ARI14 Antibody

What is the AM14 antibody and what makes it structurally unique?

AM14 is a human monoclonal antibody with potent RSV neutralizing capabilities that recognizes a quaternary epitope on the trimeric prefusion F (PreF) protein of Respiratory Syncytial Virus (RSV) . The most distinctive characteristic of AM14 is its exclusive capacity to bind an epitope that spans two F protomers within the prefusion conformation, making it uniquely trimer-specific . This property differentiates AM14 from other human antibodies that bind to RSV F, which typically recognize epitopes on a single F protomer . Even neutralizing antibodies like RSB1 and D25 that make minor contacts with a second F protomer still bind with high affinity to monomeric F, clearly distinguishing them from AM14's strict trimer specificity . The quaternary epitope recognized by AM14 is highly conserved among RSV A and B strains, explaining its similar neutralization potency toward both subtypes (RSV subtype A: IC₅₀ = 15.1 ng/mL; RSV subtype B: IC₅₀ = 11.0 ng/mL) .

How does AM14 compare to other RSV F-targeting antibodies?

AM14 differs significantly from other RSV F-targeting antibodies in its binding properties and epitope recognition. While many antibodies recognize antigenic sites shared between prefusion (PreF) and postfusion (PostF) conformations of RSV F (such as site II bound by palivizumab and motavizumab, or site IV bound by 101F), AM14 specifically recognizes a quaternary epitope only present in the prefusion conformation . This contrasts with other PreF-specific antibodies like D25 (targeting site Ø) and hRSV90, CR9501, and RSB1 (targeting site V), which bind to single-protomer epitopes . The unique trimer-specific binding property of AM14 makes it particularly valuable for assessing the trimeric state and conformational integrity of PreF-based immunogens during vaccine development . Studies have shown that sites present exclusively on PreF are targeted by the most potent neutralizing antibodies, while sites present on either PreF only or both PreF and PostF encompass the majority of the human B-cell repertoire for RSV .

What are the main applications of AM14 in RSV research and vaccine development?

AM14 serves critical functions in RSV research and vaccine development due to its unique trimer-specific binding properties:

• Conformational Probe: AM14 acts as a key reagent for probing the trimeric state of the RSV F protein during vaccine development, ensuring antigens maintain the desired prefusion conformation .

• Epitope Mapping: The antibody provides insights into critical quaternary epitopes on the prefusion F protein, guiding rational design of antigens that can elicit desired neutralizing responses .

• Quality Assessment: AM14 can be used to assess the quality and functionality of PreF-based immunogens during vaccine production .

• Structural Vaccinology: By understanding the AM14 epitope at high resolution, researchers can apply principles of structural vaccinology to design improved RSV vaccine candidates .

• Antibody Design Template: The unique specificity profile of AM14 provides a template for designing antibodies with customized specificity profiles using computational approaches .

The importance of AM14 is highlighted by the fact that PreF-specific antibodies (like AM14) have been shown to be more potently neutralizing than those targeting epitopes present in both PreF and PostF conformations .

What structural methods have improved our understanding of the AM14-RSV F interaction?

Recent structural studies have significantly enhanced our understanding of the AM14-RSV F interaction through complementary high-resolution techniques:

X-ray Crystallography: Researchers determined the crystal structure of DS-Cav1 (a prefusion-stabilized form of RSV F) bound by Fab AM14 at 3.6 Å resolution, which represents a ~2 Å improvement over previously published data (5.5 Å) . This higher resolution enabled clear visualization of side-chain densities and accurate mapping of the AM14 epitope on DS-Cav1 .

Cryo-electron Microscopy (cryo-EM): Parallel cryo-EM studies achieved a 3.4 Å resolution structure of the same complex, providing complementary data that confirmed and extended the X-ray crystallography findings . The combination of both methods allowed researchers to:

• Precisely delineate antibody/antigen contacts

• Map the complete epitope spanning two F protomers

• Understand the molecular basis for monoclonal antibody-resistant mutants (MARMs)

• Reveal flexibility for key antigenic residues on PreF

The key methodological advancement in these studies was the use of orthogonal structure determination methods to achieve improved resolution, providing the basis for comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens .

What is the molecular basis for AM14's epitope recognition and monoclonal antibody-resistant mutants?

The high-resolution structural studies of AM14 binding to DS-Cav1 have revealed detailed molecular interactions at the epitope and explained how certain mutations can confer resistance:

Key Epitope Interactions:
AM14 uses a combination of heavy- and light-chain CDRs to simultaneously contact portions of two DS-Cav1 protomers . The epitope on the membrane-proximal protomer (protomer 1) encompasses 531 Ų of surface area . Several somatic hypermutations (SHMs) in AM14 make critical interactions with DS-Cav1, including:

• Glu56 HCDR2 (mutated from serine)

• Ser28 HCDR1 (mutated from threonine)

• His31 HCDR2 (mutated from serine), which binds between the two F protomers

Molecular Basis for MARMs:
Four monoclonal antibody-resistant mutants (MARMs) have been identified that interfere with key AM14 interactions: L160S, N183K, N426D, and R429S . The molecular mechanisms for these resistance mutations include:

• Leu160: Located on the loop connecting helices ⍺2 and ⍺3, it recognizes a hydrophobic pocket formed by AM14 residues. Mutation to serine would disrupt this hydrophobic interaction .

• Asn426Asp: Located on the loop connecting β17-β18, it forms extensive hydrogen bonding interactions with AM14. The mutation would lead to a loss of charge complementarity and likely loss of a salt bridge .

• Arg429Ser: Also located on the loop connecting β17-β18, it participates in hydrogen bonding with AM14. Mutation would disrupt these interactions .

• N183K: Part of the epitope recognition, the mutation would interfere with antibody binding .

These findings provide a structural basis for understanding RSV escape mechanisms from AM14-like antibodies and highlight critical residues for PreF antigenicity .

How can computational approaches be used to design antibodies with AM14-like specificity?

Recent advances in computational biology have enabled the design of antibodies with tailored specificity profiles similar to AM14:

Biophysics-informed Modeling Approach:
Researchers have developed approaches combining high-throughput sequencing and machine learning to predict antibody properties beyond experimentally observed sequences . These models:

• Associate distinct binding modes with different potential ligands

• Enable prediction and generation of specific variants beyond those observed experimentally

• Allow for the design of antibodies with customized specificity profiles

Experimental Validation Process:
The computational approach has been validated through phage display experiments where:

• Data from one ligand combination is used to predict outcomes for another

• The model generates antibody variants not present in the initial library that are specific to given combinations of ligands

Applications for AM14-like Specificity:
For designing antibodies with AM14-like specificity, the model can:

• Identify and disentangle multiple binding modes associated with specific ligands

• Design antibodies with either specific high affinity for a particular target ligand or cross-specificity for multiple target ligands

• Mitigate experimental artifacts and biases in selection experiments

This approach extends beyond antibody design to the broader field of protein engineering, offering a powerful toolset for designing proteins with desired physical properties, including the exquisite binding specificity exemplified by AM14 .

What methodologies can be used to assess AM14 binding to prefusion F conformations?

To assess AM14 binding to prefusion F conformations in research and vaccine development, several methodological approaches can be employed:

Binding Assays:

• ELISA: Enzyme-linked immunosorbent assays can be used to measure direct binding of AM14 to PreF antigens, with the unique trimer specificity of AM14 providing a readout of properly folded trimeric PreF .

• Surface Plasmon Resonance (SPR): This technique allows for real-time measurement of binding kinetics between AM14 and PreF, providing information on association and dissociation rates that reflect conformational integrity .

Structural Analysis:

• X-ray Crystallography: As demonstrated in the research, crystallography at 3.6 Å resolution provides detailed structural information about AM14 epitope binding to DS-Cav1 .

• Cryo-EM: Complementary to X-ray studies, cryo-EM at 3.4 Å resolution can visualize the AM14-PreF complex, confirming trimer conformation .

Functional Assays:

• Neutralization Assays: Since AM14 has potent neutralizing activity against both RSV A and B subtypes (IC₅₀ = 15.1 ng/mL and 11.0 ng/mL respectively), neutralization potency can serve as a functional readout for proper epitope presentation .

• Competition Binding Assays: Competition between AM14 and other PreF-specific antibodies can map conformational integrity of different epitopes .

By combining these methodologies, researchers can comprehensively assess the conformational integrity of PreF antigens and their suitability for vaccine development.

What is the relationship between AM14 binding and RSV neutralization mechanisms?

The relationship between AM14 binding and RSV neutralization reveals important insights into protective immunity:

Neutralization Mechanism:
AM14's neutralization mechanism is linked to its recognition of a quaternary epitope that spans two protomers in the RSV F trimer . This unique binding property suggests that AM14 may:

• Stabilize the prefusion conformation, preventing the conformational changes required for membrane fusion

• Block interactions between the F protein and cellular receptors

• Inhibit the structural rearrangements necessary for viral entry

Correlation with Protection:
The potent neutralization activity of AM14 against both RSV A and B subtypes (IC₅₀ = 15.1 ng/mL and 11.0 ng/mL respectively) demonstrates that targeting this quaternary epitope is an effective strategy for virus neutralization . The high conservation of the AM14 epitope among RSV strains suggests it represents a vulnerability in the virus that is difficult to escape through mutation without affecting viral fitness .

Implications for Vaccine Design:
Understanding AM14's neutralization mechanism has direct implications for RSV vaccine design:

• The capacity to elicit AM14-like antibodies may be an important correlate of protection for PreF-based vaccines

• The quaternary nature of the epitope highlights the importance of maintaining the trimeric prefusion conformation in vaccine antigens

• The high-resolution structure of the AM14-PreF complex provides a template for designing immunogens that specifically present this vulnerable epitope

This relationship between binding and neutralization underscores why AM14 has become a critical tool for assessing the quality and potential efficacy of PreF-based vaccine candidates.