To evaluate the effects of SARS-CoV-2 strain variation on mAb protection, we assembled a panel of infectious SARS-CoV-2 strains with sequence substitutions in the spike gene (Fig 1a-b). A B.1.1.7 isolate from the United Kingdom had signature changes in the spike gene14 including the 69-70 and 144-145 deletions, and N501Y, A570D, D614G, and P681H substitutions. A B.1.429 isolate from California contained the characteristic S13I, W152C, and L452R changes. We also used a previously generated Washington SARS-CoV-2 strain with a D614G substitution (WA1/2020 D614G), a SARS-CoV-2 strain with N501Y and D614G substitutions (WA1/2020 N501Y/D614G), and recombinant, chimeric SARS-CoV-2 strains with a South African (Wash SA-B.1.351; D80A, D215G, 242-244 deletion, K417N, E484K, N501Y, D614G, and A701V) or Brazilian (Wash BR-B.1.1.28; L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F) spike genes in the Washington strain background1,15. All viruses were propagated in Vero cells expressing transmembrane protease serine 2 (TMPRSS2) to prevent the emergence of mutations at or near the furin cleavage site in the spike protein, which occurs with passage in Vero E6 cells16 and can impact virulence17. All viruses were deep-sequenced to confirm the presence of expected mutations prior to use in vitro or in vivo (Supplementary Table S1).
We first assessed the impact of SARS-CoV-2 spike variation on antibody neutralization in Vero-TMPRSS2 cells (Fig 1c-d) using the WA1/2020 D614G, WA1/2020 N501Y/D614G, B.1.1.7, Wash SA-B.1.351, Wash BR-B.1.1.28, and B.1.429 viruses. We tested individual and cocktails of mAbs in clinical development that target the RBD including 2B04/47D11 (AbbVie), S309/S2E12 (Vir Biotechnology), COV2-2130/COV2-2196 (Vanderbilt University Medical Center with engineered derivatives being evaluated by AstraZeneca), REGN10933/REGN10987 (synthesized based on casirivimab and imdevimab sequences from Regeneron), and LY-CoV555 (synthesized based on bamlanivimab sequences from Lilly). All individual mAbs tested efficiently neutralized the WA1/2020 D614G, WA1/2020 N501Y/D614G, and B.1.1.7 strains, and several mAbs (COV2-2130, COV2-2196, S309, S2E12, and 47D11) showed little change in potency against the Wash SA-B.1.351, Wash BR-B.1.1.28, and B.1.429 strains (Fig 1c-d). In comparison, REGN10987 or LY-CoV555 respectively showed a ~10-fold or complete loss in inhibitory activity against the B.1.429 strain, which is consistent with studies identifying L452 and adjacent residues as interaction sites for these mAbs (Table 1). Moreover, REGN10933, LY-CoV555, and 2B04 exhibited a marked loss or complete absence of neutralizing activity against Wash SA-B.1.351, Wash BR-B.1.1.28, and viruses containing the E484K mutation (Fig 1c-d and Extended Data Fig 1), which corresponds with structural and mapping studies (Table 1). Analysis of mAb cocktails showed that COV2-2130/COV2-2196, S309/S2E12, and REGN10933/REGN10987 neutralized all virus strains tested, with the latter combination retaining potency corresponding to the mAb with inhibitory activity in the cocktail for a given virus. In comparison, while the 2B04/47D11 mAb combination efficiently neutralized WA1/2020 D614G, WA1/2020 N501Y/D614G, B.1.1.7, and B.1.429 strains, its inhibitory activity against Wash SA-B.1.351 and Wash BR-B.1.1.28 reflected the less potent 47D11 mAb component (EC50 of 384-431 ng/mL) (Fig 1c-d).
To evaluate the efficacy of the mAb combinations in vivo, we initially used the K18-hACE2 transgenic mouse model of SARS-CoV-2 pathogenesis in which human ACE2 expression is driven by the cytokeratin-18 gene promoter18,19. In prior studies, we established that low (2 mg/kg) doses of several different anti-RBD neutralizing human mAbs provide a threshold of protection against the WA1/2020 strain when administered as prophylaxis20. Accordingly, we gave K18-hACE2 mice a single 40 μg (~2 mg/kg total) dose of mAb combinations (2B04/47D11, S309/S2E12, COV2-2130/COV2-2196, or REGN10933/REGN10987) or LY-CoV555 as monotherapy by intraperitoneal injection one day prior to intranasal inoculation with SARS-CoV-2 (103 focus-forming units [FFU] of WA1/2020 N501Y/D614G, B.1.1.7, Wash SA-B.1.351 or Wash BR-B.1.1.28). For these in vivo studies, we used a recombinant version of WA1/2020 that encodes N501Y for direct comparison to B.1.1.7, Wash SA-B.1.351 or Wash BR-B.1.1.28, all of which naturally contain this residue. This substitution increases infection and pathogenicity in mice21,22 yet did not substantively impact neutralization of the mAbs we tested (Fig 1c). We monitored weight change for six days, and then euthanized animals and harvested tissues for virological and immunological analyses.
Compared to a control human mAb (anti-West Nile virus hE1623), a single 40 mg prophylaxis dose of the anti-SARS-CoV-2 mAbs conferred substantial protection against WA1/2020 N501Y/D614G-induced weight loss and viral burden in the lungs, nasal washes, brain, spleen, and heart in the K18-hACE2 mice at 6 days post-infection (dpi) (Fig 2a-d, Extended Data Fig 2 and 3a). While all of the anti-SARS-CoV-2 mAb cocktails conferred protection against weight loss caused by B.1.1.7, Wash SA-B.1.351 or Wash BR-B.1.1.28, LY-CoV555 monotherapy protected only against the B.1.1.7 strain (Fig 2e, i, and m). Some of the antibodies provided less virological protection against the B.1.1.7, Wash SA-B.1.351 or Wash BR-B.1.1.28 strains in specific tissues. Whereas all mAb groups protected against B.1.1.7 infection in the lung, 2B04/47D11 and LY-CoV555 failed to perform as well in nasal washes, and LY-CoV555 showed reduced protection against infection in the brain (Fig 2f-h). Sanger sequencing analysis of the RBD region of viral RNA of brain, nasal wash, and lung samples from animals treated with these mAbs did not show evidence of neutralization escape (Supplementary Table S2). 2B04/47D11 and LY-CoV555-treated animals also showed greater virus breakthrough than the other tested antibodies when challenged with Wash SA-B.1.351 or Wash BR-B.1.1.28 viruses: 2B04/47D11 reduced viral burden in the lungs, nasal washes, and brain (500-10,000-fold) much less efficiently than other mAb cocktails, and LY-CoV555 mAb treatment conferred virtually no virological protection in any tissue analyzed (Fig 2j-l and n-p and Extended Data Fig 3b). Compared to the COV2-2130/COV2-2196 and S309/S2E12 combinations, REGN10933/REGN10987 also showed less ability to reduce viral RNA levels in nasal washes of K18-hACE2 mice infected with Wash SA-B.1.351 or Wash BR-B.1.1.28 viruses.
An excessive pro-inflammatory host response to SARS-CoV-2 infection is hypothesized to contribute to pulmonary pathology and severe COVID-1924. To evaluate further the extent of protection conferred by the different mAb groups against the SARS-COV-2 variant viruses, we measured pro-inflammatory cytokine and chemokines in lung homogenates harvested at 6 dpi (Fig 2q and Extended Data Fig 4). This analysis showed a strong correspondence with viral RNA levels in the lung: (a) compared to the control mAb, S309/S2E12, COV2-2130/COV2-2196, and REGN10933/REGN10987 combinations showed markedly reduced levels of pro-inflammatory cytokines and chemokines (G-CSF, IFN-g, IL-6, CXCL10, LIF, CCL2, CXCL9, CCL3, and CCL4) after infection with WA1/2020 N501Y/D614G, B.1.1.7, Wash SA-B.1.351 or Wash BR-B.1.1.28; (b) prophylaxis with 2B04/47D11 or LY-CoV555 resulted in reduced inflammatory cytokine and chemokine levels in mice infected with WA1/2020 N501Y/D614G and B.1.1.7, with substantially less improvement in animals infected with Wash SA-B.1.351 and Wash BR-B.1.1.28.
Given that a 40 mg dose of S309/S2E12, COV2-2130/COV2-2196, and REGN10933/REGN10987 combinations prevented infection and inflammation caused by the different SARS-CoV-2 strains, we next tested a ten-fold lower 4 mg dose (~0.2 mg/kg) to assess for possible differences in protection. Prophylaxis with COV2-2130/COV2-2196, S309/S2E12, REGN10933/REGN10987, or 2B04/47D11 protected K18-hACE2 mice against weight loss caused by all four viruses (Extended Data Fig 5a-d). Whereas the COV2-2130/COV2-2196, S309/S2E12, and REGN10933/REGN10987 mAb combinations reduced viral RNA levels in the lung at 6 dpi in K18-hACE2 mice infected with WA1/2020 N501Y/D614G, B.1.1.7, Wash SA-B.1.351, or Wash BR-B.1.1.28, the 2B04/47D11 treatment conferred protection against B.1.1.7 and WA1/2020 N501Y/D614G but not against Wash SA-B.1.351 and Wash BR-B.1.1.28 viruses at this lower dose (Extended Data Fig 5e-h). In comparison, in nasal washes, all four mAb cocktails resulted in relatively similar reductions in viral RNA levels at 6 dpi of animals inoculated with WA1/2020 N501Y/D614G, B.1.1.7, Wash SA-B.1.351 or Wash BR-B.1.1.28 (Extended Data Fig 5i-l). Even at this low treatment dose, with the exception of some substantive breakthrough events (>6 log 10 copies of N/mg: COV2-2130/COV2-2196 [2 of 24 mice]; S309/S2E12 [6 of 24 mice]; REGN10933/REGN10987 [1 of 24 mice]; and 2B04/47D11 [6 of 24 mice]), the mAb combinations generally prevented viral dissemination to and high-level infection of the brain (Extended Data Fig 5m-p and Supplementary Table S2).
Although K18-hACE2 mice have been used extensively to test vaccines and therapeutics against SARS-CoV-220,25-28, the high level and distinct pattern of transgene expression in these animals could impact entry pathways, and neutralization and protection conferred by anti-RBD antibodies. As an alternative model for evaluating mAb efficacy, we tested immunocompetent, inbred 129S2 mice, which are permissive to infection by SARS-CoV-2 strains encoding an N501Y substitution without ectopic hACE2 expression21,22; presumably, the N501Y adaptive mutation enables efficient engagement of murine (m)ACE2. We administered a single 40 µg (~2 mg/kg) dose of mAb cocktails (COV2-2130/COV2-2196, S309/S2E12, or REGN10933/REGN10987) or a control mAb via intraperitoneal injection one day prior to intranasal inoculation with 103 FFU of WA1/2020 N501Y/D614G, Wash SA-B.1.351, or Wash BR-B.1.1.28, and 105 FFU of B.1.1.7 (Fig 3). A higher inoculating dose of B.1.1.7 was required to obtain equivalent levels of viral RNA in the lung compared to the other three viruses. At 3 dpi, we harvested tissues for viral burden analyses; at this time point, reproducible weight loss was not observed. All three mAb cocktails tested (COV2-2130/COV2-2196, S309/S2E12, and REGN10933/REGN10987) protected 129S2 mice against infection in the lung by all SARS-CoV-2 strains as judged by reductions in viral RNA levels (Fig 3a-d); despite some variability, we observed a trend toward less complete protection in animals infected with Wash SA-B.1.351 and Wash BR-B.1.1.28 strains (Fig 3c-d and Extended Data Fig 3c-f). When we evaluated the nasal washes, reductions in viral RNA levels were diminished with the Wash SA-B.1.351 virus, especially for the COV2-2130/COV2-2196 and REGN10933/REGN10987 combinations (Fig 3e-h). Sequencing analysis of lung samples from the infected 129S2 mice also did not reveal evidence of acquisition of mutations in the RBD (Supplementary Table S2).
The immunocompetent Syrian golden hamster also has been used to evaluate mAb activity against SARS-CoV-2 infection in the upper and lower respiratory tracts29,30. We used this animal model to assess independently the inhibitory activity and possible emergence of resistance of one of the mAb combinations (COV2-2130/COV2-2196) against viruses containing the B.1.351 spike protein at threshold doses of protection. One day prior to intranasal inoculation with 5 x 105 FFU of Wash SA-B.1.351 or WA1/2020 D614G, we treated hamsters with a single 800 µg (~10 mg/kg) or 320 µg (~4 mg/kg) dose of the COV2-2130/COV2-2196 cocktail or isotype control mAb by intraperitoneal injection (Fig 4). Weights were followed for 4 days, and then tissues were harvested for virological and cytokine analysis. At the 800 µg mAb cocktail dose, hamsters treated with COV2-2130/COV2-2196 and infected with WA1/2020 D614G or Wash SA-B.1.351 showed protection against weight loss (Fig 4a) and reduced viral burden levels in the lungs but not nasal swabs compared to the isotype control mAb (Fig 4b-d). Correspondingly, RT-qPCR analysis of a previously described set of cytokines and inflammatory genes20 showed reduced mRNA expression in the lungs of hamsters treated with COV2-2130/COV2-2196 (Fig 4e-h). Consensus sequencing of the RBD region of viral RNA samples from the lungs of hamsters treated with COV2-2130/COV2-2196 and inoculated with WA1/2020 D614G or Wash SA-B.1.351 did not show evidence of mutation or escape (Supplementary Table S2). When the lower 320 µg dose of COV2-2130/COV2-2196 was administered, we observed a trend toward protection against weight loss in hamsters infected with WA1/2020 D614G and Wash SA-B.1.351 (Fig 4i). Consistent with a partially protective phenotype, hamsters treated with the lower 320 µg dose of COV2-2130/COV2-2196 and inoculated with either WA1/2020 D614G and Wash SA-B.1.351 showed a trend towards reduced viral RNA in the lungs at 4 dpi and markedly diminished (~104 to 105-fold) levels of infectious virus as determined by plaque assay (Fig 4j-k). The reduction in lung viral load conferred by the lower dose COV2-2130/COV2-2196 corresponded with diminished inflammatory gene expression after infection with either WA1/2020 D614G or Wash SA-B.1.351 (Fig 4m-p). In contrast to the protection seen in the lung, differences in viral RNA were not observed in nasal washes between COV2-2130/COV2-2196 and isotype control mAb-treated animals regardless of the infecting strain (Fig 4l). Sequencing of the RBD of viral RNA from the lungs of COV2-2130/COV2-2196 or isotype mAb-treated hamsters also did not detect evidence of escape mutation selection after infection with WA1/2020 D614G or Wash SA-B.1.351 (Supplementary Table S2). Overall, these studies in hamsters with near threshold dosing of the COV2-2130/COV2-2196 mAb cocktail establish equivalent protection and an absence of rapid escape against SARS-CoV-2 containing spike proteins from historical or variant strains.
As mAbs are being developed clinically as therapeutics, we assessed their post-exposure efficacy against the SARS-CoV-2 strain expressing the B.1.351 spike protein using the stringent K18-hACE2 model. We administered a single, higher 200 µg (~10 mg/kg) dose of COV2-2130/COV2-2196, S309/S2E12, REGN10933/REGN10987 or 2B04/47D11 by intraperitoneal injection one day after inoculation with 103 FFU of WA1/2020 N501Y/D614G or Wash SA-B.1.351, and then monitored the mice for six days prior to necropsy and virological analysis (Fig 5). We did not test the LY-CoV555 mAb in these therapeutic experiments, since it failed to protect against Wash SA-B.1.351 as prophylaxis. Compared to the control mAb-treated animals, which lost at least 15% of their starting weight over the 6 days of the experiment, each of the mAb cocktails prevented weight loss induced by WA1/2020 N501Y/D614G or Wash SA-B.1.351 infection (Fig 5a and e). COV2-2130/COV2-2196, S309/S2E12, and REGN10933/REGN10987 mAb cocktail treatments resulted in reduced infectious virus and viral RNA levels in lung homogenates, and viral RNA levels in nasal washes and brain homogenates from animals infected with either WA1/2020 N501Y/D614G or Wash SA-B.1.351 (Fig 5b-d, f-h and Extended Data Fig 3g-h). In comparison, while the 2B04/47D11 mAb cocktail reduced viral RNA levels in the lungs, it showed less protection in the nasal washes of WA1/2020 N501Y/D614G and Wash SA-B.1.351 infected mice.