Elsevier

Vaccine

Volume 36, Issue 16, 12 April 2018, Pages 2086-2092
Vaccine

Expression and characterization of a novel truncated rotavirus VP4 for the development of a recombinant rotavirus vaccine

https://doi.org/10.1016/j.vaccine.2018.03.011Get rights and content

Abstract

The outer capsid protein VP4 is an important target for the development of a recombinant rotavirus vaccine because it mediates the attachment and penetration of rotavirus. Due to the poor solubility of full-length VP4, VP8 was explored as candidate rotavirus vaccines in the past years. In previous studies, it has been found that the N-terminal truncated VP8 protein, VP8-1 (aa26-231), could be expressed in soluble form with improved immunogenicity compared to the core of VP8 (aa65-223). However, this protein stimulated only a weak immune response when aluminum hydroxide was used as an adjuvant. In addition, it should be noted that the protective efficacy of VP4 was higher than that of VP8 and VP5. In this study, it was found that when the N-terminal 25 amino acids were deleted, the truncated VP4 (aa26-476) containing VP8 and the stalk domain of VP5 could be expressed in soluble form in E. coli and purified to homogeneous trimers. Furthermore, the truncated VP4 could induce high titers of neutralizing antibodies when aluminum adjuvant was used and conferred high protective efficacy in reducing the severity of diarrhea and rotavirus shedding in stools in animal models. The immunogenicity of the truncated VP4 was significantly higher than that of VP8 and VP5 alone. Taken together, the truncated VP4 (aa26-476), with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development and has the potential to become a parenterally administered rotavirus vaccine.

Introduction

Globally, rotavirus is one of the leading causes of acute diarrhea in infants and children under 5 years old, with an estimated annual mortality of 146,480 and most of the mortality occurs in low income countries in Africa and Southeast Asia [1]. Currently, two live attenuated rotavirus vaccines Rotarix® (GlaxoSmithKline) and RotaTeq® (Merck) have been approved by the FDA and are widely used throughout the world [2]. After the introduction of rotavirus vaccines, the annual morbidity and mortality were significantly decreased [3]. Nevertheless, the protective efficacy of these rotavirus vaccines is lower in low-income countries where the rotavirus vaccines are most needed [4], [5]. In addition, there is a higher risk of intussusception under vaccination with Rotarix® and RotaTeq® [6]. Furthermore, the attenuated vaccine strains could reassort with other rotaviruses, restore virus virulence and then transmit to unvaccinated contacts [7], [8]. Therefore, it is necessary to develop a more effective and safer alternative rotavirus vaccine.

Recombinant subunit vaccines have been proven to be effective and safe in the case of hepatitis B virus and human papillomavirus [9], [10], and a series of recombinant antigens derived from rotavirus proteins have been shown to be effective in animal models [11], [12], [13], [14], [15]. Among these proteins, the neutralizing antigen VP4 was proven to be pivotal in rotavirus attachment and internalization [16]. VP4 can be cleaved into VP8 and VP5 in the presence of trypsin [17], [18]; VP8 can bind to cell surface receptors and mediates the attachment of rotavirus, while VP5 mediates the cellular penetration of rotavirus [19]. Both VP8 and VP5 can stimulate neutralizing antibodies, and most of the neutralizing antibodies against VP8 block virus attachment, while the antibodies specific for VP5 blocks virus penetration [20].

In the early years, the full-length VP4 was expressed in the baculovirus system and it was found that the protective efficacy of the full-length VP4 was higher than that of VP5 and VP8 [21], [22]. The low yield of VP4 in the baculovirus system limits further development of VP4 as a vaccine. High-level expression and low cost were the characteristics of the bacterial expression system, however, due to the poor solubility of full-length VP4 when expressed in bacteria and the poor immunogenicity of VP5, VP8 was explored as s candidate rotavirus vaccine in the past years [23]. Wen et al. found that the core region of VP8 (ΔVP8, aa65-223) could be expressed in soluble form in E. coli with a high yield and elicited high titers of neutralizing antibodies in guinea pigs; however, it only induced a weak immune response in mice [11]. The CD4+ T cell epitope P2 improves the immunogenicity of ΔVP8 [24], and in a clinical trial, the fusion protein P2-VP8 was proven to be highly immunogenic in adults; however, the seroconversion of neutralizing antibodies was only 50–66.7% [24]. Considering the existents of neutralizing epitopes located in the N-terminus of VP8 [25], the N-terminus of ΔVP8 was extended, and we found that VP8-1 (aa26-231) could stimulate higher levels of neutralizing antibodies than ΔVP8 when Freund’s adjuvant was used [26].

As shown in the atomic structure of rotavirus, VP4 could be divided into four major domains, the lectin domain (head), the β-barrel domain (body and stalk, VP5Ag), and the C-terminal domain (VP5 foot) [17]. Till now, most of the identified neutralizing epitopes were mapped to be located within VP8 and the antigen domain of VP5 [27]. Simultaneously, it was found that deletion of the N-terminal 25 amino acids resulted in the expression of soluble VP8 [26]. Thus, it was speculated that deletion of the N-terminal 25 amino acids and the C-terminal foot domain of VP5 would result in the expression of soluble VP4 without significantly decreasing its protective efficacy. In this study, the truncated VP4 (aa26-476, containing the VP8 domain and the VP5 antigen domain) was expressed in E. coli in soluble form and was purified into homogeneous trimers. The truncated VP4 stimulated higher levels of neutralizing antibodies than VP8 and VP5 in aluminum adjuvant and conferred protection against rotavirus induced diarrhea and fecal shedding of rotavirus after challenges in a mouse model. These results suggested that the truncated VP4 protein could be a better candidate than VP8 to further develop subunit rotavirus vaccines.

Section snippets

Viruses and cell culture

The lamb rotavirus strain LLR (G10P[15]), kindly provided by Beijing Wantai Biological Pharmacy Enterprise Co. Ltd. (Beijing, China), was cultured in MA-104 cells (ATCC® CRL2378.1) [28]. Murine rotavirus EDIM (G16P[16]) was kindly provided by the Institute of Pathogen Biology at Chinese Academy of Medical Sciences (Beijing, China) and was propagated in suckling mice. The infectious titers of the viruses were determined by an enzyme-linked immune-spot assay as described previously [29].

Construction of the expression plasmids

The

Expression, purification and digestion of the truncated rotavirus VP4 proteins

VP8-1 was expressed in soluble form in E. coli and was purified to homogeneity as described previously [26]. The LLR-VP4 and EDIM-VP4 were expressed in both soluble and insoluble form, and the target proteins were purified from the supernatant (approximately 20% of total expression for both strains) using two-step chromatography. After purification, the purity of the truncated VP4 proteins was greater than 90%, as analyzed by SDS-PAGE (∼50 kDa) and HPSEC (Fig. 2). The retention time of VP4

Discussion

In this study, it was found that the truncated VP4 protein containing VP8 and the antigenic domain of VP5 (aa248-476) could be solubly expressed in E. coli BL21 (DE3) and purified to homogeneous trimers (Fig. 2). In a mouse model, VP4 could stimulate high titers of neutralizing antibodies and confer protection against rotavirus infection induced diarrhea and virus shedding when aluminum adjuvant was used. In accordance with previous studies, the protective efficacy of the truncated VP4 was

Acknowledgments

This work was supported by the Scientific Research Foundation of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (Grant No. 2016ZY005). Financial support was provided by the National Natural Science Foundation of China (81501741) and Fujian Province Science and Technology Innovation Platform (2014Y2004).

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  • Cited by (0)

    1

    Current address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43202, USA.

    2

    Current address: SINOCHEM OIL ZHEJIANG CO., LTD., Hangzhou 310000, China.

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