No evidence for increased cell entry or antibody evasion by Delta sublineage AY.4.2

Since the beginning of the COVID-19 pandemic, multiple SARS-CoV-2 variants have emerged. While some variants spread only locally, others, referred to as variants of concern, disseminated globally and became drivers of the pandemic. All SARS-CoV-2 variants harbor mutations relative to the virus circulating early in the pandemic, and mutations in the viral spike (S) protein are considered of particular relevance since the S protein mediates host cell entry and constitutes the key target of the neutralizing antibody response. As a consequence, mutations in the S protein may increase SARS-CoV-2 infectivity and enable its evasion of neutralizing antibodies. Furthermore, mutations in the S protein can modulate viral transmissibility and pathogenicity.


Sequence analysis and protein models
All S protein sequences and the underlying information (collection date, location) were obtained from the GISAID (global initiative on sharing all influenza data) database (https://www.gisaid.org/).
Protein models were are based on a template in which the SARS-2 S sequence was modelled on PDB: 6XR8 (5) using the SWISS-MODEL online tool (https://swissmodel.expasy.org) and further processed using YASARA (http://www.yasara.org/index.html).

Immunoblot
To investigate S protein cleavage and particle incorporation, vesicular stomatitis virus (VSV) pseudotypes bearing S proteins with a C-terminal HA-epitope tag were concentrated by high-speed centrifugation (13,300 rpm, 90 min, 4 °C) through a sucrose cushion (20% w/v sucrose in PBS) and subsequently lysed in 2x SDS-sample buffer (0.03 M Tris-HCl, 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.2% bromophenol blue, 1 mM EDTA) by incubation at 96 °C for 15 min. Quantification of protein bands was performed using the ImageJ software (version 1.53C, https://imagej.nih.gov/ij/). For the analysis of S protein incorporation into VSV particles, total S protein signals (uncleaved, S0 and cleaved, S2) were normalized against their respective VSV-M signals and the resulting values were further normalized against the B.1 S protein (set as 1). For quantification of S protein cleavage, total S protein signals (uncleaved, S0 and cleaved, S2) were set as 100% for each S protein and the respective portions of S0 and S2 were calculated.

Production of VSV pseudotypes
Viral particles pseudotyped with the SARS-CoV-2 S proteins were produced as described previously (6). In brief, 293T cells were transfected with plasmids encoding S protein or VSV-G, or empty plasmid (control) using the calcium phosphate method. At approximately 30 h posttransfection, cells were inoculated with VSV-G-transcomplemented VSV*ΔG(FLuc), a replication-deficient vesicular stomatitis virus (VSV) that lacks the genetic information for its own glycoprotein (VSV-G) and instead codes for two reporter proteins, enhanced green fluorescent protein (eGFP) and firefly luciferase (kindly provided by Gert Zimmer) (7). After 1 h of incubation, the inoculum was removed and cells were washed with phosphate-buffered saline (PBS). Thereafter, to neutralize residual input virus, all cells received DMEM medium containing anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no. CRL-2700) except for cells expressing VSV-G, which received medium without antibody. After an incubation period of 16-18 h, the culture supernatant was harvested, clarified from cellular debris by centrifugation at 4,000 x g for 10 min, aliquoted and stored at -80 °C until further use.

Transduction of target cells
For transduction, target cells seeded in 96-well plates were inoculated with equal volumes of pseudotypes and transduction efficiency was evaluated at 16-18 h post transduction by measuring luciferase activity in cell lysates. For this, cells were lysed in PBS containing 0.5% Triton X-100 (Carl Roth) for 30 min at room temperature. Subsequently, cell lysates were transferred into white 96-well plates and mixed with luciferase substrate (Beetle-Juice, PJK) before luminescence was measured using a Hidex Sense plate luminometer (Hidex).

VSV pseudotype-based neutralization assay
Collection of convalescent plasma and vaccinee serum samples and corresponding patient information have been described before (3,4,8). All serum and plasma samples were heatinactivated at 56 °C for 30 min and pre-screened for their ability to neutralize transduction of Vero cells by particles pseudotyped with SARS-CoV-2 B.1 S. Neutralization assays were conducted as described (4,8). In brief, S protein bearing particles were pre-incubated for 30 min at 37 °C with  a Pseudotype entry data normalized against the assay background (related to Fig. 1f). The experimental procedure is outlined in the legend of Figure 1. The assay background is defined signals obtained from cell inoculated with particles harboring no viral glycoprotein (background, set as 1). Green bars further indicate pseudotype entry mediated by VSV-G. Error bars indicate the standard error of the mean. b and c Individual neutralization data for each convalescent plasma (b) and vaccinee (BNT162b2/ BNT162b2) serum (c). Circles indicate mean values from four technical replicates and error bars indicate the standard deviation. Curves were calculated using a non-linear regression model (variable slope).