Elsevier

Virus Research

Volume 284, 15 July 2020, 197984
Virus Research

Combination of three adjuvants enhances the immunogenicity of a recombinant protein containing the CTL epitopes of non-structural proteins of hepatitis C virus

https://doi.org/10.1016/j.virusres.2020.197984Get rights and content

Highlights

  • A protein comprising epitopes of non-structural proteins of HCV was designed.

  • T-helper epitope PADRE, bacterial lipopeptide and IL-2 were included as adjuvants.

  • Recombinant fusion proteins stimulate cell-mediated immunity in immunized mice.

  • Proliferation of T cells, induction of cytokines and IFN-γ synthesis was observed.

  • Recombinant protein comprising three adjuvants showed the highest immunogenicity.

Abstract

Hepatitis C virus (HCV) can cause chronic infection and evade the immune response. The generation and maintenance of an effective T-cell response is important for immune-mediated control of HCV infection. The purpose of this study was to obtain recombinant mosaic proteins containing the cytotoxic T lymphocyte (CTL) epitopes of HCV fused with different adjuvants and analyse their immunogenicity. A recombinant polyepitope protein comprising HLA-A2-restricted CTL epitopes of the NS3, NS4ab and NS5a proteins of HCV was designed. Adjuvant compounds, the T-helper (Th) epitope PADRE, lipopeptide from Neisseria meningiditis and interleukin 2 (IL-2) were included in the fusion proteins. Three proteins differing in their adjuvant content were expressed in Escherichia coli and purified. The purified proteins formed nanosized particles. The proteins were characterized by their ability to cause proliferation of spleen cells, induce expression of cytokine genes and production of interferon gamma by T lymphocytes of immunized mice. The obtained recombinant vaccine proteins effectively stimulate dendritic cells, which in turn specifically activate Th1 and Th2 lymphocytes. Adjuvant components act additively to enhance the stimulation of dendritic cells and polarize them in the direction of Th1 lymphocyte activation. Analysis of spleen cell proliferation, expression of Th1 and Th2 cytokines and production of interferon gamma by lymphocytes of immunized mice after specific stimulation in vitro revealed that recombinant protein comprising CTL epitopes of HCV, Th epitope PADRE, lipoprotein and IL-2 induced the highest response of T-lymphocytes.

Introduction

Hepatitis C virus (HCV), a positive-stranded RNA virus belonging to the Flaviviridae family, is a major cause of chronic liver disease worldwide. A distinctive feature of this virus is that it frequently leads to the development of chronic hepatitis C (CHC) after the acute phase of infection. CHC can result in the development of cirrhosis and hepatocellular carcinoma (Cohen, 1999).

Although HCV infection elicits the production of antiviral antibodies, chronic infection is characterized by a lack of effective cellular CD4+ and CD8+ T-cell immune responses (Lasarte et al., 1998; Shin and Wherry, 2007). Interaction of viral proteins within infected cells with components of the cell machinery may constitute an important mechanism that would allow HCV to evade the immune response (Lasarte et al., 1998; Rehermann et al., 1996). There is evidence that T cells play a fundamental role in the control of HCV infection, since viral clearance observed during acute transient infection is correlated with the activation of CD4+ T helper lymphocytes and, importantly, with a broad and sustained response by cytotoxic CD8+ T lymphocytes (Thimme et al., 2001, 2002).

It has been demonstrated that the generation and maintenance of an effective Th-cell response is an important factor in immune-mediated control of HCV infection (Gerlach et al., 1999). Th cells recognize their antigens as small, linear peptides derived from the original protein by proteolytic cleavage and bind to autologous major histocompatibility complex (MHC) class I and II molecules on the surface of antigen-presenting cells which are dendritic cells in particular. However, Th cells of most patients with chronic hepatitis C respond only weakly. It was proposed that HCV-specific CD4+ Th-cell responses are critical for infection resolution and control (Gerlach et al., 1999; Lechmann et al., 1999). Th lymphocytes produce cytokines that stimulate the effector functions of CTL (Lechmann et al., 1999). Chronic HCV infection is mainly attributed to uncontrolled HCV replication due to CD8+ T-cell dysfunction (Neumann-Haefelin et al., 2008; Zabaleta et al., 2007; Shin and Wherry, 2007). Perhaps sufficient stimulation of CTLs could improve the effectiveness of therapeutic treatments for hepatitis C. An efficient vaccine against HCV infection requires the induction of broad cellular and humoral immune responses against several viral proteins (Wong et al., 2001).

Immunization with purified protein antigens typically results in the induction of a modest antibody response with little or no T-cell response. Therefore, vaccine developers include adjuvants in candidate vaccines to enhance the efficacy of weak antigens. Adjuvants are now considered to be a key component of modern vaccines (Day et al., 2002).

The immunogenicity of recombinant proteins could be enhanced by combination with adjuvant compounds such as Th epitopes (Reed et al., 2013; Alexander et al., 1994; Huang et al., 2013; Alexander et al., 2000), interleukins (Faulkner et al., 2001; Zhang et al., 2014), lipopeptides (Bessler and Jung, 1992; Chen et al., 2009; Jackson et.al. 2004) and others.

DCs represent a crucial target of most vaccine adjuvants in both preventive and therapeutic vaccination strategies (Gluckman et al., 2002). DCs play an essential role in the immune response by communicating between the peripheral and lymphoid tissues (Banchereau and Steinman, 1998). They also instruct T-cell responses, being capable of polarizing T-cell development or inducing T-cell tolerance (Lanzavecchia and Sallusto., 2001). Immunotherapy with activated DCs can be used to fight with chronic hepatitis C (Jackson et al., 2005; Zhou et al., 2012). It was shown that lipopeptide-based vaccines have the capacity to induce DC maturation (Zeng et al., 2002). Such lipopeptide-based recombinant protein comprising influenza virus epitopes was able to induce a protective immune response against a challenge with the influenza virus (Jackson et al., 2004). Thus, lipopeptide binding to the toll-like receptors (TLRs) on the DC surface can stimulate the immune response. TLRs are important for the stimulation of innate immunity (Kaisho and Akira, 2002).

The purpose of this study was to design and analyse the immunogenicity of recombinant mosaic proteins containing CTL epitopes of NS3, NS4a, NS4b and NS5a antigens of HCV and combination of the Th epitope PADRE with IL-2 and lipopeptide of Neisseria meningiditis as adjuvants. We studied their ability to stimulate mouse T cell response. It was shown that a recombinant fusion protein comprising three adjuvants, the Th epitope PADRE, lipopeptide and IL-2, has the highest immunostimulatory effect.

Section snippets

Expression and purification of recombinant proteins

Three artificial genes encoding multiepitope recombinant HCV proteins were cloned into the plasmid pQE30Xa. Recombinant proteins carrying N-terminal polyhistidine tags were expressed in E. coli DLT1270 cells. The strains were grown in 2x YT medium (Y1003, Sigma-Aldrich) supplemented with 50 μg/mL ampicillin to OD600 ∼0.7, then IPTG was added to 0.5 mM, and the culture was grown for another 18 h at 28 °C. Protein expression in induced cells was analysed by SDS-PAGE.

Cells from 50 mL were

Design and expression of recombinant proteins containing HCV epitopes

Recombinant polyepitope protein (PE) was designed on the basis of immunogenic peptides selected from NS3, NS4a, NS4b and NS5a non-structural proteins of HCV subtype 1b. Selection of immunogenic CTL peptides (restricted by HLA-A2) of these proteins was carried out on the basis of both literature data (Thimme et al., 2001, 2002; Neumann-Haefelin et al., 2008; Wong et al., 2001; Day et al., 2002; Ward et al., 2002; Capone et al., 2006; Fournillier et al., 2006; Urbani et al., 2001; Guo et al., 2012

Discussion

The efficacy of treatment for HCV infection has improved considerably with the use of inhibitors of different HCV proteins (Hoofnagle, 2009). Nevertheless, new therapeutic options are not sufficient to limit the rapid spread of hepatitis C worldwide. This goal could be achieved by prophylactic vaccination. Vaccines based on recombinant proteins can be cost-effective due to their high efficiency of expression in bacterial systems and the low cost of purification of the expressed protein.

Key

Conclusions

The obtained results suggest that proposed recombinant vaccine proteins act efficiently and specifically stimulated DCs, which in turn activate lymphocytes and polarize them in the direction of activation Th1 lymphocytes. The effect of adjuvant compounds was summarized, eliciting a stronger immune response. We suppose that further research in this direction will lead to the development a prototype vaccine against HCV.

CRediT authorship contribution statement

Victor V. Kuprianov: Project administration, Conceptualization, Investigation, Writing - original draft, Writing - review & editing. Liudmila I. Nikolaeva: Conceptualization, Methodology, Investigation, Writing - original draft. Anna A. Zykova: Investigation, Visualization. Anna V. Dedova: Investigation. Alexander E. Grishechkin: Investigation. Ivan V. Kapustin: Investigation. Roman Y. Kotlyarov: Investigation. Nikolai V. Ravin: Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

This work was partly supported by the Russian Foundation for Basic Research (projects 16-04-00450 and 20-04-00705).

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