key: cord-0781852-wzy2jupp authors: Kruglova, Natalia; Siniavin, Andrei; Gushchin, Vladimir; Mazurov, Dmitriy title: SARS-CoV-2 cell-to-cell infection is resistant to neutralizing antibodies date: 2021-05-05 journal: bioRxiv DOI: 10.1101/2021.05.04.442701 sha: 240564042add906f003ce946381820a4857e4046 doc_id: 781852 cord_uid: wzy2jupp The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. Based on available studies, this is the first attempt to quantitatively measure SARS-CoV-2 cell-to-cell infection, for which the mechanisms are largely unknown. The findings suggest that this route of SARS-CoV-2 transmission could be of great importance for treatment and prevention of COVID-19. Importance Immune surveillance of viral or bacterial infections is largely mediated by neutralizing antibodies. Antibodies against the SARS-CoV-2 spike protein are produced after vaccination or infection, but their titers only partly reflect the degree of protection against infection. To identify protective antibodies, a neutralization test with replicating viruses or pseudoviruses (PVs) is required. This study developed lentiviral-based PV neutralization assays that, unlike similar systems reported earlier, enable quantitative measurement of SARS-CoV-2 neutralization in cell cocultures. Using both PVs and replication-competent virus, it was demonstrated that SARS-CoV-2 cell-to-cell infection is considerably more resistant to serum neutralization than infection with purified viral particles. The tests are easy to set up in many labs, and are believed to be more informative for monitoring SARS-CoV-2 collective immunity or entry inhibitor screening. This was achieved with replication-dependent reporter vectors that were developed earlier. 37, 38 The key 83 feature of these vectors is that the reporter is silent in the pseudovirus-producing cells, but active after 84 infection of the target cells and completion of one cycle of viral replication. This enables infectious events 85 to be measured directly in cocultures of producer and target cells at zero background level. The concept 86 was effectuated by placing a reporter cassette in reverse orientation relative to HIV-1 genomic RNA and 87 through interruption it with an intron, that prevented a functional reporter protein expression from LTR HIV-1 gp41 increased the level of infectious lentiviral particle production. 40 Later on, the spike protein 105 from SARS-CoV-2 with a cytoplasmic portion deleted was used in a number of pseudoviral test 106 systems. 6,32,41-43 The furin cleavage site that was present in SARS-CoV-2 S but not in the SARS-CoV S 107 protein is thought to be involved in spike maturation, virus entry, and syncytium formation 5 and, 108 therefore, can also affect infectivity measured with PVs. 109 In order to establish an HIV-based infection system, the SARS-CoV-2 S protein was modified by 110 deleting the last 19 amino acids (∆C19) or substituting them with eight amino acids from HIV-1 gp41 (H2). 111 These modifications were either combined with the mutation in the furin cleavage site RRAR to A (∆F), or 112 left uncombined, to generate the six variants of spike protein indicated in Figure 1A . Next, a SARS-CoV-2 113 permissive HEK 293T cell line was established with a stable expression of the human ACE2 receptor via 114 lentiviral transduction and FACS sorting ( Figure 1B) . A cell-free infectivity assay was set up, as 115 schematically illustrated in Figure 1C . PVs were generated by co-transfecting 293T cells with one of the S 116 protein coding plasmids, HIV-1 packaging vector pCMV-dR8-2, and an improved intron-regulated reporter 117 vector pUCHR-inLuc-mR, capable of measuring both cell-free and cell coculture infections using the mean 118 of luciferase activity. 37,38 Additionally, the pUCHR-IR-GFP reporter plasmid without an intron was used to normalized to p24 levels, and presented relative to the values obtained for the wild-type S protein. As 124 shown in Figure 1D , ∆C19 moderately increased the level of infection, while the H2 modification had no 125 or little effect on infectivity. By contrast, the ∆F mutation resulted in about a 1.5 log increase in PV 126 infectivity. On ∆F background, however, the improving effect of ∆C19 was much less pronounced than 127 detected without ∆F. PV titration was used to confirm a substantial effect of the ∆F mutation on the level 128 of PV transduction (~20 fold enhancement in many PV dilutions) ( Figures 1E and F) . The increased 129 infectivity of the ∆F mutant PVs was not accompanied by an increase in S protein expression on PV-130 producing 293T cells ( Figure 1G ). Thus, it was unclear whether ∆F infectivity was enhanced from S 131 incorporation into PVs or if this was a feature of the 293T cellular system, in which S processing by furin is 132 important during the fusion step of the viral life cycle. In summary, a SARS-CoV-2 cell-free infection test was developed in a 24-well plate format with a 134 high level of sensitivity. Using the ∆F∆C19 modification of the SARS-CoV-2 S protein, 50-60% GFP 135 transduction and about 4 log over the background elevation of luciferase activity was achieved, making 136 consecutive inhibitory analysis accurate and reproducible. Development of a SARS-CoV-2 pseudoviral system to quantify cell-to-cell infection. 150 In order to evaluate SARS-CoV-2 cell-to-cell infection, a one step transfection-infection assay with 151 the inLuc-mR reporter vector described earlier was set up. 37,38 Briefly, 293T/ACE2 cells were co-152 transfected with viral vectors, as outlined above, for cell-free infection. In approximately 12-16 hours, 153 transfected cells started to produce PVs, which infected nearby 293T/ACE2 cells. At 48 hours post-154 transfection, one cycle of replication was complete and luciferase activity can be measured (Figure 2A ). Using this assay, the levels of infection with one of the six variants of the S protein were quantified. Wild 156 type, ∆C19, and H2 proteins did not mediate infection at all, however, all three variants bearing the ∆F 157 mutation supported a good level of infection. The addition of ∆C19 to ∆F increased the level of infectivity 158 by 0.5 log, while the H2 modification had no effect on the signal ( Figure 2B) . We have previously The differences between ΔF mutants were calculated by one-way ANOVA with the Tukey's multiple comparison test, and are significant at reproduced at the level of ~10 6 RLU, giving an opportunity to detect a wide range of inhibitory activity. Five COVID-19 convalescent sera with high neutralizing activity were selected and evaluated in the cell-194 free infection test with ∆F∆C19. As shown in Figure 3A and E, all samples demonstrated NT 50 in a range 195 between 1/1500 and 1/12000 dilution, whereas a non-immune serum had no inhibitory activity. 196 Additionally, in order to determine whether the furin cleavage site mutation influenced neutralization 197 titer, ∆C19 variant per se was tested. The inhibition rates against ∆C19 were slightly higher than those for 198 ∆F∆C19 ( Figure 3B ), including NT 50 values ( Figure 3C ). Nevertheless, similar titration curves for all tested 199 sera were observed with both ∆F∆C19 and ∆C19. Thus, the ΔF mutation did not dramatically change S 200 protein neutralization in the cell-free test, but was absolutely necessary for measuring cell coculture 201 infectivity and making the correct comparison between two types of infection. The cell-to-cell neutralization test was designed to be as similar as possible to settings used for the 203 cell-free PV inhibition analysis. To generate SARS-CoV-2 producer cells, non-permissive 293T cells were 204 co-transfected with pCMV-Δ8.2R, pUCHR-inLuc-mR, and pCG1-SARS-2-SΔFΔC19 plasmids, as described for In summary, by using the developed pseudoviral single cycle replication assay with the intron-220 regulated reporter vector, it was demonstrated that SARS-CoV-2 cell coculture infection was much more 221 resistant to neutralization by convalescent sera than infection with purified PVs. Cell-to-cell SARS-CoV-2 spreading assay was initiated by infecting 2.6x10 4 Vero E6 cells with fully 257 competent virus for 24 hours, followed by PBS washing and preincubating with a serum before it was 258 added to 5.6x10 4 uninfected Vero E6 cells. The level of CPE was measured five days later using the MTT 259 test. In stark contrast to the cell-free infectious test, the majority of the serum samples, even at minimal 260 dilution, did not prevent cytopathic effect of fully-competent SARS-CoV-2 ( Figure 4C) , with the exception 261 of serum 9611, which at 1/20 dilution displayed ~50% inhibitory activity. In conclusion, it was demonstrated that cell-to-cell spread of the fully competent wild type SARS- CoV-2 is almost completely resistant to convalescent serum neutralization. This effect was even more 264 pronounced than the resistance detected using pseudoviruses. The aim of this study was to not just replicate a PV system but rather, develop a lentivirus-based 287 PV test capable of measuring SARS-CoV-2 infection, both with purified PVs and in cell cocultures. The 288 latter has not been appreciated previously or measured accurately. The use of identical vectors to initiate 289 both types of infection makes comparative analysis of PV infectivity more rigorous. First, our system was 290 optimized by pseudotyping PVs with S protein mutants. Since the cytoplasmic portion of the S protein has 291 an ER-Golgi retention signal needed for incorporation into coronaviruses that bud from endosomal 292 membranes, 48 ,49 but which may not be optimal for efficient pseudotyping of lenti-or retroviral particles 293 that assemble predominantly at the plasma membrane, this signal should be removed. An early study on CoV-2. 6,32,41-43,50 The truncation of last 18-19, 43,50,51 or even 13, amino acids 41 enhanced PV infectivity 297 from 10 to 100 fold. Consistent with the data reported above, our study has shown that the ΔC19 298 mutation improved cell-free infection by 15 fold, and one-step infection by 5 fold (Figure 1D and 2B) . 299 Nevertheless, a few studies did not find a substantial influence from ΔC19 42 or point mutations in the ER 300 retention signal 33,52 on PV infectivity. In agreement with published papers, 33,39,41 we did not observe 301 substantial differences between wt and ΔC19 S protein levels expressed on the surface of PV producing 302 cells ( Figure 1G) . Thus, the mechanism of enhanced infectivity for ΔC19 PVs remains unclear, and can be 303 related to improved incorporation of this mutant into PVs 40,41,52 and/or stabilization of S1-S2 subunit Next, the generated PV system was validated in a neutralization test with convalescent sera. As 326 the ΔF mutation is localized in the external part of the S protein, it can potentially influence serum 327 neutralization activity. A comparison of the ΔFΔC19 and ΔC19 mutants revealed that ΔF required ~ 328 twofold higher serum concentration for PV neutralization than without ΔF ( Figure 3C ). This is consistent CoV-2, which transmits in airway epithelium. In conclusion, we developed lentivirus-based single round pseudoviral infection assays suitable for 364 quantitatively measuring SARS-CoV-2 entry in cell-free and cell coculture conditions. Using this system, as 365 well as the SARS-CoV-2 spreading assay, our study has shown that cell-to-cell infection of SARS-CoV-2 is 366 considerably more resistant to serum neutralization than infection with purified viral particles. These To measure neutralizing activity of sera from COVID-19 patients, sera were serially four-fold diluted in 431 growth medium and preincubated with pseudoviruses in the total volume of 400 µl for 1 h at room 432 temperature before addition to target cells. 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