A chimeric HIV-1 envelope glycoprotein trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain induces enhanced antibody and T cell responses

Abstract

An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric Env(GM-CSF) enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines.

FIGURE 1.

Design of chimeric Env_hGM-CSF. A, linear representation of Env_hGM, Env_ΔV1V2, and Env_GM-CSF. Clade B JR-FL gp140 (amino acids 31–681) contains several modifications that have been described elsewhere (31). The amino acid sequences of the Env-hGM-CSF junctions are shown. The linker residues are indicated in italics and GM-CSF residues in bold type. A potential site for N-linked glycosylation is underlined. B, schematic of Env_hGM-CSF compared with Env_wt and Env_ΔV1V2. The V1V2 loop present in Env_wt was replaced by amino acids 26–139 of hGM-CSF or amino acids 26–136 of mGM-CSF.

FIGURE 2.

Env_GM-CSF is expressed as a trimer. Shown are reducing SDS-PAGE (upper panel) and Blue native PAGE (lower panel) analyses of Env_wt, Env_ΔV1V2, and Env_GM-CSF proteins secreted from transiently transfected 293T cells. Recombinant purified JR-FL gp120 (50 ng) was included for comparison.

FIGURE 3.

Chimeric Env_hGM-CSF is recognized by conformational Env Ab and receptor mimics. ELISA analysis of the binding of Env variants to: HIV-Ig and 2F5 (A); glycan-dependent DC-SIGN-Fc and 2G12 (B); 39F (V3), CD4-IgG2, b12, and VRC01 (CD4BS) (C); and 17b, 48d, and 412d (CD4i) in the absence and presence of soluble CD4 (sCD4) (D). Culture supernatant from mock transfected 293T cells was used as a negative control.

FIGURE 4.

The GM-CSF domain of chimeric Env_hGM-CSF is functional. A, ELISA analysis of the binding of a neutralizing, conformation-dependent anti-hGM-CSF Ab. B, proliferation of TF-1 cells in response to culture supernatant containing Env_GM-CSF, Env_wt, or rhGM-CSF. The numbers of cells present after 5 days of culture in a volume of 75 μl are given. Culture supernatant from mock transfected 293T cells was used as a negative control (∼15,000 cells) and deducted from the test values.

FIGURE 5.

Three different models of Env_hGM-CSF trimers. hGM-CSF (magenta) is shown in the up (top panels), side (middle panels), and down (lower panels) orientation attached to gp120 (cyan). The models were generated using Chimera (35), RosettaDesign (36), and RosettaRemodel as described under “Experimental Procedures” and rendered using PyMOL (version 1.3, Schrödinger, LLC).

FIGURE 6.

Env_mGM-CSF induces better anti-Env Ab responses in mice. A, mouse immunization scheme. B, end point anti-gp120 IgG titers obtained from mice immunized with different Env immunogens at day 42 as determined by ELISA. C, end point anti-gp120 IgG titers over the course of the immunization experiment as determined by ELISA. The individual end point titer is shown for each mouse. Note that both comparison groups (Env versus Env containing embedded mGM-CSF) contain 15 mice. Within these groups of 15, five mice were immunized with Env, Env-BAFF, or Env-CD40L or, for comparison, with Env_GM-CSF, Env_GM-CSF-BAFF, or Env_GM-CSF-CD40L. The mean end point titers of the 15 mice/group are shown ±S.E. Statistical significance was determined using a one-tailed Mann-Whitney test, and significant p values are represented with asterisks: *, p ≤ 0.05; ***, p ≤ 0.001.

FIGURE 7.

Env_mGM-CSF induces enhanced Env-specific T helper responses. Splenocytes were incubated with control media, gp120, or anti-CD3 stimuli for 96 h in vitro. Release of cytokines in the supernatant of gp120 (A)- or anti-CD3 (B)-stimulated splenocytes was measured. Cytokine responses from splenocytes treated with media provided background values that were subtracted from the values obtained with anti-CD3 or gp120 stimulation. Statistical significance was determined using a two-tailed Student's t test, and significant p values are represented with asterisks: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 8.

Env_mGM-CSF induces enhanced neutralizing Ab responses that are partially dependent on V3. A, the percentage of SF162 infectivity in the absence or presence of serial dilutions of pooled sera from the Env_wt or Env_mGM-CSF mice was measured in a single-cycle infectivity assay using TZM-bl cells. Significance was determined using a two-way analysis of variance test. B, the percentage SF162 infectivity in the presence of pooled sera diluted 20× was measured in the absence or presence of a mixture of three overlapping V3 peptides or of an unrelated peptide. To assess whether the V3 peptide pool had nonspecific effects, we measured whether they affected neutralization by b12.