Epitope mapping of the monoclonal antibody IP5B11 used for detection of viral haemorrhagic septicaemia virus facilitated by genome sequencing of carpione novirhabdovirus.

The monoclonal antibody (mAb) IP5B11, which is used worldwide for the diagnosis of viral haemorrhagic septicaemia (VHS) in fish, reacts with all genotypes of VHS virus (VHSV). The mAb exceptionally also reacts with the carpione rhabdovirus (CarRV). Following next generation genome sequencing of CarRV and N protein sequence alignment including five kinds of fish novirhabdoviruses, the epitope recognized by mAb IP5B11 was identified. Dot blot analysis confirmed the epitope of mAb IP5B11 to be associated with the region N219 to N233 of the N protein of VHSV. Phylogenetic analysis identified CarRV as a new member of the fish novirhabdoviruses.

A novel monoclonal antibody efficiently blocks the infection of duck adenovirus 3 by targeting Fiber-2.

Duck adenovirus 3 (DAdV-3), identified as the causative agent of a disease characterized by swelling and hemorrhage of liver and kidney, has caused substantial economic losses to duck industry in China. However, the neutralizing epitopes and the infection mechanism of DAdV-3 have not been extensively elucidated. In this study, a novel monoclonal antibody (mAb) targeting Fiber-2 protein of DAdV-3 was generated and designated as mAb 3E7. Indirect immunofluorescence assay showed that mAb 3E7 specifically reacted with the Fiber-2 in LMH cells transfected with pcDNA3.1-Fiber-2 or infected with DAdV-3. Moreover, mAb 3E7 could immunoprecipitate the Fiber-2 and efficiently inhibit the infection of DAdV-3 in vitro. Further epitope mapping revealed mAb 3E7 recognized the epitope 108LALGDGLE115 in Fiber-2, which was highly conserved among DAdV-3 strains. These findings not only identified a novel neutralizing epitope in Fiber-2, but also paved the way for further elucidating the vital roles of Fiber-2 in the infection and pathogenesis of DAdV-3.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

Identification of B-cell linear epitopes in domains 1-3 of pyolysin of Trueperella pyogenes using polyclonal antibodies.

Trueperella pyogenes is an important opportunistic pathogen. Pyolysin (PLO) makes important contributions to the pathogenicity of T. pyogenes. However, the structure and function of PLO has not been well documented. In the current study, epitopes in domain 1-3 of PLO have been mapped using rabbit anti-recombinant PLO (rPLO) polyclonal antibodies, and then the results were re-checked by using mouse and chicken anti-rPLO polyclonal antibodies, respectively. The results indicated that the region of aa 281-393 in PLO could not elicit antibodies against linear epitopes. A total of six B cell linear epitopes have been found in domain 1 of PLO. Two of the six epitopes (EP1 and EP2) were used to immunize mice and chicken. Chicken anti-EP1 and anti-EP2 serum and mouse anti-EP2 serum could react with rPLO and corresponding epitope polypeptide in western blot assay; however, only mouse anti-EP2 serum shows weak anti-hemolysis effect in the rPLO and sheep red blood system. Our results provide some new information to the research field of PLO structure and function.

Identification of two conserved B-cell epitopes in the gp90 of reticuloendothelial virus using peptide microarray.

Since the gp90 protein of Reticuloendotheliosis virus (REV) plays vital roles in virus neutralization, so detailed analysis of REV-gp90 epitopes is important for the development of epitope based marker vaccines and diagnostic tools for REV infections. In this study, two monoclonal antibodies (mAbs) namely 6C12 and 09980 were used to map the epitopes in REVgp90 using peptide microarray and ELISA. Peptide microarray revealed that mAbs 6C12 and 09980 recognized 216YHPLA220 and 230DPQTSDILEA239 motifs, respectively. This result was confirmed by ELISA using synthetic peptides. Moreover, homology analysis indicated that mAbs defined epitopes are highly conserved among REV strains used in this study. The mAbs and their epitopes identified in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines for control of REV infections.

Development of CDV-specific monoclonal antibodies for differentiation of variable epitopes of nucleocapsid protein.

The highly contagious canine distemper viruses (CDVs) are still a major threat to a wide range of natural susceptible hosts. The nucleocapsid (N) protein plays various roles in the virus-induced immune response. But precise mapping of epitopes and antigenic variations in N protein of CDV are still scant. In this study, two monoclonal antibodies (MAbs), designated as F8N and G3N, against the N protein of CDV were generated and characterized. The epitopes recognized by the two MAbs were mapped by truncated N protein fragments expressed in E.coli based on western blotting. The 470ESRYDTQ476 and 385GITKEEAQL393 were identified as the minimal linear epitopes recognized by F8N and G3N, respectively. The amino acid residues of the epitope (385-393aa) were highly conserved in a variety of CDV strains from the databases as well as five CDV strains in this study, indicating that MAb G3N can detect various CDV strains. However, MAb F8N was found not to react with an older CDV 851 strain of the five CDV strains due to both of two amino substitution (S471P and Y473H) in the epitope, whereas either single mutant S471P or Y473H did not eliminate the binding of F8N. Further, the variable epitopes existed in the N protein of six CDV strains resembling CDV3 in phylogenic tree by alignment with sequences from the databases. This is the first record of a precise epitope affecting antigenity of N protein of CDV. These results may facilitate future investigations into the function of NP of CDV and diagnostic methods for CDV infection.

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan-1) for nuclear medicine preclinical trials on spontaneous tumours.

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B-cell lymphoma, CD138 expression is associated with the 'non-germinal center' phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour-bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio-immunotherapy.

Immunodiagnosis of canine visceral leishmaniasis using mimotope peptides selected from phage displayed combinatorial libraries.

ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.

Fine mapping and conservation analysis of linear B-cell epitopes of peste des petits ruminants virus nucleoprotein.

Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays.

Epitope mapping of bovine viral diarrhea virus nonstructural protein 3.

Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea virus (BVDV) were cloned into pMAL-c2X plasmid vector and expressed in Escherichia coli cells (BL21 strain). The recombinant proteins were then purified by amylose resin to determine the most immunogenic domain(s) of the NS3 molecule. Evaluation of the recombinant proteins was carried out by indirect ELISAs using several bovine sera (previously characterized by virus neutralization test, a commercial ELISA kit, and a newly developed NS3-ELISA) and 6 monoclonal antibodies. The experiments showed that the most immunogenic domain of the NS3 protein was the fourth designed fragment (F4), a 122 amino-acid (AA) region of about 13.5 kDa (nucleotide 1003-1368; residue 335-456). Purified recombinant F4 was also evaluated as single ELISA antigen (F4-ELISA) for the detection of anti-BVDV antibodies in sera of infected cattle. Although this small recombinant fragment of NS3 protein was almost completely soluble and expressed more efficient respect to whole NS3 molecule, it did not show enough sensitivity and specificity to be a proper substitute for NS3 as ELISA antigen to detect specific antibodies against BVDV. However, statistical analyses showed a medium correlation between the results of the developed F4-ELISA and virus neutralization test (kappa coefficient=0.63, P<0.001), with the relative sensitivity and specificity of 78.05% and 84.91%, respectively, suggesting the potential use of this fragment as an ELISA antigen along with other antigens or monoclonal antibody(s) in a competitive ELISA.

IgE and IgG epitope mapping by microarray peptide-immunoassay reveals the importance and diversity of the immune response to the IgG3 equine immunoglobulin.

The presence of whole horse IgG in therapeutic snake antivenom preparations of high purity is a contamination that can cause IgE-mediated allergic reactions in patients. In this study, the immunodominant IgE and IgG-binding epitopes in horse heavy chain IgG3 were mapped using arrays of overlapping peptides synthesized directly onto activated cellulose membranes. Pooled human sera from patients with and without horse antivenom allergies were used to probe the membrane. We have demonstrated that, for both cases, individuals produce antibodies to epitopes of sequential amino acids of horse heavy chain IgG3, although the signal strength and specificity appear to be distinct between the two groups of patients. A single region was found to contain the dominant allergic IgE epitope. The critical residues involved in the binding of human IgE to the epitope were determined to include four hydrophobic amino acids followed by polar and charged residues that formed a coil structure. This is the first study to describe the specific amino acid sequences involved with the immune recognition of human IgG and IgE to horse antivenom.

Accumulation profiles of PrP(Sc) in hemal nodes of naturally and experimentally scrapie-infected sheep.

BACKGROUND: In classical scrapie, the disease-associated abnormal isoform (PrP(Sc)) of normal prion protein accumulates principally in the nervous system and lymphoid tissues of small ruminants. Lymph nodes traffic leukocytes via lymphatic and blood vasculatures but hemal nodes lack lymphatic vessels and thus traffic leukocytes only via the blood. Although PrP(Sc) accumulation profiles are well-characterized in ovine lymphoid tissues, there is limited information on such profiles in hemal nodes. Therefore, the objective of this study was to compare the follicular accumulation of PrP(Sc) within hemal nodes and lymph nodes by prion epitope mapping and western blot studies. RESULTS: Our studies found that PrP(Sc) accumulation in 82% of animals' abdominal hemal nodes when PrP(Sc) is detected in both mesenteric and retropharyngeal lymph nodes collected from preclinical and clinical, naturally and experimentally (blood transfusion) scrapie-infected sheep representing all three major scrapie-susceptible Prnp genotypes. Abdominal hemal nodes and retropharyngeal lymph nodes were then used to analyze immune cell phenotypes and PrP(Sc) epitope mapping by immunohistochemistry and PrP(Sc) banding patterns by western blot. Similar patterns of PrP(Sc) accumulation were detected within the secondary follicles of hemal nodes and retropharyngeal lymph nodes, where cellular labeling was mostly associated with macrophages and follicular dendritic cells. The pattern of PrP(Sc) accumulation within hemal nodes and retropharyngeal lymph nodes also did not differ with respect to epitope mapping with seven mAbs (N-terminus, n = 4; globular domain, n = 2; C-terminus, n = 1) in all three Prnp genotypes. Western blot analysis of hemal node and retropharyngeal lymph node homogenates revealed identical three banding patterns of proteinase K resistant PrP(Sc). CONCLUSION: Despite the anatomical difference in leukocyte trafficking between lymph nodes and hemal nodes, the follicles of hemal nodes appear to process PrP(Sc) similarly to lymph nodes.

Epitope mapping of the nucleocapsid protein of sendai virus and application of antigenic epitopes for the ELISA-based diagnosis of sendai virus infection.

Sendai virus (SeV) is one of the most prevalent viral pathogens infecting laboratory mice and rats. To date, mature SeV virions have been used as antigens for serological diagnosis. To develop antigens that are more specific and easier to prepare for diagnosis, we examined the antigenic sites in the nucleocapsid protein (NP) of SeV with antisera from experimentally SeV-infected mice and a peptide array membrane containing overlapping 10-mer peptides covering the entire NP. We found antigenic linear sequences in two regions, amino acids 120-160 and 420-500, of the SeV-NP. From these antigenic sequences, we applied two synthesized peptides, IVKTRDMEYERTTEWL and FVTLHGAERLEEETNDE, which correspond to positions 119-134 and 458-474 of the SeV-NP, respectively, as antigens in an enzyme-linked immunosorbent assay (ELISA). Evaluation of the ELISAs using these peptides revealed that they were specific to anti-SeV antisera. Furthermore, the ELISAs using these peptides were able to distinguish between SeV-positive and SeV-negative mouse sera to the same extent as a commercial ELISA kit. These results indicate that these peptides are useful for the serological diagnosis of SeV infection.

Epitopes from two soybean glycinin subunits are antigenic in pigs.

BACKGROUND: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of basic and acidic portions joined by disulfide bridges. There are five glycinin subunit isoforms designated Gy1-Gy5. The purpose of this study is to identify epitopes from selected glycinin subunits that are antigenic in pigs. RESULTS: Twenty-seven out of 30 pigs had antibodies against glycinin in their sera. Ten of these sera had immunoglobulin G (IgG) against the Gy4 (A5A4B3) or Gy1 (A1aBx) subunit. Three sera recognised overlapping regions between the two subunits tested, though no serum stained both A5A4B3 and A1aBx. Two sera stained a highly conserved region between A5A4B3 and A1aBx, though again neither serum stained both peptides. The basic part of the A1aBx subunit was not recognised by any of the sera tested even though immunoblot data indicated that the basic and acidic subunits of glycinin are nearly equally antigenic. CONCLUSION: Two antigenic regions of A5A4B3 and A1aBx were identified that bound antibodies in half of the sera that reacted with these two proteins. Half of the sera reacted with unique regions of A5A4B3 and A1aBx. The failure of the basic portion of A1aBx to bind pig antibodies may indicate that it is less antigenic than the basic portion of A5A4B3 and other glycinin subunits.

Epitope mapping of a monoclonal antibody against the Gp85 of avian leukosis virus subgroup J.

Avian leukosis virus subgroup J poses a great threat to the poultry industry in China. To reduce the economic losses, a quick method for detection of ALV-J antigen is required for diagnosis and identification of the congenitally transmitting hens. In this study, we report the production and evaluation of one monoclonal antibody (MAb) suitable for achieving these goals. The gp85 gene of avian leukosis virus subgroup J CAUHM01 China isolates was subcloned into the expression vectors pGEX-6p-1 and pET28a and successfully expressed in E. coli. After immunizing BALB/c mice with recombinant His-Jgp85 protein, splenic cells from immunized mice were fused with SP2/0 myeloma cells to produce hybridomas. We isolated and characterized one ALV-J gp85-specific MAb by determining its titer, affinity and IgG subclass. In addition, we performed epitope mapping and determined the epitope for the MAb 1E3 to be 81-92 aa of ALV-J gp85 protein (LPWDPQELDILG). Bioinformatics analysis and IFA studies revealed that this epitope is conserved among all ALV-J isolates and that this antibody could serve as a useful reagent for ALV-J detection and diagnosis.

Localization of linear B-cell epitopes on goose parvovirus structural protein.

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, a highly contagious and lethal disease in goslings and muscovy ducklings, leading to a huge economic loss. However, little is known about the localization of B-cell epitopes on GPV structural protein. To address the issue, the structural protein of GPV was dissected into sets of partially overlapping fragments and expressed in Escherichia coli. Then Western blot reactivity of these glutathione S-transferase (GST) fusion short peptides to viral infected sera was surveyed. The results showed linear immunodominant epitopes, which were found in seven fragments covering amino acid residues 35-71, 123-198, 423-444, 474-491, 531-566, 616-669, 678-732. Our findings may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for Derzsy's disease.

Phage display identifies two Caprine Arthritis Encephalitis Virus env epitopes.

Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein.

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum.

REASONS FOR PERFORMING STUDY: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. HYPOTHESIS: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. METHODS: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n=100) and sera (n=100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n=95) and horses (n=50). RESULTS: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K63 and K238 of the EGF-like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 x biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. CONCLUSIONS AND POTENTIAL RELEVANCE: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.