Circularized Nanodiscs for Multivalent Mosaic Display of SARS-CoV-2 Spike Protein Antigens

Abstract

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin-phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

Keywords: COVID-19; nanodisc; vaccine.

Figure 1

Formation of stable nanodisc using cNW50 incorporating CoPoP as a His-tag protein-binding lipid, and QS-21 and MPLA as immune stimulants. (A) Size exclusion chromatogram of empty nanodiscs containing MPLA, QS-21, and CoPoP, with a schematic insert of the proposed structure of the formed nanodisc. (B) Negatively stained transmission electron micrograph of formed nanodiscs, scale bar = 100 nm. (C) Stability of formed nanodisc at 4 °C for 28 days as tested by size exclusion chromatography.

Figure 2

Recombinant, His-tagged spike proteins bind to CoPoP nanodiscs. (A) Size exclusion chromatogram of spike bound nanodisc using a Superose 6 column, with schematic insert of the proposed structure of the formed nanodisc. (B) Binding kinetics of a fluorescent labeled spike protein to CoPoP nanodisc. (C) Binding stability of a fluorescent labeled spike decorated nanodisc when incubated with 20% serum for 48 h in the dark at 4 degrees. (D) Dot blot detection of ACE2 binding to adsorbed spike or gp160 (an unrelated control antigen) in soluble or particulate form. (E) Nickel- nitrilotriacetic acid (Ni-NTA) bead competition assay; spike protein antigen was incubated with CoPoP nanodisc for various times, and then Ni-NTA beads were added and then isolated for analysis. Protein that was stably bound to nanodisc is in the supernatant (“S”) lanes, whereas the unbound protein is in the bead (“B”) fraction. (F) TEM image of spike decorated nanodisc, scale bar = 100 nm.

Figure 3

Spike/CoPoP nanodisc is uptaken by THP-1 macrophage cells and favors M1 differentiation. (A) Microscopy photos showing polarization of PMA differentiated THP-1 from M0 to M1 cells in response to ND and/or spike addition, yellow arrows show M1-polarized macrophages. (B) Flowcytometry histogram of CD 80 indicating polarization of M0 macrophages to M1 due to MPLA and QS-21 incorporated into nanodisc. (C) Analysis of mean fluorescence intensity of M1-polarized cells. (D) Flowcytometry histogram of uptake of PE-labeled nanodisc and/or FITC-labeled spike protein. (E) Analysis of mean fluorescence intensity of uptake of PE-labeled nanodiscs (left y-axis, red bars and stats) and/or FITC-labeled spike protein (right y-axis, green bars and stats). (F) TNF-α release from macrophages as measured by ELISA in response to addition of nanodisc alone, spike alone and nanodisc-spike. Analysis was performed by one-way ANOVA test, followed by Tukey’s comparisons, * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.001.

Figure 4

Broadly neutralizing antibodies induced by spike nanodiscs. (A) Schematic representation of immunization scheme and immunogens used. CD-1 mice (n = 5 per group) were immunized as indicated. (B) Anti-spike ELISA IgG titer using WT, BA.1, or BA.2 as coating antigen (C) WT, BA.1, or BA.2 spike/ACE-2 interaction inhibition by immunized mice sera after 1:20 dilution. (D) Pseudo-typed virus neutralization assay using WT, BA.1, or BA.2 decorated pseudo-typed virus. For (B,D), log10-transformed titer was analyzed by one-way ANOVA test, followed by Tukey’s comparisons. For (C), data were analyzed by one-way ANOVA test, followed by Tukey’s comparisons. * p < 0.05, ** p < 0.01 and **** p < 0.001 against WT control; ^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.005, and ^$$$$ p < 0.001 against BA.1 Control; ^# p < 0.05, ^### p < 0.005, and ^#### p < 0.001 against BA.2 Control.