key: cord-0720209-770fvz57
authors: Widera, Marek; Wilhelm, Alexander; Hoehl, Sebastian; Pallas, Christiane; Kohmer, Niko; Wolf, Timo; Rabenau, Holger F; Corman, Victor M; Drosten, Christian; Vehreschild, Maria J G T; Goetsch, Udo; Gottschalk, Rene; Ciesek, Sandra
title: Limited neutralization of authentic SARS-CoV-2 variants carrying E484K in vitro
date: 2021-07-05
journal: J Infect Dis
DOI: 10.1093/infdis/jiab355
sha: d58f38c3ff19b17f81a243e122d24c39fe26c0a3
doc_id: 720209
cord_uid: 770fvz57

Whether monoclonal antibodies are able to neutralise SARS-CoV-2 variants of concern has been investigated using pseudoviruses. In this study we show that bamlanivimab, casirivimab, and imdevimab efficiently neutralise authentic SARS-CoV-2 including variant B.1.1.7 (Alpha) but variants B.1.351 (Beta) and P.2 (Zeta) were resistant against bamlanivimab and partially to casirivimab.

M a n u s c r i p t 4

As vaccination campaigns against COVID-19 are ongoing, the majority of the world´s population remains unimmunized, and many at risk for severe disease. The availability of both therapeutic and prophylactic agents with proven efficacy are still urgently needed. IgG1 monoclonal antibodies (mAb) prevent viral attachment and entry into human cells by blocking attachment to the ACE2 receptor. However, since several SARS-CoV-2 variants of concern (VoCs), that emerged late 2020, have been associated with increased transmissibility or immune evasion, it is of particular importance to evaluate the effectiveness of mAb against these variants. In particular, substitutions E484K and K417N in S have been associated with immune escape, or increased binding to the ACE-2 receptor (e.g., N501Y) [1] .

To evaluate whether variants harboring these substitutions might be also effectively neutralized is critical for effective treatment. So far, studies have been conducted with artificial pseudoviruses indicating that B.1.351 (Beta), P.1 (Gamma) and P.2 (Zeta) variants are expected to be resistant to therapeutic monoclonal antibodies. Furthermore, these variants have been proposed to be partially resistant to neutralization by convalescent sera obtained from COVID-19 patient as well as by vaccine-elicted sera obtained from individuals after immunization with BNT2b2 or mRNA1273. However, since in real world settings additional substitutions that might determine replication efficiency define each VoC, there is still lacking evidence that these artificially performed studies can also be transferred to authentic viruses. Hence, in this study we analysed the ability of bamlanivimab, casirivimab, and imdevimab to neutralize authentic SARS-CoV-2 variants of concern including B.1.1.7 (Alpha), B.1.351 (Beta), and P.2 (Zeta) in infectious cell culture. Additionally, we analyzed vaccine-elicited sera after immunization with BNT162b2 and mRNA1273, and convalescent sera for their ability to neutralize authentic SARS-CoV-2 variants.

Bamlanivimab and REGN-CoV-2 are IgG1 monoclonal antibody (mAb) preparations to treat COVID-19. Bamlanivimab (LY-CoV555) has been demonstrated to accelerate the decline in viral load [2] and has been authorized by the FDA for emergency use in early mild to A c c e p t e d M a n u s c r i p t 5 moderate COVID-19 disease. REGN-CoV-2 consists of two potent mAb REGN10933 (Casirivimab) and REGN10987 (Imdevimab), both binding non-competitively in different regions of Spike S [3] .It is also approved by the FDA for emergency use. Class 1 mAb REGN10933 has an ACE2 blocking property, while class 3 mAB REGN1087 binds outside of the receptor binding domain (RBD).

Emerging SARS-CoV-2 "variants of concern" (VoCs) and "variants of interest" (VoIs) referred to as B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and P.2 (Zeta), initially observed in the United Kingdom, South Africa, and Brazil, respectively, are in the process of fixation in the population. Mutations in the spike´s receptor binding domain, in particular N501Y, were associated with increased infectivity due to enhanced receptor binding [4] . Further studies have shown that of E484K represents an immunodominant site on the RBD since E484K reduced naturalization capacity of human convalescent sera by >100-fold [1] [2] [3] [4] . These amino acid substitutions have also been observed to evade the antibody response elicited by an infection with other SARS-CoV-2 variants, or vaccination.. So far, it is still under debate whether mAb preparations are equally effective against these variants.

In this study, we examined the ability of bamlanivimab, casirivimab, imdevinab, vaccineelicited sera after immunization with BNT162b2 and mRNA1273, respectively, and convalescent sera, to neutralize authentic SARS-CoV-2 variants B.1.1.7 (Alpha, mutations include N501Y and Δ69/70), B.1.351 (Beta, mutations include E484K and N501Y), and P.2 (Zeta, mutations include E484K in the absence of a 501 mutation). These isolates have been obtained from travelers from Great Britain, South Africa and Brazil, respectively. Two SARS-CoV-2 isolates collected in early 2020 were also tested (B, FFM1; B.1, FFM7) [5, 6] . with artificial pseudoviruses published previously [7] . In addition, P.2 (Zeta) virus was resistant to bamlanivimab and partly resistant against casirivimab (>4-fold). However, using the clinically used combination of REGN-CoV-2, full neutralization was observed indicating unrestricted effectiveness of a therapeutic treatment.

To determine the neutralisation efficiency of convalescence and vaccine-elicited sera, SARS-CoV-2 specific antibodies were quantified (table 1) slightly lower compared to preceding viral isolates, however, the neutralisation sensitivity of variant B.1.351 was shown to be significantly reduced [8] . Our in vitro findings using authentic SARS-CoV-2 confirms that, in contrast to vaccine-elicited sera, bamlanivimab and casirivimab may not provide efficacy against SARS-CoV-2 variants B.1.351 (Beta) and P.2 (Zeta) both harboring the E484K substitution. Since imdevimab was able to efficiently neutralize both variants, therapeutical treatment with the REGN-COV-2 combination is unrestricted effective. In agreement, previous studies using artificial pseudoviruses described that LY-CoV555 and REGN10933 are ineffective against B.1.351 (Beta) but is still effective against B.1.1.7 (Alpha) [7, 9] . This and previous work revealed a lower neutralizing activity against E484K harboring variants B1.351 (Beta) and P.2 (Zeta), which may facilitate re-infection with emerging variants [8, 10, 11] . Amino acid substitutions hinders spike proteins to be bound by antibodies, resulting in reduced protection against SARS-CoV-2 infection [12]. Hence, a higher antibody titer is needed, which might be provided by a second vaccination dose inducing the formation of a critical amount of neutralizing antibodies [13, 14] . Even a single immunization already increased the neutralising titers of convalescents, although with reduced efficiency for B.1.351 (beta) [8] . Of note, immune response after vaccination also includes a broad T cell repertoire, which might be effective despite resistance to antibody A c c e p t e d M a n u s c r i p t 9 mediated immunity [15] . Hence, viral escape of T cell immunity is unlikely. However, whether this also applicable for tested and future SARS-CoV-2 variants has to be further investigated.

Our study has the following limitations: While we used authentic SARS-CoV-2 variants rather than pseudotyped surrogate models, only a single representative isolate was studied for each variant. Hence, a broader analysis with more isolates should be expanded in future studies. We tested convalescent sera and vaccine-elicited sera (mRNA1273 and BNT162b2) for their neutralisation efficiency, however, the number of sera was relatively small and most of these convalescence sera originate from individuals infected with non-VOCs. In future, it would be also interesting to study sera from individuals infected with VOCs and thus with corresponding antibodies. In addition, sera from individuals vaccinated with vector vaccines and heterologously vaccinated should be included.

We conclude that confirmation of the SARS-CoV-2 variant, including screening for E484K, may be needed before initiating mAb treatment with bamlanivimab and to ensure both efficacious and efficient use of the antibody product. Variant-specific mAb agents may be required to treat emerging SARS-CoV-2 variants of concern. To efficiently neutralize VOCs carrying E484K, a high antibody titre is needed to inducing the formation of a critical amount of neutralizing antibodies. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 16 Figure 1 

Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies

SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid-19

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail

Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Optimized qRT-PCR Approach for the Detection of Intra-and Extra-Cellular SARS-CoV-2 RNAs

Evidence of SARS-CoV-2 Infection in Returning Travelers from Wuhan

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B

Evidence for increased breakthrough rates of SARS

CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies

A c c e p t e d M a n u s c r i p t 11