Dengue type 2 Antibody

What are the primary targets for neutralizing antibodies against DENV-2?

Neutralizing antibodies against DENV-2 primarily target the viral envelope (E) protein, which plays a critical role in virus attachment and entry into host cells. The E protein consists of three distinct domains: domain I (DI), domain II (DII), and domain III (DIII). Research has identified several specific epitopes that are particularly important for antibody neutralization:

• The lateral ridge of domain I (DI)

• The dimer interface, lateral ridge, and fusion loop of domain II (DII)

• The lateral ridge, C-C′ loop, and A strand of domain III (DIII)

Among these, antibodies targeting DIII have demonstrated particularly potent neutralizing activity. For example, the mAb DB32-6, which targets DIII, has shown strong neutralizing capacity against diverse DENV-2 strains by binding to residue K310 in the E-DIII A-strand . Similarly, another antibody, DV2-96, reacts specifically with sites along the lateral ridge of the DIII domain . These findings indicate that DIII contains critical neutralizing epitopes that make it an attractive target for therapeutic antibody development.

How are neutralizing antibodies against DENV-2 measured in laboratory settings?

Researchers employ several standardized assays to measure the neutralizing capacity of antibodies against DENV-2:

• Plaque Reduction Neutralization Test (PRNT): This gold-standard method involves incubating serial dilutions of antibodies with a fixed amount of DENV-2, then adding the mixture to susceptible cells. Plaques (areas of viral infection) are counted, and the antibody concentration that reduces plaque formation by 50% (PRNT50) is calculated using nonlinear regression analysis . PRNT values are typically expressed as nanograms per milliliter of antibody.

• Reporter Virus Particle (RVP) Assays: DENV-2 RVPs can be produced by transfecting cells (typically HEK-293T) with plasmids encoding DENV-2 C-prM-E proteins and a viral replicon. Neutralization is measured by flow cytometry approximately 48 hours after infection, with EC50 values calculated using nonlinear regression analysis .

• Pre- and Post-attachment Assays: Modified PRNT assays can determine whether antibodies inhibit at pre- or post-attachment steps of viral entry. In pre-attachment assays, antibodies and virus are mixed prior to addition to cells, while in post-attachment assays, virus is allowed to bind to cells first at 4°C before antibodies are added .

These methodologies allow for precise quantification of neutralizing antibody potency and provide insights into their mechanisms of action.

What is antibody-dependent enhancement (ADE) and how is it measured for DENV-2?

Antibody-dependent enhancement (ADE) is a phenomenon where sub-neutralizing concentrations of antibodies can enhance DENV infection, potentially contributing to severe disease during secondary infections with heterologous serotypes. ADE occurs when antibodies bind to virus particles but fail to neutralize them, instead facilitating viral entry into cells expressing Fcγ receptors, such as monocytes and macrophages.

ADE is typically measured using Fcγ receptor-bearing K562 cells, which are not directly permissive to DENV infection but become infected in the presence of enhancing antibodies. The standard methodology involves:

• Serial dilution of serum or antibody samples in culture medium

• Addition of a standardized amount of DENV-2 to allow antibody-virus complex formation

• Introduction of K562 cells to the antibody-virus complexes

• Incubation and subsequent measurement of infection levels, typically by immunofluorescence or flow cytometry

ADE activity typically follows a bell-shaped curve, with peak enhancement occurring at intermediate antibody dilutions. For example, in one study, ADE peaked at a serum dilution of 1:90 for both immune serum globulin (ISG) and the monoclonal antibody Mab11/wt . Importantly, engineered antibodies with mutations in their Fc regions (like Mab11/mutFc) can maintain neutralizing activity while preventing ADE, demonstrating the potential for designing therapeutic antibodies that avoid this adverse effect .

What are the temporal patterns of antibody responses following DENV-2 infection?

The kinetics of antibody responses following DENV-2 infection follow distinct patterns depending on whether the infection is primary or secondary:

In secondary DENV infections, anti-DENV IgG titers, neutralizing antibodies, and ADE titers against the infecting serotype typically:

• Peak around day 10 post-infection

• Maintain relatively stable levels until at least day 60 post-infection

Secondary infections generally elicit higher antibody titers and stronger functional antibody responses compared to primary infections . This enhanced response is attributed to the anamnestic response from prior exposure to a heterologous DENV serotype.

How does antibody strain-specificity impact DENV-2 neutralization across different genotypes?

DENV-2 exhibits significant genetic diversity with distinct genotypes circulating globally. This diversity presents a challenge for antibody-based therapeutics, as neutralizing efficacy can vary substantially across genotypes.

Research has demonstrated that several monoclonal antibodies (mAbs) with strong neutralizing activity against the homologous DENV-2 strain fail to efficiently neutralize at least one DENV-2 strain of a distinct genotype . This suggests that recognition of neutralizing epitopes varies among DENV-2 genotypes, despite their classification within the same serotype.

When developing therapeutic antibodies, researchers should:

• Test neutralization against a panel of genetically diverse DENV-2 strains

• Target epitopes that are highly conserved across genotypes

• Consider potential combinations of antibodies targeting different epitopes to broaden coverage

The correlation between antibody potency and specificity presents another challenge - antibodies with the highest neutralizing capacity often display narrowed genotype and serotype specificity . This creates a tension between selecting for broad coverage versus maximal potency that must be carefully balanced in therapeutic antibody development.

For optimal cross-genotype protection, researchers might consider targeting the most conserved epitopes on the E protein, such as certain regions of DIII, or developing antibody cocktails that collectively provide broader coverage against diverse DENV-2 strains.

What strategies can be employed to engineer therapeutic antibodies that prevent ADE while maintaining neutralizing capacity?

Engineering therapeutic antibodies that maintain strong neutralizing activity while preventing ADE is a critical goal for dengue treatment development. Several successful strategies have emerged:

• Fc Region Modifications: Introducing mutations in the Fc region of antibodies can disrupt their interaction with Fcγ receptors while preserving antigen binding and neutralization. For example, the antibody Mab11/mutFc retained neutralizing capacity against DENV-2 but did not enhance infection in in vitro ADE assays, unlike its wild-type counterpart (Mab11/wt) .

• Epitope Selection: Targeting specific epitopes that enable potent neutralization can minimize the risk of ADE. Antibodies like DB32-6 that target domain III (DIII) of the E protein show strong neutralizing capacity against diverse DENV-2 strains and can be engineered to reduce ADE risk .

• Humanization Process: When developing therapeutic antibodies from mouse monoclonals, careful humanization is essential to maintain potency while reducing immunogenicity. The successful humanization of DB32-6 retained its potent neutralization of DENV-2 without enhancing viral infection .

• Validation in Multiple Models: Comprehensive testing of engineered antibodies should include:

  • In vitro neutralization assays against diverse DENV-2 strains

  • ADE assays using Fcγ receptor-bearing cells

  • Animal protection studies in both prophylactic and therapeutic settings

These approaches have yielded promising results. For instance, in animal models, antibody prophylaxis showed complete protection against viremia in groups receiving immune serum globulin, and significant reduction in viremia duration (89% and 83%) in groups receiving Mab11/wt and Mab11/mutFc, respectively . These findings suggest that properly engineered antibodies can effectively reduce viral loads, potentially minimizing disease progression without enhancing infection.

How do in vitro neutralization assays correlate with in vivo protection for anti-DENV-2 antibodies?

Understanding the correlation between in vitro neutralization and in vivo protection is crucial for developing effective therapeutic antibodies. Research has revealed several important patterns:

• Predictive Value of PRNT: Plaque reduction neutralization test (PRNT) results generally correlate with protective efficacy, but the relationship is not always linear. Antibodies with the strongest in vitro neutralizing activity (lowest PRNT50 values) typically show the best protection in animal models, but exceptions exist .

• Epitope Dependence: The location of the targeted epitope influences the correlation between in vitro and in vivo results. Antibodies targeting certain regions of DIII (particularly the lateral ridge) often show stronger correlation between neutralization potency and protective efficacy compared to antibodies targeting other domains .

• Post-exposure Efficacy: Some antibodies demonstrate therapeutic efficacy even when administered post-exposure. For example, the mAb DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models, even in post-exposure trials . This suggests that strong neutralizing capacity in vitro may translate to therapeutic potential in vivo.

• Viremia Reduction: In animal studies, effective antibodies can reduce or eliminate viremia. Complete protection against viremia was observed in groups receiving immune serum globulin, with significant reduction in viremia duration in groups receiving monoclonal antibodies . This viremia reduction correlates with survival and reduced disease severity.

• Methodological Considerations: When evaluating in vivo protection, researchers should consider:

  • Using multiple animal models (when possible)

  • Testing against diverse viral strains

  • Evaluating both prophylactic and therapeutic applications

  • Measuring multiple endpoints (survival, viremia, clinical signs)

These findings underscore the importance of comprehensive testing that includes both in vitro characterization and in vivo validation to accurately predict the therapeutic potential of anti-DENV-2 antibodies.

What novel epitope mapping strategies are most effective for DENV-2 antibody characterization?

Accurate epitope mapping is essential for understanding antibody function and developing improved therapeutics. Several innovative approaches have proven particularly effective for DENV-2 antibody characterization:

• Combined Phage Display and Virus-Like Particles (VLPs): This hybrid approach has successfully identified critical binding residues. For example, researchers used this method to determine that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding . The strategy involves:

  • Displaying fragments of the E protein on phage

  • Testing antibody binding to these fragments

  • Confirming findings using VLPs with specific mutations

  • Validating results with neutralization assays

• Yeast Surface Display: This technique allows for the expression of DENV-2 E protein domains on yeast cell surfaces, facilitating rapid screening of antibody binding sites. Combined with neutralization escape studies, this approach has successfully mapped binding sites to specific regions including the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of domain II (DII), and the lateral ridge, C-C′ loop, and A strand of domain III (DIII) .

• Neutralization Escape Mutant Analysis: By selecting for viral variants that escape neutralization by specific antibodies, researchers can identify critical residues involved in antibody binding. When combined with structural analysis, this approach provides insights into the molecular basis of neutralization.

• Structure-Function Correlations: Integrating epitope mapping with functional assays (neutralization, ADE, etc.) allows researchers to correlate specific epitopes with particular antibody functions. This has revealed that antibodies targeting certain regions (like the DIII lateral ridge) typically show stronger neutralizing activity .

These complementary approaches provide a comprehensive understanding of antibody-epitope interactions, guiding the development of improved therapeutic antibodies with enhanced potency and reduced risk of ADE.

How do antibody responses differ between primary and secondary DENV-2 infections, and what are the implications for disease severity?

The antibody response to DENV-2 differs substantially between primary and secondary infections, with important implications for disease severity:

These findings suggest that qualitative aspects of the antibody response, beyond simply titer or neutralizing capacity, may influence clinical outcomes. The heightened risk associated with secondary infections underscores the importance of developing therapeutic antibodies that can effectively neutralize virus without enhancing infection through ADE mechanisms.

For researchers developing vaccines or therapeutic antibodies, these differences highlight the need to consider how interventions might perform differently in dengue-naive versus dengue-experienced individuals.

What cell-based systems are optimal for evaluating anti-DENV-2 antibody functions?

Selecting appropriate cell systems is crucial for accurate characterization of anti-DENV-2 antibodies. Different cell types provide complementary information about antibody functions:

• Vero Cells:

  • Widely used for PRNT assays due to their high susceptibility to DENV infection

  • Allow for clear plaque formation, facilitating quantification of neutralizing activity

  • Used for direct plaque assays to quantify virus from serum samples

  • Limitations: lack Fcγ receptors, thus cannot demonstrate ADE

• K562 Cells:

  • Express Fcγ receptors but are nonpermissive for direct DENV infection

  • Ideal for ADE assays, as they become infected only in the presence of enhancing antibodies

  • Standard protocol involves incubating diluted serum samples with DENV-2, adding to K562 cells, and measuring infection after incubation

  • Provide critical information about potential enhancement activity of antibodies

• HEK-293T Cells:

  • Used for producing DENV-2 reporter virus particles (RVPs)

  • Typically transfected with plasmids encoding DENV-2 C-prM-E and a viral replicon

  • RVPs produced in these cells enable high-throughput neutralization assays

• Raji-DC-SIGN Cells:

  • Express the dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) receptor

  • Highly susceptible to DENV infection

  • Used as a reference for determining viral dilutions that yield specific infection rates

• C6/36 Cells:

  • Mosquito cell line used for virus isolation and propagation

  • Less prone to introducing adaptive mutations during viral growth

  • Useful for maintaining viral stocks for neutralization assays

For comprehensive antibody characterization, researchers should employ multiple cell systems to evaluate different functional aspects, including neutralization potency, mechanism of action (pre- vs. post-attachment inhibition), and potential for enhancement. Combining results from these complementary systems provides a more complete understanding of antibody properties relevant to therapeutic development.

What animal models best predict therapeutic efficacy of anti-DENV-2 antibodies?

Animal models play a critical role in evaluating the protective and therapeutic potential of anti-DENV-2 antibodies before clinical testing. Several models offer valuable but distinct insights:

• AG129 Mice (Lacking Type I and II Interferon Receptors):

  • Highly susceptible to DENV infection

  • Develop viremia and disease symptoms similar to human dengue

  • Used to evaluate both prophylactic and therapeutic efficacy of antibodies

  • Support antibody-dependent enhancement, allowing assessment of both protective and potentially harmful antibody effects

  • The DB32-6 antibody showed therapeutic efficacy against mortality induced by different strains of DENV-2 in this model, even in post-exposure trials

• STAT1-Deficient Mice:

  • Another immunodeficient model susceptible to DENV infection

  • Useful for evaluating antibody protection against lethal DENV challenge

  • Provides a complementary system to AG129 mice

  • DB32-6 demonstrated efficacy in both this model and AG129 mice, suggesting robust therapeutic potential

• Humanized Mouse Models:

  • Mice engrafted with human immune system components

  • More accurately reflect human immune responses

  • Allow testing of fully human or humanized antibodies in a more relevant context

  • More expensive and technically challenging than conventional models

• Non-Human Primates:

  • Most closely resemble human infection and immune responses

  • Develop viremia but typically not severe disease

  • Valuable for evaluating antibody pharmacokinetics and effects on viral load

  • Expensive and logistically challenging, typically reserved for late-stage therapeutic candidates

For robust evaluation, researchers should consider:

• Testing antibodies in multiple animal models

• Evaluating both prophylactic (pre-exposure) and therapeutic (post-exposure) administration

• Challenging with different DENV-2 strains to assess breadth of protection

• Measuring multiple endpoints including survival, viremia, and clinical signs

The strong correlation between results in these animal models and potential human efficacy provides confidence in the therapeutic potential of antibodies like DB32-6, which showed protection in multiple models and against different DENV-2 strains .

What are the prospects for anti-DENV-2 antibodies as prophylactic and therapeutic agents?

Antibody-based interventions show significant promise for both prophylactic and therapeutic applications against DENV-2 infection, with several key advances:

• Prophylactic Potential:

  • Complete protection against viremia has been demonstrated in animal models receiving immune serum globulin

  • Significant reduction in viremia duration (89% and 83%) in groups receiving monoclonal antibodies Mab11/wt and Mab11/mutFc, respectively

  • These findings suggest antibody prophylaxis could effectively eliminate or reduce viral loads, potentially minimizing disease progression

• Therapeutic Applications:

  • Some antibodies have shown efficacy even when administered post-exposure

  • The mAb DB32-6 demonstrated therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models

  • This suggests potential for treating ongoing infections, not just prevention

• Engineering Advances:

  • Successful conversion of mouse antibodies to humanized versions that retain neutralizing potency

  • Development of Fc-modified antibodies that avoid ADE while maintaining protective functions

  • Identification of key epitopes that confer broad protection against diverse DENV-2 strains

• Remaining Challenges:

  • Ensuring coverage against genetically diverse DENV-2 strains

  • Balancing broad serotype coverage with high neutralizing potency

  • Optimizing dosing and timing for therapeutic applications

  • Addressing production and cost considerations for global implementation

The most promising approaches likely involve humanized or fully human antibodies targeting conserved epitopes in DIII, with Fc modifications to prevent ADE. Combination approaches, either multiple antibodies targeting different epitopes or antibodies combined with antiviral drugs, may provide synergistic benefits for both prophylaxis and therapy.