Elsevier

Vaccine

Volume 18, Issue 25, 15 June 2000, Pages 2878-2885
Vaccine

Recombinant polioviruses expressing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes

https://doi.org/10.1016/S0264-410X(00)00060-8Get rights and content

Abstract

T-cell immune response to recombinant poliovirus expressing foreign antigens has not been elucidated well. In order to investigate the potential use of poliovirus as a T-cell vaccine vector, we constructed recombinant polioviruses expressing HBV-derived CTL epitopes, HBsAg28–39 (Ld-restricted; IPQSLDSWWTSL) and HBc93–100 (Kb-restricted; MGLKFRQL) at the junction between the P2 and P3 regions, designated V3CDs and V3CDc, respectively. The V3CDs and V3CDc recombinant viruses replicated efficiently in HeLa cells and showed a similar infection profile to that of the wild-type Mahoney strain in one-step growth kinetics. Genetic stability analysis showed that V3CDc retained the foreign insert over twelve successive passages examined, whereas V3CDs lost part of the foreign insert after four passages. Our results indicated that the stability of the inserted foreign sequences was rather affected by their nucleotide sequence than by their length when located between the P2 and P3 regions. The junction between these nonstructural protein-coding regions is a novel site for the construction of replication-competent recombinant poliovirus. Immunization of BALB/c (H-2d) and C57BL/6 mice (H-2b) with V3CDs and V3CDc, respectively, elicited significant antigen-specific CTL responses to HBsAg28–39 but not to HBcAg93–100.

Introduction

Understanding roles of T-lymphocytes in immune response has demonstrated that specific cytotoxic T-lymphocytes may be critical to cure not only patients who have contracted autoimmune diseases and cancers, but also who have been infected with several infectious agents such as HIV, HBV, etc. [1]. In this regard, studies for the development of T-cell vaccine have been undertaken using various types of antigens, e.g. synthetic peptides, recombinant proteins containing foreign T-cell epitopes, recombinant viruses, naked DNA. Among these approaches, recombinant viruses have been considered as an attractive type of antigen because specific peptides can be presented via the MHC class I pathway from the inserted minigene encoding a T-cell epitope, and thereby induce specific CTL responses. Moreover, the use of recombinant viruses gives the advantage of inducing a protective immunity not only against the foreign epitopes, but also against the carrier virus itself.

For construction of those recombinant viruses, poxvirus and adenovirus have been frequently utilized. Recently, poliovirus was recognized as a promising candidate for use as a vaccine vector. This is because the live-attenuated poliovirus vaccine strains have many advantages, including extensive use in humans, verified higher safety, ability to induce both systemic humoral immunity and local intestinal mucosal immunity and easy oral administration at a sufficiently low cost [2].

There have been many reports describing recombinant polioviruses which could induce specific B-cell immune responses [3], [4], [5], [6]. However, there have been few reports demonstrating that recombinant polioviruses can induce antigen-specific cellular immune responses. Recently, it has been just reported that Polio-Ova recombinant expressing the C-terminal half of chicken ovalbumin elicited antigen-specific CTLs in poliovirus-susceptible transgenic mice (H-2b) and protected the immunized mice against challenge with a lethal dose of malignant melanoma cells expressing ovalbumin [7].

The poliovirus genome consists of a positive single-stranded RNA molecule of approx. 7500 nucleotides that encodes a single large polyprotein precursor [8]. The polyprotein is proteolytically processed by virus-encoded proteases 2A, 3C and 3CD into the mature structural and nonstructural proteins [9], [10]. The 2A protease cleaves cotranslationally at the junction between the P1 and P2 proteins to release the P1 precursor [9]. The major viral protease 3C recognizes and cleaves at specific amino acid sequences (AXXQ▾G) to generate viral capsid proteins VP0, VP3 and VP1 from the P1 precursor [10], and nonstructural proteins involved in the replication process from the P2 and P3 precursors [11]. The junction between 5′UTR and P1 or between the P1 and P2 regions was reported successful as an insertion site of foreign antigens by many investigators. In this study, we manipulated the junction between the P2 and P3 nonstructural protein-coding regions to construct recombinant polioviruses. Function of 2C and 3A proteins and their precursors flanking the insertion site has not been thoroughly understood yet, although there are some reports suggesting their putative roles in RNA replication [12], [13], [14], [15] and encapsidation [16].

Hepatitis B virus (HBV) infection is known to be controlled by the strength and multispecificity of HBV class I- and class II-restricted T-cell responses [17]. HBV can be successfully cleared in a large number of adult patients with acute viral hepatitis who mount a multi-specific polyclonal CTL response to several gene products of HBV. But a small number of acute patients fail to eliminate HBV and develop chronic hepatitis who primed just a weak CTL response. Since progress of HBV-caused hepatitis is closely associated with CTL induction, the HBV-specific CTL epitopes could be utilized for development of therapeutic HBV vaccines.

In order to assess the poliovirus as a T-cell vaccine vector, we introduced HBV-specific CTL epitopes of different haplotypes, HBsAg28–39 (H-2d) and HBcAg93–100 (H-2b) into the poliovirus genome to construct recombinant polioviruses V3CDs and V3CDc, respectively. The recombinant viruses replicated efficiently in HeLa cells and were genetically stable with some difference between each recombinant. Immunization of BALB/C with V3CDs induced significant HBV-specific CTLs.

Section snippets

Plasmids construction

Recombinant polioviruses were constructed by subcloning the HBV genome fragments encompassing CTL epitopes into pW3CD, a derivative of pT7PVM (kindly provided by E. Wimmer, State University of New York). The pW3CD was constructed by inserting an in-frame polylinker at the junction between the P2 and P3 regions of the infectious poliovirus cDNA. The synthetic polylinker included 3C protease (3Cpro) recognition sequence (ALAQ▾G) and a multiple cloning site (MCS) to facilitate the processing and

Construction of recombinant polioviruses delivering murine CTL epitope from HBV

We constructed two recombinant polioviruses carrying HBV-specific CTL epitope at the junction between the P2 and P3 regions, both nonstructural protein-coding regions. A schematic diagram of the genomic structure of these viruses is depicted in Fig. 1. pW3CDs and pW3CDc were constructed by inserting the coding sequence for murine CTL epitopes derived from HBsAg and HBcAg, respectively, into SalI/SacI sites of the MCS of pW3CD. The coding sequences for the CTL epitopes included 22 amino acids

Discussion

To date, various recombinant polioviruses have been constructed to express foreign proteins by introducing foreign inserts in front of the P1 region or at the junction between the P1 and P2 regions.

In this article, we described several recombinant polioviruses expressing foreign sequences at the novel site of P2/P3 junction. The 2C and 3A proteins, flanking foreign peptides, as well as their precursors 2BC and 3AB, have been reported to play roles in RNA replication, encapsidation, etc.

Acknowledgements

We thank Dr J.M. Park for critically reviewing this manuscript. We also thank Dr E. Wimmer for the gift of plasmid pT7PVM. This work was supported in part by the Korea Green Cross Corporation and the Korean Ministry of Science and Technology.

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