Monocytes transduced with lentiviral vectors expressing hepatitis C virus non-structural proteins and differentiated into dendritic cells stimulate multi-antigenic CD8⁺ T cell responses

Abstract

Halting the spread of hepatitis C virus (HCV) and also eradicating HCV in subjects with chronic infection are major goals for global health. To this end, several years of research on HCV vaccine development have led to the conclusion that multi-antigenic and multi-functional vaccine types are necessary for effectiveness against HCV infection. In this study, we evaluated lentiviral vectors (LV) expressing clusters of HCV structural (LV-HCV-S) and non-structural (LV-HCV-NS) genes for future vaccine development. Batches of high titer LV were used to transduce differentiated dendritic cells (DC) and monocytes. We report successful delivery of HCV gene clusters, particularly into monocytes, leading to >80% LV-HCV-NS and >70% LV-HCV-S and transduced cells, respectively. Intracellular expression of HCV proteins in monocyte-derived DC resulted in immunophenotypic changes, such as downregulation of CD83 and CD86. Monocytes expressing NS proteins and differentiated into DC stimulated allogeneic and autologous CD8⁺ and CD4⁺ T cells in vitro and resulted in antigen-specific CD8⁺ T cell responses against NS3, NS4a and NS5b. Hence, lentiviral-mediated expression of the multi-antigenic HCV-NS cluster in monocytes subsequently differentiated into DC is a novel potential anti-HCV vaccine modality.

Introduction

Approximately 130 million chronic hepatitis C virus (HCV) infections have been estimated worldwide. The standard treatment consisting of recombinant IFN-α alone or in combination with ribavirin, has proven efficacious in approximately half of HCV-infected patients [1]. However, many patients cannot be treated using this standard regimen as IFN-α is associated with frequent and sometimes severe side effects. Moreover, between 10% and 50% of individuals infected with HCV are unable to clear the virus following an acute infection and, as a result, become persistently infected leading to chronic infection, which can lead to liver cirrhosis and development of hepatocellular carcinoma. Therefore, prophylactic vaccines (to hinder HCV epidemics) and therapeutic adjuvant immunotherapy approaches (to prevent disease progression or to ultimately cure chronic HCV patients) have been actively explored in past years but have not yet ultimately succeeded in clinical trials [2], [3], [4].

HCV contains a single-stranded, positive-sense RNA genome of approximately 9500 nucleotides. The genome consists of a single open reading frame (ORF), which encodes a large polyprotein of approximately 3000 amino acid residues. The polyprotein is cleaved by cellular and viral proteases into at least ten different products consisting in structural (the core, the E1 and E2 envelope proteins and p7) and non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a and NS5b) [5], [6]. Although some structural proteins can stimulate antibody and T cell responses, a major drawback in their use for vaccination strategies is their high mutability, which may be involved in evasion of the innate and adaptive host immune response and seroconversion [6], [7]. In contrast, studies in acute or persistently HCV-infected humans have demonstrated that immunity against conserved domains of HCV-NS proteins are generally correlated with viral clearance [4], [8], [9], [10].

It is not clearly known how an individual develops into a chronic hepatitis virus carrier state; however, a defective immune response of the host is thought to play a critical role in the underlying pathogenic mechanism. Peripheral blood DC from HCV-infected patients compared to normal controls have shown decreased expression of CD86, decreased production of IL-12 and lower allostimulatory capacity [11], [12], [13]. The apparent defects in DC correlate with an impairment of the effector function of HCV-specific CD8⁺ T cells in chronic HCV infection. HCV-specific tetramer positive T cells are frequently found in PBMCs from chronically infected patients, but they display an impaired proliferative capacity [14]. This phenomenon may be the consequence of “helpless” stimulation of CD8⁺ T cells during the inefficient presentation of HCV antigens by DC, leading to anergy or ultimately tolerance. Thus, the defective functions of HCV-specific CD8⁺ T cells might contribute to viral persistence in chronically infected patients, and approaches to avoid or revert their dysfunction may facilitate the development of prophylactic and immunotherapeutic vaccines.

DC provide the most potent pathway for initiating T and B cell immune responses [15]. Thus, the use of DC-based vaccines in the treatment of patients with acute and chronic infections is a field with vast applications. Since blood is the most accessible tissue for clinical studies, various groups have developed protocols using peripheral blood mononuclear cells (PBMC) for the in vitro production of DC. GM-CSF and IL-4 added to peripheral blood monocytes in culture promote the generation of “immature DC”, the most prevalent form of DC in tissues [16], [17]. DC differentiated in vitro and genetically manipulated with cDNA, RNA or engineered viral vectors have been evaluated in several clinical trials. These strategies have proven to be feasible, safe and effective to activate both CD8⁺ CTL and CD4⁺ T-helper cells [18]. In addition, genetic manipulations of DC in order to ectopically express immune modulators may potentially help to overcome immune dysfunctions that occurred in vivo, as is the case for chronic HCV infections.

LV are a subtype of retroviral vectors that were intensively developed during the last decade. Unlike non-integrating viral vectors such as adenovirus and vaccinia, LV integrate in the genome and offer an approach by which efficient, long lasting, non-toxic and non-immunogenic gene delivery into monocytes and DC may be obtained. In contrast to previously developed onco-retroviral vectors (such as Moloney Murine Leukemia Virus), lentiviruses are able to infect non-proliferating cells, due to the karyophilic properties of the lentiviral pre-integration complex, which allows recognition by the cell nuclear import machinery. Therefore, LV can transduce primary quiescent cells, cells that are growth-arrested in culture, as well as terminally differentiated cells. The lentiviral packaging system was originally developed as a tripartite transient transfection procedure [19] and later evolved into further generations where the four accessory genes of HIV (vif, vpr, vpu and nef) were deleted from the viral packaging system [20] and a 400-nucleotide deletion in the 3′ long terminal repeat resulted into self-inactivating (SIN) LV. The risk of vector mobilization and production of replication competent LV is drastically reduced for the SIN vectors [20]. In most cases, LV are pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), which is a rhabdovirus envelope protein that is reported to bind to ubiquitous cell surface phospholipids, thereby achieving a wide host range. Previous observations from other groups and from our own research demonstrate that lentiviral vector transduction is a suitable methodology for efficient gene delivery into DC or monocytes [21], [22], [23], [24]. Due to their robust infectivity and persistent transgene delivery capabilities into APCs, LV have emerged as a novel potent approach for genetically engineered DC [24].

Of considerable interest for vaccine development, intravenous or subcutaneous LV administration has resulted consistently into potent CTL responses specific against several cancer antigens such as Melan-A [25], [26], NY-ESO 1 [27], [28] and TRP2 [29], [30]. LV vaccines are also currently in preclinical testing for protection or treatment of human immunodeficiency virus (HIV). Several routes of LV administration have been explored in mice, leading to consistent and persistent anti-HIV/SIV immune responses [31], [32].

In this report, we demonstrate the high capability of LV to transfer whole sets of HCV structural or non-structural gene clusters in vitro into monocytes prior to their differentiation into DC. Notably, gene delivery of the HCV-NS cluster into monocytes resulted in its persistent expression in differentiated DC leading to potent stimulation of CD4⁺ and CD8⁺ allogeneic and autologous responses.