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Maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection.

ABSTRACT: Multiple bacterial and protozoal pathogens utilize gene conversion to generate rapid intrahost antigenic variation. Both large- and small-genome pathogens expand the size of the variant pool via a combinatorial process in which oligonucleotide segments from distinct donor loci are recombined in various combinations into expression sites. Although the potential combinatorial diversity generated by this segmental gene conversion mechanism is quite large, the functional variant pool depends on whether immune responses against the recombined segments are generated and maintained, regardless of their specific combinatorial context. This question was addressed by tracking the Anaplasma marginale variant population and corresponding segment-specific immunoglobulin G (IgG) antibody responses during long-term infection. Antibody was induced early in A. marginale infection, predominately against the surface-exposed hypervariable region (HVR) rather than against the invariant conserved flanking domains, and these HVR oligopeptides were most immunogenic at the time of acute bacteremia, when the variant population is derived via recombination from a single donor locus. However antibody to HVR oligopeptides was not consistently maintained during persistent infection, despite reexpression of the same segment, although in a different combinatorial context. This dynamic antibody recognition over time was not attributable to the major histocompatibility complex haplotype of individual animals or use of specific msp2 donor alleles. In contrast, the position and context of an individual oligopeptide segment within the HVR were significant determinants of antibody recognition. The results unify the genetic potential of segmental gene conversion with escape from antibody recognition and identify immunological effects of variant mosaic structure.

SUBMITTER: Zhuang Y

PROVIDER: S-EPMC2168278 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection.

Zhuang Yan Y Futse James E JE Brown Wendy C WC Brayton Kelly A KA Palmer Guy H GH

Infection and immunity 20070904 11

Multiple bacterial and protozoal pathogens utilize gene conversion to generate rapid intrahost antigenic variation. Both large- and small-genome pathogens expand the size of the variant pool via a combinatorial process in which oligonucleotide segments from distinct donor loci are recombined in various combinations into expression sites. Although the potential combinatorial diversity generated by this segmental gene conversion mechanism is quite large, the functional variant pool depends on whet ...[more]

PMID: 17785476

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Project description:Phage display technology is a common approach for discovery of therapeutic antibodies. Drug candidates are typically isolated in two steps: First, a pool of antibodies is enriched through consecutive rounds of selection on a target antigen, and then individual clones are characterized in a screening procedure. When whole cells are used as targets, as in phenotypic discovery, the output phage pool typically contains thousands of antibodies, binding, in theory, hundreds of different cell surface receptors. Clonal expansion throughout the phage display enrichment process is affected by multiple factors resulting in extremely complex output phage pools where a few antibodies are highly abundant and the majority is very rare. This is a huge challenge in the screening where only a fraction of the antibodies can be tested using a conventional binding analysis, identifying mainly the most abundant clones typically binding only one or a few targets. As the expected number of antibodies and specificities in the pool is much higher, complementing methods, to reach deeper into the pool, are required, called deep mining methods. In this study, four deep mining methods were evaluated: 1) isolation of rare sub-pools of specific antibodies through selection on recombinant proteins predicted to be expressed on the target cells, 2) isolation of a sub-pool enriched for antibodies of unknown specificities through depletion of the primary phage pool on recombinant proteins corresponding to receptors known to generate many binders, 3) isolation of a sub-pool enriched for antibodies through selection on cells blocked with antibodies dominating the primary phage pool, and 4) next-generation sequencing-based analysis of isolated antibody pools followed by antibody gene synthesis and production of rare but enriched clones. We demonstrate that antibodies binding new targets and epitopes, not discovered through screening alone, can be discovered using described deep mining methods. Overall, we demonstrate the complexity of phage pools generated through selection on cells and show that a combination of conventional screening and deep mining methods are needed to fully utilize such pools. Deep mining will be important in future phenotypic antibody drug discovery efforts to increase the diversity of identified antibodies and targets.

Dataset Information

Maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection.

Publications

Maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection.

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