Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display.

Peptide phage display has historically been used to epitope map monoclonal antibodies. More recently, by coupling this method with next-generation sequencing (so-called next-generation phage display, NGPD) to mass screen peptide binding events, the methodology has been successfully applied to map polyclonal antibody responses to infection. This leads to the identification of panels of mimotopes that represent the pathogen's epitopes. One potential advantage of using such an approach is that the mimotopes can represent not just linear epitopes but also conformational epitopes or those produced from post-translational modifications of proteins or from other non-protein macromolecules. The mapping of such complex immunological recognition of a pathogen can inform novel serological assay development and vaccine design. Here, we provide detailed methods for the application of NGPD to identify panels of mimotopes that are recognized specifically by antibodies from individuals with a particular infection.

Next-Generation Phage Display to Identify Peptide Ligands of Deubiquitinases.

Phage display (PD) is a powerful method and has been extensively used to generate monoclonal antibodies and identify epitopes, mimotopes, and protein interactions. More recently, the combination of next-generation sequencing (NGS) with PD (NGPD) has revolutionized the capabilities of the method by creating large data sets of sequences from affinity selection-based approaches (biopanning) otherwise challenging to obtain. NGPD can monitor motif enrichment, allow tracking of the selection process over consecutive rounds, and highlight unspecific binders. To tackle the wealth of data obtained, bioinformatics tools have been developed that allow for identifying specific binding sequences (binders) that can then be validated. Here, we provide a detailed account of the use of NGPD experiments to identify ubiquitin-specific protease peptide ligands.

A Simple Whole-Plasmid PCR Method to Construct High-Diversity Synthetic Phage Display Libraries.

Phage display technology utilises peptide and antibody libraries with very high diversities to select ligands with specific binding properties. The production of such libraries can be labour intensive and technically challenging and whilst there are commercial sources of libraries, the exploitation of the resulting binders is constrained by ownership of the libraries. Here, a peptide library of ~ 1 × 109 variants for display on gene VIII was produced alongside three VHH antibody libraries with similar diversity, where 12mer, 16mer or 21mer CDR3s were introduced into the highly stable cAbBCII10 scaffold displayed on gene III. The cloning strategy used a simple whole-plasmid PCR method and type IIS restriction enzyme assembly that facilitate the seamless insertion of diversity into any suitable phage coat protein or antibody scaffold. This method reproducibly produced 1 × 109 variants from just 10 transformations and the four libraries had relatively low bias with 82 to 86% of all sequences present as single copies. The functionality of both peptide and antibody libraries were demonstrated by selection of ligands with specific binding properties by biopanning. The peptide library was used to epitope map a monoclonal antibody. The VHH libraries were pooled and used to select an antibody to recombinant human collagen type 1.

A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes.

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-ß RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-ß RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-ß RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages-again dependent on TGF-ß RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targeting juvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection.

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

Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of differentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identified in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and specificity. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.

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

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.

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrP(Sc) over time.

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP(Sc) phenotypes were present: G338 or Apl338ii. Thirteen months later some divergent PrP(Sc) phenotypes were seen: a mixture of Apl338ii with either G338 or P338, and a completely novel PrP(Sc) deposition, designated Cag338. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP(Sc) biological/biochemical properties.

Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets.

Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.

Temperature influences the interaction of ruminant PrP (TSE) with soil.

Ovine scrapie and cervid chronic wasting disease can be transmitted in the absence of animal-to-animal contact, and environmental reservoirs of infectivity have been implicated in their spread and persistence. Investigating environmental factors that influence the interaction of disease-associated PrP with soils is imperative to understanding what is likely to be the complex role of soil in disease transmission. Here, we describe the effects of soil temperature on the binding/desorption and persistence of both ovine scrapie- and bovine BSE-PrP (TSE) . Binding of PrP (TSE) to a sandy loam soil at temperatures of 4°C, 8-12°C and 25-30°C demonstrated that an increase in temperature resulted in (1) a decrease in the amount of PrP (TSE) recovered after 24 h of interaction with soil, (2) an increase in the amount of N-terminal cleavage of the prion protein over 11 d and (3) a decrease in the persistence of PrP (TSE) on soil over an 18 mo period.

The interaction of ruminant PrP(Sc) with soils is influenced by prion source and soil type.

The persistence of prions within the environment is implicated in the horizontal transmission of ovine scrapie and cervid chronic wasting disease. Description of the interaction of prion strains derived from their natural hosts with a range of soil types is imperative in understanding how prions persist in the environment and, therefore, the characteristics of prion transmission. Here, we demonstrate that all detectable ovine scrapie and bovine BSE PrP(Sc) bind to a range of soil types within 24 h. This highly efficient binding of prions to soils is characterized by truncation of desorbed PrP(Sc) in a soil-dependent manner, with clay-rich soils resulting in N-terminal truncation of the PrP(Sc) and sand-rich soils yielding full length PrP(Sc) species. PrP(Sc) did not migrate through soil columns during incubation for up to 18 months, and for all combinations of soil and prion types, a decrease in recoverable PrP(Sc) was seen over time. Persistence of PrP(Sc) within soil and their interaction with soil particles of distinct sizes was dictated by both the soil type and the source of the prion, with ovine scrapie being apparently more persistent in some soils than cattle BSE. These data indicate that natural ruminant prion strains are stable in the soil environment for at least 18 months and that PrP(Sc)-soil interaction is dictated by both the soil properties and the strain/host species of PrP(Sc).

Concentration of disease-associated prion protein with silicon dioxide.

Reagents that can precipitate the disease-associated prion protein (PrP(Sc)) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP(C)) and ovine scrapie PrP(Sc). The precipitation of prion protein with silicon dioxide is unaffected by PrP(Sc) strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP(Sc) (PrP(res)) derived from 1.69 microg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO(2) precipitation step into the immunological detection of PrP(res) increased detection sensitivity by over 1,500-fold. Minerals such as SiO(2) are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.

Molecular profiling of ovine prion diseases by using thermolysin-resistant PrPSc and endogenous C2 PrP fragments.

Disease-associated PrP fragments produced upon in vitro or in vivo proteolysis can provide significant insight into the causal strain of prion disease. Here we describe a novel molecular strain typing assay that used thermolysin digestion of caudal medulla samples to produce PrPres signatures on Western blots that readily distinguished experimental sheep bovine spongiform encephalopathy (BSE) from classical scrapie. Furthermore, the accumulation of such PrPres species within the cerebellum also appeared to be dependent upon the transmissible spongiform encephalopathy (TSE) strain, allowing discrimination between two experimental strains of scrapie and grouping of natural scrapie isolates into two profiles. The occurrence of endogenously produced PrP fragments, namely, glycosylated and unglycosylated C2, within different central nervous system (CNS) regions is also described; this is the first detailed description of such scrapie-associated fragments within a natural host. The advent of C2 fragments within defined CNS regions, compared between BSE and scrapie cases and also between two experimental scrapie strains, appeared to be largely dependent upon the TSE strain. The combined analyses of C2 fragments and thermolysin-resistant PrP species within caudal medulla, cerebellum, and spinal cord samples allowed natural scrapie isolates to be separated into four distinct molecular profiles: most isolates produced C2 and PrPres in all CNS regions, a second group lacked detectable cerebellar C2 fragments, one isolate lacked both cerebellar PrPres and C2, and a further isolate lacked detectable C2 within all three CNS regions and also lacked cerebellar PrPres. This CNS region-specific deposition of disease-associated PrP species may reflect the natural heterogeneity of scrapie strains in the sheep population in the United Kingdom.

Use of thermolysin in the diagnosis of prion diseases.

The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrPC from PrPSc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala, and Met, residues that are absent from the protease accessible aminoterminal region of PrPSc. Therefore, although thermolysin readily digests PrPC into small protein fragments, full-length PrPSc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrPSc species is produced, PrP27-30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrPSc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrPSc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrPSc to disease pathogenesis.

Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria.

Several isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria. Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G+C and Sulfobacillus acidophilus sp. nov. with 55-57 mol% G+C. Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract. Autotrophic growth on sulphur was consistently obtained only with S. acidophilus.