In a recent article published in the Cell Journal, researchers demonstrated that enveloped virus-like particles (eVLPs) self-assembling into an endosomal sorting complex required for transport (ESCRT)- and ALG-2-interacting protein X (ALIX)-binding region (EABR) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) cytoplasmic tail elicited more potent antibody responses than conventional SARS-CoV-2 S-based messenger ribonucleic acid (mRNA) vaccines in mice.
Study: ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines. Image Credit: CoronaBorealisStudio/Shutterstock.com
Several clinical studies have demonstrated the higher efficacy of mRNA vaccines for coronavirus disease 2019 (COVID-19) in preventing over 90% of COVID-19 cases by eliciting B and T cell responses.
However, their prime-boost regimens have failed to combat Omicron and its subvariants, thus raising the need for frequent boosters to maintain neutralizing antibody (nAb) levels.
Unlike protein nanoparticle-based vaccines, e.g., NVX-CoV2373, that mimic viruses by presenting S protein arrays, mRNA vaccines display viral peptides (formed by mRNA translation) on major histocompatibility complex (MHC) class I molecules to activate T cells.
To stimulate B cell activation, mRNAs formulated in lipid nanoparticles (LNP) mimic an infected cell and express SARS-CoV-2 S protein on the cell surface.
Both mRNA and protein nanoparticle-based COVID-19 vaccines elicit comparable nAb titers, a correlate of protection conferred by vaccines.
Achieving higher nAb titers is crucial as antibody levels gradually decline/wane over time. This makes an individual susceptible to breakthrough infection by SARS-CoV-2 variants of concern (VOCs) less sensitive to the vaccine- and natural infection-induced nAbs.
Thus, the research for an optimal vaccine continues that combine attributes of S-encoding mRNA-lipid nanoparticle (LNP) and protein nanoparticle-based vaccines, delivers a genetically encoded SARS-CoV-2 S protein that triggers self-assembly and subsequent release of S-presenting eVLPs for the activation of T cells.
About the study
In the present study, researchers first fused EABRs from different sources, e.g., human centrosomal protein 55 (CEP55), and joined it to the shortened cytoplasmic tail of the SARS-CoV-2 S protein's C terminus.
The unit remained separated by a short linker fragment composed of glycine (Gly) and serine(Ser). Notably, CEP55 binds tumor susceptibility gene 101 protein (TSG101) and ALIX during cytokinesis.
For comparisons, they used three domains that recruit ESCRT proteins early during the viral-budding process, namely, residues 1–44 of the Ebola virus (EBOV) VP40 protein, Equine infectious anemia virus (EIAV) p9 protein, and the human immunodeficiency virus (HIV)-1 p6 protein.
In addition, they added an endocytosis prevention motif (EPM) to extend the duration of interaction between EABR-fusion proteins and ESCRT proteins at the cell surface and, as expected, enhanced eVLP production.
Next, the team evaluated the abilities of all fused constructs to generate eVLPs by transfecting Expi293F cells and estimating eVLP concentration in supernatants purified by ultracentrifugation.
Further, they used a quantitative Western blot (WB) to detect the presence and concentration of SARS-CoV-2 S on purified eVLPs for in vivo studies. Multiplying S1 protein concentrations of S-EABR eVLP samples by a factor of 1.8 showed the difference in molecular weights (MW) of S1 and the full-length S protein.
The team also performed cryogenic-electron tomography (cryo-ET) to illustrate the three-dimensional form of S-EABR eVLPs.
Furthermore, the researchers evaluated the potential of purified S-EABR eVLPs as a vaccine candidate for SARS-CoV-2 in C57BL/6 mice. To this end, first, they administered 0.1 μg doses of S-EABR eVLPs subcutaneously on days 0 and 28 in mice.
Next, they evaluated serum antibody responses by enzyme-linked immunosorbent assays (ELISAs) and in vitro pseudovirus neutralization assays.
The EABR from CEP55 generated eVLPs 10-fold more efficiently than the EIAV late domain p9, suggesting that efficient eVLP assembly required the recruitment of both ESCRT proteins, ALIX and TSG101.
WB analysis demonstrated that purified S-EABR eVLP fractions had nearly 10-fold more S protein than eVLPs developed by other approaches. Since S-HIV-1 p6 samples had little or no S protein due to lower affinities for ESCRT proteins, its eVLP assembly was inefficient.
S-EABR eVLPs elicited robust antibody binding and neutralization responses in mice against the authentic SARS-CoV-2 variant, WA1. However, neutralization titers dropped four- and two-fold against the SARS-CoV-2 Beta and Delta VOCs.
Strikingly, S-EABR eVLPs improved neutralizing titers by >10-fold against Omicron subvariants for up to three months post-boosting.
Indeed, EABR technology enhanced the potency and breadth of vaccine-induced responses enabling longer-lasting protection against SARS-CoV-2 and other viruses.
3D reconstructions showed that purified S-EABR eVLPs were surrounded by a lipid bilayer densely coated with spikes (10 to 40 around each eVLP), and their diameters ranged between 40 to 60 nm. Intriguingly, the spike densities on most eVLPs exceeded those of authentic viruses.
The authors deployed the novel EABR technology that recruited ESCRT proteins to induce eVLP budding from cells in a mouse model.
They demonstrated how easily this technology generated eVLPs presenting a wide range of surface proteins for COVID-19 vaccines and other therapeutic applications for fighting SARS-CoV-2 infections.
To summarize, the EABR technology exhibited several attractive features compared to conventional protein nanoparticle-based and mRNA-based vaccine approaches, as follows:
i) S-EABR constructs could be delivered as mRNA vaccines since eVLP assembly and cell surface expression only required the expression of a single genetically encoded component;
ii) retention of transmembrane proteins in their native membrane-associated conformation ensured optimal protein expression and stability;
iii) fully assembled eVLPs could be purified directly from culture supernatants with no need for detergent-mediated cell lysis and separation of membrane protein antigens from cell lysates; and
iv) eVLPs lipid bilayer prevented off-target antibody responses against a nanoparticle scaffold.
Hoffmann, M.A.G., Yang, Z., Huey-Tubman, K.E., Cohen, A.A., Gnanapragasam, P.N.P., Nakatomi, L.M., Storm, K.N., Moon, W.J., Lin, P.J.C., West, A.P. & Bjorkman, P.J. (2023) ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines. Cell, 186 (11), pp.2380–2391.e9. doi: 10.1016/j.cell.2023.04.024. https://www.cell.com/cell/fulltext/S0092-8674(23)00420-8