identification-mouse-human-antibody-repertoires-next-generation

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

National Institute of Infectious Diseases

The H5N1 subtype of the highly pathogenic (HP) avian influenza virus has been recognized for its ability to cause serious pandemics among humans. In the present study, new monoclonal antibodies (mAbs) against viral proteins were established for the immunological detection of H5N1 influenza virus for research and diagnostic purposes. B-cell hybridomas were generated from mice that had been hyperimmunized with purified A/Vietnam/1194/2004 (NIBRG-14) virion that had been inactivated by UV-irradiation or formaldehyde. After screening over 4,000 hybridomas, eight H5N1-specific clones were selected. Six were specific for hemagglutinin (HA) and had in vitro neutralization activity. Of these, four were able to broadly detect all tested clades of the H5N1 strains. Five HA-specific mAbs detected denatured HA epitope(s) in Western blot analysis, and two detected HP influenza virus by immunofluorescence and immunohistochemistry. A highly sensitive antigen-capture sandwich ELISA system was established by combining mAbs with different specificities. In conclusion, these mAbs may be useful for rapid and specific diagnosis of H5N1 influenza. Therapeutically, they may have a role in antibody-based treatment of the disease.

Newly established monoclonal antibodies for immunological detection of H5N1 influenza virus.

Vast diversity and high specificity of antigen recognition by antibodies are hallmarks of the acquired immune system. Although the molecular mechanisms that yield the extremely large antibody repertoires are precisely understood, comprehensive description of the global antibody repertoire generated in individual bodies has been hindered by the lack of powerful measures. To obtain holistic understanding of the antibody-repertoire space, we used next-generation sequencing (NGS) to analyze the deep profiles of naive and antigen-responding repertoires of the IgM, IgG1, and IgG2c classes formed in individual mice. The overall landscapes of naive IgM repertoires were almost the same for each mouse, whereas those of IgG1 and IgG2c differed considerably among naive individuals. Next, we immunized mice with a model antigen, nitrophenol (NP)-hapten linked to chicken γ-globulin (CGG) carrier, and compared the antigen-responding repertoires in individual mice. To extract the complete antigen response, we developed an intelligible method for detecting common components of antigen-responding repertoires. The major responding antibodies were IGHV1-72/IGHD1-1/IGHJ2 for NP-hapten and IGHV9-3/IGHD3-1/IGHJ2 for CGG-carrier protein. The antigen-binding specificities of the identified antibodies were confirmed through ELISA after antibody-gene synthesis and expression of the corresponding NGS reads. Thus, we deciphered antigen-responding antibody repertoires by inclusively analyzing the antibody-repertoire space generated in individual bodies by using NGS, which avoided inadvertent omission of key antibody repertoires.

Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis.

The pre-B cell receptor (pre-BCR), consisting of the μ heavy chain (μHC) and the surrogate light chain (SLC, Vpre-B and λ5), plays important roles during B cell development. The formation of the pre-BCR, which enables the nascent immunoglobulin HC to associate with the SLC, is considered a prerequisite for B cell development. However, a significant number of peripheral mature (leaky) B cells exist in SLC-deficient mice. These leaky B cells develop in the absence of pre-BCR and do not undergo the pre-BCR checkpoint. The antibody repertoires of leaky B cells thus reflect the absence of pre-BCR function. To investigate how the absence of the pre-BCR is circumvented by these leaky-B cells and examine the effect of the pre-BCR checkpoint on the antibody system, we analyzed the antibody repertoires of λ5-deficient (λ5) mice using next-generation sequencing. In λ5 mice, spleen B cells displayed different patterns of VDJ-usage, relative to those in wild-type (WT) mice. Moreover, leaky B cells were neither derived from unusual B2 cells, characterized by particular LC gene rearrangements in the absence of pre-BCR signaling, nor from B1 cells, originating from different B cell progenitors. Analysis of the CDR-H3 amino acid sequences of μ-chain repertoires revealed that certain bone marrow B cells with particular CDR-H3 profiles undergo clonal expansion in λ5 mice. Part of these CDR-H3s contain arginine(s) in the middle of the CDR-H3 loop in λ5 mice, whereas few arginine(s) exist in this middle loop in WT CDR-H3s in the absence of clonal expansion. This CDR-H3 feature in λ5 mice presumably reflects the role of the pre-BCR in autoantibody regulation, since arginine(s) are often found in the antigen-binding site of autoantibodies. Here, we present a unique viewpoint on the role of pre-BCR, by assessing the whole antibody repertoire formed in SLC-deficient mice.

Naoko Kono

Center for Influenza Virus Research

National Institute of Infectious Diseases

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing