Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining

Abstract

The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138+ antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported.

Immunoglobulin gene analysis as a tool for investigating human immune responses

“…Indeed, there are several reports where the predominant clones in a response have been shown to bind the antigen 103. In mice these experiments have been particularly successful 104. However, the assumption of largest clone providing best protection may be too simple, and many different immunoglobulin genes can respond to a single challenge.…”

Antibodyomics: bioinformatics technologies for understanding B‐cell immunity to HIV‐1

“…This work helped reveal the structural similarities between different antibody solutions to broad V1/V2‐based HIV neutralization and outlined new strategies for V1/V2‐targeting vaccine designs. Paired V H :V L sequencing has also been applied to address several problems beyond HIV 56, 57…”

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VLlibraries from immunized animals

“…Interestingly, we found that antibodies isolated by yeast display from combinatorial libraries in which the V H and V L from spleen or bone marrow had been randomly paired were clonal relatives of antibodies identified via mining of the draining lymph node repertoire, and comprised authentic, natively paired, V H and V L sequences. Thus, in hyperimmune animals where antigen-specific antibodies comprise a significant portion of the repertoire, 29,[32][33][34][35] flow cytometric screening of libraries constructed via the random pairing of V H and V L genes can nonetheless result in the isolation of native antibodies.…”

“…30,31 More recently, we have shown that antibody secreting B cells (CD138 C plasmablasts) within the draining lymph node are overwhelmingly antigen-specific, and that antibodies derived from these cells exhibit high binding affinity. 32 In this report, we sought to compare the isolation of highaffinity anti-ricin antibodies via mining of the draining lymph node repertoire and via yeast display of immune combinatorial libraries constructed from antibody mRNAs obtained from spleen or bone marrow cells. Overall, both approaches yielded strong ricin A chain binders (the lowest K d values were 0.97 and 0.58 nM, respectively, for these 2 methods).…”

“…[21][22][23]27,28 Our lab has pioneered methods for the discovery of high affinity antigen-specific antibodies directly via mining of the immunoglobulin repertoire by capitalizing on next-generation sequencing technologies without the need for screening. [29][30][31][32] Specifically, we developed methods for high-throughput determination of the natively paired V H :V L repertoire from single B cells. 30,31 More recently, we have shown that antibody secreting B cells (CD138 C plasmablasts) within the draining lymph node are overwhelmingly antigen-specific, and that antibodies derived from these cells exhibit high binding affinity.…”