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. 2022 May 4:acsnano.2c02794.
doi: 10.1021/acsnano.2c02794. Online ahead of print.

Antibody-Dependent Complement Responses toward SARS-CoV-2 Receptor-Binding Domain Immobilized on "Pseudovirus-like" Nanoparticles

Hanmant Gaikwad  1 , Yue Li, Guankui Wang  1 , Ronghui Li, Shaodong Dai, Cody Rester  2 , Ross Kedl  2 , Laura Saba, Nirmal K Banda  3 , Robert I Scheinman  1 , Casey Patrick, Krishna M G Mallela, S Moein Moghimi  1   4   5 , Dmitri Simberg  1
Affiliations

Antibody-Dependent Complement Responses toward SARS-CoV-2 Receptor-Binding Domain Immobilized on "Pseudovirus-like" Nanoparticles

Hanmant Gaikwad et al. ACS Nano. .

Abstract

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce "pseudovirus-like" nanoparticles with ∼70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all vaccinated, convalescent, and naı̈ve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization via anti-RBD antibodies is not an efficient process: on average, each bound antibody promotes binding of less than one C3 molecule. C3 deposition is exclusively through the alternative pathway. C3 molecules bind to protein deposits, but not IgG, on the nanoparticle surface. Lastly, "pseudovirus-like" nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement opsonization and immune recognition.

Keywords: SARS-CoV-2; antibody; complement; iron oxide nanoparticle; opsonization; receptor-binding domain.

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Figures

Figure 1.
Figure 1.. Synthesis and characterization of “pseudovirus-like” nanoparticles.
A) Purified His-tagged RBD (from left to right: non-reduced and reduced forms); B) synthesis of CLIO-RBD starting from crosslinked dextran iron oxide nanoworms (CLIO NW); C) transmission electron microscopy of CLIO-RBD shows electron-dense iron oxide cores (the shell and the ligand are not visible); D) high magnification confocal microscopy of CLIO(Cy5)-RBD(Cy3). Size bar, 0.5μm. Nanoparticles appear larger than the optical resolution limit due to the fluorescence halo; E) binding of anti-RBD single chain antibody to nanoparticles in full serum (prepandemic). ***p<0.001; F) uptake of control and RBD-modified particles (Cy5 labeled) by human ACE2-expressing A549 cells by fluorescence microscopy and G) Prussian blue staining. Incubation conditions are in Methods. The experiment was repeated twice.
Figure 2.
Figure 2.. Anti-RBD and anti-N-protein levels in donors’ sera.
A) anti-RBD and anti-N-protein antibody (IgG) levels (geometric mean fluorescence intensity gMFI, average of 3 technical replicates) by flow cytometry-based immunoassay (Methods); B) comparison of anti-RBD IgG levels in 3 donor groups (full statistical analysis in Supplemental Table 1; n=10 vaccinated, 10 convalescent and 11 naïve donors; ***p<0.001; ****p<0.0001).
Fig. 3.
Fig. 3.. RBD-dependent C3 deposition on nanoparticles.
A) Study design. C3, IgG, and IgM binding were quantified by dot-blot assay; B) Levels of bound C3 (μg C3/mg Fe, each dot is the mean value of 3 technical replicates) were calculated after subtracting C3 deposition on control CLIO-NTA-Ni2+ particles. Full raw data are in Supplementary Fig. S1. On average, the deposition was increased in vaccinated sera compared to naïve sera (p-value = 0.0047, statistical analysis in Supplementary Table 2). Only some of the vaccinated and convalescent samples had higher RBD-dependent C3 deposition; C-D) Deposition of C3 (C) and release of fluid phase marker C5a (D) after incubation of CLIO-RBD and CLIO-NTA-Ni2+ in vaccinated (VAC) and naïve (NC) sera (means of 3 technical replicates). Both assays demonstrate RBD-dependent complement activation, enhanced in vaccinated sera; E) C3 deposition on CLIO-RBD is decreased in the presence of 0.2 mg/mL soluble RBD protein (means of 3 technical replicates, ****p<0.0001).
Fig. 4.
Fig. 4.. RBD-dependent immunoglobulin deposition on nanoparticles and association with anti-RBD levels and C3 deposition.
A) RBD dependent IgG deposition (μg IgG/mg Fe, each dot is the mean value of 3 technical replicates) is significantly higher in vaccinated and in post-COVID19 sera than in naïve sera. The baseline (control particle) values were subtracted from CLIO-RBD values. **p<0.01. Raw data are in Supplementary Fig. S3, statistical analysis in Supplementary Table 3; B) association between anti-RBD IgG levels measured with the immunoassay and RBD-dependent IgG binding in full serum. Since values for anti-RBD IgG were right skewed, a log base 2 transformation was used prior to correlation analysis. Parametric Pearson correlation coefficients were used for determining association. The cluster of subjects with higher IgG deposition (red rectangle) can be identified; C) subjects with higher RBD-dependent IgG deposition (red rectangle) have higher C3 deposition, and vice versa (blue rectangle); D) RBD-dependent IgM deposition (μg IgM/mg Fe, each dot is the mean value of 3 technical replicates) did not show significant differences between groups. The baseline (control particle) values were subtracted from CLIO-RBD values. Note a much greater deposition of IgG vs IgM (μg/mg) in vaccinated and post-COVID19 sera. Full statistical analysis in Supplementary Table 3.
Fig. 5.
Fig. 5.. C3 deposition via IgG is alternative pathway-driven.
A) Three complement pathways converge into C3 cleavage and nanoparticle opsonization by C3 fragments (C3b/iC3b/C3c/C3d). Inhibitors for each pathway are shown in red; B) Western blot analysis of nanoparticle-deposited C3 in vaccinated serum. Lane 1: serum 1:200 dilution shows native C3; Lane 2: CLIO-RBD after incubation in serum; Lane 3; after incubation in serum/EDTA; Lane 4: SPIO NW after incubation in serum/EGTA/Mg2+. Intact α-chain (115kDa) and β-chain (75kDa) are detectable in serum, β-chain and α’2 (43kDa) are detectable on the particles. Some other α-chain fragments (e.g., α’1-chain) are likely to be in the high molecular weight fraction bound to other proteins via amide or ester bonds, and therefore could not be identified by their molecular weight; C) complement inhibition results (% of serum control) in donors with highest RBD-dependent C3 deposition (means of 3 donors per group, 3 technical replicates per donor) show that CP and LP are not involved in C3 opsonization. C1INH, 100μM; sCR1, 1μM; mannose, 250μM; D) dot blot analysis of binding of C1q shows increased binding to CLIO-RBD in vaccinated sera, but the binding was extremely low and did not lead to activation of the classical pathway; E) molar ratio of RBD-dependent C3 over RBD-dependent IgG deposition for vaccinated and convalescent donors shows a relatively inefficient enhancement of complement opsonization; F) analysis of association between C3 and IgG on particles in vaccinated serum (VAC M54). Proteins were eluted with 5% SDS and the eluted fraction and the nanoparticle-bound fraction were run in non-reducing SDS-PAGE and analyzed by anti-IgG/anti-C3 Western blot. C3 in the eluted fraction is mostly not associated with IgG but appears to be bound to other proteins (high molecular weight bands above 250kDa). Repeated twice.
Fig. 6.
Fig. 6.. Variable uptake of “pseudovirus-like” nanoparticles by leukocytes in lepirudin blood.
A) blood donors with high and low anti-RBD titers measured with the microbead assay (mean of triplicate run); B) RBD-dependent C3 deposition in lepirudin plasma (mean and SD of triplicate); C) uptake of nanoparticles (mean fluorescence intensity) by total leukocytes and effect of sCR1 in 4 blood donors. D) Flow analysis of uptake by leukocytes in blood from donor F61. Ly=lymphocytes; Mo=monocytes; Gr=granulocytes; E) uptake efficiency by different leukocyte types (determined from FSC/SSC plots) in 2 donors with high anti-RBD titers.
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