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Clinical Trial
. 2022 Jan 12;18(1):e1010242.
doi: 10.1371/journal.ppat.1010242. eCollection 2022 Jan.

SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

Dina Khateeb  1 , Tslil Gabrieli  1 , Bar Sofer  1 , Adi Hattar  1 , Sapir Cordela  1 , Abigael Chaouat  2 , Ilia Spivak  3 , Izabella Lejbkowicz  4 , Ronit Almog  4 , Michal Mandelboim  5 , Yotam Bar-On  1
Affiliations
Clinical Trial

SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

Dina Khateeb et al. PLoS Pathog. .

Abstract

In-depth analysis of SARS-CoV-2 quasispecies is pivotal for a thorough understating of its evolution during infection. The recent deployment of COVID-19 vaccines, which elicit protective anti-spike neutralizing antibodies, has stressed the importance of uncovering and characterizing SARS-CoV-2 variants with mutated spike proteins. Sequencing databases have allowed to follow the spread of SARS-CoV-2 variants that are circulating in the human population, and several experimental platforms were developed to study these variants. However, less is known about the SARS-CoV-2 variants that are developed in the respiratory system of the infected individual. To gain further insight on SARS-CoV-2 mutagenesis during natural infection, we preformed single-genome sequencing of SARS-CoV-2 isolated from nose-throat swabs of infected individuals. Interestingly, intra-host SARS-CoV-2 variants with mutated S genes or N genes were detected in all individuals who were analyzed. These intra-host variants were present in low frequencies in the swab samples and were rarely documented in current sequencing databases. Further examination of representative spike variants identified by our analysis showed that these variants have impaired infectivity capacity and that the mutated variants showed varied sensitivity to neutralization by convalescent plasma and to plasma from vaccinated individuals. Notably, analysis of the plasma neutralization activity against these variants showed that the L1197I mutation at the S2 subunit of the spike can affect the plasma neutralization activity. Together, these results suggest that SARS-CoV-2 intra-host variants should be further analyzed for a more thorough characterization of potential circulating variants.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Study design and participants demographics.
(A) Detailed description of SARS-CoV-2-infected individuals from whom nose-throat swabs were isolated. The infected individuals are represented by the study ID. The geographic location where the individuals were infected is shown in the table. (B) Ct values (right y-axis) and viral titers (left y-axis) of SARS-CoV-2 in nose-throat swabs. Values were obtained by Real-Time Reverse Transcriptase-Polymerase Chain Reaction measurements that was performed during the clinical diagnostic of the swabs. The study ID of the infected individuals is shown in the x-axis. (C) Schematic representation SARS-CoV-2 SGS. RNA isolated from nose swabs was reverse transcribed into cDNA using SARS-CoV-2 specific primer (N-Out-3’). cDNA was diluted to single copy per well and subjected to a nested PCR to amplify single copies of SARS-CoV-2 S gene and N gene. PCR products were then sequenced by Illumina MiSeq. Figure was generated using biorender.com.
Fig 2
Fig 2. Single genome analysis reveals SARS-CoV-2 spike diversity.
Phylogenetic trees depicting S genes isolated from nose-throat swabs by SGS. IDs of individuals from whom swabs were isolated are indicated in the figure. Black rectangles depict the dominant S gene isolated from each swab sample. Red rectangles depict S genes with missense mutations, grey rectangles depict S genes with silent mutations and blue rectangles depict S genes with nonsense mutations. Mutations that are not documented in the GSAID are marked with an asterisk. For each S gene variant, the nucleotide substitution is indicated (based on Wuhan-Hu-1 numbering). The segment with the number below each phylogenetic tree shows the length of branch that represents an amount genetic change. The amount of genetic change is the number of nucleotide substitutions divided by the length of the spike sequence.
Fig 3
Fig 3. Intra-host SARS-CoV-2 N-gene diversity.
(A) Phylogenetic trees depicting N genes isolated from nose-throat swabs by SGS. IDs of individuals from whom swabs were isolated are indicated in the figure. Black rectangles depict the dominant N gene isolated from each swab sample. Red rectangles depict N genes with missense mutations, grey rectangles depict N genes with silent mutations and blue rectangles depict N genes with nonsense mutations. Mutations that are not documented in the GSAID are marked with an asterisk. For each N-gene variant, the nucleotide substitution is indicated (based on Wuhan-Hu-1 numbering). The segment with the number below each phylogenetic tree shows the length of branch that represents an amount genetic change. The amount of genetic change is the number of nucleotide substitutions divided by the length of the N-gene sequence. (B) Pie charts showing the proportion of all N-gene sequences isolated from each swab sample. The White pie slice depicts unmutated sequences. The Colored pie slices depict mutated N genes. The different mutations are indicated in the figure. The number in the middle of the pie chart depicts the total number of N-gene variants that were sequenced. (C) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The x-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the y-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated N proteins and unmutated (Wuhan-Hu-1) N protein (WT). The x-axis depicts the mutation in the N proteins of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, p < 0.005). Figure was generated using biorender.com.
Fig 4
Fig 4. Reduced infectivity of SARS-CoV-2 pseudoviruses expressing mutated spikes.
(A) Schematic representation of the ten spike variants that were tested for infectivity. Amino acids substitution in each spike mutants is shown in the figure. NTD = N-terminal domain, RBD = receptor binding domain, FP = fusion peptide, HR1 = heptad repeat 1, HR2 = heptad repeat 2, TM = transmembrane domain, CT = cytoplasmic domain. (B) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The y-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the x-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. (C) FACS staining of 293T-ACE2 cells. The gray histogram shows the staining of the 293T-ACE2 cells with secondary antibody only. The empty black histograms depict the staining anti-ACE2 antibody or RBD-Ig. Shown is one representative experiment out of three preformed. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated spikes, unmutated (Wuhan-Hu-1) spike protein and bald pseudovirus. The x-axis depicts the mutation in the spike of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, *p < 0.05, **p < 0.005). Figure was generated using biorender.com.
Fig 5
Fig 5. Levels of anti-spike IgG and neutralization activity of plasma samples.
(A, C) OD values of ELISA against spike S1 subunit of plasma from vaccinated individuals (A) and of convalescent plasma (C). Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. OD values of control plasma (unvaccinated healthy individual) are shown in the red dashed graph. (B) The luminescence values derived from 293T-ACE2 cells 48 hours after infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with increasing concentrations of plasma from vaccinated individuals. The plasma dilution is shown in the x-axis. Luminescence values after incubation with control (unvaccinated healthy individual) is shown in the red-dashed graph. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown. (D) The luminescence values derived from 293T-ACE2 cells 48 hours post infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with convalescent plasma at 10−3 dilution. The study ID of the plasma samples is shown in the x-axis. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown.
Fig 6
Fig 6. SARS-CoV-2 pseudovirus neutralization assay.
(A-B) Neutralization assays, comparing the sensitivity of pseudotyped viruses with unmutated Wuhan-Hu-1 spike (WT) and the indicated spike mutations to plasma from vaccinated individuals (A) and convalescent plasma (B). Plasma dilutions are shown in the x-axis. The y-axis depicts the normalized relative luminescence units (RLU). Values were normalized to the RLU values seen with the 10−4 plasma dilution or the 10−5 dilution for VAC05 or VAC07. Mean values and standard errors are shown; representative of three independent experiments is shown. (C) NT50 values for neutralization by plasma from vaccinated individuals (n = 7) and convalescent plasma (n = 2) against pseudotyped viruses with unmutated Wuhan-Hu-1 spike (WT) and the indicated spike mutations. Mean values and standard errors are shown. Statistically significant differences NT50 values are indicated (student’s t test, *p < 0.05). NS = not significant.
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Y.B received the ISF-2029450 grant from the Israel Science Foundation https://www.isf.org.il/#/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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