Impact of Dengue Virus Serotype 2 Strain Diversity on Serological

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Impact of dengue virus serotype 2 strain diversity on serological immune responses to dengue Sarah Keasey, Jessica Smith, Stefan Fernandez, Anna Durbin, Bryan Zhao, and Robert G Ulrich ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00185 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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ACS Infectious Diseases

Impact of dengue virus serotype 2 strain diversity on serological immune responses to dengue

Sarah L. Keasey1,2, †, Jessica L. Smith1, †, Stefan Fernandez3, Anna P. Durbin4, Bryan M. Zhao1, and Robert G. Ulrich1*

1Molecular

and Translational Sciences Division, U.S. Army Medical Research Institute of

Infectious Diseases, 1425 Porter St, Frederick, Maryland 21702, USA 2Department

of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop

Circle, Baltimore, Maryland 21250, USA 3Armed

Forces Research Institute of Medical Sciences, Bangkok, 10400, Thailand

4Center

for Immunization Research, Johns Hopkins Bloomberg School of Public Health, 624

North Broadway, Room 251, Baltimore, Maryland 21205, USA

†Both

authors contributed equally.

*correspondence: [email protected]; 301 619-4232

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Dengue is a mosquito-borne disease caused by four dengue virus serotypes (DENV1-4) that are loosely categorized by sequence commonalities and antibody recognition profiles. The highly variable envelope protein (E) that is prominently displayed on the surface of DENV is an essential component of vaccines currently under development, yet the impact of using single strains to represent each serotype in tetravalent vaccines has not been adequately studied. We synthesized chimeric E by replacing highly variable residues from vaccine strain PUO-218 with those from 16 DENV2 lineages spanning 60 years of antigen evolution. Examining sera from human and rhesus macaques challenged with single strains of DENV2, antibody-E interactions were markedly inhibited or enhanced by residues mainly focused within a 480Å2 footprint displayed on the E backbone. The striking impact of E diversity on polyclonal immune responses suggests that frequent antigen updates may be necessary for vaccines to counter shifts in circulating strains.

Keywords. Dengue virus, dengue virus envelope, dengue vaccine, antibody response, dengue strain variability, protein microarray


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The clinical symptoms of dengue range from mild febrile illness to severe hemorrhagic fever and shock syndrome with capillary leakage1. Four serotypes of the mosquito-borne dengue virus (DENV1-4) circulate in most tropical or semi-tropical regions of the world, and this extensive distribution leads to greater than 500,000 severe cases and 22,000 deaths annually2, 3. DENV are members of the Flavivirus genus of single-stranded RNA viruses, which also includes Zika, yellow fever, and West Nile viruses. Long-lasting immunity to homologous DENV infections is mediated by antibody and T-cell responses to primary infections by one of the DENV serotypes4, while immunity against other serotypes (heterotypic immunity) may be shortlived5-8 or lower in magnitude9, resulting in continuous cycles of infection within susceptible human populations. A further concern is that secondary infection with a heterologous serotype can in some cases lead to increased risk of severe dengue disease, possibly because antibodies to one serotype may enhance infections with heterologous serotypes by promoting viral entry and infection through Fc receptor-expressing cells (antibody-dependent enhancement or ADE)10. Despite similar disease presentations, DENV1-4 differ11 at the proteomic level by 25-40%, and by 3-6% within serotypes12, 13, while nucleic acid substitution rates are generally consistent across serotypes. The circulation of new viral isolates is driven by selection pressures14, 15 that are imposed during the natural cycling of DENV between mosquito vectors and human hosts. The emerging viral isolates often harbor structural variants of the envelope (E) protein7, 16, 17, the dominant surface antigen of DENV. The E and membrane (M) structural proteins of DENV assemble into 180 heterodimers that form a sheath around the nucleocapsid core. The E monomer folds into three domains (EDI, EDII, and EDIII) comprising the ectoprotein and two alpha helices of a membrane-proximal stem. Three parallel E dimers are complexed with the M



protein (prM), which contains a transmembrane anchor peptide that is cleaved during maturation of the virus18-20. The extensive diversity of virus strains moving in and out of human populations presents a significant challenge to the development of effective dengue vaccines that must first incorporate antigenic components from all serotypes. There are several competing tetravalent dengue vaccines (TDV) that are approaching licensure for immunization and distribution21. The first TDV approved for use in a limited number of DENV-endemic countries is a live-attenuated virus with prM and E proteins from one prototype strain of each DENV serotype inserted into the remaining proteome of the yellow fever virus 17D vaccine (CYD-TDV). A limited, overall efficacy rate of 56.5-60.8% was reported in the first advanced clinical trials of CYD-TDV, especially for previously seronegative subjects, and the lowest efficacy was observed with the DENV2 serotype (18.7-55.2%)22-24. Whereas new sequence variants of DENV2 are reported almost every year, the monoclonal prM and E proteins of DENV2 that were used for CYD-TDV originated from a virus (PUO-218) that was isolated in 1980 from a patient in Thailand. Although the basic E protein structure is highly conserved among DENV, amino acid residues that are permissive to mutations are likely to play a role in driving adaptation to selection pressures imposed by host immune responses. Here we examine the relationship between serological immune responses to DENV2 strains representative of two separate lineages and antibody interactions with naturally occurring variants of the DENV2 E protein. Chimeric E proteins were constructed by grafting solvent-exposed amino acid residues from strains that represent major branch points in the evolution of DENV2 onto the PUO-218 backbone, and these recombinant antigens were then used to evaluate human and rhesus macaque antibody responses to disparate DENV2 strains.


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Results E protein evolution of DENV2 The molecular evolution of DENV2 was examined with 266 non-redundant E proteins encoded by fully-sequenced strains that circulated in 36 countries between 1944 and 2010 (Supplementary File 1). The minimal amino acid sequence identity by pair-wise comparison was 91.92%, and similar to levels observed across DENV2 genotypes12, 25. Bayesian methods26-30 were used to determine the specific lineages that emerged from the most recent common DENV2 ancestor (Fig. 1), which was estimated to have originated in the late 1800s (ca 1897) at a time that was approximately 47 years before the oldest (ca 1944) documented strain of DENV2 (New Guinea C (NGC)) was isolated in Papua, New Guinea (Accession No. AF038403)31. Because the early genomic records were sparse, our estimate of the most recent common ancestor is likely to represent an upper boundary to the date of DENV2 emergence. Higher order phylogenetic relationships of amino acid sequences clustered E proteins into specific clades that mapped to discrete geographical origins and previously described lineages (Fig. 1; Supplementary File 2) of (i) Asian I, (ii) Asian II, (iii) Cosmopolitan, (iv) American, (v) Asian/American, and (vi) sylvatic genotypes32, 33. The clustering of lineages indicated robust support (posterior probabilities >0.9) for the evolutionary divergence of these groups and the constraints of DENV2 E amino acid diversity based on geographical distributions34. For example, a sylvatic clade encompassing isolates primarily from West Africa and Malaysia clustered independently of all other lineages, and diverged much earlier (Fig. 1). Further, Asian/American strains principally originated from the Caribbean region and nearby Latin American countries, but also from geographically distant Southeast Asia (Fig. 1). While distinct strains formed location-specific clusters within the



Asian/American clade32, the geographical diversity among DENV2 strains may be due to rapid spread of mosquito vectors by urbanization and increased global travel34.

High surface entropy of the DENV2 E antigen Because antibodies resulting from immune responses to DENV interact with amino acid residues that are exposed to solvent on the surface of E proteins35-37, it follows that conserved positions are less likely to drive viral evolution, while selection pressures imposed by host antibodies may lead to changes in residues that are permissive to mutations. We evaluated the evolutionary stability of solvent-exposed positions by using entropy (H) as a measure of amino acid variation for each E residue over time (Fig. 2a). Only 22% of residues were completely conserved across all E proteins of DENV2 (Fig. 2a), suggesting a great amount of tolerance to mutations within each of the three E protein domains (EDI, II, and III). Approximately 30% of E monomer residues (146 of 495 total residues) are exposed38 (Fig. 2b) on the surface of the mature DENV2 virus39 (PDB 3J27; Supplementary Fig. 1), including the conserved glycosylation sites at positions N67 and N153. The remaining side chains are buried in contact surfaces between monomers or adjacent units of symmetry (Supplementary Fig. 1). Further, amino acid residues positioned as buried contacts between subunits (Fig. 2b, c) are highly conserved (34% H > 0). In contrast, 44% of surface exposed residues displayed H > 0, indicating that variability is focused on solvent accessible residues of E proteins (Fig. 2b, c). Notably, DENV2 harbors the least evolutionary stability in E protein surfaces among serotypes, with 32 solvent-accessible residues that are highly variable compared to ≤ 23 for each of the other 3 serotypes (Supplementary Fig. 2).


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Antibody recognition of E proteins across 60 years of DENV2 evolution Modeling the cumulative distribution function of the exponentially distributed number of strains (Scl) and total entropy (Hsecl) per clade (Supplementary Fig. 3), thresholds for each determinant were established as the value that represented 90% of the population (Scl = 30 and Hsecl = 0.2). Sixteen clades that represented major branch points in the evolution of five DENV2 lineages were identified by using values of Scl 0.2 (Fig. 1; Supplementary Fig. 3). A small cluster of E proteins from seven American strains that represented the sixth DENV2 lineage did not exceed Hsecl > 0.2, and thus did not meet a sufficient level of diversity. However, one of the sixteen selected strains harbored all amino acid substitutions present in the American sequences, except residue 71, which is A in Cosmopolitan strains, D in American, and E in all others. A representative isolate was selected from each of the sixteen DENV2 clades (Fig. 1; Table 1), and the variable E surface residues of the isolates (designated here as variants) were grafted onto the backbone of the PUO-218 E protein by designing synthetic genes of the E soluble ectodomain. The DENV2 proteins were produced by in vitro expression (Supplementary Fig. 4), including all essential components for protein glycosylation and translation from DNA templates. To evaluate the contribution of buried residues, the E variants 2C and 5C were synthesized with surfaces of PUO-218 on backbones that exhibited the lowest (variant 2) and highest (variant 5) total sequence identity to PUO-218 (Table 1). The recombinant proteins from E variants, the prototype strains of DENV1-4, and PUO-218 were isolated by affinity chromatography, and printed in microarrays for analysis of antibody recognition, using previously described methods40-43. Residues Gly104, Gly106, and Leu107 of the fusion-loop peptide of E domain II are highly conserved among the flaviviruses, and site-specific mutations of these residues prevent binding of monoclonal antibody 4G2 to bind to the E glycoprotein monomer44, 45. The



printed E proteins were equally recognized by 4G2 and a rabbit polyclonal antibody that was raised against the fusion-loop peptide that harbors the 4G2 epitope (Supplementary Fig. 4). We reasoned that an examination of polyclonal B-cell responses to monoclonal E antigens should allow us to describe a global perspective of adaptive immune responses to dengue that may complement results obtained with monoclonal antibodies35-37. Serological immune responses were studied with a non-human primate model that is used to evaluate efficacy of dengue vaccines40, 46. Asian rhesus macaques (Macaca mulatta) were subcutaneously challenged with representative strains of each DENV serotype, and blood sera were collected for analysis. The DENV2 challenge strain (S16803) was an Asian I lineage that differs from the full-length E polypeptide of PUO-218 by a single conservative substitution of aspartic for glutamic acid (PUO-218) at the surface accessible residue of position 383 (Supplementary File 1). The E protein of DENV4 was recognized ( 3.5), were removed and pixel counts from replicate spots were averaged to obtain mean fluorescence intensities (MFI). Relative binding was calculated with equation 2: (2)

Relative binding = ( x / xi )100,

where x is MFI origination from microarrayed antigens, and i is the infecting virus species. Percent signal change was calculated using equation 3: (3)

% signal change =

( ) × 100, 𝑦 ― 𝑦𝑗 𝑦𝑗

where y is the MFI originating from DENV2 E variant proteins and j is the E variant exhibiting the highest sequence identity to the infecting strain. Statistically significant antibody binding was



determined by using Student’s two-tailed t-tests, performed with the Data Analysis add-in for Microsoft Excel. Histograms and exponential curve fitting were performed using OriginPro v9.0 (Origin Lab Corporation, Northampton, MA). For threshold determinations of number of strains and entropy per clade, we selected the value that represented 90% of the distribution based on modeling the cumulative distribution function with equation 4: (4)

0.9 = 1- 𝑒( ― 𝜆𝑥),

where λ = 4.84 (Hsecl) and 0.06206 (Scl) and x is the selected threshold value.

DENV2 E proteins and neutralizing antibodies. A cell-based infection assay was used to evaluate the relationship between virus-neutralizing antibodies and the recombinant E protein variants. To produce virus for infection assays, Vero cells (ATCC® CCL-81TM) grown at 37oC, 5% CO2 in Dulbecco's Modification of Eagle's Medium (DMEM) (Mediatech, Inc, Manassas, VA) containing 10% HycloneTM (GE Healthcare Life Sciences, Logan, UT) fetal bovine serum (FBS) were seeded in T-75 flasks 24 h before infection. DENV2 NGC (BEI Resources NR-84) was diluted in DMEM containing 2% FBS and Vero cells were infected with 0.14 TCID50 units per cell (~MOI of 0.1). Cell culture supernatants harvested 8 days post-infection were centrifuged (1200 x g, 10 min) and passed through a 0.2 µm HT Tuffryn® Acrodisc® filter (Pall Life Sciences, Ann Arbor, MI) prior to storage (-80oC). For infection assays, Vero cells (3.8 x 104/well) were seeded in 24-well plates using DMEM with 10% FBS 24 h before infection. Viral titration experiments were performed in infection media (DMEM containing 2% heatinactivated FBS) to determine the amount of virus (2.5 µL) to infect up to 15% of the cells. A previously described flow cytometry-based infectivity assay71 was used with modifications to examine neutralizing antibody interactions with recombinant variant E proteins. Sera from


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human DENV2 challenge subjects were heat-inactivated (55oC, 30 min) and 50% neutralizing (NT50) antibody titers were calculated as the sera dilution factor required to neutralize 50% of the virus. To determine the inhibitory effect of soluble E proteins on antibody neutralization of cell infection, titrated dilutions of the purified recombinant E (2-200 nM) were incubated (37oC, 1 h) with diluted sera (2x NT50) from the DENV2 challenge subjects. Equal volumes of infection media containing no virus (Vero uninfected control) or virus diluted in infection media were incubated (37oC, 1 h) with the sera and E mixtures. Cell culture media were replaced with fresh media containing penicillin-streptomycin (1%), and the Vero cells were cultured for 1.5 h (37oC, 5% CO2,). The supernatants were aspirated and the cells were washed once before incubating (48 h, 37oC) with fresh infection media containing penicillin-streptomycin (1%). DENV2infected cells were washed (2 x) with PBS (Mediatech, Inc, Manassas, VA) and trypsinized to detach from the plastic surfaces. PBS containing 10% FBS was added, and the cells were resuspended and transferred to polystyrene tubes. The cells were centrifuged (500 x g, 5 min), washed in PBS (1 x), treated with 1x BD FACS™ Permeabilizing Solution 2 (22oC, 10 min), washed in PBS (1 x), blocked with 1x PBS/BSA (5%) (22oC, 20 min), and washed again (1 x PBS). The cells were incubated (22oC, 20 min) with anti-dengue virus complex antibody, clone D3-2H2-9-21 (MilliporeSigma, Burlington, MA) diluted (10 µg/mL) in PBS/1% BSA, and washed with PBS (1 x). To detect the primary antibody, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies) diluted 1:1000 in PBS (22oC, 20 min), centrifuged, and stored in PBS (4oC) containing 2% formaldehyde (ThermoFisher Scientific). Flow cytometry data were acquired on a BD FACSCalibur™ instrument, using BD CellQuestTM Pro software v 5.2.1, and analyzed using the Super-Enhanced Dmax Subtraction (SED) algorithm in FlowJo v10.3, with uninfected Vero cell histograms used



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as controls in population comparisons. Infected cell populations were quantile normalized by group (pre- and post-challenge), and the percentage of virus neutralized by post-challenge antibodies was calculated as follows with equation 5: (5)

% neutralization =

(

𝑦𝑝𝑟𝑒

―

) × 100,

𝑦𝑝𝑜𝑠𝑡

𝑦𝑝𝑟𝑒

where y is the percentage of infected cells by virus pre-incubated with sera from each time point. Neutralizing antibody inhibition by recombinant E variants was calculated as the change in the % of virus neutralized, relative to the level of neutralization achieved in the presence of recombinant PUO-218 envelope protein.

Dengue challenge sera. Sixteen healthy, flavivirus naïve rhesus macaques (M. mulatta) were subcutaneously injected with 105 PFU of either DENV1 (West Pac 74), DENV2 (S16803), DENV3 (CH53489), or DENV4 (341750) (n=4 per challenge group), derived from low passage, near wild-type virus isolates. Sera were collected prior to and 30 days post-infection (DPI)40. Sera from human primary DENV2 infections (n = 10) were collected as part of a DENV human challenge model originally developed by the Laboratory of Infectious Diseases at the U. S. National Institutes of Health47, 48. Sera from participants that had no prior history or serological evidence of flavivirus infection were collected prior to and 28 days post-challenge with 103 PFU of rDEN2∆30. rDEN2∆30 induced viremia in all participants by 5 DPI.

Ethics Statement This research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and adhered principles stated in the Guide for the Care and Use of Laboratory Animals, National Research


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Council, 1996, under facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International, by an approved protocol through the Institutional Animal Care and Use Committee (IACUC) of the Walter Reed Army Institute of Research (WRAIR). Animal care procedures complied with the regulations detailed under the Animal Welfare Act and in the Guide for the Care and Use of Laboratory Animals, in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research”. Animals were kept at a constant temperature (22 °C ± 2 °C) and relative humidity (50%), with environmental enrichment and 12 h of artificial light per day. If the animal was in pain on the basis of their observations of animal behavior, analgesics were subsequently administered, through a single i.m. injection of 5 mg/kg flunixine (Finadyne®, Schering Plough) in the days after interventions. Research on human subjects was conducted in full compliance with DoD, NIH, federal, and state statutes and regulations relating to the protection of human subjects and adheres to principles identified in the Belmont Report (1979). Sera from adult human (18-50 years of age) primary DENV2 (rDEN2∆30) challenges were collected under approval from the Institutional Review Boards of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Regulatory Compliance and Human Subjects Protection Branch (RCHSPB), and also the Western Institutional Review Board® (Olympia, Washington). All subjects participated willingly in the study as evidenced by signing the informed consent document.



Ancillary Information Supporting Information: Supplemental Figures S1-S5 and Supplemental Files 1 and 2.

Corresponding Author Information: [email protected]; 301 619-4232

Present/Current Author Addresses: 1Molecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702; 2Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250; 3Armed

Forces Research Institute of Medical Sciences, Bangkok, 10400, Thailand; 4Center for

Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA

Author Contributions: SLK and JLS contributed equally as first authors. Conceived study (RGU, SF); prepared and reviewed manuscript (SLK, JLS, SF, APD, BMZ, RGU); performed laboratory experiments (SLK, JLS); protein modeling (SLK, BMZ); data analysis (SLK, JLS, SF, APD, BMZ, RGU).

Acknowledgment: Funding was provided to RGU and SF by the Military Infectious Disease Research Program (https://midrp.amedd.army.mil/), project S0453_14_AF. This research was supported in part by the National Academies of Sciences, Engineering, and Medicine through a National Research Council (NRC) research associateship awarded to JLS, and by an appointment of SLK and BZ to the Postgraduate Research Participation Program administered by Oak Ridge Institute for Science and Education through an interagency agreement with the U.S. Department


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of Energy. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by any Agency or branch of the U.S. government.

Abbreviations Used: ADE, antibody-dependent enhancement; CYD-TDV, recombinant chimeric yellow fever-17D-dengue virus, live attenuated, tetravalent dengue vaccine; DENV1-4, dengue virus serotypes 1-4; DENV2-NGC, dengue virus serotype 2 strain New Guinea C; E, envelope protein; EDI, EDII, and EDIII, envelope protein domains I, II, and III; ESS, effective sample size; HPD, highest posterior density; Hsecl, total entropy per clade; IMAC, immobilized metal affinity chromatography; IVT, in vitro translation; MCC, maximum clade credibility tree; MCMC, Markov Chain Monte Carlo; MFI, mean fluorescence intensities; NHP, nonhuman primate; NT50, 50% neutralizing antibody titers; PFU, plaque forming unit; prM, pre-membrane; PRNT, virus plaque reduction neutralization titer; PUO-218, dengue virus serotype 2 vaccine strain; Scl, distribution function of the exponentially distributed number of strains; TVD, tetravalent dengue vaccines; TM, transmembrane; ViPR, Virus Pathogen Database and Analysis Resource

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Figure Legends Figure 1. Molecular phylogeny of DENV2 envelope protein sequences. The evolutionary history of 266 unique global DENV2 envelope (E) protein sequences isolated between 1944 and 2010 was inferred using the maximum clade credibility tree obtained using a Bayesian method26, 27.

Clade branches are colored according to geographical area of isolation, which correspond to

the colored country regions in the continent maps. For clarity, triangles represent large clades of geographically related isolates. Triangle size is proportional to the number of isolates contained within the clade. The horizontal length of the triangle reflects the age of the clade, and begins at



the estimated MRCA of the clade and extends to the date of isolation of the most recently isolated strain. Probability values >0.9 are represented by (*), >0.95 by (**), and >0.99 by (***), inside the nodes. Each of the six DENV2 genotypes are represented in the tree and cluster distinctly, as indicated by the bold black vertical bars to the right of the tree. Numbered clades represent major branch points in the evolution of DENV2 E that exhibit elevated diversity in solvent accessible E amino acids displayed in macromolecular complexes on the virion surface.

Figure 2. Solvent accessible residues of the envelope monomer displayed on the surface of DENV2 exhibit increased diversity in amino acid sequences. Surface exposed envelope (E) residues that exhibited amino acid variability in a multiple sequence alignment of 266 unique E amino acid sequences representing isolates originating between 1944 and 2010 from diverse geographic regions and genotypes were mapped on to E protein structures. (a) Amino acid variability was calculated as Shannon entropy (H) for each column of the E sequence alignment. E residues are colored by E protein domain (red, DI; yellow, DII; royal blue, DIII; cyan, transmembrane (TM) domain). (b) Proportions of total and variable amino acids of the fulllength E polypeptide that are solvent accessible or non-solvent accessible. (c) Entropy values shown in a for each E amino acid were mapped on to an E monomer (space filling) in the structural model of the DENV2 E-M-M-E heterotetramer (PDB 3J2P) viewed from the exterior of the virus (top), side (middle), and interior (bottom). Residue entropy is gradient colored from low (white) to high (non-solvent accessible, red; solvent accessible, blue) according to the key shown at the right. For clarity, only ribbon structures of E DI-III (colored as in a) are shown in exterior and interior views, while E-TM domain (cyan) and M (orange) are included in the side view of the complex.


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Figure 3. Specificity of antibodies from primary challenges with DENV. Relative binding of convalescent serum antibodies from rhesus macaques and human primary DENV infections to microarrayed DENV envelope (E) proteins (DENV1-Hawaii, DENV2-NGC, DENV3-H87, DENV4-H241). Antibody specificity for E from representative DENV1-4 strains by sera from (a) rhesus macaques challenged independently with DENV1, DENV3, DENV4, or (b) rhesus macaques challenged with Asian I lineage and humans challenged with American lineage DENV2. Values are shown for antibody binding to E relative to the challenge serotype.

Figure 4. Polyclonal antibody recognition of solvent-accessible residues from current and historical DENV2 strains displayed on the PUO-218 envelope backbone. Serum antibodies from rhesus macaques challenged with the Asian-lineage strain S16803 (a, b) or humans challenged with the American-lineage strain Tonga/1974 (c, d). DENV2 lineages of residues displayed: Asian I, AsI; sylvatic, Syl; Asian II, AsII; Cosmopolitan, Cosmo; Asian/American, As/Am. In a and c, antibody binding is shown relative to the envelope (E) variant exhibiting the highest sequence identity to the strain used for challenge: V (PUO-218) for Asian-lineage antibodies; average of variants 12 and 13 for American-lineage antibodies. Antibody interactions (a-e) are shown as decreased (hashed), unchanged (open), or increased (shaded) bars. E proteins that exhibited changes in antibody recognition are indicated (* p < 0.05, ** p < 0.001, or *** p < 0.00001). E surface residues that contributed to differential antibody binding are indicated in b and d. Columns are labeled with residues of the challenge strains, and amino acid substitutions representing each DENV2 clade are indicated within rows. Amino acids are colored by charge (blue, positive; red, negative; unshaded, uncharged). (e) Antibody recognition of variants 2C and



5C that display surface residues of PUO-218 on backbones that exhibited the lowest (variant 2) and highest (variant 5) total sequence identity to PUO-218. Non-solvent accessible residues Y59F and T236M of 2C, as well as G228E of 5C, alter antibody binding compared to complementary variants 2 and 5.

Figure 5. Antibodies primarily target solvent-accessible residues within a 480Å2 region on the surface of the DENV2 envelope protein monomer. Surface exposed residues of the E monomer that contributed to differential recognition of envelope (E) protein variants by convalescent antibodies from non-human primates and humans after primary infection with DENV2 were mapped on to E protein structures. E monomers are colored by domain (EDI, red; EDII, yellow; EDIII, blue). (a) The soluble ectodomain structure of the DENV2 E dimer as viewed from the outside of the virus. Solvent accessible residues that were targeted by polyclonal antibodies are shown as green spheres (ribbon) or are green surfaces (space filling monomer), whereas buried residues that presumably affect E surfaces are shown in orange (ribbon). The residues form a hexagonal footprint (dashed line and inset) of ~480Å2. (b) An E protein raft consisting of three E dimers (PDB 3J27). Surface accessible residues associated with polyclonal antibody interactions are shown as green spheres. (c) Potential antibody recognition orientations based on surface accessible residues identified in our study: binding that spans surfaces of a single E dimer (green shading) or bridges neighboring dimers (blue shading).


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Table 1. DENV2 sequences selected for construction of chimeric envelope proteins. Cladea/ Variant

Accession Date of No. Strain Location isolation Genotype AF038403 NGC Papua New Guinea 1944 Asian II V D00345d PUO-218 Thailand 1980 Asian I 1 EF105385 Dak Ar D20761 Senegal 1974 Sylvatic 2 EF105382 Dak Ar 2039 Burkina Faso 1980 Sylvatic 3 EF105390 Dak Ar 141070 Senegal 1999 Sylvatic 4 HQ891024 V5056 Taiwan 2008 Asian II 5 FJ024454 V1675 Vietnam 2007 Asian I 6 EU482676 V739 Vietnam 2006 Asian I 7 FJ196852 GD01/01 China 2001 Cosmopolitan 8 JN851128 0232Y06 Singapore 2006 Cosmopolitan 9 GU131843 V3502 Burkina Faso 1986 Cosmopolitan 10 EU569707 V1368 USA/PRe 1995 As/Amf 11 EU677144 V1427 USA/PR 1999 As/Am 12 EU482720 V588 USA/PR 2006 As/Am 13 EU482726 V595 USA/PR 2006 As/Am 14 FJ639822 V2262 Venezuela 2006 As/Am 15 FJ182012 V1597 Columbia 2005 As/Am 16 FJ639698 V2021 Cambodia 2002 As/Am aClade number corresponds to numbering in Figure 1. bHse corresponds to the total entropy of surface exposed residues per clade cl cSurface exposed dPUO-218 included in CYD-TDV ePuerto Rico fAsian/American


Hseclb

0.42434 0.34658 0.69315 0.42434 0.21217 0.22528 0.34658 0.34658 0.21217 0.34658 0.34658 0.20016 0.20016 0.23468 0.34658 0.34658

% Sequence Identity Total SEc Residues Residues 100 94.34 93.94 94.93 97.98 98.59 98.59 97.78 98.18 98.18 97.98 98.18 98.59 97.17 97.78 97.76 97.78

100 90.41 87.67 90.28 95.21 97.95 97.95 95.89 96.58 95.89 95.89 97.26 97.26 97.26 96.58 96.5 95.89


Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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For Table of Contents Use Only



Dengue virus 2 envelope surface variants

Protein microarray

Detect antibody bound to proteins

Y Y

Y Y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

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Digital antibody binding data


Impact of Dengue Virus Serotype 2 Strain Diversity on Serological

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