Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles

Abstract

It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR—a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

“…VLPs were precipitated with 40% ammonium sulfate and resuspended in 1× PBS (140 mM NaCl, 2 mM KCl, 10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , pH 7.4), then purified via iodixanol density gradient centrifugation. The VLP-containing 20% iodixanol fraction was concentrated on a 300 kDa molecular weight cut-off membrane (Sartorius) and quantified via immunoblot for immunizations as described in (Kessans et al, 2016). A typical total yield of a highly enriched VLP preparation in terms of its HIV-1 protein constituents was 275 mg/kg and 92 mg/kg for Gag and dgp41, respectively.…”

“…into the thigh. Plant-produced, gradient-purified VLPs were prepared in 1× PBS pH 7.4 with 2 μg p24 and 1.2 μg MPER and mixed with Ribi adjuvant (Sigma) per manufacturer’s protocol to reach a final concentration of 2% oil [described previously in (Kessans et al, 2016)]. Mock mice were injected i.m.…”

“…Serum, fecal, and vaginal lavage samples were collected as previously described (Kessans et al, 2016) every 2 weeks at days 0, 14, 28, 42, 56, 80, and at the endpoint of day 97 for analysis of Ab production. One week post the final boost (day 97), spleens were collected for analysis of CD8 T-cell responses as described below.…”

“…Such VLPs display the MPER of gp41 without steric hindrance from gp120, without the immunodominant epitopes on both Env subunits, and with a higher antigen load per VLP than what exists in an HIV virion (Kessans et al, 2016). A similar construct has been shown by us to be a likely trimer with its bnAb epitopes exposed (Gong et al, 2015).…”

“…A similar construct has been shown by us to be a likely trimer with its bnAb epitopes exposed (Gong et al, 2015). When administered to mice, these VLPs elicit both serum IgG and mucosal IgA to Gag and dgp41 antigens (Kessans et al, 2016). …”