Figures and data in Evolution of a functionally intact but antigenically distinct DENV fusion loop

Version of Record

The authors declare this version of their article to be the Version of Record.

About eLife's process

Download
Cite
Share
CommentOpen annotations (there are currently 0 annotations on this page).

Version of Record published: September 19, 2023 (This version)
Reviewed preprint version 2: September 8, 2023 (Go to version)
Reviewed preprint version 1: July 12, 2023 (Go to version)
Preprint posted: March 27, 2023 (Go to version)
Sent for peer review: March 22, 2023

Figures
Tables
Additional files

3 figures, 1 table and 1 additional file

Figures

Figure 1

Download asset Open asset

Generation of DENV2 fusion loop mutants via directed evolution.

(A) Alignment of top: dengue virus fusion loops; bottom: mosquito-borne flavivirus fusion loops, including yellow fever virus (YFV), Zika virus (ZIKV), West Nile virus (WNV), Kunjin virus (KUNV), Murray Valley encephalitis virus (MVEV), Japanese encephalitis virus (JEV), Usutu virus (USUV), and Saint Louis encephalitis virus (SLEV). Amino acids are colored by functional groups: negatively charged (red), positively charged (blue), nonpolar (yellow), polar (green), aromatic (pink), and sulfide (dark red). (B) Schematic of directed evolution procedure. Saturation mutagenesis libraries were used to produce viral libraries, which were passaged three times in either C6/36 or Vero 81 cells. At the end of the selection, viral genomes were isolated and mutations were identified by high-throughput sequencing. (C) Left: bubble plot of the sequences identified from either the unselected or selected (passage 3) C6/36 DENV libraries. Right: pie chart of the sequences from passage 3 C6/36 DENV libraries. (D) Structure of the DENV envelope with the fusion loop mutations highlighted in red. (E) Sequences of the fusion loop and furin cleavage site of DENV2, D2-FL, and D2-FLM.

Figure 2

Download asset Open asset

Biological and physical properties of mature DENV2 fusion loop mutants.

(A) Multistep growth curves [Multiplicity of infection (MOI) = 0.05–0.1] of DV2-WT, D2-FL, and D2-FLM on C6/36 cells (left) or Vero 81 cells (right). (B) Thermostability assay on DV2-WT, D2-FL, and D2-FLM. (C) Western blot of virions, blotted against Envelope and prM proteins. Equal volumes of supernatants from viral infected C6/36 cells at 5 days post-infection (dpi) (A) were loaded into each lane. The prM:E ratio was determined and normalized to the DV2-WT ratio. High exposure of D2-FLM (right) illustrates the relative abundance of prM protein to E protein. Averages of three biological replicates are shown. The data are graphed as means ± standard deviations (Error bars). Two-way ANOVA was used for statistical comparison of growth curves and thermostability: ns, not significant; *<0.05; **<0.005; ***<0.0005.

Figure 2—source data 1 Raw gel images of the western blot shown in Figure 2C.: https://cdn.elifesciences.org/articles/87555/elife-87555-fig2-data1-v1.zip
Download elife-87555-fig2-data1-v1.zip

Figure 3

Download asset Open asset

Fusion loop (FL) mutant is insensitive to FL monoclonal antibodies (mAbs), the major target for cross-reactive Abs in non-human primates (NHPs).

(A) Left: FRNT₅₀ values for neutralization of DV2-WT, D2-FL, and D2-FLM with mAbs against the FL (1M7, 1N5, 1L6, 4G2). All Abs were tested in at least n = 3 independent experiments, except 1N5 due to limited Ab. Right: average neutralization curves for neutralization of DV2-WT, D2-FL, and D2-FLM with mAbs against the DENV2 FL. (B) FRNT₅₀ values for neutralization of DV2-WT and D2-FLM with mAbs against DENV2 prM (2H2, 1E16, 5M22), EDI (3F9), EDE (C10, B7), and EDIII (2D22). All Abs were tested in at least n = 3 independent experiments. The data are graphed as means ± standard deviations (Error bars). (C) Neutralization of DV2-WT, D2-FL, and D2-FLM with sera from NHPs infected with either DENV4 or DENV2. FRNT₅₀s were compared using Student’s t-test. Significant symbols are as follows: *p<0.05; **p<0.005; ***p<0.0005; ****p<0.00005. The data are graphed as means ± standard deviations.

Tables

Table 1

Summary of FRNT₅₀ values of human convalescent serum and non-human primates (NHP) infection serum against DV2, D2-FL (not determined in human serum) and D2-FLM.

FRNT₅₀ values were derived from an eight-point dilution curve of serum started from 1:40 with subsequent threefold dilutions. FRNT₅₀ values were calculated/extrapolated from the neutralization curves. For human serum, the average FRNT₅₀ values were shown from two independent experiments with technical replicates. For NHP serum, FRTN₅₀ values were average of three independent experiments. ND, not determined. * denotes the Hill slopes of all the neutralization curve of them sample are <0.5 (low confidence of neutralization).

Human #	Infected serotype	Collection tme	DV2	D2-FL	D2-FLM
DS1500	DENV1	Convalescent serum	1:75	ND	1:53
DS2499	DENV1		1:80	ND	1:52
DS1136	DENV3		1:133	ND	1:45
DS1160	DENV3		1:86	ND	1:208
DS0275	DENV4		1:94	ND	1:94
DS2239	DENV4		1:156	ND	1:42
NHP #	Infected serotype	Collection time (dpi)	DV2	D2-FL	D2-FLM
R628	DENV4	30	<1:40	<1:40	<1:40
		60	<1:40	<1:40	<1:40
		90	1:42*	<1:40	<1:40
R737		30	<1:40	<1:40	<1:40
		60	<1:40	<1:40	<1:40
		90	<1:40	<1:40	<1:40
R1132		30	<1:40	<1:40	<1:40
		60	<1:40	<1:40	<1:40
		90	<1:40	<1:40	<1:40
R1160		30	1:58*	<1:40	<1:40
		60	1:57*	1 : 50*	<1:40
		90	<1:40	1 : 57*	1:91*