Pagina | 7

2

Abstract

The hepatitis C virus (HCV) is a virus with small pericapsid, a single-stranded RNA molecule with positive polarity and belonging to the family Flaviviridae.

The virus replicates predominantly in hepatocytes, causing chronic liver damage that can lead to the development of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.

HCV triggers both a cell-mediated and humoral immune response which is frequently not sufficient to eradicate the infection and prevent the progression of the disease. One of the main roles in preventing the disease seems to be played by neutralizing antibodies (nAb) able to bind to HCV receptors (E1-E2 glycoproteins on HCV envelope) and to block the entry into target cells.

The real contribution of these antibodies is still uncertain due to the impossibility to grow HCV in vitro and therefore to develop an effective neutralization assay.

On these premises, my thesis has focused on the set up of a neutralization assay with the aim of measuring the neutralizing antibody activity in serum of patients infected with HCV.

In order to do that, I have produced viral particles pseudotyped with HCV envelope E1-E2 glycoproteins using a murine leukemia virus (MLV) derived vector. This vector is based on a split-component system with 3 plasmids: a packaging construct expressing gag and pol genes necessary for structural and enzymatic viral proteins production, a vector construct that maintains the encapsidation signal and contains an expression cassette expressing the green fluorescent protein (GFP), and an envelope construct encoding HCV surface glycoproteins E1 E2.

Similarly, and only by replacing the envelope construct, particles were also produced with the G protein of vesicular stomatitis virus (VSV-G). VSV-G binds to a phospholipid that can be found on the surface of every cell and therefore has been used as a control of particles production and for evaluating antibodies aspecific neutralizing activity during the assay.

Pagina | 8 The three constructs were co-transfected into 293T cells, a line of human embryonic kidney fibroblasts, to form pseudotyped particles that were then used for infecting human hepatocellular carcinoma cell line Huh-7, on which we have set up a neutralization assay.

This consists in the use of fixed quantities of pseudovirions incubated with serial dilutions of serum, allowing the link between nAb and E1-E2; then the mixtures were incubated with Huh-7 cells.

After seventy-two hours, the samples were analyzed by flow cytometry in order to quantify transduced cells, recognizable because GFP-positive.

Finally, the reduction of fluorescent cells number in samples incubated with serum was compared to that of controls (cells incubated with only vector) and expressed as a percentage.