An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

<div><p>Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.</p></div

Antigenic characterization of Env_IL-4 and Env_IL-21 molecules.

<p>ELISA reactivity of Env_IL-4 and Env_IL-21 with 2G12 and HIV-Ig (A); b12 and CD4-IgG2 (B); and 48d (CD4i) in the absence and presence of sCD4 at 1 µg/ml (C). All ELISA results are representative for at least three independent experiments using proteins derived from three independent transfections.</p

Immunoglobulin production by B cells stimulated with Env_wt, Env_IL-4 and Env_IL-21 molecules and controls.

<p>IgG, IgA and IgM levels secreted by the B cells from human PBMCs cultured with (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10. Data are representative of three independent experiments showing similar results. Immunoglobulin secretion by B cells from different donors cultured with Env_wt and Env_IL-21 molecules in (C) CD40L/IL-10 and (D) CD40L/IL-4/IL-10 milieu. Culture supernatant from mock transfected 293T cells was used as a negative control and the values were deducted from the test values. Data represent the fold change values compared to Env_wt from at least 12 donors and each donor sample was tested in duplicate. (E) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env_wt, Env_IL-21, Env_ChimIL-21/4 supernatants and controls in CD40L/IL-10 milieu. Data are representative of three experiments using B cells from three different donors. (F) The expression of CD38 cell surface marker treated with different Env_IL-21 constructs and controls in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six different donors.</p

Immunoglobulin secretion from B cells cultured with Env_wt, Env_IL-21, clEnv and clEnv_IL-21 molecules in the presence of (A) CD40L/IL-10 and (B) CD40L/IL-4/IL-10.

<p>Data are representative of two independent experiments using B cells from two different donor, each tested in duplicate. (C) The expression of cell surface markers CD38 and CD27 on B cells cultured with Env_wt, Env_IL-21, clEnv and clEnv_IL-21supernatants in medium supplemented with CD40L/IL-10. Data are representative of three independent experiments using B cells from three donors. (D) The expression of CD38 cell surface marker treated with different cleaved Env (clEnv) and cleaved Env_IL-21 (clEnv_IL-21) constructs in CD40L/IL-10 milieu. Data are representative of six experiments using B cells from six donors.</p

Schematic and expression of the cleavable Env_IL-21.

<p>Linear (A) and cartoon (B) representation of the Env_IL-21 and clEnv_IL-21 proteins. Cleaved proteins were created by introducing a stop codon in front of the isoleucine zipper (IZ) trimerization domain in the Env_IL-21. (C) SDS-PAGE analysis of chimeric uncleaved and cleaved Env_IL-21 constructs.</p

Cross-linked gp120 sensitizes DC through DC-SIGN and MCLRs for CD40L-mediated apoptosis.

<p>(<b><i>A</i></b>, <b><i>B</i></b>) moDC were pretreated with anti-DC-SIGN mAbs or isotype control Ab before pulse with cross-linked gp120_ADA and co-culture for 3 d with autologous activated CD4 T cells, and subsequently subjected to cell viability assay. Data are representative of 3 experiments and are expressed as mean ± SD from 3 experiments in <b><i>B</i></b>. (<b><i>C, D</i></b>) moDC were treated with cross-linked recombinant gp120_ADA with or without pre-treatment by soluble ICAM-3-Fc chimeric protein, anti-CD4 plus anti-CCR5 mAbs, or anti-DC-SIGN mAbs. Cells were subsequently co-cultured with mock- or CD40L-transfected (CD40L Tf) cells for 3 d. Data are representative of 7 experiments in panel <b><i>C</i></b> and are expressed as mean ± SD (n = 7) in <b><i>D</i></b>; ***p<0.005. (<b><i>E, F</i></b>) Recombinant gp120_ADA were treated with or without EndoH, and then cross-linked with anti-His Ab before use to pulse moDC. Prior to gp120 pulsing, moDC were pre-treated with or without mannan or FcR blocking reagent, or anti-DC-SIGN mAbs for 30 minutes. After gp120 pulsing, DC were subsequently cocultured with CD40 Tf for 3 days. DC without any pre-treatment and only pulsed with anti-His Ab were used as a control (control DC). Data are representative of 3 experiments and expressed as mean ± SD from 3 experiments in <b><i>F</i></b>; *p<0.05, **p<0.01.</p

Freshly-isolated DC-SIGN(+) blood DC underwent DC-SIGN-dependent CD40L-mediated apoptosis and DC-SIGN(+) cells from HIV-1-infected individuals are pre-sensitized for CD40L-mediated apoptosis.

<p>(<b><i>A</i></b>) PBMCs from normal HIV(−) individuals were labelled with anti-CD14 plus either isotype control (left panel) or anti-DC-SIGN (right panel) mAbs and analysed by flow cytometry for cell isolation. Data are representative of 4 experiments. (<b><i>B</i></b>) Purified CD14(+)DC-SIGN(+) cells were treated with anti-His mAb alone (Control) or anti-His cross-linked recombinant gp120_ADA, in the absence or presence of anti-DC-SIGN mAbs, and subsequently co-cultured with CD40L Tf for 3 d. The non-adherent DC were then harvested and subjected to cell viability assay. Data are representative of 4 experiments. (<b><i>C, D</i></b>) freshly isolated DC-SIGN(+) cells from HIV(+) and HIV(−) blood were cocultured with mock Tf or CD40L Tf for 3 days and subjected to cell viability assay. Data are representative of 4 experiments in <b><i>C</i></b> and expressed as mean ± SD in <b><i>D</i></b>. ***p<0.005.</p

Sera from HIV-1(+) individuals can sensitize moDC for DC-SIGN dependent CD40L-mediated apoptosis.

<p>(<b><i>A</i></b>) moDC were treated with HIV(+) serum before or after immunoprecipitation (IP) with anti-gp120 mAbs, or with or without anti-DC-SIGN or isotype control mAbs, and subsequently co-cultured with autologous activated CD4 T cells. After 3 d, cells were harvested and subjected to TUNEL assays. Cell death was assessed as the percentage of cells expressing terminal deoxynucleotidyl transferase (TdT). DC pulsed with HIV(+) serum without coculture with activated CD4 T cells (top panel) were also used as a control. Data are representative of 4 experiments. (<b><i>B</i></b>) MoDCs were treated with anti-DC-SIGN mAbs, isotype control Ab, or anti-CD40L mAb before pulse with HIV serum (before or after immunoprecipitation of gp120) and cocultured with activated CD4 T cells. Data are expressed as mean ± SD (n = 4); *p<0.05 and **p<0.01 compared with ‘HIV(+) serum-DC plus isotype Ab’. (<b><i>C</i></b>,<b><i>D</i></b>) moDC were treated with normal AB serum or HIV-1(+) serum with viral RNA copies >400,000/ml (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003100#ppat.1003100.s013" target="_blank">Table S1</a>), with or without pre-treatment with anti-CD4 plus chemokine receptor or anti-DC-SIGN mAbs, or with gp120-depleted (immunoprecipitated, IP) HIV(+) serum, and co-cultured with CD40L Tf for 3 d. Data are representative of 4 experiments in panel <b><i>C</i></b> and individual datum with the mean is shown in panel <b><i>D</i></b>; †: P<0.001 between with and without IP of gp120 from the HIV serum, **: P<0.01 between with and without pre-treatment with anti-DC-SIGN mAbs.</p

Cross-linked recombinant gp120 sensitizes moDC for CD40L-mediated apoptosis after co-culture with activated CD4 T cells.

<p>(<b><i>A</i></b>) moDC were treated for 24 h with anti-His mAb alone (control DC, left panels) or with 25 nM gp120_ADA cross-linked with anti-His mAb (gp120-DC, right panels), and co-cultured with autologous activated (upper panels) or naïve (lower panels) CD4 T cells for 3 d. The moDC (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003100#ppat.1003100.s001" target="_blank">Fig. S1</a>) were analyzed for Annexin V (AV) and propidium iodide (PI) expression to assess the extent of apoptosis, as manifested by the percentage of the AV-positive [AV(+)] cells. Data are representative of 5 experiments. (<b><i>B</i></b>) Apoptosis of moDC was analyzed after treatment with different concentrations of cross-linked recombinant gp120_ADA or gp120_HXBc2 and co-culture with activated CD4 T cells for 3 d. DC treated with monomeric gp120 (not cross-linked with anti-His or anti-FLAG mAb) were used as a control. Data represent mean ± SD from 5 experiments; **p<0.01. The use of cross-linked recombinant gp120_BAL gave similar results (not shown). (<b><i>C</i></b>) Apoptosis of moDC was analyzed after treatment with the indicated cross-linked recombinant gp120, or appropriate mAb controls, at the indicated time points after co-culture with activated CD4 T cells. Data represent mean ± SD from 5 experiments (data for anti-His and anti-FLAG controls were indistinguishable); *P<0.05 and **P<0.01 compared with control group (DC with no gp120 pulse or DC plus anti-His/FLAG Ab). (<b><i>D</i></b>) moDC were respectively not treated (Control DC), or treated with gp120_ADA cross-linked with mouse IgG2a anti-His mAb (dimeric gp120-DC), or treated with cross-linked gp120_ADA supplemented with isotype control mouse IgG (gp120-DC+isotype IgG), and subsequently co-cultured with autologous activated CD4 T cells for 3 days before AV/PI staining. Data are representative of 3 experiments. (<b><i>E</i></b>) moDC were treated with cross-linked gp120_ADA and co-cultured for 3 d with autologous activated or naïve CD4 T cells, that had been pre-treated without or with 10 µg/ml isotype control or anti-CD40L mAb, before cell viability analysis. Data are expressed as mean ± SD from 5 experiments. **p<0.01.</p

CD40L-mediated apoptosis of HIV(+) serum-pulsed DC is proportional to viral loads and predominantly induced by the 100–1000 kDa fraction.

<p>(<b><i>A</i></b>) moDC were treated with HIV-1(+) sera with viral copy numbers >400,000/ml or <100,000/ml or normal AB serum (control) and co-cultured with CD40L Tf cells for 3 d. Data are expressed as individual datum with the mean. N = 4 for each condition; *p<0.05 and **p<0.01. (<b><i>B, C</i></b>) moDC were treated for 24 h with normal AB serum or with >1000 kDa or 100–1000 kDa fractions from HIV(+) serum and were co-cultured with CD40L Tf for 3 d. Data are representative of 5 experiments in <b><i>B</i></b> and expressed as mean ± SD in <b><i>C</i></b>; ** p<0.01.</p