1. Activated suicide gene modified lymphocytes (SGMLs) inhibit Hepatitis C virus (HCV) replication multiple in vitro models of infection.
2. At low effector:target cell ratios, SGMLs inhibit HCV replication through non-cytotoxic mechanisms and are resistant to calcineurin inhibitors.
Evidence Rating Level: 2 (Good)
Study Rundown: Chronic HCV infections are strongly associated with severe late stage liver disease, including liver cirrhosis and hepatocellular carcinoma, both of which may lead to the need for liver transplantation (LT). Unfortunately, the recurrence of HCV-caused liver disease often follows LT as a result of graft reinfection. Existing treatments for transplant patients are often not ideal as they exhibit poor efficacy, toxicity, and may be prohibitively expensive. Previous work demonstrated the injection of activated graft lymphocytes three days after transplantation reduced HCV viral load in the patients. Unfortunately, in some patients reinfection occurred. It was hypothesized that earlier injection of activated lymphocytes may reduce the chance of reinfection.
In this current proof-of-concept study, healthy, un-related donor lymphocytes were activated ex vivo (or after isolation), and then cultured with target cells recently inoculated with HCV. The activated lymphocytes were also genetically modified to express a vector for a drug-inducible suicide gene to avoid graft-verus-host-disesase (GVHD), which may be induced via the injection of non-self, non-graft lymphocytes.
While in vivo evidence was not demonstrated, in vitro studies demonstrated SGMLs inhibited HCV replication through non-cytotoxic, or non-cell-killing, mechanisms. This anti-viral activity was, at least in part, attributed to soluble factors, and was IFN-γ-dependent. Graft rejection prevention drugs, cyclosporine A (CsA) and Tacrolimus (FK506), did not inhibit antiviral activity of SGMLs, suggesting SGMLs utility in transplant patients. As expected, SGMLs were depleted with addition of a prodrug targeting the suicide vector, and this depletion eliminated the antiviral effect of SGMLs. While the models of HCV infection used in this work are well accepted, they use a cell line rather than primary hepatocytes as their target cells, which may reduce the heterogeneity of results. Additionally, many of the robust antiviral effects were observed for relatively high effector:target cell ratios, which may be difficult to replicate in vivo.
In-Depth [in vitro study]: This study used CD3 or CD3/CD28 activated lymphocytes, which were transduced with retroviral vectors expressing the suicide gene iCasp9, to investigate the anti-viral potential of these effector cells when cultured in the presence of virus-transfected target cells. The SGMLs were incubated with two separate models of HSV infection: the HCV replicon or HCVcc model in Huh7.5.1 cells.
At an effector:target cell ratio of 2:1, SGML presence drove an approximate 3-log reduction in HCV replication after three days of co-culture. No strong cytotoxic effects were found, and a transwell experiment that separated the two cell types confirmed the anti-viral effect of the SGMLs was at least partially through soluble factors. Antiviral activity was more potent in the presence of the activating interleukin-2 (IL-2) in the HCVcc model of infection. The soluble antiviral effect was reversed when IFN-γ blocking antibodies were added to culture media. When SGMLs were introduced on the same day as HCV infection in the HCVcc model of viral replication, inhibition was more pronounced than if the SGMLs were introduced 1-3 days after infection of the target cells. Cryopreserved SGMLs exhibited antiviral activity similar to fresh SGMLs. Calcineurin inhibitors, CsA and FK506, were shown to not affect the antiviral activity of SGMLs at concentrations up to 100 ng/ml (CsA) or 10 ng/ml (FK506). Administration of the prodrug, a chemical inducer of death via the iCasp9 suicide gene, to a cell culture of low target:effector cell ratios, drove SGML depletion and subsequent abrogation of the antiviral activity.
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