Attenuation of tick-borne encephalitis virus using large-scale random codon re-encoding.

Lauriane de Fabritus,Xavier de Lamballerie,Fabien Aubry,Antoine Nougairède,Ernest A Gould,Jens H. Kuhn

doi:10.1371/journal.ppat.1004738

Abstract

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.105 or 2.106 TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.

Highlights

The genus Flavivirus includes important human pathogens such as yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus and tick-borne encephalitis virus (TBEV)
The TBEVs are subdivided into three sub-types, namely Siberian, Western European and Far Eastern viruses [8,9], the latter being responsible for the most severe forms of central nervous system (CNS) disorders associated with high fatality rates (5–20%) [10]
Our studies demonstrate that this re-encoded TBEV Oshima 5–10 strain exhibited an attenuated phenotype in vivo and induced robust protective immunity in mice subsequently infected with the WT virus

Summary

Introduction

The genus Flavivirus (family Flaviviridae) includes important human pathogens such as yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus and tick-borne encephalitis virus (TBEV). Some mosquito-borne flaviviruses harbour sequences that induce a proportion of translating ribosomes to shift-1 nt and continue translating in the new reading frame to produce a ‘transframe’ fusion protein [3,4]. TBFVs include a heterogeneous group called seabird tick-borne flavivirus group (S-TBFV) [6] and the mammalian tick-borne flavivirus group (M-TBFV), with all known pathogenic TBFVs causing febrile illness, encephalitis and/or haemorrhagic fever in humans. In the latter group, TBEVs are recognised in 25 European and 7 Asian countries and transmitted by Ixodes species ticks [7]. Despite the availability of several licensed inactivated vaccines and vaccination programmes [1,11], the incidence of TBEV infections is increasing across much of Central and Eastern European countries, currently with an estimated 9,000 cases per year [12,13,14]

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