Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or CCR5 Fused to the C34 Peptide from gp41 HR2

Background: HIV-1 entry into CD4+ T cells requires binding to CD4 and either the CCR5(R5)or CXCR4 (X4) co-receptor. Thus, strategies that disable productive co-receptor (CoR) engagement should provide potent protection from HIV infection. Previously we described a 34 amino acid peptide from the C-terminal heptad repeat-2 domain of gp41 (C34) which, when fused to the amino terminus (NT) of either R5 or X4, inhibits HIV-1 infection in transformed cells in vitro. Moreover, our initial studies suggested that C34-R5 or C34-X4 fusions provided trans-dominant resistance to infection irrespective of viral tropism (i.e. either C34-R5 or C34-X4 could inhibit entry of R5, X4 or dual-tropic isolates). Methodology: Using lentiviral vectors, C34-R5 or C34-X4 fusion constructs were transduced into anti-CD3/CD28-stimulated primary human CD4 T-cells. Controls included GFP alone and C34-conjugated to CD4. Susceptibility to infection by R5, X4, or dual tropic HIV-1 from different clades was assessed by flow cytometry and reverse transcriptase activity (RT). Functionality of the transduced T-cells was determined by evaluating their expansion and intracellular cytokine levels after restimulation with PMA/ionomycin or anti-CD3/CD28 beads. Results: In CD4 T-cells from multiple donors, when C34-R5 or C34-X4 were expressed, there was almost complete inhibition (>98%) of HIV-1 infection by intracellular p24 levels and RT. GFP-only and C34-CD4 expressing cells were infected at levels similar to untransduced T-cells. C34/CoR expression was >90% on Day 0 and stable during the 14 days of culture. Trans-dominant inhibition by C34-R5 or C34-X4 occurred for X4, R5 and dual-tropic primary isolates from clades B and A/E. Remarkably, when C34-CoR transduced and untransduced cells were titrated (1:4, respectively) and challenged with diverse HIV isolates, a condition that provides a more sustained exposure to HIV, selective enrichment of C34-CoR expressing cells occurred from the expected starting levels of ~25% up to 60% C34-CoR+ cells during viral replication. Lastly, PMA/ionomycin and anti-CD3/CD28 stimulation of C34-R5 and C34-X4 expressing T-cells grew normally and produced levels of intracellular IFNg, MIP-1b, TNFa, and IL-2 that were indistinguishable from untransduced cells. Conclusions: Engineering primary CD4 T-cells with C34-modified chemokine coreceptors provides potent trans-dominant and heterologous resistance to diverse HIV-1 isolates. This cross-clade protection results in the survival and selective expansion of C34-CoR transduced cells. This novel method of engineering HIV-resistant, functional CD4 T-cells that can be expanded ex vivo and adoptively reinfused represents a promising and innovative approach with the potential to control HIV infection in humans.