Overcoming immunotolerance in chronic viral hepatitis with lentivector vaccine immunotherapy.
Lead Research Organisation:
University College London
Department Name: Infection
Abstract
Chronic hepatitis B and hepatitis C affect around 350 and 170 million people worldwide respectively. A far greater number have been infected but have successfully cleared the virus. In chronic infection, however, the virus has been able to evade the immune system by changing rapidly or interfering with normal immunity. In these patients, persistent infection often progresses to cirrhosis of the liver and liver cancer. Unfortunately, current treatments have limited success rates and frequent side-effects.
Our goal is to develop a means of clearing viral infection by targeted activation of the immune system in such a way that bypasses inhibitory signals from the virus. We have already successfully used this technique to stimulate the immune system in cultures of human immune cells and to shrink tumours in mice. This approach involves delivering genes to cells of the immune system which enhance their function and stimulate responses against chosen targets - in this instance the hepatitis viruses.
We will test the efficacy of this approach in a mouse model as well as in blood samples from patients with chronic hepatitis B and C. This will be a crucial first step towards developing an effective targeted therapy for chronic viral hepatitis.
Our goal is to develop a means of clearing viral infection by targeted activation of the immune system in such a way that bypasses inhibitory signals from the virus. We have already successfully used this technique to stimulate the immune system in cultures of human immune cells and to shrink tumours in mice. This approach involves delivering genes to cells of the immune system which enhance their function and stimulate responses against chosen targets - in this instance the hepatitis viruses.
We will test the efficacy of this approach in a mouse model as well as in blood samples from patients with chronic hepatitis B and C. This will be a crucial first step towards developing an effective targeted therapy for chronic viral hepatitis.
Technical Summary
Background
Successful immunotherapy for chronic hepatitis B (CHB) and C (CHC) must overcome both endogenous immunotolerant mechanisms and also viral inhibitory factors that directly down-regulate dendritic cell (DC) and T-cell function. Loading DCs with key antigens can recruit naive multispecific T-cell responses, but sustained activation of DCs is essential for reconstitution of exhausted T-cells and expansion of T-cell populations with underrepresented specificities, thus broadening the response. We have developed lentiviral vectors which can efficiently transduce DCs in vivo and in vitro with both antigen and also constitutive activators of the NF-KB and MAPK pathways (vFLIP and MEKK6 respectively). This dramatically increases DC-activation markers, antigen presentation and subsequent antigen-specific CD8 responses (up to 10-fold). In vivo, co-expression of MEKK6 or vFLIP with tumour antigens has a potent anti-tumour effect prolonging survival in murine models. We recently reproduced these results with non-integrating lentivectors which minimise the theoretical risk of insertional mutagenesis in target cells.
Aims
The goal of this project is to develop a lentivector-based immunotherapy for CHB and CHC. We will test the ability of vFLIP- or MEKK6-expressing lentivectors to induce a more potent, multispecific CTL response to viral hepatitis antigens. This will be assessed in vivo using an HLA A2-transgenic murine model and in vitro with DCs and T-cells from patients with chronic viral hepatitis.
Methods
1) HLA-A2 transgenic mice will be injected subcutaneously with lentiviral vectors encoding viral hepatitis antigen (HBV or HCV nucleoprotein) and either vFLIP or MEKK6. Control groups will include unvaccinated mice and vaccination with vectors encoding mutant inactive vFLIPa57l or MEKK6 K82A. Immune responses will be measured by in vivo killing assays of peptide pulsed splenocytes from naïve mice and FACS quantification of specific CD8 T-cells (pentamer staining), IFN-producing CD8 T-cells and markers of CD8 cytotoxicity and DC activation.
2) DCs from patients with chronic viral hepatitis and those who have cleared infection will be isolated and treated with lentivector preparations in vitro. After rounds of stimulation with lentivector-transduced DCs or DCs treated with peptide pulsing alone, autologous T-cell responses will be measured as above.
Further development
We will assess the immunogenicity of a range of HBV and HCV antigens in lentivector constructs, with the aim of using the most promising in phase I clinical trials. We will also test HBV antigen-expressing lentivectors in the HBV transgenic mouse model in which T-cell responses to HBV antigens are tolerised.
Successful immunotherapy for chronic hepatitis B (CHB) and C (CHC) must overcome both endogenous immunotolerant mechanisms and also viral inhibitory factors that directly down-regulate dendritic cell (DC) and T-cell function. Loading DCs with key antigens can recruit naive multispecific T-cell responses, but sustained activation of DCs is essential for reconstitution of exhausted T-cells and expansion of T-cell populations with underrepresented specificities, thus broadening the response. We have developed lentiviral vectors which can efficiently transduce DCs in vivo and in vitro with both antigen and also constitutive activators of the NF-KB and MAPK pathways (vFLIP and MEKK6 respectively). This dramatically increases DC-activation markers, antigen presentation and subsequent antigen-specific CD8 responses (up to 10-fold). In vivo, co-expression of MEKK6 or vFLIP with tumour antigens has a potent anti-tumour effect prolonging survival in murine models. We recently reproduced these results with non-integrating lentivectors which minimise the theoretical risk of insertional mutagenesis in target cells.
Aims
The goal of this project is to develop a lentivector-based immunotherapy for CHB and CHC. We will test the ability of vFLIP- or MEKK6-expressing lentivectors to induce a more potent, multispecific CTL response to viral hepatitis antigens. This will be assessed in vivo using an HLA A2-transgenic murine model and in vitro with DCs and T-cells from patients with chronic viral hepatitis.
Methods
1) HLA-A2 transgenic mice will be injected subcutaneously with lentiviral vectors encoding viral hepatitis antigen (HBV or HCV nucleoprotein) and either vFLIP or MEKK6. Control groups will include unvaccinated mice and vaccination with vectors encoding mutant inactive vFLIPa57l or MEKK6 K82A. Immune responses will be measured by in vivo killing assays of peptide pulsed splenocytes from naïve mice and FACS quantification of specific CD8 T-cells (pentamer staining), IFN-producing CD8 T-cells and markers of CD8 cytotoxicity and DC activation.
2) DCs from patients with chronic viral hepatitis and those who have cleared infection will be isolated and treated with lentivector preparations in vitro. After rounds of stimulation with lentivector-transduced DCs or DCs treated with peptide pulsing alone, autologous T-cell responses will be measured as above.
Further development
We will assess the immunogenicity of a range of HBV and HCV antigens in lentivector constructs, with the aim of using the most promising in phase I clinical trials. We will also test HBV antigen-expressing lentivectors in the HBV transgenic mouse model in which T-cell responses to HBV antigens are tolerised.