Gag Virus-like Particles Functionalized with SARS-CoV-2 Variants: Generation, Characterization and Recognition by COVID-19 Convalescent Patients' Sera

Abstract

The robustness, safety, versatility, and high immunogenicity of virus-like particles (VLPs) make them a promising approach for the generation of vaccines against a broad range of pathogens. VLPs are recombinant macromolecular structures that closely mimic the native conformation of viruses without carrying viral genetic material. Particularly, HIV-1 Gag-based VLPs are a suitable platform for the presentation of the SARS-CoV-2 Spike (S) protein on their surface. In this context, this work studies the effect of different rationally engineered mutations of the S protein to improve some of its characteristics. The studied variants harbored mutations such as proline substitutions for S stabilization, D614G from the early dominant pandemic form, the elimination of the S1/S2 furin cleavage site to improve S homogeneity, the suppression of a retention motif to favor its membrane localization, and cysteine substitutions to increase its immunogenicity and avoid potential undesired antibody-dependent enhancement (ADE) effects. The influence of the mutations on VLP expression was studied, as well as their immunogenic potential, by testing the recognition of the generated VLP variants by COVID-19 convalescent patients' sera. The results of this work are conceived to give insights on the selection of S protein candidates for their use as immunogens and to showcase the potential of VLPs as carriers for antigen presentation.

Keywords: COVID-19; D614G; SARS-CoV-2; furin cleavage; spike; variants; virus-like particles.

Figure 1

(A): SARS-CoV-2 virion scheme. M, N, E and S proteins are represented. S1 subunit of the S protein interacts with the host cell receptor membrane protein ACE2 to bind and promote viral internalization. (B): Schematic representation of the Spike protein. S1/S2 cleavage site is indicated with an arrow. Abbreviations: NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; FP, fusion peptide; HR1 and HR2, heptad repeat 1 and 2; CH, central helix; CD, connector domain; TM, transmembrane domain; CT, cytoplasmic tail.

Figure 2

Schematic representation of the S_WT protein and the S_mut2 and S_mut3 variants with its mutations indicated. Furin cleavage site (CS) is indicated by an arrow. ER-Golgi intermediate compartment retention signal (ERGIC ret. signal) is represented as a white box.

Figure 3

(A): Cell concentrations (solid lines) and viabilities (dotted lines) of HEK293 cells transfected for the production of different VLP variants, described in Table 1. (B): Transfected populations, analyzed by ICC at 72 hpt. (C): Particle size distribution of the purified S VLP variants, analyzed by nanoparticle tracking analysis (NTA), with the mode diameter of each variant indicated.

Figure 4

Relative pixel densities of the VLP recognition assays by negative (blue) and positive (red) tested sera samples. Horizontal dashed line indicates the unspecific recognition threshold, marked by the pixel density of the G-VLP negative control. Saturated pixel densities are indicated with an “s”. Soluble spike recombinant protein (S rec.) was used as positive control.