Antibody repertoire dynamics in the changing landscape of infection

Abstract

Antibody responses are fundamentally important to effector and memory mechanisms of disease resistance. In order to respond to a nearly infinite array of possible antigens, the antibody repertoire must be suitably diverse. To achieve this necessary high level of diversity, the antibody repertoire has evolved a unique recombinatorial system consisting of a large number of gene segments that can recombine in different combinations to yield an astronomical array of potential antigen-binding structures. Understanding the antibody repertoire of swine (Sus scrofa) can inform about host genetic differences that may affect disease susceptibility and resistance. Also, it may allow identification of antibody molecules that are important in the host immune response against specific pathogens. Such knowledge could potentially be used in the future to develop selective breeding programs for animals that possess desirable immunological traits, and to screen for specific antibody molecules that are of either therapeutic or diagnostic importance. Knowledge of antibody repertoire diversity in swine has heretofore been lacking. While most previous studies have focused heavily on understanding the heavy chain repertoire by analyzing hundreds of cDNA clones, there have been few investigations of the porcine light chain repertoire. This study was designed to characterize the organization and complexity of both the kappa and lambda light chain loci in the pig genome. Findings revealed extensive allelic variation between both homologous pairs of chromosomes in a single sow and suggested non-crossover homologous recombination (i.e. gene conversion) as a potential evolutionary mechanism to explain at least part of that variation. Armed with this new information, and with that from the previously characterized heavy chain locus, antibody variable region amplicon libraries were generated from lymphoid tissues of pigs either infected (n=2) or mock-infected (heavy chain, n=2; light chain, n=3) with the major swine pathogen, porcine reproductive and respiratory syndrome virus (PRRSV). It is hypothesized that the major anti-PRRSV antibody responses would be detectible in infected animals compared to their control counterparts. Approximately a half-million reads for each heavy and light chain library were generated. From this data, diversity of the expressed antibody repertoire was assessed, including gene segment usage and allelic variability, and anti-PRRSV responses. As predicted, due to biological necessity, the heavy and light chain repertoires possessed a rich array of putatively functional antibody transcripts (heavy chain richness estimate, 3x105 molecules; kappa light chain, 1.5x105; lambda light chain, 2.3 x 105), despite being restricted in their germline to a small number of functional D and J gene segments, a single heavy chain V gene segment family and four light chain V gene families. Interestingly, a power-law distribution of antibody abundances was detected similar to what has previously been reported in zebrafish (Danio rerio), whereby a small number of antibody sequences are exceptionally common and the vast majority are exceptionally rare. Substantial allelic variation was also detected, most notably in the lambda locus. Four out of 5 pigs possessed a functional copy of a previously undescribed V gene segment (IGLV3-1-1) which substantially contributed to the expressed repertoire of the animals that possessed a copy. Importantly, a small number of antibody sequences were detected which were incredibly abundant (>1% of the entire repertoire) in PRRSV-infected pigs and rare in uninfected pigs. It is hypothesized that these highly abundant antibody molecules are PRRSV-specific. Using the knowledge obtained from these studies, future investigations will examine the repertoire for specific heavy and light chain pairs from PRRSV-infected pigs that can neutralize PRRSV using an antibody yeast-display system. In addition, specific heavy and light chain pairs identified in our expression analysis and deemed putatively PRRSV-specific are to be tested for epitopic specificity against labeled PRRSV as well as individual PRRSV recombinant antigens. This last method represents a potential novel and non-lethal means of generating antigen-specific recombinant antibodies derived from lymphoid tissue of immunized animals.