Characterization of dengue complex-reactive epitopes on dengue 3 virus envelope protein domain III

The disease dengue (DEN) is caused by four genetically and serologically related viruses termed DENV-1, -2, -3, and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3. The ED3 contains the DENV type-specific and DENV complex-reactive (epitopes shared by DENV 1-4) antigenic sites. In this study the epitopes recognized by four DENV complex-reactive monoclonal antibodies (MAbs) with neutralizing activity were mapped on the DENV-3 ED3 using a combination of physical and biological techniques. Amino acid residues L306, K308, G381, I387, and W389 were critical for all four MAbs, with residues V305, E309, V310, K325, D382, A384, K386, and R391 being critical for various subsets of the MAbs. A previous study by our group (Gromowski, G.D., Barrett, N.D., Barrett, A.D., 2008. Characterization of dengue complex-specific neutralizing epitopes on the envelope protein domain III of dengue 2 virus. J. Virol 82, 8828-8837) characterized the same panel of MAbs with DENV-2. The location of the DENV complex-reactive antigenic site on the DENV-2 and DENV-3 ED3s is similar; however, the critical residues for binding are not identical. Overall, this indicates that the DENV complex-reactive antigenic site on ED3 may be similar in location, but the surprising result is that DENV 2 and 3 exhibit unique sets of residues defining the energetics of interaction to the same panel of MAbs. These results imply that the amino acid sequences of DENV define a unique interaction network among these residues in spite of the fact that all flavivirus ED3s to date assume the same structural fold.     

Mutations of an antibody binding energy hot spot on domain III of the dengue 2 envelope glycoprotein exploited for neutralization escape

Previous crystallographic studies have identified a total of 11 DENV-2 envelope protein domain III (ED3) residues (K305, F306, K307, V308, V309, K310, I312, Q325, P364, K388, and N390) that interacted, through both side- and main-chain contacts, with the Fab of a dengue virus (DENV) subcomplex-specific neutralizing monoclonal antibody (MAb) 1A1D-2 (Lok et al., 2008). Here, we used DENV-2 recombinant ED3 mutants of the MAb 1A1D-2 structural epitope residues to determine the functional epitope of this MAb. The side-chains of residues K307, K310 and I312 were determined to be functionally critical for MAb binding, and thus constitute a hot spot of binding energy for MAb 1A1D-2 on the DENV-2 ED3. Overall, these findings demonstrate that only a subset of the amino acid residue side-chains within the structural epitope of MAb 1A1D-2 define a functional epitope on the DENV-2 ED3 that is essential for MAb binding and neutralization escape.     

Solution structure of the envelope protein domain III of dengue-4 virus

The disease dengue (DEN) is caused by four serologically related viruses termed DEN1, DEN2, DEN3 and DEN4. The structure of the ectodomain of the envelope protein has been determined previously for DEN2 and DEN3 viruses. Using NMR spectroscopic methods, we solved the solution structure of domain III (ED3), the receptor-binding domain, of the envelope protein of DEN4 virus, human strain 703-4. The structure shows that the nine amino acid changes in ED3 that separate the sylvatic and human DEN4 strains are surface exposed. Important structural differences between DEN4-rED3 and ED3 domains of DEN2, DEN3 and other flaviviruses are discussed.     

Role of BC loop residues in structure, function and antigenicity of the West Nile virus envelope protein receptor-binding domain III

Site-directed mutagenesis of residues in the BC loop (residues 329-333) of the envelope (E) protein domain III in a West Nile virus (WNV) infectious clone and in plasmids encoding recombinant WNV and dengue type 2 virus domain III proteins demonstrated a critical role for residues in this loop in the function and antigenicity of the E protein. This included a strict requirement for the tyrosine at residue 329 of WNV for virus viability and E domain III folding. The absence of an equivalent residue in this region of yellow fever group viruses and most tick-borne flavivirus suggests there is an evolutionary divergence in the molecular mechanisms of domain III folding employed by different flaviviruses.     

Antibodies targeting dengue virus envelope domain III are not required for serotype-specific protection or prevention of enhancement in vivo

The envelope (E) protein of dengue virus (DENV) is composed of three domains (EDI, EDII, EDIII) and is the main target of neutralizing antibodies. Many monoclonal antibodies that bind EDIII strongly neutralize DENV. However in vitro studies indicate that anti-EDIII antibodies contribute little to the neutralizing potency of human DENV-immune serum. In this study, we assess the role of anti-EDIII antibodies in mouse and human DENV-immune serum in neutralizing or enhancing DENV infection in mice. We demonstrate that EDIII-depleted human DENV-immune serum was protective against homologous DENV infection in vivo. Although EDIII-depleted DENV-immune mouse serum demonstrated decreased neutralization potency in vitro, reduced protection in some organs, and enhanced disease in vivo, administration of increased volumes of EDIII-depleted serum abrogated these effects. These data indicate that anti-EDIII antibodies contribute to protection and minimize enhancement when present, but can be replaced by neutralizing antibodies targeting other epitopes on the dengue virion.     

Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes

The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGΔ) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGΔ in Vero cells at 37 °C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope.     

Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.     

Dengue virus neutralization by human immune sera: Role of envelope protein domain III-reactive antibody

Dengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes 1 through 4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes, the long-term protective antibody response is restricted to the serotype responsible for infection. Cross reactive antibodies appear to enhance infection during a second infection with a different serotype. The goal of the present study was to characterize the binding specificity and functional properties of human DENV immune sera. The study focused on domain III of the viral envelope protein (EDIII), as this region has a well characterized epitope that is recognized by strongly neutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate that EDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Human antibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype cross reactive epitope on EDIII after secondary infection. However, EDIII binding antibodies constituted only a small fraction of the total antibody in immune sera binding to DENV. Studies with complete and EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies play a minor role in DENV neutralization. We propose that human antibodies directed to other epitopes on the virus are primarily responsible for DENV neutralization. Our results have implications for understanding protective immunity following natural DENV infection and for evaluating DENV vaccines.     

Differential expression of domain III neutralizing epitopes on the envelope proteins of West Nile virus strains

Neutralization of flaviviruses by antibody is primarily mediated via epitopes in the viral envelope (E) protein. Comparative studies using neutralizing monoclonal antibodies revealed differential expression of epitopes within the E protein domain III of ten naturally occurring West Nile virus strains representing major subtypes of genetic lineages 1 and 2. Residues that defined these subtype-specific determinants were identified by mutational studies and found to be surface exposed in the domain III structure. Mutations of residue 332 had the most significant effects on variation of domain III neutralizing epitopes among strains.     

West Nile virus is neutralized by HOCl-modified human serum albumin that binds to domain III of the viral envelope protein E

The outbreaks of West Nile virus (WNV), an emerging flavivirus recently implicated in outbreaks of fatal encephalitis, necessitate the development of effective anti-WNV drugs. In this study, it is demonstrated that human serum albumin is transformed into a WNV antiviral substance by hypochlorite (HOCl) modification. The HOCl-modified albumin efficiently neutralized WNV in vitro (EC50=300 nM) and showed binding to a recombinant protein, representing the domain III of the WNV external envelope E glycoprotein.     

我国登革2型病毒43株包膜E蛋白MBP-B165抗原性的研究

目的 通过对我国登革2型病毒株包膜E蛋白包括B区在内的第267－432氨基酸编码基因片段的表达,研究MBP－B165蛋白的抗原性。方法 首先采用PCR方法扩增了编码B165蛋白的基因片段,并将其插入到pMal－C2核载体进行融合表达。采用蛋白印迹和ELISA法对表达产物进行特异性鉴定。用表达的融合蛋白MBP－B165免疫大白兔,产通过ELISA法检测兔免疫血清中登革2型病毒特异的抗体。结果 表达的融合蛋白MBP－B165可与我国登革2型病毒株抗体特异结合,而且与登革1、3和4型病毒参考株的多克隆抗体均具有较高的反应性。用表达蛋白免疫大白兔可产生针对我国登革2型病毒株E蛋白的特异抗体,并且该抗体与基他3个型病毒参考株有交叉反应。结论 我国登革2型病毒43株E蛋白包括B区在内的第267－432氨基酸序列具有一定的抗原性,而且具有黄病毒亚组特异的反应性表位,即4个型登革病毒的结构保守性表位。     

Characterization of a peptide domain within the GB virus C envelope glycoprotein (E2) that inhibits HIV replication

GB virus C (GBV-C) infection is associated with prolonged survival in HIV-infected cohorts, and GBV-C E2 protein inhibits HIV entry when added to CD4+ T cells. To further characterize E2 effects on HIV replication, stably transfected Jurkat cell lines expressing GBV-C E2 or control sequences were infected with HIV and replication was measured. HIV replication (all 6 isolates studied) was inhibited in all cell lines expressing a region of 17 amino acids of GBV-C E2, but not in cell lines expressing E2 without this region. In contrast, mumps and yellow fever virus replication was not inhibited by E2 protein expression. Synthetic GBV-C E2 17mer peptides did not inhibit HIV replication unless they were fused to a tat-protein-transduction-domain (TAT) for cellular uptake. These data identify the region of GBV-C E2 protein involved in HIV inhibition, and suggest that this GBV-C E2 peptide must gain entry into the cell to inhibit HIV.     

Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coronavirus: importance for designing SARS vaccines

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.     

登革2型病毒NS3蛋白NTPase/RNA解旋酶结构域的原核表达及活性研究

目的 表达登革病毒非结构蛋白NS3 NTPase／RNA解旋酶结构域（dNS3），纯化表达产物并对其活性作初步研究。方法提取感染登革2型病毒的BHK-21细胞总RNA，RT-PCR扩增目的基因，经NcoⅠ、HindⅢ双酶切后连入表达载体pET28a，转化宿主菌BL21（DE3），优化诱导表达条件，SDS-PAGE和免疫印迹鉴定表达蛋白。表达产物经亲和层析纯化，超滤浓缩脱盐，孔雀绿钼酸铵法检测NTPase活性。结果成功构建重组表达载体pET28a-dNS3并在大肠杆菌中获得可溶性表达，纯化产物经测定具有良好的NTease活性及抗原性。体外条件下证实登革2型病毒非结构蛋白NS5（RDRP）对dNS3的ATPase活性有促进作用，提示在病毒复制时NS3与NS5间的相互作用对NS3的生物活性及对病毒复制过程可能具有调控作用。结论本研究为基于抑制NS3 NTPase／RNA解旋酶活性的抗病毒药物的筛选提供了实验依据。     

Generation and characterization of a monoclonal antibody that cross‐reacts with the envelope protein from the four dengue virus serotypes

Dengue viruses (DENVs; serotypes 1–4) are members of the flavivirus family. The envelope protein (E) of DENV has been defined as the principal antigenic target in terms of protection and diagnosis. Antibodies that can reliably detect the E surface glycoprotein are necessary for describing and mapping new DENV epitopes as well as for developing more reliable and inexpensive diagnostic assays. In this study, we describe the production and characterization of a monoclonal antibody (mAb) against a recombinant DENV‐2 E protein that recognizes a sequential antigen in both native and recombinant form located in domain II of the E protein of all four DENV serotypes. We confirmed that this mAb, C21, recognizes a sequence located in the fusion peptide. In addition, C21 does not have neutralizing activity against DENV‐2 in an in vitro system. Furthermore, the C21 mAb is an ideal candidate for the development of research reagents for studying DENV biology because it cross‐reacts with the four dengue serotypes.     

Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31

Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.     

A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4⁺ and CD8⁺ cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.     

The capsid-coding region hairpin element (cHP) is a critical determinant of dengue virus and West Nile virus RNA synthesis

Dengue virus (DENV) and West Nile virus (WNV) are members of the Flavivirus genus of positive-strand RNA viruses. RNA sequences and structures, primarily in the untranslated regions, have been shown to modulate flaviviral gene expression and genome replication. Previously, we demonstrated that a structure in the DENV coding region (cHP) enhances translation start codon selection and is required for viral replication. Here we further characterize the role of the cHP in the DENV life cycle. We demonstrate that the cHP is required for efficient viral RNA synthesis in a sequence-independent manner. Viruses with a disrupted cHP are rescued by a spontaneous compensatory mutation that restabilizes the structure. Furthermore, the cHP, which is predicted to be conserved among arthropod-borne flaviviruses, is required for WNV replication. We propose that the cHP is a multifunctional determinant of flavivirus replication, functioning in both translation and RNA synthesis.     

Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein,gp120

Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of additional antibodies to GDEs.