Qualitatively distinct modes of Sputnik V vaccine-neutralization escape by SARS-CoV-2 Spike variants
Summary (3 min read)
INTRODUCTION.
- The recent emergence of novel SARS-CoV-2 variants has reignited concerns that the pandemic may not be so easily brought under control.
- The P.2 lineage, originally detected in Rio de Janeiro, carries only the E484K mutation in the RBD and has spread to other parts of South America, including Argentina 30 .
- Since the Sputnik vaccine is now in use not only in Russia, but also in countries like Argentina, Mexico, and Hungary, where some of the VOC and emerging lineages bearing the E484K mutation are more widespread, it is critical to assess the neutralizing activity of Sputnik vaccine elicited antibody responses against these cognate VOC and mutant spikes.
SARS-CoV-2 Spike proteins.
- Several groups have now generated replication-competent VSV expressing SARS-CoV-2 spike in place of VSV-G (rcVSV-CoV2-S) [35] [36] [37].
- To generate rcVSV-CoV2-S containing different variants or mutants on demand, without the need for extensive passaging, the authors developed a robust reverse genetics system and VNA which leverages the cell lines they previously developed for a standardized SARS-CoV-2 VNA that correlates well with live virus neutralization 41 .
- The characteristics of the vaccine recipient cohort (n=12) receiving the twodose regimen of the Sputnik vaccine are given in Table 1 .
- The Hill slope of their neutralization curves for B.1.351 were extremely shallow (h<0.40), resulting in a low IC50s and maximal neutralization of 50-60% even when extrapolated to full serum strength (Fig. 3E and Fig. 4 ).
- This 1.5 to 2-fold increase in RBD-Fc Experimental measurements of both RBD and trimeric spike binding to ACE2 have revealed that the E484K mutation alone does not confer increase binding affinity for ACE2 unlike N501Y 44, 46 .
DISCUSSION
- A key public health concern related to emergent SARS-CoV-2 variants is that by incrementally accruing mutations that escape neutralizing antibodies, they will penetrate herd immunity and spread to reach unvaccinated individuals, some of whom will be susceptible to severe or fatal disease.
- Even more revealing is their dose-response curves.
- The neutralization curves for B.1.351 in their study are not classically sigmodal and have significantly shallower slopes than WT, B.1.17 and E484K, which result in ≤ 90% neutralization for all but one sample when extrapolated to full serum strength.
- While the E484K substitution appears to be a common route of escape from many RBD-targeting monoclonal antibodies, it is somewhat surprising that a single mutation can confer a significant degree of neutralization resistance from polyclonal responses.
- Even in the case of infection by VOC, their data reveal a concerning potential of B.1.351, and to a lesser extent, any variant carrying the E484K substitution (e.g. P.2), to escape the neutralizing antibody responses that this immunization elicits.the authors.
Cell lines
- The HEK 293T-hACE2-TMPRSS2 cells were plated on collagen coated plates or dishes.
- BSR-T7 cells 61 , which stably express T7-polymerase were maintained in DMEM with 10% FBS.
- These accessory plasmids were a kind gift from Dr. Benjamin tenOever.
Generation of VSV-CoV2 spike from cDNA
- 30 min later, transfection mixture was applied to 293T-hACE2-TMPRSS2 cells in a dropwise fashion.
- Cells were maintained with medium replacement every day for 4 to 5 days until GFP positive syncytia appeared.
- Rescued viruses were amplified in Vero-CCL81 TMPRSS2 cells 41 , then titered and used for the assay.
Virus neutralization assay
- 5 x 10E4 293T-hACE2-TMPRSS2 cells per well were seeded onto collagen-coated 96 well cluster plates one day prior to use in viral neutralization assays.
- Virus stocks were mixed with serially diluted serum for 10 minutes at room temperature, then infected to cells.
- At 10 h post infection, GFP counts were counted by Celigo imaging cytometer .
- For calculation of IC50, GFP counts from "no serum" conditions were set to 100%; GFP counts of each condition (serum treated) were normalized to no serum control well.
- Inhibition curves were generated using Prism 8.4.3 (GraphPad Software) with 'log vs normalized response -variable slope' settings.
Design of RBD-Fc producing Sendai virus
- Sendai virus (SeV) Z strain cDNA sequence (AB855655.1) was generated and cloned into pRS vector with the addition of eGFP transcriptional unit at the head of SeV genome.
- The sequence of F transcriptional unit was from SeV fushimi strain (KY295909.1) due to the cloning reason.
- The authors refer to the pRS-based plasmid coding this sequence as pRS-SeVZ-GFP-F fushimi in this paper.
- RBD-Fc construct was generated as below; codon optimized DNA sequence of from SARS-CoV-2 spike (MN908947) in pCAGGS a gift of Dr. Florian Krammer 64 .
Generation of recombinant Sendai virus from cDNA.
- 2x10E5 BSR-T7 cells per well were seeded onto 6-well cluster plates.
- At one day post transfection, medium was replaced with DMEM + 0.2 µg/ml of TPCK-trypsin (Millipore Sigma, #T1426), with subsequent medium replacement each day until infection reached 100% cytopathic effect.
- Supernatants were stored at -80˚C until use in experiments.
Titration of viruses.
- GFP positive foci were counted at 24 hours post infection using a Celigo imaging cytometer (Nexcelom, Inc.).
- Infectivity is presented in infectious units (IU) per mL. For VSV-CoV2 titration, 5 x 10E4 293T-hACE2-TMPRSS2 cells per well were seeded onto a collagen-coated 96 well plate.
- Serially diluted virus stocks were then applied to the cells, and GFP positivity was scored at 10 h post infection using a Celigo imaging cytometer.
Production of proteins and purification.
- Cells were infected by SeV at MOI of 0.1 for one hour, followed by replacement of medium with DMEM supplemented with 0.2 mg/mL TPCK-trypsin.
- Supernatant including RBD-Fc were applied to Protein G Sepharose (Millipore Sigma, #GE17-0618-01) containing column (5ml Polypropylene Columns ;ThermoFisher, #29922), followed by wash and elution.
- Authorities who make SARS-CoV-2 sequence data available in a timely manner via the GISAID initiative 66, 67 .
- No statistically significant differences were detected between WT and VOC spikes in the size of GFP+ syncytia at any given time point (two-way ANOVA as above, 'ns' not indicated in graph).
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