Development of bivalent RBD adapted COVID-19 vaccines for broad sarbecovirus immunity

Abstract

COVID-19 vaccine adaptation is critical to respond to continuously emerging SARS-CoV-2 variants with enhanced immune evasion. The ARVAC protein subunit vaccine, based on the receptor binding domain of the spike protein of SARS-CoV-2, has been adapted to XBB.1.5 and JN.1 variants, as monovalent and bivalent formulations. Preclinical studies in mice showed that ARVAC XBB.1.5 and JN.1 monovalent vaccines induced strong neutralizing antibodies against XBB and JN.1 lineages, though with limited efficacy against phylogenetically distant variants. By contrast, bivalent formulations combining Gamma antigen with either XBB.1.5 or JN.1 antigens demonstrated superior cross-neutralizing activity, covering variants from Ancestral to JN.1. Additionally, Gamma-containing bivalent vaccines elicited neutralizing antibodies against SARS-CoV-1, highlighting their potential for broad-spectrum immunity. Cellular immune studies confirmed robust CD4⁺ T cell activation across all formulations. These findings support the continued adaptation of ARVAC to current circulant variants and propose ARVAC bivalent vaccines containing the Gamma antigen as a strategy for induction of pan-sarbecovirus immunity.

Fig. 1. Prime immunization with monovalent and bivalent adapted XBB.1.5 vaccines induce nAbs and Ag-specific T cell immune responses.

BALB/c mice were i.m. immunized on days 0 and 14 with ARVAC XBB.1.5 (n = 6), ARVAC Omicron BA.4/5 (n = 6), ARVAC Gamma/Omicron BA.4/5 (n = 6) or ARVAC Gamma/XBB.1.5 (n = 6). a Neutralizing antibody titers against JN.1, XBB.1.18, Omicron BA.5, Gamma and ancestral SARS-CoV-2 were evaluated at 42 days post prime (dpp). Neutralization titer was defined as the serum dilution that reduces 50% the cytopathic effect (NT50). Bars are GMT ± SD. *p < 0.05, ***p < 0.001. Kruskal–Wallis test. b Radar charts were drawn based on the geometric mean titers (GMTs) of nAbs against live viruses at 42 dpp. c Antigenic map was generated from the nAb titers in serum after the second dose of vaccination. Squares represented nAb titers from mice vaccinated with XBB.1.5 (red squares), Omicron BA.4/5 (green squares), Gamma/BA.4/5 (blue squares) and Gamma/XBB.1.5 (purple squares) formulations. Black, blue, green, red and purple circles correspond to the Ancestral, Gamma, Omicron BA.5, XBB.1.18 and JN.1 SARS-CoV-2 variants, respectively. Each grid square corresponds to a two-fold dilution in the neutralization assay. The antigenic distance is interpretable in any direction. d Splenocytes from immunized mice were obtained 1 month after last dose and were stimulated with medium or Omicron BA.4/5 RBD-peptides pool plus recombinant Omicron BA.4/5 RBD for 18 h and then brefeldin A was added for 5 h. Afterward, cells were harvested and stained with anti-CD4 specific Abs, fixed, permeabilized, and stained intracellularly with anti-IFN-γ and anti-TNF-α. Results are presented as percentage of IFN-γ or TNF-α-producing CD4⁺ T lymphocytes. Bars are means ± SEM. Each dot is an individual mouse. *p < 0.05, **p < 0.01 vs. placebo. Kruskal–Wallis. e Splenocytes from immunized mice were stimulated with complete medium or recombinant Omicron BA.4/5 RBD for 5 d and then IL-5 was measured in the supernatant by ELISA. Results are presented as concentration of IL-5 in pg/ml. Bars are means ± SEM. Each dot is an individual mouse. *p < 0.05, **p < 0.01 vs. medium. Kruskal–Wallis. f Lung cells from immunized mice were stimulated with medium or recombinant Omicron BA.4/5 RBD for 3 d and then IL-5, IFN-γ and TNF-α was measured in the supernatant by ELISA. Results are presented as concentration of the cytokine in pg/ml. Bars are means ± SEM. Each dot is an individual mouse. *p < 0.05, **p < 0.01 vs. medium. Kruskal–Wallis.

Fig. 2. Monovalent and bivalent adapted XBB.1.5 vaccines induce nAbs against XBB and JN.1 lineages in booster immunization schedules.

BALB/c mice were i.m. immunized on days 0 and 14 with bivalent mRNA vaccine Spikevax (Prototype/Omicron BA.5) and boosted on day 67 with: ARVAC XBB.1.5 (n = 6), ARVAC Omicron BA.4/5 (n = 6), ARVAC Gamma/Omicron BA.4/5 (n = 6), ARVAC Gamma/XBB.1.5 (n = 6) or Spikevax (Prototype/Omicron BA.5) (n = 6). a Neutralizing-antibody titers against XBB.1.18, JN.1, Omicron BA.5, Gamma and Ancestral SARS-CoV-2 were evaluated at 63 (preboost) and 93 dpp (postboost). Neutralization titer was defined as the serum dilution that reduces 50% the cytopathic effect (NT50). Bars are GMT ± SD. *p < 0.05, ***p < 0.001. Kruskal–Wallis test. b Radar charts were drawn based on the GMTs of nAb in serum pre and post boost against live viruses. c Antigenic maps were generated from nAb titers in serum pre and after the booster dose. Squares represented nAb titers from mice vaccinated with XBB.1.5 (red squares), Omicron BA.4/5 (green squares), Gamma/BA.4/5 (blue squares) and Gamma/XBB.1.5 (purple squares) formulations. Black, blue, green, red and purple circles correspond to the Ancestral, Gamma, Omicron BA.5, XBB.1.18 and JN.1 variants, respectively. Each grid square corresponds to a two-fold dilution in the neutralization assay. The antigenic distance is interpretable in any direction.

Fig. 3. Neutralizing antibodies against SARS-CoV-1 virus induced after booster vaccination.

Titers of nAbs to SARS-CoV-1 (a) and SARS-CoV-2 (b) virus by PSVNA at day 93 (28 days post boost) after administration of ARVAC vaccines. Positive control in a is a human IgG1 anti SARS-CoV-2 Spike CR3022 antibody and the positive control in b is the International Standard (NIBSC, code: 21/340). Bars are GMT ± SD. The GMT value is indicated inside bars. *p < 0.05, ***p < 0.001. Kruskal–Wallis test.

Fig. 4. Monovalent and bivalent adapted JN.1 vaccines induce nAbs against JN.1, KP.2 and KP.3 lineages and specific T cell responses in the spleen and lung.

BALB/c mice were immunized at day 0 and day 14 via i.m. with: ARVAC JN.1 (n = 6), ARVAC XBB.1.5 (n = 6), ARVAC Gamma/BA.4/5 (n = 6), ARVAC Gamma/JN.1 (n = 6) and Spikevax XBB.1.5 mRNA vaccine. a Neutralizing antibody titers against KP.3.1.1, KP.2.3, JN.1, XBB.1.5, Omicron BA.5, Gamma and Ancestral SARS-CoV-2 were evaluated at 42 dpp. Neutralization titer was defined as the serum dilution that reduces 50% the cytopathic effect (NT50). Bars are GMT ± SD. *p < 0.05, ***p < 0.001. Kruskal–Wallis test. b Radar charts were drawn based on the GMTs of nAb in serum 42 dpp against live viruses. c Splenocytes from immunized mice were obtained a month after last immunization and stimulated with medium or JN.1 RBD-peptides pool plus recombinant JN.1 RBD for 18 h and then brefeldin A was added for 5 h. Afterward, cells were harvested and stained with anti-CD4 and anti-CD8 specific Abs, fixed, permeabilized, and stained intracellularly with anti-IFN-γ and anti-TNF-α. Results are presented as percentage of IFN-γ or TNF-α-producing CD4⁺ or CD8⁺ T lymphocytes^. Bars are means ± SEM. Each dot is an individual mouse. *p < 0.05, **p < 0.01, ***p < 0.001 vs. placebo. Kruskal–Wallis. d Splenocytes from immunized mice were stimulated with medium or recombinant JN.1 RBD for 3 d and then IL-5 was measured in the supernatant by ELISA. Results are presented as concentration of IL-5 in pg/ml. Bars are means ± SEM. Each dot is an individual mouse. *p < 0.05, ***p < 0.001 vs. placebo. Kruskal–Wallis. e Lung cells obtained from immunized mice were stimulated with medium or recombinant JN.1 RBD for 3 days and then IFN-γ, TNF-α and IL-5 were measured in the supernatant by ELISA. Results are presented as cytokine concentration in pg/ml. Bars are means ± SEM. Each dot is an individual mouse. *p < 0.05, **p < 0.01 vs. placebo. Kruskal–Wallis.

Fig. 5. Monovalent and bivalent adapted JN.1 vaccines induce nAbs against JN.1 and KP.2, KP.3 and XEC subvariants in booster immunization schedules.

BALB/c mice were i.m. immunized on days 0 and 14 with mRNA XBB.1.5 vaccine (Spikevax) and boosted on day 75 with: ARVAC JN.1 (n = 6), ARVAC XBB.1.5 (n = 6), ARVAC Gamma/Omicron BA.4/5 (n = 6) and ARVAC Gamma/JN.1 (n = 6) or Spikevax XBB.1.5 (n = 6). a Neutralizing-antibody titers against XEC, KP.2, KP.3, JN.1, XBB.1.5, Omicron BA.5, Gamma and Ancestral SARS-CoV-2 were evaluated at 75 (preboost) and 103 dpp (postboost). Neutralization titer was defined as the serum dilution that reduces 50% the cytopathic effect (NT50). Bars are GMT ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Kruskal–Wallis test. b Radar charts were drawn based on the geometric mean titers (GMTs) of neutralizing antibody in serum pre and post boost against live viruses for each vaccinated group.