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

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

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