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In vitro reconstitution of B cell receptor–antigen interactions to evaluate potential vaccine candidates

Nature Protocols volume 11, pages 193–213 (2016)Cite this article

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Abstract

Predicting immune responses before vaccination is challenging because of the complexity of the governing parameters. Nevertheless, recent work has shown that B cell receptor (BCR)-antigen engagement in vitro can prove a powerful means of informing the design of antibody-based vaccines. We have developed this principle into a two-phased immunogen evaluation pipeline to rank-order vaccine candidates. In phase 1, recombinant antigens are screened for reactivity to the germline precursors that produce the antibody responses of interest. To both mimic the architecture of initial antigen engagement and facilitate rapid immunogen screening, these antibodies are expressed as membrane-anchored IgM (mIgM) in 293F indicator cells. In phase 2, the binding hits are multimerized by nanoparticle or proteoliposome display, and they are evaluated for BCR triggering in an engineered B cell line displaying the IgM sequences of interest. Key developments that complement existing methodology in this area include the following: (i) introduction of a high-throughput screening step before evaluation of more time-intensive BCR-triggering analyses; (ii) generalizable multivalent antigen-display platforms needed for BCR activation; and (iii) engineered use of a human B cell line that does not display endogenous antibody, but only ectopically expressed BCR sequences of interest. Through this pipeline, the capacity to initiate favorable antibody responses is evaluated. The entire protocol can be completed within 2.5 months.

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Figure 1: BCR antigen-recognition pipeline.
Figure 2: Inferring a germline-reverted antibody.
Figure 3: A leader sequence should precede the variable region of the germline-reverted antibody.
Figure 4: Overlap PCR to generate the membrane-anchored IgM configuration of germline-reverted antibodies.
Figure 5: Membrane presented antibody as a means to prescreen candidate immunogens for the capacity to engage germline BCR sequences of interest.
Figure 6: Lentiviral-mediated expression of BCR in Ramos cells.
Figure 7: BCR activation in response to multimerized candidate immunogen.
Figure 8: Proteoliposome array of candidate immunogen.

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Acknowledgements

The authors thank members of the Lingwood laboratory for critical reading of the manuscript, S. Perfetto (Vaccine Research Center, National Institutes of Health (NIH)) for flow cytometry advice, and J. Boyington and J. Whittle (Vaccine Research Center, NIH) for key discussions. This work was supported by the following awards to D.L.: a Harvard University Center for AIDS Research (CFAR) grant (P30 AI060354); a Broad-Ragon ENDHIV Catalytic grant; the William F. Milton Fund; and the Gilead Sciences Research Scholars Program in HIV.

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Authors and Affiliations

  1. The Ragon Institute of Massachusetts General Hospital, The Massachusetts Institute for Technology and Harvard University, Cambridge, Massachusetts, USA

    Grant C Weaver, Rina F Villar & Daniel Lingwood

  2. Vaccine Research Center, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Masaru Kanekiyo & John R Mascola

  3. Sanofi, Cambridge, Massachusetts, USA

    Gary J Nabel

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  1. Grant C Weaver
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Contributions

G.C.W., R.F.V., M.K., G.J.N., J.R.M. and D.L. designed the research; G.C.W., R.F.V., M.K. and D.L. performed the research; G.C.W., R.F.V., M.K., G.J.N., J.R.M. and D.L. analyzed the data and wrote the paper.

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Correspondence to Daniel Lingwood.

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Weaver, G., Villar, R., Kanekiyo, M. et al. In vitro reconstitution of B cell receptor–antigen interactions to evaluate potential vaccine candidates. Nat Protoc 11, 193–213 (2016). https://doi.org/10.1038/nprot.2016.009

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