key: cord-0983102-7pvphbt2
authors: Saunders, Kevin O.; Lee, Esther; Parks, Robert; Martinez, David R.; Li, Dapeng; Chen, Haiyan; Edwards, Robert J.; Gobeil, Sophie; Barr, Maggie; Mansouri, Katayoun; Alam, S. Munir; Sutherland, Laura L.; Cai, Fangping; Sanzone, Aja M.; Berry, Madison; Manne, Kartik; Kapingidza, Anyway B.; Azoitei, Mihai; Tse, Longping V.; Scobey, Trevor D.; Spreng, Rachel L.; Rountree, R. Wes; DeMarco, C. Todd; Denny, Thomas N.; Woods, Christopher W.; Petzold, Elizabeth W.; Oguin, Thomas H.; Sempowski, Gregory D.; Gagne, Matthew; Douek, Daniel C.; Tomai, Mark A.; Fox, Christopher B.; Seder, Robert; Wiehe, Kevin; Weissman, Drew; Pardi, Norbert; Acharya, Priyamvada; Andersen, Hanne; Lewis, Mark G.; Moore, Ian N.; Montefiori, David C.; Baric, Ralph S.; Haynes, Barton F.
title: SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys
date: 2021-02-17
journal: bioRxiv
DOI: 10.1101/2021.02.17.431492
sha: 20d445a317cb09ea2f1cdde45fa48b7523ff7241
doc_id: 983102
cord_uid: 7pvphbt2

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

addition of mutations from mink strains of SARS-CoV-2, the South African B1.351, Brazilian P.1 138 strain, and UK B.1.1.7 strains did not change binding magnitude for DH1047 nor macaque 139 plasma IgG (Fig. 2c,d) . However, the addition of these mutations slightly increased ACE2 140 binding compared to D614G alone (Fig. 2c) . Next we tested binding to recombinant RBD 141 monomers with and without the K417N, E484K, and N501Y mutations present in the B1.351 142 variant. ACE2 binding was decreased by K417N and increased by N501Y, while the RBD with 143 all three mutations showed no change in ACE2 binding (Fig. 2e) . RBD neutralizing antibody 144 DH1041 is focused on the ACE2 binding site, and was knocked out by the E484K change ( Fig.   145 2e). Importantly, the binding to SARS-CoV-2 RBD for cross-reactive antibody DH1047 was 146 unchanged to RBDs with E484K or other mutations (Fig. 2f) . Also, RBD-scNP-vaccinated 147 macaque plasma IgG and S-2P mRNA-LNP-immunized macaque plasma IgG were unaffected 148 by the RBD mutations (Fig. 2e,f) . 41 batCoV-SHC014 (Fig. 3a, Extended data Fig. 2) . Neutralization was more potent for 159 replication-competent SARS-CoV-2 virus compared to the other three SARS-related viruses 160 ( Fig. 3a, Extended data Fig. 2) . Among the three immunogens, RBD-scNP elicited the highest 161 neutralization titers and mRNA-LNP expressing monomer RBD elicited the lowest neutralization 162 titers (Fig. 3a, Extended data Fig. 2) . Also, RBD-scNP immunization elicited cross-reactive IgG binding against SARS-CoV-2, SARS-CoV-1, batCoV-RaTG13, batCoV-SHC014, pangolin CoV-164 GXP4L Spike proteins ( Fig. 3c and Extended data Figs. 3a,c). RBD-scNP immune plasma IgG 165 did not bind the S ectodomain of four endemic human CoVs, nor did it bind MERS-CoV S 166 ectodomain (Extended data figs. 3a,c) . The lack of binding by plasma IgG to these latter five S 167 ectodomains was consistent with RBD sequence divergence among groups 1, 2a, 2b and 2c 168 coronaviruses ( Fig. 3e and Extended data figs. 4-6) . Nonetheless, the SARS-CoV-2 spike 169 induced cross-reactive antibodies against multiple group 2b SARS-related betaCoVs, with the 170 highest titers induced by RBD-scNP.

The immune sera from RBD-scNP-immunized exhibited a similar cross-neutralizing profile as 173 the cross-neutralizing antibody DH1047. DH1047 binds with <0.02 nM affinity to monomeric 174 SARS-CoV-2 RBD (Extended data fig. 3b) , and bound the RBD-scNP (Fig. 1b) . DH1047 (Fig. 3d) . Antibodies targeting near the DH1047 epitope would be predicted to be 180 cross-reactive with group 2b betaCoVs given the high sequence conservation present in and 181 immediately proximal to the DH1047 epitope (Fig. 3e) . Comparison of RBD sequences showed 182 them to be relatively conserved within betaCoV group 2b, but minimally conserved between 183 groups 2b and 2c ( Fig. 3f and Extended data figs. 4-6). To examine whether RBD-scNP-184 induced antibodies bound near the DH1047 epitope, we assessed plasma antibody blocking of 185 DH1047 binding to SARS-CoV-2 S-2P ectodomain. Plasma from all RBD-scNP immunized 186 macaques blocked the binding of ACE2 and DH1047 to SARS-CoV-2 S-2P ectodomain (Fig. 3g   187 and Extended data fig. 3d ). Since DH1047-blocking plasma antibodies could be SARS-CoV-2-188 specific, we also examined plasma IgG blocking of DH1047 binding to batCoV-SHC014. Similar to SARS-CoV-2 S binding, RBD-scNP plasma also blocked DH1047 binding to batCoV-SHC014 190 (Fig. 3g) . While 96% of COVID-19 patients made antibodies that blocked ACE2 as a dominant 191 RBD response, only 31% made antibodies that blocked the cross-reactive antibody, DH1047 192 ( Fig. 3h) . Thus, in natural SARS-CoV-2 infection the cross-reactive DH1047 IgG blocking 193 response is subdominant, and RBD-scNP vaccination focused antibody responses to this 194 subdominant cross-reactive neutralizing epitope.

Finally, to determine whether vaccination elicited coronavirus protective immunity, we 197 challenged RBD scNP-vaccinated monkeys with 10 5 plaque forming units of SARS-CoV-2 virus 198 (Fig 4a) . To assess virus replication in the upper and lower respiratory tract, E or N sgRNA was 199 quantified in fluid from nasal swabs and bronchoalveolar lavage (BAL) two and four days after 200 challenge (Fig 4a) . In unimmunized macaques two days after challenge, there were on average 201 1.3x10 5 and 1.2x10 4 copies/mL of E gene sgRNA in the nasal swab and BAL fluids, respectively 202 ( Fig. 4b,c) . In contrast, vaccinated monkeys had undetectable levels of subgenomic envelope E 203 gene RNA in the upper and lower respiratory tract (Fig. 4b,c) . We sampled monkeys again 2 204 days later to determine if detectable virus replication was present, but again found no detectable 205 E gene sgRNA in any monkey BAL or nasal swab samples (Fig. 4b,c) . Similarly, all RBD-206 scNP-vaccinated macaques had undetectable N gene sgRNA in BAL and the nasal swab fluid, 207 except one macaque that had 234 copies/mL of N gene sgRNA detected on day 2 in nasal 208 swab fluid (Fig. 4d,e) . Viral replication was undetectable in this macaque by the fourth day after 209 challenge ( Fig. 4d,e) . Thus, RBD-scNP-induced immunity prevented virus replication, and likely 210 provided sterilizing immunity, in the upper and lower respiratory tract in all but one macaque.

This study demonstrates that immunization with SARS-CoV-2 Spike either as a protein RBD-213 scNP or as an mRNA-LNP elicits a cross-reactive antibody response capable of neutralizing 214 multiple SARS-related human and bat betaCoVs. These results demonstrate that SARS-CoV-2 vaccination with either the RBD-scNP or the stabilized transmembrane spike mRNA-LNP 216 vaccines currently approved for use in humans, will likely elicit cross-neutralizing antibodies with 217 the potential to prevent future animal CoV spillover events to humans 30, 42, 45 

Phase I clinical study using 3M-052/Alum to induce neutralizing antibody responses to an HIV 232 vaccine candidate is underway (NCT04177355). Thus, this vaccine modality represents a 233 promising first-generation pan-group 2b betaCoV vaccine with the potential to durably inhibit 234 future zoonotic transmission 25 .

The emergence of SARS-CoV-2 neutralization-resistant and highly infectious variants continues 237 to be a concern for vaccine efficacy. We found here that protein nanoparticle or mRNA-LNP 238 SARS-CoV-2 spike immunization elicited SARS-CoV-2 neutralizing antibodies capable of 239 neutralizing the predominant SARS-CoV-2 variant D614G as well as the newly-emerged B. 

Representative results from two independent experiments are shown. 

Processing of negative stain images. The RELION 3.0 program was used for all negative 648 stain image processing. Images were imported, CTF-corrected with CTFFIND, and particles 649 were picked using a spike template from previous 2D class averages of spike alone. Extracted 650 particle stacks were subjected to 2-3 rounds of 2D class averaging and selection to discard junk 651 particles and background picks. Cleaned particle stacks were then subjected to 3D classification 652 using a starting model created from a bare spike model, PDB 6vsb, low-pass filtered to 30 Å.

Classes that showed clearly-defined Fabs were selected for final refinements followed by sgRNA E (copies/mL) 10 2 10 3 10 4 10 5 10 6 10 7 10 8 sgRNA E (copies/mL)

Comparison of serum neutralization ID50 titers from cynomolgus macaques immunized with 774 recombinant protein RBD sortase conjugated nanoparticles (RBD-scNP; blue) or nucleoside-775 modified mRNA-LNP expressing S-2P (burgundy)

Horizontal bars are the group geometric mean (**P<0.01, Two-tailed Exact Wilcoxon

Fold decrease in neutralization potency between neutralization of SARS-CoV-2 D614G and 779

Fold change is shown for RBD-scNP-immunized and 780 mRNA-LNP-immunized macaques based ID50 (left) and ID80 (right) titers

ACE2 receptor and cross-neutralizing antibody DH1047 ELISA binding to SARS-CoV-2 Spike 783 ectodomain (ECD) based on a Danish mink

RBD-scNP and S-2P mRNA-LNP-immunized macaque serum IgG ELISA binding to SARS-788

CoV-2 Spike variants shown in c. Serum was tested after two immunizations. Horizontal bars 789 are the group mean

cross-neutralizing antibody DH1047 (navy), and ACE2 binding site-791 targeting neutralizing antibody DH1041 (green) ELISA binding to SARS-CoV-2 Spike RBD 792 monomers

Titers are shown as area under the log-transformed curve (log AUC)

RBD-scNP and S-2P mRNA-LNP-immunized macaque serum IgG ELISA binding to SARS-795

CoV-2 Spike RBD variants shown in e