Down-modulation of the CXCR4 co-receptor by intracellular expression of a single chain variable fragment (SFv) inhibits HIV-1 entry into primary human brain microvascular endothelial cells and post-mitotic neurons.

Our laboratories previously demonstrated that expression of a single chain variable antibody fragment (SFv), anti-CXCR4 SFv, in human lymphoid cells suppresses surface display of the chemokine co-receptor CXCR4 and inhibits infectious entry of human immunodeficiency virus type I (HIV-1). We now sought to extend these results to two types of central nervous system (CNS) cells, primary isolated human brain microvascular endothelial cells (MVECs), and post-mitotic differentiated human neurons, both of which normally express significant levels of CXCR4. The anti-CXCR4 SFv expression construct was delivered using an HIV-1-based vector, and control cells received LacZ-expressing viral particles. Upon intracellular expression of the anti-CXCR4 SFv, immunostaining revealed a marked reduction in surface display of CXCR4 on both cell types. Consequently, post-mitotic neurons expressing the anti-CXCR4 SFv were significantly protected from HIV-1 infection, as measured by HIV-1 p24 antigen production, and partial protection was observed in human brain MVECs. The ability to selectively down-modulate the surface expression of CXCR4 in CNS cells may allow for the development of clinical molecular therapy strategies against HIV-1-related neurodegenerative disorders and neuroinvasion.

Primary isolated human brain microvascular endothelial cells express diverse HIV/SIV-associated chemokine coreceptors and DC-SIGN and L-SIGN.

Chemokines have received increasing attention due to their inhibitory activities on human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV) replication and the potential for chemokine receptors to assist in HIV-1/SIV entry into permissive cells. Besides CD4, which is the major receptor for HIV-1 and SIV, a number of chemokine receptors including but not limited to APJ, CCR3, CXCR4, and CCR5 may be coreceptors for HIV-1/SIV, not only in peripheral blood and lymphoid tissues but also in the central nervous system (CNS). The present studies reveal the lack of CD4, but the significant expression of various chemokine receptors, APJ, CCR3, CXCR4, and CCR5, plus C-type lectins DC-SIGN and L-SIGN on isolated primary human brain microvascular endothelial cells (MVECs). As these MVECs do not express CD4, this suggests a CD4-independent HIV/SIV entry/infection of these cells, which are the major cells constituting the human blood-brain barrier. We also found that chemokines for cognate chemokine receptors individually were unable to block binding of HIV-1 to brain MVECs. These results reveal that in primary isolated brain MVECs viral attachment is mediated by a possible previously unknown receptor(s) or by cooperative activity of various receptors. Moreover, mRNA transcripts for DC-SIGN/L-SIGN, as well as DC-SIGN protein expression, suggest the capability of MVECs to attach viral particles on cell surfaces, even though polyclonal antisera for DC-SIGN did not affect viral binding to these cells. These data will assist in further understanding lentiviral entry into the CNS.