Viral attenuation by codon re-encoding : application to chikungunya and tick-borne encephalitis viruses

Le ré-encodage aléatoire des codons à grande échelle est une nouvelle méthode d'atténuation virale qui consiste en l'insertion d'un grand nombre de mutations synonymes, individuellement peu délétères, de façon aléatoire dans une ou plusieurs régions codantes d'un virus. Cette approche permet de diminuer de façon significative et modulable le fitness réplicatif des virus in cellulo et in vivo, ainsi que la pathogénicité du virus chez la souris, tout en induisant une protection immunitaire spécifique et efficace lors d'une nouvelle infection par le virus sauvage. Les virus ré-encodés présentent également une grande stabilité et une absence de réversion ce qui en font des candidats vaccins très prometteurs en termes d'efficacité et de fiabilité pour la conception de candidats vaccins vivants atténués contre une grande variété de virus à ARN. La combinaison du ré-encodage aléatoire et d'une nouvelle méthode de génétique inverse permettant de générer de nouveaux virus en quelques jours: ISA (Amplicon Subgenomique Infectieux), est une approche prometteuse qui pourrait aider au développement de vaccins vivants atténués de nouvelle génération en un temps record.Large-scale random codon re-encoding is a new method of viral attenuation consisting in the insertion of a high number of slightly deleterious synonymous mutations, randomly, in one or several coding regions of a virus. This approach significantly reduces the replicative fitness of re-encoded viruses in cellulo and in vivo, as viral pathogenicity, while inducing a specific and effective immune response in mice against a new infection with wild-type viruses. Re-encoded viruses also present a high stability and an absence of reversion, making them promising vaccine candidates in term of reliability and efficiency for the conception of new vaccine candidates against a wide variety of RNA viruses. Combination of random re-encoding with a new method of revers genetics allowing to generate new viruses in days : ISA (Infectious Subgenomic Amplicons) would be very helpful to develop new-generation vaccine candidates

Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding

International audienceLarge-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 2.10(5) or 2.10(6) 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 wildtype 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 reencoded 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

Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days

International audienceLarge-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines

"ISA-Lation" of Single-Stranded Positive-Sense RNA Viruses from Non-Infectious Clinical/Animal Samples.

Isolation of viral pathogens from clinical and/or animal samples has traditionally relied on either cell cultures or laboratory animal model systems. However, virus viability is notoriously susceptible to adverse conditions that may include inappropriate procedures for sample collection, storage temperature, support media and transportation. Using our recently described ISA method, we have developed a novel procedure to isolate infectious single-stranded positive-sense RNA viruses from clinical or animal samples. This approach, that we have now called "ISA-lation", exploits the capacity of viral cDNA subgenomic fragments to re-assemble and produce infectious viral RNA in susceptible cells. Here, it was successfully used to rescue enterovirus, Chikungunya and Tick-borne encephalitis viruses from a variety of inactivated animal and human samples. ISA-lation represents an effective option to rescue infectious virus from clinical and/or animal samples that may have deteriorated during the collection and storage period, but also potentially overcomes logistic and administrative difficulties generated when complying with current health and safety and biosecurity guidelines associated with shipment of infectious viral material

Random Codon Re-encoding Induces Stable Reduction of Replicative Fitness of Chikungunya Virus in Primate and Mosquito Cells

International audienceLarge-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (,0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine

Results of TBEV serology at day 43 post-inoculation.

<p>ELISA <b>(A)</b> and sero-neutralisation assays <b>(B)</b>. Black crosses represent each individual value. Grey bars represent mean values. For ELISA tests, ratios were calculated as follows: OD/background cut off (mean of OD for control group + (3 x SD of OD for control group)). Samples were considered as positive if the ratio was higher than 1.1, negative if under 0.9 and uncertain if between 0.9 and 1.1 (grey zone). * All the sera of the control group tested were negative (<i>e</i>.<i>g</i>. under the detection threshold of the method (2.25 PRNT/mL)).</p

Time distribution of the proportion of mice with at least one symptom, with a weight loss of more than 6% and with virus detected in the serum or in the brain by qRT-PCR.

<p>Mice were inoculated with 2.10⁶ TCID50 of WT_IC virus (<b>A</b>), 2.10⁵ TCID50 of WT_IC virus (<b>B</b>), 2.10⁶ TCID50 of NS5_Reenc_IC virus (<b>C</b>) or 2.10⁵ TCID50 of NS5_Reenc_IC virus (<b>D</b>).</p

Results of <i>in vivo</i> competition experiments.

<p>Competitions were performed using two initial PFU ratios (WT_IC/NS5_Reenc_IC virus: 50/50 or 10/90). Two qRT-PCR assays, specific for WT_IC or NS5_Reenc_IC virus enabled to quantify the proportion of each virus in the viral population in sera (<b>A</b>) and in brains (<b>B</b>). Dark and light grey rectangles represent the percentage of WT_IC and NS5_Reenc_IC virus in each sample (percentages are represented using a log₁₀ scale). Black diamonds represent samples for which qRT-PCR were negative for WT_IC and NS5_Reenc_IC detection.</p