Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus.

The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient's sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient's prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient's samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results.

Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease.

BACKGROUND: The envelope protein from multiple sclerosis (MS) associated retroviral element (MSRV), a member of the Human Endogenous Retroviral family 'W' (HERV-W), induces dysimmunity and inflammation. OBJECTIVE: The objective of this study was to confirm and specify the association between HERV-W/MSRV envelope (Env) expression and MS. METHODS: 103 MS, 199 healthy controls (HC) and controls with other neurological diseases (28), chronic infections (30) or autoimmunity (30) were analysed with an immunoassay detecting Env in serum. Env RNA or DNA copy numbers in peripheral blood mononuclear cells (PBMC) were determined by a quantitative polymerase chain reaction (PCR). Env was detected by immunohistology in the brains of patients with MS with three specific monoclonals. RESULTS: Env antigen was detected in a serum of 73% of patients with MS with similar prevalence in all clinical forms, and not in chronic infection, systemic lupus, most other neurological diseases and healthy donors (p<0.01). Cases with chronic inflammatory demyelinating polyneuropathy (5/8) and rare HC (4/103) were positive. RNA expression in PBMC and DNA copy numbers were significantly elevated in patients with MS versus HC (p<0.001). In patients with MS, DNA copy numbers were significantly increased in chronic progressive MS (secondary progressive MS vs relapsing-remitting MS (RRMS) p<0.001; primary progressive MS vs RRMS -<0.02). Env protein was evidenced in macrophages within MS brain lesions with particular concentrations around vascular elements. CONCLUSION: The association between MS disease and the MSRV-type HERV-W element now appears quite strong, as evidenced ex-vivo from serum and PBMC with post-mortem confirmation in brain lesions. Chronic progressive MS, RRMS and clinically isolated syndrome show different ELISA (Enzyme-Linked Immunosorbent Assay) and/or PCR profiles suggestive of an increase with disease evolution, and amplicon sequencing confirms the association with particular HERV-W elements.

Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR.

BACKGROUND: The principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or ß2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses. RESULTS: Stool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID(50/ml)). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis. CONCLUSIONS: In this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.

Endogenous retrovirus type W GAG and envelope protein antigenemia in serum of schizophrenic patients.

BACKGROUND: Recent and independent molecular studies have shown an association between human endogenous retroviruses type "W" family (HERV-W) and schizophrenia, mostly by polymerase chain reaction studies, but none has yet addressed specific antigen detection in living patients. METHODS: Forty-nine schizophrenic patients and an equivalent number of healthy control subjects were included in the present exploratory study. The HERV-W GAG and envelope (ENV) proteins were quantified in the serum with a dedicated immunoassay set-up with specific monoclonal antibodies to either antigen. RESULTS: In schizophrenic patients, positive antigenemia for ENV was found in 23 of 49 (47%) and for GAG in 24 of 49 (49%). Only 1 of 30 (3%) for ENV and 2 of 49 (4%) for GAG were positive in blood donors (p < .01 for ENV; p < .001 for GAG). Interestingly, bioclinical data analyses revealed significant correlation between GAG or ENV antigenemia (a protein causing dysimmune inflammatory effects) and C-reactive protein (CRP) levels (a systemic inflammation biomarker). CONCLUSIONS: Frequently elevated CRP has previously been described in schizophrenic patients and has been shown to match with an evolution toward cognitive deficit and neuronal loss. Elsewhere viruses such as influenza, long-associated with risk for schizophrenia through perinatal infections, have been shown to activate HERV-W elements in human cells. We therefore discuss a relationship between environment factors long-associated with early risk, genetic factors represented by this endogenous family, the production of its pro-inflammatory ENV protein and known "inflammation-mediated" neurotoxicity, as a possible hypothesis for a pathogenic cascade in association with HERV-W. Our present results thus confirm that HERV-W studies have opened a novel avenue of research in schizophrenia.

Biotinylation of glycan chains in beta2 glycoprotein I induces dimerization of the molecule and its detection by the human autoimmune anti-cardiolipin antibody EY2C9.

Binding of beta2GPI (beta2 glycoprotein I), a human plasma protein, to AnPLs (anionic phospholipids) plays a key role in the formation of antiphospholipid antibodies involved in autoimmune diseases like antiphospholipid syndrome or systemic lupus erythematosus. We recently showed that binding of beta2GPI to AnPLs was enhanced by biotinylation of its glycan chains with biotin-hydrazide. In the present study, we investigated why this chemical modification of beta2GPI increased both its affinity for AnPLs and its recognition by anti-cardiolipin antibodies. Electrophoretic analysis showed that: (i) high molecular mass beta2GPI (dimers and other oligomers) covalently coupled by imine bonds, were present in variable amounts in oxidized beta2GPI and in beta2GPI-bh (beta2GPI-biotin-hydrazide), but were absent in native beta2GPI; (ii) binding of beta2GPI-bh to phosphatidylserine-coated microtitre plates generated high molecular mass polymers in a time-dependent manner. Native beta2GPI did not polymerize in these conditions. These polymers did not bind more strongly to AnPLs than the monomer beta2GPI. However, in solution at 1 microM beta2GPI-bh essentially appeared as a dimer as revealed by light-scattering analysis. SPR (surface plasmon resonance) analysis showed that the increased affinity of beta2GPI-bh for AnPL monolayers was due to a lower dissociation rate constant compared with native beta2GPI. Finally, the monoclonal human aCL (auto-immune anti-cardiolipin antibody) EY2C9 bound to beta2GPI-bh but did not bind to monomeric native and oxidized beta2GPI. It is likely that the dimeric quaternary structure of beta2GPI-bh is in fact responsible for the appearance of the epitopes targeted by the EY2C9 antibody.

Oxidation and biotinylation of beta 2 glycoprotein I glycan chains induce an increase in its affinity for anionic phospholipids similar to that obtained by the addition of anti-beta 2 glycoprotein I or anti-cardiolipin antibodies.

Binding of beta 2 glycoprotein I (beta2GPI) to apoptotic cells plays a key role in the opsonization of apoptotic bodies and the formation of antiphospholipids antibodies. Here, we describe the binding of beta2GPI to apoptotic cells using beta2GPI labelled with biotin-hydrazide (beta2GPI-bh) after oxidation of its glycan chains. Flow cytometry analyses and confocal microscopy showed that beta2GPI-bh, contrary to native beta(GPI, bound to apoptotic cells, either permeable or non-permeable to propidium iodide (PI), as did annexin-V-FITC. But, in the absence of divalent ions, beta2GPI-bh, contrary to annexin V, was still able to bind to apoptotic cells. Binding equilibrium studies, performed on solid-state anionic phospholipids (AnPL), revealed that beta2GPI-bh had a greater apparent affinity for AnPL than native beta2GPI. In presence of the anti-beta2GPI mAb 8C3, the ability of native beta2GPI to bind to AnPL was increased and binding to apoptotic PI+ and PI- CEM cells was observed whereas binding of beta2GPI-bh was barely affected by the addition of 8C3. However, the 8C3-enhanced ability of native beta2GPI to bind to AnPL was still weaker than that of beta2GPI-bh. It is not clear why the oxidation and biotinylation of glycan chains of beta2GPI increases its affinity for AnPL, but it seems that if such oxidative process occurs naturally, it could participate in enhancing antiphospholipid formation.