Infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels

Dengue viruses are distributed widely in the tropical and subtropical areas of the world and cause dengue fever and its severer form, dengue hemorrhagic fever. While neutralizing antibodies are considered to play a major role in protection from these diseases, antibody-dependent enhancement (ADE) of infection is an important mechanism involved in disease severity, in addition to the involvement of T lymphocytes. Here, we analyzed relationships between neutralizing and enhancing activities at a clonal level using models of dengue type 2 virus (DENV2) and dengue type 4 virus (DENV4). Totals of 33 monoclonal antibodies (MAbs) against DENV2 and 43 against DENV4 were generated, all directed to the envelope protein. In these MAbs, enhancing activities were shown at subneutralizing doses under normal ADE assay conditions where test samples were heat inactivated. However, the inclusion of commercial rabbit complement or fresh sera from healthy humans in the ADE assay system abolished the enhancing activities of all these MAbs. The reductive effect of fresh sera on enhancing activities was significantly reduced by their heat inactivation or the use of C1q- or C3-depleted sera. In some fresh sera, enhancing activities were shown within a range of 20 to 80% of normal complement levels in a dose-dependent fashion. These results indicate that a single antibody species possesses two distinct activities (neutralizing/enhancing), which are controlled by the level of complement, suggesting the involvement of complement in dengue disease severity. Fresh human sera also tended to reduce enhancing activities more effectively in homologous than heterologous combinations of viruses (DENV2/DENV4) and MAbs (against DENV2/DENV4).     

Comparison of infection-neutralizing and -enhancing antibody balance induced by two distinct genotype strains of dengue virus type 1 or 3 DNA vaccines in mice

Dengue viruses have spread throughout tropical and subtropical countries, and vaccine development is urgently needed. However, one concern is that induction of insufficient levels of neutralizing antibodies in vaccines may increase disease severity because of a hypothetical mechanism termed antibody-dependent enhancement of infection. This study used two distinct genotype strains of dengue virus types 1 and 3 (DENV1 and DENV3, respectively) to compare antibody responses in a mouse-DNA vaccine model. As expected, a conventional neutralization test using Vero cells showed higher antibody titers in homologous rather than heterologous combinations of genotype strains used for mouse immunization and the neutralization test, for each of DENV1 and DENV3. However, our assay system using K562 cells to measure the balance of neutralizing and enhancing antibodies indicated that Vero cell-neutralizing antibody titers did not always correlate with enhancing activities observed at subneutralizing doses. Rather, induction of enhancing activities depended on the genotype strain used for mouse immunization. The genotype/strain difference also affected IgG subclass profiles and potentially the composition of antibody species induced in mice. This study suggests that enhancing activities of dengue virus-induced neutralizing antibodies may vary according to the genotype and has implications for vaccine antigen development.     

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection‐enhancing antibody response

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection‐enhancing activities at sub‐neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM‐E) gene‐based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM‐E gene backbone showed high neutralizing antibody titers with less DENV infection‐enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.     

A Mouse Monoclonal Antibody against Dengue Virus Type 1 Mochizuki Strain Targeting Envelope Protein Domain II and Displaying Strongly Neutralizing but Not Enhancing Activity

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans.     

Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose–response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.     

Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.     

An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2

Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.     

Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells

Background

Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo.

Methods and Findings

We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT₅₀≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT₅₀≥10), but not when determined by using FcγR-expressing cells.

Conclusion

Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.     

Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.     

High-Avidity and Potently Neutralizing Cross-Reactive Human Monoclonal Antibodies Derived from Secondary Dengue Virus Infection

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.     

The Synergistic Effect of Combined Immunization with a DNA Vaccine and Chimeric Yellow Fever/Dengue Virus Leads to Strong Protection against Dengue

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.     

The Type-Specific Neutralizing Antibody Response Elicited by a Dengue Vaccine Candidate Is Focused on Two Amino Acids of the Envelope Protein

Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination.     

Dengue Viruses Are Enhanced by Distinct Populations of Serotype Cross-Reactive Antibodies in Human Immune Sera

Dengue viruses (DENV) are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE) is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE) protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to the development of safe, non-enhancing vaccines against dengue.     

Seroepidemiologic study on convalescent sera from dengue fever patients in Jinghong,Yunnan

After dengue virus (DENV) infection, antibody-dependent enhancement (ADE) is easy to occur when the neutralizing antibody (NAb) gradually decreases to a sub-neutralizing concentration. In this cohort surveillance, we utilized sera samples collected from dengue fever patients at different convalescent phases in Jinghong City, to investigate the dynamic change rule of DENV-specific antibodies, and to analyze the risk of ADE caused by secondary infection with heterologous serotypes DENVs. For baseline serosurvey, 191 four-year and 99 six-year sera samples during convalescence were collected in 2017 and 2019, respectively. The positive rate of DENV-specific immunoglobulin G was 98.4% in 2017, which significantly decreased to 82.8% in 2019. The geometric mean titer (GMT) of NAb decreased from 1:155.35 to 1:46.66. Among 290 overall samples, 73 paired consecutive samples were used for follow-up serosurvey. In four-year sera, the GMTs of NAb against DENV-3 and cross-reactive antibodies against DENV-1, DENV-2 and DENV-4 were 1:167.70, 1:13.80, 1:18.54 and 1:45.26, respectively, which decreased to 1:53.18, 1:10.30, 1:14.60 and 1:8.17 in six-year sera. In age-stratified analysis, due to the increasing number of ADE positive samples from 2017 to 2019 in 31–40 and 51–60 years groups, the risk of ADE in DENV-4 infection was positively associated with the extension of convalescent phase, and the odd ratio was higher than other groups. With the recovery period lengthened, the risk of secondary infection with DENV-1 and DENV-2 was reduced. Our results offer essential experimental data for risk prediction of severe dengue in hyper-endemic dengue areas, and provide crucial scientific insight for the development of effective dengue vaccines.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

Recombinant dengue type 2 viruses with altered e protein domain III epitopes are efficiently neutralized by human immune sera

Humans develop polyclonal, serotype-specific neutralizing antibody responses after dengue virus (DENV) infection. Many mouse antibodies that neutralize DENV bind to the lateral ridge or A strand epitopes on domain III of the viral envelope (EDIII) protein. It has been assumed that these epitopes are also the main target of human neutralizing antibodies. Using recombinant dengue serotype 2 viruses with altered EDIII epitopes, we demonstrate that EDIII epitopes are not the main target of human neutralizing antibody.     

Development,Characterization and Application of Monoclonal Antibodies against Brazilian Dengue Virus Isolates

Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV) detection through the production and characterization of 22 monoclonal antibodies (mAbs) against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus (YFV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV). None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV). Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA) for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.     

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.     

Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay

Background

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.

Methodology/Principal Findings

We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer.

Conclusions/Significance

Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.