Characterization of an attenuated TE3L-deficient vaccinia virus Tian Tan strain
Highlights
► We construct an attenuated TE3L deficient vaccinia virus Tian Tan strain. ► Cell toxicity of the attenuated virus is significantly decreased. ► The in vivo virulence of the attenuated virus is reduced. ► The attenuated virus is a candidate vector with increased safety.
Introduction
Vaccinia virus is a double-stranded DNA virus that replicates and multiplies in the cytoplasm of infected cells using both virally encoded proteins and resources of the host cells. The attenuated form of vaccinia virus (VACV) has been used as a vaccine against smallpox to successfully eradicate this human disease. However, it has been reported that the use of VACV has resulted in several adverse events, which include encephalitis, eczema after vaccination, generalized and progressive poxvirus infections, as well as some less severe reactions (Wollenberg and Engler, 2004). Furthermore, cardiac complications have been reported to occur after vaccination (Cassimatis et al., 2004). Increased numbers of individuals have impaired immune functions due to organ transplantation, human immunodeficiency virus (HIV) infection and cancer treatments, which may affect the development and clinical application of VACV or VACV-based vaccines in the future. All these consequences mean that there is a need to develop a safer VACV vector.
VACV has widespread application in biological and medical investigations, therefore scientific researchers have committed considerable efforts to study attenuated VACV vectors in order to overcome potential adverse reactions following vaccination. Some attenuated strains have been obtained so far, such as the modified Ankara strain (MVA) (Coulibaly et al., 2005, Meyer et al., 1991, Nigam et al., 2007), NYVAC (Tartaglia et al., 1992), the Lister mutant strain of VACV (Hiley et al., 2010, Tysome et al., 2009), Lister clone 16m8 (LC16m8) (Kenner et al., 2006) and Dairen I strain (Someya et al., 2004). The MVA, LC16m8 and Dairen I strain were obtained by passage in alternative hosts, while the third-generation attenuated strains, NYVAC and the Lister mutant, were generated by deletion of certain genes (Jacobs et al., 2009, Rosenwirth et al., 2001). Most of these vectors are replication-defective viruses in most mammalian cells, except for LC16m8 which has a limited capacity to replicate in human cells. Although non-replicating virus vectors have the advantage of increased safety, the use of high doses and multiple immunizations is generally required to induce a strong immune response (Meseda et al., 2005).
The VACV E3L gene, a host range and virulence gene, encodes a protein that inhibits the innate immune response (Beattie et al., 1996, Langland and Jacobs, 2002, Vijaysri et al., 2008). Recently, a study also shows that the amino terminus of the vaccinia virus E3L protein is necessary to inhibit the interferon response (White and Jacobs, 2012). We have noted previously that the full E3L deletion mutation in the Copenhagen, Western Reserve (WR) and NYCBH strains of VACV are highly attenuated and more appropriate for use in humans (Fenner et al., 1988, Jentarra et al., 2008, Vijaysri et al., 2008). However, currently there are no reports of vaccinia virus Tian Tan strain deleted TE3L gene, and the pathogenesis and immunogenicity of TianTan strain vaccinia virus with deletion of TE3L (TE3L−VTT) are not known. To test the efficacy of the TE3L mutation in the TianTan strain, we constructed and recovered the mutant strain of VTT by deletion of the TE3L region, which has been found to contribute to pathogenesis and is necessary for full virulence in mouse models. The genetic stability of the mutant strain was analyzed by passage in baby hamster kidney (BHK)-21 cells. Importantly, we evaluated its safety and immunogenicity in both mouse and rabbit models. These systematic studies provided scientific support for the development of the mutant strain as a live virus vaccine vector with increased safety.
Section snippets
Viruses, cells and animals
The vaccinia virus Tian Tan strain, kindly provided by Dr. Kuoshi Jin (Academy of Military Medical Sciences, Changchun, China), was propagated and titered in BHK-21 (baby hamster kidney) cells. BHK-21 and PK15 (porcine kidney) cells were maintained in minimal essential medium (MEM) with 5% fetal bovine serum (FBS) and 1% penicillin (10,000 U/mL)/streptomycin (10 mg/mL) solution (P/S). HeLa (human cervical carcinoma) and Vero (African green monkey kidney) cells were cultured in improved RPMI-1640
Construction of recombinant virus and virus morphogenesis in BHK-21 cells
The plasmid vector was constructed and inserted to the genome of recombinant vaccinia viruses as shown in Fig. 1. Virus was collected after cytopathic effect (CPE) appearing, plaques were isolated for expression of green fluorescent protein under a fluorescence microscope. Pure recombinant viruses were obtained after 10 rounds of plaque purification (Fig. 2). The genome of the recombinant vaccinia virus was extracted as the template to determine whether the TE3L gene had been removed from the
Discussion
The E3L protein is synthesized early during the virus infection cycle and contains a C-terminal consensus dsRNA-binding domain and an N-terminal consensus Z-nucleic acid-binding domain (Chang et al., 1992). The E3L-encoded protein is a potent inhibitor of the antivirus activity of IFN-γ and is required for virulence in the mouse model. The deletion of E3L in VACV (Beattie et al., 1996, Langland and Jacobs, 2002) has been shown to cause replication deficiency in HeLa cells, while retaining full
Acknowledgments
This work was supported by grants from the Genetically Modified Organisms Breeding Major Projects (2009ZX08006-002B), the Key Technologies R&D Programme of Jilin Province (10ZDGG007) and the National Natural Science Foundation of China (81101140).
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