RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge

Abstract

The ongoing COVID-19 pandemic has contributed largely to the global vaccine disparity. Development of protein subunit vaccines can help alleviate shortages of COVID-19 vaccines delivered to low-income countries. Here, we evaluated the efficacy of a three-dose virus-like particle (VLP) vaccine composed of hepatitis B surface antigen (HBsAg) decorated with the receptor binding domain (RBD) from the Wuhan or Beta SARS-CoV-2 strain adjuvanted with either aluminum hydroxide (alum) or squalene in water emulsion (SWE). RBD HBsAg vaccines were compared to the standard two doses of Pfizer mRNA vaccine. Alum-adjuvanted vaccines were composed of either HBsAg conjugated with Beta RBD alone (β RBD HBsAg+Al) or a combination of both Beta RBD HBsAg and Wuhan RBD HBsAg (β/Wu RBD HBsAg+Al). RBD vaccines adjuvanted with SWE were formulated with Beta RBD HBsAg (β RBD HBsAg+SWE) or without HBsAg (β RBD+SWE). Both alum-adjuvanted RBD HBsAg vaccines generated functional RBD IgG against multiple SARS-CoV-2 variants of concern (VOC), decreased viral RNA burden, and lowered inflammation in the lung against Alpha or Beta challenge in K18-hACE2 mice. However, only β/Wu RBD HBsAg+Al was able to afford 100% survival to mice challenged with Alpha or Beta VOC. Furthermore, mice immunized with β RBD HBsAg+SWE induced cross-reactive neutralizing antibodies against major VOC of SARS-CoV-2, lowered viral RNA burden in the lung and brain, and protected mice from Alpha or Beta challenge similarly to mice immunized with Pfizer mRNA. However, RBD+SWE immunization failed to protect mice from VOC challenge. Our findings demonstrate that RBD HBsAg VLP vaccines provided similar protection profiles to the approved Pfizer mRNA vaccines used worldwide and may offer protection against SARS-CoV-2 VOC. IMPORTANCE Global COVID-19 vaccine distribution to low-income countries has been a major challenge of the pandemic. To address supply chain issues, RBD virus-like particle (VLP) vaccines that are cost-effective and capable of large-scale production were developed and evaluated for efficacy in preclinical mouse studies. We demonstrated that RBD-VLP vaccines protected K18-hACE2 mice against Alpha or Beta challenge similarly to Pfizer mRNA vaccination. Our findings showed that the VLP platform can be utilized to formulate immunogenic and efficacious COVID-19 vaccines.

Keywords: COVID-19; HBsAg; RBD; SARS-CoV-2; SWE; SpyCatcher; SpyTag; VLP; vaccines.

FIG 1

Characterization of RBD IgG antibody responses against 10 SARS-CoV-2 VOC RBDs. (A) Depiction of assembly of β or Wuhan RBD on HBsAg using SpyTag and SpyCatcher technology. (B) Schematic of K18-hACE2 mouse immunization and serological assessment schedule. (C) MSD V-PLEX SARS-CoV-2 IgG panel 11 was used to determine RBD IgG levels. The heat map depicts mean values of IgG (AU/mL) generated from each mouse. RBD IgG titers were measured against 10 VOC RBDs at 2 weeks post prime. (D) Four weeks post-second dose RBD IgG titers against 10 VOC RBDs. (E to H) RBD IgG titers from 2 weeks post prime and 4 weeks post-second dose against Wuhan, Alpha, Beta, and Delta RBD VOC, respectively. IgG titers are represented as log AU/mL. Two-way ANOVA with Sidak’s multiple-comparison test was performed for statistical analysis. ****, P < 0.0001, The dotted line represents the limit of detection for the specific RBD variant.

FIG 2

Evaluation of RBD-VLP and mRNA vaccine protection against VOC challenge. (A) Vaccine and challenge experimental timeline in K18-hACE2 mice. Mice were intramuscularly administered three doses of either β RBD HBsAg+Al, β/Wu RBD HBsAg+Al, β RBD HBsAg+SWE, or β RBD+SWE. Pfizer mRNA-vaccinated mice were administered 2 doses of vaccine. Vaccinated mice were bled every 2 weeks post-vaccine dose. Mice were intranasally challenged with 10⁴ PFU/dose of either Alpha or Beta variant and monitored for 11 days after challenge. (B) The Kaplan-Meier survival curve shows the percent survival of NVNC (n = 5), NVC (n = 5), β RBD HBsAg+Al (n = 5; P = 0.0143), β/Wu RBD HBsAg+Al (n = 5; P = 0.0027), β RBD HBsAg+SWE (n = 5; P = 0.0027), β RBD+SWE (n = 5), and Pfizer mRNA (n = 5; P = 0.0027) mice challenged with Alpha. (C) Kaplan-Meier survival curve of NVNC (n = 5), NVC (n = 5), β RBD HBsAg+Al (n = 5; P = 0.0411), β/Wu RBD HBsAg+Al (n = 4; P = 0.0237), β RBD HBsAg+SWE (n = 5; P = 0.0143), β RBD+SWE (n = 5), and Pfizer mRNA (n = 5; P = 0.0143) mice challenged with Beta. A log-rank (Mantel-Cox) test determined the statistical significance between NVC compared to the respective vaccine groups. (D and E) Daily disease scores of Alpha- and Beta-challenged mice, respectively.

FIG 3

Determination of viral RNA burden in VOC-challenged mice. Lung and brain (100 ng) were assessed for nucleocapsid RNA copies. (A and B) Violin plots depict SARS-CoV-2 RNA copies in the (A) right lobe of the lung and (B) brain. Left side of the bold vertical dotted line represents mice challenge with Alpha, and the right side represents mice challenged with Beta. The horizontal dotted line represents the limit of detection calculated with the NVNC viral copy numbers. Ordinary one-way ANOVA with Tukey’s multiple-comparison test was performed for statistical analysis among the Alpha- and Beta-challenged groups. Asterisks denote significant differences compared to NVC, and the # symbol indicates a significant difference compared to RBD+SWE. For Alpha challenged lung: ****, P < 0.0001; **, P = 0.0035 (NVCα vs β/Wu RBD HBsAg+Al) and **, P = 0.0014 (NVCα vs β RBD HBsAg+SWE), and ##, P = 0.0014 (β RBD HBsAg+SWE vs. β RBD+SWE). For Beta challenged lung: *, P = 0.0187 (NVCβ vs β RBD HBsAg+Al); **, P = 0.0089 (NVCβ vs β/Wu RBD HBsAg+Al); **, P = 0.0052 (NVCβ vs β RBD HBsAg+SWE). For Alpha challenged brain: *, P = 0.0231 (NVCα vs mRNA); **, P = 0.0035 (NVCα vs β/Wu RBD HBsAg+Al), **, P = 0.0014 (NVCα vs β RBD HBsAg+SWE) and #, 0.0452 (β RBD HBsAg+SWE vs RBD+SWE). For Beta challenged brain: *, 0.0495 (NVCβ vs mRNA); **, P = 0.0089 (NVCβ vs β/Wu RBD HBsAg+Al), and **, P = 0.0052 (NVCβ vs β RBD HBsAg+SWE).

FIG 4

RBD HbsAg+SWE induced broadly neutralizing antibodies against VOC RBD. MSD V-PLEX SARS-CoV-2 Panel 22 (ACE2) kit with 5 VOC and Wuhan RBD was used to measure serum antibody neutralization in Beta challenged mice in immunized and non-immunized mice. All values were depicted as % inhibition. Negative % inhibition values were not represented in the analysis. Percent inhibition of neutralizing antibodies measured against A) Wuhan, B) Alpha, C) Beta, D) Gamma, E) Delta, and F) Omicron respectively. Results represented as mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparisons test were conducted for statistical analysis. Asterisks denote significant difference compared to NVC and # symbol indicates significant difference compared to RBD+SWE. Wuhan: *, P = 0.0063. Alpha: *, P = 0.00319 (NVC vs RBD HBsAg+SWE); **, P = 0.0019 (NVC vs mRNA). Beta: *, P = 0.0081 (NVC vs mRNA). Gamma: *, P = 0.0151 (NVC vs mRNA), #, P = 0.0461 (RBD HBsAg+SWE vs RBD+SWE). Delta: *, P = 0.0319 (NVC vs RBD HBsAg+SWE), **, P = 0.0045(NVC vs mRNA). Omicron: *, P = 0.0213 (NVC vs mRNA), **, P = 0.0029 (NVC vs RBD HBsAg+SWE).

FIG 5

Histopathological analysis of the lung in vaccinated mice challenged with VOC. (A) H&E-stained lungs from vaccinated groups that were Alpha challenged. (B) H&E-stained lungs from vaccinated groups that were Beta challenged. (C) No vaccine, no challenge (NVNC) H&E-stained lungs at ×100 magnification. (D) Alpha-challenged total inflammation scores (chronic plus acute inflammation scores). #, P = 0.0213 (NVNC versus β RBD+SWE); *, P = 0.0251 (NVCα versus mRNA). (E) Beta-challenged total inflammation scores. #, P = 0.0341 (NVNC versus NVC-β); ##, P = 0.0062 (NVNC versus β RBD+SWE). Red dots represent mice that were euthanized due to morbidity before the termination of the study at day 11. Results are represented as the mean ± SD. The Kruskal-Wallis test with Dunn’s multiple-comparison test was performed for statistical analysis. Asterisks denote significant differences compared to NVC, and # symbols indicate significant differences compared to NVNC.