Immunogen design

For designing an S2-based SARS-CoV-2 immunogen, S2 subunit residues interacting with the S1 subunit were identified by accessibility calculations using the Naccess program on PDB 6VXX. Accessible surface areas (ASAs) of all residues in Spike were calculated in the absence and presence of the S1 subunit. Residues of the S2, which had a total side-chain ASA difference of >5 Å2, were identified as S1 interacting residues. The following mutations were made to mask newly exposed hydrophobic residues to prevent S2 aggregation in the absence of S1, namely F855S, L861E, L864D, V976D, and L984Q39,40.

Proline substitutions in the loop between HR1 (heptad repeat 1) and the central helix in fusion proteins from viruses like SARS-CoV-2, SARS-CoV, MERS-CoV, and RSV are known to retain prefusion conformation, and enhance expression yields41,42,43,44. More recently, HexaPro Spike, a S-6P variant, with six proline substitutions (F817P, A892P, A899P, A942P, K986P, V987P) displayed 9.8-fold increased protein expression and ~5 °C increase in Tm relative to S-2P (Spike with two proline mutation)45. Hence, we additionally included all these six proline substitutions in the present S2 immunogen (S2) comprising residues 698–1211 of the SARS-CoV-2 Spike. Since RBD contains the major neutralizing antibody epitopes on Spike and S2 is well conserved, we designed RS2 and S2R immunogens (Fig. 1a). For this, an S2 fragment comprising residues 698–1163 of the SARS-CoV-2 was genetically fused with our previously reported high-yielding, thermostable RBD containing A348P, Y365W, and P527L mutations20,22.

Fig. 1: Characterization of S2, RS2, S2R, and Spike immunogens.

a Schematic representation of designed immunogen sequences. b Reducing SDS-PAGE profile of protein samples. SEC profile of purified (c) S2, (d) RS2, (e) S2R and (f) Spike immunogens.

Biophysical characterization of S2, RS2, S2R and Spike

The designed immunogens were transiently expressed as secreted proteins in Expi293F suspension cells. Recombinant proteins S2, RS2, and S2R were purified in high yields of ~120 mg/L, ~850 mg/L, and ~800 mg/L, respectively, using nickel affinity chromatography (Fig. 1b). The oligomeric state of the immunogens was determined using size exclusion chromatography (SEC), and revealed that S2 exists as homogeneous nonamers in the solution (Fig. 1c). RS2 and S2R exist as a mixture of monomers and trimers, and the calculated molecular weights were in good agreement with the theoretical molecular weight (Fig. 1d, e).

For comparison, the stabilized trimeric Spike ectodomain (1-1208) containing six proline mutations (F817P, A892P, A899P, A942P, K986P, and V987P), along with the additional three RBD stabilizing mutations (A348P, Y365W, and P527L) described above, was also expressed and purified from Expi293F cells (Fig. 1a, b, f) with a purified yield of ~150 mg/L22,45.

The apparent melting temperature (Tm) and the thermal unfolding profile of designed immunogens and Spike was determined using Nano-DSF. The RS2 and S2R displayed similar thermal unfolding profiles and comparable Tm of ~50 °C (Fig. 2a, b). The S2 immunogen exhibited Tm of 52.2 °C while the stabilized trimeric spike demonstrated two Tm, Tm1 of 50.4 °C and Tm2 of 61.5 °C, which implied that trimeric Spike has different structural components of varying stability (Fig. 2c, d). RS2, S2R, and S2, when subjected to 37 °C for 1 h, exhibited similar thermal unfolding profiles to those protein samples stored at 4 °C (Fig. 2e–g). However, the Spike showed a slightly broadened thermal unfolding profile after incubation at 37 °C, with more noticeable broadening at 50 °C (Fig. 2h). Moreover, RS2 and S2R were found to be stable even after incubation at 50 °C, although a slight decrease was observed. On the other hand, S2 was thermally unstable at 50 °C, which suggested that RS2 and S2R are more resistant to transient thermal stress than Spike and S2. (Fig. 2e–h). Compared to the other designed immunogens and Spike, RS2 was more rapidly digested by TPCK-trypsin at both 4 °C and 37 °C, (Fig. 2i–l). This data suggested that RS2 is more susceptible to proteolytic degradation than S2R, S2, and Spike, which showed highest proteolytic stability. Whether the apparent enhanced proteolytic resistance of Spike (Fig. 2l) is due to aggregation following initial proteolytic cleavage, remains to be elucidated.

Fig. 2: Equilibrium thermal unfolding, transient thermal stability and limited trypsin proteolytic profiles of designed immunogens.

Thermal unfolding profiles. Apparent melting temperature of (a) RS2, (b) S2R, (c) S2 and (d) Spike were measured using Nano-DSF. e–h Transient thermal stability profiles. Protein samples were subjected to different temperatures (4, 37, and 50 °C) for one hour. Thermal stability of (e) RS2, (f) S2R, (g) S2 and (h) Spike was monitored using Nano-DSF. Normalized first derivative of fluorescence at 350 nm is plotted as function of temperature. i–l Proteolytic stability profile. Coomassie stained SDS-PAGE profiles of purified (i) RS2, (j) S2R, (k) S2 and (l) Spike subjected to TPCK-Trypsin proteolysis at 37 °C and 4 °C.

The binding of S2, RS2, and S2R immunogens to its cognate receptor, ACE2-hFc, a panel of RBD conformation-specific (CR3022, S309, ADG-2, and H014) and S2-specific (B6 and CC40.8) antibodies were probed using surface plasmon resonance (SPR). Spike, RS2, and S2R bound well with ACE2-hFc, RBD-specific, and S2-specific antibodies (Tables 1 and 2). S2 binds only to S2-specific antibodies with high affinity (Table 2). This indicated the proper folding of designed immunogens.

Table 1 Kinetic parameters of immunogens for binding to different RBD conformation-specific ligands in PBS pH 7.4 at 25 °C.Table 2 Kinetic parameters of immunogens for binding to different S2-specific ligands in PBS pH 7.4 at 25 °C.Immunogenicity and protective efficacy of S2R relative to S2 and RBD immunogens against heterologous challenge

Since in the initial characterizations, S2R showed higher proteolytic stability and comparable thermal stability to RS2, the immunogenicity and protective efficacy of S2R, relative to RBD and S2 immunogens, was evaluated in hACE-2 expressing C57BL/6 transgenic mice. Our previously reported mammalian cell expressed, stabilized RBD containing A348P, Y365W, and P527L mutations was expressed and purified20,22. Mice were intramuscularly immunized with 2 μg immunogens (S2R, S2 or RBD) formulated with SWE in a prime-boost regimen 3 weeks apart. SWE is equivalent to MF59, a very safe adjuvant that has been used for many years in the context of human influenza vaccines46. While there are other more potent adjuvants available, stronger adjuvant mediated immune responses can be associated with unfavorable side effects46. Two weeks post-boost, RBD, S2 and Spike-specific IgG titers in sera of immunized mice were measured using ELISA. Relative to RBD and S2, the S2R immunogen elicited significantly higher RBD, S2 and Spike-specific ELISA endpoint titers (Fig. 3a–c). S2R immunized mice elicited significantly higher neutralizing antibody titers against B.1 pseudovirus compared to RBD immunized mice, while the sera from S2 immunized mice failed to neutralize B.1 pseudovirus (Fig. 3d). At this administered dose, sera from RBD-immunized mice did not show neutralization against BA.1. However, S2R immunized mice sera showed neutralization against BA.1, BA.5, and BF.7 albeit at significantly reduced levels (Fig. 3e, f). Furthermore, these sera exhibited substantial cross-neutralization against heterologous clade1a SARS-CoV-1 viruses, which demonstrates the potential of S2R to elicit broadly protective antibodies against sarbecoviruses (Fig. 3f). Although the neutralization ID50 was too low to be measured in mice immunized with SWE formulated S2, addition of these S2 elicited sera enhanced neutralization potency of the broadly neutralizing antibody S309 (Fig. 3g).

Fig. 3: Immunogenicity of RBD, S2 and S2R in hACE-2 expressing mice.

Three groups of hACE-2 expressing transgenic mice were primed and boosted with 2 μg of RBD, S2 and S2R respectively, followed by an intranasal challenge with 105 pfu of the beta variant of SARS-CoV-2. a–c ELISA endpoint titers against RBD, S2, and spike ectodomain respectively two weeks post-boost. d, e Neutralizing antibody titers elicited by RBD, S2, and S2R against B.1 and BA.1 Omicron SARS-CoV-2 pseudovirus. No neutralization was seen with S2 immunized animals. f Neutralizing antibody titers elicited by S2R against various pseudoviruses. Lines connect the neutralizing titers for different variants in a sera sample from an individual animal against different variants. g Neutralization curves of pooled S2 immunized mice sera, monoclonal antibody S309, and S2 immunized mice sera in presence of S309. The sera sample was tested in five technical repeats. Each point represents the median of five independent values. h Survival Curve. i Average weight changes up to 5 days post-challenge. j Lung viral titer. k Histopathology scores of lungs. l Histology of lung tissue sections from unimmunized-unchallenged control (UC), unimmunized- Beta variant challenged control (Unimmunized-Beta) and mice immunized with RBD, S2 and S2R at 4X magnification. The scale bar indicates 50 µm. Titers are shown as geometric mean with geometric SD. The ELISA binding, neutralization titer, lung viral titer and histopathology score data were analyzed with a two-tailed Mann–Whitney test. Neutralizing titers elicited by individual sera in S2R immunized animals against various pseudoviruses (3f) were analyzed with non-parametric Kruskal–Wallis test with Dunn’s multiple correction. Weight changes (3i) were analyzed with a Multiple Student’s t test with Bonferroni Dunn’s correction method. (ns indicates non-significant, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).

Next, the protective efficacy of RBD, S2, and S2R formulations was assessed against the Beta variant of SARS-CoV-2. Unimmunized-unchallenged, and unimmunized-Beta variant challenged mice were used as control groups. Three weeks post boost, mice were intranasally challenged with 105 plaque-forming units (pfu) of Beta variant virus, and weight change was monitored for up to five days. Only 33% of unimmunized mice survived, while 72% of S2 immunized mice survived Beta-variant challenge. In contrast, all RBD and S2R immunized mice survived the Beta variant challenge (Fig. 3h). Post challenge, no weight change was seen in S2R immunized mice. Mice immunized with RBD, and S2 showed a significant weight reduction of ~10% and ~17% respectively. As expected, no weight reduction was observed in the unimmunized group, while 22–25% weight reduction was seen in the unimmunized-Beta variant challenged group (Fig. 3i). Mice immunized with either RBD, S2 or S2R showed significantly lower lung viral titers than unimmunized mice challenged with the Beta variant (Fig. 3j). Despite lack of neutralization, lung viral titers were significantly reduced in the S2-immunized group, suggesting that S2 provides protection by non-neutralizing mechanisms. The lung tissue sections obtained from S2R immunized mice showed clear interstitial spaces within lung epithelium and reduced immune cell infiltration compared to unimmunized-Beta variant challenged group, RBD and S2 immunized group (Fig. 3k, l).

RS2 is more immunogenic than S2R in mice

At the same time, we also compared the immunogenicity of RS2 and S2R in BALB/c mice. BALB/c mice were vaccinated twice intramuscularly with either 20 μg of RS2 or S2R formulated with SWE adjuvant. Two weeks after the second vaccination, RBD and Spike specific IgG titers and neutralizing immune responses were measured in the sera samples of immunized mice. Both RS2 and S2R immunized mice elicited equivalent RBD and Spike-specific ELISA endpoint titers (Supplementary Fig. 1a, b). RS2 elicited significantly higher neutralizing titers against B.1 pseudovirus (Supplementary Fig. 1c). Neutralizing titers against Delta variant were also higher in RS2 immunized mice sera but differences did not reach statistical significance (Supplementary Fig. 1d). Thus, the data indicated that RS2 is more immunogenic than S2R. Hence for all further studies, including formulation stability and comparative immunization studies with Spike, RS2 was used.

RS2 induces an equivalent neutralizing immune response to Spike ectodomain in mice and protects against mouse-adapted SARS-CoV-2 challenge

Considering that most currently licensed COVID-19 vaccines have the Spike as the sole immunogen, we compared the immunogenicity and protective efficacy of RS2 with the stabilized Spike ectodomain. BALB/c mice were intramuscularly immunized with 2 μg of RS2 or Spike formulated with SWE adjuvant in a prime-boost regimen 21 days apart. Fourteen days post-second immunization, both RS2 and Spike immunized mice elicited high RBD, S2 and Spike-specific IgG titers (Fig. 4a–c). Although the neutralizing titers against B.1, Beta and Delta variant pseudoviruses were comparatively higher in RS2 immunized mice sera than Spike immunized mice sera, the differences did not reach statistical significance (Fig. 4d–f). Sera from RS2 and Spike immunized mice also showed neutralization against BA.1, BA.5 and BF.7 pseudoviruses, albeit with lower titers (Fig. 4g–i). Compared to Spike, RS2 also exhibited substantial cross-neutralization against heterologous clade1a SARS-CoV-1 and WIV-1 viruses, which demonstrates the potential of RS2 to elicit broadly protective antibodies against sarbecoviruses (Fig. 4j, k). Three weeks post-boost, all mice were intranasally challenged with 105 pfu of mouse-adapted SARS-CoV-2 MA10 virus. One-day post-challenge, mice immunized with either RS2 or spike showed a slight body weight reduction of ~5%, and from day two, all mice regained their initial weight. In contrast, unimmunized-MA10 challenge control mice showed ~25% weight loss by day four, post-challenge. No weight change was seen in the unimmunized-unchallenged control group (Fig. 4l). RS2 and Spike immunized mice showed significantly reduced lung viral titers compared to unimmunized-MA10 mice (Fig. 4m). Analysis of lung tissue sections of mice immunized with RS2 showed clear lung epithelial interstitial spaces and lower immune cell infiltration compared to both Spike immunized and unimmunized MA10 challenged group (Fig. 4n, o). This data suggests that RS2 elicits a broadly neutralizing humoral immune response and protects against SARS-CoV-2 MA10 virus challenge.

Fig. 4: Immunogenicity of RS2 and Spike in BALB/c mice.

BALB/c mice were immunized twice with either 2 µg RS2 or 2 µg Spike, followed by intranasal challenge with 105 pfu of MA-10 mouse adapted SARS-CoV-2. Two weeks following the boost, RBD, S2 and Spike specific IgG and neutralizing titers in immunized mice sera were measured (a–c) ELISA endpoint titers against RBD, S2 and spike ectodomain, respectively. Comparison of neutralizing antibody titers elicited by 2 µg of RS2 and Spike against (d) B.1, (e) Beta, (f) Delta, (g) BA.1, (h) BA.5, (i) BF.7, (j) WIV-1, and (k) SARS-CoV-1 pseudoviruses. l Average weight changes up to six days post-MA10 challenge. m Lung viral titers. n Histopathology scores of lungs. o Histology of lung tissue sections from unimmunized-unchallenged control (UC), Unimmunized-MA10 virus challenged control (Unimmunized MA-10), mice immunized with Spike or RS2 at 4X magnification. The scale bar indicates 50 µm. Titers are shown as geometric mean with geometric SD. The ELISA binding, neutralization titer, lung viral titer, and histopathology score data were analyzed with a two-tailed Mann–Whitney test. Weight changes were analyzed with a Multiple Student’s t test with Bonferroni Dunn’s correction method. (ns indicates non-significant, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).

Thermal stability of RS2

As with our previously reported RBD immunogens, our newly designed RS2 immunogen showed identical thermal unfolding profiles before and after lyophilization and solubilization, suggesting that lyophilization did not affect the thermal stability of these immunogens (Supplementary Fig. 2a)20,22. Further, the effect of transient thermal stress on the thermal stability of lyophilized RS2 was studied by performing nano-DSF. Following incubation at different temperatures (4 °C, 37 °C, 50 °C, 70 °C and 90 °C) for 60 min, no change in thermal unfolding profiles was observed (Supplementary Fig. 2b). Moreover, the Tm and conformational integrity of lyophilized RS2 protein remained unchanged after a month of storage at 37 °C (Supplementary Fig. 2c) (Table 3). In addition, the antigenicity of RS2 was assessed by performing SPR using a panel of RBD and S2-specific antibodies. Interestingly, following thermal stress, RS2 retained binding with the cognate receptor ACE2-hFc, RBD-specific antibodies (CR3022, S309), and S2-specific antibodies (B6, CC40.8). This suggests that lyophilized RS2 immunogen is resistant to transient thermal stress (Supplementary Fig. 2d–h). The stability of SWE adjuvanted RS2 in PBS buffer was also evaluated at 5 °C and 40 °C over a period of one month. Antigen integrity and physiochemical characterization of SWE adjuvanted RS2 formulations was carried out by performing ELISA, particle size, polydispersity, zeta potential, pH, osmolality and squalene content measurements. The RS2 formulation in SWE is stable at 5 °C and 40 °C in both polypropylene tubes and glass vials for at least one month (Fig. 5a–k).

Table 3 Kinetic parameters for binding of lyophilized and resolubilized RS2 after incubation of lyophilized protein at 37 °C for 1 month to different RBD-specific and S2-specific ligands in PBS pH 7.4 at 25 °C.Fig. 5: Stability of RS2.

Characterization of lyophilized and resolubilized RS2 after incubation at 37 °C for 1 month. a–h Physiochemical characterization of RS2 in 1X PBS with equal amount (v/v) of SWE adjuvant incubated at 5 and 40 °C in polypropylene (PP) and glass vials (GV) for one month. Adjuvant properties were measured on day 0, day 7, day 14 and day 30. a Particle size, b Polydispersity Index, c Zeta Potential, d pH, e Osmolality, f Squalene content. g, h Antigenic integrity of RS2 in PBS and SWE adjuvant was measured based on binding to CR3022 using ELISA. i Day 7, j Day 14, k Day 30. Freshly thawed RS2 sample without any external modification ‘RS2 ctrl extemp’ was used as a control for undegraded antigen.

Protective efficacy of lyophilized RS2 formulation after month long storage at 37 °C

The immunogenicity of the lyophilized RS2 protein stored at 37 °C for a month was evaluated in hACE-2 expressing C57BL/6 transgenic mice. Mice were intramuscularly immunized with 20 μg immunogen extemporaneously formulated with SWE adjuvant in a prime-boost regimen. Two weeks following the boost, the formulation showed high RBD, S2 and Spike-specific binding titers, and neutralizing titers against B.1, Beta, Delta, and BA.1 pseudoviruses (Fig. 6a, b). The above formulation elicited equivalent neutralization titers to a non-lyophilized freshly prepared RS2 formulation stored at 4 °C, against B.1 and BA.1 pseudoviruses (Fig. 6c). Twenty-one days following the boost, mice were challenged with 104 pfu of Beta and Delta variants of SARS-CoV-2. None of the unimmunized control mice survived Beta variant challenge, while 57% of unimmunized control mice survived Delta variant challenge and the remaining mice showed 20 % weight loss, nine days post-challenge. In contrast, all RS2 immunized mice survived the Beta and Delta variant SARS-CoV-2 challenges and showed no weight loss (Fig. 6d–f). Lung viral titers of RS2-immunized mice challenged with Beta and Delta variants were below the detection limit and lung tissues showed minimal pathology (Fig. 6g–k). Lung viral titers and tissue sections from the unimmunized mice challenged with the Beta variant were not examined because none of the mice survived. These findings indicate that RS2 is stable and immunogenic even after storage at 37 °C for at least one-month.

Fig. 6: Immunogenicity of lyophilized RS2, that had been incubated at 37 °C for 1 month, in hACE-2 expressing transgenic mice.

hACE-2 expressing transgenic mice immunized twice with 20 μg of lyophilized RS2 that was previously incubated at 37 °C for over a month and then formulated in SWE adjuvant. This was followed by intranasal challenge with 104 pfu Beta and Delta variants. a ELISA endpoint titers against RBD, S2, and spike ectodomain. b Neutralizing antibody titers against B.1, Beta, Delta and BA.1 pseudoviruses. c Neutralizing antibody titers elicited by lyophilized RS2 subjected to 37 °C for over a month (Lyo), and non-lyophilized RS2 (Non-lyo) stored at 4 °C, against B.1 and BA.1 Omicron SARS-CoV-2 pseudovirus (d, e) Average weight change up to nine days post-Beta and Delta virus challenge respectively. f Survival curve. g, h Lung viral titers in RS2 immunized mice, challenged with Beta VOC and Delta VOC respectively. i, j Histopathology scores of lungs. k Histology of lung tissue sections from unimmunized-unchallenged control (UC), mice immunized with 20 μg RS2 challenged with Beta variant (RS2-Beta challenged), unimmunized Delta virus challenged control (Unimmunized-Delta), mice immunized with 20 μg RS2 and challenged with Delta variant (RS2-Delta challenged), at 4X magnification. The scale bar indicates 50 µm. None of the unimmunized controls survived the Beta virus challenge (Unimmunized-Beta). Titers are shown as geometric mean with geometric SD. The ELISA binding, neutralization titer, lung viral titer and histopathology score data were analyzed with a two-tailed Mann–Whitney test. Neutralization titers for individual sera against B.1, Beta, Delta and BA.1 pseudoviruses (6b) were analyzed with non-parametric Kruskal–Wallis test with Dunn’s multiple correction. Weight changes were analyzed with a Multiple Student’s t test with Bonferroni Dunn’s correction method. (ns indicates non-significant, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).

RS2 shows superior immunogenicity and protective efficacy to stabilized Spike ectodomain in hamsters

Protective efficacy of RS2 was compared with the stabilized Spike against challenge with the Beta variant of SARS-CoV-2 in Syrian hamsters. Female Syrian Golden hamsters were immunized with 5 μg of stabilized Spike or RS2 formulated with SWE on day 0 and day 21, while the control group of hamsters was immunized with SWE adjuvant in PBS. Both Spike and RS2 immunized hamsters elicited high RBD, S2 and Spike-specific ELISA endpoint titers (Fig. 7a–c). Notably, two immunizations with 5 μg SWE adjuvant formulated RS2 elicited significantly higher neutralizing titers against B.1, Beta, Delta and Omicron BA.1, BA.5 and BF.7 variants than corresponding titers elicited by SWE formulated Spike in hamsters (Fig. 7d–i). Consistent with the BALB/c mice study, RS2 immunized hamsters exhibited substantially higher cross-neutralizing activity against clade 1a sarbecoviruses, WIV-1 and SARS-CoV-1 compared to those of Spike immunized hamsters (Fig. 7j, k). Moreover, hamsters immunized with RS2 showed initial transient weight loss (up to 3 %) and regained weight at 3 days post Beta variant infection. In contrast, Spike-immunized hamsters showed comparatively higher lung viral titers and weight loss, while no weight regain was observed (Fig. 7l, m). Both RS2 and Spike immunized hamsters showed significantly reduced lung viral titers compared to unimmunized-Beta challenged hamsters (Fig. 7m). Analysis of RS2 and Spike immunized hamster lung tissue sections showed clear lung epithelial interstitial spaces and lower immune cell infiltration compared to the unimmunized Beta-challenged groups (Fig. 7n, o). Overall, the data shows that RS2 is more immunogenic and efficacious than Spike in hamsters.

Fig. 7: Comparative protective efficacy of RS2 and spike in hamsters.

Syrian hamsters were immunized twice with 5 µg of RS2 or Spike, followed by intranasal challenge with 105 pfu of the Beta VOC. a–c ELISA endpoint titers against RBD, S2 and spike ectodomain, respectively. Comparative neutralizing antibody titers elicited by 5 µg of RS2 and spike against (d) B.1, (e) Beta, (f) Delta, (g) BA.1, (h) BA.5, (i) BF.7, (j) WIV-1 and, (k) SARS-CoV-1 pseudovirus. l Average weight change up to five days post-beta variant virus challenge. m Lung viral titers. n Histology of lung tissue sections from unimmunized-unchallenged control (UC), unimmunized-Beta challenged control (Unimmunized-Beta) and hamsters immunized with Spike and RS2 at 4X magnification. The scale bar indicates 50 µm. o Histopathology scores of lungs. The ELISA binding, neutralization titer, lung viral titer and, histopathology score data were analyzed with a two-tailed Mann–Whitney test. Weight changes were analyzed with a Multiple Student’s t test with Bonferroni Dunn’s correction method. (ns indicates non-significant, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001).