Lamivudine treatment enhances allostimulatory function of HBV-infected dendritic cells in vitro

Category 5: Viral hepatitis: basic aspects ~ 0 - ~ CHARACTERIZATION OF HCV RNA PARTICLES FROM THE SERUM OF A PATIENT WITH COMMON VARIABLE IMMUNODEFIClENCY ON ISOTONIC IODIXANOL (OPTIPREP) GRADIENTS. ASSOCIATION WITH APOLIPOPROTEIN-S100 Soren Nielsen, Wanna Pumeechockchai, Alistair Burt, Margaret Bassendine, Geoffrey Toms. Centrefor Liver Research, The

University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK Objective: To investigate the association of HCV RNA and lipoprotein in the serum of an antibody negative patient with common variable immunodeficiency. Methods: The serum collected six weeks post-orthotopic liver transplant from a patient with common variable immunodeficiency and chronic HCV infection was fractionated by sequential density centrifugation on sodium bromide and by isopycnic centrifugation on isotonic iodixanol (optiprep). Results: Whilst sequential density centrifugation yielded HCV RNA fractions of high (> 1.2 g/ml), intermediate (1.063-1.21 g/ml) and low (< 1.063 g/ml) density of approximately equal titre, on iodixanol gradients all the RNA was recovered at the top of the gradient with a density below 1.13 g/ml. Following precipitation with anti-apoB100 or manganese chloride and heparin the majority of the HCV RNA was removed from the serum leaving only a minor peak with a density of 1.13 g/ml on iodixanol gradients. Treatment of sera with sodium desoxycholate released further particles with a density of 1.13 g/ml in iodixanol gradients, suggesting that this is the density of the virus particle stripped of serum lipoproteins. Treatment with 0.18% NP40 produced a single peak of HCV RNA in a fraction with a density of 1.23 g/ml and a sedimentation coefficient of 150S in iodixanol gradients, taken to be naked viral cores. Conclusions: Fractionation of HCV RNA on iodixanol in isotonic conditions indicates that essentially all RNA is present in lower density fractions and is associated with low density or very low density lipoproteins.

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~ 2 - 7 YELLOW FEVER VACCINATION IS ASSOCIATED WITH FALSE-POSITIVE SEROLOGY TO HEPATITIS C VIRUS Eric Bassetti-Soares 1,2, Erika Mota Pereira I , Marcelo Eduardo Lima Souza I , Rosangela Teixeira 2. 1Hemominas Foundation, Research

Department, Belo Horizonte, Minas Gerais; 2Liver Ambulatory, GEN/CAD, InternalMedicine Department, UFMG, Belo Horizonte, Minas Gerais, Brazil Introduction: The HCV prevalence (EIA) among blood donations at Hemominas is 0.25%. From October 1998 to December 1999 occurred an increase in this prevalence (up to 700%), following a massive population vaccination against yellow fever. Therefore, we sought to investigate the cross-reaction positivity of anti-HCV test after yellow fever vaccine. Methods: 110 blood donors with two consecutive anti-HCV positive tests were submitted to a 3rd serology test (3rd generation EIA) six months after. Demographic data and date of yellow fever vaccination were collected. Results: Ninety-two out of 110 donors (83.6%) switched to a 3rd negative anti-HCV exam, and 18 out of 110 donors (16.4%) remains antiHCV positive. There was no difference in demographic variables between these groups. Fifty-six out of 92 3rd anti-HCV negative (60.9%) and 6/18 (33.3%) 3rd anti-HCV positive serology had been vaccinated against yellow fever (Qui-square = 4.64; p = 0.03), almost all in the first semester before donation (mean 4.1 months). Overall, among the yellow fever vaccinated donors, 56/62 (90.3%) had 3rd serology negative. Conclusions: Anti-HCV test can be false-positive after yellow fever vaccination, probably related to HCV and yellow fever cross-reaction due to the similar structure genomic of these flaviviridae viruses. Based on this report, we would like to call the attention of physicians working in endemic areas of yellow fever and blood banks to this interesting issue.

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LAMIVUDINE TREATMENT ENHANCES ALLOSTIMULATORY FUNCTION OF HBV-INFECTED DENDRITIC CELLS IN VITRO

~ 0 - ~ CHARACTERIZATION OF THE STRUCTURAL PROTEINS OF HCV ISOLATED FROM HUMAN LIVER

Susanne Beckebaum 1,2, Vito Cicinnati3, Xia Zhang l , Anne Achterfeld 1, Donna Stolz 4, Andrea Frilling 2, Christoph E. Broelsch 2, Guido Gerken ~.

Soren Nielsen, Margaret Bassendine, Alistair Burt, Geoffrey Toms. The

t Department of Gastroenterology and Hepatology, University of Essen; 2Department of Surgery, University of Essen; 3Departmentof Pathology, University of Essen, Germany; 4Departmentof CeU Biology and Physiology, University of Pittsburgh, USA

Centrefor Liver Research, The University of Newcastle upon Tyne, Newcastle upon Tyne, UK Objective: To determine the molecular weights of the structural protein of HCV recovered from infected human liver. Methods: Macerates of a six weeks post-orthotopic liver transplant from a patient with common variable immunodeficiency and chronic HCV infection were shown to contain 9 LogIU of HCV RNA/g. Macerates were analysed by isopycnic centrifugation on iodixanol density gradients. HCV RNA was precipitated from crude macerates with manganese chloride and heparin and the solubilised precipitate was analysed by SDS-PAGE and western blotting with monoclonal antibodies to the viral structural proteins. Results: Following fractionation of the liver macerate on iodixanol density gradients all the HCV RNA was recovered in fractions of density of 1.13 g/ml and below suggesting that the RNA is associated with host lipoprotein. In line with this, manganese/heparin treatment precipitated essentially all of the HCV RNA. SDS-polyacrylamide gel electrophoresis of the proteins present in manganesehaeparin precipitates and western blotting revealed a single band of core protein of molecular weight 21 kDa and an E1 band of 31 kDa which migrated in approximately the same position in the gel as the corresponding proteins transiently expressed from a recombinant vaccinia virus system. A single E2 band, however, migrated with a molecular weight of 62 kDa some 8 kDa smaller than the equivalent band expressed from recombinant vaccinia virus. Conclusions: The molecular weights of the HCV structural proteins recovered from beta-lipoprotein associated virions suggest that processing of the virus polyprotein in the liver may differ from that in recombinant vaccinia virus expression systems.

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a central role in the induction of antiviral immune response. Recently we have shown that monocyte-derived DCs (mDCs) from patients with chronic hepatitis B infection are functionally impaired. In this study we established an in vitro model and investigated the ability of hepatitis B virus (HBV) to infect mDCs from healthy volunteers. Monocyte - derived DCs from healthy subjects were propagated in vitro and inoculated with HBV particles. Viral infection has been confirmed by PCR and electron microscopy. Non-infected mDC cultures propagated from the same healthy volunteers in parallel served as controls. MLR assays revealed an impaired allostimulatory capacity of HBV-infected mDCs (HBV-mDCs) in comparison to controls. Interferon-g production by responder T ceUs in MLR assays with HBV-mDCs was lower than with non-infected mDCs and correlated to lower expression of 1L- 12 p35 and p40 transcripts in HBV-mDCs. Presence of Lamivudine restored impaired allostimulatory function of HBV-mDCs. This effect was not due to a direct T cell stimulatory activity in vitro. These results show that HBV infection compromises antigen presenting function of mDCs and thus may play an important role in viral escape from immune surveillance. However, Lamivudine treatment can overcome mDC related T cell hyporeactivity and underlines its important role for an enhanced immune response against HBV.