An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV.Abbreviations: BV, B virus (Macacine herpesvirus 1); EU, ELISA units; g, glycoprotein; HSV, herpes simplex virus; tELISA, titration ELISA; UN, uninfected; WBA, western blot analysisB virus (BV; Macacine herpesvirus 1) is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. The virus occurs naturally in macaques (Macaca spp.) and causes a lethal zoonotic infection in 80% of untreated humans. Because biomedical professionals working with macaques, their cells, or tissues are at risk for becoming infected with BV, it is important to know the status of macaques involved in potential BV exposures. Although cases of BV infection after encounters between tourists and macaques have not been reported, any event that involves direct or fomite-associated contact with macaques has inherent risks. Identification of zoonotic BV infection through the detection of antibodies enables timely antiviral intervention, which is critical to reduce or prevent morbidity and mortality. Similarly rapid detection is important to maintain the biointegrity of SPF captive macaque colonies. The identification of BV in clinical specimens is achieved by using cell culture, PCR, or antibody detection methods. Because BV is shed only rarely from peripheral sites, the identification of BV infection in monkeys and humans currently is based on antibody detection (serology).^14,23,28In our laboratory, current serological diagnosis for B virus infections has been based on 2 principal tests: a titration-based (that is, traditional) ELISA (tELISA) as a screening test and western blot analysis (WBA) as a confirmatory test. Each test uses quality-controlled BV antigens that are prepared from lysates of infected cells.^20,22,23 Because BV is the only simplex virus in the Alphaherpesvirinae subfamily that is known to infect macaques,^14,28 antibodies interacting with BV antigens are used to indicate BV infection and not an infection due to a crossreacting virus. In practice, tELISA has identified numerous BV antibody-positive sera, the majority of which are low-titer sera from SPF colonies, which fail to be confirmed by WBA, and therefore, are classified as false positives.²³ We, therefore, searched for other approaches that could be used for confirmation of tELISA results. One reasonable option was the use of BV recombinant proteins as antigens. Numerous investigators have used recombinant-based assays for routine diagnosis of infections with viruses, including cytomegalovirus,³⁶ Epstein–Barr,⁶ herpes simplex (HSV1 and HSV2)^{2,3,17,31,32,34} Crimean–Congo hemorrhagic fever,¹⁰ HIV,³⁶ dengue,^5,11,27 hepatitis C,²⁴ hepatitis B,⁸ West Nile,²⁶ influenza,¹⁶ Ebola, and Marburg³³ viruses.Screening for the presence of serum IgG molecules against an array of defined and purified recombinant antigens has distinct advantages over assays that use the entire complement of viral antigens that are present in virus-infected cells. This is particularly true for pathogens that require BSL4 laboratories.^28,33 The pattern of reactivity obtained against each individual recombinant protein may have diagnostic value, by enabling identification of the stage of infection and the prediction of the prognosis of the disease.^3,4,18 However, using a single or only a few recombinant proteins as ELISA antigens can lead to a false-negative result if the antibody repertoire produced after BV infection reacts with other antigenic determinants that are not represented by the particular recombinant antigens used in the test.^{3,18,28,31,34}Several laboratories have examined the efficacy of using a single BV recombinant antigen (that is, glycoprotein D [gD]) for diagnosing BV infections in macaques^25,37 and humans,¹⁵ and we previously reported the diagnostic potential of an ELISA that incorporated several recombinant BV antigens.²⁸ We chose 4 recombinant BV glycoproteins as candidate antigens: peptides corresponding to the full-length extracellular domain of gB, gC, and gD and the membrane-associated segment of gG (gGm). Among these antigens, gGm was the most BV-specific, because it failed to crossreact with antibodies induced by HSV1 and HSV2. To validate the use of the recombinant BV antigens for the purpose of BV antibody detection, a panel of antibody-negative (n = 40) and antibody-positive (n = 75) macaque sera that were confirmed to be positive by tELISA and WBA were tested against the panel of the 4 B virus recombinant antigens, all of which showed fairly high sensitivity for detecting antibodies to BV.²⁸Here, we examine the performance of the recombinant-based ELISA (rELISA) for BV detection by using numerous (>1000) macaque sera, which have a broad range of antibody titers as determined by tELISA. Because manual ELISA to identify antibodies against an array of antigens are too laborious to be cost-effective, we adapted a previously described high-throughput automated single-antigen ELISA performed in 384-well plates to detect antibodies in macaque sera to multiple BV antigens.²³ This assay format has been adapted to include antigens from other alphaherpesviruses²³ and can be easily modified further for other viruses. We then compared the performance of the rELISA with that of whole-virus tELISA and WBA. The main goal of this study was to determine whether the 384-well rELISA is an effective alternative to WBA as a confirmatory assay for tELISA.     

Fungus mediated synthesis of gold nanoparticles and their conjugation with genomic DNA isolated from Escherichia coli and Staphylococcus aureus

Biological systems employing microorganisms have been used as an alternative to conventional chemical techniques for synthesizing gold nanoparticles. In the present study, gold nanoparticles have been synthesized from the supernatant broth (SB) and live cell filtrate (LCF) of the industrially important fungus Penicillium rugulosum. Additionally, potato dextrose broth (PDB) medium which is used for the growth of the fungus has also been able to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification as well as after purification to find the difference in morphology pattern of the nanoparticles. Different characterization techniques like X-ray diffraction (XRD), infra-red (FTIR), X-ray photoelectron (XPS) and UV–vis spectroscopy have been used for analysis of the particles. SB of the fungus has yielded nanoparticles with better morphology and hence further optimization studies were conducted for controlling the size and shape of the above by altering pH and concentration of gold salt. A pH range of 4–6 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. The optimized nanoparticles have been used to conjugate with isolated genomic DNA of bacteria Escherichia coli and Staphylococcus aureus. Visual observation of agarose gel electrophoresis images confirmed the binding of gold nanoparticles (4 μL and 6 μL) with isolated DNA (2 μL) fragments of both the organisms. The slight red shift of the surface plasmon (SP) band and minor aggregations noticed in Bio-TEM images for the DNA conjugated gold nanoparticles indicates that the genomic DNA could stabilize the particles against aggregation owing to negatively charged phosphate backbone.     

Differential activities of the two closely related withanolides, withaferin a and withanone: bioinformatics and experimental evidences

BACKGROUND AND PURPOSE: Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have employed bioinformatics tools on four genes, i.e., mortalin, p53, p21 and Nrf2, identified by loss-of-function screenings. We examined the docking efficacy of Wi-N and Wi-A to each of the four targets and found that the two closely related phytochemicals have differential binding properties to the selected cellular targets that can potentially instigate differential molecular effects. We validated these findings by undertaking parallel experiments on specific gene responses to either Wi-N or Wi-A in human normal and cancer cells. We demonstrate that Wi-A that binds strongly to the selected targets acts as a strong cytotoxic agent both for normal and cancer cells. Wi-N, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. The present molecular docking analyses and experimental evidence revealed important insights to the use of Wi-A and Wi-N for cancer treatment and development of new anti-cancer phytochemical cocktails.     

Long-Term Engraftment of Human Natural T Regulatory Cells in NOD/SCID IL2rγcnull Mice by Expression of Human IL-2

Regulatory T cells are essential to maintain immune homeostasis and prevent autoimmunity. Therapy with in vitro expanded human nT_Regs is being tested to prevent graft versus host disease, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate appropriately and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model for studying the in vivo reconstitutive capability of human nT_Regs is a major impediment for investigating the behavior of adoptively transferred nT_Regs in vivo. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nT_Regs in NOD-SCID IL2rγc^null mice. We also demonstrate that these in vivo reconstituted T_Regs traffic to different organs of the body and retain suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted T_Regs are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel ‘hu-T_Reg mouse’ model offers a pre-clinical platform to study the in vivo function and stability of human nT_Regs and their ability to modulate autoimmune diseases and GVHD.     

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.     

Metabolic effects of chronic obestatin infusion in rats

Obestatin is purported to be a peptide hormone encoded in preproghrelin. We studied the metabolic effects of continuous infusion of obestatin via subcutaneously implanted osmotic mini-pumps. Administration of up to 500nmol/kg body weight/day obestatin did not change 24h cumulative food intake or body weight in rats. Similarly, no effects were observed when obestatin was infused at 1000nmol/kg body weight/day for seven days. This dose of obestatin infused during a 24h fast did not alter weight loss, suggesting that obestatin has no effect on energy expenditure, and this dose did not alter glucose or insulin responses during an IPGTT. Obestatin was originally proposed to interact with GPR39 and subsequently the receptor for GLP-1. While both receptors are expressed in pancreatic islets, incubation with obestatin did not alter insulin release from islets in vitro. Moreover, obestatin did not bind to INS-1 beta-cells or HEK cells overexpressing GLP-1 receptors or displace GLP-1 binding to these cells. Our findings do not support the concept that obestatin is a hormone with metabolic actions.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 degrees C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 degrees C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

Thermodynamic characterization of cytosolic phospholipase A2 alpha inhibitors

Cytosolic phospholipase A₂ alpha (cPLA₂α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA₂α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA₂α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA₂α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA₂α with a 1:1 stoichiometry, and the dissociation constant K_d of the inhibitors calculated from the ITC experiments correlated well with the IC₅₀ values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA₂α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA₂α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA₂α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.     

Enhanced thermal stability of chemically deglycosylated human choriogonadotropin

Exposure of aqueous solutions of native human choriogonadotropin (hCG), asialo-hCG (A-hCG), and chemically deglycosylated hCG (DG-hCG) to heat treatment revealed significant differences in their stability. Solutions of hCG and A-hCG were rapidly inactivated above 50 degrees C. On the other hand, solutions of DG-hCG were comparatively more stable under similar conditions as shown by the retention of significant receptor binding, immunological, and hormonal antagonistic activities. Heated solutions (100 degrees C) of hCG and A-hCG quickly lost their ability to enhance the fluorescence of the probe 1-anilino-8-naphthalenesulfonate (1,8-ANS) indicating dissociation into subunits. DG-hCG solutions were more stable in this respect suggesting significant preservation of conformational features required for the interaction with 1,8-ANS. Solutions of hCG and A-hCG which had been thermally denatured (100 degrees C, 10 min) required almost 48 h at 37 degrees C to regain complete ANS binding ability as well as receptor binding activity. Under the same conditions, heated solutions of DG-hCG completely regained these abilities in less than 2 h. A similar pattern was observed with acid (pH 2.0)-dissociated hCG, A-hCG, and DG-hCG. While heated solutions of hCG had no effect on the action of native hCG in vitro, heated DG-hCG solutions still retained their ability to antagonize the cyclic AMP accumulation or steroidogenesis induced by native hCG in rat interstitial cells. Thus, removal of carbohydrate residues (approximately 75% loss) from hCG renders the hormone more resistant to thermal denaturation.