Mapping polyclonal antibody responses to bacterial infection using next generation phage display

Abstract

Mapping polyclonal antibody responses to infectious diseases to identify individual epitopes has the potential to underpin the development of novel serological assays and vaccines. Here, phage-peptide library panning coupled with screening using next generation sequencing was used to map antibody responses to bacterial infections. In the first instance, pigs experimentally infected with Salmonella enterica serovar Typhimurium was investigated. IgG samples from twelve infected pigs were probed in parallel and phage binding compared to that with equivalent IgG from pre-infected animals. Seventy-seven peptide mimotopes were enriched specifically against sera from multiple infected animals. Twenty-seven of these peptides were tested in ELISA and twenty-two were highly discriminatory for sera taken from pigs post-infection (P < 0.05) indicating that these peptides are mimicking epitopes from the bacteria. In order to further test this methodology, it was applied to differentiate antibody responses in poultry to infections with distinct serovars of Salmonella enterica. Twenty-seven peptides were identified as being enriched specifically against IgY from multiple animals infected with S. Enteritidis compared to those infected with S. Hadar. Nine of fifteen peptides tested in ELISA were highly discriminatory for IgY following S. Enteritidis infection (p < 0.05) compared to infections with S. Hadar or S. Typhimurium.

Antibody Engineering for Pursuing a Healthier Future

“…Phage display panning methods have failed to yield specific results because of the presence of parasitic phage clones that are often not removed by washing. To overcome these limitations, next generation sequencing (NGS) technology is used to sequence sub-libraries in biopanning experiments (Naqid et al, 2016). Next generation phage display (NGPD) can sequence 3,000 up to a million reads per panning round.…”

Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals

“…We recently reported an effective NGPD method to map B-cell responses to infection 17 . The method is based on the parallel binding of phage-peptide libraries to polyclonal IgY/IgG from a target cohort (e.g.…”

“…Enteritidis (19 birds in total) and S . Typhimurium (16 birds in total) challenge experiments were conducted and are described in detail elsewhere 17 . All chickens were challenged via oral gavage between 98 and 140 days old and blood samples taken 13 or 14 days later.…”

“…Bacteria was grown overnight in 2YT with ampicillin and the phagemid DNA extracted, amplified by PCR and sequenced using an Ion Torrent PGM service (University of Pennsylvania, US) on a 318 chip exactly as previously described 17 . During this procedure, phagemid originating from selection against each IgY sample were tagged with a unique DNA barcode.…”

Identification of highly selective MMP‐14 inhibitory Fabs by deep sequencing

“…Next-generation sequencing (NGS) technologies have revolutionized multiple aspects of biological researches (Margulies et al, 2005; Pushkarev et al, 2009; Metzker, 2010; Georgiou et al, 2014), with profound impacts on discovery of specific and functional mAbs (Reddy et al, 2010; Reddy et al, 2011; Zhua et al, 2013; Naqid et al, 2016). By high-resolution profiling of an antibody library’s diversity, with sequence and frequency information on virtually all clones during screening process, NGS followed by in-depth analysis has been employed to discover many valuable mAbs not found by ELISA screenings (Ravn et al, 2010; Ravn et al, 2013; Turner et al, 2016).…”