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    Antiviral strategies for targeting the HIV-1 Nef and envelope proteins



    Rationale and objectives


    Even if the use of highly active antiretroviral therapies leads to a net decrease of morbidity and mortality of HIV-1 infected patients, these treatments are still unable to eradicate virus infection. Thus, identification of new viral targets and new technological approaches for inhibition of structural and regulatory viral proteins are still required for development of new antiviral strategies. Our team recently used the specific characteristics and advantages of the single-domain antibodies (sdAbs), derived from camelid antibodies naturally devoid of light chains, for selection of specific tools which target the viral surface envelope glycoprotein (gp120) and the regulatory Nef protein from HIV-1 (Bouchet, 2011, 2012; Lulf, 2014; Matz, 2013). While the viral gp120 glycoprotein plays a key role in the virus entry process through sequential binding to host CD4 and chemokine receptors expressed at the surface of the HIV-1 target cells, the Nef regulatory protein is absolutely required for maintenance of high virus load and is essential for AIDS pathogenesis (Laguette, 2010, for review).

    Taking advantage of the characteristics of camelid single-domain antibody fragments, we have selected and characterized a panel of sdAbs targeting gp120 and displaying a broad neutralizing activity against HIV-1 strains carrying envelopes from different HIV-1 subtypes (Matz, 2013). In addition, we have also isolated and characterized an sdAb targeting Nef with a high affinity, and this anti-Nef sdAb (sdAb19) is able to counteract, both in vitro and in vivo in transgenic mice expressing Nef, all the functions of this viral protein in HIV-1 infected cells (Bouchet, 2011, 2012; Lulf, 2014). Therefore, these sdAbs constitute original tools for investigation of new antiviral strategies targeting the gp120 glycoprotein and the Nef virulence factor.


    1) Development of Nef inhibitors using anti-Nef sdAb


    While all inhibitory effects of the anti-Nef sdAb19 inhibitor revealed so far have been analyzed by direct expression in HIV-1 infected T cells and macrophages (Bouchet, 2011, 2012), we want now to develop the best formulation strategy for intracellular drug-delivery of these anti-Nef inhibitors. This strategy will consist in designing new constructs of recombinant mutated forms of sdAb19 for intracellular delivery in HIV-1 infected cells. Within the process of developing the best delivery system, it will be necessary to verify that the new formulations of sdAb19 and Neffins does not alter the anti-Nef inhibitory activity of Nef inhibitors. This will provide the basis for the preclinical pharmacokinetic and safety/toxicity studies and for analysis of in vivo activity in a relevant animal mode such as HIV-1 infected humanized mice in collaboration with the Laboratory of Victor Garcia (University of North Carolina, Chapel Hill).





    2) Development of anti-HIV-1 neutralizing approach using anti-envelope sdAbs


    This part of the project is conducted in collaboration with the team of P. Zhou (Institut Pasteur Shanghai-CAS, China) who developed an original and very potent strategy for expression of antibody fragments by expression as glycosyl-phosphatidylinositol(GPI)-anchored polypeptides at the surface of HIV-1 target cells. Two different anti-env sdAbs displaying broad neutralizing anti-HIV-1 activities, and targeting either the CD4- or the co-receptor binding sites on HIV-1 envelope (Matz, 2013), have been selected for further characterization using this strategy. Promising results have been already obtained regarding expression of JM4, an anti-env sdAb that neutralizes diverse HIV-1 strains and targets the co-receptor binding site of gp120, when it is expressed as a GPI-anchored protein at the surface of human T cells. We show that the GPI-sdAb JM4 is targeted to lipid rafts of the plasma membrane, and neutralizes diverse HIV-1 strains from different subtypes more potently that the soluble recombinant JM4 counterpart. Additional investigations are in progress to analyze whether expression of JM4 can also be able to neutralize virus entry in the primary target cells of HIV-1, such as primary CD4+ T lymphocytes purified from healthy blood donors, when it is expressed as a GPI-anchored form at the cell surface of the target T cells using lentiviral expression vectors. Finally, we will explore whether JM4 would be able to block direct cell-to-cell transfer of HIV-1 between primary T lymphocytes, or from infected macrophages towards T cells. Then, the in vivo inhibitory activity of GPI-sdAb JM4 will be evaluated in the HIV-1 infected BLT ("bone marrow liver thymus") “humanized” mouse model developed in the Laboratory of Victor Garcia (University of North Carolina, Chapel Hill, USA). BLT mice will be reconstituted prior to HIV-1 infection with human CD34+ progenitor cells expressing GPI-sdAb JM4, and then infected with HIV-1 in order to assess the impact of its inhibitory activity on the establishment of infection.




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