DEGP8 Antibody

What are the primary target epitopes for neutralizing antibodies against dengue virus?

Neutralizing antibodies against dengue virus primarily target several key epitopes on the envelope (E) protein:

• E Dimer Epitope (EDE): This epitope forms at the interface between two E protein monomers and is recognized by broadly neutralizing antibodies that can neutralize all DENV serotypes .

• Domain III (EDIII) Lateral Ridge: This region is targeted by serotype-specific antibodies such as 2C8 and 3H5, which show potent neutralization against DENV-2 . This epitope often elicits type-specific responses due to sequence variability among the four DENV serotypes.

• Domain I Quaternary Epitopes: These epitopes are formed in the context of the assembled virion and represent another target for neutralizing antibodies .

The specificity of antibody binding to these epitopes significantly affects whether they will be broadly neutralizing or serotype-specific, and whether they might contribute to antibody-dependent enhancement.

How do antibodies 2C8 and 3H5 differ despite targeting similar epitopes?

Both 2C8 and 3H5 are potently neutralizing, serotype-specific murine antibodies that target the lateral ridge epitope of DENV-2 EDIII, but they exhibit striking differences in their functional properties:

• Neutralization capacity: Both antibodies demonstrate potent neutralization of DENV-2 with 50% neutralization titers in the sub-nanomolar range (0.025 nM for 2C8 and 0.044 nM for 3H5) .

• ADE properties: 2C8 demonstrates typical infection enhancement with peak titers of over 1000-fold enhancement and a wide concentration range where enhancement occurs. In contrast, 3H5 shows dramatically reduced enhancement of DENV-2 at a very narrow concentration range or no enhancement at all .

• Fcγ receptor interaction: 3H5-DENV2 immune complexes show weak or no interaction with Fcγ receptors, unlike 2C8, which explains the low ADE activity of 3H5 .

• pH sensitivity: 2C8 shows decreased avidity at endosomal pH (from 0.03 nM at neutral pH to 0.1 nM at low pH), while 3H5 maintains consistent binding affinity across pH ranges (0.31 nM at neutral pH to 0.35 nM at low pH) .

• Binding mode: 2C8 likely binds bivalently to the virus (significant difference between Fab and full IgG Kd values: 4.76 nM vs 0.03 nM), whereas 3H5 appears to bind monovalently (similar Kd values for Fab and full IgG: 0.2 nM vs 0.3 nM) .

These differences highlight how subtle variations in epitope recognition can dramatically impact antibody function, despite targeting the same general region.

What is antibody-dependent enhancement (ADE) and why is it a concern in dengue antibody research?

Antibody-dependent enhancement (ADE) is a phenomenon where sub-neutralizing concentrations of antibodies facilitate virus entry into Fcγ receptor-bearing cells, potentially increasing infection severity. For dengue research, ADE represents a significant concern:

• Mechanistic basis: ADE occurs when antibodies bind to viral particles but fail to fully neutralize them. These immune complexes then engage Fcγ receptors on monocytes/macrophages, enhancing viral uptake and replication .

• Clinical relevance: ADE is purported to drive higher virus loads in secondary heterologous infections, and is associated with severe manifestations such as dengue hemorrhagic fever (DHF) .

• Research challenge: Most antibodies generated during dengue infection are non-neutralizing and can cause ADE, making therapeutic antibody development particularly challenging .

• Experimental evaluation: ADE is typically evaluated using Fcγ receptor-expressing cell lines such as U937 or K562, which resist dengue infection in the absence of antibodies but become highly susceptible in the presence of enhancing antibodies .

Understanding why certain antibodies like 3H5 exhibit minimal ADE while others like 2C8 promote strong enhancement is crucial for developing safe therapeutic antibodies and vaccines.

What structural characteristics of the 3H5 antibody contribute to its unusual minimal ADE profile?

The 3H5 antibody demonstrates an exceptional profile with potent neutralization but minimal ADE, which stems from several structural characteristics:

• Epitope targeting: 3H5 targets residues that are buried between E dimers and located close to the viral membrane, which appears to influence how immune complexes interact with Fcγ receptors .

• Immune complex orientation: When 3H5 binds to virus particles, the resulting immune complexes demonstrate inefficient interaction with Fcγ receptors, likely due to the specific binding mode and orientation that prevents the Fc region from engaging FcγR .

• Resilient binding at endosomal pH: Unlike many antibodies that show pH-dependent affinity reductions, 3H5 maintains consistent binding affinity at endosomal pH conditions, potentially allowing continued neutralization in the endosomal environment where fusion occurs .

• Neutralization at low occupancy: 3H5 appears capable of neutralizing at lower occupancy levels compared to other antibodies, meaning fewer antibodies are needed to neutralize each virion .

The passive transfer of 3H5 has been shown to protect mice from lethal DENV infection without discernible clinical symptoms, highlighting its therapeutic potential . These structural insights are valuable for designing antibodies that maximize neutralization while minimizing enhancement.

How has structural knowledge been leveraged to engineer improved anti-dengue antibodies?

Structural insights have been crucial for engineering antibodies with improved properties:

• Structure-guided mutagenesis: Analysis of the epitope-paratope interface has allowed researchers to identify key residues that can be modified to improve binding. For instance, the antibody Ab513 was engineered from 4E11 by introducing six affinity-enhancing point mutations and an affinity-enhancing deletion at position 26 (VH), along with humanization changes .

• Network theory application: Adapting from network (graph) theory, researchers developed a framework to compute inter-residue atomic interactions between interacting amino acid pairs of an antigen-antibody interface. This approach guided the deletion of residues in the 25ASGF28 region of CDR-H1, which increased shape complementarity between the interacting surfaces by approximately 8% .

• Electrostatic optimization: Engineering efforts focused on creating a positively charged surface on the antibody VH domain (using Arg99 of CDR-H3 and Lys3 of FR1) to better engage negatively charged residues 360-363 of EDIII across multiple serotypes .

• Cross-serotype binding enhancement: The engineered antibody Ab513 exhibited significant affinity improvements, with 13-fold and 22-fold higher affinity to DENV-3 and DENV-4 respectively, compared to its predecessor 4E5A .

These engineering approaches demonstrate how structural knowledge can be harnessed to develop broadly neutralizing antibodies with enhanced properties against dengue virus.

What role does antibody avidity play in broad neutralization of dengue serotypes?

Antibody avidity (the combined strength of multiple binding interactions) plays a complex role in dengue neutralization:

• Diverse contributions to neutralization: Despite sharing an epitope, antibodies can exhibit different neutralization mechanisms based on avidity. For example, when comparing bivalent IgGs and monovalent Fab fragments of EDE antibodies A11 and C10, both showed more potent neutralization in IgG form, but only C10's enhanced activity was associated with bivalent binding .

• pH-dependent avidity effects: Some antibodies like 2C8 show significant reduction in avidity under acidic conditions that mimic the endosomal environment, while others like 3H5 maintain consistent binding. This differential response suggests varied neutralization mechanisms even among antibodies targeting similar epitopes .

• Monovalent versus bivalent binding: Comparison of Kd values between full-length antibodies and their Fab fragments can reveal binding modes. A minor difference between Fab and IgG (e.g., 0.2 nM vs. 0.3 nM for 3H5) suggests monovalent binding, while large differences (e.g., 4.76 nM vs. 0.03 nM for 2C8) indicate bivalent interaction .

• Neutralization potency correlation: For antibodies employing bivalent binding, Fab fragments often show greatly reduced neutralization potency compared to full-length antibodies, while antibodies that function monovalently show similar neutralization between Fab and IgG forms .

Understanding these avidity effects is crucial for predicting antibody behavior across different physiological conditions and for engineering antibodies with improved neutralization breadth.

How can glycan-masking strategies be employed to elicit 3H5-like antibodies while avoiding ADE-inducing responses?

Glycan-masking represents a promising approach for focusing antibody responses toward specific epitopes:

• Concept and mechanism: N-glycans can function as molecular shields to prevent antibody binding to specific epitopes on antigen proteins. By strategically introducing glycosylation sites, researchers can mask immunodominant but poorly neutralizing epitopes while preserving access to potent neutralizing epitopes .

• Target epitope selection: For dengue, the ideal strategy would focus on eliciting 3H5-like antibodies (potently neutralizing with minimal ADE) while blocking 2C8-like antibodies that cause significant ADE despite targeting similar EDIII regions .

• Previous successes: This approach has been successfully implemented for other viruses, including HIV and influenza. For instance, a glycan-masked version of HIV vaccine candidate eOD-GT8 successfully focused antibody responses to the targeted CD4-binding site .

• Implementation for dengue: An engineered N-glycosylated dengue envelope protein domain III could potentially mask the epitopes that elicit ADE-inducing or weakly neutralizing antibodies while preserving accessibility to the epitopes recognized by 3H5 .

By carefully designing glycan shields based on the subtle differences in binding epitopes between 3H5 and 2C8, researchers could potentially develop immunogens that selectively elicit antibodies with 3H5-like properties - potent neutralization without significant ADE.

What methodologies are being used to map epitope-specific antibody responses following DENV infection or vaccination?

Researchers employ several advanced techniques to characterize epitope-specific responses:

• Structure-guided immunogen design: By using structural information about antibody-antigen interactions, researchers can design modified immunogens with specific alterations to probe epitope importance .

• Reverse genetics approaches: Generation of recombinant viruses containing amino acid alterations and epitope transplants between different serotypes allows for fine mapping of antibody binding sites .

• Competition assays: These assays determine whether antibodies compete for binding to the same or overlapping epitopes, helping to categorize antibody responses .

• Panel of modified viruses: Testing antibody binding and neutralization against a panel of viruses with specific mutations helps identify critical residues involved in antibody recognition .

• Epitope transplantation: Moving epitopes between serotypes creates chimeric viruses that help determine if a specific epitope is sufficient for antibody recognition .

• Polyclonal sera dissection: Using engineered viruses with modified epitopes to test binding and neutralization by polyclonal sera helps quantify the fraction of the antibody response directed toward specific epitopes .

These methodologies have revealed that antibodies following DENV2 infection or vaccination circulate as separate populations that neutralize by occupying domain III and domain I quaternary epitopes, with the fraction of neutralizing antibodies directed to different epitopes varying between individuals .

How can the unique properties of antibody 3H5 be harnessed for therapeutic development?

The exceptional properties of 3H5 make it a valuable template for therapeutic development:

• Minimal ADE liability: The unusually low enhancement profile of 3H5 addresses one of the major safety concerns in dengue antibody therapeutics. Understanding the structural basis for this property could guide development of antibodies with similar safety profiles .

• Protective efficacy: Passive transfer of 3H5 has been shown to protect mice from lethal DENV infection without discernible clinical symptoms, demonstrating its potential effectiveness as a therapeutic .

• Correlation with protection: Blockade of the 3H5 epitope has been correlated with serum antibody neutralization in DENV-2-infected/immunized nonhuman primates, suggesting this epitope is a key target for protective immunity .

• Engineering approaches: Using 3H5 as a template, researchers could employ structure-guided design to extend its specificity to other serotypes while maintaining its favorable safety profile. Alternatively, the unique binding characteristics of 3H5 could inform the design of novel immunogens .

• Cocktail potential: While 3H5 is serotype-specific for DENV-2, it could be combined with other minimally enhancing antibodies targeting different serotypes to create a therapeutic cocktail with broad coverage .

Leveraging these unique properties could lead to safer and more effective antibody-based therapies for dengue infections.

What experimental models are most appropriate for evaluating anti-dengue antibody efficacy and safety?

Various experimental models provide complementary insights for evaluating anti-dengue antibodies:

• In vitro neutralization assays: Focus reduction neutralization tests (FRNT) determine the potency of antibody-mediated virus neutralization, with results reported as the concentration achieving 50% neutralization .

• Pre- and post-attachment neutralization assays: These differentiate whether antibodies block attachment to cells or inhibit a post-attachment step such as fusion, providing mechanistic insights .

• ADE assessment models:

  • U937 cells: Human monocytic cell line expressing Fcγ receptors that resist dengue infection without antibodies but become susceptible with enhancing antibodies .

  • K562 cells: Another Fcγ receptor-expressing cell line used to evaluate ADE .

• Animal models:

  • Humanized mouse models: Evaluate effects on thrombocytopenia, which is a clinical feature of dengue disease .

  • Maternal transfer model: Assesses protection against lethal antibody-mediated enhancement, crucial for safety evaluation .

  • Vascular leakage models: Measure the ability of antibodies to resolve this key pathological feature of severe dengue .

• Fcγ receptor binding assays: Direct measurement of immune complex interaction with Fcγ receptors helps predict ADE potential .

• pH-dependent binding studies: ELISA-based assays at neutral and endosomal pH evaluate antibody resilience across physiologically relevant conditions .

Using multiple models provides a comprehensive assessment of both efficacy (neutralization breadth/potency) and safety (ADE potential), which is crucial given the complex pathogenesis of dengue.

How can in silico approaches accelerate the development of improved anti-dengue antibodies?

Computational methods offer powerful tools for antibody engineering and evaluation:

• Epitope-paratope interface analysis: Computational frameworks can analyze atomic interactions between interacting amino acid pairs to identify critical residues for binding. This guided the successful engineering of Ab513 with improved affinity to multiple serotypes .

• Shape complementarity calculations: Computational assessment of shape complementarity between antibody and antigen surfaces (using metrics like "Sc") can predict how modifications will affect binding. An 8% increase in shape complementarity was achieved through structure-guided deletion in Ab513 development .

• Electrostatic interaction modeling: Computational analysis of charged residue interactions helps optimize antibody-antigen binding. This approach identified opportunities to engineer electrostatic complementarity between positively charged antibody surfaces and negatively charged EDIII residues .

• In silico mutant library screening: As demonstrated for the scFv against conserved dengue regions, virtual libraries of antibody mutants can be computationally screened for improved binding before experimental validation .

• Molecular dynamics simulations: These can predict the stability of antibody-antigen complexes and how mutations might affect binding, as shown in the scFv-FuBc complex analysis .

• Conserved epitope identification: Computational analysis of sequence conservation across serotypes identifies potential targets for broadly neutralizing antibodies, as was done to identify conserved regions of dengue envelope protein .

These computational approaches can significantly reduce the experimental burden by focusing wet-lab efforts on the most promising candidates, accelerating the development timeline for new anti-dengue therapeutics.

How might epitope-specific antibody responses be used as correlates of protective immunity?

Epitope-specific antibody responses could serve as valuable correlates of protection:

• Polyclonal response characterization: Research has shown that following DENV2 infection or vaccination, antibodies circulate as separate populations targeting different epitopes (domain III and domain I quaternary epitopes). The relative proportion of these populations varies between individuals and may correlate with protection .

• Epitope-specific assays: Developing standardized assays to measure antibody responses to specific epitopes (like those recognized by 3H5 or Ab513) could provide more predictive information than total binding or neutralizing antibody titers .

• Minimal-enhancement epitopes: Monitoring antibody responses specifically to epitopes associated with minimal ADE (like the 3H5 epitope) could help predict not only protection but also safety of the immune response .

• Longitudinal studies: Tracking epitope-specific responses over time and correlating them with protection from natural infection could identify which epitopes elicit durable protective immunity .

• Post-vaccination assessment: Following vaccination, epitope-specific antibody profiling could potentially predict which individuals might remain at risk despite developing antibody responses .

This approach could potentially improve vaccine evaluation by moving beyond simple measurement of neutralizing antibody titers to more sophisticated analysis of the quality and specificity of the antibody response.

What strategies might overcome the challenge of developing antibodies effective against all dengue serotypes?

Several approaches show promise for developing broadly effective anti-dengue antibodies:

• Structure-guided engineering: Using structural information to modify existing antibodies can improve cross-reactivity. Ab513, engineered from 4E11, showed dramatically improved binding to DENV-4 (22-fold) and DENV-3 (13-fold) while maintaining strong binding to DENV-1 and DENV-2 .

• Targeting conserved epitopes: Identifying and targeting highly conserved regions of the dengue envelope protein can lead to broader protection. In silico analysis has identified such conserved regions that could serve as targets for broadly neutralizing antibodies .

• E Dimer Epitope (EDE) focus: Antibodies recognizing the E dimer epitope show promise for broad neutralization of all DENV serotypes and represent promising templates for vaccine design .

• Bispecific antibody formats: Developing bispecific antibodies that simultaneously target different epitopes or serotypes could provide broader coverage than traditional monoclonal antibodies.

• Cocktail approaches: Combining multiple antibodies with complementary specificities, each with minimal enhancement potential, could provide comprehensive coverage of all serotypes .

• Fc engineering: Modifying the Fc region to prevent Fcγ receptor binding (as demonstrated with LALA mutations) can eliminate ADE risk while preserving neutralizing capacity, potentially allowing use of antibodies that would otherwise cause enhancement .