Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP119, PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP119. Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against Anaplasma marginale in cattle

The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA.

Recombinant expression of molluscan hemocyanin (KLH) substructures in a prokaryotic system: E. coli

The recombinant expression of 19 different substructures of KLH in the prokaryotic sys-tem E. coli has been successfully achieved: each one of the eight single FUs a to h of both isoforms, KLH1 and KLH2, two substructures consisting of two consecutive FUs (KLH1-bc and KLH1-gh) as well as a cDNA encompassing KLH1-abc. All recombinant proteins, fused to an N-terminal 6xHis tag, have successfully been detected by immuno precipitation using monoclonal α-His-antibodies and polyclonal α-KLH1- and α-KLH2-antibodies. One exception remained: SP-KLH2-a, which was not detected by the α-His-antibodies. This allows speculations as to whether the coexpressed signal peptide can lead, at one hand, to the secretion of the recombinant protein, and on the other to the simultaneous cut-off of the leader peptide, which results in the splitting off of even more N-terminal 6xHis tag, leading to failed recognition by the appropriate antibodies. The comparison of native KLH with recombinantly expressed prokaryotic (E. coli) and eukaryotic (Sf9 insect cells) KLH was done using FU-1h. The weak detection by the polyclonal α-KLH1-antibodies of both recombinantly expressed proteins showed that the native protein was the best recognized. For the prokaryotic one, both the denaturation applied for solubilisation of the bacterial inclusion bodies and the inability of bacterial cells to add N-linked glycosylation, are the reason for the poor hybridization. In contrast, KLH1-h expressed in eukaryotic insect cells is likely to be glycosylated. The incubation with the α-KLH1-antibodies resulting in the same weak detection, however, revealed that the linked carbohydrate side chains are not those expected. The establishment of SOE-PCR, together with further improvement, has enabled the generation of a clone encompassing the complete subunit KLH1-abcdefgh. The se-quence analysis compared to the original KLH1 sequence showed, however, that the resulting recombinant protein is defective in two histidines, required for the copper bind-ing sites in FU-1b and FU-1d and in three disulfide bridges (FU-1a, FU-1b and FU 1g). This is due to polymerase-related nucleotide exchanges, resulting in a changed amino acid sequence. Nevertheless, all eight potential N-glycosylation sites are present, leading to the speculation that the recombinant protein can in theory be fully glycosylated, which is the most important aspect for the clinical applicability of recombinant KLH as an im-munotherapeutic agent. The improvement of this method elaborated during the present work indicates bright prospects for the future generation of a correct cDNA sequence encoding for the complete KLH2 subunit.

Recombinant ADAMTS13 normalizes von Willebrand factor-cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non-inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme.

Distinct reactivities of interleukin-4-specific antibodies with recombinant and native canine interleukin-4 in various assays

Interleukin 4 (IL-4) plays a central role in immune responses to parasites and allergens. IL-4 drives the differentiation of naive T cells into Th2 cells and regulates immunoglobulin class switching to IgE.Little is known about the role of IL-4 in canine allergies and parasite infections. Most of the information derives from measurement of IL-4 mRNA expression in dog tissues, but detection of IL-4 protein has been difficult so far, probably due to low sensitivity of available methods. Antibodies (Ab) specific for canine IL-4 are available from various sources, but these Ab have been produced against recombinant Escherichia coli-expressed canine IL-4 and there is only limited information on their reactivities with native canine IL-4. Therefore, in the present study, we tested six available canine IL-4-specific Ab for their reactivities with recombinant canine IL-4 expressed in E. coli (rec.IL-4) or in mammalian cells (mam.IL-4), and with supernatants from stimulated canine peripheral blood mononuclear cells (PBMCs) using several detection methods, including Western blotting, ELISA, cytokine bead assay, and intracellular IL-4 staining. Additionally, we tested a bovine IL-4-specific antibody that has been previously shown to cross-react with canine IL-4. All tested Ab except anti-bovine IL-4 reacted with rec.IL-4, and most of them reacted with mam.IL-4. However, only the cytokine bead assay was sensitive enough to allow the detection of IL-4 in supernatants of canine PBMCs.

Production of antibodies to canine IL-1beta and canine TNF to assess the role of proinflammatory cytokines

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1beta and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1beta and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1beta or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISA's was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1beta as an antigen allowed the detection of lower concentrations of rcanIL-1beta (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1beta-specific and TNF-specific ELISA's, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-gamma combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1beta and TNF production in vitro, and the neutralization of these cytokines.

Virus neutralization by germ-line vs. hypermutated antibodies

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Cκ) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in VH increased by 300-fold the binding strength of monovalent scFv-Cκ. The multivalent binding avidity of germ-line scFv-Cκ was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Cκ did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show “affinity maturation” effects of up to 1000-fold.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Molecular cloning and characterization of a cDNA encoding monoclonal nonspecific suppressor factor.

The monoclonal nonspecific suppressor factor (MNSF) is a lymphokine product of a murine T-cell hybridoma that inhibits the generation of lipopolysaccharide-induced immunoglobulin-secreting cells in an antigen-nonspecific manner. A cDNA clone encoding MNSF beta (an isoform of MNSF) was isolated and expressed in bacteria. The sequence obtained is virtually identical to the Fau protein, a product of the ubiquitously expressed fau gene with unknown function. Northern blot analysis demonstrated a single, 0.6-kb transcript. Specific polyclonal antibodies against synthetic peptides corresponding to the deduced amino acid sequences were elicited in rabbits. Immunoprecipitation experiments with these antibodies showed that MNSF beta is released extracellularly in an aggregate form, albeit it lacks a signal peptide sequence. The anti-MNSF beta affinity eluate from the MNSF-producing murine hybridoma (E17) and concanavalin A-activated splenocyte culture supernatants inhibited the immunoglobulin production by lipopolysaccharide-activated splenocytes. Recombinant MNSF beta also showed a similar biologic activity. Thus, ubiquitin-like protein(s) may be involved in the regulation of the immune responses.

Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column. Results: There were 12 hybridoma strains selected into the polyol-responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA. Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8 +/- 25.2 nM and 167.39 +/- 60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4 +/- 15.9 nM to laminin and of 290.8 +/- 11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.