The Molecular Virology and Vaccinology 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.

1. Key findings in fundamental virology

a. Formation of the central DNA Flap facilitates HIV-1 uncoating

The laboratory has previously shown that a (+) strand overlap of the HIV-1 genome, called the central DNA Flap and generated during reverse transcription between the central polypurine tract (cPPT) and the central termination sequence (CTS), acts as a cis-determinant of HIV-1 genome nuclear import in both dividing and non-dividing cells (Zennou et al., Cell 2000). We sought to understand the mechanisms by which the central DNA Flap mediates HIV-1 nuclear import. We found that HIV-1 reverse transcription occurs within an intact capsid (CA) core, independently of the routing process towards the nuclear membrane (Arhel et al., EMBO J 2007). Although early work suggested that uncoating (loss of viral capsid) occurs immediately following viral entry in the cell, thus attributing a trivial role for the capsid in infected cells, our data suggest that uncoating occurs several hours later and that capsid has an all-important role in the cell that it infects: for transport towards the nucleus, reverse transcription and nuclear import. We show thatuncoating occurs after completion of reverse transcription and formation of the central DNA Flap (Arhel et al., EMBO J 2007).In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore.This is concordant with other works that show that premature uncoating leads to abortive infection, that unsuccessful reverse transcription precludes uncoating, and that timely uncoating underlies the ability of HIV-1 to infect non-dividing cells (reviewed in Arhel, Retrovirology 2010).

b. Lack of endogenous TRIM5alpha-mediated restriction in rhesus macaque dendritic cells

Primate cells can resist certain retroviral infections by expressing a dominant restriction factor named TRIM5alpha. This member of the tripartite motif (TRIM) protein family interferes specifically with the replication of various retroviruses in a species-specific manner. In Old World monkey (such as rhesus macaque) cells, TRIM5alpha is responsible for the post-entry block of HIV-1. This endogenous restriction, originally termed Lv1, was observed in several rhesus macaque cell lines, including LL-Cmk2, FRhK4, and FrHL-2 cells, or in primary cells, such as primary lung fibroblasts. Thus, although no systematic study has been conducted so far, endogenous TRIM5alpha-mediated HIV-1 restriction is thought to occur identically in all rhesus macaque cell. We compared TRIM5alpha-mediated restriction of HIV-1 infection in primary cell types that are important in vivoreplication sites for HIV-1, namely, T lymphocytes, dendritic cells (DCs), and macrophages. Results revealed that, unlike primary lymphocytes, macaque DCs do not mediate TRIM5alpha restriction against HIV-1 (Arhel et al., Blood 2008). Perhaps the role of DCs as professional antigen-presenting cells (APCs) and their importance in stimulating host adaptive immunity preclude them from maintaining cellular restriction.

2. Lentiviral vectors-based vaccines

The goal of the lab is to develop lentiviral vectors-based vaccines stimulating the T cell arm and the B cell arms of the immune system. 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 more recently a vaccine against malaria in the Plasmodium yoelii/BALB/c mice model. We aim at optimizing lentiviral vector-based vaccines both at the efficacy and safety levels. In particular, we have designed non-integrative lentiviral vectors.

Lentiviral vectors derived from HIVwere initially used for gene therapy purposes. Their use as vaccine vector is more recent : it has boomed after the demonstration of their capacity to efficiently transduce dendritic cells.

a. AIDS vaccine

HIV derived LV-based vaccine can be used against HIV infection itself. We have shown that a lentiviral vector encoding a HIV-1 polyepitope induced strong, diverse, and long-lastingcytotoxic T lymphocytes responses in HLA-A2 and -B7 transgenic mice (Iglesias et al., Mol Ther 2007). We have also performed the first pilot study in non human primates. We have demonstrated that lentiviral vector can confer protection against viral replication and CD4+ T cells depletion in Mauritius cynomolgus macaques infected with SIV. After 2 injections of vector encoding a single viral antigen, animals have mounted strong cellular immunity. After intrarectal viral challenge with a high dose of SIVmac251 their viremia were reduced at the peak of acute infection (a mean of over 2 log10 fold reduction) and their blood memory CD4+T cells were fully preserved during the acute phase, in contrast to non-vaccinated monkeys (Beignon et al., J Virol 2009).

b. Non integrative lentiviral vectors

Non-integrative lentiviral vectors carry a defective integrase, hence they obviate the risk of insertional oncogenesis. They mediate stable transgene expression in nondividing cells and transient gene expression in proliferating cells because of the loss of the episomes as the cells divide. Since the main targeted cells in vaccination procedure are the non-dividing dendritic cells, we reasoned that it may be possible to develop safe vaccine strategies with non-integrative lentiviral vectors. We have shown that non-integrative lentiviral vectors efficiently transduce conventional and plasmacytoid dendritic cells and that a single injection of non-integrative vectors encoding a secreted form of the envelope of a virulent strain of West Nile Virus (WNV) induces a robust B cell response in mice. Animals were fully protected from a challenge with a lethal dose of WNV (Coutant et al., PLoS One 2008).

c. Malaria vaccine

More recently, we have developed a vaccine against malaria. Our preliminary results are very encouraging. They indicate that immunization of mice with vectors encoding a single antigenic protein specific for the pre-erythrocytic stage confers high protection against rodent malaria.

Keywords: HIV-1 nuclear import – reverse transcription – HIV-1 capsid lentiviral vectors, vaccine, AIDS, 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.

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.

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.

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.

Iglesias MC, Mollier K, Beignon AS, Souque P, Adotevi O, Lemonnier F, Charneau P. Lentiviral vectors encoding HIV-1 polyepitopes induce broad CTL responses in vivo. Mol Ther. 2007 Jun;15(6):1203-10.