Recently, a new SARS-CoV-2 variant named VOC-202012/01 and classified as lineage B.1.1.7 has been reported to spread unexpectedly fast in the United Kingdom1. This variant attracted people's attention also due to its unusually large number of genetic changes on the spike protein, which are speculated to increase infectivity and cause immune escape. Eight of 17 mutations in VOC-202012/01 variant is located at the spike protein including 69-70del, 144/145del, N501Y, A570D, P681H, T716I, S982A and D1118H (COVID-19 Genomics Consortium UK). The 69-70del has been described to become a dominant mutation in an immune suppressed individual treated with convalescent plasma2-5 and also exists in several natural variants including those transmitted in mink6. The N501Y mutation in the receptor binding domain (RBD) has been identified in the mouse-adapted strain, which potentially associated with the increased virulence in mice7. It is also suggested that theN501Y mutation increased its binding affinity to human ACE28. Furthermore, as P681H mutation is immediately adjacent to the Furin cleavage site (682-685 RRAR), the proteolytic activity of the enzyme assumed to be changed as well9.
Up to Jan 13th, 2021, there are 359, 302 SARS-CoV-2 sequences in the GISAID database, of which 44% were from the UK. We first investigated the growth trend of all the mutations with the frequency above 1% globally and in the UK (Fig 1A). The most popular mutations globally or in the UK are D614G, A222V, L18F, and S477N. Since December 2020, the VOC-202012/01 variant including multiple mutations 69-70del, 144/145del, N501Y, A570D, P681H, T716I, S982A and D1118H is found to increase very fast. As the recent SARS-CoV-2 variants appeared as combinations of different mutations, we then chose the five most frequency natural variants happened in the UK for further study, which including D614G+L18F+A222V, D614G+A222V, D614G+S477N, D614G+69-70del+N439K and the VOC-202012/01 strain (Fig1B). To determine whether their infectivity and antigenicity had been altered, we constructed pseudotyped viruses for the five variants and 12 single mutants (L18F, A222V, S477N, 69-70del, 144/145del, N501Y, A570D, P681H, T716I, S982A, D1118H and N439K) composed those variants based on D614G virus using VSV vector system
The infectivity of the five variants
The infectivity of the five variants was first tested in four SARS-CoV-2 susceptible cell lines, including two human cell lines Huh-7 and 293T-ACE2, and two non-human primate cell lines LLC-MK2 and Vero. The results showed that the infectivity of VOC-202012/01 variant increased at least 4-fold (Fig2A) compared to the reference strain 614G. Furthermore, the infectivity of D614G +A222V, D614G+69-70del+N439K and D614G+L18F+A222V also slightly increased, whereas the infectivity of D614G+S477N is decreased. To figure out whether any single mutant could determine the infectivity of the variants, we investigated the impact of single mutation that composed the variants on infectivity. As shown in Figure 2B, of the eight mutants composed VOC-202012/01, the infectivity of 69-70del, 144/145del, A570D, P681H, S982A, D1118H were all increased to some extent, except N501Y and T716I (Fig2B). Therefore, the accumulative effects of different mutations may cause the increased infectivity of VOC-202012/01. The enhancement effect of 69-70del might be the reason of the increased infection in D614G+69-70del+N439K. Moreover, the decreased infectivity of L18F may result in the relative low counts of D614G+L18F variant (Fig 1) and the decreased infectivity of D614G+L18F+A222V mutant compared to D614G+A222V (Fig2B). To our surprise, we did not find the increased infectivity in the N501Y single mutated variant, which was not in compliance with other study using yeast-display platform8. This may be due to the different glycan between yeast versus human cells. In addition, RBD is just one domain of the viral spike, while dynamic movements of viral entry is complicated in the physiological environment.
As the mutation P681H is immediately adjacent to the Furin or TMPRSS2 cleavage site, the structure changes of the mutation are predicted to influence the function of the enzymes9. We subsequently investigated the proteolytic activity of the enzyme by examining the proportion of S1 and S2 protein in the 293T cells expressed with the spike protein of the variants. As shown in the Fig 2D, there is no difference of the S2 proportion in the VOC-202012/01 variant compared to D614G. Furthermore, the infectivity of the variants in the Furin/TMPRSS2/Cathepsin L overexpressed cells were tested, the results showed that the infectivity of VOC-202012/01 was increased in the Furin and TMPRSS2 and Cathepsin L overexpressed cells which was comparable to the reference D614G variant (fig 2C). Therefore, our results suggested the P681H mutation or other mutations VOC-202012/01 did not change the proteolytic activity of Furin as well as TMPRSS2 and Cathepsin L.
The animal tropism of the five variants
To investigate whether the tropism of VOC-202012/01 and the other four epidemic variants to difference species are changed, 14 ACE2s from different species were overexpressed in 293T cells (Fig3A and B). We found that the infectivity of VOC-202012/01 was significantly changed only in the mouse ACE2 overexpressed cell lines, with enhancement of 79.5-fold compared to 614G. Single mutation tests showed that N501Y and A570D increased their infectivity only in mouse ACE2 overexpressed cell lines. That would be the reason for this dramatic increased infectivity of VOC-202012/01 (Fig3C), which contained both N501Y and A570D mutations. This result was consistent with the previous reported about the mouse-adapted strain containing the N501Y mutation1. Meanwhile, the infectivity of other variants was not found to be changed in all overexpressed cell lines with different ACE2.
The neutralization activity of mAbs to the five variants.
To find out if the existing neutralizing monoclonal antibodies are still effective to the five variants, the neutralizing activity of 17 monoclonal antibodies (mAbs) targeting different area of receptor-binding domain was tested. Nine out of 17 mAbs, including H00S022, A157, A247, 2F7, 11D12, 1F9, 2H10, 10D12 and 10F9 displayed significantly reduced neutralizing activity against the VOC-202012/01 variants as well as variants carrying single N501Y mutant (Fig 4A and4B). Furthermore, mAbs H00S022 and A157 also lost most of their neutralizing activity against D614G+69-70del+N439K and N439K +D614G mutation (Fig 4A and Fig S1). The S477N variant show decreased susceptibility to mAb 7B8, while most of the antibodies were still effective (Fig 4A and Fig S1). No decreased neutralization to all the mAbs was observed for the variants without mutation in RBD. The results indicated the mutations N439K, S477N and N501Y in RBD can affect the neutralizing property of SARS-CoV-2 variants.
The neutralization activity of convalescent sera and immunized sera to the five variants
In order to predict whether the five variants could change their neutralizing property to polyclonal antibodies, we examined the neutralization ability of sera from convalescent patients with COVID-19, as well as sera from animals immunized with purified RBD protein (mouse, horse) and recombinant DNA containing full-length spike gene (guinea pig). As shown in Figure 4B and4C, the neutralization ability of VOC-202012/01 against most of the convalescent sera was not changed. However, the convalescent sera showed increased neutralization ability against D614G+L18F+A222V to some extent, and decreased neutralization ability to D614G+S477N. To our surprise, the pattern of neutralization was different in animal immunized sera compared to convalescent human sera. VOC-202012/01 showed the decreased neutralization in sera from both RBD protein immunized mice and horses and spike DNA immunized guinea pigs. The results indicate that composition of the sera from convalescent patients and immunized animals may be different, which need to be studied further.
In this study, the biological functions of top five variants of SARS-CoV-2 in the UK were analyzed. We found that the VOC-202012/01 variant does enhance virus infectivity. This variant also decreases the neutralization activity of some monoclonal antibodies but does not affect significantly the neutralization effect of convalescent sera. As the epidemic progresses, the complicated variants of SARS-CoV-2 would occur. It is very important to closely monitor their functional change for prevention and control of SARS-CoV-2 epidemic.