ABSTRACT
We present a comprehensive vaccine strategy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by combining antigen optimization and nanoparticle display. We first developed a receptor binding domain (RBD)-specific antibody column for purification and displayed the RBD on self-assembling protein nanoparticles (SApNPs) using the SpyTag/SpyCatcher system. We then identified the heptad repeat 2 (HR2) stalk as a major cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on three SApNPs with high yield, purity, and antigenicity. Compared to the RBD, the RBD-ferritin SApNP elicited a more potent murine neutralizing antibody (NAb) response on par with the spike. S2GΔHR2 elicited two-fold-higher NAb titers than the proline-capped spike (S2P), while S2GΔHR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3-01v9 SApNP also induced critically needed T-cell immunity, thereby providing a next-generation vaccine candidate to battle the COVID-19 pandemic.
ONE-SENTENCE SUMMARY The receptor binding domain and stabilized SARS-CoV-2 spike were displayed on nanoparticles as vaccine antigens and elicited potent immune responses.
INTRODUCTION
Three beta-coronaviruses (β-CoVs) have caused outbreaks in humans, including the severe acute respiratory syndrome CoV-1 (SARS-CoV-1), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, which is the causative agent of COVID-19 (1–3) and has resulted in more than 920,000 deaths worldwide (4). Enormous efforts are being undertaken to develop effective therapeutics and prophylactics for SARS-CoV-2. Small molecules that can block the host receptor, angiotensin-converting enzyme 2 (ACE2), and the transmembrane protease serine 2 (TMPRSS2) (5), which is required to process the spike (S) protein, are considered treatment in addition to other interventions (6). While the immunology underlying COVID-19 is yet to be fully understood (6–8), vaccine development is well underway (9, 10). Two inactivated vaccines have exhibited robust neutralizing antibody (NAb) responses in animals (11, 12), whereas vectored vaccines based on human adenovirus (Ad) type-5/26 and chimpanzee Ad (ChAdOx1) have been tested in nonhuman primates (NHPs) and human trials (13–16). Nucleic acid vaccines have further accelerated the response to the pandemic (17). Both DNA (18–20) and mRNA (21) vaccines have been rapidly developed, with moderate NAb titers observed for the mRNA vaccine in medium and high dose groups (21). A recombinant spike adjuvanted with lipid nanoparticles (NPs), NVX-CoV2373, was reported to elicit high NAb titers in human trials that were on average four-fold greater than in convalescent patients (22, 23). As of now, no vaccine has yet been approved by the U.S. Food and Drug Administration (FDA) for human use.
The SARS-CoV-2 spike protein is a trimer of S1-S2 heterodimers. The S1 subunit contains a receptor-binding domain (RBD) that binds to ACE2 on host cells to initiate infection. The S2 subunit consists of a fusion peptide (FP) and heptad repeat regions 1 and 2 (HR1 and HR2). Upon endocytosis of the virion, the S1 subunit is cleaved off to facilitate the FP insertion into the host membrane, while the remaining S2 refolds to bring HR1 and HR2 together to fuse the viral and host cell membranes (24). The spike protein harbors all NAb epitopes and is the main target for vaccine development against SARS-associated CoVs (25). Convalescent plasma (CP) has been used to treat COVID-19 patients in severe conditions (26), highlighting the importance of NAbs in protection (27). Due to sequence conservation of RBD (but only around 73%), some previously identified NAbs targeting the SARS-CoV-1 RBD have been shown to bind and cross-neutralize SARS-CoV-2 (28, 29). Using single-cell technologies and SARS-CoV-2 RBD or spike as a bait, potent NAbs have now been isolated from COVID-19 patients (30–36). Camelid-derived single-chain NAbs have also been obtained by panning naïve or immune llama single-chain antibody (VHH) libraries (37, 38). Structures of SARS-CoV-2 spike and RBD in unliganded (39, 40), ACE2-bound (41, 42), and antibody-bound (43–45) states determined by x-ray crystallography and cryo-electron microscopy (cryo-EM) have paved the way for rational vaccine design. Cryo-EM and cryo-electron tomography (ET) have revealed the inherent spike metastability and the co-existence of pre/post-fusion spikes on virions (46). A double-proline mutation, S2P (46), has been used in most soluble constructs and all but inactivated vaccines, although a HexaPro version with greater yield and stability is now available (47). Cryo-ET analysis has also uncovered a dynamic, triple-hinged HR2 stalk that facilitates viral entry and immune evasion (48–50).
In this study, we design and optimize SARS-CoV-2 RBD/spike antigens and display them on self-assembling protein nanoparticles (SApNPs) as COVID-19 vaccine candidates. To facilitate vaccine purification, we developed an immunoaffinity column based on antibody CR3022 that binds to both SARS-CoV-1/2 RBDs (29, 44). We first designed a scaffolded RBD trimer to mimic the “RBD-up” spike conformation. For multivalent display, RBDs were attached to SApNPs using the SpyTag/SpyCatcher system (51), providing a robust and practical strategy for generating RBD SApNP vaccines. We probed the spike metastability by comparing two uncleaved spike antigens, S2P (K986P/V987P) and S2G (K986G/V987G). The SARS-CoV-2 S2G spike exhibited abnormal behavior, suggesting that an unidentified facet of the spike can promote conformational change and block antibody access to the RBD. An HR2-deleted spike, S2GΔHR2, produced high-purity trimers, suggesting that the HR2 stalk may be a trigger of spike metastability consistent with recent findings (48–50). We next displayed S2GΔHR2 on ferritin (FR), E2p, and I3-01 SApNPs (52, 53), of which the latter two contain locking domains (LD) and helper T-cell epitopes within the protein shell (54). In mouse immunization, the S2P spike elicited the lowest level of NAb response. In contrast, the scaffolded RBD trimer registered two-to-three-fold higher NAb titers, with another five-fold increase in NAb titer achieved by multivalent display on FR. S2GΔHR2 elicited up to seven-fold higher NAb titers, while the two large, multilayered SApNPs induced up-to-10-fold higher NAb titers compared to S2P. Further analysis indicated that the S2GΔHR2-presenting I3-01v9 SApNP can elicit a strong Th1 response as well as other types of T-cell response needed for protective cellular immunity. Our study thus identifies the HR2 stalk as a major cause of spike metastability, validates an HR2-deleted spike design, and provides a set of RBD- and spike-based virus-like particles (VLPs) as potential effective vaccine candidates against SARS-CoV-2.
RESULTS
Rational design of scaffolded RBD trimer and RBD-presenting SApNPs
RBD binding to the ACE2 receptor initiates the membrane fusion process (5). The crystal structure of SARS-CoV-2 RBD/ACE2 complex revealed atomic details of receptor recognition (55). The SARS-CoV-2 RBD has been used as a bait to isolate monoclonal antibodies (mAbs) from patient samples (30–36). For SARS-CoV-1 and MERS-CoV, RBD-based vaccines have induced potent NAbs that effectively block viral entry (25). Therefore, the RBD represents a major target for the humoral response during viral infection and can be used to develop epitope-focused vaccines.
We first hypothesized that RBD attached to a trimeric scaffold could mimic the “RBD-up” spike conformation and elicit NAbs that block ACE2 binding. To test this possibility, we designed a fusion construct containing SARS-CoV-1/2 RBD, a short 5-aa G4S linker (with a 2-aa restriction site), and a trimeric viral capsid protein, SHP (PDB: 1TD0) (Fig. 1A). Structural modeling showed that the three tethered RBDs form a triangle of 92 Å (measured at L492), which is 14 and 18 Å wider than the SARS-CoV-1 “two-RBD-up” spike (PDB: 6CRX, measured at L478) (56) and the MERS-CoV “all-RBD-up” spike (PDB: 5X59, measured for L506) (57), respectively, allowing NAb access to each RBD. We then developed an immunoaffinity chromatography (IAC) column to facilitate tag-free purification. Previously, NAb-derived IAC columns have been used to purify HIV-1 Env trimers/NPs (52, 53, 58, 59), hepatitis C virus (HCV) E2 cores/NPs (60), and Ebola virus (EBOV) GP trimers/NPs (54). Tian et al. reported that a SARS-CoV-1 NAb, CR3022, can bind SARS-CoV-2 RBD (29). The SARS-CoV-2 RBD/CR3022 structure revealed a conserved cryptic epitope that is shared by the two SARS-CoVs, suggesting that transient breathing motions of the spike protein enable CR3022 binding to RBD (44). Here, we examined the utility of CR3022 in IAC columns. The SARS-CoV-1/2 RBD-5GS-1TD0 constructs were transiently expressed in 100-ml ExpiCHO cells and purified on a CR3022 column prior to size-exclusion chromatography (SEC) using a Superdex 200 10/300 GL column. While the SARS-CoV-1 RBD construct showed both aggregate (∼8.6 ml) and trimer (∼12.7 ml) peaks in the SEC profile, the SARS-CoV-2 RBD construct produced a single, pure trimer peak at ∼12.8 ml (Fig. 1B). In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a monomer band of ∼37 kD and a trimer band of ∼100 kD were observed under reducing and non-reducing conditions, respectively (fig. S1A). Antigenicity was assessed for the two scaffolded RBD trimers in enzyme-linked immunosorbent assay (ELISA) after CR3022/SEC purification (Fig. 1C). RBD-specific NAbs targeting SARS-CoV-1 (CR3022 (61), m396 (62), 80R (63), and S230 (64)) and SARS-CoV-2 (B38 (33), CB6 (32), S309 from a SARS survivor (28), and P2B-2F6 (31)), were tested in ELISA. Overall, similar half maximal effective concentration (EC50) values were observed for the two RBD trimers binding to their respective NAbs (Fig. 1C). The SARS-CoV-1 RBD trimer showed greater affinity for CR3022 than its SARS-CoV-2 counterpart with a 1.3-fold difference in the EC50 value, consistent with previous findings (29, 44). Of the SARS-CoV-2 NAbs, B38 yielded a similar EC50 value to CR3022. Antibody binding kinetics was measured using biolayer interferometry (BLI) (Fig. 1D and fig. 1B). Overall, all tested antibodies exhibited a fast on-rate but with visible differences in their off-rates. B38 showed a faster off-rate than other SARS-CoV-2 NAbs, while CR3022, the antibody used to purify SARS-CoV-1/2 RBD proteins, exhibited a comparable kinetic profile.
We then hypothesized that the SpyTag/SpyCatcher (or simply SPY) system can be used to conjugate RBD to SApNPs to create multivalent RBD vaccines capable of eliciting a more potent NAb response (Fig. 1E). The 13-aa SpyTag spontaneously reacts with the SpyCatcher protein to form an irreversible isopeptide bond (51). The SPY system has been successfully used to attach antigens to VLPs (65). Here, SpyTag was fused to the C terminus of RBD, while SpyCatcher was fused to the N terminus of an SApNP subunit, both with a 5-aa G4S linker. This design was first tested for the 24-meric ferritin (FR) used in our previous studies (52–54, 60). We compared two production strategies – co-expression of RBD-5GS-SpyTag and SpyCatcher-5GS-FR versus supernatant mix after separate expression – and performed purification on a CR3022 column. Protein obtained from transient transfection in 50-ml ExpiCHO cells was analyzed by SEC on a Superose 6 10/300 GL column (Fig. 1F). Both production strategies produced a peak (12 ml) corresponding to SApNPs. While the SARS-CoV-2 construct outperformed its SARS-CoV-1 counterpart in particle yield (0.6-1.0 mg versus 0.3-0.5 mg after CR3022/SEC), the supernatant mix appeared to be superior to co-expression for yield in both cases. Nonetheless, the results suggest that both strategies can be used to produce RBD SApNPs in Good Manufacturing Practice (GMP)-compatible Chinese hamster ovary (CHO) cells. Antigenicity was assessed for SEC-purified RBD-5GS-SPY-5GS-FR SApNPs. In ELISA, RBD SApNPs showed slightly improved mAb binding compared to the RBD trimers, as indicated by lower EC50 values (Fig. 1G). In BLI, a more pronounced effect of multivalent display on antigenicity was observed, showing notably increased binding signals and plateaued dissociation (Fig. 1H and fig. 1C). Structural integrity of various RBD SApNPs was analyzed by negative stain EM (nsEM) (Figs. 1I and 1J). For SARS-CoV-1, an RBD-10GS-FR construct was included for comparison that produced very few SApNPs (Fig. 1I, left). In contrast, the RBD-5GS-SPY-5GS-FR construct produced a high yield of SApNPs with visible surface decorations (Fig. 1I, right). For SARS-CoV-2, the purified RBD-5GS-SPY-5GS-FR SApNPs, irrespective of the production strategy, showed morphologies corresponding to well-formed nanoparticles (Fig. 1J). Following a similar strategy, SARS-CoV-1/2 RBDs were also attached to a multilayered I3-01v9 SApNP (54) (Fig. 1K). Despite the modest yield (Fig. 1L), large SApNPs were readily observed in the EM images (Fig. 1M). In summary, we illustrate the utility of the SPY system for rapid development of RBD-based SApNP vaccines. Compared to the recently reported two-component RBD SApNPs (66), the SPY-linked RBD SApNPs presented here may be more advantageous in terms of stability and manufacturability.
Rational design of prefusion spike through minimizing metastability
In addition to the RBD, the SARS-CoV-1/2 spikes contain other NAb epitopes (25), which are all presented in a trimeric context (Fig. 2A). A double-proline mutation (2P) between HR1 and the central helix (CH) has been used to stabilize the MERS-CoV (67) and SARS-CoV-1 spikes (56). A similar 2P mutation (K986P/V987P) was introduced into the SARS-CoV-2 spike (termed S2P), which has been used to isolate and characterize NAbs (28, 30, 35, 37–40, 43) and is the antigen in almost all current vaccine candidates (13–16, 18–23). However, a recent cryo-EM study revealed an unexpected packing of S1 in the S2P spike, positioned ∼12Å outwards, compared to the full-length native spike, as well as a more ordered FP proximal region (FPPR) in S2 (46). New designs have been generated to control the spike conformation (68) or to further stabilize it with more prolines (HexaPro) (47). Recent cryo-EM and cryo-ET studies revealed diverse spike orientations on native virions due to the highly flexible HR2 stalk (48–50). Previously, we identified an HR1 bend as the cause of HIV-1 Env metastability (52, 69) and probed the contribution of an equivalent HR1 bend and the HR2 stalk to EBOV GP metastability (54) to facilitate rational vaccine design. It is therefore imperative to understand SARS-CoV-2 spike metastability, and then design an optimized spike as a vaccine immunogen.
We first created His-tagged, uncleaved spike ectodomain (SECTO) constructs for SARS-CoV-1/2, both containing the 2P mutation (K986P/V987P) and a trimerization motif (1TD0) fused to the C terminus with a 5-aa G4S linker. The two constructs were transiently expressed in 50-ml ExpiCHO cells followed by purification on either a Nickel column or a CR3022 column. The S2PECTO-5GS-1TD0-His6 protein was characterized by SEC on a Superose 6 10/300 GL column (Fig. 2B, panels 1 and 2). After the Nickel column, both S2PECTO constructs showed a trimer peak (∼12 ml) with shoulders to the left and right indicative of aggregate and dimer/monomer species, respectively. CR3022 purification resulted in a consistent trimer peak and less dimer/monomer species. We then tested a pair of SECTO constructs containing a double glycine mutation (K986G/V987G, termed 2G). The 2G mutation had little effect on the SARS-CoV-1 spike but produced abnormal SEC profiles and showed no yield for the SARS-CoV-2 spike after purification by Nickel and CR3022 columns, respectively (Fig. 2B, panels 3 and 4). Lastly, we tested a pair of S2G variants without the HR2 stalk (E1150-Q1208), termed S2GΔHR2. Deletion of the HR2 stalk restored the SARS-CoV-2 trimer peak and reduced aggregates for both SARS-CoVs, as shown by the SEC profiles upon CR3022 purification (Fig. 2B, panel 5). Since the triple-hinged HR2 stalk can generate diverse spike orientations on native virions (48–50), and the fusion core is formed by HR1 and HR2, we hypothesized that HR2 may be a key determinant of SARS-CoV-2 spike metastability (Fig. 2C, left). It is possible that the interactions between HR1 and HR2 of two neighboring spikes may facilitate the pre-to-post-fusion transition in addition to ACE2 binding and S1 dissociation. Given the extensive sequence difference in HR1 (9 amino acids in total) compared to SARS-CoV-1 (Fig. 2C, right), we sought to examine the role of HR1 in SARS-CoV-2 spike metastability with two HR1-swapped spike constructs. Interestingly, while HR1 swapping proved ineffective, deletion of the HR2 stalk once again restored the trimer peak (fig. S2, A to C). Therefore, S2GΔHR2 appeared to provide a general spike design for SARS-CoV-1/2 and perhaps other CoVs. Four separate production runs of SARS-CoV-2 S2GΔHR2-5GS-1TD0 in 300-ml ExpiCHO cells resulted in nearly identical SEC profiles with a trimer yield of 0.8-1.0 mg (Fig. 2D, left). Blue native polyacrylamide gel electrophoresis (BN-PAGE) confirmed the purity of the S2GΔHR2 spike across SEC fractions (Fig. 2D, right). Antigenicity was assessed for freshly produced SARS-CoV-2 S2PECTO and S2GΔHR2 spike proteins. In ELISA, the S2GΔHR2 spike showed slightly higher affinity for the five representative mAbs than did the S2PECTO spike (Fig. 2E). When tested against three newly identified human NAbs, C105 (43) and CC12.1/CC12.3 (36), the two spikes yielded similar EC50 values (fig. S2D). In BLI, the S2GΔHR2 spike showed higher binding signals than the S2PECTO spike at the highest concentration, while exhibiting similar binding kinetics (Fig. 2F). The use of NAb P2B-2F6 (31) for spike purification resulted in higher trimer yield with similar purity to the CR3022 column across SEC fractions (fig. S2E). Altogether, we demonstrated that deletion of the HR2 stalk can improve spike properties and S2GΔHR2 may be a more effective spike antigen for vaccine development.
Rational design of single-component, multilayered, self-assembling spike nanoparticles
Although it was possible to conjugate trimeric SARS-CoV-2 spikes to an SApNP using the SPY system (70), the random, irreversible chemical linking will likely result in irregular display with unoccupied but spatially occluded anchoring sites on the surface. The SPY system is perhaps more suitable for small individual antigens, such as the RBD. Using gene fusion, we previously designed single-component SApNPs displaying stabilized HIV-1 Env trimers (52, 53) and optimized HCV E2 cores (60). Recently, we further engineered the E2p and I3-01v9 60-mers to incorporate locking domains (LDs) and helper T-cell epitopes into the constructs to create highly stable, multilayered SApNPs as multivalent carriers to develop VLP-type vaccine immunogens (54).
Native SARS-CoV-2 virions present both pre- and post-fusion spikes on the surface (46, 48, 49) (Fig. 3A, top). Here, our vaccine strategy aimed to develop single-component, multilayered SApNPs that each present 8 or 20 stable S2GΔHR2 spikes to the immune system (Fig. 3A, bottom). To explore this possibility, we modeled the S2GΔHR2 spike on ferritin (FR) with a 5-aa G4S linker, on E2p with a 5-aa G4S linker, and on I3-01v9 with a 10-aa (G4S)2 linker, resulting in SApNPs with diameters of 47.9 nm, 55.9 nm, and 59.3 nm, respectively (Fig. 3B). The three S2GΔHR2 SApNP constructs were transiently expressed in 400-ml ExpiCHO cells, followed by CR3022 purification and SEC on a Superose 6 10/300 GL column (Fig. 3C). Three production runs generated highly consistent SEC profiles for all three constructs, despite the variation of low-m.w. impurities observed for the FR and E2p SApNPs. Following CR3022/SEC purification, we obtained on average 0.3-0.4 mg, 0.15-0.25 mg, and 0.3-0.35 mg SApNP for S2GΔHR2-5GS-FR, S2GΔHR2-5GS-E2p-LD4-PADRE (or E2p-L4P (54)), and S2GΔHR2-10GS-I3-01v9-LD7-PADRE (or I3-01v9-L7P (54)), respectively. Overall, S2GΔHR2-10GS-I3-01v9-L7P appeared to perform best in terms of particle yield, purity and stability in production. The structural integrity of CR3022/SEC-purified SApNPs was further characterized by nsEM, which showed well-formed particles of 45-65 nm with spikes easily recognizable on their surface (Fig. 4D). Antigenicity of S2GΔHR2-presenting SApNPs was assessed using the same antibody panel as above. In ELISA, three SApNPs showed slightly improved binding to some, but not all, antibodies compared to the individual spike (Fig. 4E). In BLI assays, we observed a clear correlation between peak antibody-binding signal and antigen valency, with E2p/I3-01v9>FR>spike (Fig. 4F). Multivalent display on the two 60-mers significantly improved antibody binding compared to the 24-mer. In previous studies, we observed a similar correlation for HIV-1 gp140 trimer vs. gp140 SApNPs (52) and HCV E2 core vs. E2 core SApNPs (60). In summary, these VLP-size SApNPs with 8 or 20 spikes on the surface provide promising vaccine candidates for in vivo evaluation.
SARS-CoV-1/2 vaccine-induced binding antibody response
Selected SARS-CoV-1/2 RBD- and spike-based immunogens were evaluated in BALB/c mice to evaluate vaccine-induced antibody responses (Fig. 4A). Groups of five mice were immunized four times at three-week intervals. All vaccine antigens were formulated with AddaVax, an oil-in-water emulsion adjuvant (54), except for I3-01v9, which was formulated with aluminum phosphate (AP) (71). We first performed a longitudinal analysis of binding antibody response, as measured by half maximal effective dilution (ED50), in the two SARS-CoV-2 RBD vaccine groups (Fig. 4B and fig. S4). The RBD SApNP (RBD-5GS-SPY-5GS-FR) elicited significantly higher ED50 titers than the scaffolded RBD trimer (RBD-5GS-1TD0) at w2 and w5, irrespective of the coating antigen, with a P value of 0.0009 at w8 when RBD was coated. Compared to the stabilized spike (S2GΔHR2-5GS-1TD0), the RBD SApNP elicited significantly higher ED50 titers against the RBD at w2, w5, and w8 (Fig. 4B, right), demonstrating a strong “epitope-focusing” effect. Mouse sera bound the SARS-CoV-1 spike with lower ED50 titers than the SARS-CoV-2 spike but with similar patterns (fig. S4A). We then performed a longitudinal analysis of binding antibody response induced by two SARS-CoV-2 spikes, S2PECTO-5GS-1TD0 and S2GΔHR2-5GS-1TD0, and three SApNPs each displaying 8 or 20 S2GΔHR2 spikes (Fig. 4C and fig. S5). The S2GΔHR2 spike elicited 2∼3-fold higher average ED50 titers than the S2PECTO spike irrespective of the coating antigen, showing greater immunogenicity (of note, to facilitate a fair comparison, mouse sera from the two spike groups were tested against their respective spikes). Three SApNPs exhibited different temporal patterns depending on the coating antigen. Using spike as the coating antigen, the I3-01v9 group showed a steady increase in average ED50 titer over time, with the highest average ED50 titers at two time pints, w2 and w8, and significantly outperforming the S2PECTO spike at all time points. The I3-01v9 group also showed higher ED50 tiers than the S2GΔHR2 group throughout, although not with significant P values. The smaller FR SApNP exhibited a similar temporal pattern with lower average ED50 titers, but still significantly higher than the S2PECTO group. Among the three SApNPs, E2p registered the lowest average ED50 titer at w2 and reached the highest at w5, which then decreased slightly at w8. In terms of the RBD-specific response, the five groups showed a clear ranking based on their average ED50 titers, which remained consistent across time points. At w2, I3-01v9 elicited an average ED50 titer of 175, whereas all other spike-based vaccine groups showed little RBD-specific response. At w5 and w8, S2GΔHR2 elicited higher ED50 titers (on average by 2-fold) than S2PECTO, while all three SApNPs outperformed the individual S2GΔHR2 spike with a ranking of ED50 titers correlated with their size (FR<E2p<I3-01v9). Sera reacted with the SARS-CoV-1 spike similarly, albeit at a lower level (fig. S5A). Lastly, we compared binding antibody responses induced by three SARS-CoV-1 immunogens – S2PECTO spike (S2PECTO-5GS-1TD0), scaffolded RBD trimer (RBD-5GS-1TD0), and RBD SApNP (RBD-5GS-SPY-5GS-FR) (Fig. 4D and fig. S6). Based on the ED50 values, the SARS-CoV-1 S2PECTO spike appeared to be more immunogenic than the SARS-CoV-2 S2GΔHR2 spike, whereas the SARS-CoV-1 RBD SApNP was less advantageous in ED50 titer than its SARS-COV-2 counterpart. Serum reactivity with the SARS-CoV-2 S2PECTO spike was observed for all three SARS-CoV-1 vaccine groups (fig. S6A). In summary, RBD SApNPs can elicit RBD-specific antibody titers at a similar or higher level compared to the spike. Furthermore, the S2GΔHR2 spike appears to be more immunogenic than the widely used S2PECTO spike, in addition to its superior in-vitro properties. The multilayered E2p and I3-01v9 SApNPs are the best performers among the spike-based vaccines, consistent with our previous HIV-1, HCV, and EBOV SApNP vaccine studies (52, 54, 60).
SARS-CoV-1/2 vaccine-induced NAb response
One major goal in COVID-19 vaccine development is to generate a potent NAb response that can protect against SARS-CoV-2 infection. Pseudoparticle (SARS-CoV-1/2-pp) neutralization assays (72) were used to evaluate serum NAb responses elicited by different vaccine candidates. We first performed a longitudinal analysis of NAb response, as measured by half maximal inhibitory dilution (ID50), in the two SARS-CoV-2 RBD vaccine groups (Fig. 5A and fig. S7). The RBD SApNP elicited a NAb response against autologous SARS-CoV-2 as early as w2, albeit with low titers, and retained its advantage at the two later time points, suggesting that such RBD SApNP vaccines can elicit a rapid NAb response. The scaffolded RBD trimer group showed the lowest average ID50 titer at w5 but a NAb response comparable to that induced by the stabilized S2GΔHR2 spike at w8. A somewhat different pattern was observed in the SARS-CoV-1-pp assay. At w2, no vaccine group showed detectable heterologous NAb response. At w5 and w8, the S2GΔHR2 spike elicited a more potent SARS-CoV-1 NAb response than both RBD-based vaccines, suggesting that non-RBD epitopes may contribute to cross-neutralization. We then analyzed the NAb response induced by five spike-based vaccines longitudinally (Fig. 5B and fig. S8). In terms of autologous neutralization (Fig. 5B, upper panel), no spike-based vaccine elicited any SARS-CoV-2-pp NAb response at w2 after one injection. But a consistent pattern was observed for serum neutralization at w5 and w8: the S2PECTO spike showed the lowest average ID50 titers, 879 and 2481 at w5 and w8, respectively, whereas the S2GΔHR2 spike induced a stronger NAb response with 2.8-6.7-fold higher average ID50 titers, which did not reach P ≤ 0.05 due to within-group variation. Nonetheless, this result confirmed the beneficial effect of the 2P-to-2G substitution and deletion of the HR2 stalk on NAb elicitation; among the three SApNPs, E2p was the best performer at w5, showing an average ID50 titer of 8435 that is 9.6-fold higher than S2PECTO and 1.4-fold higher than S2GΔHR2, while I3-01v9 showed the most potent NAb response at w8 with an average ID50 titer of 17351 that is 7-fold and 2.5-fold higher than S2PECTO and S2GΔHR2, respectively. A similar temporal pattern was observed in the heterologous SARS-CoV-1-pp assay (Fig. 5B, lower panel). It is worth noting that the I3-01v9 SApNP elicited a SARS-CoV-1 NAb response with an average ID50 titer of 351 at w2, whereas all other groups showed no detectable serum neutralization. Nonetheless, these results suggest that the SARS-CoV-2 S2GΔHR2-based vaccines, particularly SApNPs, may provide protection against both SARS-CoV-1/2. Lastly, we performed a longitudinal analysis of NAb response for three SARS-CoV-1 vaccines (Fig. 5C and fig. S9). In the autologous SARS-CoV-1-pp assay, the S2PECTO spike and RBD SApNP induced significantly more potent NAb responses than the scaffolded RBD trimer at w2 and w5 and all three vaccine groups showed similar ID50 titers at w8. However, heterologous SARS-CoV-2 neutralization was below or at the baseline level for three SARS-CoV-1 vaccines at w2, w5, and w8. The pseudovirus neutralization assay has been validated using a panel of known SARS-CoV-1/2 NAbs (fig. S9C). As a control, the w8 mouse sera were tested against pseudoparticles bearing the murine leukemia virus (MLV) Env, MLV-pps, and did not show non-specific neutralization (fig. S9D). In summary, these results demonstrate an advantage in NAb elicitation by the S2GΔHR2 spike and S2GΔHR2-presenting SApNPs compared to the S2PECTO spike. Although SARS-CoV-2 RBD and S2GΔHR2 SApNPs are both effective at eliciting NAb responses that target SARS-CoV-2, S2GΔHR2 SApNPs may provide broader protection against SARS-associated CoVs.
SARS-CoV-2 vaccine-induced T-cell response
While humoral immunity is required to block host-virus interaction and prevent viral infection, cellular immunity is essential for eliminating infected host cells to control viral infection (73–76). Emerging evidence indicates that an early T-cell response (77, 78), as well as T-cell memory (79), is critical for protection against SARS-CoV-2. However, COVID-19 vaccines must induce a CD4+ T helper 1 (Th1), but not Th2-type, T-cell response, as the latter has been linked to vaccine-associated enhancement of respiratory disease (VAERD) (10). In addition, T follicular helper cells (Tfh) play an important role in the maturation and production of NAbs. Therefore, understanding T-cell responses is crucial for development of an effective and safe COVID-19 vaccine.
Interferon (IFN)-γ-producing Th1 cells are important for generating an optimal antibody response and for induction of cellular immunity (73–75). We first examined various SARS-CoV-2 vaccine formulations on induction of CD4+ Th1 responses specific to the vaccine antigen at w11, two weeks after the fourth immunization, when memory T cells had already developed in spleen (76). Mouse splenocytes from the S2P group and two SApNP groups (E2p and I3-01v9) were analyzed by flow cytometry using naïve samples as a negative control. The I3-01v9 group induced about 1.5- and 2.3-fold higher frequency of IFN-γ-producing CD4+ Th1 cells than the S2P and E2p groups, respectively (Fig. 6A). Notably, following re-stimulation with the respective antigens for as few as 4 hours, both E2p and I3-01v9 groups produced ∼2-fold higher frequency of CD107a-producing cytolytic CD4+ T cells than the S2P and naïve groups (Fig. 6B). IFN-γ/IL-4 (interleukin-4) double-positive cells are memory CD4+ T cells that have acquired the ability to produce IL-4 while still retaining the ability to produce IFN-γ under Th1 conditions (80). It appeared that I3-01v9 induced 3- and 5-fold more IFN-g/IL-4 double-positive memory CD4+ T cells than S2P and E2p (Fig. 6A). These results suggest that I3-01v9 can induce both CD4+ Th1 cells and IFN-γ/IL-4 double-positive memory CD4+ T cells. In addition, I3-01v9 induced more IFN-γ/GM-CSF (granulocyte-macrophage colony-stimulating factor) double-positive CD8+ effector T cells than S2P and E2p (Fig. 6C), suggesting that protective CD8+ T cell responses were also generated in mice immunized with I3-01v9. Of note, CD8+ T cells derived from mice immunized with I3-01v9, rather than those with S2P and E2p, acquired the ability to rapidly produce IFN-γ upon antigen re-stimulation (Fig.6D), suggesting generation of I3-01v9-responsive effector/memory T cells. These findings indicate that the S2GΔHR2 I3-01v9 SApNP can induce robust T-cell responses consisting of CD4+ Th1 cells, IFN-γ/IL-4 double-positive memory CD4+ T cells, and effector CD8+ T cells, thus providing protective cellular immunity in addition to a potent NAb response. Since T cell immunity against the SApNP backbone cannot be ruled out, a more detailed T-cell analysis using spike antigens, SApNP backbones, and peptides for re-stimulation may be warranted.
DISCUSSION
COVID-19 marks the first worldwide pandemic of this scale since the infamous Spanish influenza over a century ago (81), which caused ∼50 million deaths and remains a painful reminder of our vulnerability to a new virus without a protective vaccine. Therefore, the rapid spread of SARS-CoV-2 demands rapid vaccine development (10). Operation Warp Speed (OWS) aims to deliver 300 million doses of safe and effective vaccines by January 2021 through a public-private partnership, which has resulted in five vaccine candidates (82) in addition to others being tested in human trials. However, vaccine development during a pandemic against a new virus poses unique challenges, one of which is how to balance public health need and scientific rigor (83–85). The global vaccine campaign also provides a unique opportunity to compare different vaccine design strategies and platforms – especially new ones – against a common target.
Here, we approached SARS-CoV-2 vaccine development with a rational design strategy. First, the choice of antigen is key to success of a vaccine irrespective of the delivery platform. Most vaccine antigens including OWS’s vaccine candidates are based on S2P, which produces a spike structure that differs in detail from the full-length wild-type spike, e.g. in FPPR of S2 and in the relative dispositions of the S1 domains (46). These differences may complicate interpretation of vaccine outcome. S2P and other empirical spike designs (47) have attempted to constrain the spike conformation and increase trimer yield. However, as we previously found for HIV-1 Env and EBOV GP (52, 54, 69), it is important to identify and eliminate (if possible) the root cause of spike metastability. During antigen screening, we found that deletion of the HR2 stalk with a 2P- to-2G substitution renders a more stable spike, which is consistent with recent reports on a highly flexible HR2 stalk in the native spikes on SARS-CoV-2 virions (48–50). Thus, S2GΔHR2 would seem to present an advance on spike design. Second, single-component SApNPs provide a new, powerful platform for VLP-type vaccine development against diverse viral pathogens (52, 54, 60). S2GΔHR2 was genetically fused, rather than chemically linked, to three SApNPs, including two multilayered SApNPs with enhanced stability and an embedded T-help signal. Such recombinant protein vaccines should be more effective in eliciting a potent anti-SARS-CoV-2 NAb response and less likely to induce adverse responses (86). An epitope-focused vaccine strategy was also explored by designing scaffolded RBD trimers and RBD-presenting SApNPs. Third, to achieve high efficacy and ensure safety, vaccine-induced NAb and T-cell responses must be evaluated in animals prior to clinical trials. Indeed, in our mouse study, the S2GΔHR2 spike appeared to be more effective than the S2P spike in NAb elicitation, both alone and displayed on SApNPs. Of note, the S2GΔHR2-presenting I3-01v9 SApNP elicited not only high NAb titers but also desired T-cell responses. In addition to viral antigen-responsive CD4+ Th1 cells and memory CD4+ T cells, the I3-01v9 SApNP also induced CD107a-producing cytolytic CD4+ T cells, which may directly kill infected host cells, and GM-CSF-producing CD8+ effector T cells, which may promote the generation of macrophages and functional dendritic cells (DCs) to facilitate the clearance of infected cells. Lastly, expression of vaccine antigens in GMP-compatible CHO cells followed by purification using an antibody column, such as CR3022, would allow rapid and industrial-scale vaccine production. In summary, our study provides promising next-generation COVID-19 vaccine candidates that are ready for evaluation in human trials.
MATERIALS AND METHODS
Design, expression and purification of SARS-CoV-2 RBD and spike antigens
The spike (S) genes of the SARS-CoV-1 isolate Tor2 (GenBank accession #: NC_004718) and the SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank accession #: MN908947) were used to design all the RBD and spike constructs following codon-optimization for expression in mammalian cells. The RBD sequence is defined as P317-D518 and P330-N532 for SARS-CoV-1 and 2, respectively. The SECTO sequence is defined as M1-Q1190 and M1-Q1208 for SARS-CoV-1 and 2, respectively. To remove the S1/S2 cleavage site, an R667G mutation and a 682GSAGSV687 modification were introduced in the SARS-CoV-1 and 2 spikes, respectively. The 2P (or 2G) mutation was made to K968/V969 and K986/V987 in the SARS-CoV-1 and 2 spikes, respectively. The SARS-CoV-2 C-terminal region (E1150-Q1208) containing the HR2 stalk was removed from S2GECTO, resulting in an HR2-deleted spike construct termed S2GΔHR2. The viral capsid protein SHP (PDB: 1TD0) was used as a trimerization motif in spike constructs for immunization, whereas the foldon domain from the bacteriophage T4 fibritin (PDB: 1RFO) was used in coating spike antigens for ELISA to mask the 1TD0-derived antibody response. All constructs were transiently expressed in ExpiCHO cells (Thermo Fisher). Briefly, ExpiCHO cells were thawed and incubated with ExpiCHOTM Expression Medium (Thermo Fisher) in a shaker incubator at 37 °C, 135 rpm and 8% CO2. When the cells reached a density of 10×106 ml−1, ExpiCHOTM Expression Medium was added to reduce cell density to 6×106 ml−1 for transfection. The ExpiFectamineTM CHO/plasmid DNA complexes were prepared for 100-ml transfection in ExpiCHO cells following the manufacturer’s instructions. For a given construct, 100 μg of plasmid and 320 μl of ExpiFectamineTM CHO reagent were mixed in 7.7 ml of cold OptiPRO™ medium (Thermo Fisher). After the first feed on day one, ExpiCHO cells were cultured in a shaker incubator at 33 °C, 115 rpm and 8% CO2 following the Max Titer protocol with an additional feed on day five (Thermo Fisher). Culture supernatants were harvested 13 to 14 days after transfection, clarified by centrifugation at 4000 rpm for 25 min, and filtered using a 0.45 μm filter (Thermo Fisher). The CR3022 antibody column was used to extract SARS-CoV-1/2 antigens from the supernatants, which was followed by SEC on a Superdex 200 10/300 GL column (for scaffolded RBD trimer) or a Superose 6 10/300 GL column (for RBD-SPY-SApNPs, spikes, and spike-presenting SApNPs). For comparison, His-tagged SECTO-5GS-1TD0 spike protein was extracted from the supernatants using an immobilized Ni SepharoseTM Excel column (GE Healthcare) and eluted with 500 mM Imidazole prior to SEC. Protein concentration was determined using UV280 absorbance with theoretical extinction coefficients.
Blue native polyacrylamide gel electrophoresis
SARS-CoV-2 spikes and spike-presenting SApNPs were analyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE) and stained with Coomassie blue. The proteins were mixed with sample buffer and G250 loading dye and added to a 4-12% Bis-Tris NativePAGETM gel (Life Technologies). BN-PAGE gels were run for 2 to 2.5 hours at 150 V using the NativePAGETM running buffer (Life Technologies) according to the manufacturer’s instructions.
Enzyme-linked immunosorbent assay
Each well of a CostarTM 96-well assay plate (Corning) was first coated with 50 µl PBS containing 0.2 μg of the appropriate antigens. The plates were incubated overnight at 4 °C, and then washed five times with wash buffer containing PBS and 0.05% (v/v) Tween 20. Each well was then coated with 150 µl of a blocking buffer consisting of PBS, 40 mg ml−1 blotting-grade blocker (Bio-Rad), and 5% (v/v) FBS. The plates were incubated with the blocking buffer for 1 hour at room temperature, and then washed five times with wash buffer. For antigen binding, antibodies were diluted in the blocking buffer to a maximum concentration of 5 μg ml−1 followed by a 10-fold dilution series. For each antibody dilution, a total of 50 μl volume was added to the appropriate wells. For mouse sample analysis, serum or plasma was diluted by 20-fold in the blocking buffer and subjected to a 10-fold dilution series. For each sample dilution, a total of 50 μl volume was added to the wells. Each plate was incubated for 1 hour at room temperature, and then washed 5 times with PBS containing 0.05% Tween 20. For antibody binding, a 1:5000 dilution of goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Inc), or for mouse sample analysis, a 1:3000 dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories), was then made in the wash buffer (PBS containing 0.05% Tween 20), with 50 μl of this diluted secondary antibody added to each well. The plates were incubated with the secondary antibody for 1 hour at room temperature, and then washed 5 times with PBS containing 0.05% Tween 20. Finally, the wells were developed with 50 μl of TMB (Life Sciences) for 3-5 min before stopping the reaction with 50 μl of 2 N sulfuric acid. The resulting plate readouts were measured at a wavelength of 450 nm. Of note, the w2 serum binding did not reach the plateau (or saturation) to allow for accurate determination of ED50 titers. Nonetheless, the ED50 values at w2 were derived by setting the lower/upper constraints of OD450 at 0.0/3.2 to facilitate the comparison of different vaccine groups at the first time point.
Bio-layer interferometry
The kinetics of SARS-CoV-1/2 vaccine antigens, RBD versus RBD-presenting SApNPs as well as spike versus spike-presenting SApNPs, binding to a panel of known antibodies was measured using an Octet RED96 instrument (FortéBio, Pall Life Sciences). All assays were performed with agitation set to 1000 rpm in FortéBio 1× kinetic buffer. The final volume for all the solutions was 200 μl per well. Assays were performed at 30 °C in solid black 96-well plates (Geiger Bio-One). For all antigens with the exception of S2GΔHR2-NPs, 5 μg ml−1 of antibody in 1× kinetic buffer was loaded onto the surface of anti-human Fc Capture Biosensors (AHC) for 300 s. For S2GΔHR2-NPs, anti-human Fc Quantitation Biosensors (AHQ) were used. A 60 s biosensor baseline step was applied prior to the analysis of the association of the antibody on the biosensor to the antigen in solution for 200 s. A two-fold concentration gradient of antigen, starting at 950 nM for scaffolded RBD trimers, 37 nM for RBD-5GS-SPY-5GS-FR SApNP, 150 nM for spike trimers, and 9/3.5/3.5 nM for S2GΔHR2 presented on FR/E2p/I3-01v9 SApNPs, was used in a titration series of six. The dissociation of the interaction was followed for 300 s. Correction of baseline drift was performed by subtracting the mean value of shifts recorded for a sensor loaded with antibody but not incubated with antigen and for a sensor without antibody but incubated with antigen. Octet data were processed by FortéBio’s data acquisition software v.8.1. Experimental data were fitted with the binding equations describing a 2:1 interaction to achieve optimal fitting. Of note, S2GΔHR2 trimer binding was also measured using AHQ to facilitate the comparison of antibody binding with S2GΔHR2-presenting SApNPs.
Electron microscopy (EM) assessment of nanoparticle constructs
The initial EM analysis of RBD and S2GΔHR2-presenting SApNPs was conducted at the Core Microscopy Facility at The Scripps Research Institute. Briefly, SApNP samples were prepared at the concentration of 0.01 mg/ml. Carbon-coated copper grids (400 mesh) were glow-discharged and 8 µL of each sample was adsorbed for 2 min. Excess sample was wicked away and grids were negatively stained with 2% uranyl formate for 2 min. Excess stain was wicked away and the grids were allowed to dry. Samples were analyzed at 80 kV with a Talos L120C transmission electron microscope (Thermo Fisher) and images were acquired with a CETA 16M CMOS camera.
Animal immunization and sample collection
Similar immunization protocols have been reported in our previous SApNP vaccine studies. Briefly, the Institutional Animal Care and Use Committee (IACUC) guidelines were followed with animal subjects tested in the immunization study. Eight-week-old BALB/c mice were purchased from The Jackson Laboratory and housed in ventilated cages in environmentally controlled rooms at The Scripps Research Institute, in compliance with an approved IACUC protocol and AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International guidelines. Mice were immunized at weeks 0, 3, 6, and 9 with 200 μl of antigen/adjuvant mix containing 50 μg of vaccine antigen and 100 μl of adjuvant, AddaVax or Adju-Phos (InvivoGen), via the intraperitoneal (i.p.) route. Blood was collected two weeks after each immunization. All bleeds were performed through the retro-orbital sinus using heparinized capillary tubes into EDTA-coated tubes. Samples were diluted with an equal volume of PBS and then overlaid on 4.5 ml of Ficoll in a 15 ml SepMateTM tube (STEMCELL Technologies) and spun at 1200 RPM for 10 min at 20 °C to separate plasma and cells. The plasma was heat inactivated at 56 °C for 30 min, spun at 1200 RPM for 10 min, and sterile filtered. The cells were washed once in PBS and then resuspended in 1 ml of ACK Red Blood Cell lysis buffer (Lonza). After washing with PBS, peripheral blood mononuclear cells (PBMCs) were resuspended in 2 ml of Bambanker Freezing Media (Lymphotec). Spleens were also harvested and ground against a 70-μm cell strainer (BD Falcon) to release the splenocytes into a cell suspension. Splenocytes were centrifuged, washed in PBS, treated with 5 ml of ACK lysing buffer (Lonza), and frozen with 3ml of Bambanker freezing media. Sera were heat inactivated for ELISA binding and pseudovirus neutralization assays.
SARS-CoV-1/2 pseudovirus neutralization assay
Pseudoparticle (SARS-CoV-1/2-pp) neutralization assays were utilized to assess the neutralizing activity of previously reported antibodies and vaccine-induced murine antibody response. SARS-CoV-1/2-pps were generated by co-transfection of HEK293T cells with the HIV-1 pNL4-3.lucR-E-plasmid (obtained from the NIH AIDS reagent program: https://www.aidsreagent.org/) and the expression plasmid encoding the S gene of SARS-CoV-1 isolate Tor2 (GenBank accession #: NC_004718) and the SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank accession #: MN908947) at a 4:1 ratio by lipofectamine 3000 (Thermo Fisher Scientific). After 48 to 72 hours, SARS-CoV-1/2-pps were collected from the supernatant by centrifugation at 4000 rpm for 10 min, aliquoted, and stored at −80 °C before use. The mAbs at a starting concentration of 0.1-10 μg/ml, or mouse serum at a starting dilution of 100-fold, were mixed with the supernatant containing SARS-CoV-1/2-pps and incubated for 1 hour at 37°C in white solid-bottom 96-well plate (Corning). A 3-fold dilution series was used in the assay. The HEK293T-hACE2 cell line (catalogue#: NR-52511) and the vector pcDNA3.1(-) containing the SARS-CoV-2 S gene (catalogue#: NR52420) were obtained from BEI RESOURCES (https://www.beiresources.org/) and used in pseudovirus neutralization assays (72). Briefly, HEK293T-hACE2 cells at 1×104 were added to each well and the plate was incubated at 37°C for 48 hours. After incubation, overlying media was removed, and cells were lysed. The firefly luciferase signal from infected cells was determined using the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. Data were retrieved from a BioTek microplate reader with Gen 5 software, the average background luminescence from a series of uninfected wells was subtracted from each well, and neutralization curves were generated using GraphPad Prism 8.4.3, in which values from wells were compared against a well containing SARS-CoV-1/2-pp only. Due to the difference in mouse samples (serum: Pre, w2, w5, and w8; plasma: w11) and the sensitivity of SARS-CoV-2-pp assays, the w11 Nab responses were not compared but included in figs. S7-S9 for the sake of completeness. The same HIV-1 vectors pseudotyped with the murine leukemia virus (MLV) Env gene, termed MLV-pps, were produced in HEK293T cells and included in the neutralization assays as a negative control.
Dendritic cell (DC) production
Mouse bone marrow (BM) was cultured in RPMI 1640 medium containing 10% fetal bovine serum and recombinant mouse Flt3L (50 ng/mL) and SCF (10 ng/ml) for 9 days as described (87). To induce DC activation, immature DCs were incubated with lipopolysaccharide (LPS, 100 ng/mL), R848 (Resiquimod, 100 ng/mL) or CpG (ODN 1585, 1µM) overnight, which activated Toll-like receptor (TLR)4, TLR7/8 or TLR9 signaling, respectively. Cells were harvested for experiments. pDCs were sorted to isolate CD11c+B220+ cells using FACS cell sorter and magnetic beads (Miltenyi-Biotech, CA).
Antibodies and flow cytometry analysis
All antibodies used for immunofluorescence staining were purchased from eBioscience (San Diego, CA), BioLegend (San Diego, CA) or BD Biosciences (San Jose, CA). Magnetic microbead-conjugated Abs and streptavidin were purchased from Miltenyi-Biotech (Auburn, CA). Recombinant human IL-2 protein was purchased from R&D Systems (Minneapolis, MN). Recombinant mouse Flt3 ligand (Flt3L) and mouse SCF were purchased from Shenandoah Biotech (Warwick, PA). Cells were stained with appropriate concentrations of mAbs. Dead cells were excluded using Fixable Viability Dye from eBioscience (San Diego, CA). Flow cytometry (FC) analyses were performed using LSRII (BD Bioscience, CA) and Canto cytometers (Becton Dickinson, NJ). Cells were sorted on BD FACSAria II (BD Bioscience, CA).
T cell culture and activation
Splenic mononuclear cells from each group of immunized mice were cultured in the presence of DCs pulsed with or without S2P, E2P or I3-01v9 SApNP (1 × 10−7 μM) in complete IMDM medium containing IL-2 (5.0 ng/ml). Cells were collected 16 hours and 4 hours later for intracellular cytokine staining and flow cytometric analysis.
Statistics
In antibody analysis, comparison of different vaccine groups was performed in GraphPad Prism 8.4.3 using the two-tailed unpaired Student’s t test. In T cell analysis, comparison of means was done using the two-tailed unpaired Student’s t test, ANOVA and then post-hoc t test. P values of 0.05 or less were considered significant.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://xxx/xxx/xxx.
fig. S10. T-cell response induced by S2PECTO and S2GΔHR2-presenting SApNPs.
Funding
This work was funded by NIH Grants AI129698 and AI140844 (to J.Z.), Ufovax/SFP-2018-0416, Ufovax/SFP-2018-1013 and Ufovax/SFP-2020-0111 (to J.Z.).
Author contributions
Project design by L.H., Y.Z., I.A.W. and J.Z; construct design of RBD and spike-based immungoens by L.H. and J.Z.; plasmid design and processing by L.H. and C.S.; antigen production, purification and basic characterization by L.H., X.L., and T.N.; antibody production and column packing by L.H., X.L., and T.N.; antibody-antigen ELISA and BLI by L.H. and C.S.; serum-antigen ELISA by L.H., X.L., and C.S.; antibody and mouse serum neutralization by L.H. and X.L.; vaccine-induced T-cell response analysis by Y.W., C.A., and Y.Z.; Manuscript written by L.H., Y.Z., I.A.W. and J.Z. All authors were asked to comment on the manuscript. The TSRI manuscript number is 30028.
Competing interests
Authors declare no competing interests.
Data and materials availability
All data are available in the main text or in the supplementary materials. Additional data related to this paper may be requested from the corresponding author.
Acknowledgements
References
- 1.↵
- 2.
- 3.↵
- 4.↵
- 5.↵
- 6.↵
- 7.
- 8.↵
- 9.↵
- 10.↵
- 11.↵
- 12.↵
- 13.↵
- 14.
- 15.
- 16.↵
- 17.↵
- 18.↵
- 19.
- 20.↵
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.↵
- 34.
- 35.↵
- 36.↵
- 37.↵
- 38.↵
- 39.↵
- 40.↵
- 41.↵
- 42.↵
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.↵
- 65.↵
- 66.↵
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.↵
- 72.↵
- 73.↵
- 74.
- 75.↵
- 76.↵
- 77.↵
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.
- 85.↵
- 86.↵
- 87.↵