key: cord-0262489-qlxel9zo
authors: Ren, Wenlin; Ju, Xiaohui; Gong, Mingli; Lan, Jun; Yu, Yanying; Long, Quanxin; Zhang, Yu; Zhong, Jin; Zhong, Guocai; Wang, Xinquan; Huang, Ailong; Zhang, Rong; Ding, Qiang
title: Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor
date: 2021-09-03
journal: bioRxiv
DOI: 10.1101/2021.09.03.458829
sha: 20a1c024bf25ed8929312328867b556136746f1f
doc_id: 262489
cord_uid: qlxel9zo

Recently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations Δ145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, Δ145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

Since its emergence in late 2019, the severe acute respiratory syndrome coronavirus 50 6 L452R and T478K mutations. In contrast, Kappa did not increased cell entry into 120

HeLa-marmoset or HeLa-koala cells, and B.1.618 only showed enhanced entry into 121

HeLa-koala cells, which is attributable to E484K. 122

Taken together, our results demonstrated that the spike protein of Kappa, Delta or 123 B.1.618 with distinct mutations have altered their ability in utilizing ACE2 orthologs for cell 124 entry. Delta and B.1.618 variants gained an enhanced ability to use human ACE2 receptor 125 for cell entry. Remarkably, Delta variant gained the function to utilize mouse, New World 126 monkey or koala ACE2 orthologs, which cannot be engaged with WT virus, for cell entry, 127 with potential to extend its host range to these species. 128

The spike protein of Kappa, Delta and B.1.618 gained an increased binding affinity 130 with human ACE2 and other othologs. 131

As the Kappa, Delta and B.1.618 spike mediate increased cell entry efficiency, which 132 could be caused by the increased binding affinity for ACE2. We employed a cell-based 133 assay that uses flow cytometry to assess the binding of RBD of spike protein to human 134 ACE2 (Fig. S1A) . We cloned the cDNA of human ACE2 into a bicistronic lentiviral vector 135 (pLVX-IRES-zsGreen1) that expresses the fluorescent protein zsGreen1 via an IRES 136 element and can be used to monitor transduction efficiency. Next, WT or variants derived 137 RBD-His (a purified fusion protein consisting of the RBD and a polyhistidine tag at the 138 C-terminus) was incubated with HeLa cells transduced with the human ACE2. Binding of 139 RBD-His to ACE2 was then quantified by flow cytometry (Fig. S1 and Fig. 2A (98.48%) were higher than WT (89.6%), suggesting the RBD of variants bind human 143 ACE2 with a higher affinity ( Fig. 2A) . 144

To test whether the spike proteins of the variants have altered in binding with mouse, 7 RBD-His to ACE2 ortholog was quantified by flow cytometry (Fig. S1 and Fig. 2A) .The 148

WT RBD-His cannot bind with mouse, marmoset or koala ACE2 as previously 149 reported 2,7,29 ; In contrast, RBD-His of Kappa, Delta, and B.1.618 bind with mouse, 150 marmoset and koala ACE2 with a varying affinity, suggesting that these variants have 151 evolved to gain the function for binding with non-human ACE2 orthologs. 152

As Delta variant has now become the most dominant strain of the coronavirus 153 circulating globally, we expressed and purified recombinant human and ACE2, as well as 154

WT and Delta variant's RBDs, and directly assayed the protein binding in vitro by surface 155 plasmon resonance (SPR) analysis (Fig. 2B) . The dissociation constant (Kd) for human 156 ACE2 binding the WT RBD was 5.50 nM while that of Delta RBD was 2.67 nM, about 157 2-fold higher than WT RBD. As expected, the WT RBD cannot bind with mouse ACE2; 158 strikingly, the Delta RBD could bind mouse ACE2 with Kd of 65.93 nM. 159

Taken together, our results demonstrated that the spike proteins of Kappa, Delta and 160 B.1.618 have evolved to enhance their binding affinity with human ACE2. Remarkably, the 161 spike proteins of these variants also gain the function to bind with mouse, marmoset and 162 koala ACE2, with potential to extend SARS-CoV-2 host range. 163 164 P681R mutation in Kappa and Delta variants with enhanced spike protein cleavage. 165 SARS-CoV-2 spike harbors a multibasic furin cleavage site (residues 681-686; 166 PRRARS) at the S1/S2 junction, and the proteolytic processing of the spike by furin and 167 TMPRSS2 proteases is important for SARS-CoV-2 infection 5,30-33 . Kappa and Delta 168 variants contain a P681R substitution ( Fig. 1A and 2C) , potentially optimizing the furin 169 cleavage site, which prompted us to examine the effect of the P681R substitution on furin 170 cleavage. To do this, we produced MLV viral particles pseudotyped with WT spike, Kappa 171 spike, or P681R spike. Viruses in the cell culture supernatants were harvested and 172 concentrated for immunoblot analysis of spike protein cleavage by polyclonal antibody 173 against spike protein (Fig. 2C) . Interestingly, our data showed significantly increased was 2.1-(Kappa) or 3.0-(P681R) folds higher than WT, and the cleaved S2/S0 was 2.4-177 (Kappa) or 1.8 (P681R)-folds higher than WT. These results suggest that P618R 178 substitution in the Kappa and Delta variants could enhance spike cleavage, and 179

subsequently facilitate viral entry and transmission. neutralizing antibodies. Therefore, we sought to determine the sensitivity of these variants 188 to neutralization by convalescent serum. We chose plasma from COVID-19 patients 189 (Table S1, as well as other orthologs and the sensitivity to neutralization by convalescent plasma and 225 recombinant ACE2-lg decoy receptor (Fig. 5) . 226

We found that the Kappa variant has not increased in cell entry of HeLa-human ACE2 227 cells and slightly increased in cell entry of HeLa-mouse ACE2 cells. Delta variant has 228 significantly increased in cell entry of HeLa-human ACE2, HeLa-mouse ACE2, 229

HeLa-marmoset ACE2 and HeLa-koala ACE2 cells; consistently, Delta RBD binds human 230 ACE2 with a higher affinity. Remarkably, Delta RBD gained the ability to bind with mouse, 231 marmoset and koala ACE2 orthologs, which exhibited limited binding affinity with WT RBD. usages into mouse, koala and New World monkeys (Fig. 1B) , raising a potential risk of 243 mice or other rodents becoming the reservoirs for SARS-CoV-2, and the virus could 244 potentially spillback to humans as the mice are living closed to human (Fig. 5) . Thus, we 245

recommend that the host range should be closely monitored along the continued evolution with human ACE2 (Fig. 2B) . In addition, it has been demonstrated that human ACE2 262 peptidase activity and viral receptor activity could be uncoupled 42,43 , thus it is possible that 263 the enzymatic inactivated ACE2 was developed as the antivirals in clinics to avoid the 264 potential ACE2 side effected mediated by its enzymatic activity. 265

New variants of concern will continue to emerge as the COVID-19 pandemic persists, triplicate, and the data are representative of three independent experiments (mean ± standard deviation). ns, no significance; *, P < 0.05, **, P < 0.01, ***, P < 0.001. 375

Significance assessed by one-way ANOVA. Table S1 . Summary of sera information of COVID-19 patients for evaluation of 468 

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