Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop.
ABSTRACT: 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.
Project description:Dengue, caused by the four serotypes of dengue virus (DENV), represents an expanding global health challenge. The potential for serotype-cross-reactive antibodies to exacerbate disease during a secondary infection with a heterologous DENV serotype has driven efforts to study human DENV-specific antibodies. Most DENV-specific antibodies generated in humans are serotype-cross-reactive, weakly neutralizing, and directed against the immature pre-membrane (prM), envelope (E), and nonstructural 1 (NS1) proteins. To broaden the characterization of human DENV-specific antibodies, we assessed B-cell responses by ELISpot assays and isolated B cells from the peripheral blood of a human subject with previous DENV infection. Forty-eight human IgG monoclonal antibodies (hMAbs) were initially characterized by their potential to bind to an inactivated lysate of DENV-infected cells. Subsequently, most DENV-specific hMAbs were found to bind soluble, recombinant E protein (rE). Two hMAbs were unable to bind rE, despite strongly binding to the DENV-infected cell lysate. Further analyses showed that these two hMAbs bound conformation-dependent, reduction-sensitive epitopes on E protein. These data shed light on the breadth of DENV-specific hMAbs generated within a single immune donor.
Project description:UNLABELLED:Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. IMPORTANCE:Dengue virus causes fever and dengue hemorrhagic fever. Dengue serotype 2 (DENV2) is widespread and frequently responsible for severe epidemics. Natural DENV2 infections stimulate serotype-specific neutralizing antibodies, but a leading DENV vaccine did not induce a similar protective response. While groups have identified epitopes of single monoclonal antibodies (MAbs), the molecular basis of DENV2 neutralization by polyclonal human immune sera is unknown. Using a recombinant DENV displaying serotype 2 epitopes, here we map the main target of DENV2 polyclonal neutralizing antibodies induced by natural infection and a live DENV2 vaccine candidate. Proper display of the epitope required the assembly of viral envelope proteins into higher-order structures present on intact virions. Despite the complexity of the epitope, it was possible to transplant the epitope between DENV serotypes. Our findings have immediate implications for evaluating dengue vaccines in the pipeline as well as designing next-generation vaccines.
Project description:Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.
Project description:Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.
Project description: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.
Project description:Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies1-3 that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fc? receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE)1,4,5. ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.
Project description:Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV). At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs) and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.
Project description:Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner; however, most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with a virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies, using viral and recombinant antigens, mainly led to the isolation of dominant nonneutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells by using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput, flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype-cross-neutralizing anti-DENV monoclonal Abs (MAbs), including ultrapotent inhibitory antibodies with neutralizing activity concentrations of <10 ng/ml. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein, including epitopes present only on the intact, fully assembled viral particle. We also isolated a number of serotype-cross-neutralizing MAbs, most of which recognized a region in E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines.Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. Our studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. These studies provide important new information about protection from dengue virus infection that will be useful in the design and testing of new experimental dengue vaccines for humans.
Project description:UNLABELLED:Following natural dengue virus (DENV) infection, humans produce some antibodies that recognize only the serotype of infection (type specific) and others that cross-react with all four serotypes (cross-reactive). Recent studies with human antibodies indicate that type-specific antibodies at high concentrations are often strongly neutralizing in vitro and protective in animal models. In general, cross-reactive antibodies are poorly neutralizing and can enhance the ability of DENV to infect Fc receptor-bearing cells under some conditions. Type-specific antibodies at low concentrations also may enhance infection. There is an urgent need to determine whether there are conserved antigenic sites that can be recognized by cross-reactive potently neutralizing antibodies. Here, we describe the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the DENV domain II fusion loop (FL) envelope protein region from subjects following vaccination or natural infection. Most of the FL-specific antibodies exhibited a conventional phenotype, characterized by low-potency neutralizing function and antibody-dependent enhancing activity. One clone, however, recognized the bc loop of domain II adjacent to the FL and exhibited a unique phenotype of ultrahigh potency, neutralizing all four serotypes better than any other previously described MAb recognizing this region. This antibody not only neutralized DENV effectively but also competed for binding against the more prevalent poor-quality antibodies whose binding was focused on the FL. The 1C19 human antibody could be a promising component of a preventative or therapeutic intervention. Furthermore, the unique epitope revealed by 1C19 suggests a focus for rational vaccine design based on novel immunogens presenting cross-reactive neutralizing determinants. IMPORTANCE:With no effective vaccine available, the incidence of dengue virus (DENV) infections worldwide continues to rise, with more than 390 million infections estimated to occur each year. Due to the unique roles that antibodies are postulated to play in the pathogenesis of DENV infection and disease, there is consensus that a successful DENV vaccine must protect against all four serotypes. If conserved epitopes recognized by naturally occurring potently cross-neutralizing human antibodies could be identified, monovalent subunit vaccine preparations might be developed. We characterized 30 DENV cross-neutralizing human monoclonal antibodies (MAbs) and identified one (1C19) that recognized a novel conserved site, known as the bc loop. This antibody has several desirable features, as it neutralizes DENV effectively and competes for binding against the more common low-potency fusion loop (FL) antibodies, which are believed to contribute to antibody-mediated disease. To our knowledge, this is the first description of a potent serotype cross-neutralizing human antibody to DENV.
Project description:The four dengue virus serotypes (DENV1-4) cause major public health problems worldwide. Highly neutralizing type-specific human monoclonal antibodies (hmAbs) target conformation-dependent epitopes on the DENV envelope protein, including 1F4, a DENV1 type-specific hmAb. Using a recombinant DENV2 virus displaying the DENV1 1F4 epitope (rDENV2/1), we measured the proportion and kinetics of DENV1 neutralizing antibodies targeting the 1F4 epitope in individuals living in Asia and the Americas where different DENV1 genotypes were circulating. Samples from 20 individuals were analyzed 3 and 18 months post-primary DENV1 infection, alongside samples from 4 individuals collected annually for four years post-primary DENV1 infection, from two studies in Nicaragua. We also analyzed convalescent post-primary DENV1 plasma samples from Sri Lankan individuals. We found that neutralizing antibodies recognizing the 1F4 epitope vary in prevalence across both populations and were detected from 20 days to four years post-infection. Additionally, both populations displayed substantial variability, with a range of high to low proportions of DENV1 type-specific neutralizing antibodies recognizing the 1F4 epitope seen across individuals. Thus, the 1F4 epitope is a major but not exclusive target of type-specific neutralizing antibodies post-primary infection with different DENV1 genotypes in Asia and Latin America, and additional epitopes likely contribute to type-specific neutralization of DENV1.