Homotypic antibodies target novel E glycoprotein domains after natural DENV 3 infection/vaccination

Abstract

The envelope (E) glycoprotein is the primary target of type-specific (TS) neutralizing antibodies (nAbs) after infection with any of the four distinct dengue virus serotypes (DENV1-4). nAbs can be elicited to distinct structural E domains (EDs) I, II, or III. However, the relative contribution of these domain-specific antibodies is unclear. To identify the primary DENV3 nAb targets in sera after natural infection or vaccination, chimeric DENV1 recombinant encoding DENV3 EDI, EDII, or EDIII were generated. DENV3 EDII is the principal target of TS polyclonal nAb responses and encodes two or more neutralizing epitopes. In contrast, some were individuals vaccinated with a DENV3 monovalent vaccine-elicited serum TS nAbs targeting each ED in a subject-dependent fashion, with an emphasis on EDI and EDIII. Vaccine responses were also sensitive to DENV3 genotypic variation. This DENV1/3 panel allows the measurement of serum ED TS nAbs, revealing differences in TS nAb immunity after natural infection or vaccination.

Keywords: RNA; antibody; dengue virus; immunity; monoclonal; natural infection; polyclonal; sera; structural targets; vaccine.

Figure 1.. DENV1/3 E glycoprotein recombinant viruses

(A) Design of DENV1/3 glycoproteins. Structural glycoprotein design. DENV1/3 EDI, EDII, and EDIII chimeras are depicted in a hexameric raft formation with 6 antiparallel E glycoprotein monomers forming 3 parallel dimers (PDB: 3J27, for visualization); DENV1 = dark blue, DENV3 = pink, DENV1 1F4/14C10 epitopes =cyan on the EDII chimera and residue mutations in the EDIII chimera = lime green. The premembrane (prM) protein is pictured to the right of the EDII and EDIII chimeras (PDB: 6IDI, for visualization). For prM, DENV1= black, DENV3 = teal. (B) DENV1/3 particle symmetry. DENV3 E protein 3-fold, 2-fold, and 5-fold symmetry are represented on the whole virion with DENV1/3 EDI, DENV1/3 EDII, and DENV1/3 EDIII respectively, with DENV3 = pink, DENV1 = dark blue (PDB: 3J6S_*60, for visualization). (c) Primary sequence variation in chimeric viruses. E glycoprotein DENV3 original encoded changes into DENV1 are represented for DENV1/3 EDII and EDIII chimeras, DENV3 = pink, DENV1 = dark blue.

Figure 2.. Phenotypic analysis of DENV1/3 E glycoprotein recombinant viruses

(A) Virus growth. Vero81 NHP cells were inoculated in triplicate and virus titers were measured by fluorescent foci units (FFU) at 48 h after infection, blue circles = triplicate titers. (B) hmAB neutralization phenotypes. Confirmation of DENV1 and 3 epitopes in the DENV1/3 EDI, EDII, and EDIII panel plus parental strains using DENV1 and 3 mAb arrays. (C) Western blots. E and prM proteins were visualized through western blot and (D) Virus Maturation Status. prM/E ratios determined, with lower numbers indicating greater maturity.

Figure 3.. DENV3 vaccine-elicited hmAB neutralize multiple DENV3 genotypes

Isolated from vaccinee’s, novel EDII-targeting mAbs-P3D05 and -P7C07 were tested via FRNT for neutralization with a panel of DENV3/3 envelope genotype I–IV chimeras, displayed on a DENV3 G-III backbone, and exhibited similar potency between strains.

Figure 4.. DENV3 hmAbs-115 and −419 target clustered residues in EDII

(A) DENV3 EDII epitope mapping strategy. DENV3/3 EDII, G-III into G-IV parent (9 residues) and 4 DENV3/3, G-III into G-IV cluster chimeras (C1–C4) encoding between 2 and 6 DENV3 G-III residues are depicted, G-III changed residues = blue spheres (PDB: 3J27, for visualization). C2 and C3 together make the residues encoded into C4. (B) hmAB neutralization phenotypes. DENV1/3 EDII-targeting mAbs-115 and −419 neutralize C3 and C4, with the critical residues lying in C3. (C) Annotated location of nAb epitopes in EDII. The critical mAb-115 and −419 epitope residues are depicted on the whole viron and are located at the EDII-EDII dimer interface (E62, G63, Q120, P124) (PDB: 3J6S_*60, for visualization).

Figure 5.. Primary DENV3 TS serum neutralization responses primarily target EDII after infection

(A) ED-targeting of late convalescent primary infection sera. Six DENV3 primary natural serum samples, 3 from the adult Arbovirus Travelers Cohort (ATC A–C) and three from the Nicaraguan Pediatric Dengue Cohort Study (NPDCS A–C) were depleted with bovine serum albumin (BSA), DENV1, 2, and 4, or DENV3 virus on a magnetic bead, and the remaining sera tested for neutralization with the DENV1/3 EDI-EDIII panel. (B) Wild-type and genotype outcomes. Late convalescent sera were used to neutralize wild-type viruses (DENV1, 2, 4, and DENV3 genotypes G-II and G-III). The BSA control represents total neutralization, the middle column (DENV1, 2, 4 depletion) represents the DENV3 TS nAb response, and the DENV3 depleted column represents any background neutralization which was then subtracted from the other columns. (C) ED specific neutralization phenotypes. The mean DENV3 %TS antibody responses and mean DENV3 TS ID₅₀ titers were calculated from the BSA and 1, 2, 4 depleted fractions to each virus across the late convalescent sera and represent the mean percentage or ID₅₀ titer to each virus that is DENV3 TS compared with the control depletions, which contain both TS and CR antibody fractions.

Figure 6.. Monovalent DENV3 vaccine serum preferentially targets EDIII and EDI over EDII

(A) ED-targeting of DENV3 monovalent vaccine serum. Four DENV3 NIH monovalent vaccinee serum samples (NIH A, B, D, and F) were depleted with BSA, DENV1, 2, and 4 or DENV3 on a magnetic bead and the remaining sera tested for neutralization with the DENV1/3 EDI-EDIII panel. (B) Wild-type and genotype outcomes. DENV3 vaccine sera were used to neutralize wild-type viruses (DENV1, 2, 4, and DENV3 genotypes G-II and G-III). The BSA-depleted denotes the control where total antibodies are present. The DENV1, 2, 4 column denotes DENV3 TS antibodies and the DENV3 column denotes any background neutralization, which was then subtracted from the BSA and DENV1, 2, and 4 columns. (C) ED specific neutralization phenotypes. The mean DENV3 %TS antibody responses and mean DENV3 TS ID₅₀ titers were calculated from the BSA and 1, 2, 4 depleted fractions to each virus across NIH monovalent and represent the percentage or ID₅₀ titer to each virus that is DENV3 TS compared with the control depletions, which contain both TS and CR antibody fractions.

Figure 7.. Cartography reveals distinct clusters of natural infection and vaccine serum responses

Antigenic cartography of the DENV wild-type and recombinant viruses and serum ID₅₀s are depicted using the Smith et al. method. Viruses are shown as circles and serum samples are shown as triangles, corresponding to the colored key on the right. Convalescent sera are grouped together in the black circle, and further separated into pediatric sera (blue oval) and adult sera (rust colored oval). Vaccine sera are grouped together in the purple oval. Each grid box represents a 2-fold change in titer.