A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein.

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell's receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.

The Adjuvant of α-Galactosylceramide Presented by Gold Nanoparticles Enhances Antitumor Immune Responses of MUC1 Antigen-Based Tumor Vaccines.

BACKGROUND: Therapeutic tumor vaccines are one of the most promising strategies and have attracted great attention in cancer treatment. However, most of them have shown unsatisfactory immunogenicity, there are still few available vaccines for clinical use. Therefore, there is an urgent demand to develop novel strategies to improve the immune efficacy of antitumor vaccines. PURPOSE: This study aimed to develop novel adjuvants and carriers to enhance the immune effect of MUC1 glycopeptide antigen-based antitumor vaccines. METHODS: An antitumor vaccine was developed, in which MUC1 glycopeptide was used as tumor-associated antigen, α-GalCer served as an immune adjuvant and AuNPs was a multivalent carrier. RESULTS: Immunological evaluation results indicated that the constructed vaccines enabled a significant antibody response. FACS analysis and immunofluorescence assay showed that the induced antisera exhibited a specific binding with MUC1 positive MCF-7 cells. Moreover, the induced antibody can mediate CDC to kill MCF-7 cells. Besides stimulating B cells to produce MUC1-specific antibodies, the prepared vaccines also induced MUC1-specific CTLs in vitro. Furthermore, the vaccines significantly delayed tumor development in tumor-bearing mice model. CONCLUSION: These results showed that the construction of vaccines by presenting α-GalCer adjuvant and an antigen on gold nanoparticles offers a potential strategy to improve the antitumor response in cancer immunotherapy.

Rescuing AAV gene transfer from neutralizing antibodies with an IgG-degrading enzyme.

Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.

Peptidorhamnomannan from Lomentospora prolificans modulates the inflammatory response in macrophages infected with Candida albicans.

BACKGROUND: Peptidorhamnomannan is a glycoconjugate that consists of a peptide chain substituted by O- and N-linked glycans, present on the cell surface of Lomentospora prolificans, a saprophytic fungus which is widely distributed in regions with temperate climates. O-linked oligosaccharides from peptidorhamnomannan isolated from Lomentospora prolificans conidia are recognized by macrophages mediating macrophage - conidia interaction. In this work, peptidorhamnomannan was isolated from L. prolificans mycelium cell wall and its role in macrophage - Candida albicans interaction was evaluated. RESULTS: Purified peptidorhamnomannan inhibits the reactivity of rabbit immune sera to mycelial and conidia forms of L. prolificans, indicating that this glycoconjugate is exposed on the fungal surface and can mediate interaction with host immune cells. We demonstrated that peptidorhamnomannan leads to TNF-α production in J774 macrophages for 1, 2 and 3 h of incubation, suggesting that this glycoconjugate may have a beneficial role in the response to fungal infections. In order to confirm this possibility, the effect of peptidorhamnomannan on the macrophage - C. albicans interaction was evaluated. Macrophages treated with peptidorhamnomannan led to a lower fungal survival, suggesting that peptidorhamnomannan induces an increased fungicidal activity in macrophages. Furthermore, TNF-α levels were measured in supernatants after macrophage - C. albicans interaction for 1, 2 and 3 h. Peptidorhamnomannan treatment led to a higher TNF-α production at the beginning of the interaction. However, the release of TNF-α was not maintained after 1 h of incubation. Besides, peptidorhamnomannan did not show any inhibitory or fungicidal effect in C. albicans when used at 100 µg/ml but it was able to kill C. albicans at a concentration of 400 µg/ml. CONCLUSION: We suggest that peptidorhamnomannan acts as a molecular pattern on the invading pathogen, promotes TNF-α production and, thus, increases macrophage fungicidal activity against Candida albicans.

Differential distribution and potential regulatory roles of estrogen receptor 2a and 2b in the pituitary of ricefield eel Monopterus albus.

Estrogens play important regulatory roles in the pituitary of vertebrates. Two forms of estrogen receptor 2 (Esr2), namely Esr2a and Esr2b, are identified in teleosts, but their differential roles remain to be fully elucidated. In the present study, expression and potential functional roles of Esr2a and Esr2b were characterized in ricefield eels. esr2a and esr2b mRNA were broadly distributed in tissues, with high levels observed in the brain, pituitary, and gonads. In order to examine the cellular localization of Esr2a and Esr2b in the pituitary, specific antisera against ricefield eel Esr2a and Esr2b were generated, respectively. Interestingly, immunohistochemistry and Western blot analysis revealed that Esr2a and Esr2b were differentially distributed in the pituitary, with the former localized to the adenohypophysis while the latter to the neurohypophysis. Dual fluorescent immunostaining showed that immunoreactive Esr2a was present in Gh and Prl cells, but not in Lh and Fsh cells. Estradiol (E2) stimulated lhb and prl gene expression in dispersed pituitary cells of intersexual ricefield eels, but had no effects on gh, fshb, and gnrhr2 gene expression and Gh release. Results of the present study are helpful for further understanding the roles and mechanisms of estrogen signals in the pituitary.

Expression of DOCK10.1 protein revealed with a specific antiserum: insights into regulation of first exon isoforms of DOCK10.

DOCK10, a guanine-nucleotide exchange factor (GEF) for Rho GTPases, represents the example of a gene that gives rise to alternative first exon mRNA isoforms, named DOCK10.1 and DOCK10.2. Expression of human DOCK10.2 protein in cell lines, and its induction by interleukin-4 (IL-4) in normal B lymphocytes and chronic lymphocytic leukemia (CLL) cells, were previously demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.2. Here, expression of human DOCK10.1 protein was demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.1. Specificity of the DOCK10.1 and DOCK10.2 antisera for their respective isoforms was demonstrated using transfected human 293 T cells. Their specificity for endogenous DOCK10 was strongly suggested by the high significance of the correlations between the levels of their expected signals at the molecular size of 250 kDa and the levels of DOCK10.1 and DOCK10.2 mRNAs, respectively, in human hematopoietic cell lines. Specificity of the DOCK10.1 antiserum for DOCK10 was also demostrated in mouse using the DOCK10 knockout model. The DOCK10.1 protein was induced by IL-4 in CLL cells, which demonstrates that the mechanism by which IL-4 regulates DOCK10 is not isoform-specific. Last, to get insights into differential regulation of the DOCK10 isoforms, their protein levels in cell lines were compared with their gene expression profiles retrieved from the Cancer Cell Line Encyclopedia (CCLE), leading to the identification of BCL3 and KLF12 as potential transcriptional regulators of DOCK10.1 and DOCK10.2, respectively.

The soluble fraction of Neospora caninum treated with PI-PLC is dominated by NcSRS29B and NcSRS29C.

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.

Molecular events contributing to successful pediatric cardiac transplantation in HLA sensitized recipients.

Antibodies to HLA resulting in positive cytotoxicity crossmatch are generally considered a contraindication for cardiac transplantation. However, cardiac transplantations have been performed in children by reducing the Abs and modifying immunosuppression. To identify mechanisms leading to allograft acceptance in the presence of Abs to donor HLA, we analyzed priming events in endothelial cells (EC) by incubating with sera containing low levels of anti-HLA followed by saturating concentration of anti-HLA. Pre-transplant sera were obtained from children with low levels of Abs to HLA who underwent transplantation. EC were selected for donor HLA and exposed to sera for 72 h (priming), followed by saturating concentrations of anti-HLA (challenge). Priming of EC with sera induced the phosphatidylinositol 3-kinase/Akt mediated by the BMP4/WNT pathway and subsequent challenge with panel reactive antibody sera increased survival genes Bcl2 and Heme oxygenase-1, decreased adhesion molecules, induced complement inhibitory proteins and reduced pro-inflammatory cytokines. In contrast, EC which did not express donor HLA showed decreased anti-apoptotic genes. Primed EC, upon challenge with anti-HLA, results in increased survival genes, decreased adhesion molecules, induction of complement inhibitory proteins, and downregulation of pro-inflammatory cytokines which may result in accommodation of pediatric cardiac allografts despite HLA sensitization.

Inner membrane complex 1l protein of Plasmodium falciparum links membrane lipids with cytoskeletal element 'actin' and its associated motor 'myosin'.

The inner membrane complex (IMC) is a defining feature of apicomplexans comprising of lipid and protein components involved in gliding motility and host cell invasion. Motility of Plasmodium parasites is accomplished by an actin and myosin based glideosome machinery situated between the parasite plasma membrane (PPM) and IMC. Here, we have studied in vivo expression and localization of a Plasmodium falciparum (Pf) IMC protein 'PfIMC1l' and characterized it functionally by using biochemical assays. We have identified cytoskeletal protein 'actin' and motor protein 'myosin' as novel binding partners of PfIMC1l, alongside its interaction with the lipids 'cholesterol' and 'phosphatidyl-inositol 4, 5 bisphosphate' (PIP2). While actin and myosin compete for interaction with PfIMC1l, actin and either of the lipids (cholesterol or PIP2) simultaneously bind PfIMC1l. Interestingly, PfIMC1l showed enhanced binding with actin in the presence of calcium ions, and displayed direct binding with calcium. Based on our in silico analysis and experimental data showing PfIMC1l-actin/myosin and PfIMC1l-lipid interactions, we propose that this protein may anchor the IMC membrane with the parasite gliding apparatus. Considering its binding with key proteins involved in motility viz. myosin and actin (with calcium dependence), we suggest that PfIMC1l may have a role in the locomotion of Plasmodium.

Fermitin family homolog-2 (FERMT2) is highly expressed in human placental villi and modulates trophoblast invasion.

BACKGROUND: Integrins are transmembrane receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesion and trophoblast cells undergo changes in integrin expression as they differentiate. However, the mechanism(s) of integrin activation leading to integrin-mediated signaling in trophoblast cell differentiation is unknown. The Fermitin family proteins are integrin activators that help mediate integrin-mediated signaling, but have never been studied in detail within the human placenta. Thus, we examined the spatiotemporal pattern of expression of Fermitin family homolog-2 (FERMT2) in human chorionic villi throughout gestation and its role in trophoblast-substrate adhesion and invasion. METHODS: Placental villous tissue was obtained from patients undergoing elective terminations by dilatation and curettage at weeks 8-12 (n = 10), weeks 13-14 (n = 8), as well as from term deliveries at weeks 37-40 (n = 6). Tissues were fixed, processed and sections utilized for immunofluorescence analysis of FERMT2 expression during gestation. Additionally, HTR8-SVneo human trophoblast cells were transfected by electroporation with FERMT2-specific siRNAs or non-targeting siRNAs (control) and used in cell-substrate adhesion as well as invasion assays. RESULTS: FERMT2 was more commonly expressed in the basal domain of villous cytotrophoblast cells and prominently localized around the periphery of individual extravillous trophoblast cells. siRNA-mediated knockdown of FERMT2 in HTR8-SVneo cells resulted in significantly decreased trophoblast-substrate attachment (p < 0.05) as well as significantly decreased trophoblast invasion (p < 0.05) relative to control cells. CONCLUSIONS: The detection of FERMT2 throughout extravillous trophoblast columns and the results of invasion assays demonstrated that this protein is likely an important regulator of integrin activation in extravillous cells to modulate migration and invasion.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

Establishment of a Screening System to Identify Novel GATA-2 Transcriptional Regulators.

Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is essential for cell proliferation and differentiation. Heterozygous germline GATA2 mutations induce GATA-2 deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia and acute myeloid leukemia, and a profoundly reduced dendritic cell (DC) population, which is associated with increased susceptibility to viral infections. Because patients with GATA-2 deficiency syndrome could retain a wild-type copy of GATA-2, boosting residual wild-type GATA-2 activity may represent a novel therapeutic strategy for the disease. Here, we sought to establish a screening system to identify GATA-2 activators using human U937 monocytic cells as a potential model of the DC progenitor. Enforced GATA-2 expression in U937 cells induces CD205 expression, a marker of DC differentiation, indicating U937 cells as a surrogate of human primary DC progenitors. Transient luciferase reporter assays in U937 cells reveals a high promoter activity of the -0.5 kb GATA-2 hematopoietic-specific promoter (1S promoter) fused with two tandemly connected GATA-2 +9.9 kb intronic enhancers. We thus established U937-derived cell lines stably expressing tandem +9.9 kb/-0.5 kb 1S-luciferase. Importantly, forced GATA-1 expression, a repressor for GATA-2 expression, in the stable clones caused significant decreases in the luciferase activities. In conclusion, our system represents a potential tool for identifying novel regulators of GATA-2, thereby contributing to the development of novel therapeutic approaches.

Interactions of antisera to different Chlamydia and Chlamydophila species with the ribosomal protein RPS27a correlate with impaired protein synthesis in a human choroid plexus papilloma cell line.

Chlamydia trachomatis (CT) and the Chlamydophila species (CS) Chlamydophila pneumoniae (CPn), and Chlamydophila psittaci (CPs) are suggested to induce autoantibodies causative of several human autoimmune disorders like rheumatoid arthritis and systemic lupus erythematosus (SLE). The aim of the present study was therefore to identify cellular protein interaction partners with antisera to CT (α-CT) or CS (α-CS) and to identify functional consequences of such interaction in vitro. As detected with a commercial first trimester human prenatal brain multiprotein array (hEXselect, Engine, Germany), the most frequent interaction partner with both α-CT and α-CS was the ribosomal small subunit protein RPS27a. This could be confirmed by Western blot analysis with a recombinant RPS27a sample. In addition, immunocytochemistry with both antisera in the human choroid plexus papilloma cell line HIBCPP revealed a granular cytoplasmic staining, and Western blot analysis with whole-cell protein samples of HIBCPP cells revealed both antisera to label protein bands of different molecular weights and intensity. By 2D Western blot analysis and mass spectrometry, one of the protein spots interacting with α-CT could be identified as the RPS27a. Finally, two different methods for the detection of protein synthesis activity, the SUnSET technique and an HPG fluorescence assay revealed both antisera to cause reduced translational activity in HIBCPP cells. Together with previous findings of RPS27a as an autoimmune target in a mouse model of systemic lupus erythematosus (SLE), these results suggest that infections with CT and/or CS could induce SLE-associated immune modifications. However, direct evidence for a pathogenic role of these interactions for SLE demands further investigations.

Peptides mimicking viral proteins of porcine circovirus type 2 were profiled by the spectrum of mouse anti-PCV2 antibodies.

BACKGROUND: Porcine circovirus 2 (PCV2) is a small, non-enveloped DNA virus causing swine lymphocyte depletion and severe impact on the swine industry. The aim of this study was to evaluate the antigenicity and immunogenicity of specific peptides, and seeking the potential candidate of PCV2 peptide-based vaccine. It's initiating from peptides reacting with PCV2-infected pig sera and peptide-immunized mouse sera. RESULTS: The data showed that the sera from PCV2-infected pigs could react with the N-terminal (C1), middle region (C2), and C-terminal peptide (C3) of PCV2 capsid protein (CP), ORF3 protein (N1), ORF6 protein (N2) and ORF9 protein (N3). This study demonstrated that anti-PCV2 mouse antisera could be generated by specific synthetic peptides (C3 and N2) and recognized PCV2 viral protein. We found that the tertiary or linear form C-terminal sequence (C3) of PCV2 capsid peptide only appeared a local distribution in the nucleus of PCV2-infected PK cells, virus-like particles of PCV2 major appeared a local distribution in the cytoplasm, and ORF 6 protein of PCV2 were shown unusually in cytoplasm. Furthermore, most residues of the C1 and the C3 were presented on the surface of PCV2 CP, in the view of 3-D structure of the CP. Our data demonstrated that PCV2-infected pigs had higher OD405 value of anti-C3 IgG on Day 1, Month 3 and Month 6 than in Month 1. These pigs had higher anti-C3 IgM level in Month 3 and Month 6 than on Day 1 (P < 0.01). CONCLUSIONS: We demonstrated that the key peptide (C3) mimic the C-terminal of PCV2 capsid protein which were capable of inducing antibodies. The specific antibody against the C3 were confirmed as the serological marker in PCV2-infected pigs.

Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin.

Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 µM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

Structural and biochemical characterisation of VaF1, a P-IIIa fibrinogenolytic metalloproteinase from Vipera ammodytes ammodytes venom.

A high molecular mass metalloproteinase with α-fibrinogenolytic activity, termed VaF1, was purified from nose-horned viper (Vipera ammodytes ammodytes) venom. Subcutaneous injection of 9 µg of VaF1 did not induce bleeding in rats. Nevertheless, in vitro it degraded collagen IV, nidogen and fibronectin, components of the extracellular matrix, although with low efficacy and narrow specificity. VaF1 would be expected to exert anti-coagulant action, due to its hydrolysis of fibrinogen, factor X, prothrombin and plasminogen, plasma proteins involved in blood coagulation. The enzyme is a single-chain glycoprotein with a molecular mass of 49.7 kDa, as determined by mass spectrometry, and multiple isoelectric points centred at pH 5.8. The complete amino acid sequence of the precursor of VaF1 was deduced by cloning and sequencing its cDNA. Composed of metalloproteinase, disintegrin-like and cysteine-rich domains, VaF1 is a typical P-IIIa subclass snake venom metalloproteinase. Although it possesses a collagen-binding sequence in its disintegrin-like domain, VaF1 displayed no effect on collagen-induced platelet aggregation in vitro. Two consensus N-glycosylation sites are present in the sequence of VaF1, however, the extent of its glycosylation is low, only 5.2% of the total molecular mass. Interestingly, in standard experimental conditions VaF1 is not recognised by antiserum against the whole venom, so it can contribute to post-serotherapy complications, such as ineffective blood coagulation, in the envenomed patient.

Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis.

The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using test serum samples. Immunolocalisation showed that T. pisiformis FABP (TpFABP) localised to the parenchyma of the bladder wall of the cysticercus and perinuclear cytoplasm of parenchyma of the adult parasite. After cloning and expression, recombinant TpFABP (rTpFABP) protein was used for serodiagnosis of T. pisiformis infection in rabbits by dot-ELISA. The antibody was detected 14 days post-infection in rabbits experimentally infected with T. pisiformis. Based on the necropsy results, the sensitivity and specificity of 169 serum samples tested by rTpFABP dot-ELISA were found to be 98.2% (54/55) and 92.1% (105/114), respectively. These data suggest that the dot-ELISA developed in this study has potential for detection of T. pisiformis infection in rabbits.

Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.

Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection.

Synthetic peptides for in vitro evaluation of the neutralizing potency of Loxosceles antivenoms.

An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.

Using immunoproteomics to identify alpha-enolase as an autoantigen in liver fibrosis.

Liver fibrosis results from extracellular matrix accumulation during the wound healing process when the liver is insulted with chronic viral infection, inflammation, or alcoholic diseases. The current diagnosis of liver fibrosis is mainly dependent on biopsy, which is an invasive approach. Identification of serological biomarkers has been considered as the most promising way for early detection of the disease. Although several biomarkers in liver fibrosis have been identified, the problem is that these markers can be also detected in fibrogenesis that occurred in other organs. In this study, we have identified and characterized some cellular proteins that can be recognized by autoantibodies in the sera from patients with precirrhotic stage of liver fibrosis. Among 180 sera from patients with liver fibrosis, 14.4% (26/180) of sera contained autoantibody against a protein migrating around 47 kDa on SDS-PAGE gel. Indirect immunofluorescence assay using purified autoantibody against the 47-kDa protein showed that this protein mainly localized in the cytoplasm. Using immunoproteomic approach, the 47-kDa protein was identified as alpha-enolase. In further study, the frequency of antialpha-enolase antibody in sera from patients with precirrhotic stage of liver fibrosis (21.6%, 27/125) was significantly higher than that in sera from patients with cirrhosis (9.1%, 5/55) and liver cancer (14.3%, 12/84), as well as in sera from healthy individuals (4.1%, 3/74). Therefore, alpha-enolase is an autoantigen that elicits autoimmune response in liver fibrosis and can be a potential prognostic factor for liver fibrosis diagnosis.