Isolation of Human Antibody Repertoires with Preservation of the Natural Heavy and Light Chain Pairing

Meijer, Per-Johan; Andersen, Peter S.; Hansen, Margit Haahr; Steinaa, Lucilla; Jensen, Allan; Lantto, Johan; Oleksiewicz, Martin B.; Tengbjerg, Kaja; Poulsen, Torben; Coljee, Vincent W.; Bregenholt, Søren; Haurum, John S.; Nielsen, Lene

doi:10.1016/j.jmb.2006.02.040

Cited by 119 publications

(104 citation statements)

References 16 publications

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“…8 Briefly, the cognate antibody heavy and light chain pairs were cloned using the Symplex TM technology, 43 followed by transfection of mammalian cells to enable IgG expression. 44 Antibodies against WTA were selected based on primary screens for binding to cell wall preparations of S. aureus USA300 or Newman, followed by secondary screens for binding to whole bacteria from a variety of clinically relevant S. aureus strains and to S. aureus immediately after isolation from infected mouse kidneys.…”

Section: Methodsmentioning

confidence: 99%

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Fong¹,

Kajihara²,

Chen³

et al. 2018

mAbs

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Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.

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Section: Methodsmentioning

confidence: 99%

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Fong¹,

Kajihara²,

Chen³

et al. 2018

mAbs

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“…Repertoires from the first and the second vaccinations were prepared as previously described (5,7). For the third vaccination, the procedure for sorting single B cells had been optimized to include a marker for CD27.…”

Section: Generation and Analysis Of Ab Repertoiresmentioning

confidence: 99%

“…Recent advances in cloning of Ab genes from single human B cells have provided important new insights into the molecular composition of Ab responses from single individuals (5)(6)(7)(8)(9)(10)(11). What remain to be determined are the actual limits of the repertoire diversification processes and their functional outcome in terms of affinity maturation of Abs.…”

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confidence: 99%

Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans

Poulsen

Jensen

Haurum

et al. 2011

The Journal of Immunology

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The immune system is known to generate a diverse panel of high-affinity Abs by adaptively improving the recognition of pathogens during ongoing immune responses. In this study, we report the biological limits for Ag-driven affinity maturation and repertoire diversification by analyzing Ab repertoires in two adult volunteers after each of three consecutive booster vaccinations with tetanus toxoid. Maturation of on-rates and off-rates occurred independently, indicating a kinetically controlled affinity maturation process. The third vaccination induced no significant changes in the distribution of somatic mutations and binding rate constants implying that the limits for affinity maturation and repertoire diversification had been reached. These fully matured Ab repertoires remained similar in size, genetically diverse, and dynamic. Somatic mutations and kinetic rate constants showed normal and log-normal distribution profiles, respectively. Mean values can therefore be considered as biological constants defining the observed boundaries. At physiological temperature, affinity maturation peaked at kon = 1.6 × 104 M−1 s−1 and koff = 1.7 × 10−4 s−1 leading to a maximum mean affinity of KD = 1.0 × 10−9 M. At ambient temperature, the average affinity increased to KD = 3.4 × 10−10 M mainly due to slower off-rates. This experimentally determined set of constants can be used as a benchmark for analysis of the maturation level of human Abs and Ab responses.

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“…These technologies often also have the capacity to produce single cell transcriptomic data (scRNA‐seq), the estimated prices for some of the more popular methods are included in Table 2 and see also Ziegenhain et al85 for a more comprehensive list on scRNA‐seq. The first of these technologies to be applied extensively used FACS or a microfluidic devise to deposit single cells into a well allowing for Ig specific RT‐PCR of a single cell86, 87, 88, 89, 90; the major drawback being low throughput. These techniques have, however, rapidly been succeeded with microfluidic technologies which have massively increased throughput, allowing thousands of heavy and light chains to be bound together and sequenced as a single entity.…”

Section: Repertoire Analysis Approachesmentioning

confidence: 99%

“…Human studies have the additional challenge that only a sampled snapshot of the repertoire can be examined. One of the earliest reports of heavy/light chain repertoire in human tetanus vaccine response illustrated the breadth of responding genes across the repertoire 86. While different people can share similarities of repertoire, there are aspects of an individual repertoire that are unique to that person105 and they may not always expand the same Ig genes in response to challenge.…”

Section: Clonality Analysismentioning

confidence: 99%

Immunoglobulin gene analysis as a tool for investigating human immune responses

Dunn-Walters

Townsend

Sinclair

et al. 2018

Immunological Reviews

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SummaryThe human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self‐reactive B cells removed. Later antigen‐dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases, and cancer. It can also be used to identify antigen‐specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub‐speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.

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Isolation of Human Antibody Repertoires with Preservation of the Natural Heavy and Light Chain Pairing

Cited by 119 publications

References 16 publications

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans

Immunoglobulin gene analysis as a tool for investigating human immune responses

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