In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains

Rapidly-emerging variants jeopardize antibody-based countermeasures against SARS-CoV-2. While recent cell culture experiments have demonstrated loss of potency of several anti-spike neutralizing antibodies against SARS-CoV-2 variant strains1-3, the in vivo significance of these results remains uncertain. Here, using a panel of monoclonal antibodies (mAbs) corresponding to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron, and Lilly we report the impact on protection in animals against authentic SARS-CoV-2 variants including WA1/2020 strains, a B.1.1.7 isolate, and chimeric strains with South African (B.1.351) or Brazilian (B.1.1.28) spike genes. Although some individual mAbs showed reduced or abrogated neutralizing activity against B.1.351 and B.1.1.28 viruses with E484K spike protein mutations in cell culture, low prophylactic doses of mAb combinations protected against infection in K18-hACE2 transgenic mice, 129S2 immunocompetent mice, and hamsters without emergence of resistance. Two exceptions were mAb LY-CoV555 monotherapy which lost all protective activity in vivo, and AbbVie 2B04/47D11, which showed partial loss of activity. When administered after infection as therapy, higher doses of mAb cocktails protected in vivo against viruses displaying a B.1.351 spike gene. Thus, many, but not all, of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing SARS-CoV-2 variant strains.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global coronavirus disease 2019 (COVID- 19) pandemic and resulted in more than 140 million con rmed infections and over 3 million deaths. The sustained nature of the COVID-19 pandemic and its accompanying extensive morbidity and mortality have made the development and immediate deployment of therapeutics and vaccines an urgent global health priority 4 . Indeed, the emergency use authorization (EUA) of several monoclonal antibody (mAb) therapies and mRNA, inactivated, and viral-vectored vaccines has provided hope for controlling infection and curtailing the pandemic.
Currently authorized antibody countermeasures against SARS-CoV-2 target the spike protein from strains circulating during the early phases of the pandemic in 2020. The SARS-CoV-2 spike protein binds the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) to facilitate viral entry into and infection of human cells 5 . Upon cell attachment, SARS-CoV-2 spike proteins are cleaved by host proteases into S1 and S2 fragments. The S1 protein includes the Nterminal (NTD) and receptor binding (RBD) domains, whereas the S2 protein promotes membrane fusion. The RBD, in particular, is the target of many potently neutralizing monoclonal 6-10 and serum polyclonal antibodies 11 . and authentic infectious SARS-CoV-2 strains suggest that neutralization by a substantial fraction of previously generated antibodies may be diminished against variants expressing mutations in the spike gene, especially at position E484 [1][2][3]12,13 . However, the in vivo signi cance of this loss of mAb neutralizing activity remains uncertain, particularly for combination mAb therapies, as high doses could compensate for changes in neutralization potency. Here, using mice and hamsters, we assessed the protective activity of clinically relevant mAbs against WA1/2020 strains and a panel of SARS-CoV-2 variants including a B.1.1.7 isolate, and chimeric strains with South African (B.1.351) or Brazilian (B.1.1.28) spike genes. We tested cocktails of mAbs from AbbVie (2B04/47D11) and Vir Biotechnology (S309/S2E12) as well as ones corresponding to those from AstraZeneca (COV2-2130/COV2-2196), Regeneron (REGN10933/REGN10987), and Lilly (LY-CoV555) as prophylaxis or therapy against SARS-CoV-2 in K18-hACE2 transgenic mice, 129S2 immunocompetent mice, and Syrian hamsters. Whereas several antibody combinations conferred protection in both mouse models with all variant strains tested, the 2B04/47D11 combination and LY-CoV555 showed reduced or complete loss of protective activity. One of the combinations (COV2-2130/COV2-2196) also showed equivalent protective activity in hamsters against WA1/2020 D614G and the chimeric strain with a B.1.351 spike.
We rst assessed the impact of SARS-CoV-2 spike variation on antibody neutralization in Vero-TMPRSS2 cells (Fig 1c-d) (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 e ciently neutralized WA1/2020 D614G, WA1/2020 N501Y/D614G, B.  (Fig 1c-d).
To evaluate the e cacy 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 promoter 18, 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 prophylaxis 20 . 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 ( 21,22 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 hE16 23 ), 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- 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 e ciently than other mAb cocktails, and LY-CoV555 mAb treatment conferred virtually no virological protection in any tissue analyzed ( An excessive pro-in ammatory host response to SARS-CoV-2 infection is hypothesized to contribute to pulmonary pathology and severe COVID-19 24 . To evaluate further the extent of protection conferred by the different mAb groups against the SARS-COV-2 variant viruses, we measured pro-in ammatory 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: Given that a 40 mg dose of S309/S2E12, COV2-2130/COV2-2196, and REGN10933/REGN10987 combinations prevented infection and in ammation 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  Table S2).
Although K18-hACE2 mice have been used extensively to test vaccines and therapeutics against SARS-CoV-2 20,25-28 , 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 e cacy, we tested immunocompetent, inbred 129S2 mice, which are permissive to infection by SARS-CoV-2 strains encoding an N501Y substitution without ectopic hACE2 expression 21,22 ; presumably, the N501Y adaptive mutation enables e cient 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 10 3 FFU of WA1/2020 N501Y/D614G, Wash SA-B.1.351, or Wash BR-B.1.1.28, and 10 5 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 tracts 29,30 . 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 10 5 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 in ammatory genes 20 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 (~10 4 to 10 5 -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 in ammatory 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 e cacy 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 10 3 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 34 , showed complete neutralization escape in cell culture and failed to confer any protection against viruses containing E484K substitutions. In contrast, all cocktails of two neutralizing mAbs conferred protection to varying degrees even if one of the constituent mAbs showed reduced activity due to resistance. Moreover, the higher doses of mAbs used in patients (e.g., 2.4 g or ~ 35 mg/kg for casirivimab and imdevimab [REGN mAbs]) could compensate for loss in neutralization potency.
Combination therapy with multiple mAbs in our study (COV2-2130/COV2-2196, S309/S2E12, REGN10933/REGN10987, or 2B04/47D11) was protective in mice and hamsters against the variant strains, highlighting the importance of using multiple mAbs recognizing distinct epitopes rather than monotherapy to control SARS-CoV-2 infection. Indeed, the emergency use authorization for bamlanivimab (LY-CoV555) as monotherapy recently was revoked, since the antibody does not e ciently reduce SARS-CoV-2 infection of several variants of concern that are spreading globally 35 ; instead, a combination of bamlanivimab and etesevimab is now recommended even though some strains containing E484 and K417 mutations (e.g., B.1.351 and B.1.1.28) likely will have resistance to both mAb components ( 2,36 and Extended Data Fig 6). In our study, combination therapy with two mAbs including one (2B04) that failed to neutralize a virus containing the E484K mutation still protected when administered at higher doses, although the reduction in viral burden was less than with other mAb cocktails at equivalent doses. Beyond a loss of potency against already circulating resistant variants, antibody monotherapy can be compromised within an individual by rapid selection of escape mutations de novo or enrichment of pre-existing mutants in the quasispecies present at low frequency. Consistent with this idea, in other animal experiments with SARS-CoV-2, we have observed the rapid emergence of resistance against antibody monotherapy, resulting in the accumulation of mutations at RBD residues 476, 477 484, and 487, only some of which were detectable in our parental virus stocks by next generation sequencing ( 37 and M. Diamond, A. Boon, and A. Ellebedy, unpublished data). Remarkably, and despite amplifying the RBD sequence from 96 brain, nasal wash, and lung samples from mice and hamsters treated with the different mAb combinations, we did not detect a single escape mutant. Although further study is warranted, combination mAb treatment may prevent escape through synergistic interactions in vivo or by driving selection of mutants with compromised tness.
At the lower doses of mAbs tested, we observed some differences in mAb cocktail e cacy between rodent models, which could be due to host variation, viral variation, or both. For example, mutations in the RBD can affect mAb binding as well as ACE2 binding 38 . Mutation at position 501 of the spike is of particular interest, since it enables mouse adaptation 21,22 and is present in many variants of concern (e.g., B. 1.1.7, B.1.351, and B.1.1.28). The N501Y change associated with infection of conventional laboratory mice could facilitate virus engagement with murine ACE2 or possibly other putative target receptors 39 . Beyond this, polymorphisms in or differences of expression of host receptors on key target cells also could impact SARS-CoV-2 infection in different hosts and the inhibitory effects of neutralizing antibodies. As both viral and host sequences determine the interface between SARS-CoV-2 spike and its cell entry receptors like ACE2, mAb interactions and potency could be affected in different species of animals. Changes in the a nity of interaction between spike proteins and receptors can impact the stoichiometry of neutralizing antibody binding required to inhibit infection 40 . Although further study of antibody-based countermeasures in vivo is required, the complexity of antibody-spike protein-receptor interactions likely explains some of the variation in protection K18-hACE2 mice, 129S2 mice, and hamsters. Alternatively, the pharmacokinetics and/or biodistribution of antibodies in these animals also could vary and affect e cacy. In the animal models we tested, we did not observe marked differences in serum antibody levels in the context of viral challenge (Supplementary Table S3). in Vero-TMPRSS2 cells and subjected to next-generation sequencing as described previously 1 to con rm the introduction and stability of substitutions (Supplementary Table S1). All virus experiments were performed in an approved biosafety level 3 (BSL-3) facility.
Mouse experiments. Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381-01). Virus inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.
For antibody prophylaxis and therapeutic experiments, animals were administered the indicated mAb dose by intraperitoneal injection one day before or after intranasal inoculation with the indicated SARS-CoV-2 strain.
Hamster experiments. Six-month-old male Syrian hamsters were purchased from Charles River Laboratories and housed in microisolator units. All hamsters were allowed free access to food and water and cared for under United States Department of Agriculture (USDA) guidelines for laboratory animals. Hamsters were administered by intraperitoneal injection mAbs COV2-2130 + COV2-2196 or isotype control (4 or 10 mg/kg depending on the experiment). One day later, hamsters were given 5 x 10 5 FFU of SARS-CoV-2 (2019-nCoV/USA-WA1/2020) by the intranasal route in a nal volume of 100 μL. All hamsters were monitored for body weight loss until humanely euthanized at 4 dpi. Nasal swabs were collected 3 dpi. All procedures were approved by the Washington University School of Medicine (assurance number A3381-01). Virus inoculations and antibody transfers were performed under anesthesia that was induced and maintained with 5% iso urane. All efforts were made to minimize animal suffering.
Measurement of viral burden. Tissues were weighed and homogenized with zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1000 μL of DMEM medium supplemented with 2% heat-inactivated FBS. Tissue homogenates were clari ed by centrifugation at 10,000 rpm for 5 min and stored at −80°C. RNA was extracted using the MagMax mirVana Total RNA isolation kit (Thermo Fisher Scienti c) on the King sher Flex extraction robot (Thermo Fisher Scienti c). RNA was reverse transcribed and ampli ed using the TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher Scienti c). Reverse transcription was carried out at 48°C for 15 min followed by 2 min at 95°C. Ampli cation was accomplished over 50 cycles as follows: 95°C for 15 s and 60°C for 1 min. Copies of SARS-CoV-2 N gene RNA in samples were determined using a previously published assay 53 . Brie y, a TaqMan assay was designed to target a highly conserved region of the N gene (Forward primer: ATGCTGCAATCGTGCTACAA; Reverse primer: GACTGCCGCCTCTGCTC; Probe: /56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/). This region was included in an RNA standard to allow for copy number determination down to 10 copies per reaction. The reaction mixture contained nal concentrations of primers and probe of 500 and 100 nM, respectively.
Plaque assay. Vero-TMPRSS2-hACE2 cells were seeded at a density of 1×10 5 cells per well in 24-well tissue culture plates. The following day, medium was removed and replaced with 200 μL of material to be titrated diluted serially in DMEM supplemented with 2% FBS. One hour later, 1 mL of methylcellulose overlay was added. Plates were incubated for 72 h, then xed with 4% paraformaldehyde ( nal concentration) in PBS for 20 min. Plates were stained with 0.05% (w/v) crystal violet in 20% methanol and washed twice with distilled, deionized water.
Cytokine and chemokine protein measurements. Lung homogenates were incubated with Triton-X-100 (1% nal concentration) for 1 h at room temperature to inactivate SARS-CoV-2. Homogenates then were analyzed for cytokines and chemokines by Eve Technologies Corporation (Calgary, AB, Canada) using their Mouse Cytokine Array / Chemokine Array 31-Plex (MD31) platform.
Data availability. All data supporting the ndings of this study are available within the paper and are available from the corresponding author upon request.
Statistical analysis. All statistical tests were performed as described in the indicated gure legends using Prism 8.0. Statistical signi cance was determined using an ordinary one-way ANOVA with Dunnett's post-test when comparing three or more groups. to antibodies described in this paper. The Ellebedy laboratory has received funding support in sponsored research agreements from AbbVie Inc. and Emergent BioSolutions. The Boon laboratory has received funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences, AbbVie, and Nano targeting & Therapy Biopharma. The Shi laboratory has received sponsored research agreements from P zer, Gilead, Merck, and IGM Sciences Inc. D.C. and L.P. are employees of Vir Biotechnology and may hold equity in Vir Biotechnology. L.P. is a former employee and may hold equity in Regeneron Pharmaceuticals. W.B.S. is an employee of AbbVie and may hold equity.