Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice

Design and expression of chimeric spike constructs to cover pandemic and zoonotic SARS-related coronaviruses

Sarbecoviruses exhibit considerable genetic diversity (Fig. 1A), and SARS-like bat CoVs (Bt-CoVs) are recognized threats to human health (6, 8). Because potent neutralizing antibody epitopes exist in each of the modular structures on CoV spikes (21), we hypothesized that chimeric spikes that encode NTD, RBD, and S2 domains into “bivalent” and “trivalent” vaccine immunogens have the potential to elicit broad protective antibody responses against clades I to III Sarbecoviruses. We designed four sets of chimeric spikes. Chimera 1 included the NTD from clade II Bt-CoV Hong Kong University 3-1 (HKU3-1), the clade I SARS-CoV RBD, and the clade III SARS-CoV-2 S2 (Fig. 1B). Chimera 2 included SARS-CoV-2 RBD and SARS-CoV NTD and S2 domains (11). Chimera 3 included the SARS-CoV RBD and SARS-CoV-2 NTD and S2, whereas chimera 4 included the RsSHC014 RBD and SARS-CoV-2 NTD and S2. We also generated a monovalent SARS-CoV-2 spike furin knockout (KO) vaccine, partially phenocopying the Moderna and Pfizer mRNA vaccines in human use, and a negative control norovirus GII capsid vaccine (Fig. 1, B and C). We generated these chimeric spikes and control spikes as lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines with LNP adjuvants (mRNA-LNP), as described previously (22). This mRNA LNP stimulates robust T follicular helper cell activity, germinal center B cell responses, durable long-lived plasma cells, and memory B cell responses (23, 24). We verified their chimeric spike expression in human embryonic kidney (HEK) cells (fig. S1B). To confirm that scrambled coronavirus spikes are biologically functional, we also designed and recovered several high-titer recombinant live viruses of RsSHC014/SARS-CoV-2 NTD, RBD, and S2 domain chimeras that included deletions in nonessential, accessory open reading frame 7 (ORF7) and ORF8 that encoded nanoluciferase (fig. S1C). SARS-CoV-2 ORF7 and -8 antagonize innate immune signaling pathways (25, 26), and deletions in these ORFs are associated with attenuated disease in humans (27, 28).

Immunogenicity of mRNAs expressing chimeric spike constructs against coronaviruses

We next sought to determine whether simultaneous immunization with mRNA-LNP expressing the chimeric spikes of diverse Sarbecoviruses was a feasible strategy to elicit broad binding and neutralizing antibodies. We immunized aged mice with the chimeric spikes formulated to induce cross-reactive responses against multiple divergent clades I to III Sarbecoviruses, a SARS-CoV-2 furin KO spike, and a GII.4 norovirus capsid negative control. Group 1 was primed and boosted with chimeric spikes 1, 2, 3, and 4 (fig. S1A). Group 2 was primed with chimeric spikes 1 and 2 and boosted with chimeric spikes 3 and 4 (fig. S1A). Group 3 was primed and boosted with chimeric spike 4 (fig. S1A). Group 4 was primed and boosted with the monovalent SARS-CoV-2 furin KO spike (fig. S1A). Last, group 5 was primed and boosted with a norovirus capsid GII.4 Sydney 2011 strain (fig. S1A). We then examined the binding antibody responses by means of enzyme-linked immunosorbent assay (ELISA) against a diverse panel of CoV spike proteins that included epidemic, pandemic, and zoonotic coronaviruses.

Mice in groups 1 and 2 generated the highest-magnitude responses to SARS-CoV Toronto Canada isolate (Tor2), RsSHC014, and HKU3-1 spike as compared with group 4 (Fig. 2, A, G, and H). Whereas mice in group 2 generated lower-magnitude binding responses to both SARS-CoV-2 RBD (Fig. 2C) and SARS-CoV-2 NTD (Fig. 2D), mice in group 1 generated similar-magnitude binding antibodies to SARS-CoV-2 D614G (in which aspartic acid at position 614 is replaced with glycine) as compared with that of mice immunized with the SARS-CoV-2 furin KO spike mRNA-LNP (Fig. 2B). Mice in groups 1 and 2 generated similar-magnitude binding antibody responses against SARS-CoV-2 D614G, Pangolin GXP4L, and RaTG13 spikes (Fig. 2, B, E, and F) compared with those of mice from group 4. Mice in group 1 and group 4 elicited high-magnitude levels of hACE2 blocking responses, as compared with those of groups 2 and 3 (Fig. 2J). Because binding antibody responses after boost mirrored the trend of the after-prime responses, it is likely that the second dose is boosting immunity to the vaccine antigens in the prime (Fig. 2). Last, we did not observe cross-binding antibodies against common-cold CoV spike antigens from HCoV-HKU1, HCoV-NL63, and HCoV-229E in most of the vaccine groups (fig. S2, A to D), but we did observe low binding levels against more distant group 2C MERS-CoV (Fig. 2I) and other Betacoronaviruses such as group 2A HCoV-OC43 in vaccine groups 1 and 2 (fig. S2B). These results suggest that chimeric spike vaccines elicit broader and higher-magnitude binding responses against pandemic and bat SARS-like viruses as compared with those of monovalent SARS-CoV-2 spike vaccines.

Neutralizing antibody responses against live Sarbecoviruses and variants of concern

We then examined the neutralizing antibody responses against SARS-CoV, Bt-CoV RsSHC014, Bt-CoV WIV-1, and SARS-CoV-2 including variants of concern using live viruses as previously described (Fig. 3, A to D) (17). Group 4 SARS-CoV-2 S mRNA–vaccinated animals mounted a robust response against SARS-CoV-2; however, responses against SARS-CoV, RsSHC014, and WIV-1 were 18-, >300- or 116-fold decreased, respectively (Fig. 3, A to D, and fig. S3, G and H). By contrast, aged mice in group 2 showed a 42- and twofold increase in neutralizing titer against SARS-CoV and WIV1 and less than onefold decrease against RsSHC014 relative to SARS-CoV-2 neutralizing titers (Fig. 3, A to D, and fig. S3, C and D). Mice in group 3 elicited thee- and sevenfold increased neutralizing titers against SARS-CoV and RsSHC014 yet showed a threefold decrease in WIV-1 neutralizing titers relative to SARS-CoV-2 (Fig. 3, A to D, and fig. S3, E and F). Last, mice in group 1 generated the most balanced and highest neutralizing titers, which were 13- and 1.2-fold increased against SARS-CoV and WIV-1 and less than onefold decreased against RsSHC014 relative to the SARS-CoV-2 neutralizing titers (Fig. 3, A to D, and fig. S3, A and B). The serum of mice from groups 1 and 4 neutralized the dominant D614G variant with similar potency as that of the wild-type D614 nonpredominant variant, and both groups had similar neutralizing antibody responses against the UK B.1.1.7 and the mink cluster 5 variant as compared with the D614G variant (Fig. 3, E and F). Despite the significant but small reduction in neutralizing activity against the B.1.351 variant of concern (VOC), we did not observe a complete ablation in neutralizing activity in either group. Mice from groups 1 and 2 elicited lower binding and neutralizing responses to SARS-CoV-2 as compared with those of group 4, perhaps reflecting a decreased amount of mRNA vaccine incorporated into multiplexed formulations; the monovalent vaccines may drive a more focused B cell response to SARS-CoV-2, whereas chimeric spike antigens lead to more breadth against distant Sarbecoviruses. Thus, both monovalent SARS-CoV-2 vaccines and multiplexed chimeric spikes elicit neutralizing antibodies against newly emerged SARS-CoV-2 variants, and multiplexed chimeric spike vaccines outperform the monovalent SARS-CoV-2 vaccines in terms of breadth against multiclade Sarbecoviruses.

In vivo protection against heterologous Sarbecovirus challenge

To assess the ability of the mRNA-LNP vaccines to mediate protection against previously epidemic SARS-CoV, pandemic SARS-CoV-2, and Bt-CoVs, we challenged the different groups and observed the mice for signs of clinical disease. Mice from group 1 or group 2 were completely protected from weight loss and lower- and upper-airway virus replication as measured with infectious virus plaque assays after 2003 SARS-CoV mouse-adapted (MA15) challenge (Fig. 4, A, B, and C). Similarly, these two vaccine groups were also protected against SARS-CoV-2 mouse-adapted (MA10) challenge. By contrast, group 3 showed some protection against SARS-CoV MA15–induced weight loss but not against viral replication in the lung or nasal turbinates. Group 3 was fully protected against SARS-CoV-2 MA10 challenge. By contrast, group 5 vaccinated mice developed severe disease, including mortality in both SARS-CoV MA15 and SARS-CoV-2 MA10 infections (fig. S5, B and C). Monovalent SARS-CoV-2 mRNA vaccines were highly efficacious against SARS-CoV-2 MA10 challenge but failed to protect against SARS-CoV MA15–induced weight loss and replication in the lower and upper respiratory tract (Fig. 4, A, B, and C), suggesting that SARS-CoV-2 mRNA-LNP vaccines are not likely to protect against future SARS-CoV emergence events. Mice from groups 1 to 4 were completely protected from weight loss and lower airway SARS-CoV-2 MA10 replication (Fig. 4, D, E, and F). Using both a Bt-CoV RsSHC014 full-length virus and a more virulent RsSHC014-MA15 chimera in mice (6), we also demonstrated protection in groups 1 to 3 against RsSHC014 replication in the lung and nasal turbinates (fig. S4) but not in mice that received the SARS-CoV-2 mRNA vaccine. Group 5 control mice challenged with RsSHC014-MA15 developed disease, including mortality (fig. S5D). Group 3 mice, which received a SARS-CoV-2 NTD/RsSHC014 RBD/SARS-CoV-2 S2, were fully protected against both SARS-CoV-2 and RsSHC014 challenge, whereas group 4 mice were not, demonstrating that a single NTD and RBD chimeric spike can protect against more than one virus compared with a monovalent spike.

We then performed a heterologous challenge experiment with the bat preemergent WIV-1-CoV (7). Mice from groups 1 and 2 were fully protected against heterologous WIV-1 challenge, whereas mice that received the SARS-CoV-2 mRNA vaccine had breakthrough replication in the lung (Fig. 4, G, H, and I). We also challenged with a virulent form of SARS-CoV-2 VOC B.1.351, which contains deletions in the NTD and mutations in the RBD, and observed full protection in vaccine groups 1, 2, and 4 compared with that in controls, whereas breakthrough replication was observed in group 3, further indicating the importance of the NTD in vaccine-mediated protection (Fig. 4, J, K, and L). The reduced protection against the B.1.351 variant containing NTD deletions indicates that the NTD is a clear target of protective immunity and that its inclusion in vaccination strategies, as opposed to RBD-alone vaccines, may be required to achieve full protection. Moreover, the SARS-CoV-2 mRNA vaccine protected against SARS-CoV-2 B.1.351 challenge in aged mice despite a reduction in the neutralizing activity against this VOC.

Lung pathology and cytokines in mRNA-LNP–vaccinated mice challenged with epidemic and pandemic coronaviruses

To quantify the pathological features of acute lung injury (ALI) in mice, we used a tool from the American Thoracic Society (ATS). We similarly scored lung tissue sections for diffuse alveolar damage (DAD), the pathological hallmark of ALI (29, 30). We observed significant lung pathology with both the ATS and DAD scoring tools in groups 4 and 5 vaccinated animals. By contrast, multiplexed chimeric spike vaccine formulations in groups 1 and 2 provided complete protection from lung pathology after SARS-CoV MA15 challenge (Fig. 5, A and B). Mice immunized with the SARS-CoV-2 mRNA vaccine that showed breakthrough infection with SARS-CoV MA15 developed similar lung inflammation as that of control vaccinated animals, potentially suggesting that future outbreaks of SARS-CoV may cause disease even in individuals vaccinated with SARS-CoV-2. Because eosinophilic infiltrates have been observed in vaccinated, 2003 SARS-CoV–challenged mice previously (31), with immunohistochemistry we analyzed lung tissues in protected versus infected animals with SARS-CoV MA15 for eosinophilic infiltrates (fig. S6). Groups 1 and 2 contained rare, scattered eosinophils in the interstitium. Group 3 showed bronchus-associated lymphoid tissue. By contrast, group 4 and group 5 contained frequent perivascular cuffs with prevalent eosinophils. All groups challenged with SARS-CoV-2 MA10 were protected against lung pathology compared with the norovirus capsid-immunized control group, supporting the hypothesis that the SARS-CoV-2 NTD present in the chimeric spike from group 3 is sufficient for protection (Fig. 5, C and D).

We measured lung proinflammatory cytokines and chemokines in the different vaccination groups. Groups 1 and 2 had baseline levels of macrophage-activating cytokines and chemokines, including interleukin-6 (IL-6), chemokine (C-C motif) ligand 2 (CCL2), IL-1α, granulocyte colony-stimulating factor (G-CSF), and CCL4, compared with group 5 after SARS-CoV MA15 challenge (fig. S7A). Group 3 and group 4 showed high and indistinguishable levels of IL-6, CCL2, IL-1α, G-CSF, and CCL4 compared with those of group 5 mice after SARS-CoV MA15 challenge. After SARS-CoV-2 MA10 challenge, group 4 and group 1 showed the lowest levels of IL-6 and G-CSF relative to that in group 5 controls (fig. S7B), and we only observed significant reductions in CCL2, IL-1α, and CCL4 lung levels in groups 3 and 4 compared with the group 5 control, despite full protection from both weight loss and lower-airway viral replication.