SHC014 spike to mediate infection of the human airway, we examined 2B4 Calu-3 cells, a human epithelial airway cell line8 , and found robust SHC014-MA15 replication comparable to SARS-CoV Urbani (Fig. 1c). To extend these findings, primary human airway epithelial cultures (HAEs) were infected and indicated robust replication of both viruses (Fig. 1d). Together, the data confirm the ability Menachery et al. Page 2 Nat Med. Author manuscript; available in PMC 2016 June 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript of SHC014 spike to infect human airway cells and underscore the threat of cross-species transmission. We next evaluated in vivo infection of 10-week old BALB/c mice with 104 plaque-forming units (PFU) of either SARS-MA15 or SHC014-MA15 (Fig. 1e–h). Animals infected with SARS-MA15 experienced rapid weight loss and lethality by four days post infection (DPI); in contrast, SHC014-MA15 produced substantial weight loss (10%), but no lethality (Fig. 1e). Examination of viral replication revealed nearly equivalent titers from lungs of mice infected with SARS-MA15 and SHC014-MA15 (Fig. 1f). While SARS-CoV MA15 produced robust staining in both the terminal bronchioles and the lung parenchyma 2 DPI (Fig. 1g), SHC014-MA15 had a deficit in airway antigen staining (Fig. 1h). In contrast, no equivalent deficit was observed in the parenchyma or overall histology scoring, suggesting differential infection following SHC014-MA15 (Supplementary Table 2). Shifting to more susceptible aged animals, SARS-MA15 infected animals rapidly lost weight and succumb to infection (Supplementary Fig. 3 a, b); SHC014-MA15 induced robust and sustained weight loss, but had minimal lethality. Histology and antigen staining trends observed in young mice were conserved in the older animals (Supplementary Table 3). We excluded use of an alterative receptor based on Ace2−/− mice infection, which did not produce weight loss or antigen staining following SHC014-MA15 infection (Supplementary Fig. 4a, b; Supplementary Table 2). Together, the data indicate that viruses utilizing SHC014 spike are capable of inducing considerable disease in mice in the context of a virulent CoV backbone. Given the efficacy of Ebola monoclonal antibody therapies like ZMApp9 , we next sought to determine the efficacy of SARS-CoV monoclonal antibodies against SHC014-MA15. Four broadly neutralizing human monoclonal antibodies had been previously reported and are likely reagents for immunotherapy10–12. Examining percent inhibition, wild-type SARSCoV Urbani was strongly neutralized by all four antibodies at relatively low antibody concentrations (Fig. 2a–d). In contrast, neutralization varied for SHC014-MA15. Fm6, an antibody generated by phage display and escape mutants10,11, achieved only background levels of inhibition of SHC014-MA15 (Fig. 2a). Similarly, antibodies 230.15 and 227.14, derived from memory B cells of SARS-CoV infected patients12, also failed to block SHC014-MA15 (Fig. 2b, c). For all three antibodies, differences between SARS and SHC014 spikes corresponded to direct or adjacent residue changes found in escape mutants (fm6 - N479R; 230.15 - L443V; 227.14- K390). Finally, monoclonal antibody 109.8 was able to achieve 50% neutralization of SHC014-MA15, but only at very high concentrations (Fig. 2d). Together, the results demonstrate that despite the development of broadly neutralizing antibodies against SARS-CoV, these reagents may only have marginal efficacy against emergent SARS-like CoV strains like SHC014. To evaluate existing vaccines against SHC014-MA15, aged mice were vaccinated with double-inactivated whole SARS-CoV (DIV). Previously, DIV had shown neutralization and protection from homologous virus challenge13, but vaccine failure and augmented immune pathology in aged animals indicated a possibility for harm due to vaccination14. In this study, DIV provided no protection from SHC014-MA15 in regards to weight loss or viral titer (Supplementary Fig. 5a, b). Consistent with previous reports14, serum from DIVvaccinated aged mice also failed to neutralize SHC014-MA15 (Supplementary Fig. 5c). Menachery et al. Page 3 Nat Med. Author manuscript; available in PMC 2016 June 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Perhaps most importantly, DIV vaccination resulted in robust immune pathology (Supplementary Table 4) and eosinophilia (Supplementary Fig. 5d–f). Together, these results confirm DIV vaccine failure and illustrated augmented disease for the aged vaccinated group. In contrast to DIV, SHC014-MA15 challenge as a vaccine showed promise, but with important caveats. Utilizing a high dose, we infected young mice with SHC014-MA15 and followed over 28-days; the mice were subsequently challenged with SARS-MA15 (Supplementary Fig. 6a). Prior high-dose infection with SHC014-MA15 conferred protection against lethal SARS-MA15 challenge, but only minimal SARS-CoV neutralization response from SHC014-MA15 antisera (Supplementary Fig. 6b, 1/200) implying diminished protection over time. Similar results were observed in aged BALB/C mice in terms of weight loss and viral replication (Supplementary Fig. 6c, d). However, this infection dose induced > 10% weight loss and lethality in some