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.     

Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed in Escherichia coli

Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni-NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.     

登革病毒四型联合重组包膜蛋白III区的表达和免疫原性鉴定

登革热在全球范围内广泛流行,但是目前为止却仍然没有疫苗上市,疫苗的开发迫在眉睫。抗体依赖增强感染效应是登革病毒疫苗开发中遇到的一个瓶颈问题。研究表明登革病毒的包膜蛋白III区能够介导中和抗体产生,且诱导产生较少的交叉抗体或无交叉抗体,能够大大减弱抗体依赖增强感染效应,因而是登革热重组蛋白疫苗的首选靶标。通过酵母密码子优化后合成同时包含4种血清型登革病毒包膜蛋白III区的四价联合DV EDIII蛋白序列,随后构建酵母表达质粒,并获得酵母表达菌株,经诱导后四联DV EDIII蛋白获得高效表达。通过Western blot、ELISA检测及蛋白质免疫原性鉴定,结果表明登革病毒四联DV EDIII蛋白表达质粒构建成功,重组蛋白在毕赤酵母获得高效表达,免疫小鼠后能够介导产生较高水平的血清效价。这表明已获得了能引起有效免疫反应的四型登革病毒EDIII蛋白,为登革病毒疫苗的研究提供了良好的基础。     

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.     

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.     

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.     

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.     

Characterization of a Novel Dengue Serotype 4 Virus-Specific Neutralizing Epitope on the Envelope Protein Domain III

The dengue virus (DENV) envelope protein domain III (ED3) has been suggested to contain receptor recognition sites and the critical neutralizing epitopes. Up to date, relatively little work has been done on fine mapping of neutralizing epitopes on ED3 for DENV4. In this study, a novel mouse type-specific neutralizing antibody 1G6 against DENV4 was obtained with both prophylactic and therapeutic effects. The epitope was mapped to residues 387–390 of DENV4 envelope protein. Furthermore, site-directed mutagenesis assay identified two critical residues (T388 and H390). The epitope is variable among different DENV serotypes but is highly conserved among four DENV4 genotypes. Affinity measurement showed that naturally occurring variations in ED3 outside the epitope region did not alter the binding of mAb 1G6. These findings expand our understanding of the interactions between neutralizing antibodies and the DENV4 and may be valuable for rational design of DENV vaccines and antiviral drugs.     

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.     

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.     

Induction of Neutralizing Antibodies against Four Serotypes of Dengue Viruses by MixBiEDIII,a Tetravalent Dengue Vaccine

The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1–2 and type 3–4) of DENV connected by a Gly-Ser linker ((Gly₄Ser)₃) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.     

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).     

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.     

C5a receptor-targeting ligand-mediated delivery of dengue virus antigen to M cells evokes antigen-specific systemic and mucosal immune responses in oral immunization

Oral mucosal immunization is a feasible and economic vaccination strategy. In order to achieve a successful oral mucosal vaccination, antigen delivery to gut immune inductive site and avoidance of oral tolerance induction should be secured. One promising approach is exploring the specific molecules expressed on the apical surfaces of M cells that have potential for antigen uptake and immune stimulation. We previously identified complement 5a receptor (C5aR) expression on human M-like cells and mouse M cells and confirmed its non-redundant role as a target receptor for antigen delivery to M cells using a model antigen. Here, we applied the OmpH ligand, which is capable of targeting the ligand-conjugated antigen to M cells to induce specific mucosal and systemic immunities against the EDIII of dengue virus (DENV). Oral immunization with the EDIII–OmpH efficiently targeted the EDIII to M cells and induced EDIII-specific immune responses comparable to those induced by co-administration of EDIII with cholera toxin (CT). Also, the enhanced responses by OmpH were characterized as Th2-skewed responses. Moreover, oral immunization using EDIII–OmpH did not induce systemic tolerance against EDIII. Collectively, we suggest that OmpH-mediated targeting of antigens to M cells could be used for an efficient oral vaccination against DENV infection.     

Defining levels of dengue virus serotype-specific neutralizing antibodies induced by a live attenuated tetravalent dengue vaccine (TAK-003)

The four dengue virus serotypes (DENV1-4) infect several hundred million people each year living in tropical and sub-tropical regions. Clinical development of DENV vaccines is difficult because immunity to a single serotype increases risk of severe disease during a second infection with a new serotype. Leading vaccines are based on tetravalent formulations to induce simultaneous and balanced protective immunity to all 4 serotypes. TAK-003 is a tetravalent live attenuated dengue vaccine candidate developed by Takeda Vaccines Inc, which is currently being evaluated in phase 3 efficacy trials. Here, we use antibody depletion methods and chimeric, epitope transplant DENVs to characterize the specificity of neutralizing antibodies in dengue-naïve adults and non-human primates immunized with TAK-003. Our results demonstrate that TAK-003 induced high levels of DENV2 neutralizing antibodies that recognized unique (type-specific) epitopes on DENV2. In contrast, most vaccinated subjects developed lower levels of DENV1, DENV3 and DENV4 neutralizing antibodies that mainly targeted epitopes that were conserved (cross-reactive) between serotypes.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02425098","term_id":"NCT02425098"}}NCT02425098.     

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.     

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.     

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.     

Dengue virus infection-enhancing and neutralizing antibody balance in children of the Philippines and Indonesia

Dengue fever and dengue hemorrhagic fever are important diseases worldwide. Although antibody-dependent enhancement of infection has been proposed as a mechanism for increased disease severity, enhancing antibodies in endemic people have not been thoroughly investigated. Recently, we established a serological assay system to measure the balance of enhancing and neutralizing activities, which provides useful information for estimating in vivo antibody status. We measured the balance of these activities against four dengue virus (DENV) types in endemic populations, and analyzed the proportion of sera containing enhancing and neutralizing antibodies. Predominantly healthy Filipino children were used for analysis, although a population of Indonesian children was also investigated. In the Filipino population, the highest proportion of neutralizing activities was shown against DENV2, followed by DENV1. A greater proportion of sera exhibited enhancing rather than neutralizing antibodies against other virus types. Neutralizing activities were complement-dependent, while enhancing activities were complement-independent. The Indonesian population showed a similar dengue antibody status. Our results indicate that a relatively high proportion of endemic children possessed complement-independent enhancing antibodies against some DENV types.     

A conserved set of mutations for stabilizing soluble envelope protein dimers from dengue and Zika viruses to advance the development of subunit vaccines

Dengue viruses (DENV serotypes 1–4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.