Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation

Abstract

We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an 18.6-24% increase in transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in the B.1.1.7, B.1.351, and P.1 variants. Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.

Figure 1.. Increasing frequency of the B.1.427/B.1.429 variant in California from September 1. 2020 to January 29, 2021.

(A) County-level representation of the 2,172 newly sequenced SARS-CoV-2 genomes in the current study. Counties from which at least 1 genome were sequenced are colored in sky blue. The size of the circle is proportionally to the number of genomes sequenced from each county, while points designate counties for fewer than 10 genomes were sequenced. Logistic growth curves fitting the 5-day rolling average of the estimated proportion of B.1.427/B.1.429 variant cases in (B) California, (C) San Francisco County, (D) Alameda County, and (E) Santa Clara County. The predicted time when the growth curve crosses 0.5 is indicated by a vertical red line. A vertical black dotted line denotes the transition from 2020 to 2021. The increase in transmission rate is defined as the change in the relative proportion of B.1.427/B.1.429 variant cases relative to circulating non-B.1.427/B.1.429 variant lineages as estimated from the logistic growth model (Volz et al., 2020; Washington et al., 2021).

Figure 2.. Genomic, phylogenetic, and molecular clock analyses of the B.1.427/B.1.429 variant in California.

(A) A multiple sequence alignment of 6 representative B.1.427/B.1.429 genomes, 3 from the B.1.427 lineage and 3 from the B.1.429 lineage, using the prototypical Wuhan Hu-1 genome as a reference. Defining single nucleotide polymorphisms (SNPs) in the B.1.427 and B.1.429 lineages are compared to each other and to other SARS-CoV-2 viruses in Nextstrain clade 20C. The SNPs are color coded as follows: black SNPs are shared between the B.1.427 and B.1.429 lineages, blue SNPs are specific to B.1.427, red SNPs are specific to B.1.429, brown SNPs are shared among nearly all clade 20C viruses, and gray SNPs are specific to individual viruses. (B) Bayesian phylogenetic tree of 1,166 subsampled genomes constructed using molecular clock analysis from a complete dataset consisting of the 2,172 genomes recovered in the current study and 347 representative global genomes. The left panel shows a radial view of the tree, with marking of segments corresponding to the major clades. The right panel shows the divergence dates and associated 95% highest posterior density (HPD) distributions, or confidence intervals, for the B.1.427/B.1.429 variant (D1), B.1.427 lineage (D2), and B.1.429 lineage (D3), as estimated from TMRCA (time to most recent common ancestor) calculations. The B.1.427 lineage is colored in blue, and the B.1.429 lineage in red. The red asterisk denotes a UK B.1.1.7 variant genome. The red dot denotes the first reported genomic sequence of the B.1.429 variant from Los Angeles County from a sample collected July 13, 2020.

Figure 3.. Higher viral loads in infections from the B.1.427/B.1.429 variant as compared to non-B.1.427/B.1.429 variant lineages.

Box and whisker plots of available PCR cycle threshold (C_t) values for B.1.427/B.1.429 variant (left) and non-variant (right) samples sequenced in the current study. Note that a Ct difference of 1 represents a 2-fold difference in the virus concentration (Drew et al., 2020). The dashed horizonal line in the box denotes the median value, the solid horizontal lines the mean value. The interquartile ranges are calculated with respect to the median value.

Figure 4.. Increased infectivity of L452R-carrying pseudoviruses

(A) Upper panel: Ribbon diagram of the SARS-CoV-2 spike RBD in cyan bound to ACE2 receptor in magenta (PDB ID 6M0J). The receptor-binding motif of RBD is colored in dark cyan with L452 in solid spheres and F490 and L492 with dotted spheres. Sugars and Zn2+ are shown in grey. The position of N501 in direct contact with the ACE2 receptor is also shown for purposes of comparison. Lower panel: Surface representation of the spike RBD showing the hydrophobic patch outlined by L452, F490, and L492. (B) Levels of infection of SARS-CoV-2 spike pseudoviruses carrying D614G alone or D614G with N501Y, L452R, or W152C mutations in 293T cells stably expressing ACE2 and TMPRSS2. 293T cells were seeded in 96-well plates and infected with high (6 ng, left) or low (3 ng, right) concentrations of the indicated pseudoviruses for 48 h. Two biological replicates were assessed in two independent experiments, with 3 technical replicates per experiment. (C) Levels of infection in human lung airway organoids (HAO) stably expressing ACE2. HAO were seeded in 24-well plates and infected with high (4 ng, left) or low (2 ng, right) concentrations of the indicated pseudoviruses for 72 h. Pseudovirus cell entry was measured with a luciferase assay. The error bars represent the standard deviation of 3 technical replicates. Dunn’s multiple comparisons test was used to determine significance. Note that each of the N501Y, L452R, and W152G pseudoviruses also carries D614G. Abbreviations: NS, not significant.

Figure 5.. B.1.427/B.1.429 variant resistance to antibody neutralization in vitro.

(A) Antibody neutralization titers from 9 convalescent patients and 12 vaccine recipients against cultured WA1 (control), D614G (control), and B.1.429 viral isolates were assessed using a PRNT assay. Lines connect the individual plasma samples tested pairwise for neutralization (top row). Only a subset of the plasma samples were tested with the WA1 and D614G head-to-head comparisons (top row, right). The dotted lines denote the upper and lower bounds for the PRNT assay (1:100 to 1:3200). Plasma samples that did not exhibit detectable neutralizing activity at titers above the lower threshold are shown as transparent. Individual PRNT₅₀ measurements are plotted along with error bars denoting the median and standard deviation (bottom row). (B) Antibody neutralization titers from 10 convalescent patients against cultured WA1 (control), D614G (control) and B.1.427 viral isolates were assessed by 50% CPE endpoint dilution. Lines connect the individual plasma samples tested pairwise for neutralization (top row). Individual TCID₅₀ measurements are plotted along with error bars denoting the median and standard deviation (bottom row). A Wilcoxon matched pairs signed rank test was used to determine significance. Abbreviations: NS, not significant; PRNT, plaque-reduction neutralization test; CPE, cytopathic effect; TCID, tissue culture infective dose.