key: cord-0997138-hwbb3z9i
authors: Kuzmina, Alona; Wattad, Seraj; Khalaila, Yara; Ottolenghi, Aner; Rosental, Benyamin; Engel, Stanislav; Rosenberg, Elli; Taube, Ran
title: SARS CoV-2 Delta variant exhibits enhanced infectivity and a minor decrease in neutralization sensitivity to convalescent or post-vaccination sera
date: 2021-11-15
journal: iScience
DOI: 10.1016/j.isci.2021.103467
sha: 9104966195d908e1cef8f64efecf678aacf44f18
doc_id: 997138
cord_uid: hwbb3z9i

Since their identification, SARS-CoV-2 Kappa and Delta have rapidly spread to become globally dominant. However, their infectivity and sensitivity to administered vaccines have not been documented. We monitored neutralization potential of convalescent or BNT162b2-post-vaccination sera against Kappa and Delta SARS-CoV-2 pseudoviruses. We show that both variants were successfully neutralized by convalescent and post-vaccination sera, exhibiting a mild decrease in their neutralization sensitivity. Of the two variants, Delta presented enhanced infectivity levels compared to Kappa or wild-type SARS-CoV-2. Nevertheless, both variants were not as infectious or resistant to post-vaccination sera as the Beta variant of concern. Interestingly, the Delta plus variant (AY.1/B.1.617.2.1) exhibited high resistance to post-vaccination sera, similar to that of the Beta SARS-CoV-2. However, its infectivity levels were close to those of wild-type SARS-CoV2. These results account for the worldwide prevalence of Delta variant of concern, and confirm the efficacy of the BNT162b2-vaccine against circulating other Delta variants.

In late 2019, an emergent betacorinavirus, Severe Acute Respiratory Syndrome Coronavirus 2 41 (SARS-CoV-2) was identified as the cause for severe respiratory disease in humans. One year 42 into this outbreak, COVID19 is a global pandemic forcing most countries to adopt a lockdown 43 mode, causing economic burden and human suffering with more than 178M cases and 3.8M 44 deaths. (Zhou et al., 2020; Zhu et al., 2020) . SARS-CoV-2 infection of its target cells occurs 45 via the spike glycoprotein, S, a trimeric class I fusion transmembrane glycoprotein with S1 and 46 S2 subunits that are non-covalently associated. Within S1, the receptor binding domain (RBD; 47

We acquired blood samples from a cohort of convalescent sera (n=35) drawn from recovered 116 COVID19 patients who presented severe disease symptoms (n=14) as well as post-vaccination 117 individuals who received two doses (9-11 days post second dose; n=19) of the BNT162b2-118

Pfizer vaccine (see supplementary Table 1 and Taube 2 for additional details on sera samples 119 and timing of collection). All convalescent sera were originated from patients that were 120 infected with the Alpha-SARS variant. We employed neutralization assays to monitor the 121 potency of each of our sera samples to neutralize pseudoviruses that exhibit spike of wild-type 122 SARS CoV-2, or its Kappa-B.1.617.1 variant. This variant harbors L452R; E484Q, D614G 123 and P681R spike mutations. Our data showed that both convalescent and post vaccination sera 124 neutralized B.1.617.1, with only a modest reduction in neutralization potential that was 125 detected in both sera. For convalescent sera, we observed relatively low titers of neutralizing 126 antibodies (nAb), with a x1.78-fold reduction in neutralization sensitivity relative to wild-type 127 SARS-CoV-2 ( Figure 1A) . Upon vaccination, titers of nAb increased, and successfully 128 We next employed neutralization assays and monitored the potency of each of our sera samples 143 to neutralize pseudoviruses that exhibit spike of Delta-B.1.617.2 variant. This variant harbors 144 L452R; T478K, D614G and P681R spike mutations. Our data showed that both convalescent 145 and post vaccination sera neutralized B.1.617.2, with only a modest reduction in neutralization 146 potential that was detected in both sera. For convalescent sera, we observed relatively low titers 147 of neutralizing antibodies (nAb), with a x2.35-fold reduction in neutralization sensitivity 148 relative to wild-type SARS-CoV-2 (Figure 2A ). Upon vaccination, titers of nAb increased, 149 and successfully neutralized B.1.617.2. Herein, a x2.11-fold decrease in neutralization 150 sensitivity of Delta-B.1.617.2 to post vaccination sera was observed relative to wild type 151 SARS-CoV-2 ( Figure 2B ). We conclude that both convalescent and the BNT162b2 vaccine 152 provide neutralization protection against pseudoviruses that carry the Delta-B.1.617.2 RBD 153 mutations, with only a moderate reduction of about x2 fold in neutralization potential. 154

We also tested the infectivity levels of Delta-B. we were interested in elucidating the role of this mutation in neutralization sensitivity and 166 infectivity of the two variants and the effects of single and combined spike mutations on 167 neutralization sensitivity and infectivity. We thus tested the neutralization potential of post-168 vaccinated sera towards single or combined RBD mutations that preset within the spike of 169 B.1.617 variants. For this, we generated pseudoviruses that carried a single L452R spike 170 mutation, combined L452R/E484Q or L452/E484K -RBD spike mutations as well as single 171

T478K mutants. Our analysis demonstrated that relative to wild-type SARS CoV-2, L452R-172 pseudoviruses exhibited x2.14-fold reduction in their neutralization sensitivity titers against 173 post-vaccination sera ( Figure 3A) . Furthermore, combining the L452R with E484Q (i.e. Kappa 174 B.1.617.1), confirmed the decrease in neutralization sensitivity against post-vaccination sera, 175 presenting x2.45-fold reduction in neutralization sensitivity relative to wild type SARS CoV-176 2. Interestingly, switching E484Q with E484K, resulted in a slight decrease of x2.6-fold of 177 neutralization sensitivity titers relative to SARS-CoV-2 pseudoviruses. Indeed, E484 is known 178 to serve as a key residue in RBD that promotes neutralization resistance. Thus, pseudoviruses 179 that caried E484K spike mutation exhibited a x2.8-fold decrease in neutralization sensitivity, 180 relative to wild-type pseudoviruses. Similarly, L452/E484K pseudoviruses, showed a similar 181 reduction in neutralization sensitivity of x2.6-fold. The Delta-B.1.617.2, which carries T478K 182 mutation also exhibited a x2.18-fold reduction in its neutralization sensitivity towards post 183 vaccination sera. Furthermore, single T478K pseudoviruses showed no effects on 184 neutralization sensitivity relative to wild type or Kappa-B.1.617.1 pseudoviruses.

Finally, we also tested neutralization sensitivity of the Delta Plus variant and compared it to 189 the other indicated viruses. Surprisingly, our analysis demonstrated that this variant displayed 190 a x5.35 increase in its sensitivity to post vaccination sera, similar levels that were observed 191 with Beta variant of concern ( Figure 3A) . 192

The infectivity levels of pseudovirues that carried single L452R, combined L452R/E484Q or 193 L452/E484K was also determined by transducing HEK-ACE2 ( Figure 3B ). We showed that 194 pseudoviruses that carried L452R, pseudoviruses presented an enhanced infectivity levels 195 which were x2.3-fold above wild type SARS CoV-2 ( Figure 3B ). L452R/E484Q (i.e. Kappa-196 B.1.617.1) exhibited similar infectivity levels as wild type SARS-CoV-2 pseudoviruses. 197

Interestingly, L452R/E484K also presented similar infectivity levels as wild type SARS CoV- variant, is of a major concern. Most infections occur in young adults and children who have 215 not been vaccinated. Moreover, to some degree, vaccinated individuals are also found to carry 216 the Delta B.1.617.2 variant, as it rapidly dominates other variants of concern. Nevertheless, 217 despite increase in numbers of infected individuals, disease symptoms are not severe, as cases 218 of hospitalized patients slowly rise. Selective pressure on the virus to adapt its new host, led to 219 the early rise of new variants that carry unique mutations within the spike RBD that exhibit 220 either high affinity to the ACE2 receptor on human target cells, and efficient escape from 221 neutralizing antibodies (Figure 4) . As vaccination programs expand, viral evolution finds ways 222 to the emergence of evolutionary improved variants that present high transmissibility that 223 promote reinfection. These variants are defined as a concern by the WHO, as they rapidly 224 spread and potentially compromise vaccine efficiency. We can thus confirm that the Pfizer vaccine neutralizes the newly emerged B.1.617 variants. 303

Nevertheless, the mild decrease in neutralization sensitivity and primarily the enhanced 304 infectivity of the Delta-B.1.617.2 variant, provide an explanation for the rapid spread of this 305 variant, and call for measures that will limit viral spread and advance vaccination programs, as 306 its features can harm individuals who are not vaccinated. 307 308

There are several limitations of our system that need to be noted. Our work relies on 310 pseudoviruses, which are only used to characterize the first step of the virus life cycle, i.e. 311 binding of the viral particle to the host cell receptor and entry into the target cells that is to neutralize (inhibit viral attachment and entry) B.1.617 variants. We conclude that the spread 314 of the Delta-B.1.617.2 occurs mainly due to its increased infectivity relative to Kappa-315 B.1.617.1 Furthermore, the pseudovirus system has been broadly used in the literature for 316 testing vaccine neutralization efficiency against circulating variants of SARS Numerous studies have demonstrated by now high correlation between vacine-neutralization 318 titers measured against pseudovirus and live SARS- CoV-2 (Crawford et al., 2020; Garcia-319 Beltran et al., 2021; Ju et al., 2020; Kuzmina et al., 2021; Pinto et al., 2020; Wang et al., 2021; 320 Wang et al., 2020; Yan et al., 2020) . Moreover, the contribution of additional mutations outside 321 of the spike, may also affect resistance to neutralization, infectivity levels, or pathogenesis of 322 SARS CoV-2. Additionally, it is worth stating that our findings are relevant only to the tested 323 sera. However, the mid-sized cohort that was analyzed in our work, combined with other 324 reports with similar conclusions validates our findings. 325

Our work also implies that in pseudotype SARS-CoV-2 that transduce HEKACE-2 target cells, 326 the role of the Furin Cleavage Site (FCS) in the viral spike is non-essential. Thus, we performed 327 our transduction experiments in the presence of TMPRSS2 or in its absence ( Figure 3C) . 328

According to our data, we could not detect an effect of the TMPRSS2 protease on transduction 329 levels in our hands. We assume that the reason for this is the use of pseudovirus that cannot 

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Pseudotyped viruses were generated in HEK293T cells. Briefly, LTR-PGK luciferase 491 lentivector was transfected into cells together with other lentiviral packaging plasmids coding 492for Gag, Pol Tat Rev, and the corresponding wild type or mutate spike envelopes. Transfections 493 were done in a 10cm format, as previously described and supernatant containing virus were 494 harvested 72hr post transfection, filtered and stored at -80 0 C (Krasnopolsky et al., 2020). 495Neutralization assays were performed in a 96 well format, in the presence of pseudotyped 496 viruses that were incubated with increasing dilutions of the tested sera (1:2000; 1:8000: 497 1;32000: 1:128000) or without sera as a control. Cell-sera were for 1hr. at 37 o C, followed by 498 transduction of HEK-ACE2 cells for additional 12 hr. 72hr post transduction, cells were 499 harvested and analyzed for luciferase readouts according to the manufacturer protocol 500 (Promega). Neutralization measurements were performed in triplicates using an automated 501Tecan liquid handler and readout were used to calculate NT50 -50% inhibitory titers 502 concentration. 503