ARR14 Antibody

What is AM14 antibody and what is its target specificity?

AM14 is a prefusion-specific human IgG1 monoclonal antibody that specifically recognizes and binds to the respiratory syncytial virus (RSV) fusion (F) glycoprotein. Unlike other RSV-targeting antibodies, AM14 exhibits exclusive binding to the trimeric, furin-cleaved prefusion F conformational state, which represents the mature form of F on infectious virions . This high specificity makes AM14 particularly valuable for characterizing RSV F-based vaccine antigens and for therapeutic applications targeting the prefusion conformation.

How does AM14 differ from other anti-RSV antibodies in terms of binding characteristics?

AM14 differs from other anti-RSV antibodies in several critical ways:

• Conformational specificity: AM14 binds exclusively to trimeric prefusion F, while other antibodies like D25 can bind to both monomeric and trimeric forms.

• Binding kinetics: AM14 exhibits significantly faster association rates (1.87 × 10^7 M^−1s^−1) compared to D25, which binds approximately 10 times slower. This unique kinetic profile may contribute to AM14's enhanced neutralization efficiency.

• Epitope recognition: AM14 recognizes a quaternary epitope that spans two protomers, requiring the intact trimer structure, whereas other antibodies target epitopes present on monomeric forms of F .

These differences highlight AM14's unique value in RSV research and therapeutic development.

What is the structural basis for AM14's neutralization activity?

The neutralization activity of AM14 is based on its recognition of a quaternary epitope that spans two protomers of the prefusion F trimer. This epitope includes three critical regions that undergo extensive conformational changes during the pre- to postfusion F transition :

• The loop between α2 and α3 helices

• The loop between β3 and β4 strands

• The α4 helix near antigenic site Ø

Key residues critical for binding have been identified through monoclonal antibody-resistant mutants (MARMs):

By binding to this complex epitope, AM14 effectively blocks the conformational changes necessary for viral fusion, thus preventing viral entry at a post-attachment stage.

How can AM14 be used to validate prefusion-stabilized F antigens in vaccine development?

AM14's unique specificity for the trimeric prefusion F conformation makes it an excellent tool for quality control in vaccine development. Researchers can use AM14 to:

• Confirm proper folding: As AM14 binds exclusively to correctly folded trimeric prefusion F, positive binding confirms that recombinant antigens maintain the desired conformation.

• Monitor stability: During vaccine formulation and storage, AM14 binding assays can track the stability of prefusion F conformations over time and under various conditions.

• Validate antigen display: For nanoparticle or virus-like particle (VLP) vaccines, AM14 can verify that antigens maintain their native prefusion conformation when displayed on carrier platforms.

• Quality control: In manufacturing processes, AM14-based assays can ensure batch-to-batch consistency of prefusion F antigens.

Methodologically, researchers should incorporate appropriate controls, including known prefusion-stabilized antigens (e.g., DS-Cav1) and postfusion F proteins to ensure assay specificity.

What methodological approaches can be used to assess AM14's neutralization potency against clinical RSV isolates?

When evaluating AM14's neutralization potency against clinical RSV isolates, researchers should consider these methodological approaches:

• Plaque reduction neutralization test (PRNT): This gold-standard method allows quantification of neutralization activity by measuring the reduction in viral plaque formation. Data should be reported as IC50 values (ng/mL or nM).

• Microneutralization assay: This higher-throughput alternative measures the inhibition of cytopathic effects in cell cultures using colorimetric or luminescent readouts.

• Flow cytometry-based neutralization: This method quantifies the percentage of infected cells using fluorescent markers.

• Reference standards: Include palivizumab as a comparative control, as AM14 has shown approximately 100-fold higher potency (IC50 = 0.18-2.1 ng/mL for AM14 versus 209 ng/mL for palivizumab).

• Strain diversity: Test against both RSV-A and RSV-B clinical isolates to confirm broad neutralization activity.

This comprehensive approach provides robust assessment of neutralization potency and breadth.

How can surface plasmon resonance (SPR) be optimized to characterize AM14-RSV F interactions?

Optimizing SPR for AM14-RSV F binding studies requires careful consideration of several parameters:

• Surface preparation:

  • Immobilize AM14 antibody using amine coupling chemistry to CM5 sensor chips

  • Aim for low surface density (200-400 response units) to minimize mass transport limitations

  • Include a reference surface with an irrelevant IgG antibody

• Analyte preparation:

  • Use highly purified prefusion F trimers (>95% purity by SDS-PAGE)

  • Prepare a concentration series (typically 0.1-50 nM) in running buffer containing 0.005% surfactant

• Kinetic analysis:

  • Use a multicycle kinetic approach with regeneration between cycles (10 mM glycine pH 1.5)

  • Include sufficiently long association (120-180s) and dissociation phases (600-1200s)

  • Apply a 1:1 Langmuir binding model for data fitting

• Controls and validation:

  • Run prefusion F monomers as negative controls (should show no binding)

  • Include D25 antibody as a positive control that binds both monomeric and trimeric F

  • Validate trimeric state of F protein by analytical size exclusion chromatography

This approach will enable accurate determination of binding kinetics, including the characteristic rapid association rate (1.87 × 10^7 M^−1s^−1) and dissociation rate (3.4 × 10^−3 s^−1) of AM14.

What strategies can be employed to study AM14 epitope accessibility during the viral fusion process?

Studying AM14 epitope accessibility during viral fusion requires sophisticated approaches that capture this dynamic process:

• Temperature-jump experiments:

  • Pre-bind AM14 to RSV at 4°C

  • Rapidly increase temperature to 37°C to trigger fusion

  • Assess binding retention at various timepoints using flow cytometry or imaging

• Stopped-flow kinetic analysis:

  • Mix RSV with target cells in a stopped-flow apparatus

  • Introduce AM14 at defined time points after mixing

  • Monitor fluorescence changes associated with fusion inhibition

• Single-particle tracking:

  • Label AM14 with quantum dots or fluorophores

  • Track binding to individual virions during fusion using super-resolution microscopy

  • Correlate binding with fusion events marked by lipid mixing dyes

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium incorporation in free F protein versus AM14-bound F

  • Identify regions protected by AM14 during fusion-triggering conditions

  • Map temporal changes in epitope accessibility

• Cryo-electron tomography:

  • Visualize RSV fusion intermediates in the presence and absence of AM14

  • Create 3D reconstructions of fusion complexes at nanometer resolution

These methodologies provide complementary insights into how AM14's quaternary epitope changes during the fusion process, informing both basic virology research and therapeutic development.

What crystallization approaches have been successful for the AM14-RSV F complex, and how can they be optimized?

Successfully crystallizing the AM14-RSV F complex requires overcoming several challenges inherent to antibody-antigen complexes, particularly those involving conformationally sensitive glycoproteins:

• Protein engineering strategies:

  • Use Fab fragments rather than whole IgG to reduce conformational heterogeneity

  • Remove flexible glycans from both RSV F and the antibody by enzymatic treatment or expression in GnTI-deficient cells

  • Consider creating disulfide-stabilized prefusion F constructs to maintain conformational homogeneity

• Complex formation optimization:

  • Isolate the complex by size exclusion chromatography after mixing purified components

  • Verify complex formation by analytical ultracentrifugation

  • Assess monodispersity using dynamic light scattering prior to crystallization trials

• Crystallization conditions:

  • Employ sparse matrix screening at multiple protein concentrations (5-15 mg/mL)

  • Explore both vapor diffusion and lipidic cubic phase methods

  • Include small molecules that enhance crystal contacts (e.g., specific metal ions)

  • Test crystallization at both 4°C and 20°C

• Data collection considerations:

  • Use microfocus beamlines for small crystals

  • Consider multiple crystal averaging to improve data quality

  • Implement anisotropic diffraction correction if necessary

Notably, the AM14-RSV F complex structure determination has previously succeeded by focusing on the unique quaternary epitope that spans two protomers, providing insights into its exclusive recognition of the prefusion trimer conformation .

How can computational modeling be integrated with experimental data to enhance our understanding of AM14 binding?

Integrating computational modeling with experimental data provides comprehensive insights into AM14 binding mechanisms:

• Homology modeling workflow:

  • Generate initial antibody models using tools like PIGS server or AbPredict

  • Refine models through molecular dynamics simulations

  • Validate structural predictions against experimental binding data

• Epitope mapping integration:

  • Incorporate mutational data from monoclonal antibody-resistant mutants (MARMs)

  • Use saturation transfer difference NMR (STD-NMR) to define antigen contact surfaces

  • Employ hydrogen-deuterium exchange mass spectrometry to identify protected regions

• Docking and validation approach:

  • Generate thousands of potential binding poses through automated docking

  • Filter poses using experimental constraints from mutagenesis data

  • Rank remaining models using binding energy calculations

  • Select optimal models based on agreement with multiple experimental metrics

• Molecular dynamics refinement:

  • Conduct extended simulations of the antibody-antigen complex

  • Analyze stability of key interactions over time

  • Identify water-mediated hydrogen bonds that may not be evident in static models

This integrative approach has been successfully applied to antibody-antigen systems where traditional structural methods like crystallography face challenges , providing structural insights that can guide both basic understanding and therapeutic development.

How does AM14 compare with other prefusion-specific antibodies in terms of escape mutant generation?

When comparing AM14 with other prefusion-specific antibodies regarding escape mutant generation, researchers should consider these methodological approaches and findings:

• Escape mutant selection protocols:

  • Perform serial passage of RSV in the presence of sub-neutralizing concentrations of AM14

  • Gradually increase antibody concentration over passages

  • Sequence the F gene from resistant viruses to identify mutations

• Comparative analysis with other antibodies:

  • AM14 targets a quaternary epitope spanning two protomers, which presents a higher genetic barrier to escape compared to antibodies targeting single protomer epitopes

  • Mutations affecting AM14 binding (L160S, N183K, N426D/R429S) are distinct from those affecting site Ø antibodies like D25

  • The requirement for maintaining prefusion trimer stability constrains potential escape pathways

• Functional impact assessment:

  • Evaluate how escape mutations affect viral fitness and fusion capacity

  • Determine if AM14-resistant variants remain susceptible to other prefusion-specific antibodies

  • Assess cross-resistance patterns to inform combination therapy approaches

• Structural context analysis:

  • Map escape mutations onto the F protein structure to understand their mechanistic effects

  • Analyze if mutations directly disrupt antibody contact or indirectly alter epitope conformation

  • Identify regions under immune pressure that may benefit from stabilization in vaccine design

This comparative framework helps researchers understand the unique resistance profile of AM14 and informs strategies to minimize escape through antibody combinations or improved immunogen design.

What synergistic effects might be observed when combining AM14 with other anti-RSV antibodies or antivirals?

Investigating synergistic effects between AM14 and other anti-RSV agents requires systematic evaluation:

• Combination neutralization assays:

  • Use checkerboard matrix testing with varying concentrations of AM14 and partner agents

  • Calculate combination indices (CI) to quantify synergy, additivity, or antagonism

  • Apply both Loewe additivity and Bliss independence models for robust analysis

• Partner selection rationale:

  • Combine with antibodies targeting non-overlapping epitopes (e.g., palivizumab targeting site II)

  • Test with antibodies binding different conformational states (e.g., prefusion and postfusion)

  • Evaluate combinations with small-molecule fusion inhibitors or entry inhibitors

• Mechanism investigation:

  • Assess whether combinations enhance coverage against escape mutants

  • Determine if certain combinations block multiple steps in the viral entry process

  • Investigate if AM14's quaternary epitope binding creates conformational constraints that enhance binding of other antibodies

• In vivo validation:

  • Test promising combinations in animal models of RSV infection

  • Measure viral load reduction, pathology scores, and inflammatory markers

  • Evaluate dose-sparing potential of combination approaches

• Clinical translation considerations:

  • Design combination regimens that balance potency with manufacturing and regulatory complexity

  • Consider pharmacokinetic matching to ensure consistent coverage

  • Evaluate formulation compatibility for co-administration

This systematic approach identifies optimal combinations that might provide superior protection through complementary mechanisms of action, particularly valuable for high-risk populations requiring enhanced prophylaxis.

What are the critical quality attributes to monitor when working with AM14 in research applications?

When working with AM14 antibody for research applications, several critical quality attributes should be monitored to ensure reliable results:

• Conformational integrity assessment:

  • Verify binding specificity using prefusion-stabilized F versus postfusion F in ELISA

  • Monitor thermal stability through differential scanning fluorimetry

  • Assess aggregation state by size exclusion chromatography

• Functional activity verification:

  • Determine neutralization potency (IC50) against reference RSV strains

  • Compare with historical values (e.g., 0.18-2.1 ng/mL range)

  • Validate trimer-specific binding through competitive binding assays

• Storage and handling considerations:

  • Store antibody (in aliquots) at -20°C to maintain stability

  • Avoid repeated freeze-thaw cycles to prevent aggregation

  • Monitor post-thaw activity periodically

• Batch-to-batch consistency:

  • Implement reference standard comparisons for each new lot

  • Establish acceptance criteria for binding affinity (KD = 0.18 nM)

  • Document association (1.87 × 10^7 M^−1s^−1) and dissociation rates (3.4 × 10^−3 s^−1)

• Contaminant evaluation:

  • Ensure SDS-PAGE purity >95%

  • Verify low endotoxin levels for cell-based assays

  • Confirm absence of azide in functional studies

Implementing these quality control measures ensures that experimental results with AM14 are reproducible and accurately reflect the antibody's unique prefusion-specific binding properties.

How can researchers address potential false negative results when using AM14 as a conformational probe?

When using AM14 as a probe for prefusion F conformation, researchers may encounter false negative results that require systematic troubleshooting:

• Sample preparation factors:

  • Buffer composition: Ensure pH 7.0-7.5 and physiological salt concentration

  • Detergent interference: Minimize detergent usage or switch to less disruptive alternatives

  • Cryptic epitope exposure: Test gentle fixation methods that preserve quaternary structure

• Assay optimization strategies:

  • Titrate both antibody and antigen concentrations

  • Extend incubation times to allow for complete binding equilibrium

  • Optimize washing steps to reduce disruption of conformational epitopes

  • Include positive controls (e.g., prefusion-stabilized DS-Cav1 constructs)

• Alternative detection methods:

  • If direct ELISA fails, try sandwich ELISA with a capture antibody targeting a different epitope

  • Employ native PAGE followed by Western blotting instead of denaturing conditions

  • Consider flow cytometry for cell-surface expressed F protein

  • Use bio-layer interferometry as an alternative to plate-based assays

• Confirmatory approaches:

  • Test binding with additional prefusion-specific antibodies (e.g., D25)

  • Employ negative stain electron microscopy to directly visualize trimer morphology

  • Use thermal shift assays to assess conformational stability

• Interpretation guidelines:

  • Consider threshold adjustment based on signal-to-noise ratio

  • Implement quantitative criteria for positive versus negative results

  • Document all experimental conditions to facilitate troubleshooting

These approaches help distinguish true negative results (genuine absence of prefusion F) from technical artifacts, ensuring reliable use of AM14 as a conformational probe in various research contexts.

What novel applications of AM14 might emerge in RSV vaccine development and therapeutic design?

AM14's unique properties open several promising avenues for future research:

• Structure-guided immunogen design:

  • Engineer stabilized RSV F proteins that preferentially display the AM14 quaternary epitope

  • Create nanoparticle displays that present multiple copies of the AM14 epitope in the correct orientation

  • Develop epitope-focused vaccines targeting the specific regions recognized by AM14

• Antibody engineering opportunities:

  • Generate bispecific antibodies combining AM14's binding domain with other RSV-targeting specificities

  • Modify Fc regions to enhance effector functions while maintaining neutralization capacity

  • Engineer single-domain antibody derivatives with improved tissue penetration

• Diagnostic applications:

  • Develop rapid conformational assays to evaluate vaccine quality

  • Create point-of-care tests distinguishing RSV strains based on prefusion F epitope variations

  • Establish imaging probes for in vivo tracking of RSV infection sites

• Combination therapy platforms:

  • Design cocktail formulations containing AM14 with complementary antibodies

  • Create single-molecule multi-specific antibodies incorporating AM14 binding domains

  • Develop synergistic combinations with small-molecule antivirals

These innovative applications leverage AM14's quaternary epitope recognition to advance both preventive and therapeutic interventions against RSV infection.

How might advances in structural biology techniques further elucidate the AM14-RSV F complex?

Emerging structural biology techniques offer new opportunities to further characterize the AM14-RSV F complex:

• Cryo-electron microscopy advancements:

  • Apply single-particle cryo-EM with 2-3Å resolution to visualize the complete AM14-F trimer complex

  • Implement time-resolved cryo-EM to capture conformational changes during binding

  • Use cryo-electron tomography to study AM14 binding to authentic virions

• Integrative structural approaches:

  • Combine X-ray crystallography of domain fragments with cryo-EM of the full complex

  • Implement cross-linking mass spectrometry to map interface residues

  • Apply small-angle X-ray scattering to analyze solution dynamics

• Advanced spectroscopic methods:

  • Use single-molecule FRET to track conformational changes upon AM14 binding

  • Implement advanced HDX-MS protocols with improved spatial and temporal resolution

  • Apply site-specific infrared spectroscopy to probe hydrogen bonding networks

• Computational advances:

  • Implement enhanced sampling molecular dynamics to explore conformational space

  • Apply machine learning approaches to predict epitope accessibility during fusion

  • Develop physics-based scoring functions for more accurate binding energy prediction

• In situ structural biology:

  • Study AM14-F interactions in cellular membranes using correlative light and electron microscopy

  • Apply cellular cryo-electron tomography to visualize binding in a native context

  • Implement super-resolution microscopy to track AM14-F complexes during viral entry