Biology subjects

Goo, L.

Publications and source records attributed to Goo, L..

7 recordsLinked to original sources

bioRxiv2025

Structural basis for antibody cross-neutralization of dengue and Zika viruses

Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus (ZikV) and the four serotypes of Dengue virus (DenV1-4) must elicit broadly neutralizing antibodies (bnAbs) to prevent the risk of enhancement of infection by non-neutralizing antibodies. We recently discovered new orthoflavivirus-directed bnAbs, including F25.S02, which neutralizes DenV1-4 and ZikV with comparable or superior potency to the previously characterized E dimer epitope (EDE) bnAbs. Mutagenesis studies of viral envelope proteins showed that the epitope specificity of F25.S02 is distinct from EDE1 bnAbs. Here, we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F25.S02 at the molecular level. We obtained a ~4.2 [A] cryoEM structure of F25.S02 Fab bound to a stabilized DenV3 soluble E protein dimer and a 2.3 [A] crystal structure of F25.S02 Fab bound to ZikV soluble E protein dimer. Like previously described EDE1 bnAbs, the structural epitope of F25.S02 is at the E dimer interface, encompassing predominantly conserved regions in domain II, including the fusion loop. However, unlike EDE1 bnAbs, F25.S02 binding is almost entirely dependent on the heavy chain and is shifted slightly away from the dimer symmetry axis. Our findings emphasize the importance of this cross-neutralizing site of vulnerability for DenV and ZikV that can facilitate rational design of vaccines and therapeutics.

immunology↗

bioRxiv2024

Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection

Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.

microbiology↗

bioRxiv2023

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies

Zika virus and dengue virus are co-circulating flaviviruses with a widespread endemic range. Eliciting broad and potent neutralizing antibodies is an attractive goal for developing a vaccine to simultaneously protect against these viruses. However, the capacity of viral mutations to confer escape from broadly neutralizing antibodies remains undescribed, due in part to limited throughput and scope of traditional approaches. Here, we use deep mutational scanning to map how all possible single amino acid mutations in Zika virus envelope protein affect neutralization by antibodies of varying breadth and potency. While all antibodies selected viral escape mutations, the mutations selected by broadly neutralizing antibodies conferred less escape relative to those selected by narrow, virus-specific antibodies. Surprisingly, even for broadly neutralizing antibodies with similar binding footprints, different single mutations led to escape, indicating distinct functional requirements for neutralization not captured by existing structures. Additionally, the antigenic effects of mutations selected by broadly neutralizing antibodies were conserved across divergent, albeit related, flaviviruses. Our approach identifies residues critical for antibody neutralization, thus comprehensively defining the as-yet-unknown functional epitopes of antibodies with clinical potential. ImportanceThe wide endemic range of mosquito-vectored flaviviruses - such as Zika virus and dengue virus serotypes 1-4 - places hundreds of millions of people at risk of infection every year. Despite this, there are no widely available vaccines, and treatment of severe cases is limited to supportive care. An avenue towards development of more widely applicable vaccines and targeted therapies is the characterization of monoclonal antibodies that broadly neutralize all these viruses. Here, we measure how single amino acid mutations in viral envelope protein affect neutralizing antibodies with both broad and narrow specificities. We find that broadly neutralizing antibodies with potential as vaccine prototypes or biological therapeutics are quantifiably more difficult to escape than narrow, virus-specific neutralizing antibodies.

microbiology↗

bioRxiv2023

Defining the impact of flavivirus envelope protein glycosylation on sensitivity to broadly neutralizing antibodies

Antibodies targeting the so-called envelope dimer epitope (EDE) cross-neutralize Zika virus (ZIKV) and all four dengue virus (DENV) serotypes and have thus inspired an epitope-focused vaccine design against these flaviviruses. There are two EDE antibody subclasses (EDE1, EDE2) distinguished by their dependence on viral envelope (E) protein N-linked glycosylation at position N153 (DENV) or N154 (ZIKV) for binding. Here, we determined how E glycosylation affects neutralization by EDE and other broadly neutralizing antibodies. Consistent with structural studies, mutations abolishing the N153/N154 glycosylation site increased DENV and ZIKV sensitivity to neutralization by EDE1 antibodies. Surprisingly, these mutations also increased sensitivity to EDE2 antibodies although they occurred at predicted contact sites. Despite preserving the glycosylation site motif (N-X-S/T), substituting the threonine at ZIKV E residue 156 with a serine resulted in loss of glycan occupancy accompanied with increased neutralization sensitivity to EDE antibodies. For DENV, the presence of a serine instead of a threonine at E residue 155 retained glycan occupancy, but nonetheless increased sensitivity to EDE antibodies, in some cases to a similar extent as mutation at N153, which abolishes glycosylation. E glycosylation site mutations also increased ZIKV and DENV sensitivity to other broadly neutralizing antibodies, but had limited effects on ZIKV-or DENV-specific antibodies. Thus, E protein glycosylation is context-dependent and modulates the potency of broadly neutralizing antibodies in a manner not predicted by existing structures. Manipulating E protein glycosylation could be a novel strategy for engineering vaccine antigens to elicit antibodies that broadly neutralize ZIKV and DENV. IMPORTANCEAntibodies that can potently cross-neutralize Zika (ZIKV) and dengue (DENV) viruses are attractive to induce via vaccination to protect against these co-circulating flaviviruses. Structural studies have shown that viral envelope protein glycosylation is important for binding by one class of these so-called broadly neutralizing antibodies, but less is known about the determinants of neutralization. Here, we investigated how envelope protein glycosylation impacts broadly neutralizing antibody potency. By characterizing a panel of ZIKV and DENV variants encoding envelope protein glycosylation site mutations, we found that glycan occupancy was not always predicted by an intact N-X-S/T sequence motif. Moreover, envelope protein glycosylation status alters the neutralization potency of broadly neutralizing antibodies in a manner unexpected from their predicted binding mechanism as determined by existing structures. We highlight the complex role and determinants of envelope protein glycosylation that should be considered in the design of vaccine antigens to elicit broadly neutralizing antibodies.

microbiology↗

bioRxiv2023

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasure strategies that avoid infection enhancement associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following secondary DENV infection. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

microbiology↗

bioRxiv2021

Norstictic acid is a selective allosteric transcriptional regulator

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (~900 [A]2) and have little topology, and thus do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprised of a highly dynamic loop flanking one canonical binding surface and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

biochemistry↗

bioRxiv2019

Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To delineate bNAb targets, we characterized 28 monoclonal antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies revealed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against flaviviruses with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested a memory origin and divergent somatic hypermutation pathways for these bNAbs, and a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a novel epitope that can be exploited for vaccine design.

microbiology↗