Construction of Chicken and Ostrich Antibody Libraries.

Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage-displayed scFvs for use in veterinary diagnostics and research. To add diversity to the scFv repertoire, we expanded the library to include genes from the ostrich, indigenous to southern Africa. The libraries described in this chapter are based on the chicken and ostrich variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.

Theileriosis in naturally infected roan antelope (Hippotragus equinus).

Cases of Theileria-associated mortality are rarely reported in African wild artiodactyls. Descriptions of lesions are limited, particularly in endangered hippotraginids. Here, we analyzed retrospectively the gross and histologic findings in 55 roan antelope (Hippotragus equinus) with fatal natural theileriosis. The most frequently recorded gross findings in 40 cases were widespread petechiae and ecchymoses (72.5%), probable anemia (67.5%), icterus (60%), splenomegaly (60%), hepatomegaly (52.5%), and pulmonary edema (50%). Histologic lesions in 34 cases were characterized by multi-organ infiltrates of parasitized and nonparasitized mononuclear leukocytes (MLs), and fewer multinucleate giant cells (MNGCs). Liver, lung, kidney, adrenal gland, and heart were most consistently infiltrated, followed by spleen and lymph nodes. Leukocytes were phenotyped in lung, liver, kidney, and heart specimens from 16 cases, using immunohistochemistry to detect CD20, CD3, myeloid/histiocyte antigen (MAC387), IBA-1, and CD204 surface receptors. A roan polyclonal anti-Theileria sp. (sable) antibody was applied to the same tissues to identify intraleukocytic parasite antigens. Similar proportions of intravascular and extravascular IBA-1-, CD204-, and MAC387-reactive putative monocyte-macrophages and fewer CD3-positive putative T-lymphocytes were identified in all organs, especially the lungs in infected roan. CD20-positive putative B-lymphocytes were significantly scarcer than in uninfected controls. Intraleukocytic Theileria parasites labeled consistently in affected tissues. Some parasitized and nonparasitized MLs and the MNGCs failed to label with selected leukocyte markers. Fatal theileriosis in roans may largely be the result of multi-organ monocyte-macrophage activation with associated tissue injury and overwhelming systemic inflammation. The identity of the parasitized leukocytes and characteristics of the lymphohistiocytic response require further clarification in roans.

Polyclonal antibody-based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes.

Theileria parasites commonly infect African wild artiodactyls. In rare roan (Hippotragus equinus) and sable (H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other Theileria spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of Cryptosporidium. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.

Diagnostic and Epitope Mapping Potential of Single-Chain Antibody Fragments Against Foot-and-Mouth Disease Virus Serotypes A, SAT1, and SAT3.

Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the Nkuku ® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.

The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies.

Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto-MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains-CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.

Construction of Chicken Antibody Libraries.

Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage displayed scFvs for use in veterinary diagnostics and research. The libraries described in this chapter are based on chicken variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage, or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.

B-cell epitopes of African horse sickness virus serotype 4 recognised by immune horse sera.

Identifying antigenic proteins and mapping their epitopes is important for the development of diagnostic reagents and recombinant vaccines. B-cell epitopes of African horse sickness virus (AHSV) have previously been mapped on VP2, VP5, VP7 and NS1, using mouse, rabbit and chicken monoclonal antibodies. A comprehensive study of the humoral immune response of five vaccinated horses to AHSV-4 antigenic peptides was undertaken. A fragmented-genome phage display library expressing a repertoire of AHSV-4 peptides spanning the entire genome was constructed. The library was affinity selected for binders on immobilised polyclonal immunoglobulin G (IgG) isolated from horse sera collected pre- and post-immunisation with an attenuated AHSV-4 monovalent vaccine. The DNA inserts of binding phages were sequenced with Illumina high-throughput sequencing. The data were normalised using preimmune IgG-selected sequences. More sequences mapped to the genes coding for NS3, VP6 and VP5 than to the other genes. However, VP2 and VP5 each had more antigenic regions than each of the other proteins. This study identified a number of epitopes to which the horse's humoral immune system responds during immunisation with AHSV-4.

Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus).

An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic avian influenza H5N2 in a single sample was designated for culling, despite no evidence of sero-conversion as assessed by haemagglutination inhibition (HI) tests. A month later and immediately prior to culling, all birds were bled and tested with an IDEXX avian influenza virus (AIV) nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and a high sero-prevalence was detected. To address the question of whether the NP-specific antibodies detected indicated exposure to H5 or non-H5 subtypes (H6N2 and H1N2 strains were also circulating regionally at the time), we developed two H5-specific ELISAs, both based on a recombinant H5 HA1 antigen. The H5 indirect ELISA used a horseradish peroxidase ostrich IgY conjugate that we produced in chicken eggs. The single-chain variable fragment (scFv) competitive ELISA (H5 scFv cELISA) used a scFv derived from an H5-immune chicken scFv library. By comparing IDEXX AIV ELISA results with those of the two H5-specific ELISAs and HI tests, we determined that up to 89% of the flock had been exposed to H5N2 AIV. We also detected evidence of suspected vaccination, since 17% of sera contained antibodies against the H5 glycoprotein but not the NP protein. Comparative analytical sensitivity indicated that HI tests are likely to miss up to 35% of H5-positive samples, and thus we consider that H5/H7-specific ELISAs should replace HI tests for ostrich testing in future.

Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs.

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv.

Recombinant antibodies can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa antigen. Three fusion phages which bound specifically to the antigen were selected, each of which produced low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an attempt to improve its binding. Firstly, a mutant sublibrary was created by random mutagenesis. High stringency panning of this sublibrary yielded binders which produced ELISA signals up to eleven times higher than the parent scFv. An increase in the intrinsic affinity was confirmed by surface plasmon resonance. Secondly, the flexible linker between the heavy and light chains of the parent scFv was either shortened to one glycine residue or deleted entirely. No ELISA signal was obtained when the linker was absent, but the glycine-linked scFv showed enhanced binding. Size exclusion chromatography revealed that the enhanced binder had aggregated to form tetramers. This study confirms that the strategies used to improve the binding of human and mouse scFvs can also enhance chicken scFvs.

Chicken scFvs and bivalent scFv-C(H) fusions directed against HSP65 of Mycobacterium bovis.

Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heat-shock protein (HSP65) of Mycobacterium bovis were selected from a large semi-synthetic phage displayed library. Both recognised HSP65 in indirect enzyme-linked immunosorbent assay (ELISA) and immunoblots and retained their activity during storage. Neither, however, could function as the capture reagent in a sandwich ELISA when immobilised on polystyrene. To establish whether they could be engineered for general use in immunotests, the genes coding for these scFvs were subcloned in expression vectors that contained sequences encoding chicken IgY heavy-chain constant region domains. This resulted in larger bivalent constructs which more closely resembled IgY molecules. The engineered fragments were evaluated in ELISAs and gold-conjugated immunochromatographic tests (ICTs). In contrast to their previous behaviour as scFvs, the modified fragments (designated "gallibodies") could be used for immunocapture in ELISA and could be readily conjugated to colloidal gold nanoparticles. A sandwich ICT that could detect recombinant HSP65 was also devised. Although converting the recombinant single-chain monomeric antibody fragments to bivalent immunoglobulin-like molecules did not entirely 'standardise' the behaviour of the scFvs, this approach remains potentially useful for developing practical, robust, immunodiagnostic reagents.

A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes.

BACKGROUND: Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. RESULTS: With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA. CONCLUSION: The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.