key: cord-0831829-xan2tz4f
authors: Tang, Juanjie; Lee, Youri; Ravichandran, Supriya; Grubbs, Gabrielle; Huang, Chang; Stauft, Charles; Wang, Tony; Golding, Basil; Golding, Hana; Khurana, Surender
title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19
date: 2021-03-19
journal: bioRxiv
DOI: 10.1101/2021.03.19.436183
sha: cb4dd88f829d8b42388bdfffce4c0914bf37cd2f
doc_id: 831829
cord_uid: xan2tz4f

Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 (≤2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.

An expedited access to treatment of COVID-19 patients with convalescent plasma was 45 issued by FDA via Emergency Use Authorization on August 23, 2020. Additional studies, 46 including randomized, controlled trials, have provided data to further inform the safety and The phage display technique is suitable to display properly folded and conformationally 61 active proteins, as it has been widely used for display of large functionally-active antibodies, 62 enzymes, hormones, and viral and mammalian proteins. We have adapted this Genome Fragment In the current study we probed the antibody epitope repertoires of 6 hCoV-2IG products 79 The spike protein is the antigen of choice for development of vaccines and therapeutics 80 against SARS-CoV-2. To decipher the epitope-specificity of the SARS-CoV-2 spike-specific 81 antibodies in an unbiased manner, we subjected the six hCoV-2IG lots to antibody epitope 82 profiling with a highly diverse SARS-CoV-2 spike GFPDL with >10 7.1 unique phage clones 83 displaying epitopes of 18-500 amino acid residues across the SARS-CoV-2 spike. During GFPDL 84 characterization, GFPDL based epitope mapping of monoclonal antibodies (MAbs) targeting 85 SARS-CoV-2 spike or RBD identified the expected linear or conformation-dependent epitopes 86 recognized by these MAbs. Recently, we showed that SARS-CoV-2 spike GFPDL can recognize 87 5 both linear, conformational and neutralizing epitopes in the post-vaccination sera of rabbits (11) 88 and post-SARS-CoV-2 infection sera in the adults and elderly (10, 12). 89 Six hCoV-2IG lots were used for SARS-CoV-2 GFPDL based epitope mapping as 90 previously described (7-11, 13). Similar numbers of phages were bound by IgG of these hCoV-91 2IG batches (3.4 x 10 4 -2.1 x 10 5 ) (Fig. 1A) . The bound phages demonstrated a diverse epitope 92 repertoire spanning the entire SARS-CoV-2 spike protein including N-terminal domain (NTD) and 93 RBD in S1, and the fusion peptide (FP), β-rich connector domain (CD), heptad repeat 1 (HR1) and the SARS-CoV-2 spike peptides. In aggregate, the convalescent plasma showed lower binding to 99 epitopes spanning the entire spike in comparison with the hCoV-2IG (Fig. 1C ). In agreement with 100 GFPDL analysis, the hCoV-2IG demonstrated highest antibody binding to the spike peptide 790-101 834 that contains the fusion peptide sequence (residues 788-806), which is unchanged among the 102 major VOCs (( Fig. S2 and Table S1 ). Most of the GFPDL-identified antigenic site sequences 103 recognized by hCoV-2IGs are conserved in the spike protein of various SARS-CoV-2 VOC (Table   104   S1 ). Table   113 S2.

All sixteen pre-pandemic 2019-IVIG preparations demonstrated titers of <20 PsVNA50 115 against SARS-CoV-2 strains ( Fig. 2A and Table S3 ). Among the nine CP lots tested against WA-116 1, variable PsVNA50 titers were observed, including one negative, one low (<1:80), six medium 117 (>1:160<1:640) and two high (>1:640). In contrast, all six hCoV-2IG lots exhibited high 118 PsVNA50 titers against WA-1 ranging between 1:1238-1:3309. PsVNA80 titers for hCoV-2IG 119 ranged between 1:168-1:593, but none of the CP lots showed PsVNA80 titers above 1:80 (range 120 <20 to 1:74) against WA-1 (Table S3) . Neutralization of the VOCs showed gradual loss of titers 121 as determined by either PsVNA50 or PsVNA80 for the hCoV-2IG and the CPs with greatest 122 reduction in titers measured against the SA VOC ( Fig. 2A and Table S3 ).

For confirmation of the PsVNA neutralization titers, the six hCoV-2IG lots were also 124 evaluated in a classical PRNT assay using VERO-E6 cells against authentic SARS-CoV-2 viruses Table S4 ). Correlation of 127 hCoV-2IG neutralization titers between PRNT50 and PsVNA50 were observed, as well as a 128 similar decline in neutralization titers against the UK and SA VOC compared with WA-1 strain 129 (Table S4 and Fig. 2B-C) . Since all hCoV-2IG lots contain 100 mg/mL of IgG, it allowed 130 calculation of ID50 values for the six hCoV-2IG lots (Table S5 and Fig 2D) . Compared to the WA-1 strain, the average PsVNA50 of the hCoV-2IG against CA, UK, 132 JP and SA VOC were reduced by 1.7, 1.9, 3.5, and 9.2-fold respectively (Fig. 2E ). Since the 133 amount of SARS-CoV-2 specific IgG in CP lots is more variable and 5-10 fold lower compared 134 with the hyperimmune hCoV-2IG, the CPs exhibited greater loss of neutralizing activities against 135 the variants in comparison with hCoV-2IG. The average PsVNA50 of the CP against the CA, UK, 136 JP, and SA VOC were reduced by 3.1, 3.3, 3.9, and 18.7-fold, respectively (Table S3 and Fig. 2F ).

PsVNA80 titers against the UK and JP VOC for all 9 CPs were lower (~2-fold) and were minimal 138 or negligible against the SA VOC ( Fig. 2A and Table S3 ). For hCoV-2IG, the PsVNA80 titers Table S2 ), but a few key mutations among these strains are shared by VOCs as shown in 146   Table S2 . N501Y is shared among the UK, JP, and SA variants. E484K is shared between the JP 147 and SA variants, and K417 is mutated to T in the JP variant, and to N in the SA variant. These key identified that targeted the RBD, S1-NTD, S2, and S protein trimer(16-18). The most potent 168 neutralizing antibodies that target directly the RBM/ACE2 interface were isolated at low frequency 169 (19).

In light of the rapid spread of SARS-CoV-2 variants of concern around the globe, it is (Table S1 ). The PsVNA using 293-ACE2-TMPRSS2 250 cell line was described previously (10, 11) . 251 Briefly, human codon-optimized cDNA encoding SARS-CoV-2 S glycoprotein of the WA- phage clones were amplified, the inserts were sequenced, and the sequences were aligned to the 302 SARS-CoV-2 spike gene, to define the fine epitope specificity in these polyclonal hCoV-2IG lots.

The GFPDL affinity selection was performed in duplicate (two independent experiments 304 by research fellow in the lab, who was blinded to sample identity). Similar numbers of bound 305 phage clones and epitope repertoire were observed in the two GFPDL panning. The difference within each group were performed using one-way ANOVA using Tukey's pairwise 331 multiple comparison test. The differences were considered statistically significant with a 95% The numbers above the peptides show the mean value for each respective group antibody binding 384 to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue).

All SPR experiments were performed twice and the researchers performing the assay were blinded 

2019-IVIG-1 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-2 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 2019-IVIG-3 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-4 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 2019-IVIG-5 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-6 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-7 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-8 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-9 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-10 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 2019-IVIG-11 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 2019-IVIG-12 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-13 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-14 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-15 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

2019-IVIG-16 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 Convalescent plasma batches produced from COVID-19 survivors hCoV- 

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