27 research outputs found
Isolation of antigenic peptides of Cowdria ruminantium and their encoding genes using a genome-derived phage display library
The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics
Chicken single-chain antibody fragments directed against recombinant VP7 of bluetongue virus
VP7, the major structural core protein of bluetongue virus, is conserved among the 24 bluetongue virus serotypes. The gene encoding VP7 of serotype 4 was expressed in Escherichia coli. A semi-synthetic chicken antibody library was screened with the resulting protein. Six single-chain antibody fragments (scFvs) were isolated. Immune sera blocked the binding of four of the six scFvs in enzyme-linked immunosorbent assays. These scFvs recognised recombinant VP7 coated directly onto a plastic surface. Their behaviour therefore differs from that of scFv F10 which was selected earlier on directly immobilised bluetongue virus and which binds to VP7 only when it is captured by an immobilised immunoglobulin directed against bluetongue virus.Department of Science and Technologyhttp://www.tandfonline.com/loi/cfai20ab201
Cowdria ruminantium DNA is unstable in a SuperCos1 library
A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of
easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library
contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns
indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not
detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected.
Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli
host, the C. ruminantium clones were unstable in the SuperCos 1 vector and most clones did not grow
reproducibly. The library was contaminated with E. coli clones and these clones were maintained with
greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We
have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two
of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be
cloned and represent almost 10% of the C. ruminantium genome.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi.
Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201
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.The Economic Competitiveness
Support Programme, ARC-OVI, South Africahttp://www.ojvr.orgam2017Veterinary Tropical Disease
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.S1 Fig. Sequences of gallibody clones produced by antibody engineering. 12) Anti-MA 12,
16) Anti-MA 16, 18) Anti-MA 18, CH1-4 = full length constant region, CH2-4 = truncated constant region, VH = variable heavy chain, VL = variable light chain.S2 Fig. SDS-PAGE analysis illustrating gallibody purification using Ni-NTA affinity columns.
A) 12CH1-4, B) 16CH1-4, C) 18CH1-4, D) 12CH2-4, E) 16CH2-4, F) 18CH2-4. Gel lanes 1)
Marker, 2) Culture supernatant, 3) Flow through 1, 4) Flow through 2, 5) Washes, 6) Elution
1, 7) Elution 2, 8) Elution 3, 9) Elution 4. Successful purification is demonstrated by the comparable
thickness of the 67 kDa band obtained with the culture supernatant (2) and the elutions
(6±9).S1 Dataset. Experimental data used for producing Figs 3 and 4.S2 Dataset. Experimental data used for producing Fig 5.S3 Dataset. Experimental data used for producing Fig 6.The
Council for Scientific and Industrial Research
(CSIR) parliamentary grants (YL) and the National
Research Foundation of South Africa for the grants,
unique grant numbers: 99386 (HR), 88622, 80577
(YL) and TTK1206281756 (LN).http://www.plosone.orgam2018Biochemistr
Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis
Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heatshock
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
Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries
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 antigenwere 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
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
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
Agricultural Research Council (ARC), Onderstepoort Veterinary
Research (OVR) Institute of South Africa, and the U.S.
Department of Agriculture, Agricultural Research Service,
entitled Antigenic structure of FMDV capsid proteins and the
selection of appropriate candidate vaccine strains in emerging
FMD outbreaks.https://www.frontiersin.org/journals/veterinary-science#am2020BiochemistryGeneticsMicrobiology and Plant PathologyProduction Animal StudiesVeterinary Tropical Disease