Étude de deux vecteurs d'expression endogène dans le cadre de la vaccination contre les coronavirus félins

Les Coronavirus félins (FCoV) provoquent chez le chat une péritonite infectieuse (PIF) létale ou une infection entérique bénigne. Les agents étiologiques respectifs de ces pathologies sont nommés FIPV et FECV. Ces virus sont étroitement liés : le FIPV est un variant pathogène du FECV, apparaissant de façon sporadique chez les chats porteurs de FECV. Le développement d'un vaccin FIPV est problématique car la présence d'anticorps induit l'accélération de la maladie. Cependant, un antigène membranaire (la protéine M) exprimé par des virus recombinants confère une protection partielle. Dans nos travaux, cet antigène a été présenté par 2 systèmes d'expression endogène : l'ADN plasmidique et le Poxvirus MVA. Ces vecteurs recombinants n'ont pas reproduit la protection partielle vis à vis d'une épreuve létale sur l'espèce cible. Nos vaccins candidats ont donc été modifiés pour optimiser la présentation de l'antigène : l'insertion du gène E des FCoV permet la sécrétion de pseudoparticules virales par co-expression des protéines M et E. Devant les difficultés à reproduire la protection vis à vis d'une épreuve létale, un nouveau modèle d'infection expérimentale a été développé. Son but est de se rapprocher des conditions naturelles d'infection par les FCoV. Cette nouvelle épreuve avec le FECV met en évidence un effet bénéfique de la vaccination par les vecteurs exprimant les pseudoparticules virales. L'effet protecteur de la protéine M seule n'est pas validé, mais la présentation sous forme de pseudoparticules ouvre de nouvelles perspectives dans la vaccination contre les FCoV du terrain. Mots clefs : Coronavirus félins, FIPV, FECV, vecteur d'expression, Poxvirus recombinant, MVA, vaccination ADN, infection expérimentale.Feline Coronaviruses (FCoVs) are responsible of a lethal infectious peritonitis (FIP) or a mild enteric infection in the cat. The etiologic agents of these pathologies are respectively FIPV and FECV. Both viruses are strongly related : FIPV is a pathogenic mutant of FECV and appears sporadically in FECV carriers. Vaccination against FIPV is problematic due to an antibody-dependent enhancement of the disease. However, a membrane antigen (the M protein) is partially protective when expressed by recombinant viruses. In our studies, this antigen was presented by two endogenous expression systems : plasmid DNA and the Poxvirus MVA. This recombinant vectors were not protective against a lethal challenge in the cat model. Our candidate vaccines were consequently modified in order to ameliorate the antigen presentation : insertion of the FCoV E gene and thus co-expression of M and E proteins induce the release of Coronavirus-like particles. Because the partial protection against a lethal challenge was not reproducible, we developed a novel model of experimental infection? This aims at resembling the natural conditions of an FCoV infection. This new challenge with FECV showed a positive activity of the vaccination with the virus-like particles-expressing vectors. The protective effect of the M protein alone is not validated, but the virus-like particles presentation offers new prospect to vaccinate against the FCoVs.NICE-BU Sciences (060882101) / SudocSudocFranceF

High level protein expression in mammalian cells using a safe viral vector: Modified vaccinia virus Ankara.

Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production

A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products

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Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing

International audienceRecent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles