Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections

Tomer Hertz, P. Robert Beatty, Zachary MacMillen, Sarah S. Killingbeck, Chunling Wang and Eva Harris
J Immunol May 15, 2017, 198 (10) 4025-4035; DOI: https://doi.org/10.4049/jimmunol.1700029

Abstract

Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

Introduction

The four dengue virus (DENV) serotypes (DENV1-4) are transmitted by Aedes aegypti and Ae. albopictus mosquitoes, causing up to an estimated 390 million infections and 96 million cases of dengue annually (1). DENV is an enveloped flavivirus whose positive-sense 10.7-kb RNA genome encodes a polyprotein that is co- and posttranslationally cleaved by host and viral proteases into three structural (capsid, membrane [prM/M], envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins. DENV infections in humans range from asymptomatic to dengue fever (DF) to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (2). DF is an acute febrile illness with headache, retro-orbital pain, myalgia, arthralgia, rash, hemorrhagic manifestations, and/or leukopenia. The hallmark features of DHF consist of thrombocytopenia, hemorrhagic manifestations, and signs of plasma leakage, which can lead to hypotensive shock (DSS) and death (3). A primary infection with one DENV serotype is thought to lead to homologous protective immunity; however, subsequent infection with a different serotype (secondary heterologous DENV infection) is a major risk factor for severe disease. The Ab response to DENV in both primary and secondary infection is dominated by anti-E Abs (4, 5), with some anti-prM/M and anti-NS1 Abs (6–11). Ab-dependent enhancement is thought to occur when cross-reactive anti-E and anti-prM Abs facilitate entry of DENV into Fcγ receptor-bearing cells, leading to increased viral load, immune activation, and more severe disease. In contrast, Abs to NS1 should not enhance infection as they do not target structural proteins on the virion.

NS1 is the only viral protein secreted by DENV-infected cells, and it is conserved ∼79% across the four DENV serotypes (12–14). NS1 has been reported to play an immunomodulatory role via its interaction with the complement component C4b (15, 16). Although natural infection with DENV has been shown to elicit a potent NS1 Ab response in both humans and mice, some of these Abs have been shown to cross-react with platelets or endothelial cells (17–19). Although some groups have suggested that these cross-reactive Abs to NS1 contribute to pathogenesis (8), mice vaccinated with recombinant DENV NS1 are protected against lethal infection without evidence of pathogenesis from Abs to NS1 (20–22). Overall, NS1 vaccines have been shown to be highly immunogenic in mice, and Abs to NS1 prevent the pathogenic effects of secreted NS1 (22). In addition, several immunodominant B-cell NS1 epitopes in naturally infected mice are conserved across all four DENV serotypes (23).

A principal challenge in vaccine development is eliciting long-lived memory CD8+ T cells and protective Abs (24, 25). One effective strategy is the use of adjuvants that enhance vaccine immunogenicity. For example, monophosphoryl lipid A adjuvant is a TLR4 agonist that has been shown to elicit strong Ab and CD8+ T cell immune responses and has been approved for use in the human papillomavirus vaccine (26).

Ag microarrays are high-throughput Ab binding assays that build upon the technology of DNA microarrays. Ag microarrays have previously been used in several application domains, including identifying immunogenic responses to pathogens with large genomes (27, 28) and biomarkers for cancers (29, 30) and autoimmune diseases (31, 32). Although Ags spotted on the arrays can include peptides, proteins, or virions, we and others have recently shown that peptide Ag microarrays (hereafter referred to as peptide microarrays) spotted with overlapping peptides of influenza glycoproteins can be used to profile vaccine-induced responses in mice and that the Ab profiles generated by distinct vaccines differ from one another (33–35).

In this study, we used a peptide microarray spotted with overlapping 20-mer peptides that span the NS1 proteins of three DENV serotypes (DENV1-3) to obtain Ab profiles of mice vaccinated with recombinant DENV2 NS1 combined with three different adjuvants. For comparison, we also mapped the Ab responses to NS1 in the sera of mice infected with DENV2. We then used the same peptide microarrays to profile the NS1 Ab response in humans naturally infected with DENV. We identified five immunodominant regions of Ab reactivity, three of which were shared across DENV serotypes and two of which were recognized by immunized mice, infected mice, and infected humans.

Materials and Methods

Ethics statement

Animal experiments were performed strictly following the guidelines of the American Veterinary Medical Association and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The University of California, Berkeley’s Animal Care and Use Committee preapproved all experiments. The protocols for the studies involving human subjects were reviewed and approved by the Institutional Review Boards of the University of California, Berkeley, and the Nicaraguan Ministry of Health. Parents or legal guardians of all participants provided written informed consent, and participants 6 y of age and older provided assent.

Viruses

All viruses were propagated in the Ae. albopictus C6/36 cell line (American Type Culture Collection), titered by plaque assay on baby hamster kidney cells (BHK21, clone 15) and expressed as PFUs per milliliter. The parental strain DENV2 PL046 was obtained originally from H.Y. Lei (National Cheng Kung University, Taiwan), and DENV2 D220 was produced from PL046 as previously described (36, 37).

Recombinant NS1 proteins

Recombinant DENV2 NS1 protein was obtained from Merck. The NS1 working stocks were tested using the Endpoint Chromogenic Limulus Amebocyte Lysate QCL-1000TM kit (Lonza) and confirmed to be bacterial endotoxin-free (<0.1 EU/ml per 25 μg of protein).

Mouse NS1 vaccination experiments

Six- to eight-wk old IFN-α/β receptor–deficient 129 mice (A129) were bred and maintained in specific pathogen-free conditions at the University of California, Berkeley Animal Facility. A129 mice were injected i.p. three times (days 0, 14, and 42) with 20 μg NS1 or 20 μg of OVA protein in combination with 1 μg monophosphoryl lipid A (InvivoGen, San Diego, CA) and AddaVax (0.5% sorbitan trioleate, 5% squalene, 0.5% Tween-80 in 10 mM sodium citrate buffer; InvivoGen) or Sigma adjuvant system (SAS) (Sigma-Aldrich) with 20 μg CpG or 120 μg aluminum hydroxide and magnesium hydroxide (alum; Pierce). Two weeks after the third immunization (day 56), mice were challenged i.v. with a lethal dose of 107 PFU of DENV2 strain D220. A separate group of mice were infected with 105 PFU DENV2 strain PL046 at day 0 as a primary infection control and challenged at day 56. After challenge, mice were observed every 12 h using a morbidity scoring system on a scale of 1–5 (36). Mice were euthanized immediately when they became moribund (score of 5). Immune serum was collected by submandibular bleed from NS1- or OVA-immunized mice or DENV2 PL046–infected mice on day 49.

NS1 ELISA

To determine levels of NS1-specific Abs in immunized mice prior to challenge, 50 μl of 0.5 μg/ml NS1 was coated on a Nunc Immulon polystyrene plate. After blocking with 5% nonfat dry milk in PBS+0.05% Tween-20, 50 μl of a 1:100 dilution of each test serum was added and incubated for 24 h. After washing with PBS+0.05% Tween-20, biotinylated goat anti-mouse IgG (Jackson ImmunoResearch) was added, followed by washing and addition of streptavidin-alkaline phosphatase (Life Technologies), and para-nitrophenyl phosphate (Sigma-Aldrich) substrate. The reaction was terminated with 0.5 M EDTA after 10–15 min, and the OD was read at 405 nm.

Human samples

A prospective study was conducted from 2005 to the present in the Infectious Disease Ward of the Hospital Infantil Manuel de Jesús Rivera in Managua, Nicaragua, to study clinical, immunological and viral risk factors for severe dengue. Infants and children between 6 mo and 14 y of age with fever or history of fever <7 d and one or more of the following signs and symptoms: headache, arthralgia, myalgia, retro-orbital pain, positive tourniquet test, petechiae, or signs of bleeding were eligible to participate in the study. Both inpatients and outpatients were enrolled each year during the peak of the dengue season (August 1–January 31) and followed clinically through the acute phase of illness. A blood sample was collected daily for 3 d for complete blood counts with platelets, blood chemistry, and serological, virological, and molecular biological tests for DENV infection. A convalescent blood sample (14–28 d post-onset of illness) was also collected for paired serological testing. After undergoing a separate informed consent, subjects could choose to participate in a longitudinal arm of the study, wherein blood samples were collected 3, 6, 12, and 18 mo post-onset of illness. A participant was considered positive for DENV infection when laboratory tests met one or more of the following criteria: 1) dengue viral RNA was detected by RT-PCR (38, 39); 2) DENV was isolated (38); 3) seroconversion of DENV-specific IgM was detected by MAC-ELISA in paired acute and convalescent samples (38, 40); and/or 4) DENV-specific Ab titer by Inhibition ELISA (41–43) demonstrated a 4-fold or greater increase between acute and convalescent sera. Primary DENV infections were considered those in which the convalescent Ab titer was <2560, and secondary infections were considered those in which the convalescent Ab titer was ≥2560 as determined by Inhibition ELISA. The severity of laboratory-confirmed dengue cases was classified using a computerized algorithm that compiled all clinical data meeting the criteria for DF, DHF, and DSS, defined according to the 1997 World Health Organization classification criteria (44, 45). Specifically, we tested patient samples collected in 2006–2010, consisting of 10 DF cases (four DENV2 primary, three DENV3 primary, and four DENV3 secondary infections), seven DHF cases (two DENV3 primary and five DENV3 secondary), and three DSS cases (DENV2 secondary).

The Nicaraguan Pediatric Dengue Cohort Study is a prospective cohort study of ∼3500 active participants 2–14 y of age in Managua, Nicaragua, which has been ongoing since 2004 (46). Every year, a blood sample is collected from all participants to test for and identify any DENV infections that have occurred in the preceding year, whereas all suspected dengue cases and undifferentiated fevers are worked up to diagnose symptomatic DENV infections. A child whose paired annual serum samples demonstrate seroconversion (a titer of <1:10 to ≥1:10) or a ≥4-fold rise in Ab titer as determined by Inhibition ELISA is determined to have experienced a DENV infection. If a child entered the cohort DENV-naive and experienced no DENV infections during the study, they were considered to be DENV-negative, and their serum samples were used as negative controls in this study.

Peptide synthesis and microarray printing

Twenty-aa peptides with a 15-aa overlap between adjacent peptides spanning the DENV NS1 proteins were generated from consensus sequences computed from an alignment of 42 DENV1 (2004–2009), 181 DENV2 (1999–2009), and 123 DENV3 (2008–2010) sequences from viruses collected in Nicaragua. The peptide arrays were designed to map human responses from Nicaraguan patients, and as there is very little circulation of DENV4 in Nicaragua, we generated peptides only to DENV1, DENV2, and DENV3. A total of 204 peptides (68 for each DENV serotype) were synthesized with a MultiPep RS (Intavis AG) peptide synthesizer using a modified SPOT synthesis protocol. The peptides were lyophilized in a 384-well microtiter plate and stored at −20°C. Peptides were resuspended in 12.5 μl DMSO and 12.5 μl of ultra-pure water to create a working solution of ∼1 mg/ml. Peptide stocks were diluted 1:4 in protein printing buffer (3× saline sodium citrate, 0.1% polyvinyl alcohol, and 0.05% Tween-20). Peptide integrity was confirmed using two positive controls, hemagglutinin A (HA) (YPYDVPDYA) and FLAG (DYKDDDDK), included in multiple replicate wells for the initial optimization of the array printing conditions. Peptide stocks were diluted 1:2 in protein printing buffer (PBS with 0.005% Triton X-100) and printed in triplicate on N-hydroxysuccinimide ester-derivatized glass slides (H slides; Schott/Nexterion AG) using a Q-array II microarrayer (Genetix) with contact microarray pins (SMP2.5B; TeleChem). During printing, relative humidity was maintained at 50–60%. Following printing, slides were left to dry overnight. Arrays were stored at −20°C.

Microarray hybridization

Printed grids were outlined with a PAP hydrophobic pen (Research Products International). Slides were chemically blocked using 4 ml of 50 mM borate, 50 mM ethanolamine for 1 h. Slides were then washed twice with PBS containing 0.05% Tween-20, twice with PBS, and once in deionized water and then spun to dry at 1000 × g for 5 min at room temperature. Mouse serum samples were diluted 1:25 in 1% BSA and 0.025% Tween-20, incubated on slides for 2 h in a humidified chamber at 25°C, then washed twice with PBS containing 0.05% Tween-20 and twice with PBS. Bound IgG was detected for 45 min with Alexa Fluor 647 goat anti-mouse IgG (115-605-008; Jackson ImmunoResearch). Human plasma samples were diluted 1:100 in 1% BSA and 0.025% Tween-20, incubated on slides for 2 h in a humidified chamber at 25°C, then washed twice with PBS containing 0.05% Tween-20 and twice with PBS. Bound Abs were detected for 45 min with Alexa Fluor 647 goat anti-human IgG (Jackson ImmunoResearch). Arrays were washed and spun dry as described above.

Slide scanning

Slides were scanned on a two-laser GenePix 4400A scanner (Molecular Devices) to detect IgG responses. Images were analyzed by GenePix version 7.2 to obtain the mean fluorescence intensity (MFI) for each probe. Responses below 500 MFI after subtraction of background were considered negative (MFI range, 0–65,000). Subsequently, all data were analyzed with Matlab (MathWorks) and Python. For background normalization, samples from naive mice and DENV-naive humans (annual samples from the Nicaraguan Pediatric Dengue Cohort Study, see above) were used as negative controls to adjust for background. For each probe, we computed the median naive response and subtracted it from the median responses in NS1-vaccinated mice or from the dengue-positive human samples, accordingly.

Unsupervised data clustering

To analyze clustering of Ab profiles in the mice, the responses to the 68 NS1 peptides of DENV2 Nicaragua consensus sequence were used to define a response vector for each animal. Response vectors were compared using the Spearman rank-order correlation measure to create a similarity matrix in which each entry measures the similarity between the pair of response vectors from a specific pair of animals. The samples were then clustered using complete-linkage, a hierarchical unsupervised technique for clustering data points using a given similarity measure (47). Because there were four experimental groups of mice, the number of clusters was arbitrarily set to six, allowing the existence of outlier clusters. The same methodology was also used to cluster the human samples.

Identifying Ags with differential responses between groups

To compare response rates for specific NS1 Ags from the three DENV serotypes, we first filtered the set of Ags in a treatment-blinded manner, removing all Ags with an overall response rate of <20%. The remaining Ags were then individually tested for differences using Fisher exact test. Nonresponders were any response below 5000 MFI after subtraction of background. To adjust for multiplicity, we computed the family-wise error rates using the Bonferroni correction. Ags with adjusted p values < 0.05 were reported. We used this methodology to compare the responses of mice in the NS1+alum versus NS1+monophosphoryl lipid A + AddaVax (MA) groups and also to compare responses of acute, convalescent, and 12-mo samples from human subjects.

Summary statistics of peptide array data

The Ab profile generated by the peptide microarrays is a multidimensional measurement of the Ab responses to a large set of overlapping peptides from NS1. To compare the Ab responses of each sample as measured by the peptide microarray to NS1 in different treatment groups of mice and in different human clinical severity groups across time, we defined the magnitude and breadth of responses to each protein as follows.

We denote the normalized array measurements by Embedded Image where:

  • i denotes subject, Embedded Image; s denotes consensus sequence for each DENV serotype, S = {DENV1, DENV2, DENV3}; as denotes peptide Ags from the consensus sequence of each serotype s, Embedded Image; Embedded Image denotes treatment assignment (alum, MA, SAS+CpG DNA [SCpG], DENV2, etc.) of subject i; yi denotes outcome of subject i (vaccine-induced Ab titer/infection status/disease status). The observed data for each subject are Embedded Image, for Embedded Image s = {DENV1, DENV2, DENV3} and Embedded Image

    • 1) Embedded Image denotes the magnitude of responses to all peptide Ags from the consensus sequence of each serotype s.

    • 2) Embedded Image denotes the breadth of response to peptide Ags from the consensus sequence of each serotypes (where positivity is determined using responses of negative controls).

Statistical analysis

To assess baseline levels of responses to the NS1 peptide arrays, we profiled the responses of samples from naive mice and DENV-negative human subjects. We then used these profiles to normalize the responses of all samples by subtracting the median naive response for each Ag. This was done separately for mouse and human samples. Statistical comparisons between the response magnitudes of the different groups were performed using a Wilcoxon rank sum test. In the mouse challenge experiment, comparison of survival rates was conducted using a nonparametric log rank (Mantel–Cox) test and graphed as Kaplan–Meier survival curves.

Results

Specific adjuvants combined with NS1 provide protection against lethal DENV infection

To evaluate the effect of various adjuvants on the ability of NS1 to provide protection, we vaccinated mice three times with 20 μg DENV2 NS1 combined with MA, SCpG, alum, or 20 μg OVA combined with MA and then challenged the mice with a lethal dose of DENV2 (D220 strain) and measured survival (Fig. 1A). Mice immunized with NS1 in combination with MA or SCpG were protected from lethal challenge, whereas mice immunized with NS1+alum or OVA+MA were not protected. Mice infected with 1 × 105 PFU of DENV2 (PL046 strain) were protected against lethal challenge because a primary infection with DENV2 serves as a homotypic immunization and protects against a secondary infection even with a lethal challenge dose. Therefore, the vaccine-induced protection was specific to NS1 plus MA or SCpG adjuvant combinations.

FIGURE 1.
FIGURE 1.

Protective immunization with DENV NS1 results in the robust production of NS1-specific Abs. (A) Survival curve of mice immunized with NS1 in combination with different adjuvants, after lethal DENV2 challenge. Six- to eight-week old A129 mice (n = 8 per group) were immunized i.p. with 20 μg recombinant DENV2 NS1 combined with MA, SCpG, or alum, or 20 μg OVA combined with MA on day 0, boosted on day 14 and day 42, and challenged i.v. on day 56 with a lethal dose of 107 PFU of DENV2 D220. In parallel, mice were infected with a sublethal dose (105 PFU) of DENV2 strain PL046, which provided protection from a lethal infection. A p value < 0.0001 was observed between NS1+MA or NS1+SCpG and OVA by Mantel–Cox log-rank test. (B) NS1 IgG ELISA using mouse serum collected on day 49 after NS1 or OVA vaccination or sublethal DENV2 infection. Total anti-NS1 IgG was measured by direct ELISA, with sNS1 coated at 0.25 μg/ml and serum samples diluted 1:100. Biotinylated anti-mouse IgG secondary Ab was used for detection. (C) Boxplots of overall magnitude of the DENV2 NS1 response to the peptide array in each experimental group. The overall magnitude is defined by the sum of magnitudes to all NS1 peptides (see Materials and Methods for details). Horizontal bars represent the median response of each group. Box limits represent the 25th and 75th percentiles, and the whiskers denote the data range. Individual responses are plotted as filled circles. Responses above the whiskers are outliers. (D) Clustering dendrogram of all mouse samples generated using complete-linkage clustering over the DENV2 NS1 Ab profiles of each mouse (see Materials and Methods for details). Mice from different experimental groups were separated into distinct clusters. The MA-and SCpG-vaccinated mice cluster closer to the DENV2 naturally infected mice than to the alum-vaccinated mice.

Sera collected from the immunized mice and infected mice were tested for NS1-specific IgG by ELISA (Fig. 1B). As expected, the highest levels of NS1-specific IgG were found in mice immunized with NS1 in combination with MA or CpG; much lower levels were detected in mice immunized with NS1+alum, and mice infected with a sublethal dose of DENV2 mice generated intermediate levels of IgG to NS1.

Distinct Ab profiles were generated by NS1 vaccination with different adjuvants or DENV infection

To further characterize the Ab responses in mice immunized with recombinant NS1 combined with different adjuvants or with a low-dose DENV2 infection, we used an Ag microarray spotted with overlapping peptides covering NS1 from the consensus sequences of DENV1, DENV2, and DENV3 Nicaraguan isolates. Each NS1 protein was represented using 20-mer peptides, overlapping by 15 aa. Ab profiles were generated by applying to the arrays individual serum samples collected from mice in three experimental groups, NS1+alum, NS1+MA, and NS1+SCpG, as described above. We also profiled responses of mice immunized with OVA as a negative control, as well as mice infected with a low dose of DENV2. We found that mice immunized with NS1+alum generated weak but detectable Ab responses to the DENV2 NS1 protein, whereas the groups immunized with NS1+MA or NS1+SCpG generated robust responses to DENV2 NS1 (Fig. 1C), in accordance with the NS1 Ab titers presented in Fig. 1B. Specifically, we computed the overall magnitude of Ab responses in the serum of each mouse, combined the responses in all the mice (see Materials and Methods), and compared the three different vaccine groups. We found that mice immunized with NS1+MA had significantly higher responses as compared with mice in the NS1+alum group (p = 0.00005, Fig. 1C). The sera from DENV2-infected mice demonstrated lower reactivity to NS1 peptides than NS1+MA or NS1+SCpG–vaccinated mice.

To further illustrate the differences in Ab profiles of the different treatment groups, we used an unsupervised clustering algorithm to group the Ab profiles of the mice to the DENV1, 2, and 3 NS1 peptide Ags. The algorithm uses the vaccine-induced Ab profiles of each animal in the study to identify mice with similar Ab profiles, and does not make use of the treatment assignment of each animal (see Materials and Methods for details). We found that animals in different treatment groups formed distinct clusters (Fig. 1D, Supplemental Fig. 1). Overall, the clusters of mice immunized with NS1+MA and NS1+SCpG were closely related in a separate branch from DENV2 infection; in turn, this entire branch was separated from the alum cluster.

NS1 vaccine–induced Ab responses target specific NS1 epitopes

Because we observed that the overall magnitude of responses in the NS1+MA group was significantly higher than the NS1+alum group, we next turned to identify the specific peptide Ags that were targeted more robustly in the NS1+MA group. Comparisons were performed using Fisher exact test, and values were adjusted for multiplicity for each strain separately, using the Bonferroni correction. The set of Ags tested for each strain was blindly filtered to include only regions in which the response rates were >20% (see Materials and Methods). Because there were only two animals in the SCpG group, we did not perform statistical comparisons with this group. Using the DENV1, DENV2, and DENV3 peptide arrays, we identified five DENV NS1 regions in which responses of the NS1+MA group were significantly stronger than those of the NS1+alum vaccinated mice (Fig. 2A–C, Table I). The five regions of NS1 Ab reactivity are: Region 1 (aa 21–40), Region 2 (aa 101–135), Region 3 (aa 156–175), Region 4 (aa 231–255), and Region 5 (aa 296–335).

FIGURE 2.
FIGURE 2.

DENV NS1 Ab profiles of NS1-vaccinated mice. Ab reactivity to peptides derived from NS1 from: (A) DENV1; (B) DENV2; and (C) DENV3. Individual bars represent the average response of each experimental group to a specific NS1 peptide (n = 68). The height of each bar represents the MFI. For each serotype, responses of five groups are plotted: DENV2 infection—mice infected sublethally with PL046 (positive control, n = 9); NS1+MA—mice vaccinated with NS1 adjuvanted with MA (n = 11); NS1+SCpG—mice vaccinated with NS1 adjuvanted with SAS+CpG (n = 2); NS1+alum—mice vaccinated with NS1 adjuvanted with alum (n = 12); and OVA—mice vaccinated with OVA adjuvanted with MA (negative control, n = 7). The five dominant regions statistically identified are highlighted by the numbered rectangles across all serotypes and groups. Robust responses to Region 2 are observed in the DENV-infected, NS1+MA and NS1+SCpG groups across all three DENV serotypes. Responses to Regions 1 and 3 were observed only in NS1+MA and NS1+SCpG vaccinated mice, and not in DENV-infected mice.

View this table:
Table I. NS1 regions identified with peptide arrays probed with mouse and human sera

DENV2 NS1 vaccination generated cross-reactive responses to all three DENV serotypes and increased the breadth of the Ab response compared with DENV2 infection

Although mice were vaccinated with DENV2 NS1, we profiled their responses to the NS1 protein of DENV1-3 to detect serotype cross-reactive responses. We compared NS1+alum– and NS1+MA–induced responses to each of the three DENV serotypes separately. Several of the regions were independently identified in more than one serotype. Most notably, Ab responses to Region 2 were cross-reactive to all three serotypes tested and also to DENV2-infected mice (Supplemental Fig. 2). The Ab responses to Region 1 were also cross-reactive across all three DENV serotypes, but were statistically identified in DENV1 and DENV3 only. The responses to Regions 3 and 5 were immunodominant in DENV2 serotype only, with only weak reactivity to these regions in DENV1, and responses to Region 4 were only observed in DENV3. To assess whether these cross-reactive responses are due to sequence conservation in these regions, we generated sequence alignments of the three serotypes used in the study (Supplemental Fig. 3). We found that, overall, Regions 1–5 tended to be in conserved regions of the NS1 protein. In Region 1, to which responses were more potent in DENV1 and DENV3, we found that these serotypes shared seven aa that were different from DENV2. Similarly, serum reactivity to Region 4 was only observed in DENV3 arrays, and there were five aa differences from DENV1 and DENV2.

We had observed increased overall magnitude of Ab responses in the NS1 vaccination sera compared with natural infection in Fig. 1C. We next asked whether this increase in magnitude was a result of increased breadth (i.e., greater number of regions recognized). In comparing the responses to the five immunodominant regions identified in the NS1+MA–vaccinated mice to those elicited by DENV2 infection, we found that although Regions 2, 4, and 5 were strongly targeted in both groups, DENV2-infected mice generated weak responses to Region 1 and no detectable responses to Region 3 (Fig. 2). Furthermore, no additional epitopes were detected in the DENV2-infected mice that were not present in the NS1-vaccinated mice.

Mapping of peptide regions on the NS1 structure

The five regions of Ab reactivity were mapped to the molecular structure of the NS1 dimer (48), as shown in Fig. 3. Region 1 is exposed on the dimer surface and near the membrane-binding greasy-finger domain of NS1. The immunodominant Region 2 is in the surface-exposed disordered distal tip of the wing domain. Responses to four peptides in Region 2 were cross-reactive in the NS1+MA group across all three DENV serotypes strains and were also observed in the NS1+SCpG vaccinated mice (Supplemental Fig. 2). Region 3 mapped to the greasy-finger domain of NS1 that in the dimeric form of NS1 is thought to interact with the cell membrane (49). Region 4 is in the surface-exposed spaghetti loop region above the β-ladder (48). Region 5 is on the outer surface of the dimer perpendicular to the wing domain. In conclusion, all five regions of Ab reactivity are surface exposed in the dimeric form of NS1.

FIGURE 3.
FIGURE 3.

Structural model of NS1 showing key epitopes recognized by sera from NS1-immunized mice. NS1 dimer structures highlighting the five peptide regions of Ab reactivity, with monomer 1 in green and monomer 2 in yellow and the peptide regions colored as follows: blue, Region 1, aa 21–40; red, Region 2, aa 101–130; orange, Region 3, aa 156–175; light blue, Region 4, aa 231–255; purple, Region 5, aa 296–325. (A) Perpendicular view of NS1 from the end of the β-ladder. The mobile greasy finger and the disordered tip of the wing domain are indicated by dotted lines. (B) Perpendicular view of NS1 from the edge of the β-ladder. Full-length DENV2 NS1 structure is based on Protein Data Bank (PDB) code 4O6B (48).

NS1 Ab profiles of human subjects naturally infected with DENV2 reveal significant differences across timepoints

We used the same DENV peptide microarray to profile human Ab responses in plasma obtained from children naturally infected with DENV2 or DENV3 collected in the Nicaraguan hospital-based dengue study. We profiled the Ab responses of 10 subjects with DF, seven subjects with DHF, and three subjects with DSS. A total of 16 of the subjects had samples available during the acute phase of infection, and all 20 had convalescent-phase samples as well as 12-mo samples. As expected, we found overall stronger total anti-peptide Ab responses to DENV2 at convalescence compared with the acute phase (p = 0.0005, Fig. 4A). The responses at the 12-mo timepoint were lower than the convalescent phase, and this difference trended toward significance (p = 0.0547, Fig. 4A). The responses at the 12-mo timepoint were higher than in the acute phase, but not significantly (p = 0.074). We then analyzed the differences in magnitude stratified by clinical phenotype (DF, DHF, and DSS) (Fig. 4B–D). Analysis of response magnitude and breadth was conducted separately for primary (n = 4) and secondary (n = 16) infections. We observed that in primary infections in both DF and DHF groups, the magnitude of responses at the 12-mo timepoint did not wane and the response breadth was increased compared with the responses at the convalescent timepoint (Supplemental Fig. 4B, 4C, data not shown). However, in secondary infections, this was only the case in the DHF group.

FIGURE 4.
FIGURE 4.

DENV2 NS1 magnitude (pooled reactivity) boxplots of samples from human subjects naturally infected with DENV2 or DENV3 across timepoints. Horizontal bars represent the median response of each group. Box limits represent the 25th and 75th percentiles, and the whiskers denote the data range. Individual responses are plotted as filled circles. Responses above the whiskers are outliers. (A) Pooled reactivity to NS1 peptides (magnitude) is increased in convalescent sera and declines in samples collected 12 mo post-illness. (B–D) Boxplots of the magnitude of the NS1-specific response across time stratified by clinical phenotype: (B) DF, (C) DHF, and (D) DSS.

Human subjects infected with DENV2 generate responses to Regions 2 and 5

To identify the epitopes targeted during the convalescent phase, we used the same statistical methodology as in the mouse vaccination studies to compare the responses of the acute versus convalescent timepoints. We found that the responses to Region 2 peptides were observed almost solely in the convalescent group (Fig. 5A–C, Table I), whereas the response to this region almost completely waned at the 12-mo timepoint (Fig. 5, Supplemental Fig. 4A). Comparing the acute response to Region 2 peptides across clinical phenotypes, we observed no responses in DF patients, weak responses in DHF patients and strong responses in DSS patients, similar to convalescent responses (Fig. 5). Responses to Region 5 were weaker overall than Region 2 and also waned at the 12-mo timepoint.

FIGURE 5.
FIGURE 5.

Ab responses to DENV2 NS1 over time stratified by clinical phenotype. In each plot, the bars represent the average response of subjects with a given clinical phenotype. Responses are plotted at the acute, convalescent and 12-mo timepoints. The number of subjects in each group and timepoint is indicated in parentheses. (A) DF responses; (B) DHF responses; and (C) DSS responses. Regions 2 and 5 identified in vaccinated mice were independently identified in the human natural infection responses.

Discussion

In this study, we found that mice vaccinated with recombinant DENV2 NS1 in combination with MA or SAS+CpG generated robust Ab responses to NS1 that provided protection from a lethal DENV2 challenge, whereas mice vaccinated with NS1 in combination with alum generated weak Ab responses that were not protective. To further characterize the Ab repertoire generated by vaccination and infection, we used peptide microarrays. We profiled the responses of vaccinated mice and compared these responses to mice infected with a sublethal dose of DENV2. Overall, we found that mice vaccinated with NS1 combined with different adjuvants generated distinct NS1 Ab profiles that were different from those induced by natural infection. By comparing the responses of the alum group to the MA group, we identified five regions of DENV NS1 with significant levels of Ab reactivity in the MA group. Responses to some of these regions were cross-reactive across all three DENV serotypes tested, whereas others were only partially cross-reactive, or only reacted significantly to a single DENV serotype. Furthermore, Ab responses to two of these regions were only observed in mice vaccinated with DENV2 NS1. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 using plasma samples collected longitudinally at acute, convalescent, and 12-mo timepoints. We found that the responses across time differed in magnitude, with the strongest responses at the convalescent phase. When comparing the acute and convalescent responses we found that the reactivity to Region 2 was significantly targeted in the convalescent phase.

A fundamental aspect of this work is the comparison of Ab responses to NS1 following NS1 vaccination with Ab responses to DENV infection. We tested sera collected from DENV2 NS1-vaccinated mice, DENV2-infected mice, and DENV2- and DENV3-infected humans. Region 2, spanning aa 101–135, was immunodominant, with reactivity across all three DENV serotypes. This high reactivity of four adjacent peptides across the different serotypes may suggest that multiple epitopes are being detected within Region 2. Because the reactivity was independently found in both vaccinated and infected mice as well as DENV-infected humans, the data support the use of the mouse model for analyzing NS1-specific immune responses. Region 2 mapped in the structure to the wing domain, which has been suggested to be exposed in both the dimeric and hexameric forms of NS1 (48, 50). Our data identifying this crucial region as immunodominant is in agreement with previous work that found Ab reactivity to this region in both mice and humans (23, 51–53). Region 5 contains aa 296–335, and part of this sequence was independently found to be reactive in NS1-vaccinated mice, infected mice, and convalescent sera in humans.

We found differential serotype cross-reactivity to each of the five regions identified in the vaccinated mice. Whereas four of the five regions were immunodominant in DENV2, Region 4 (aa 231–255) was only recognized in DENV3. In addition, responses to Regions 3 and 5 were only significant in DENV2. There are three possible reasons for these observed differences in cross-reactivity profile. First, the lack of cross-reactivity may be due to differences in the amino acid sequence conservation of these regions (Supplemental Fig. 3). Second, the differences in serotype binding may reflect inherent specificity of the Abs due to differences in their binding affinity. Third, they may also result from differences in the peptide attachment to the microarrays, because different amino acids have different binding propensities to the hydrogel surface that may affect the peptide orientation on the array.

Several of the regions with peptide reactivity in the NS1-immunized mice were similar to those recognized by infected mice and humans, which supports the idea of eliciting protective epitopes from vaccination that are similar to infection. However, additional epitope reactivity was found only with vaccination. Regions 1 and 3 are located along the more hydrophobic region of the NS1 dimer, which faces in toward the lipid core of the hexamer and in the cell-associated dimeric form of NS1 is thought to appose the cell’s plasma membrane (48, 50). Therefore, Abs to these regions could block the formation of the NS1 hexamer and/or inhibit NS1 binding to cells, thus potentially inhibiting the pathogenic effects of NS1. In fact, we previously found that passive transfer of polyclonal sera from NS1-vaccinated mice as well as a mAb (1H7.4) directed to Region 1 (54) were able to completely protect mice against lethal challenge with DENV2 plus DENV2 NS1 (22). Therefore, immunization with recombinant NS1 may generate Abs to regions that are not usually exposed in natural infection and could provide additional protection against a lethal DENV infection. Furthermore, DENV2 NS1 vaccination elicited serotype-specific and cross-reactive responses that could potentially protect against all three DENV serotypes. This is consistent with previous data, where we observed partial protection against lethal DENV2 infection after immunization with DENV1 NS1, DENV3 NS1, or DENV4 NS1 protein (22).

When comparing the longitudinal responses to the NS1 peptide array in DENV-infected human populations, we found that overall, responses were higher in the convalescent phase than the acute phase. In primary infections, the magnitude of responses at the 12-mo timepoint did not wane, and we observed an increase in the response breadth, i.e., the number of targeted Ags. However, in secondary infections, we found that only responses in the DHF group retained magnitude and increased breadth. Due to the limited number of human samples tested in this study, additional studies are required to validate these findings.

The peptide microarrays used in this study allowed us to obtain high-resolution epitope mapping of vaccine- and natural infection–induced Ab responses. We have previously shown that such arrays can be used to profile vaccine-induced responses of mice vaccinated with influenza vaccines (33, 34). We further found that immunodominant responses detected by our peptide arrays were in surface-exposed, functionally important regions of the NS1 protein. This is in agreement with our previous findings in an influenza vaccination study in mice, which reported that the immunodominant responses to the HA protein were all surface-exposed on the HA trimer structure (34). In this study, by using overlapping 20-mer peptides with 15-aa overlap, we identified specific regions in DENV NS1 that are epitope hotspots. Coupled with a rigorous statistical approach that identified immunodominant regions in each of the DENV serotypes independently, we were able to identify a small number of regions to which NS1 vaccines and natural DENV infection generated Ab responses. These findings further highlight the feasibility of using these peptide microarrays for Ab profiling in studies of dengue vaccines and natural infections.

One limitation of our approach is the use of linear peptides that only capture part of the Ab repertoire; therefore, we may not have detected conformational or discontinuous epitopes. Nonetheless, we found that all of the identified peptides mapped to exposed regions of the NS1 protein (48, 50), some of which have been previously identified as Ab binding sites and regions of interest (23, 48, 53). There are several additional limitations of our current study. Because our peptide arrays were initially designed to map human responses from Nicaraguan patients, where there is very little circulation of DENV4, we only analyzed responses to three out of four globally circulating DENV serotypes. The number of DSS patients in this study was very small, and additional studies are required on a larger set of patients to address potential differences in peptide reactivity according to disease severity. Finally, we only mapped the responses of two SCpG NS1–vaccinated mice and thus could not statistically evaluate the responses to vaccination with this adjuvant combination.

Despite these limitations, we were able to identify a small number of immunodominant regions targeted following vaccination and natural infection, which were in functionally important locations on the NS1 protein both in its dimeric and hexameric forms. Combined with our previous data showing that passive transfer of polyclonal sera from NS1-immunized mice protected mice against DENV NS1-mediated lethal disease (22), we suggest that Ab responses to the regions identified in this study may play a role in protection and should be further studied in both animal models and humans.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dustin Glasner for invaluable assistance with figure preparation, Susana Orozco and Simona Zompi for initial work on NS1 vaccination and for scientific discussions, Poornima Parameswaran for help with selection of strains and generation of consensus sequences, and Magelda Montoya for managing and shipping the samples. We thank Beth-Ann Coller for helpful scientific advice and donation of recombinant NS1 protein. We are grateful to past and present members of the study team based at the Hospital Infantil Manuel de Jesús Rivera, the Centro de Salud Sócrates Flores Vivas, the National Virology Laboratory in the Centro Nacional de Diagnóstico y Referencia of the Nicaraguan Ministry of Health, and the Sustainable Sciences Institute in Managua, Nicaragua, for dedication and high-quality work, as well as to the study participants and families.

Footnotes

  • This work was supported by Grants U54 AI65359 (E.H.), K25 AI087397 (T.H.) and U19 AI109761 Center for Research in Diagnostics and Discovery (E.H., Program Director I. Lipkin) from the National Institute of Allergy and Infectious Diseases.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    alum
    aluminum and magnesium hydroxide
    DENV
    dengue virus
    DF
    dengue fever
    DHF
    dengue hemorrhagic fever
    DSS
    dengue shock syndrome
    E
    envelope
    HA
    hemagglutinin A
    MA
    monophosphoryl lipid A + AddaVax
    MFI
    mean fluorescence intensity
    NS1
    nonstructural protein 1
    SAS
    Sigma adjuvant system
    SCpG
    Sigma adjuvant system+CpG DNA.

  • Received January 6, 2017.
  • Accepted March 6, 2017.

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