Natural strain variation and antibody neutralization of dengue serotype 3 viruses - PubMed (original) (raw)
Natural strain variation and antibody neutralization of dengue serotype 3 viruses
Wahala M P B Wahala et al. PLoS Pathog. 2010.
Abstract
Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Informative sites in the envelope protein of dengue serotype 3.
One hundred and seventy five DENV3 envelope protein sequences were aligned and 32 informative/variable sites were identified. The figure displays the informative sites and the variability within and between the different genotypes (I, II, III and IV) of DENV3 . The envelope protein is divided into four domains indicated by the coloring on the position numbers. Red, domain I; yellow, domain II; blue, domain III; and cyan, transmembrane domain (TM). Residues that are unique to a given genotype are indicated by unique colors. Brown, genotype I; teal, genotype III; green, genotype IV; pink, unique polymorphisms; light yellow, predominant residues shared among multiple genotypes; and gray, variation shared among multiple genotypes.
Figure 2. Location of MAb epitopes and informative sites on DENV3 E protein.
The figure is based on the structure of the ectodomain of DENV3 E protein solved by Modis and colleagues. A. The flavivirus E protein consist of three beta-barrel domains designated domains I (red), II (yellow) and III (blue). The native protein is a homodimer that lies flat on the surface of the virus. The top image depicts the major antigenic sites on domains I and II (see Table 1 for details). The bottom image displays the location of informative sites on domains I and II (pink). B. An enlarged view of domain III displaying antigenic sites and informative sites. The left image displays the lateral ridge and A strand epitopes. The right image displays the domain III informative sites (pink).
Figure 3. Binding of mouse MAbs to recombinant EDIII from the 4 serotypes of DENV.
MAb binding was detected by ELISA. MAbs 8A1 and 1H9 bound to EDIII from DENV3 only. MAb 14A4 bound to EDIII from DENV3 and to a lesser extent to EDIIII from DENV1. MAbs 8A5 and 12C1 bound to EDIII from all 4 serotypes.
Figure 4. Mapping EDIII epitopes for MAbs 8A1, 1H9 and 14A4.
The Figure depicts the positions of mutations that reduced MAb binding by >80%. Many mutations, mainly on the lateral ridge, reduced binding of 8A1 and 1H9. In contrast only three mutations inhibited binding of 14A4.
Figure 5. Binding of mouse MAbs to recombinant EDIII from the 4 genotypes of DENV3.
MAb binding was detected by ELISA. MAbs 8A1 and 1H9 bound to EDIII from DENV3 genotypes I, II and III but not to IV. MAbs 14A4, 8A5 and 12C1 bound to all 4 genotypes.
Figure 6. Binding of mouse MAbs to DENV3 genotypes.
DENV3 genotype I (DV3-I), genotype II (DV3-II), genotype III (DV3-III) and genotype IV (DV3-IV) viruses were purified and used in binding assays with MAb 8A1, 14A4 and 1H9. MAb 14A4 bound to all 4 genotypes with similar apparent affinity. MAbs 8A1 and 1H9 bound to DENV3, genotypes I, II and III with similar apparent affinity, while no binding was detected with genotype IV virus.
Figure 7. Identification of naturally occurring mutations that reduce binding of MAb 8A1.
The table displays the EDIII amino acid differences between the different genotypes of DENV3. MAb 8A1 bound to EDIII from DENV3 genotype II (DV3-II) but not genotype IV (DV3-IV). Binding was partially restored when positions 301 and 302 were changed from the genotype IV to genotype II (DV3-IV SG301-2LN)). Complete binding was restored when positions 301, 302 and 380 were changed (DV3-IV SG301-2LN, T380I).
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