Shchelkunov Sergei N, Yakubitskiy Stanislav N, Titova Kseniya A, Pyankov Stepan A, Sergeev Alexander A
State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, Koltsovo, Novosibirsk 630559, Russia.
Pathogens. 2021 Mar 21;10(3):377. doi: 10.3390/pathogens10030377.
Following the WHO announcement of smallpox eradication, discontinuation of smallpox vaccination with vaccinia virus (VACV) was recommended. However, interest in VACV was soon renewed due to the opportunity of genetic engineering of the viral genome by directed insertion of foreign genes or introduction of mutations or deletions into selected viral genes. This genomic technology enabled production of stable attenuated VACV strains producing antigens of various infectious agents. Due to an increasing threat of human orthopoxvirus re-emergence, the development of safe highly immunogenic live orthopoxvirus vaccines using genetic engineering methods has been the challenge in recent years. In this study, we investigated an attenuated VACV LIVP-GFP (TK) strain having an insertion of the green fluorescent protein gene into the viral thymidine kinase gene, which was generated on the basis of the LIVP (Lister-Institute for Viral Preparations) strain used in Russia as the first generation smallpox vaccine. We studied the effect of gene modification and gene deletion on the immunogenic and protective properties of the LIVP-GFP strain. The obtained data demonstrate that intradermal inoculation of the studied viruses induces higher production of VACV-specific antibodies compared to their levels after intranasal administration. Introduction of two point mutations into the gene, which increase the yield of extracellular enveloped virions, and deletion of the gene, the protein product of which inhibits presentation of antigens by MHC II, enhances protective potency of the created LIVP-TK-A34R*-dA35R virus against secondary lethal orthopoxvirus infection of BALB/c mice even at an intradermal dose as low as 10 plaque forming units (PFU)/mouse. This virus may be considered not only as a candidate attenuated live vaccine against smallpox and other human orthopoxvirus infections but also as a vector platform for development of safe multivalent live vaccines against other infectious diseases using genetic engineering methods.
在世界卫生组织宣布根除天花后，建议停止使用痘苗病毒（VACV）进行天花疫苗接种。然而，由于通过定向插入外源基因或在选定的病毒基因中引入突变或缺失来对病毒基因组进行基因工程的机会，人们很快又对痘苗病毒产生了兴趣。这种基因组技术能够生产出稳定的减毒痘苗病毒株，这些毒株可产生各种传染原的抗原。由于人类正痘病毒重新出现的威胁日益增加，近年来利用基因工程方法开发安全且免疫原性高的活正痘病毒疫苗一直是一项挑战。在本研究中，我们研究了一种减毒痘苗病毒LIVP - GFP（TK）株，该毒株在病毒胸苷激酶基因中插入了绿色荧光蛋白基因，它是在俄罗斯用作第一代天花疫苗的LIVP（李斯特病毒制备研究所）株的基础上构建的。我们研究了基因修饰和基因缺失对LIVP - GFP株免疫原性和保护特性的影响。获得的数据表明，与鼻内给药后的水平相比，皮内接种所研究的病毒可诱导产生更高水平的VACV特异性抗体。在A34R基因中引入两个点突变可增加细胞外包膜病毒粒子的产量，而删除A35R基因（其蛋白产物可抑制MHC II对抗原的呈递），即使在皮内剂量低至10个蚀斑形成单位（PFU）/小鼠的情况下，所构建的LIVP - TK - A34R* - dA35R病毒对BALB/c小鼠继发性致死性正痘病毒感染的保护效力也会增强。这种病毒不仅可被视为一种针对天花和其他人类正痘病毒感染的减毒活疫苗候选物，还可作为一个载体平台，用于利用基因工程方法开发针对其他传染病的安全多价活疫苗。
