Vaccination with ancestral SARS-CoV-2 spike adjuvanted with TLR agonists provides cross-protection against XBB.1

Abstract

Many different platforms have been used to develop highly protective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans. However, protection has eroded over time due to the emergence of antigenically diverse viral variants, especially the Omicron subvariants. One successful platform for the generation of SARS-CoV-2 vaccines are recombinant spike protein vaccines, of which two are licensed in the United States and Europe. Typically, purified recombinant protein antigens are poorly immunogenic and adjuvants must be included in the formulation. Here, we adjuvanted recombinant ancestral SARS-CoV-2 Wuhan-Hu-1 spike proteins with an emulsion formulation combined with synthetic Toll-like receptor (TLR) 4 and 7/8 agonists. This combination led to the induction of a Th1-skewed immune response that included high titers of antibodies against Wuhan-Hu-1 spike. These serum antibodies included neutralizing and cross-reactive antibodies that recognized the spike from multiple SARS-CoV-2 variants, as well as the receptor binding domain (RBD) from SARS-CoV-1. Despite an absence of robust cross-neutralization, vaccination against Wuhan-Hu-1 spike in the context of TLR-containing emulsions provided complete cross-protection against disease from a lethal challenge with XBB.1 in a stringent K18-hACE2 mouse model. We believe that the combination of recombinant spike antigens with TLR agonist-based emulsion formulations could lead to the development of next-generation SARS-CoV-2 vaccines that provide significant protection from future emerging variants.

Fig. 1. Characterization of AS03-like emulsions, with and without TLR agonists.

A The concentrations of squalene, INI-2002, and INI-4001 in each starting emulsion created for these studies, and the mean particle size and polydispersity index for each. B Cryo-TEM images of each emulsion shown in (A), plus the resulting emulsion after mixing of the INI-4001 and INI-2002 emulsions. C Example UHPLC chromatograph showing the distinct peaks for INI-2002, INI-4001, DL-α-tocopherol, and squalene.

Fig. 2. Including TLR agonists in emulsion-based adjuvants promotes development of Th1 and Th17 cells and inhibits Th2.

A Cells from draining LNs (dLN) or spleen (SPL) isolated 21 days after booster vaccination were cultured with Wuhan-Hu-1 or XBB.1 spike for 72 h. Culture supernatants were assayed for cytokines by multiplex ELISA. B The same cell populations were cultured with Wuhan-Hu-1 spike protein and pooled overlapping spike peptides for 18 h, then analyzed for T cell cytokine production by flow cytometry. Differences between groups were analyzed by one-way ANOVA with correction for multiple comparisons by Tukey post-test. Asterisks denote significant differences versus unadjuvanted Spike antigen and # indicate differences between other groups. *^/#p < 0.05, ^##p < 0.01, ^***/###p < 0.001, ****^/####p < 0.0001.

Fig. 3. Addition of TLR agonists to emulsion adjuvants boosts the spike-specific antibody titers, increases the ratio of IgG2c to IgG1, and promotes cross-recognition of variants.

A Serum antibody IgG titers against recombinant Wuhan-Hu-1 spike protein at 21 days after booster vaccination. B The mean AUC value for IgG2c was divided by that of IgG1 for each group. C Serum antibody IgG titers against recombinant spike RBD from the indicated virus. D Control ELISA against his-tagged neuraminidase (NA) from influenza A virus (IAV); dotted line indicates the limit of detection. Differences between groups were analyzed by one-way ANOVA with multiple comparisons by Tukey post-test. Asterisks denote significant differences versus unadjuvanted spike antigen and # indicate differences between other, indicated groups. *^/#p < 0.05, ^##p < 0.01, *^**/###p < 0.001, ****^/####p < 0.0001.

Fig. 4. Neutralization potential of serum from vaccinated mice.

A Serum collected 21 days after booster vaccination (day 42) was used in a microneutralization assay against strain WA1/2020 (gray) and Omicron XBB.1 (green). Anti-viral drug remdesivir was used as a positive control. Each symbol represents pooled sera from two animals. The dotted line represents the limit of detection. B, C Serum collected 21 days after booster vaccination (day 42) was tested for the ability to block the binding of soluble human ACE2 to recombinant spike trimers from the indicated strains by multiplex competitive ELISA. Serum was diluted 1:500 in (B), and 1:100 in (C). See also Supplementary Fig. 4 for data with additional Omicron variants. Differences between groups were analyzed by one-way ANOVA with multiple comparisons by Tukey post-test. Asterisks denote significant differences versus unadjuvanted spike antigen and #’s indicate differences between other indicated groups. *^/#p < 0.05, ^##p < 0.01, *^**/###p < 0.001, ****^/####p < 0.0001.

Fig. 5. K18-hACE2 mice vaccinated with WA1/2020 spike adjuvanted with emulsions containing TLR agonists were better protected against weight loss after infection with Omicron variant XBB.1.

A Schematic of experimental timeline (created with BioRender.com). B Weight change relative to starting weight (on day of infection), as recorded daily after infection with WA1/2020 (left) or XBB.1 (right). Dotted line indicates weight loss that requires humane euthanasia. Error bars in the WA1/2002 challenge represent the SEM; these were omitted from XBB.1 for clarity. Statistical significance determined by unpaired t-test with Welch correction at each time point, with Holm-Sidak multiple comparison correction. *indicates significance versus the PBS treated control, # versus the Spike only control, and § versus the emulsion group. *^/#/§p < 0.05, **^/##/§§p < 0.01^{, ***/###}p < 0.001, ****^/####p < 0.0001. C Survival of mice as a percentage of total mice in each group, recorded daily following infection.