Deliverables
The HCV NS345Core polyprotein candidate vaccine was formulated in ISCOMATRIX?, a saponin based adjuvant, which has been demonstrated to promote antibody responses and induce T helper cell as well as cytotoxic T lymphocyte responses . Five chimpanzees received a 1 mg intramuscular dose of HCV Polyprotein in ISCOMATRIX? vaccine at 0, 1, 2, and 5 months before being challenged 6 months after the last dose with 100 CID50 of an heterologous HCV1a strain (HCV-H).
While the results from this study demonstrated the ability of the ISCOMATRIX? formulation of the NS345Core polyprotein to elicit HCV-specific CD4 and CD8 T cell responses, they also showed that these responses were not sufficient to confer protective immunity from infection with an heterologous HCV strain. In fact, while 40% of control animals cleared the virus, 80% of the polyprotein-vaccinated chimps developed chronic infection after a single intravenous challenge with HCV-H, as demonstrated by persistent HCV-RNA positive PCR results and slight, but consistent, increased ALT levels .
Nevertheless, evidence of antiviral activity was found in all vaccinated chimpanzees, which showed lower viremia and ALT levels in comparison to infected control animals, during the acute phase of infection. Since a broad CD4 and CD8 T cell response have been associated with a benign course of infection and higher number of HCV Core specific CTLs have been found to correlate with an improved response to treatment with pegylated Interferon-alpha an Ribavirin (4-6) the results of the present study encourage the exploitation of HCV-polyprotein ISCOMATRIX? as a possible therapeutic vaccine, at least for those HCV chronic patients showing low or no response to current therapies.
Invariant (inv)NKT cells are autoreactive lymphocytes recognizing endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines.
We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and showed modified effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13.
Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic viral hepatitis, and suggest that these cells contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.
This result represent one of the most important achievements within the MEMOVAX project. Infact, we have developed an improved method of Epstein-Barr virus transformation of human B cells. We have used this method to analyse the memory repertoire of a patient who recovered from severe acute respiratory syndrome coronavirus (SARS-CoV) infection and and to isolate monoclonal antibodies specific for different viral proteins. The technology is hereafter described.
After informed consent was obtained, peripheral blood was collected from a 35-year-old patient who had recovered from infection by SARS-CoV. IgG+ memory B cells were isolated by binding to CD22 microbeads (Miltenyi) followed by depletion of cells carrying IgM, IgD and IgA by cell sorting. Memory B cells were seeded at 10 or 50 cells per well in 9 U-bottom microplates in complete medium containing 2.5 g/ml CpG 2006, in the presence of EBV (30% supernatant of B95-8 cells) and irradiated allogeneic mononuclear cells (50,000 per well).
After 2 weeks, the culture supernatants were screened for specific antibodies. Positive cultures were cloned by limiting dilution in the presence of CpG 2006 and irradiated mononuclear cells. Antibody was purified from culture supernatants by affinity chromatography on protein A columns (Amersham). Out of 56 attempts, 43 (76%) led to the isolation of one or more clones producing antibodies of the selected specificity. The EBV clones were stable, and monoclonal antibodies were recovered in the culture supernatants at a concentrations of 3-20 µg/ml.
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