Enhanced protective efficacy of a thermostable RBD-S2 vaccine formulation against SARS-CoV-2 and its variants

Abstract

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly reactive immune responses that could improve protection. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. Immunization with S2 alone failed to elicit a neutralizing immune response, but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 (a genetic fusion of stabilized RBD with S2) showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 elicited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), and the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 elicited sera showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 °C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

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.

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.

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).

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).

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.

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).

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).