Abstract
Abstract
The hepatitis C virus (HCV) is subject of intense study. HCV infection affects approximately 3% of population worldwide and constitutes a serious social and health damage. The virus replicates predominantly in hepatocytes, causing chronic liver damage that can lead to the development of chronic hepatitis, cirrhosis of the liver and hepatocellular carcinoma. Moreover the infection increases the number of complications in people affected by human immunodeficiency virus. HCV triggers both a cell-mediated and humoral immune response which 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 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 effective neutralization assay.
On these premises my thesis has focused on the set up of a neutralization
assay to measure the neutralizing antibody activity in serum of patients infected with
HCV. To this aim, I have produced viral particles pseudotyped with E1 E2
glycoproteins of HCV envelope using a lentiviral vector previously produced and
designed in our laboratories and derived from feline immunodeficiency virus (FIV). As
positive control we also used a vector developed by Dr. Cosset research group
deived from murine leukemia virus (MLV). Both viral vectors are based on a system
using 3 plasmids (split-component system): a packaging construct expressing the
gag and pol genes necessary for structural and enzymatic viral proteins production
and expressed under the cytomegalovirus (CMV) promoter that allows the gene
expression in heterologous cells, vector construct containing the encapsidation
signal and an expression cassette containing the green fluorescent protein (GFP), a
envelope construct encoding the glycoprotein E1 E2 of HCV surface. Alternatively the
envelope construct may express the gene encoding the protein G of vesicular
stomatitis virus, used as a control because of its capability to use a
phosphatidylserine as receptor thus broadening cell tropism. The three constructs
were co-transfected into 293T cells, a line of human embryonic kidney fibroblasts, to
IIIAbstract
form pseudotyped virus that were then used for infection of human hepatocellular carcinoma cell line Huh-7. The neutralization assay has proved fast, sensitive and able to detect the presence of antibodies in some patients with chronic hepatitis C infection. For a further optimization the luciferase expressing gene was subsequently cloned in the FIV-derived vector construct in place of GFP. The use of this construct together with packaging and envelope plasmid enables the development of luciferase expressing pseudoviruses. This optimization might increase the sensitivity and spread of the assay.
IV
