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UNLABELLED - The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells.
IMPORTANCE - Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Approximately 500,000 people are hospitalized with severe dengue illness annually. Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is believed to contribute to the pathogenic cytokine storm described in severe dengue patients, but the precise signaling pathways contributing to elevated cytokine production are not elucidated. IL-1β is a potent inflammatory cytokine that is frequently elevated during severe dengue, and the unique dual regulation of IL-1β provides an informative model to study ADE-induced cytokines. This work utilizes patient-derived anti-DENV mAbs and primary human monocytes to study ADE-induced IL-1β and other cytokines. ADE of DENV serotype 2 (DENV-2) elevates mature IL-1β secretion by monocytes independent of DENV replication by 4 h postinoculation (hpi). Prior to this, DENV immune complexes activate spleen tyrosine kinase (Syk) within 1 hpi. Syk induces elevated IL1B, TNF, and IL6 mRNA by 2 hpi. Syk mediates elevated IL-1β secretion by activating ERK1/2, and both Syk and ERK1/2 inhibitors ablated ADE-induced IL-1β secretion. Maturation of pro-IL-1β during ADE requires caspase-1 and NLRP3, but caspase-1 is suboptimally increased by ADE and can be significantly enhanced by a typical inflammasome agonist, ATP. Importantly, this inflammatory Syk-ERK signaling axis requires DENV immune complexes, because DENV-2 in the presence of serotype-matched anti-DENV-2 mAb, but not anti-DENV-1 mAb, activates Syk, ERK, and IL-1β secretion. This study provides evidence that DENV-2 immune complexes activate Syk to mediate elevated expression of inflammatory cytokines. Syk and ERK may serve as new therapeutic targets for interfering with ADE-induced cytokine expression during severe dengue.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Dengue viruses (DENV) are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE) is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE) protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to the development of safe, non-enhancing vaccines against dengue.
The immunopathogenesis of severe dengue is poorly understood, but there is concern that induction of cross-reactive nonneutralizing antibodies by infection or vaccination may increase the likelihood of severe disease during a subsequent infection. We generated a total of 63 new human monoclonal antibodies to compare the B-cell response of subjects who received the National Institutes of Health live attenuated dengue vaccine rDEN1Δ30 to that of subjects following symptomatic primary infection with DENV1. Both infection and vaccination induced serum neutralizing antibodies and DENV1-reactive peripheral blood B cells, but the magnitude of induction was lower in vaccinated individuals. Serotype cross-reactive weakly neutralizing antibodies dominated the response in both vaccinated and naturally infected subjects. Antigen specificities were very similar, with a slightly greater percentage of antibodies targeting E protein domain I/II than domain III. These data shed light on the similarity of human B-cell response to live attenuated DENV vaccine or natural infection.