Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine

Abstract

Background

An effective vaccine would be a significant progress in the management of chronic HCV infections. This study was designed to examine whether different application schedules and injection routes may enhance the immunogenicity of the HCV peptide vaccine IC41.

Methods

In this randomized trial 54 healthy subjects received either subcutaneous (s.c.) or intradermal (i.d.) vaccinations weekly (16 injections) or every other week (8 injections). One group additionally received imiquimod, an activator of the toll-like receptor (TLR) 7. The T cell epitope-specific immune response to IC41 was assessed using [³H]-thymidine CD4+ T cell proliferation, interferon-gamma (IFN-γ) CD8+ and CD4+ ELIspot and HLA-A*0201 fluorescence-activated cell sorting (FACS) tetramer-binding assays.

Results

More than 60% of vaccinees responded in the CD4+ T cell proliferation assay in all groups. An HLA-A*0201 FACS tetramer-binding assay and IFN-γ CD8+ ELIspot class I response of more than 70% was induced in four and three groups, respectively. IC41 induced significant immunological responses in all groups with responder rates of up to 100%. Interestingly, topical imiquimod was not able to enhance immunogenicity but was associated with a lower immune response. Local injection site reactions were mostly transient. Intradermal injections caused more pronounced reactions compared to s.c., especially erythema and edema.

Conclusion

Compared to a previous study intensified dosing and/or i.d. injections enhanced the response rates to the vaccine IC41 in three assays measuring T cell function. Immunization with IC41 was generally safe in this study. These results justify testing IC41 in further clinical trials with HCV-infected individuals.

Introduction

The Hepatitis C virus is responsible for the majority of parenterally transmitted and community acquired non-A, non-B hepatitis virus infections. It is a positive-stranded enveloped RNA virus. The 10 kilobase genome contains a single open-reading frame encoding a polyprotein cleaved into structural (Core, E1 and E2/NS1) and non-structural (NS2, NS3, NS4 and NS5) proteins [5], [6], [7].

An estimated 120–170 million people are infected worldwide with the HCV and 3–4 million individuals are newly infected each year [1], [2]. 35,000 new infections occur in the US alone each year [3]. Current treatment with pegylated IFN and ribavirin is only effective in about 50% of subjects, shows partially severe adverse effects and is too expensive for regions with the highest HCV prevalence rates in the world. Alternative treatment modalities are urgently needed.

An infection with HCV leads to viral persistence and chronic disease in >80% of cases [8]. Sequelae contribute significantly to morbidity and mortality. Chronic HCV infection leads to liver fibrosis, cirrhosis, liver transplantation and finally hepatocellular carcinoma. Each year up to 10,000 deaths and 1000 liver transplantations are due to HCV in the US alone [4]. These pathologies occur despite significant humoral and cellular immune responses against the structural proteins of HCV [7], [9].

Although the T cell response in chronically infected patients is polyclonal and multispecific, it is not as strong as in acutely infected patients. The relatively weak immune response against HCV antigens might be responsible for the failure to eliminate HCV [5]. CD8+ T cell dysfunction during chronic HCV infection might be the main culprit for the inability of the immune system to control HCV replication and stop disease progression [10]. The role of specific antibodies against HCV in controlling the infection is more controversial. Envelope antibodies would be the prime candidates for virus neutralization, but their presence in chronically infected patients and animal experiments argue against efficient humoral virus neutralization in vivo [11], [12]. The existing antibodies are mainly specific against the hypervariable region 1 (HVR1) of the envelope protein 2. This is disadvantageous, because the heterogeneity in the envelope HVR may be accompanied by the failure of the immune system to mount an antibody response to the dominant strain and also to respond to IFN therapy [9], [14]. Finally, antibody-mediated immune pressure seems to directly correlate with an evolution of viral escape mutants during the course of infection [15]. In contrast, recently published data indicated that during acute HCV infection in vivo virus-specific neutralizing antibodies drive sequence evolution and in some individuals play a role in determining the outcome of infection [42]. An important role in viral clearance seems to be a rapid induction of neutralizing antibodies in the early phase of infection [43]. Sera from chronic HCV carriers showed broadly reactive neutralizing capacity using HCV pseudo-particles but not all viral genotypes were efficiently blocked from infecting cells [44]. Therefore, both the induction of neutralizing antibodies and a strong cellular immune response are presumably required for clearance of an acute infection, protection against chronic infection and for an efficient treatment of a chronic HCV infection [13].

In principle three design approaches for HCV vaccines are conceivable: Prevention of initial infection (sterilizing immunity); prevention of viral persistence and induction of a sustained virological response (therapeutic vaccine). So far not many therapeutic vaccine candidates besides IC41 have been tested in HCV-infected individuals in clinical trials. Recombinant envelope protein E1 adjuvanted with Aluminium hydroxide was tested in 35 patients with chronic HCV infection [26], [27]. TG4040 is currently tested in several clinical studies. This vaccine is based on the attenuated smallpox virus modified vaccinia Ankara (MVA) expressing NS3, NS4 and NS5B proteins [28]. A personalized vaccination approach using HCV-1b-derived peptides was recently tested in a Phase I study [29]. A vaccine using whole heat-killed S. cerevisieae expressing HCV NS3 and other core antigens showed preliminary promising 12 weeks results in an ongoing Phase II study compared to standard care IFN/ribavirin [30]. First data about a codon-optimized HCV NS3/4 DNA gene expressed using the cytomegalovirus immediate-early promoter have been presented recently [32]. Detailed descriptions of the different vaccine approaches and their clinical development status have been published recently [13], [16]. Clinically relevant reductions in the viral load using vaccines against HCV and correlation with immunological response parameters have been demonstrated so far only for individual patients. Data from large randomized trials are not available.

IC41, the therapeutic vaccine used in this study, is based on five synthetic peptides containing three CD4+ and five CD8+ T cell epitopes. The T helper type 1/T cytotoxic type 1 adjuvant Poly-l-Arginine is used as adjuvant. Detailed descriptions of IC41 with clinical study results from several Phase I and II trials were published recently [17], [18], [19], [31]. Briefly, IC41 was safe and well tolerated in 128 HLA A2 positive healthy subjects. Immune response analysis using CD4+ T cell proliferation, HLA-A*0201 FACS tetramer-binding and IFN-γ ELIspot assays showed that the vaccine was immunogenic with a correlation between higher responder rates with dose and number of vaccinations [17]. A Phase II study in patients chronically infected with HCV not responding to standard treatment showed that after six injections T cell proliferation was recorded in about two-thirds of patients in the IC41 groups but only in 17% treated with peptides alone [18]. ELIspot responses were only observed exclusively in the IC41 groups with response rate of up to 42%. Additional T cell analyses of the two above mentioned studies [17], [18] revealed that in healthy subjects IC41 induced both HCV-specific central memory and effector CD8+ T cells, which readily proliferated upon peptide antigen exposure in vitro [19]. In chronic HCV patients the frequency of HCV-specific CD8+ T cells was increased after IC41 administration with a decline of CD45RA-positive effector memory cells in some but not all patients.

Imiquimod is an activator of TLR 7, an important component of the innate immune system. Although the adaptive and innate arm of the immune system have previously been considered relatively independent, more recently it became clear that activation of the innate arm via TLRs leads to an induction of a predominant T helper (Th1) cell response of the acquired immunity and the induction of many cytokines. In several vaccine studies imiquimod increased the immunogenicity of an array of different vaccines including peptide-based vaccine approaches and acted as a potent adjuvant in preclinical and clinical studies [33], [34], [35], [36], [41].

Compared to subcutaneous or muscular tissue the skin contains a large number of antigen-presenting cells. It has been shown that the immune response after i.d. application using 5–10-fold lower antigen dosages produced comparable results [37], [38], [39]. Consequently, local application of imiquimod, which furthermore increases the local concentration of antigen-presenting cells, was combined with i.d. application of IC41.

We hypothesized that an intensified injection schedule and/or i.d. injection route may enhance the immunogenicity of IC41.