Genetic epidemiology of dengue viruses in phase III trials of the CYD tetravalent dengue vaccine and implications for efficacy

Abstract

This study defined the genetic epidemiology of dengue viruses (DENV) in two pivotal phase III trials of the tetravalent dengue vaccine, CYD-TDV, and thereby enabled virus genotype-specific estimates of vaccine efficacy (VE). Envelope gene sequences (n = 661) from 11 DENV genotypes in 10 endemic countries provided a contemporaneous global snapshot of DENV population genetics and revealed high amino acid identity between the E genes of vaccine strains and wild-type viruses from trial participants, including at epitope sites targeted by virus neutralising human monoclonal antibodies. Post-hoc analysis of all CYD14/15 trial participants revealed a statistically significant genotype-level VE association within DENV-4, where efficacy was lowest against genotype I. In subgroup analysis of trial participants age 9-16 years, VE estimates appeared more balanced within each serotype, suggesting that genotype-level heterogeneity may be limited in older children. Post-licensure surveillance is needed to monitor vaccine performance against the backdrop of DENV sequence diversity and evolution.

Trial registration: ClinicalTrials.gov NCT01373281 NCT01374516.

Keywords: dengue vaccine; dengue virus; epidemiology; genotype; global health; molecular epidemiology; vaccine; virus; virus evolution.

Conflict of interest statement

No competing interests declared.

Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Matthew Bonaparte: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Diane van der Vliet: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Edith Langevin: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Margarita Cortes: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Betzana Zambrano: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Corinne Dunod: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Anh Wartel-Tram: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Nicholas Jackson: Employee of Sanofi Pasteur, a company engaged in the development of CYD-TDV.

Figures

Figure 1.. Sequencing flow chart for samples obtained in CYD-TDV trials.

(A) CYD14, (B) CYD15.

Figure 1—figure supplement 1.. Probability of sequencing success versus viremia.

White boxes show the range and IQR of viremia levels from successful sequencing attempts, grey boxes show the range and IQR of viremia levels from unsuccessful sequencing attempts. (A) DENV-1, (B) DENV-2, (C) DENV-3, (D) DENV-4.

Figure 2.. Distribution of DENV serotypes and genotypes sequenced in CYD14 and CYD15 vaccine trials by country.

Numbers in parentheses indicate the total number of samples of each genotype for which complete or partial E gene sequences were obtained.

Figure 2—figure supplement 1.. Genotype assignment of DENV-1 E gene sequences obtained from VCD samples in CYD14 and CYD15.

Phylogenetic trees showing the distribution of DENV-1 genotypes for which E genes were sequenced in CYD14 and CYD15 vaccine trials. E gene sequences were aligned with up to five publically available reference sequences of every known human DENV-1 genotype and a maximum likelihood phylogeny was constructed. Genotypes were assigned when sequences fell into a known genotype lineage with high bootstrap support. Bootstrap support values for nodes > 75% are indicated. Black dots at the tips indicate CYD14 sequences, grey dots indicate CYD15 sequences, and purple stars indicate the DENV CYD-TDV vaccine sequence for each serotype.

Figure 2—figure supplement 2.. Genotype assignment of DENV-2 E gene sequences obtained from VCD samples in CYD14 and CYD15.

Phylogenetic trees showing the distribution of DENV-2 genotypes for which E genes were sequenced in CYD14 and CYD15 vaccine trials. E gene sequences were aligned with up to five publically available reference sequences of every known human DENV-2 genotype and a maximum likelihood phylogeny was constructed. Genotypes were assigned when sequences fell into a known genotype lineage with high bootstrap support. Bootstrap support values for nodes > 75% are indicated. Black dots at the tips indicate CYD14 sequences, grey dots indicate CYD15 sequences, and purple stars indicate the DENV CYD-TDV vaccine sequence for each serotype.

Figure 2—figure supplement 3.. Genotype assignment of DENV-3 E gene sequences obtained from VCD samples in CYD14 and CYD15.

Phylogenetic trees showing the distribution of DENV-3 genotypes for which E genes were sequenced in CYD14 and CYD15 vaccine trials. E gene sequences were aligned with up to five publically available reference sequences of every known human DENV-3 genotype and a maximum likelihood phylogeny was constructed. Genotypes were assigned when sequences fell into a known genotype lineage with high bootstrap support. Bootstrap support values for nodes > 75% are indicated. Black dots at the tips indicate CYD14 sequences, grey dots indicate CYD15 sequences, and purple stars indicate the DENV CYD-TDV vaccine sequence for each serotype.

Figure 2—figure supplement 4.. Genotype assignment of DENV-4 E gene sequences obtained from VCD samples in CYD14 and CYD15.

Phylogenetic trees showing the distribution of DENV-4 genotypes for which E genes were sequenced in CYD14 and CYD15 vaccine trials. E gene sequences were aligned with up to five publically available reference sequences of every known human DENV-5 genotype and a maximum likelihood phylogeny was constructed. Genotypes were assigned when sequences fell into a known genotype lineage with high bootstrap support. Bootstrap support values for nodes > 75% are indicated. Black dots at the tips indicate CYD14 sequences, grey dots indicate CYD15 sequences, and purple stars indicate the DENV CYD-TDV vaccine sequence for each serotype.

Figure 3.. Average genotype-specific amino acid identity of DENV isolated in CYD-TDV trials compared to the vaccine strain of the corresponding DENV serotype.

Black bars indicate the IQR of the full sample set. Coloured dots show the geographic regions from which each genotype was collected – red: CYD14, maritime SE Asia; blue: CYD14, mainland SE Asia; grey: CYD15, Americas. Black dots indicate the genotype of the serotype-specific CYD-TDV vaccine component.

Figure 3—figure supplement 1.. Genotype-specific amino acid identity of DENV isolated in CYD-TDV trials compared to the vaccine strain of the corresponding DENV serotype versus vaccine efficacy.

Symbols indicate the intersection of mean amino acid identity to CYD-TDV components and mean genotype-specific vaccine efficacy. Bars on the x-axis indicate the range of pairwise amino acid identities of DENV isolated in the trials compared to the CYD-TDV component. Bars on the y-axis indicate the 95% confidence intervals calculated for vaccine efficacy estimates. DENV-1 genotypes are shown in red, DENV-2 in yellow, DENV-3 in aqua, DENV-4 in blue. (A) Mean amino acid identity versus observed vaccine efficacy across all age groups. (B) Mean amino acid identity versus imputed vaccine efficacy across all age groups. (C) Mean amino acid identity versus observed vaccine efficacy in subjects ≥ 9 years of age. (D) Mean amino acid identity versus imputed vaccine efficacy in subjects ≥ 9 years of age.

Figure 4.. Amino acid differences between the DENV-2 E gene vaccine sequence, DENV-2 viruses isolated in CYD14 and CYD15 vaccine trials, and representative subsets of publically available DENV-2 sequences from the vaccine trial sites.

(A) CYD14 DENV-2 phylogeny, (B) CYD15 DENV-2 phylogeny. Coloured tips on the trees show sequences isolated in the CYD-TDV trials (country of origin coloured as indicated in the key) and the vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD14/CYD15 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 4—figure supplement 1.. Amino acid differences between DENV-1 E gene vaccine sequences, DENV-1 viruses isolated in CYD14 vaccine trials, and representative subsets of publically available DENV-1 sequences from the vaccine trial sites.

Coloured tips on the trees show sequences isolated in the CYD14 trial (country of origin coloured as indicated in the key) and the respective vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD14 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 4—figure supplement 2.. Amino acid differences between DENV-1 E gene vaccine sequences, DENV-1 viruses isolated in CYD15 vaccine trials, and representative subsets of publically available DENV-1 sequences from the vaccine trial sites.

Coloured tips on the trees show sequences isolated in the CYD15 trial (country of origin coloured as indicated in the key) and the respective vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD15 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 4—figure supplement 3.. Amino acid differences between DENV-3 E gene vaccine sequences, DENV-3 viruses isolated in CYD14 vaccine trials, and representative subsets of publically available DENV-3 sequences from the vaccine trial sites.

Coloured tips on the trees show sequences isolated in the CYD14 trial (country of origin coloured as indicated in the key) and the respective vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD14 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 4—figure supplement 4.. Amino acid differences between DENV-3 E gene vaccine sequences, DENV-3 viruses isolated in CYD15 vaccine trials, and representative subsets of publically available DENV-3 sequences from the vaccine trial sites.

Coloured tips on the trees show sequences isolated in the CYD15 trial (country of origin coloured as indicated in the key) and the respective vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD15 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 4—figure supplement 5.. Amino acid differences between DENV-4 E gene vaccine sequences, DENV-4 viruses isolated in CYD14 vaccine trials, and representative subsets of publically available DENV-4 sequences from the vaccine trial sites.

Coloured tips on the trees show sequences isolated in the CYD14 trial (country of origin coloured as indicated in the key) and the respective vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD14 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 4—figure supplement 6.. Amino acid differences between DENV-4 E gene vaccine sequences, DENV-4 viruses isolated in CYD15 vaccine trials, and representative subsets of publically available DENV-4 sequences from the vaccine trial sites.

Coloured tips on the trees show sequences isolated in the CYD15 trial (country of origin coloured as indicated in the key) and the respective vaccine sequence (purple star); grey tips indicate publicly available sequences isolated from other studies in the countries of interest. Columns to the right indicate amino acid sites at which variation was observed in two or more CYD15 sequences. Numbers at the top of columns indicate the amino acid site within the E gene. Bars at the top of the figures indicate the E gene domain of the site. Amino acids at variable sites in the E gene sequence of the vaccine component are shown in colour. For all other sequences, a lack of colour indicates an amino acid identical to that of the vaccine component at that site.

Figure 5.. Sequence conservation between the DENV-2 vaccine component and wild-type DENV-2 viruses at epitope locations targeted by virus neutralising human mAbs.

Amino acid targets for five neutralising human mAbs (Fibriansah et al., 2015b; Rouvinski et al., 2015) are coloured as indicated in the key (top) and compared to the vaccine sequence and wild-type sequences obtained within the CYD14 and CYD15 trials (middle), as well as complete E gene sequences from wild-type DENV-2 available on GenBank (bottom). Sites are indicated at the top of columns. For wild-type virus populations, the darker the block, the greater the proportion of sequences with an amino acid differing from the target amino acid at that site. When disagreement between amino acids was observed between epitope targets (as at E67 and E71), wild-type sequences were compared to 2D22 as a reference, denoted by an asterisk.

Figure 5—figure supplement 1.. Sequence conservation between DENV vaccine components and wild-type DENV viruses at epitope locations targeted by virus neutralising human mAbs.

Amino acid targets for neutralising human mAbs (Fibriansah et al., 2014; Cockburn et al., 2012a; Fibriansah et al., 2015a, 2015b; Teoh et al., 2012; Rouvinski et al., 2015; Costin et al., 2013) are coloured as indicated in the key (top) and compared to the vaccine sequence and wild-type sequences obtained within the CYD14 and CYD15 trials (middle), as well as complete E gene sequences from wild-type DENV available on GenBank (bottom, numbers in parentheses indicate the number of sequences used for comparison). Sites are indicated at the top of columns. For wild-type virus populations, the darker the block, the greater the proportion of sequences with an amino acid differing from the target amino acid at that site.