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The recent outbreaks of Zika virus (ZIKV) in flavivirus-endemic regions highlight the need for sensitive and specific serological tests. Previously we and others reported key fusion loop (FL) residues and/or BC loop (BCL) residues on dengue virus (DENV) envelope protein recognized by flavivirus cross-reactive human monoclonal antibodies and polyclonal sera. To improve ZIKV serodiagnosis, we employed wild type (WT) and FL or FL/BCL mutant virus-like particles (VLP) of ZIKV, DENV1 and West Nile virus (WNV) in enzyme linked immunosorbent assays (ELISA), and tested convalescent-phase serum or plasma samples from reverse-transcription PCR-confirmed cases with different ZIKV, DENV and WNV infections. For IgG ELISA, ZIKV WT-VLP had a sensitivity of 100% and specificity of 52.9%, which was improved to 83.3% by FL/BCL mutant VLP and 92.2% by the ratio of relative optical density of mutant to WT VLP. Similarly, DENV1 and WNV WT-VLP had a sensitivity/specificity of 100%/70.0% and 100%/56.3%, respectively; the specificity was improved to 93.3% and 83.0% by FL mutant VLP. For IgM ELISA, ZIKV, DENV1 and WNV WT-VLP had a specificity of 96.4%, 92.3% and 91.4%, respectively, for primary infection; the specificity was improved to 93.7–99.3% by FL or FL/BCL mutant VLP. An algorithm based on a combination of mutant and WT-VLP IgG ELISA is proposed to discriminate primary ZIKV, DENV and WNV infections as well as secondary DENV and ZIKV infection with previous DENV infections; this could be a powerful tool to better understand the seroprevalence and pathogenesis of ZIKV in regions where multiple flaviviruses co-circulate.

Aim: This study assessed how prime-boost strategies influence the immunogenicity of a cross-reactivity reduced dengue virus 2 vaccine (DENV-2 RD). Materials & methods: Mice were immunized with DENV-2 RD vaccines in a heterologous DNA and virus-like particle (VLP) prime-boost. Elicited antibodies were analyzed for neutralization and protective efficacy against four DENV serotypes. Results: DENV-2 RD DNA-VLP had induced higher and broader levels of total IgG and neutralizing antibodies with statistically significant IgG titers against DENV-2 and -3. Only pups of DENV-2 RD DNA-VLP immunized female mice were fully protected against homotypic DENV challenge and partially protected (60% survival rate) against heterotypic DENV-3 lethal challenge. Conclusion: DENV-2 RD vaccine requires a multivalent format to effectively elicit a balanced and protective immunity across all four DENV serotypes.

The unexpectedly low vaccine efficacy of Dengvaxia®, developed by Sanofi Pasteur, and a higher risk of severe diseases after vaccination among dengue-naive children or children younger than 6 years old, have cast skepticism about the safety of dengue vaccination resulting in the suspension of school-based immunization programs in the Philippines. The absence of immune correlates of protection from dengue virus (DENV) infection hampers the development of other potential DENV vaccines. While tetravalent live-attenuated tetravalent vaccines (LATVs), which mimic natural infection by inducing both cellular and humoral immune responses, are still currently favored, developing a vaccine that provides a balanced immunity to all four DENV serotypes remains a challenge. With the recently advanced understanding of virion structure and B cell immune responses from naturally infected DENV patients, two points of view in developing a next-generation dengue vaccine emerged: one is to induce potent, type-specific neutralizing antibodies (NtAbs) recognizing quaternary structure-dependent epitopes by having four components of vaccine strains replicate equivalently; the other is to induce protective and broadly NtAbs against the four serotypes of DENV with a universal vaccine. This article reviews the studies related to these issues and the current knowledge gap that needs to be filled in.

Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. Here, we describe a robust protocol for producing DENV VLPs from transiently-transformed or stable COS-1 cells and further provide an easily adaptable antigen purification method by sucrose gradient centrifugation.

The four serotypes of dengue virus (DENV) cause the most important rapidly emerging arthropod-borne disease globally. The humoral immune response to DENV infection is predominantly directed against the immunodominant cross-reactive weakly neutralizing epitopes located in the highly conserved fusion peptide of ectodomain II of envelope (E) protein (EDIIFP). Antibodies recognizing EDIIFP have been shown to associate with immune enhancement in an ex vivo animal model. In this study, we explored how prime-boost strategies influence the immunogenicity of a cross-reactivity reduced (CRR) DENV-2 vaccine with substitutions in EDIIFP residues (DENV-2 RD) and found that mice in various DENV-2 RD prime-boost immunizations had significantly reduced levels of EDIIFP antibodies. In addition, heterologous DENV-2 RD DNA-VLP prime-boost immunization induced higher and broader levels of total IgG and neutralizing antibodies (NtAbs) although IgG titers to DENV-2 and 3 were statistically significant. Consistently, mice from DENV-2 RD DNA-VLP prime-boost immunization were fully protected from homologous DENV-2 lethal challenge and partially protected (60% survival rate) from heterologous lethal DENV-3 challenge. Our results conclude that the CRR DENV-2 RD vaccine requires a multivalent format to effectively elicit a balanced and protective immunity across all four DENV serotypes.ImportanceThe low vaccine efficacy of the live-attenuated chimeric yellow fever virus-DENV tetravalent dengue vaccine (CYD-TDV) is unexpected and there is an urgent need to develop a next generation of dengue vaccine. Antibodies against the fusion peptide in envelope protein (E) ectodomain II (EDIIFP) can potentially induce a severe disease via antibody-dependent enhancement (ADE) of infection. This study evaluated different formats of an EDIIFP-modified DENV-2 vaccine (DENV-2 RD) in its capability of inducing a reduced EDIIFP antibodies, and sculpting the immune response towards an increased DENV complex-reactive neutralizing antibodies (CR NtAb). The results from this study confirmed the poor correlate of neutralizing assay with protection as suggested by the results of CYD-TDV clinical trials. There is a urgent need to develop a biological correlate with protection while evaluating the efficacy of the next generation dengue vaccine.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

Purifying selection during dengue viral infection has been suggested as the driving force of viral evolution and the higher complexity of the intra-host quasi-species is thought to offer an adaptive advantage for arboviruses as they cycle between arthropod and vertebrate hosts. However, very few studies have been performed to investigate the viral genetic changes within (intra-host) and between (inter-host) humans in a spatio-temporal scale. Viruses of different serotypes from various countries imported to Taiwan cause annual outbreaks. During 2001–2003, two consecutive outbreaks were caused by dengue virus serotype 2 (DENV-2) and resulted in a larger-scale epidemic with more severe dengue cases in the following year. Phylogenetic analyses showed that the viruses from both events were similar and related to the 2001 DENV-2 isolate from the Philippines. We comprehensively analyzed viral sequences from representative dengue patients and identified three consensus genetic variants, group Ia, Ib and II, with different spatio-temporal population dynamics. The phylodynamic analysis suggested group Ib variants, characterized by lower genetic diversity, transmission rate, and intra-host variant numbers, might play the role of maintenance variants. The residential locations among the patients infected by group Ib variants were in the outer rim of case clusters throughout the 2001–2003 period whereas group Ia and II variants were located in the centers of case clusters, suggesting that group Ib viruses might serve as “sheltered overwintering” variants in an undefined ecological niche. Further deep sequencing of the viral envelope (E) gene directly from individual patient serum samples confirmed the emergence of variants belonging to three quasi-species (group Ia, Ib, and II) and the ancestral role of the viral variants in the latter phase of the 2001 outbreak contributed to the later, larger-scale epidemic beginning in 2002. These findings enhanced our understanding of increasing epidemic severity over time in the same epidemic area. It also highlights the importance of combining phylodynamic and deep sequencing analysis as surveillance tools for detecting dynamic changes in viral variants, particularly searching for and monitoring any specific viral subpopulation. Such subpopulations might have selection advantages in both fitness and transmissibility leading to increased epidemic severity., Author summary Virus genomes can show changes due to transmission between hosts. In evolutionary scale, replacements with new viral lineages are usually associated with alternating clinical burdens or epidemic potentials, but few studies have integrated viral dynamics from intra-host viral variations to macro-level clade (a monophyletic group) replacements. In this study, we combined both levels of research to study the changes in viral genomes during two consecutive dengue outbreaks (2001–2003) in southern Taiwan. We identified a potential overwintering candidate virus group characterized by lower genetic diversity, transmission rate, number of intra-host variants and a more pervasive geographical distribution pattern. Deep sequencing data revealed the temporal dynamics of minor variants in different nucleotide positions, correlated with the phylogenetic analyses of consensus sequences derived from different patients. We thus propose that investigating the quasi-species complexity of viruses from intra-host and inter-host infections can provide a novel understanding of virus evolution. Such investigations could also be a useful method to monitor the dynamics of viral changes and be included in the virological surveillance.

SummaryDiagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to prevent potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both non-infectious virus-like particles (VLPs) and soluble non-structural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both VLP- and NS1-MAC-ELISAs were found to have similar sensitivity for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera. Group cross reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher P/N values than homologous wild-type VLPs. Therefore, FP-VLPs were used to develop the algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80% with no statistical significance. A novel approach to differentiate ZIKV from DENV infection serologically has been developed. The accuracy can reach up to 95% when combining both VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.

Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to mitigate potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both noninfectious wild-type (wt) virus-like particles (VLPs) and soluble nonstructural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both ZIKV-derived wt-VLP- and NS1-MAC-ELISAs were found to have similar sensitivities for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera, although lower cross-reactivity to DENV2/3-NS1 was observed. Furthermore, group cross-reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher positive-to-negative values than homologous wt-VLPs. Therefore, we used DENV-2/3 and ZIKV FP-VLPs to develop a novel, serological algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80%, with no statistical significance. The accuracy can reach up to 95% with the combination of FP-VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein we show for the first time that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our results revealed that a “epitope-resurfaced” mature-form dengue VLP has the potential to induce quaternary structure-recognizing broad CR NtAbs.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. The vaccine candidates under development require a tetravalent immunogen to induce a balanced immunity against all four serotypes of dengue virus. Herein we show that mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher and broader neutralization antibodies (NtAbs) against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. The cryo-electron microscopy reconstruction showed that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Most importantly, maternally transferred antibodies derived from mD2VLP-vaccinated female mice protected suckling mice from lethal challenge by all four serotypes of DENV. Our results support the fact that a universal dengue vaccine that protects against all four serotypes of dengue viruses can be achieved by using an immunogen such as mD2VLP.

Background: Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to prevent potential severe health outcomes. Methods: A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both non-infectious virus-like particles (VLPs) and soluble non-structural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Findings: Both VLP- and NS1-MAC-ELISAs were found to have similar sensitivity for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera. Group cross reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher P/N values than homologous wild-type VLPs. Therefore, FP-VLPs were used to develop the algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80% with no statistical significance. Interpretation: A novel approach to differentiate ZIKV from DENV infection serologically has been developed. The accuracy can reach up to 95% when combining both VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist. Funding Statement: Portions of this publication was made possible through support provided by the CDC intramural research fund and the Office of Infectious Disease, Bureau for Global Health, U.S. Agency for International Development, under the terms of an Interagency Agreement with CDC. The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of the CDC and U.S. Agency for International Development. Declaration of Interests: We declare no competing interests. Ethics Approval Statement: This study was conducted and reported in accordance with the Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. Informed consent documents for all eligible participants were waived based on the protocol number 6874. An institutional review board (IRB) waiver to use this serum panel for research purposes was approved by the CDC-human studies review board.

Flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are among the most prevalent human disease-causing arboviruses world-wide. As they continue to expand their geographic range, multivalent flavivirus vaccines may become an important public health tool. Here we describe the immune kinetics of WNV DNA vaccination and the identification of a CD4 epitope that increases heterologous flavivirus vaccine immunogenicity. Lethal WNV challenge two days post-vaccination resulted in 90% protection with complete protection by four days, and was temporally associated with a rapid influx of activated CD4 T cells. CD4 T cells from WNV vaccinated mice could be stimulated from epitopic regions in the envelope protein transmembrane domain. Incorporation of this WNV epitope into DENV-2 DNA and virus-like particle vaccines significantly increased neutralizing antibody titers. Incorporating such potent epitopes into multivalent flavivirus vaccines could improve their immunogenicity and may help alleviate concerns of imbalanced immunity in multivalent vaccine approaches.

The role of the α-helical domain (MH) of dengue virus (DENV) precursor membrane protein in replication was investigated by site-directed mutagenesis. Proline substitutions of three residues (120, 123 and 127) at the C-terminus, but not those at the N-terminus of MH domain, reduced the virus-like particles of DENV1, DENV2 and DENV4 detected in supernatants. In a DENV2 replicon trans-packaging system, these three mutations suppressed particles detected; two of them (I123P and V127P) also affected viral entry. In the context of DENV2 genome-length RNA, all three mutations reduced virion assembly and virus spreading in cell culture. Analysis of revertants showed that mutation A120P could partially support viral infection cycle; in contrast, mutations I123P and V127P were lethal, and adaptations of I123P → I123L and V127P → V127L were required to restore the viral infection cycle. These findings demonstrate that the C-terminus of the MH domain is involved in both assembly and entry of DENV.

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

Since the introduction of West Nile virus into the United States in 1999, there has been a greater awareness of arboviruses, consequently, diagnostic testing for West Nile virus and other arboviruses has increased both in U.S. and international public health laboratories. The Centers for Disease Control and Prevention/Division of Vector-Borne Infectious Diseases/Arbovirus Diagnostic and Reference Laboratory produces and provides the serodiagnostic reagents which are not available commercially. Reagents needed to conduct the enzyme-linked immunoassay (ELISA) include a virus-specific non-infectious antigen. Antigens for Japanese encephalitis and the four dengue virus serotypes have been developed from COS-1 transformed cells that secrete non-infectious, virus-like particles into the cell culture supernatant. Four methods for concentrating the supernatant are discussed here. The methods are ultracentrifugation, polyethylene glycol precipitation, and two ultrafiltration methods: the Stirred Cell (Millipore Corporation, Billerica, MA) and the Pellicon 2 (Millipore Corporation, Billerica, MA). Ultracentrifugation and the Pellicon 2 ultrafiltration system produced antigen at a sufficient concentration for use in the ELISA. Large volumes were concentrated in a shorter time in the Pellicon 2 ultrafiltration system. An additional filtration step was necessary to produce antigen of sufficient concentration for use in the microsphere-based immunoassay, which requires antigen concentrated an additional 10 times.

A multiplex real-time reverse transcriptase PCR has been developed for the rapid detection and identification of eight medically important flaviviruses from laboratory-reared, virus-infected mosquito pools. The method used involves the gene-specific amplification of yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively) by use of the flavivirus consensus amplimers located at the RNA-dependent RNA polymerase domain of nonstructural protein 5. Virus-specific amplicons were detected by four newly characterized TaqMan fluorogenic probes (probes specific for YFV, JEV, WNV, and SLEV) and four previously published probes specific for DENV-1 to -4 (L. J. Chien, T. L. Liao, P. Y. Shu, J. H. Huang, D. J. Gubler, and G. J. Chang, J. Clin. Microbiol. 44:1295-1304, 2006). This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected.

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.

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 − specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain.

The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.

Lately, the magnitude of cumulative diseases burden caused by flaviviruses, such as dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus and yellow fever virus, has reached an unprecedented level with the sizes of human and animal populations at risk increasing sharply. These diseases present highly complex medical, economic and ecologic problems, some effecting primarily human and others affecting human, livestock and wildlife. The large body of recent publications on the development of vaccines taking advantage of new generations of bio-engineering techniques clearly reflects the profound interests and deep sense of urgency in the scientific and medical communities in combating those diseases. This review reveals a collection of remarkable progresses thus far made in flaviviral vaccine research not only employing a diverse range of new strategies but also re-tooling old techniques to improve the existing vaccines. The efficacy and safety of some of the new vaccine candidates have been evaluated and proven in human clinical trials. Besides the technical advancement in vaccine development, in this review, the importance of somewhat neglected and yet critical subjects, such as adequacy of animal model, vaccine safety, vaccine formulation and delivery, complication in serodiagostics and economic factor, was examined in-depth.

The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK 2 cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication.

Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK2cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti.We used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinantOf the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Léogâne and towards the ocean.Serological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously.Différentier l'exposition au virus chikungunya, récemment introduit, de l'exposition au virus de la dengue et à d'autres pathogènes endémiques en Haïti.Nous avons procédé à une analyse multiplex à l'aide de billes pour détecter les réponses de l'immunoglobuline G (IgG) à un antigène recombinant du virus chikungunya, à deux particules pseudo-virales de la dengue et à trois antigènes recombinants deDans la cohorte longitudinale, aucun des 153 échantillons prélevés entre 2011 et 2013 n'affichait de réponse positive de l'IgG à l'antigène du virus chikungunya, mais 78,7% (48/61) de ceux prélevés en 2014 étaient en revanche positifs. Dans l'échantillon transversal, des réponses positives ont été relevées chez 96 enfants (75,6%), avec une prévalence similaire dans tous les groupes d'âge. Sur ce même échantillon, des réponses à l'antigène du paludisme n'ont été détectées que chez huit enfants (6,3%) mais la prévalence globale des réponses de l'IgG aux antigènes du virus de la dengue était de 60,6% (77/127) et augmentait progressivement avec l'âge. L'analyse géographique a révélé que la prévalence des réponses de l'IgG au virus du chikungunya et à l'une des particules pseudo-virales de la dengue diminuait à mesure que le site d'échantillonnage s'éloignait de la ville de Léogâne en direction de l'océan.Les preuves sérologiques indiquent que la propagation du virus du chikungunya en Haïti a été rapide et intense. L'analyse multiplex à l'aide de billes s'est avérée être une plate-forme sérologique appropriée pour contrôler simultanément la séroprévalence de plusieurs pathogènes.Diferenciar la exposición al nuevo virus chikungunya de la exposición al virus endémico del dengue y a otros patógenos en Haití.Se utilizó un ensayo multiplex de microesferas para detectar las respuestas de la inmunoglobulina G (IgG) ante un antígeno recombinante del virus chikungunya, dos partículas similares al virus del dengue y tres antígenos recombinantes deDe las muestras de la cohorte longitudinal, ninguna de las 153 muestras recogidas entre 2011 y 2013 dio un resultado positivo de respuestas de la IgG al antígeno del virus chikungunya, pero sí que lo dieron el 78,7% (48/61) de las muestras recogidas en 2014. En la muestra transversal, se detectaron dichas respuestas en 96 (75,6%) de los niños y se mostró una prevalencia similar en todos los grupos de edades. En la misma muestra, únicamente se detectaron respuestas al antígeno de la malaria en ocho niños (6,3%), aunque la prevalencia de las respuestas de la IgG a los antígenos del virus del dengue fue del 60,6% (77/127) en general y aumentaba de forma estable con la edad. Los análisis territoriales indicaron que la prevalencia de las respuestas de la IgG al virus chikungunya y a una de las partículas similares al virus del dengue se redujo conforme el lugar de la muestra se alejaba de la ciudad de Léogâne y se acercaba al océano.La prueba serológica indica que se ha producido una diseminación rápida e intensa del virus chikungunya en Haití. Parece que el ensayo multiplex de microesferas es una plataforma serológica adecuada para supervisar la seroprevalencia de varios patógenos al mismo tiempo.التمييز بين معدلات التعرض لفيروس شِيكُونْغُونْيا الذي تم الكشف عنه مؤخرًا ومعدلات التعرض لفيروس الضنك المتوطّن والعوامل الأخرى المسببة للأمراض في دولة هايتي.استخدمنا المقايسة المتعددة للحبيبات لاكتشاف أية استجابات من جانب الغلوبولين المناعي G (IgG) إلى مستضد مؤتلف لفيروس شِيكُونْغُونْيا، وجسيمين مشابهين لفيروس الضنك، وثلاثة مستضدات مؤتلفة لطُفيل المتصورة المنجلية. لقد تم جمع معظم عينات الدم (البالغ عددها 217) التي خضعت للاستقصاء من كل 61 طفلًا على مدى فترة طويلة تمتد بين عامي 2011 و2014، ولكن في عام 2014، تم جمع 127 عينة أخرى لقطاعات متعددة من الأطفال.لم تُظهر أي من العينات المُجمّعة فيما بين عامي 2011 و2013 والبالغ عددها 153 نتائج إيجابية عن وجود استجابات من جانب IgG إلى المستضد المؤتلف لفيروس شِيكُونْغُونْيا باستثناء ما يعادل 78.7‏% (‏48/61) من العينات التي تم جمعها في عام 2014 وذلك من أصل العينات المستخرجة من مجموعة الأطفال على مدى فترة طويلة. في العينة المستخرجة من عدة قطاعات، تم اكتشاف تلك الاستجابات في 96 طفلاً (فيما يعادل 75.6%) وكان معدل انتشارها متشابهًا في جميع الفئات العمرية. وفي نفس تلك العينة، لم يتم اكتشاف أية استجابات لمستضد الملاريا سوى في ثمانية أطفال (فيما يعادل 6.3‏%) بينما بلغ إجمالي معدل انتشار الاستجابات من جانب IgG إلى مستضدات فيروس الضنك 60.6‏% (‏77/127) واستمر في الازدياد بثبات مع ازدياد العمر. وأوضح التحليل المكاني أن معدل انتشار الاستجابات من جانب lgG إلى فيروس شِيكُونْغُونْيا وأحد الجسيمين المشابهين لفيروس الضنك قد ازداد بتغيير موقع أخذ العينات من مدينة يوغان انتقالاً باتجاه المحيط.تشير الأدلة المصلية إلى انتشار فيروس شِيكُونْغُونْيا بسرعة وبشدّة في دولة هايتي. كما تُظهر المقايسة المتعددة للحبيبات أن بإمكانها أن تصبح نظامًا مصليًا مناسبًا للإشراف على معدل الانتشار المصلي لعدة عوامل مسببة للأمراض في نفس الوقت.旨在将海地地区接触新流入的基孔肯雅病毒与接触地方性登革病毒以及其他病原体进行区分。.我们采用多重微球试验以检测免疫球蛋白 G (IgG) 对重组基孔肯雅病毒抗原、两种类似登革病毒的微粒和三种重组在纵向群组样本中，2011 年到 2013 年间采集的 153 例样本中没有一例在 lgG 对基孔肯雅病毒抗原有反应中显示阳性，但在 2014 年采集的样本中 78.7% (48/61) 显示阳性。 在横断面样本中，我们发现其中 96 位儿童 (75.6%) 存在此类反应，并且在所有年龄段都有相似概率。 在相同样本中，仅在八位儿童 (6.3%) 中检测到对疟疾抗原有反应。但整体而言，IgG 对登革病毒抗原有反应的概率是 60.6% (77/127)，并且随着年龄增长而稳步增长。 空间分析显示，IgG 对基孔肯雅病毒和其中一种类似登革病毒的微粒有反应的概率随着取样地点从莱奥甘转移至海边而有所降低。.血清学证据表明海地地区的基孔肯亚病毒已经快速密集传播。 多重微球试验似乎是适当的血清学平台，可同时监控多种病原体血清阳性率。.Провести различия между подверженностью воздействию недавно интродуцированного вируса чикунгуньи и воздействию эндемического вируса лихорадки денге и других болезнетворных микроорганизмов в Гаити.С помощью мультиплексного анализа с использованием гранул авторы статьи выявили реакцию иммуноглобулина G (IgG) на антиген рекомбинантного вируса чикунгуньи, две вирусоподобные частицы денге и три антигена рекомбинантного паразита Plasmodium falciparum. Забор большинства (217) исследованных образцов крови совершался в течение нескольких лет у 61 ребенка между 2011 и 2014 годами, а 127 образцов были взяты у детей в рамках профильной пробы в 2014 году.Ни для одного из 153 образцов, отобранных между 2011 и 2013 годами у детей из когорты долгосрочного исследования для анализа на реакцию IgG на антиген вируса чикунгуньи, не был получен положительный результат; однако 78,7% (48/61) образцов, полученных у детей из этой же группы в 2014 году, дали положительный результат. В профильной выборке реакция была обнаружена у 96 (75,6%) детей и наблюдалась со схожей частотностью во всех возрастных группах. В той же выборке реакция на малярийный антиген была обнаружена только у восьми детей (6,3%), однако общая распространенность реакции IgG на антигены вируса денге составила 60,6% (77/127) и с возрастом стабильно увеличивалась. Анализ на распределение по местности показал, что распространенность реакции IgG на вирус чикунгуньи и вирусоподобные частицы денге уменьшалась по мере перемещения места отбора проб от города Леоган в сторону океана.Выявленные серологические реакции свидетельствуют о произошедшем скоротечном и усиленном распространении вируса чикунгуньи в Гаити. Мультиплексный анализ с использованием гранул служит подходящей базой для серодиагностики для одновременного отслеживания распространенности положительных серологических реакций на несколько болезнетворных микроорганизмов.

Background Dengue viruses (DENV) are the most important arboviruses of humans and cause significant disease. Infection with DENV elicits antibody responses to the envelope glycoprotein, predominantly against immunodominant, cross-reactive, weakly-neutralizing epitopes. These weakly-neutralizing antibodies are implicated in enhancing infection via Fcγ receptor bearing cells and can lead to increased viral loads that are associated with severe disease. Here we describe results from the development and testing of cross-reactivity reduced DENV-2 DNA vaccine candidates that contain substitutions in immunodominant B cell epitopes of the fusion peptide and domain III of the envelope protein. Results Cross-reactivity reduced and wild-type vaccine candidates were similarly immunogenic in outbred mice and elicited high levels of neutralizing antibody, however mice immunized with cross-reactivity reduced vaccines produced significantly reduced levels of immunodominant cross-reactive antibodies. Sera from mice immunized with wild-type, fusion peptide-, or domain III- substitution containing vaccines enhanced heterologous DENV infection in vitro, unlike sera from mice immunized with a vaccine containing a combination of both fusion peptide and domain III substitutions. Passive transfer of immune sera from mice immunized with fusion peptide and domain III substitutions also reduced the development of severe DENV disease in AG129 mice when compared to mice receiving wild type immune sera. Conclusions Reducing cross-reactivity in the envelope glycoprotein of DENV may be an approach to improve the quality of the anti-DENV immune response.

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., Author Summary Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level.

RNA oligonucleotide fingerprinting studies on a large number of virus isolates previously demonstrated considerable genetic variation in isolates of dengue (DEN)-2 serotype. We report the entire envelope (E) glycoprotein gene and deduced amino acid sequences of 16 DEN-2 viruses and the phylogenetic relationships of these, plus 17 additional published DEN E gene sequences. Comparison of DEN-2 E glycoprotein gene sequences revealed base substitutions scattered throughout the entire gene with as much as 22% sequence divergence. Aligned E glycoprotein amino acid sequences revealed the viruses differed by as much as 10%. There appeared to be constraints on the overall structure of the E protein to maintain biological function. Clusters of amino acid substitutions were present in the hydrophobic membrane anchor region at the carboxyl terminal end of the protein. Maximum parsimony analysis of the E gene sequences allowed construction of a phylogram indicating evolutionary relationships of the virus isolates within the DEN-2 serotype. Five genetic subtypes were identified. Phylogenetic relationships of the DEN-2 serotype and other flaviviruses based on E protein sequences reflected traditional antigenic and serologic classifications.

Dengue virus (DENV) is a serious mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation dengue hemorrhagic fever (DHF). Nearly half of the world's population is at risk of dengue disease and there are estimated to be millions of infections annually; a situation which will continue to worsen with increasing expansion of the mosquito vectors and epidemic DF/DHF. Currently there are no available licensed vaccines or antivirals for dengue, although significant effort has been directed toward the development of safe and efficacious dengue vaccines for over 30 years. Promising vaccine candidates are in development and testing phases, but a better understanding of immune responses to DENV infection and vaccination is needed. Humoral immune responses to DENV infection are complex and may exacerbate pathogenicity, yet are essential for immune protection. In this report, we develop DENV-2 envelope (E) protein epitope-specific antigens and measure immunoglobulin responses to three distinct epitopes in DENV-2 infected human serum samples. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary DENV-2 infected sera. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. This report begins to tease apart complex humoral immune responses to DENV infection and is thus important for improving our understanding of dengue disease and immunological correlates of protection, relevant to DENV vaccine development and testing.

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.