cultured at 37°C and surviving colonies were expanded until reaching a high enough density to be transferred in flasks and analyzed by flow cytometry to evaluate mIgM expression. [000169] A selection of mIgM-expressing stable cell pools was evaluated by flow cytometry to confirm that the displayed engineered BCRs can bind to A-beta 1-15 antigen peptide of ACI-24 vaccine. BCR were stained with anti-kappa-PE antibody, and cell binding to biotinylated liposomes was detected by strep-488 secondary staining. For 6E10 mIgM-expressing cell pools. A-beta 1-15 binding on ACI-24 was proportional to BCR expression levels (FIG.2B). The four selected 6E10 mIgM-expressing cell pools (6E10.1.L1 and L2; 6E10.10.M2; and 6E10.10.H2) were used to develop the B-cell activation reporter assay. Example 6: B-cell Activation Assay Development A. Determination of the vaccine dilution range for B-cell activation [000170] The working vaccine dilution range was evaluated to determine the minimum antigen content necessary to reach the B-cell activation threshold, as well as the maximum antigen and liposome concentration tolerated in the assay. The high-BCR expressing cell pool 6E10.10.H2 was stimulated over 48h with decreasing concentration of ACI-24.012 and SEAP levels resulting from NF-kB / AP-1 activation were measured, as illustrated in FIG.3 (ACI-24E.012 is a negative control lacking A-beta 1-15). No SEAP secretion could be measured when using high vaccine concentration (below 1 / 40 dilution fold) while SEAP signal gradually increased with further dilutions between 1 / 80 to 1 / 640 when using an ACI-24.012 vaccine (left bar in each dilution). The absence of signal at high vaccine concentration is most likely a consequence of too concentrated liposome particles. The optimal vaccine concentration was gradually refined over the assay development, and found to be in the range of around 1 / 200-1 / 400.Attorney Docket No.01217-0002-00PCT B. B-cell priming for enhanced BCR signaling [000171] To enhance the assay window via BCR signaling, the addition of co-stimulatory signals was investigated. Several reagents were considered such as CD40L, IFNα, and human IL-4. The 6E10.10.H2 reporter cells were primed with different co-stimulatory reagents for 24h before stimulation with either ACI-24.012 or an anti-hIgM antibody (positive control inducing artificial BCR crosslinking though bivalent engagement of BCRs). SEAP levels resulting from BCR-mediated NF-κB / AP-1 activation were measured after 72h, as illustrated in FIG.4. hIL-4 priming at 5 and 10 ng / mL were able to increase BCR signaling induced by ACI-24.012 (FIG. 4) while not activating B-cells on its own as opposed to other co-stimulatory molecules such as CD40L (data not shown). hIL-4 priming at 10ng / mL was selected for further developments. C. Evaluation of cell seeding density [000172] The impact of cell density on the assay window was evaluated.6E10.10.H2 reporter cells were primed with 10ng / mL hIL-4 for 24h, seeded between 1x105cells / well to 4x105cells / well, and stimulated over 48h with ACI-24.012 vaccine. The respective SEAP secretion increase (compared to a medium-only control) is represented in FIG.5. The optimal cell density was identified between 3x105to 4x105cells / well from this experiment. The cell density was re- evaluated after further development of the assay parameters and 2.5x105cells / well was selected as the appropriate cell density. D. Diminution of liposome endocytosis [000173] As liposomes must interact with BCRs on the cell surface, it was investigated whether the endocytosis mechanism naturally occurring with liposomes could be reduced to promote liposomes-BCR interactions. The assay protocol was optimized by incubating the cells for 1h at +4°C before addition of stimulatory reagents. Vaccine samples or controls were then added, and cells were incubated for another hour at +4°C, before transferring them back at +37°C for stimulation over 72h. The respective SEAP secretion increase (compared to a medium-only control) is represented in FIG.6. While the crosslinking anti-hIgM positive control and negative control vaccine (ACI-24E.012 and ACI-EE.043) were not impacted by the incubation step at +4°C, SEAP secretion was increased for both ACI-24.012 and ACI-24.043 vaccines when compared to constant incubation at 37°C. This cold incubation step was included in further experiments. E. Effect of Cytochalasin D on B-cell activation [000174] The endocytosis inhibitor Cytochalasin D (CD) was investigated to further reduce liposome endocytosis. This reagent acts by disrupting actin filaments which also increases BCRAttorney Docket No.01217-0002-00PCT mobility and subsequently lowers the threshold for B-cell activation. Both mechanisms can contribute to enhance BCR signaling.6E10.10.H2 reporter cells were primed with hIL-4 for 24h and stimulated with ACI-24.012 or negative controls diluted in assay medium supplemented with increasing CD concentration. The respective SEAP secretion increase (compared to a medium-only control) is represented in FIG.7. A concentration of 5µM CD successfully increased B-cell stimulation by ACI-24 and shifted the dose-response curve by almost 10-fold. Increased SEAP secretion can also be observed with placebo at high liposome content (the maximum 2 µg / mL A-beta 1-15 content corresponds to a 1 / 200 dilution for negative control vaccines at equivalent liposome concentration) which is most likely a consequence of lowered B-cell activation threshold. This non-specific signal can be compensated by shifting the titration with a maximum of 1µg / mL in A-beta 1-15 content. The use of Cytochalasin D at 5µM was included in the assay protocol for further experiments. F. BCR-mediated cell survival readout [000175] Since B-cell activation is usually associated with cell proliferation and increased survival, it was investigated whether it could be used as an alternative or complementary readout to the BCR-mediated NF-kB / AP-1 reporter gene activity. As the incorporation of Cytochalasin D in the assay workflow prevents cell division - and thus proliferation – it was evaluated whether induction of BCR-mediated cell survival could be monitored using two different cell viability assays, MTT and Orangu™.6E10.10.H2 reporter B cells were primed with hIL-4 for 24h and stimulated with ACI-24.012 or negative controls diluted in assay medium supplemented with 5µM CD. Results after 48h stimulation are represented in FIG.8 as respective SEAP secretion increase (8A), and BCR-mediated cell survival increase (8B). A good correlation between both readouts was observed. Surprisingly, the assay window was better with the BCR- mediated cell survival readout, as the signal relative increase from baseline can reach up to 400% versus 100-200% for BCR-mediated NF-κB / AP-1-inducible reporter gene activity. [000176] The BCR-mediated cell survival and NF-κB / AP-1-inducible reporter gene (SEAP secretion) readouts can be complementary and are therefore both used in further development. [000177] Once suitable assay parameters were identified for B-cell activation, the isolated cell line expressing the 6E10 mIgM construct at low level (6E10.1.L2) was compared to the 6E10.10.H2 cell pool (highest expression level) used for all developments to confirm which BCR expression level was optimal.6E10 mIgM-expressing cell lines were primed with hIL-4 for 24h and stimulated with ACI-24 vaccines or negative controls diluted in assay medium supplemented with 5 µM CD.Attorney Docket No.01217-0002-00PCT [000178] Results after 48h stimulation for the two cell lines expressing high (H2) or low (L2) levels of the 6E10 mIgM are represented in FIG.9A and 9B for BCR-mediated SEAP secretion and 9C and 9D for BCR-mediated cell survival. Interestingly, the 6E10.10.H2 and 6E10.1.L2 cell lines provided very similar SEAP secretion titration curves for the four vaccine samples evaluated (FIG.9 A and B). On the contrary, the high mIgM-expressing cell line 6E10.10.H2 provided a better assay window for the BCR-mediated cell survival readout (FIG.9C – OD values between 0.4 to 1.4) in comparison to the 6E10.10.L2 cell line (FIG.9D – OD values between 0.4-0.8). The 6E10.10.H2 cell line was therefore confirmed as optimal to rank ACI-24 vaccine batches. Example 7: B-cell activation assay [000179] The theory that multivalent antigen presentation is one of the main drivers for efficient B-cell activation, especially to induce a response against a self-antigen such as A-beta was verified in this assay by comparing monomeric Abeta antigens (A-beta 1-15 and Biot-A-beta peptide) with different ACI-24 liposomal vaccine batches (ACI-24.012, ACI-24.008 and ACI- 24.043) displaying multivalent A-beta 1-15 peptides. Liposomal vaccines lacking A-beta 1-15 were used as negative controls (ACI-24E.012 and ACI-EE.043) and the anti hIgM antibody fragment was used as a positive control for BCR crosslinking (Jugloff et al., 1997, J. Immunol., 159(3): 1096-106), which may bypass the BCR activation threshold by directly engaging BCRs. [000180] As represented in FIG.10A, all ACI-24 vaccine batches efficiently activated the NF- κB / AP-1 pathway in a dose-dependent manner whereas A-beta 1-15 monomers did not induce any response. A-beta 1-42 monomers were expected to aggregate at 37°C which translated in weak activation compared to antigens with higher valency. Similarly, ACI-24 vaccines induced a robust BCR-mediated cell survival (FIG.10B) confirming the B-cell activation assay ability to assess the multivalent presentation of antigen peptides at the surface of liposomes. Example 8: Prediction of in vivo antibody-response by in vitro B cell activation assay [000181] The 6E10.10.H2 reporter cell line was incubated with different ACI-24 vaccine batches (A: immunogenic peptide A-beta 1-15 and adjuvants, B: suboptimal A-beta 1-15 presentation and adjuvants) or a negative control (C: no A-beta 1-15 antigen) at different dilutions and the BCR-mediated cell survival quantified (as described in Example 7). The area under the curve (AUC) of the BCR-mediated cell survival curve of each vaccine was normalized to a reference vaccine batch and is presented in FIG.11A. The in vivo immunogenicity of the same ACI-24 vaccine batches and negative control was assessed in mice. Mice were immunizedAttorney Docket No.01217-0002-00PCT twice and blood collected one week after the last immunization and the anti-A-beta IgG titers quantified (Fig.11 B). Vaccine batch A where the A-beta 1-15 antigen is present on the surface of intact liposomes presented a robust stimulation in the BCR-mediated cell survival assay and produced homogeneous antibody titers in mice. Suboptimal vaccine batch B presented a poor stimulation level in the survival assay which was consistent with the poor and highly unpredictable antibody responses post vaccination. As expected, the negative control batch C without target antigen did not induce BCR-mediated cell survival nor generate antibody response in vivo.Attorney Docket No.01217-0002-00PCT TABLE OF CERTAIN SEQUENCES SEQ Description Sequence ID NO 1 6E10 heavy GAGGTTCAGTTGGTGCAGTCTGGGGCAGAGCTTAAGAAGCCAGGGGCCTCAGTCAAGGT chain GTCCTGTACAGCTTCTGGTTTCAACATTAAAGACACCTATATACATTGGGTGAGGCAGGCCC variable CTGGACAGCGCCTGGAGTGGATTGGAAGGTTTGATCCTGTGAATGTTAATACTAGATATGA region DNA CTCGCGGTTCCGGGGCAGGGCCACTATAACATCAGACGCATCCACCAATACAGCCTACATG GAGCTCAGCAGCCTGAGATCTGAGGACACTGCCGTCTATTACTGTTCTAGGTCTTATTACAA sequence CGGTAGAAGACGCTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA 2 6E10 light GACATTGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCACTGGGAGAGAGGGCCACT chain ATCAACTGCAAGGCCAGTCAGAGTCTGTTAAGCAGTGGAAATCAAAAGAACTACTTGACC variable TGGTACCAACAGAAACCAGGGCAGCCTCCTAAATTGTTGATCTACTGGGCATCTATTAGGG region DNA AATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACATTTTTCACTCTCACCATC AGTAGTCTGCAGGCTGAAGACGTGGCAGTGTATTACTGTCAGAATGATTATAATTATCCATT sequence CACATTCGGCCAGGGGACAAAGTTGGAGATAAAA 3 Human IgM ATGGACTGGACCTGGAGGTTCCTCTTTGTGGTGGCAGCAGCTACAGGTGT leader DNA CCAGTCC sequence 4 Light chain ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGG leader DNA ATCCACTGGT sequence 5 Human IgM GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTC constant CCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTTCC region DNA TTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATC sequence AGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGC CACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACG AACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAAC GTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGT CCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAGCAAGTCCAAGCTCA TCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTG CGCGAGGGGAAGCAGGTCGGGTCTGGCGTCACCACGGACCAGGTGCAGGC TGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGA CCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACATGCCGCGTG GATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGTCCC CGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCA GCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTG ACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGC TGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCA GCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAG AGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCA GACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACT TGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATC ACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGAT GCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAA TGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACC GTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACATGCGTGGTGGC CCATGAGGCCCTGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCA CCGAGGGGGAGGTGAGCGCCGACGAGGAGGGCTTTGAGAACCTGTGGGCC ACCGCCTCCACCTTCATCGTCCTCTTCCTCCTGAGCCTCTTCTACAGTAC CACCGTCACCTTGTTCAAGGTGAAA 6 Human mIgκ GCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT constant AACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCA AAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCAttorney Docket No.01217-0002-00PCT region DNA GTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCAT sequence GAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCT ATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGC TTCAACAGGAATGAGTGT 6E10 mIgM MDWTWRFLFVVAAATGVQSEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHW heavy chain VKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDT AVYYCSRSYYNGRRRFTYWGQGTLVTVSSGSASAPTLFPLVSCENSPSDTSSVAV sequence, GCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQG including TDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRSKSKLICQ leader, ATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL comprising SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCL reverted VTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTG FR1-CDR1- FSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGET FR2-CDR2- YTCVVAHEALPNRVTERTVDKSTEGEVSADEEGFENLWATASTFIVLFLLSLFYS FR3 and TTVTLFKVK mature CDR3-FR4 6E10 mIgκ METDTLLLWVLLLWVPGSTGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQ light chain KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA VYYCQNDYNYPFTFGQGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF sequence, YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC including EATHKTSTSPIVKSFNRNEC leader, comprising reverted FR1-CDR1- FR2-CDR2- FR3 and mature CDR3-FR4 6E10 mIgM EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPAN heavy chain GNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSRSYYNGRRRFTYW GQGTLVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYK sequence, NNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKN without VPLPVIAELPPKVSVFVPPRDGFFGNPRSKSKLICQATGFSPRQIQVSWLREGKQ leader, VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQN comprising ASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNG reverted EAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSP FR1-CDR1- EKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTV FR2-CDR2- DKSTEGEVSADEEGFENLWATASTFIVLFLLSLFYSTTVTLFKVK FR3 and mature CDR3-FR4 Reverted EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPAN 6E10 VH GNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYY FR1-CDR1- FR2-CDR2- FR3 6E10 mature SRSYYNGRRRFTYWGQGTLVTVSS HCDR3-FR4 6E10 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPAN comprising GNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSRSYYNGRRRFTYW GQGTLVTVSS revertedAttorney Docket No.01217-0002-00PCT FR1-CDR1- FR2-CDR2- FR3 and mature CDR3-FR4 mIgM GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRG constant FPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELP PKVSVFVPPRDGFFGNPRSKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQV region QAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQD TAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNIS ESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRP DVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMP EPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTEGEVSA DEEGFENLWATASTFIVLFLLSLFYSTTVTLFKVK 6E10 mIgκ DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIY light chain WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYNYPFTFGQGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV sequence, LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC without leader, comprising reverted FR1-CDR1- FR2-CDR2- FR3 and mature CDR3-FR4 6E10 VL DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIY comprising WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYNYPFTFGQGTKL EIK reverted FR1-CDR1- FR2-CDR2- FR3 and mature CDR3-FR4 Reverted DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIY 6E10 VL WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC FR1-CDR1- FR2-CDR2- FR3 6E10 mature QNDYNYPFTFGQGTKLEIK LCDR3-FR4 Igκ constant RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS region WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Mature 6E10 CSRSYYNGRRRFTYW HCDR3 and AA JUNCTION Mature 6E10 CQNDYNYPFTF LCDR3 and AA JUNCTIONAttorney Docket No.01217-0002-00PCT 6E10 mature EVQLVQSGAELKKPGASVKVSCTASGFNIKDTYIHWVRQAPGQRLEWIGRFDPVN VH VNTRYDSRFRGRATITSDASTNTAYMELSSLRSEDTAVYYCSRSYYNGRRRFTYW GQGTLVTVSS 6E10 mature DIVMTQSPDSLTVSLGERATINCKASQSLLSSGNQKNYLTWYQQKPGQPPKLLIY VL WASIRESGVPDRFTGSGSGTFFTLTISSLQAEDVAVYYCQNDYNYPFTFGQGTKL EIK Amino acids DAEFRHDSGYEVHHQ 1-15 of β- amyloid (Aβ)

Claims

Attorney Docket No.01217-0002-00PCT What is claimed is:

1. A method of assessing a vaccine preparation, comprising: a) contacting an engineered B cell line with a vaccine preparation; and b) detecting (i) BCR-mediated cell survival and / or (ii) BCR-mediated activation of a reporter gene, wherein the engineered B cell line comprises an engineered B-cell receptor (BCR), wherein the engineered BCR comprises a heavy chain and a light chain, wherein: the heavy chain comprises: a heavy chain variable region (VH) of an antibody that binds a target antigen; and a heavy chain constant region of a membrane IgM (mIgM); and the light chain comprises: a light chain variable region (VL) of the antibody; and a light chain constant region.

2. The method of claim 1, wherein the B cell does not express endogenous surface IgM.

3. The method of claim 1 or claim 2, wherein the VH comprises a mature complementarity determining region 3 (H-CDR3).

4. The method of any one of claims 1-3, wherein the VH comprises a mature framework region 4 (H-FR4).

5. The method of any one of claims 1-4, wherein the VL comprises a mature complementarity determining region 3 (L-CDR3).

6. The method of any one of claims 1-5, wherein the VL comprises a mature framework region 4 (L-FR4).

7. The method of any one of claims 1-6, wherein the VH comprises one or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3.

8. The method of any one of claims 1-6, wherein the VH comprises a germline-reverted H- FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3.

9. The method of any one of claims 1-6, wherein the VH comprises one or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3.Attorney Docket No.01217-0002-00PCT 10. The method of any one of claims 1-9, wherein the VL comprises one or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline- reverted L-CDR2, and a germline-reverted L-FR3.

11. The method of any one of claims 1-9, wherein the VL comprises a germline-reverted L- FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3.

12. The method of any one of claims 1-9, wherein the VH comprises one or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3.

13. The method of any one of claims 1-12, wherein the engineered B cell line comprises a reporter gene operably linked to a promoter that is responsive to activation of the engineered BCR.

14. The method of claim 13, wherein the method comprises detecting BCR-mediated activation of the reporter gene.

15. The method of claim 14, wherein detecting BCR-mediated activation of the reporter gene comprises detecting expression of the reporter gene operably linked to the promoter that is responsive to activation of the engineered BCR.

16. The method of any one of claims 13-15, wherein the promoter comprises one or more enhancer elements.

17. The method of any one of claims 13-16, wherein the promoter comprises one or more nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) responsive enhancer elements.

18. The method of claim 17, wherein the promoter is a NF-κB / activator protein 1 (AP-1) inducible promoter.

19. The method of any one of claims 13-16, wherein the promoter comprises one or more NFAT-response elements.

20. The method of any one of claims 1-19, wherein the reporter gene encodes an enzyme or a fluorescent protein.

21. The method of any one of claims 1-20, wherein the reporter gene encodes a luciferase, an alkaline phosphatase, a β-lactamase, a β-galactosidase, β-glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase.

22. The method of any one of claims 1-21, wherein the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP).Attorney Docket No.01217-0002-00PCT 23. The method of any one of claims 1-22, wherein BCR-mediated activation of the reporter gene is detected using an alkaline phosphatase activity assay.

24. The method of any one of claims 1-23, wherein the method comprises detecting BCR- mediated cell survival.

25. The method of any one of claims 1-24, wherein the BCR-mediated cell survival is assessed using a cell viability assay.

26. The method of any one of claims 1-25, wherein the engineered B cell line is contacted with the vaccine preparation, and an inhibitor of cell proliferation and / or an inhibitor of actin polymerization element.

27. The method of any one of claims 1-26, wherein the engineered B cell line is contacted with the vaccine preparation and Cytochalasin D.

28. The method of any one of claims 1-27, wherein the engineered B cell line is primed with human Interleukin-4 (IL4) before being contacted with the vaccine preparation.

29. The method of any one of claims 1-28, wherein the vaccine is a liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine, or conjugate vaccine, preferably a liposomal vaccine.

30. The method of any one of claims 1-29, wherein the vaccine is a liposomal vaccine.

31. The method of any one of claims 1-30, wherein the target antigen is an antigenic peptide presented on the surface of a vaccine.

32. The method of claim 31, wherein the VH comprises a mature H-CDR3 and a mature H- FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

11.

33. The method of claim 31 or claim 32, wherein the VL comprises a mature L-CDR3 and a mature L-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

17.

34. The method of any one of claims 31-33, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

21.

35. The method of any one of claims 31-34, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22.Attorney Docket No.01217-0002-00PCT 36. The method of any one of claims 31-33 and 35, wherein the VH comprises a germline- reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

10.

37. The method of any one of claims 31-34, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

16.

38. The method of any one of claims 31-37, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

12.

39. The method of any one of claims 31-38, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

15.

40. The method of any one of claims 31-39, wherein the heavy chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

13.

41. The method of any one of claims 31-40, wherein the light chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

18.

42. The method of any one of claims 31-41, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

9.

43. The method of any one of claims 31-42, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

14.

44. The method of any one of claims 31-43, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7.Attorney Docket No.01217-0002-00PCT 45. The method of any one of claims 31-44, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

8.

46. The method of any one of claims 1-45, wherein the engineered B cell line comprises an engineered Ramos cell line, DT40 cell line, CH12 cell line, Burkitt’s lymphoma B cell line, primary B cell line, or stem cell-derived B cell line.

47. The method of any one of claims 1-46, wherein the engineered B-cell line comprises an engineered Ramos cell line.

48. The method of any one of claims 1-47, wherein the method comprises comparing BCR- mediated cell survival of the engineered B cell line contacted with the vaccine preparation to BCR-mediated cell survival of the same engineered B cell line contacted with a second vaccine preparation and / or a control vaccine preparation.

49. The method of claim 48, wherein the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated cell survival of the engineered B cell line.

50. The method of any one of claims 1-49, wherein the method comprises comparing BCR- mediated activation of the reporter gene of the engineered B cell line contacted with the vaccine preparation to BCR-mediated activation of the reporter gene of the same engineered B cell line contacted with a second vaccine preparation and / or a control vaccine preparation.

51. The method of claim 50, wherein the method comprises ranking the vaccine preparations according to their ability to induce BCR-mediated activation of the reporter gene of the engineered B cell line.

52. The method of any one of claims 48-51, wherein the vaccine preparation and the second vaccine preparation comprise the same peptide antigen.

53. The method of any one of claims 48-52, wherein the vaccine preparation and the second vaccine preparation are different preparations of the same vaccine.

54. The method of any one of claims 1-53, wherein the vaccine preparation comprises an antigenic peptide derived from a self-antigen.

55. The method of claim 54, wherein the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α-synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27.

56. The method of claim 54 or claim 55, wherein the antigenic peptide is derived from β- amyloid (Aβ).Attorney Docket No.01217-0002-00PCT 57. The method of any one of claims 1-56, wherein the vaccine preparation is a liposomal vaccine preparation and comprises a β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome.

58. The method of claim 56 or claim 57, wherein the antigenic peptide comprises amino acids 1-15 of β-amyloid (Aβ) (SEQ ID NO: 23).

59. The method of any one of claims 1-58, wherein the method comprises assessing presentation of the peptide antigen on the vaccine surface.

60. The method of any one of claims 1-59, wherein the method comprises assessing the antibody response to the vaccine preparation.

61. The method of any one of claims 1-59, wherein the method comprises assessing potency of the vaccine preparation.

62. The method of any one of claims 1-59, wherein the method comprises assessing the ability of an antigen to activate B-cell or to induce BCR signaling.

63. The method of any one of claims 1-59, wherein the method comprises predicting in vivo immunogenicity of the vaccine preparation.

64. The method of any one of claims 48-63, wherein the control vaccine preparation is a preparation of a vaccine that does not comprise a peptide antigen recognized by the BCR or wherein the control vaccine preparation is a preparation of a vaccine that has previously been determined to have immunogenicity in vivo.

65. The method of any one of claims 1-64, wherein the method comprises detecting BCR- mediated cell survival and BCR-mediated activation of the reporter gene.

66. The method of claim 65, wherein the vaccine preparation is identified as having suitable predicted in vivo immunogenicity if it induces both BCR-mediated reporter gene activation and BCR-mediated cell survival.

67. The method of any one of claims 1-66, wherein the method comprises comparing multiple batches of the vaccine preparation comprising the same antigen.

68. An engineered B-cell line comprising: an engineered B-cell receptor (BCR), wherein the engineered BCR comprises a heavy chain and a light chain, wherein: the heavy chain comprises: a heavy chain variable region (VH) of an antibody that binds a target antigen; and a heavy chain constant region of a membrane IgM (mIgM); andAttorney Docket No.01217-0002-00PCT the light chain comprises: a light chain variable region (VL) of the antibody; and a light chain constant region; and a reporter gene operably linked to a promoter that is responsive to activation of the BCR, wherein the promoter comprises one or more nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) responsive enhancer elements; and wherein the target antigen is a self-antigen.

69. The engineered B-cell line of claim 68, wherein the B-cell does not express endogenous surface IgM.

70. The engineered B-cell line of claim 68 or claim 69, wherein the VH comprises a mature complementarity determining region 3 (H-CDR3).

71. The engineered B-cell line of any one of claims 68-70, wherein the VH comprises a mature framework region 4 (H-FR4).

72. The engineered B-cell line of any one of claims 68-71, wherein the VL comprises a mature complementarity determining region 3 (L-CDR3).

73. The engineered B-cell line of any one of claims 68-72, wherein the VL comprises a mature framework region 4 (L-FR4).

74. The engineered B-cell line of any one of claims 68-73, wherein the VH comprises one or more of: a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3.

75. The engineered B-cell line of any one of claims 68-73, wherein the VH comprises a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3.

76. The engineered B-cell line of any one of claims 68-73, wherein the VH comprises one or more of: a mature H-FR1, a mature H-CDR1, a mature H-FR2, a mature H-CDR2 and a mature H-FR3.

77. The engineered B-cell line of any one of claims 68-76, wherein the VL comprises one or more of: a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline-reverted L-CDR2, and a germline-reverted L-FR3.

78. The engineered B-cell line of any one of claims 68-76, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline- reverted L-CDR2, and a germline-reverted L-FR3.Attorney Docket No.01217-0002-00PCT 79. The engineered B-cell line of any one of claims 68-76, wherein the VH comprises one or more of: a mature L-FR1, a mature L-CDR1, a mature L-FR2, a mature L-CDR2 and a mature L-FR3.

80. The engineered B-cell line of any one of claims 68-79, wherein the promoter is a NF-κB / activator protein 1 (AP-1) inducible promoter.

81. The engineered B-cell line of any one of claims 68-80, wherein the reporter gene encodes an enzyme or a fluorescent protein.

82. The engineered B-cell line of any one of claims 68-81, wherein the reporter gene encodes a encodes a luciferase, an alkaline phosphatase, a β-lactamase, a β-galactosidase, β- glucuronidase, a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or chloramphenicol acetyltransferase.

83. The engineered B-cell line of any one of claims 68-82, wherein the reporter gene encodes a secreted embryonic alkaline phosphatase (SEAP).

84. The engineered B-cell line of any one of claims 68-83, wherein the target antigen is an antigenic peptide presented on the surface of a liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine or conjugate vaccine, preferably a liposomal vaccine.

85. The engineered B-cell line of any one of claims 68-84, wherein the target antigen is an antigenic peptide presented on the surface of a liposomal vaccine.

86. The engineered B-cell line of claim 84 or claim 85, wherein the VH comprises a mature H-CDR3 and a mature H-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

11.

87. The engineered B-cell line of any one of claims 84-86, wherein the VL comprises a mature L-CDR3 and a mature L-FR4 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

17.

88. The engineered B-cell line of any one of claims 84-87, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

21.

89. The engineered B-cell line of any one of claims 84-88, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22.Attorney Docket No.01217-0002-00PCT 90. The engineered B-cell line of any one of claims 84-87 and 89, wherein the VH comprises a germline-reverted H-FR1, a germline-reverted H-CDR1, a germline-reverted H-FR2, a germline-reverted H-CDR2, and a germline-reverted H-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

10.

91. The engineered B-cell line of any one of claims 84-88, wherein the VL comprises a germline-reverted L-FR1, a germline-reverted L-CDR1, a germline-reverted L-FR2, a germline- reverted L-CDR2, and a germline-reverted L-FR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

16.

92. The engineered B-cell line of any one of claims 84-91, wherein the VH comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

12.

93. The engineered B-cell line of any one of claims 84-92, wherein the VL comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

15.

94. The engineered B-cell line of any one of claims 84-93, wherein the heavy chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

13.

95. The engineered B-cell line of any one of claims 84-94, wherein the light chain constant region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

18.

96. The engineered B-cell line of any one of claims 84-95, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

9.

97. The engineered B-cell line of any one of claims 84-96, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

14.

98. The engineered B-cell line of any one of claims 84-97, wherein the heavy chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%,Attorney Docket No.01217-0002-00PCT at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

7.

99. The engineered B-cell line of any one of claims 84-98, wherein the light chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

8.

100. The engineered B-cell line of any one of claims 68-99, wherein the engineered B cell line comprises an engineered Ramos cell line, DT40 cell line, CH12 cell line, Burkitt’s lymphoma B cell line, primary B cell line, or stem cell-derived B cell line.

101. The engineered B-cell line of any one of claims 68-100, wherein the engineered B-cell line comprises an engineered Ramos cell line.

102. The engineered B-cell line of any one of claims 84-101, wherein the antigenic peptide is derived from a self-antigen selected from β-amyloid (Aβ), Tau, α-synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27.

103. The engineered B-cell line of any one of claims 84-102, wherein the antigenic peptide is derived from β-amyloid (Aβ).

104. The engineered B-cell line of any one of claims 84-103, wherein the liposomal vaccine, DNA scaffold vaccine, nanoparticle vaccine, Virus-Like Particles (VLP) vaccine or conjugate vaccine, preferably a liposomal vaccine, comprises a β-amyloid (Aβ)-derived peptide antigen displayed on its surface.

105. The engineered B-cell line of any one of claims 84-104, wherein the liposomal vaccine comprises a β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome.

106. The engineered B-cell line of any one of claims 84-105, wherein the antigenic peptide comprises amino acids 1-15 of β-amyloid (Aβ) (SEQ ID NO: 23).

107. The engineered B-cell line of any one of claims 68-106, wherein the B-cell line is selected to present a high, medium or low expression of the engineered BCR.

108. A kit comprising: a) the engineered B-cell line of any one of claims 68-107; and b) reagents for detecting BCR-mediated activation of the reporter gene and / or BCR- mediated cell survival.

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