|Molecular Virology and Vectorology - CNRS URA 3015|
|HEAD||Dr Pierre CHARNEAU / firstname.lastname@example.org|
|MEMBERS||Dr Nathalie ARHEL (CNRS CR1) Dr Anne-Sophie BEIGNON (CNRS CR2) Andreas BECKER (étudiant en master 2) Laxmee CALEECHURN (technicienne) Pierre CHARNEAU (chercheur IP) Dr Frédéric COUTANT (post-doc) Dr Francesca DINUNZIO (post-doc) Mathieu RINGEARD (étudiant en master 2) Philippe SOUQUE (ingénieur IP) Isma ZIANI (secrétaire, 25%) Chantal BELHASSEN (aide labo, 25%) Jocelyne CREFF (aide labo, 25%)
The Molecular Virology and Vectorology Laboratory (VMV) conducts both basic and applied research projects. We study the early steps of HIV-1 replication with a particular emphasis on the mechanisms of intracellular routing and nuclear import of HIV-1 replication complexes. The understanding of some fundamental aspects of HIV-1 DNA nuclear import have brought about some important contributions in the lentiviral vector field in terms of vector design and gene transfer protocols in several cell types and tissues of major therapeutic interest. We also apply the lentiviral gene transfer technology to therapeutic gene transfer (through collaborative projects) and to vaccination.
We showed that a three-stranded DNA structure, called the central DNA Flap, created during HIV-1 reverse transcription, acts as a cis-determinant of HIV-1 DNA nuclear import. This original mechanism contributed to the understanding of HIV-1 infection in non-dividing cells, a unique feature of lentiviruses among the retrovirus family. This finding also contributed to the optimization of lentiviral gene transfer vectors. Re-insertion of the DNA Flap sequence in lentiviral vectors complemented a strong nuclear import defect to wild-type levels and consequently stimulated gene transfer efficiency in all target cells and tissues examined so far, such as hematopoietic stem cells, hepatocytes, brain and dendritic cells.... DNA Flap lentiviral vectors are now universally used in the numerous applications of lentiviral vector technology.
The VMV lab is studying the early steps of HIV-1 replication using innovative real time imaging or ultrastructural visualization methods. Among other findings, we show that the DNA Flap acts as viral promoting element for the uncoating of integral capsids, docked at the nuclear pore, allowing translocation of the viral DNA through the nuclear pore.
An important part of the VMV lab is dedicated to the application of the lentiviral vector technology for vaccination purposes. Lentiviral vectors represent very efficient vaccination tools. They induce potent humoral and cellular immunity. It is related to the quality of antigen presentation (duration, stability, intensity) after in vivo transduction by dendritic cells (among other cells). We focus our efforts on the pre-clinical development of vaccines against two major diseases of public health importance: a vaccine against HIV infection and AIDS in the SIV-macaque model and a vaccine against malaria in the Plasmodium yoleii/mice model. We have shown in a first pilot study in monkeys that a prime/boost strategy using lentiviral vectors pseudotyped with a VSV-G from two non-cross reactive serotypes was highly immunogenic, inducing strong and broad cellular response to SIV GAG. It resulted in the significant protection of the vaccinated animals against SIVmac251 challenge, with 2 log10 reduction of viral replication and the preservation of the central memory CD4+ T cells in the periphery during the acute SIVmac251 infection, in comparison with control animals. We also aim at optimizing lentiviral vector-based vaccines both at the efficacy and safety levels. In particular, we develop non-integrative lentiviral vectors.
Keywords: VIH, étapes précoces de la réplication, import nucléaire, restriction virale, imagerie, vecteurs lentiviraux, vaccination, SIDA, malaria
Beignon AS, Mollier K, Liard C, Coutant F, Munier S, Rivière J, Souque P, Charneau P. Lentiviral vector-based prime/boost vaccination against AIDS: pilot study shows protection against Simian immunodeficiency virus SIVmac251 challenge in macaques. J Virol. 2009 Nov;83(21):10963-74. Epub 2009 Aug 12. PubMed PMID: 19706700; PubMed Central PMCID: PMC2772810.
Coutant F, Frenkiel MP, Despres P, Charneau P. Protective antiviral immunity conferred by a nonintegrative lentiviral vector-based vaccine. PLoS One. 2008;3(12):e3973. Epub 2008 Dec 19. PubMed PMID: 19096527; PubMed Central PMCID: PMC2600612.
Arhel NJ, Nisole S, Carthagena L, Coutant F, Souque P, Brussel A, Estaquier J, Charneau P. Lack of endogenous TRIM5alpha-mediated restriction in rhesus macaque dendritic cells. Blood. 2008 Nov 1;112(9):3772-6. Epub 2008 Aug 14. PubMed PMID: 18703703.
Arhel NJ, Souquere-Besse S, Munier S, Souque P, Guadagnini S, Rutherford S,Prévost MC, Allen TD, Charneau P. HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore. EMBO J. 2007 Jun 20;26(12):3025-37. Epub 2007 Jun 7. PubMed PMID: 17557080; PubMed Central PMCID: PMC1894778.
Arhel N, Genovesio A, Kim KA, Miko S, Perret E, Olivo-Marin JC, Shorte S, Charneau P. Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nat Methods. 2006 Oct;3(10):817-24. PubMed PMID: 16990814.
Activity Reports 2009 - Institut Pasteur
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