Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects macaques from infection

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused an ongoing global health crisis. Here, we present as a vaccine candidate synthetic SARS-CoV-2 spike (S) glycoprotein-coated lipid vesicles that resemble virus-like particles. Soluble S glycoprotein trimer stabilization by formaldehyde cross-linking introduces two major inter-protomer cross-links that keep all receptor-binding domains in the "down" conformation. Immunization of cynomolgus macaques with S coated onto lipid vesicles (S-LVs) induces high antibody titers with potent neutralizing activity against the vaccine strain, Alpha, Beta, and Gamma variants as well as T helper (Th)1 CD4⁺-biased T cell responses. Although anti-receptor-binding domain (RBD)-specific antibody responses are initially predominant, the third immunization boosts significant non-RBD antibody titers. Challenging vaccinated animals with SARS-CoV-2 shows a complete protection through sterilizing immunity, which correlates with the presence of nasopharyngeal anti-S immunoglobulin G (IgG) and IgA titers. Thus, the S-LV approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

Keywords: B cells; COVID-19; S glycoprotein; SARS-CoV-2; antibodies; formaldehyde cross-linking; immunity; macaques; nanoparticles; protection.

Graphical abstract

Figure 1

Structural characterization of cross-linked SARS-CoV-2 S (FA-S)- and FA-S-coated lipid vesicles (LVs) (A) Right panel, cryo-EM density of FA-S with all three RBDs down; each protomer is colored differently. The structure was calculated from 126,719 particles imposing C3 symmetry. Middle panel, molecular model of FA-S refined to a resolution of 3.4 Å shown as ribbon. Modeled N-linked glycans are shown as all atom models. Right panel, two major cross-linking sites were identified that covalently link RBDs and the S2 subunits from different protomers. (B) Close-up of the cross-linking sites between RBDs. FA-cross-linked amino groups of K378 and R408 of neighboring protomers as indicated by the continuous density connecting side chains (right panel). (C) Close-up of the cross-linking sites between S2 (left panel). Continuous density between the central helix R1019 as well as S2 K776 to S2 HR1K947 suggested two alternative cross-links between protomers with equal occupancy (right panel). (D) FA-cross-linked S glycoprotein was incubated with liposomes containing 4% DGS-NTA lipids, purified by sucrose gradient density centrifugation, and analyzed by negative staining electron microscopy, revealing regular decoration of the liposomes with the S glycoprotein. Counting S on 50 S-LVs (negative staining EM 2D vision) indicated 231 ± 92 trimers. We thus estimate that approximately or at least 460 ± 184 S trimers are attached to the LVs. Scale bar, 200 nm.

Figure 2

Antibody responses induced by S-LV vaccination of cynomolgus macaques (A) Scheme of vaccination, challenge, and sampling. Syringes indicate the time points of vaccination, red drops indicate the time of serum collection, and the virus particle indicates the time point of challenge. Symbols of identifying individual macaques are used in all figures. (B) ELISA of SARS-CoV-2 S-protein-specific IgG determined during the study at weeks 0, 2, 4, 6, 8, 10, 12, 22, 24, 26, and 28. Ab titers of individual animals are shown. (C) ELISA of SARS-CoV-2 FA-S-protein-specific IgG determined during the study at the indicated weeks. (D) ELISA of SARS-CoV-2 S RBD-specific IgG determined during the study at the indicated weeks. (B–D) Differences between matched groups were compared using the Wilcoxon signed-rank test (p < 0.1). (E and F) Detection of S-specific IgG (E) and IgA (F) in nasopharyngeal fluids. Relative mean fluorescence intensity (MFI) of IgG and IgA binding to SARS-CoV-2 S measured with a Luminex-based serology assay in nasopharyngeal swabs. The background level is indicated by dotted lines. The vertical red line indicates the day of challenge. Groups were compared using the Mann-Whitney U test (∗p < 0.05). Data presented are from technical duplicates.

Figure 3

Serum neutralization of SARS-CoV-2 pseudovirus upon S-LV vaccination (A) The evolution of SARS-CoV-2 neutralizing Ab titers is shown for sera collected at weeks 0, 2, 4, 6, 8, 10, 12, and 19. Bars indicate median titers of the four animals. Differences between matched groups were compared using the Wilcoxon signed-rank test (p < 0.1). Data presented are from technical duplicates. (B) Serum from week 10 was depleted of RBD-specific Abs by affinity chromatography, and neutralization activity of the complete serum of each animal was set to 100% and compared with the RBD-depleted sera and the RBD-specific sera.

Figure 4

S-LV immunization protects cynomolgus macaques from SARS-CoV-2 infection Genomic (A) and subgenomic (sg)RNA viral loads (B) in tracheal swabs (left) and nasopharyngeal swabs (middle) of control (black) and vaccinated (red) macaques after challenge. Viral loads in control and vaccinated macaques after challenge in BAL are shown (right). Bars indicate median viral loads. Vertical red dotted lines indicate the day of challenge. Horizontal dotted lines indicate the limit of quantification. Data presented are from technical duplicates.

Figure 5

Serum antibody titers and neutralization of vaccinated and control group cynomolgus macaques after SARS-CoV-2 challenge (A–C) Antibody IgG titers were determined by ELISA at weeks 24 (challenge), 25, 26, 27, and 28 against (A) SARS-CoV-2 S, (B) SARS-CoV-2 FA-S, and (C) SARS-CoV-2 S RBD. Vaccinated animals are shown with red symbols and control animals with black symbols. (D) SARS-CoV-2 pseudovirus neutralization titers at week 24 (challenge) and 1, 2, and 4 weeks post-exposure (weeks 25, 26, and 28). The bars show the median titers. Differences between matched groups were compared using the Wilcoxon signed-rank test (p < 0.1). Neutralization data presented are from technical triplicates.

Figure 6

Antigen-specific CD4 T cell responses in S-LV-immunized cynomolgus macaques Frequency of (A) interferon gamma (IFNγ)+, tumor necrosis factor alpha (TFNα)+, and interleukin (IL)-2+, (B) Th1 (IFNγ+/–, IL-2+/−, TNFα+), and (C) IL-13+ and IL-17+ antigen-specific CD4⁺ T cells (CD154+) in the total CD4⁺ T cell population, respectively, for each immunized macaque (n = 4) at week 21 (W21) post-immunization (p.im.) (i.e., 2 weeks after the fourth immunization, pre-exposure) and 14 days post-exposure (dpe). PBMCs were stimulated overnight with medium (pink symbols) or SARS-CoV-2 S overlapping peptide pools (red symbols). Bars indicate means. Time points in each experimental group were compared using the Wilcoxon signed rank test.

Figure 7

S-vaccination induces robust neutralization of SARS-CoV-2 variants B.1.1.7 (Alpha, UK), B.1.351 (Beta, SA), and P.1 (Gamma, BR) pseudovirus neutralization titers were compared with the Wuhan vaccine strain. Titers were determined using total IgG purified from sera at weeks 8 (2 immunizations), 12 (3 immunizations), and 24 and 28 (4 immunizations). Background neutralization by IgG isolated from naive animals was <100 for all variants and is indicated by the dashed line. Data presented are from technical triplicates.