Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans

Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases

HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

<div><p>HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent <i>in vitro</i> at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.</p></div

Lentiviral backbone augments CAR expression and control over HIV replication.

<p><b>(A-D)</b> Primary human CD8 T cells were activated with αCD3/αCD28 coated beads and were either left <b>(A)</b> nontransduced (NTD), <b>(B)</b> transduced with the original MMLV-based CD4 CAR, or <b>(C)</b> transduced with the same CAR placed in a HIV-based lentiviral vector, both driven by the PGK promoter. After eight days T cells were stained for CD4 and CD8 by flow cytometry. Median fluorescence intensity (MFI) is indicated on each graph. (<b>D)</b> Overlying histograms of the data shown in <b>(A-C). (E)</b> Eight days post activation, qPCR was performed and the number of integrated vector copies per cell was calculated. (<b>F</b>) Schematic of experimental design to study the control over HIV replication by T cells expressing HIV-specific CARs. Briefly, following activation with αCD3/αCD28 coated beads, CD4 T cells were infected with HIV Bal, and 24 hours later the indicated CD8 T cells were mixed at the indicated effector to target (E:T) ratios. After 7 days of co-culture, the expression of surface CD4, CD8, and intracellular Gag was measured by flow cytometry. <b>(G)</b> Intracellular Gag staining on CD8 negative cells, and <b>(H)</b> Intracellular Gag staining on CD8 positive cells. <b>(I)</b> Summary data for a single experiment, performed in triplicate, gating on the CD8 negative cells. Error bars indicate standard error of the mean (SEM). Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s014" target="_blank">S14 Fig</a> shows each of the 3 independent experiments. (<b>J)</b> Measurement of levels of intracellular Gag in CD8 negative T cells over the time course of an experiment. Each graph represents a different E:T ratio. Error bars indicate SEM (n = 3).</p

CD4 CARs respond to Env⁺ cells and not MHC class II⁺ cells.

<p><b>(A)</b> Primary human CD8 T cells were activated with either left NTD or transduced with the indicated CD4 CARs. Two weeks post activation, the CD8 T cells were co-cultured for 6 hours at a 1:1 ratio with unmodified K562 cells, K562 cells expressing high levels of HLA-DR, or K562 cells expressing HIV-1 YU2 GP160. Intracellular IFNγ and MIP-1β expression is shown on the left, and intracellular IL-2 expression and CD107a surface mobilization is shown on the right. <b>(B)</b> A co-culture assay was designed to demonstrate that CD4 CAR⁺ CD8 T cells do not kill MHC class II-expressing target cells. Briefly, NTD or CD4 28z CAR transduced CD8 T cells from <b>(A)</b> were co-cultured with K562 cells expressing both HLA-A2 and GFP as well as K562 expressing both HLA-DR*0401 and mCherry at a 1:1:1 ratio. Flow cytometry measuring GFP and mCherry expression was performed immediately after mixing (0 hr) and after 3 days of co-culture (72 hr). <b>C)</b> Summary data for a single experiment performed in triplicate, measuring the ratio of HLA-A2/GFP-expressing cells to HLA-DR*0401/mCherry-expressing cells after 24, 48, and 72 hours of culture. Error bars indicate SEM. Data is representative of three independent experiments.</p

Re-directed T cells expressing a CD4 CAR are 100-fold more potent than re-directed T cells specific for B57-KF11.

<p><b>(A)</b> Gag staining on day 6 of co-culture for CD8 negative T cells. <b>(B)</b> Summary data for a single experiment performed in triplicate, gating on the CD8 negative T cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s016" target="_blank">S16 Fig</a> shows each of the 3 independent experiments.</p

EF1α promoter and CD8α transmembrane domains improve CAR expression and control over HIV.

<p><b>(A)</b> Schematic of the constructs compared in this figure. (<b>B)</b> CD4 CAR expression 8 days after activation. Median fluorescence intensity (MFI) is indicated on each graph. (<b>C</b>) Intracellular Gag staining on day 7 of co-culture, for CD8 negative T cells and <b>(D)</b> for CD8 positive T cells. <b>(E)</b> Summary data for a single experiment, performed in triplicate, gating on the CD8 negative cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s015" target="_blank">S15 Fig</a> shows each of the 3 independent experiments. (<b>F)</b> The levels of intracellular Gag in CD8 negative T cells over the time course of an experiment. Each graph represents a different E:T ratio. Error bars indicate SEM (n = 3).</p

CD4-based CARs control HIV more effectively than broadly neutralizing antibody-based CARs.

<p><b>(A)</b> Specific lysis of Cr⁵¹ labeled K562 target cells expressing HIV-1 YU2 GP160. Significance was detected using a 1-way ANOVA test on the 30:1 E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). Data plotted shows the average of three independent experiments. Error bars indicate SEM (n = 3). <b>(B)</b> Gag staining on day 6 of co-culture for CD8 negative T cells and <b>(C)</b> the CD8 positive T cells. The data from the best (PGT128) and one of the worst (PG9) scFv-based CARs are compared to the CD4 CAR here, but the complete construct comparison is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s006" target="_blank">S6 Fig</a>. (<b>D)</b> Summary data for a single experiment performed in triplicate, gating on the CD8 negative cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). Data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s017" target="_blank">S17 Fig</a> shows each of the 3 independent experiments.</p