key: cord-1002904-h3o9sbii
authors: Liu, Zhuoming; Wu, Hua; Egland, Kristi A.; Gilliland, Theron C.; Dunn, Matthew D.; Luke, Thomas C.; Sullivan, Eddie J.; Klimstra, William B.; Bausch, Christoph L.; Whelan, Sean P. J.
title: Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants
date: 2021-02-07
journal: bioRxiv
DOI: 10.1101/2021.02.06.430072
sha: 9523c5ef0c0ced5030d987a495295a848fc81c70
doc_id: 1002904
cord_uid: h3o9sbii

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

Ending the global pandemic caused by severe acute respiratory syndrome coronavirus 2 41 (SARS-CoV-2) requires the deployment of multiple countermeasures including therapeutics and 42 vaccines. Emergency use authorization (EUA) has already been granted by the United States 43 which exhibit resistance to multiple mAbs and are present in emerging variants of concern. We 115 also generated the dominant D614G, and the mouse adapted N501Y (Gu et al., 2020; Hou et 116 al., 2020) and E484K-N501Yvariants. To determine whether the potency of SAB-185 was 117 altered by any of these individual amino acid substitutions we performed neutralization assays 118

with the corresponding chimeric VSV (Figure 2B) . Each mutant exhibits a dose-dependent 119 inhibition of infection by Lots 1, 5 and 6 of SAB-185, at levels that were similar to wild type S. 120

We therefore tested whether the Tc-hIgG-SARS-CoV-2 also retains potency against a 121 combination of substitutions E484K-N501Y in S. The resulting chimeric virus was also potently 122 neutralized by SAB-185 with IC50 values of 9744 ng ml -1 , 1508 ng ml -1 and 468 ng ml -1 for Lots 123 1, 5, and 6 ( Figure 2C ). This data demonstrates that SAB-185 retains neutralizing potency 124 against multiple substitutions in S in chimeric VSV cell culture assays including several present 125 in circulating human variants of SARS-CoV-2. 126

We and others have previously isolated VSV-SARS-CoV-2 S gene mutants by selection 128 using mAbs and human convalescent serum that are resistant to neutralization (Baum et al., 129 2020; Weisblum et al., 2020; Z Liu., 2021). We therefore applied the same approach in an 130 attempt to isolate variants resistant to SAB-185. In contrast to the ability to readily isolate mAb 131 and convalescent serum escape mutants, we were unable to isolate mutants resistant to the 132 human immunoglobulin SAB-185 from 3 successive attempts at sub-neutralizing concentrations 133 of SAB-185 (Figure 3) . Taken together, this analysis suggests that SAB-185 poses a significant 134 barrier to immune escape. 135

We report a potently neutralizing polyclonal human IgG produced in transchromosomic 138 bovines that effectively neutralizes SARS-CoV-2 and VSV-SARS-CoV-2 chimeras that contain 139 one or two S mutations present in circulating variants of concern (Greaney et al., 2020; Li et al., 140 2020; Weisblum et al., 2020; Z Liu., 2021). This finding contrasts with earlier work which shows 141 that immune sera obtained from patients that have recovered from COVID-19 as well as several 142 mAbs show significant reductions in neutralization potency in cell culture assays particularly 143 against S substitutions E484K and S477N. The potency seen for SAB-185 with the combination 144 of E484K and N501Y likely reflects the fact that the hyperimmune IgG SAB-185 recognizes a 145 broader swath of epitopes in S such that single substitutions are unlikely to escape 146 neutralization. As a result, large scale antigenic variation that results in the appearance of new 147 serotypes may be required to evade neutralization by such a polyclonal human IgG. 148

The emergence of new SARS-CoV-2 variants in humans and other species will continue 149 to pose a threat to human health particularly if sufficient antigenic variation is achieved to blunt Methods. The negative control endpoint was calculated from six replicate wells averaged at a 216 1:20 dilution of pAb. The Lot 1 endpoint was calculated from an average of two wells on a single 217 plate (2 wells total) and Lot 5/Lot 6 were calculated from an average of two wells on three 218 separate plates (6 wells total). 219 2020). S477N and E484K were escape mutants isolated from mAbs described as previously (Z 240

Liu., 2021). N501Y and D614G were constructed using SARS-CoV-2 Wuhan-Hu-1 spike with 241 substitution at N501 or D614 site respectively and rescued by using reverse genetic system. 

After quality control testing, the qualified Tc bovine plasma was thawed, pooled, fractionated by 311 caprylic acid (CA), and clarified by depth filtration in the presence of Celpure P1000 filter aid. 312

The clarified sample containing Tc bovine-derived human IgG is further purified by affinity 313 chromatography, first using an anti-human IgG kappa light chain specific column, KappaSelect (GE Healthcare Life Sciences) to capture hIgG and remove residual non-hIgG and bovine 315 plasma proteins (BPP) followed by a low pH treatment, and, second, by passing through an 316 anti-bovine IgG heavy chain specific affinity column, Capto HC15 (GE Healthcare Life 317 Sciences). To further remove residual IgG that contains bovine heavy chain, the human IgG 318 fraction was then subjected to a Q Sepharose chromatography polishing step to further reduce 319 impurities, nanofiltration, final buffer exchange, concentration and sterile filtration. Finally, the 320 SAB-185 product was terminally filtered and filled into vials. The product protein concentration were detected colorimetrically by using the 3, 3,' 5, 5'-tetramethylbenzidine (TMB) substrate kit (SeraCare). Absorbance was read in a microplate reader at 450 nm. The titer (units/ml) is 341 defined as the reciprocal of the highest dilution of SAB-185 resulting in an optical density at 450 342 nm (OD450) reading that was 2.5-fold higher than blank. 343

Virus production and neutralization of a low passage strain of SARS-CoV-2 were as described 345

as previously (Klimstra WB, 2020). Virus growth and assays were performed at Biosafety Level 346 Neutralization assays using a recombinant VSV-SARS-CoV-2 and mutants. Briefly, the 365 initial dilution of started at 83 ug mL -1 and was three-fold serially diluted in 96-well plates over eight dilutions. Indicated dilutions of SAB-185 pAbs were incubated with 10 2 PFU of VSV-SARS-367

CoV-2 and mutants for 1 h at 37 °C. SAB-185 pAb-virus complexes then were added to Vero E6 368 cells in 96-well plates and incubated at 37 °C for 7.5 h. Cells were fixed at room temperature in 369 2% formaldehyde containing 10 µg/mL of Hoechst 33342 nuclear stain for 45 min. 

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Deployment of convalescent plasma 393 for the prevention and treatment of COVID-19

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The impact of Spike mutations 399 on SARS-CoV-2 neutralization

Neutralizing Antibody and Soluble ACE2 Inhibition of a 402

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351 and B.1.1.7 to Antibody Neutralization. bioRxiv

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