Dengue virus 4/2 envelope domain chimeric virus panel maps type-specific responses against dengue serotype 2

Abstract

The four dengue virus (DENV) serotypes infect several hundred million people each year. Although primary infection is generally mild, subsequent infection by differing serotypes increases the risk for symptomatic disease ranging from fever to life-threatening shock. Despite the availability of licensed vaccines, a comprehensive understanding of antibodies that target the viral envelope protein and protect from infection remains incomplete. In this manuscript, we develop a panel of recombinant viruses that graft each envelope domain of DENV2 onto the DENV4 envelope glycoprotein, revealing protein interactions important for virus viability. Furthermore, we map neutralizing antibody responses after primary DENV2 natural infection and a human challenge model to distinct domains on the viral envelope protein. The panel of recombinant viruses provides a new tool for dissecting the E domain-specific targeting of protective antibody responses, informing future DENV vaccine design.

Keywords: dengue; neutralizing antibodies; reverse genetics.

Fig 1

The three chimeric DENV4/2 ED chimeric viruses have each surface-exposed ED of DENV4 replaced with DENV2 residues. (A) Organization of DENV genome and schema of changes of the chimeric panel. DENV2 regions are shown in pink, and DENV4 regions are in blue-green. C, capsid; prM, pre-membrane; M, membrane; ED, envelope domain; TM, transmembrane.

Fig 2

Recovered DENV4/2 ED chimeras display ED epitopes of DENV2. The ED regions are colored in red, yellow, and blue for EDI, EDII, and EDIII, respectively. (A) Alignment of all differing amino acid residues on the surface ED between DENV4 and DENV2. Residues designed to be DENV2 are colored pink, while residues kept as DENV4 are white. This includes the additional DENV2 to DENV4 changes made for viral stability (DENV4/2-EDI M278L, S307K), and any residues within the ED that were kept DENV4 (DENV4/2-EDI Q36; DENV4/2-EDII I46, E49). Mutations that arose during passaging and then introduced into the molecular clone and rederived (DENV4/2-EDI H230N, K284R, K323R; DENV4/2-EDII N276Y and N366S) are hatched; DENV2 residues are in pink, and residues that are not shared by DENV4 and DENV2 are in gray. (B) Three monomers of each DENV4/2 ED chimera with changed residue backbones highlighted (PDB 3J27).

Fig 3

Surface projection of each DENV4/2 ED chimera (PDB 3J27) of DENV4/2-EDI, DENV4/2-EDII, and DENV4/2-EDIII shows preservation of threefold, twofold, and fivefold axes of symmetry.

Fig 4

Characteristics of DENV4/2 ED panel and parental strains. (A) Growth kinetics of ED panel on Vero 81 (mammalian) and C6/36 (insect) cells at multiplicity of infection of 0.001 in triplicate. DENV4/2-EDI had significantly lower kinetics compared to other viruses on both Vero 81 and C6/36; Ps < 0.05. All viruses had significantly different growth kinetics on Vero 81 and C6/36 cells, Ps < 0.05. (B) Western blot of virions against envelope (53 kDa) and prM (21 kDa) proteins from stocks grown on Vero 81 and Vero furin-overexpressing (VF) cells, conducted in triplicate. Each representative image consists of Vero 81 and VF stocks of parental strains DENV2 (lanes 1–2), DENV4 (lanes 3–4), and each ED chimera (lanes 5–6), from left to right, DENV4/2-EDI, EDII, and EDIII. (C) Average genome copy number per focus-forming unit (ffu) of mature stocks with RNAse treatment, conducted in duplicate.

Fig 5

Neutralization of mature chimeric DENV4/2 chimeric viruses by distinct monoclonal antibodies. (A) DENV2 (3F9 and 2D22); (B) DENV4 (5H2 and 126); (C) cross-reactive, (C10, B7, and 1M7), DENV1 (1F4), and DENV3 (5J7) antibodies. Shapes indicate the median of three to five biological replicates, and lines represent the interquartile ratio.

Fig 6

Neutralization of DENV4/2 ED panel by heterotypic DENV1 convalescent sera reflects neutralization sensitivity of parental DENV2. FRNT was conducted on Vero 81 cells in duplicate and incubated for 45–48 h at 37°C.

Fig 7

Neutralization of DENV4/2 chimeric viruses by primary DENV2 sera after natural infection and human challenge. (A) Proportion of type-specific responses to each envelope domain of DENV2 in primary sera after human challenge and natural infection. (B) Boxplot and overlaid scatterplot of DENV2, DENV4/2-EDI, DENV4/2-EDII, and DENV4/2-EDIII IC_50s in human challenge and natural infection cohorts. (C) Non-homologous residues between DENV2 parental strain S16803, human challenge strain Tonga ’74, and a contemporary strain from Southeast Asia that is relevant for the majority (10/13) of the natural infection cohort. Residues from the DENV4 parental strain are provided as a reference. Dots represent homologous residues to S16803.