Cross-clade vaccination to overcome sarbecovirus or merbecovirus neutralization gaps

Figures

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  Figure 1.. Multi-family multiplex surrogate virus neutralization test (A) Schematic illustration of the MM-sVNT platform, capable of detecting neutralizing antibodies against sarbecoviruses, merbecoviruses, and henipaviruses in a single reaction. (B–D) Phylogenetic trees of (B) sarbecovirus RBDs, (C) merbecovirus RBDs, and (D) henipavirus glycoproteins, generated using the general-time reversible (GTR) model with 1,000 bootstrap replicates. Bootstrap values > 70% are shown above or below the branches. Scale bars represent the number of substitutions per site. Viruses included in the MM-sVNT panel are indicated in blue. (E) Mean fluorescence intensity (MFI) of sarbecovirus, merbecovirus, and henipavirus RBD/glycoprotein binding to human ACE2, DPP4, and ephrin-B2 receptors, respectively. (F) Specificity of the MM-sVNT platform, tested using naïve healthy human serum samples. A cut-off dilution of 1:100 was used; samples with NT50 values below this threshold were considered negative. (G) Heatmap showing sVNT IC50 values for 12 published monoclonal antibodies against sarbecoviruses, merbecoviruses, and henipaviruses.

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  Figure 2.. Limited cross-neutralization against antigenically distinct viruses (A–C) MM-sVNT analysis was performed using serum samples collected from (A) SARS-CoV-2-vaccinated individuals, (B) SARS survivors, and (C) SARS survivors vaccinated against COVID-19. (D) A heatmap showing the percentage of amino acid identity between sarbecovirus RBDs. (E–I) Impact of the XBB booster vaccine on cross-neutralizing antibodies against SARS-CoV-2 and animal sarbecoviruses. MM-sVNT analysis was also conducted using serum samples from (F) MERS-CoV-infected and (H) NiV-infected individuals. The percentage of amino acid identity between (G) merbecoviruses and (I) henipaviruses is shown in the corresponding heat maps. Data presented in (A)–(C), (E), (F), and (H) were log10 NT50. Geometric mean NT50 values are indicated above each box plot. Samples with an NT50 value greater than 1:100 were considered positive. Statistical analysis in (A), (B), (C), (F), and (H) was performed using two-tailed unpaired t test in GraphPad Prism 10.

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  Figure 3.. Lack of cross-neutralizing antibodies against sarbecoviruses, merbecoviruses, and henipaviruses in samples collected prior to the COVID-19 pandemic MM-sVNT analysis was performed using serum samples collected before the COVID-19 pandemic from Southeast Asia (SEA), New Zealand (NZ), and Tanzania (TZ), diluted 1:20, to assess neutralization against (A) sarbecoviruses, (B) merbecoviruses, (C) henipaviruses, and (D) NeoCoV. Data are presented as violin plots, with the percentage of samples showing >40% inhibition indicated above each plot. Statistical analysis in (A), (B), and (C) was performed using two-tailed unpaired t test in GraphPad Prism 10. Two NeoCoV-positive samples were further analyzed by (E) a merbecovirus-specific MM-sVNT and (F) western blot. Western blot analysis was performed using serum diluted 1:400.

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  Figure 4.. Cross-clade prime-and-boost regime induces broad-spectrum neutralizing antibodies (A) Coronavirus family tree and schematic of the pan-sarbecovirus and pan-merbecovirus cross-clade prime-and-boost vaccination strategy. (B) Illustration of the proposed mechanism underlying the pan-sarbecovirus vaccine. (C) Graphical overview of the animal experiment involving a control group receiving either three or four doses of BNT162b2 and a test group receiving pan-sarbecovirus booster vaccines, C1.26 and C1.28. (D) MM-sVNT analysis of serum samples collected 14 days after the third and fourth booster doses. Error bars represent standard error; data are shown as mean NT50 values from five mice. (E) Graphical overview of the animal experiment involving the pan-merbecovirus vaccination strategy. Mice were primed with 10 μg of subunit homologous vaccine at day 0, 21 and 49, and boosted with 20 μg heterologous subunit vaccine. (F) Multiplex analysis of serum samples collected 14 days after the third and fourth booster doses. Error bars represent standard error; data are shown as mean NT50 values from five mice. (G) sVNT analysis of mice receiving pan-merbecovirus vaccines against BtHKU5-CoV-2.…