Neutralization and Stability of JN.1-derived LB.1, KP.2.3, KP.3 and KP.3.1.1 Subvariants

Abstract

During the summer of 2024, COVID-19 cases surged globally, driven by variants derived from JN.1 subvariants of SARS-CoV-2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and stability of these subvariants-LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2, and increased resistance to elevated temperatures. Molecular modeling suggests that the DelS31 mutation induces a conformational change that stabilizes the NTD and strengthens the NTD-Receptor-Binding Domain (RBD) interaction, thus favoring the down conformation of RBD and reducing accessibility to both the ACE2 receptor and certain nAbs. Additionally, the DelS31 mutation introduces an N-linked glycan modification at N30, which shields the underlying NTD region from antibody recognition. Our data highlight the critical role of NTD mutations in the spike protein for nAb evasion, stability, and viral infectivity, and suggest consideration of updating COVID-19 vaccines with antigens containing DelS31.

Figure 1:. Infectivity of JN.1 subvariants in 293T-ACE2 and CaLu-3 cells.

(A) Mutations that characterize JN.1-derived subvariants FLiRT, KP.2, KP.3, LB.1, KP.2.3, and KP.3.1.1. Recorded incidences of infection by relevant variants in (B) the United States and (C) globally based on data collected by the Centers for Disease Control and Prevention (CDC) and Global Initiative of Sharing All Influenza Data (GISAID). Other JN.1*: JN.1 subvariants. Infectivity of pseudotyped lentiviral vectors bearing variant spikes of interest was determined in (D) 293T-ACE2 cells and (E) CaLu-3 cells. Bars in (D and E) represent means and standard deviation from 6 independent infections (n=6). Significance was determined and displayed relative to D614G, stars represent ** p < 0.01; ***p < 0.001 and ns p > 0.05.

Figure 2:. Neutralization of JN.1 variants by antibodies in bivalent-vaccinated HCWs, XBB.1.5-vaccinated hamsters, and BA.2.86/JN.1-infected people.

NAb titers were determined against JN.1-derived variants of interest in the sera of (A-B) HCWs that received at least two doses of monovalent mRNA vaccine and a dose of bivalent (WT+BA.4/5) mRNA vaccine (n=10), (C-D) individuals that were infected during the BA.2.86/JN.1 wave of infection in Columbus, Ohio (n=10), and (E-F) golden Syrian hamsters that were vaccinated with two doses of a mumps virus-based monovalent XBB.1.5 spike vaccine (n=10). Plots in (A, C, and E) represent geometric mean nAb titers at 50% with standard errors. Geometric mean antibody titers are depicted at the top of the plots with fold changes relative to JN.1 above them. (B, D and F) Heatmaps that depict the corresponding nAb values for each cohort listed by individual samples. Significance was determined and displayed relative to JN.1 using log10 transformed values, unless otherwise indicated; stars represent *p < 0.05; **p < 0.01; ****p < 0.0001, and ns p > 0.05.

Figure 3:. Neutralization of JN.1 variants by monoclonal antibody S309.

Neutralization by class 3 monoclonal antibody S309 was determined for JN.1-derived variants of interest and plotted (A), and inhibitory concentrations at 50% (IC₅₀) was determined and displayed in (B). Raw luminescence values were normalized to untreated controls for plotting and IC₅₀ calculations.

Figure 4:. Antigenic mapping of neutralization data against JN.1 variants.

The Racmacs program was used to plot relative antigenic distances between each spike antigen (circles) and sera sample (squares) for (A) the bivalent-vaccinated HCWs, (B) the BA.2.86/JN.1-wave infected people, and (C) the XBB.1.5-vaccinated hamsters. The scale bar represents 1 antigenic distance unit (AU) which is equivalent to about a 2-fold different in nAb titer. (D) The antigenic distances of each variant relative to JN.1 from three groups of cohorts (n=3) were averaged and plotted. The scale bar represents 1 antigenic distance unit (AU).

Figure 5:. Cell-cell fusion of JN.1-derived spikes.

Fusion triggered between membranes by the spike proteins of interest was determined between 293T cells expressing the spike and 293T-ACE2 cells overexpressing ACE2 or CaLu-3 cells expressing an endogenous level of ACE2. Representative images of fusion are depicted for (A) 293T-ACE2 and (C) CaLu-3 and quantification of total areas of fusion across 3 images are represented for (B) 293T-ACE2 and (D) CaLu-3. Bars represent means with standard deviation, significance was determined relative to ancestral variants as indicated, and stars represent *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 and ns: p > 0.05.

Figure 6:. Surface expression and processing of JN.1-derived spikes.

(A-B) The surface of 293T cells used to produce pseudotyped vectors was probed with anti-S1 antibody to compare surface expression between spikes of interest. (A) Representative histograms depicting surface expression and (B) geometric mean intensities (MFIs) of surface S1 are depicted (n=3). (C) Processing of spikes into S1/S2 subunits by furin was determined by lysing 293T cells used to produce pseudotyped viruses and probed by using anti-S1, anti-S2, anti-p24, and anti-GAPDH antibodies. Relative ratios of S2/S or S1/S were quantified using NIH ImageJ, calculated by comparing to D614G, and are displayed under corresponding blots. The plot in (B) represents geometric means with standard deviation and significance was determined relative to parental FLiRT, KP.2 or KP.3 variants as indicated; stars represent **p < 0.01; ***p < 0.001; ****p < 0.0001, and ns: n > 0.05.

Figure 7:. Stability of JN.1 variant spikes and pseudotyped viral particles.

(A) Lentiviral pseudovirions were purified (without serum) and incubated at indicated temperatures (37 to 43°C) for 1 h, and viral infectivity was determined by infecting 293T-ACE2 cells. Relative percent of infection is plotted by comparing the titer at 4°C, which was set to 100%. For each variant, the temperature at which the viral infectivity was lost by 50% (T_1/2) was determined and displayed by a dashed line in (A) and listed in (B). (C) HEK293T cells were transfected with spike constructs of interest and treated with or without sACE2 (10 μg/ml) for 4 h. Cell culture media and lysates were collected, with shed S1 proteins being immunoprecipitated with an anti-S1 antibody. (D) Cell lysates were blotted with anti-S2, anti-S1 and anti-GAPDH antibodies, and relative signals were quantified by NIH ImageJ by setting the value of JN.1 to 1.0.

Figure 8:. Structural modeling of key NTD mutations in LB.1, KP.2.3, and KP.3.1.1.

(A) Structural comparisons of NTD between KP.3.1.1 and JN.1 spike proteins. The DelS31 mutation shifts F32, thus altering its side chain direction to form hydrophobic interactions with surrounding NTD core residues, including T29, R34, V62, L56, Y91, and F216, while introducing glycosylation at N30. (B) The DelS31 mutation stabilizes the NTD, reshaping its conformation and enhancing its interaction with the receptor-binding domain (RBD) to favor the RBD down conformation. (C) The down conformation reduces RBD accessibility to the ACE2 receptor (yellow surface). (D) This down conformation restricts the accessibility of class 1 and 4 antibodies, but not class 2 and 3 antibodies. Antibodies are depicted as semi-transparent surfaces. (E) and (F) Mutations at residues (shown as sticks) H146 and Q183 disrupt the epitopes of certain NTD-targeting antibodies, such as 4A8 and C1520. (G) Glycosylation (shown as sticks) at N30 interferes with the recognition of some NTD-targeting antibodies, such as C1717.