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Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells

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  • Authors: Davide Corti1, Antonio Lanzavecchia3
  • Editors: James E. Crowe Jr.4, Diana Boraschi5, Rino Rappuoli6
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Humabs BioMed SA, 6500 Bellinzona, Switzerland; 2: Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland; 3: Institute for Research in Biomedicine, 6500 Bellinzona, Switzerland; 4: Vanderbilt University School of Medicine, Nashville, TN; 5: National Research Council, Pisa, Italy; 6: Novartis Vaccines, Siena, Italy
  • Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0018-2014
  • Received 23 April 2014 Accepted 24 April 2014 Published 10 October 2014
  • : Davide Corti, davide.corti@humabs.ch
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  • Abstract:

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  • Citation: Corti D, Lanzavecchia A. 2014. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells. Microbiol Spectrum 2(5):AID-0018-2014. doi:10.1128/microbiolspec.AID-0018-2014.

Key Concept Ranking

Memory B Cell
0.7545403
B Cells
0.48158854
Plasma Cell
0.46963006
Severe Acute Respiratory Syndrome
0.46579883
0.7545403

References

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22. Rockx B, Corti D, Donaldson E, Sheahan T, Stadler K, Lanzavecchia A, Baric R. 2008. Structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge. J Virol 82:3220–3235. [PubMed][CrossRef]
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24. Sabin C, Corti D, Buzon V, Seaman MS, Lutje Hulsik D, Hinz A, Vanzetta F, Agatic G, Silacci C, Mainetti L, Scarlatti G, Sallusto F, Weiss R, Lanzavecchia A, Weissenhorn W. 2010. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41. PLoS Pathog 6:e1001195. doi:10.1371/journal.ppat.1001195. [PubMed][CrossRef]
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26. Barfod L, Bernasconi NL, Dahlbäck M, Jarrossay D, Andersen PH, Salanti A, Ofori MF, Turner L, Resende M, Nielsen MA, Theander TG, Sallusto F, Lanzavecchia A, Hviid L. 2007. Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA. Mol Microbiol 63:335–347. [PubMed][CrossRef]
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28. Lindesmith LC, Beltramello M, Donaldson EF, Corti D, Swanstrom J, Debbink K, Lanzavecchia A, Baric RS. 2012. Immunogenetic mechanisms driving norovirus GII.4 antigenic variation. PLoS Pathog 8:e1002705. doi:10.1371/journal.ppat.1002705. [PubMed][CrossRef]
29. Beltramello M, Williams KL, Simmons CP, Macagno A, Simonelli L, Quyen NTH, Sukupolvi-Petty S, Navarro-Sanchez E, Young PR, de Silva AM, Rey FA, Varani L, Whitehead SS, Diamond MS, Harris E, Lanzavecchia A, Sallusto F. 2010. The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe 8:271–283. [PubMed][CrossRef]
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32. Di Zenzo G, Di Lullo G, Corti D, Calabresi V, Sinistro A, Vanzetta F, Didona B, Cianchini G, Hertl M, Eming R, Amagai M, Ohyama B, Hashimoto T, Sloostra J, Sallusto F, Zambruno G, Lanzavecchia A. 2012. Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface. J Clin Invest 122:3781–3790. [PubMed][CrossRef]
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Abstract:

In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

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FIGURE 1

Interrogation of human memory B cells and plasma cell repertoires using high-throughput cellular screens. Shown is a timeline of the process that goes from the selection of donors to the isolation, characterization, and development of the antibodies. doi:10.1128/microbiolspec.AID-0018-2014.f1

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0018-2014
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FIGURE 2

Efficient immortalization of human memory B cells by combination of EBV and CpG and isolation of rare neutralizing antibodies. BCR stimulation mediated by antigen, T-cell help mediated by CD40L/CD40 interaction, and TLR stimulation provide three synergistic stimuli for activation of human B cells ( 15 ). EBV encodes LMP2a and LMP1 that mimic constitutively activated BCR and CD40, thus providing signal 1 and signal 2. Addition of TLR agonists potently synergizes with the viral genes leading to efficient activation and immortalization ( 8 ). Immortalization efficiency of IgG+ memory B cells plated at 1 cell/well in 384 culture plates containing irradiated allogeneic PBMC. Shown is the concentration of IgG in culture supernatants on day 10. Positive values above 2.5 OD correspond to IgG concentrations >1 µg/ml. The efficiency was calculated according to the Poisson distribution. Example of a primary screening for MPV-neutralizing antibodies. In preliminary experiments, culture conditions were defined to achieve cytopathic effect by using primary MPV isolates. Culture supernatants were incubated with MPV followed by addition of LLC-MK2 cells. Living cells were measured by using a colorimetric assay on day 8. Several antibodies were isolated, including one (MPE8) that neutralizes four different paramyxoviruses. Shown is the total numbers of cultures screened. Blood was collected from an immune donor 2 weeks after vaccination with a seasonal vaccine, and IgG memory cells were immortalized and plated at 3 cells/well in 384-well plates. The supernatants were screened for the presence of antibodies that bind to either H1 HA (CA09) present in the vaccine or to H5 HA (VN04) that represents a heterologous group 1 HA. doi:10.1128/microbiolspec.AID-0018-2014.f2

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0018-2014
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FIGURE 3

Cultures of single plasma cells are instrumental for the rapid identification of rare antibodies. Survival of single CD138 plasma cells isolated from peripheral blood (open circles) or bone marrow (filled circles) in the presence of IL-6 and stromal cell monolayers. Shown is the cumulative production of IgG in cultures containing single plasma cells. Plasma cells were isolated from peripheral blood as CD38 CD138 and plated at 0.5 cell/well in the presence of IL-6. IgG, IgA, IgM, and IgE levels were measured in each culture supernatant. The efficiency of cloning as estimated from the frequency of Ig-containing cultures was estimated to be approximately 70%. HA-specific antibodies produced by plasma cells isolated in 2009 following infection with H1N1 CA09 cross-react extensively with H5-HA (VN04). In contrast, plasma cells isolated from the same donor in 2010 following vaccination with the seasonal trivalent vaccine are largely vaccine specific . doi:10.1128/microbiolspec.AID-0018-2014.f3

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0018-2014
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FIGURE 4

A target-agnostic approach to antibody discovery and vaccine design. In analytic vaccinology, donors that have developed a protective response are identified and memory B cells/plasma cells are interrogated to identify the most effective neutralizing antibodies in terms of potency and breadth. The antibodies are then used to identify the target antigen and to probe its correct conformation when the latter is produced as a recombinant vaccine. The vaccine is expected to elicit antibodies of the same quality as those originally isolated. In addition, the recombinant molecules can be used to identify cellular receptors. doi:10.1128/microbiolspec.AID-0018-2014.f4

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0018-2014
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Tables

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TABLE 1

Examples of human monoclonal antibodies isolated by using high-throughput cellular screens

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.AID-0018-2014

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