ASK14 Antibody

What is the AM14 antibody and what viral target does it recognize?

AM14 is a recombinant human antibody capable of binding to the Respiratory Syncytial Virus (RSV). It is expressed in HEK 293 cells as a combination of a heavy chain containing VH from anti-RSV monoclonal antibody and CH1-3 region of human IgG1, along with a light chain encoding VL from anti-RSV mAb and CL of human kappa light chain. The antibody exists as a disulfide-linked dimer of the heavy chain and light chain hetero-dimer under non-reducing conditions .

What is the A14 antibody and what is its significance in viral research?

A14 antibodies target the A14 protein of vaccinia virus (VACV). A14 is a dominant antibody target in smallpox vaccine responses. Research has identified 22 monoclonal antibodies (mAbs) targeting A14, with most binding to the C-terminus region, indicating this region is the immunodominant epitope .

What are the structural characteristics of AM14 antibody?

AM14 antibody has a characteristic immunoglobulin structure composed of two heavy chains (HC) and two light chains (LC). The heavy chain contains the variable region (VH) from an anti-RSV monoclonal antibody and the constant regions (CH1-3) from human IgG1. The light chain contains the variable region (VL) from an anti-RSV monoclonal antibody and the constant region (CL) from human kappa light chain .

How can researchers validate the binding specificity of AM14 to RSV?

Researchers can validate AM14 binding specificity using several complementary approaches:

• Luminex System: Binding of AM14 to immobilized RSV F proteins can be measured using a Luminex system, which allows for multiplex analysis of protein interactions .

• Surface Plasmon Resonance: Binding of AM14 Fab fragments to immobilized prefusion RSV F can be measured by surface plasmon resonance, providing real-time binding kinetics and affinity measurements .

• Neutralization Assays: Researchers should test AM14's ability to neutralize both laboratory strains and clinical isolates of RSV from both A and B subtypes, as previous studies have shown AM14 potently neutralizes these viral strains .

What methodologies can be employed to study the internalization of antibody fragments in RSV-infected cells?

Based on experimental protocols described in the literature, researchers can study internalization using the following methodology:

• Infect HEp-2 cells with RSV

• Incubate infected cells with RSV F-specific MAbs (such as D25, AM14, 5C4, and MPE8) or corresponding monomeric Fab fragments at the same concentration for 90 minutes to induce internalization

• Fix and permeabilize the cells

• Stain with appropriate secondary antibodies (e.g., AF488 human anti-goat IgG or AF488 chicken anti-mouse IgG)

• Visualize nuclei with DAPI

• Quantify internalized vesicles in positive cells (typically 50 cells should be analyzed)

How can researchers map epitopes on the A14 protein of vaccinia virus?

Two complementary methods for epitope mapping of A14 have been successfully employed:

• Western Blot Method:

  • Create fusion proteins of Glutathione S-transferase (GST) and A14 by PCR-amplifying the viral gene from VACV WR DNA

  • Clone PCR fragments into pGEX6P-1

  • Express fusion proteins in E. coli BL21 strain using IPTG induction

  • Lyse bacteria via sonication in SDS-PAGE sample buffer

  • Use clarified cell lysates directly in Western blot analysis with anti-A14 antibodies

• ELISA Method with Infected Cells:

  • Transfect 293T cells with plasmids containing mutant alleles of the A14 gene under VACV promoter

  • Infect the cells with iA14/GFP (ITPG-inducible A14 mutant VACV)

  • Harvest cells at 24 hours post-infection

  • Use cell lysates to coat 96-well microtiter plates

  • Perform ELISA with 1 μg/ml anti-A14 antibodies

How do AM14 antibodies differ in their mechanisms from other anti-RSV antibodies?

AM14 has distinct mechanisms compared to other RSV-targeting antibodies like D25, 5C4, and MPE8:

• Quaternary Epitope Recognition: AM14 recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein, making it unique compared to other antibodies that recognize linear epitopes .

• Prefusion Specificity: AM14 preferentially binds to the prefusion conformation of the RSV F protein, which is critical for neutralization efficiency .

• Internalization Dynamics: Studies of internalization of Fab fragments showed different patterns of cellular processing between AM14 and other antibodies like D25, 5C4, and MPE8 when incubated with RSV-infected cells .

What are the challenges in evaluating in vivo protection by A14 antibodies against vaccinia virus?

Based on research findings, several challenges exist when evaluating A14 antibodies' protective effects:

• Limited Surface Accessibility: A14 is an inherently poor neutralizing target due to lack of antibody binding sites on the virion surface. EM studies showed that antibodies against both the middle region (8C6) and the C-terminus did not bind to mature virions (MV) .

• Structural Limitations: A14 has two transmembrane domains that dictate that either the middle region (residues 32-44) or the N- and C-termini are projected out of MV, but all possible antibody targets on A14 appear to be either enclosed within virions or largely inaccessible for antibody binding on the virion surface .

• Translation from in vitro to in vivo: The study demonstrated that some antibodies like 9C3 and HE6 provided protection against weight loss, death, and pox lesions in a SCID mouse model, while others like 8C6 showed modest efficacy that didn't match its in vitro neutralization results .

How can researchers distinguish between neutralizing and non-neutralizing epitopes on viral proteins?

Researchers can employ the following methodological approaches:

• Structural Analysis: Combine X-ray crystallography or cryo-EM with epitope mapping to determine if antibody binding sites are accessible on the viral surface.

• Neutralization Correlation Studies: Test panels of monoclonal antibodies against different epitopes for their neutralizing activity and correlate this with epitope location.

• Virion Binding Assays: As demonstrated in the A14 studies, electron microscopy can be used to directly visualize whether antibodies bind to mature virions, helping distinguish accessible from inaccessible epitopes .

• Mutational Analysis: Generate viral mutants with modifications in potential epitopes and assess the impact on antibody binding and neutralization.

What regions of the A14 protein are targeted by antibody responses?

Research has identified at least two regions of A14 as targets of antibody responses:

• C-terminus (residues 65-90): This 26-amino acid region is the dominant target, with an overwhelming number of antibodies from two separate screens targeting this region .

• Middle region (residues 32-44): This 13-amino acid region is also an antibody target but is relatively minor, with only 1 out of 22 monoclonal antibodies targeting this region in mouse studies .

• N-terminus (residues 1-12): This 12-amino acid region is a potential antibody target, predicted to be on the same side of the virion membrane as the C-terminus, suggesting antibodies against this region would behave similarly to those against the C-terminal region in terms of virion binding and neutralizing abilities .

Why is the AM14 antibody effective against both RSV subtypes A and B?

The AM14 antibody's effectiveness against both RSV subtypes A and B can be attributed to several factors:

• Recognition of Conserved Epitopes: AM14 targets epitopes that are conserved between the two major RSV subtypes.

• Quaternary Epitope Targeting: The antibody recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein that is maintained across subtypes .

• Prefusion Conformation Specificity: By specifically targeting the prefusion conformation of the F protein, AM14 can neutralize the virus before it undergoes the conformational changes required for membrane fusion, which is a conserved mechanism across subtypes .

What controls should be included when evaluating the protective efficacy of antibodies in animal models?

Based on the experimental design described in the vaccinia virus studies, researchers should include the following controls:

• Negative Control Antibody: Use an antibody known not to provide protection (e.g., anti-A10 mAb BG3 was used as a negative control in the vaccinia virus study) .

• Positive Control Antibody: Include an antibody with known protective effects (e.g., anti-H3 #41 was used as a positive control in the vaccinia virus study) .

• Mock-Treated Controls: Include animals treated with buffer or irrelevant antibodies to establish baseline disease progression.

• Dose-Response Assessment: Test multiple antibody concentrations to establish dose-dependent protection.

• Multiple Outcome Measurements: Assess multiple parameters (e.g., weight loss, survival, clinical lesions) to comprehensively evaluate protection .

What methodological approaches can be used to assess antibody internalization in virus-infected cells?

To properly assess antibody internalization, researchers should consider:

• Time-Course Experiments: Incubate virus-infected cells with antibodies for different time periods (e.g., 90 minutes as used in the RSV study) to capture the kinetics of internalization .

• Visualization Techniques:

  • Fix, permeabilize, and stain cells with fluorescently labeled secondary antibodies

  • Use confocal microscopy for high-resolution imaging of internalized antibodies

  • Employ dual staining with markers for different endocytic compartments to track intracellular trafficking

• Quantification Methods:

  • Count the number of internalized vesicles in a standardized number of cells (e.g., 50 positive cells)

  • Use automated image analysis software for unbiased quantification

  • Measure fluorescence intensity as a proxy for internalization efficiency

• Controls: Include non-internalizing antibodies and perform experiments at 4°C (which inhibits endocytosis) as negative controls.

How can researchers address potential discrepancies between in vitro neutralization and in vivo protection data?

Based on the findings with A14 antibodies, researchers should consider the following approaches:

• Mechanism Analysis: Investigate whether protection in vivo might be mediated by mechanisms other than direct neutralization, such as antibody-dependent cellular cytotoxicity (ADCC) or complement activation.

• Pharmacokinetics: Assess antibody stability, half-life, and tissue distribution in vivo, as these factors might influence protective efficacy independently of neutralizing potency.

• Model Appropriateness: Consider whether the in vitro neutralization assay accurately models the in vivo infection process. For example, the study with 8C6 showed modest efficacy in vivo despite promising in vitro neutralization results .

• Antibody Concentration: Evaluate whether differences in achievable antibody concentrations between in vitro and in vivo settings might explain discrepancies.

• Fc-Mediated Effects: Investigate whether Fc-dependent functions contribute to in vivo protection but are not captured in standard neutralization assays.

What factors might affect the reproducibility of epitope mapping experiments?

Several factors can influence epitope mapping reproducibility:

• Protein Conformation: Expression systems (bacterial vs. mammalian) may affect protein folding, potentially altering epitope presentation. When mapping A14 epitopes, researchers used both bacterial expression (GST fusion proteins) and mammalian expression (transfected 293T cells) .

• Fragment Selection: The choice of protein fragments for mapping can impact results if they disrupt conformational epitopes or create artificial binding sites at fragment junctions.

• Assay Conditions: Differences in pH, salt concentration, temperature, and detergent use can affect antibody-epitope interactions.

• Cross-Reactivity: Antibodies may cross-react with similar epitopes, as observed with moderate cross-reactivity of anti-MMP14 antibodies with other metalloproteinases .

• Detection Methods: Different detection systems (Western blot vs. ELISA) may have varying sensitivities and could lead to different interpretations, which is why the A14 study employed both methods .

What are promising research avenues for improving antibody-based therapies against RSV and vaccinia virus?

Based on the current literature, promising research directions include:

• Structure-Guided Antibody Engineering: Using structural information about AM14's interaction with the RSV F protein to engineer antibodies with enhanced binding affinity and neutralizing capacity.

• Combination Therapy Approaches: Investigating the potential synergistic effects of combining antibodies targeting different epitopes, such as combining AM14 with other RSV-neutralizing antibodies like D25.

• Improving Delivery Systems: Developing delivery methods that can enhance antibody penetration into respiratory tissues for RSV or relevant tissues for vaccinia virus.

• Novel Epitope Discovery: Further characterizing the viral proteome to identify additional antibody targets beyond the currently known epitopes.

• Germline Antibody Studies: Investigating whether antibodies with germline configuration and specificity for tumor antigens exert antitumoral effects and whether they can trigger loss of self-tolerance, as referenced in cancer antibody research .

How might new methodologies enhance our understanding of antibody-virus interactions?

Emerging technologies that could advance antibody-virus interaction studies include:

• Single-Particle Cryo-EM: This technique can provide high-resolution structures of antibody-virus complexes in their native state without crystallization.

• Super-Resolution Microscopy: Technologies like STORM or PALM can provide nanometer-scale resolution of antibody binding and internalization events within infected cells.

• Single-Cell Antibody Sequencing: This approach can identify rare but potentially important antibody sequences within polyclonal responses.

• In Situ Structural Analysis: Techniques like cryo-electron tomography can visualize antibody-virus interactions directly within infected cells.

• AI-Assisted Epitope Prediction: Machine learning algorithms trained on existing antibody-antigen interaction data could predict novel epitopes and optimize antibody design.