Biological and biochemical characterization of HIV-1 Gag/dgp41 virus-like particles expressed in Nicotiana benthamiana.

The transmembrane HIV-1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4⁺ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus-based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.

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

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.