Infectious DiseaseStructural BiologyCompleted

In Silico Target Discovery for Invariant Antigens: Structural Epitope Screening

A high-throughput structural bioinformatics screening pipeline predicting conformational stability, epitope exposure, and mutational preservation in viral surface glycoproteins under evolutionary antibody pressure.

STATUSCompleted

STARTEDJan 2025

FIELDInfectious Disease

KEYWORDSStructural Biology, Epitope Discovery, AlphaFold, Molecular Dynamics, Antigen Design, Immunoinformatics

TOOLSPython, AlphaFold2, PyMOL, FoldX, BioPython, OpenMM, Scikit-learn

THE QUESTION

Can structural thermodynamic stability scores and solvent accessibility profiling predict which pathogen surface epitopes resist conformational distortion during antigenic drift?

BACKGROUND

Vaccine and therapeutic monoclonal antibody efficacy frequently deteriorates due to antigenic escape mutations in pathogen surface proteins. Identifying invariant conformational epitopes that cannot easily mutate without catastrophic loss of viral structural stability (fitness penalty) is the cornerstone of universal vaccine engineering. This project established a computational screening workflow combining AlphaFold structural models, molecular dynamics relaxation, and FoldX in silico alanine scanning to discover structurally indispensable antigenic patches.

Macromolecular conformational energy landscape and thermodynamic state transitions

APPROACH OVERVIEW

STRUCTURAL ENSEMBLE MODELING

Multi-state structural modeling of viral trimeric envelope glycoproteins using AlphaFold2-multimer and experimental cryo-EM coordinates (PDB).

SOLVENT ACCESSIBILITY & BINDING SITE MAPPING

Computation of Relative Solvent Accessibility (RSA) and residue contact surface area across open and closed pre-fusion conformations.

SATURATION IN SILICO MUTAGENESIS

Systematic deep in silico mutagenesis (19 non-native amino acids per locus) using FoldX to calculate free energy changes (delta-delta-G) upon mutation.

MOLECULAR DYNAMICS RELAXATION

100 ns all-atom molecular dynamics simulations in explicit solvent (OpenMM) evaluating root-mean-square fluctuation (RMSF) of candidate epitope loops.

IMMUNOGENIC PATCH RANKING

Multi-parameter ranking scoring candidate epitopes by high structural essentiality (delta-delta-G > 2.5 kcal/mol) and immune antibody accessibility (RSA > 25%).

METHODS

•Structure Generation: Cryo-EM coordinates refined with AlphaFold2-multimer predicting full trimeric viral spike assemblies.
•Thermodynamic Free Energy Calculations: Systematic FoldX alanine and saturation scanning evaluating thermodynamic destabilization ( $\Delta\Delta G = \Delta G_{\text{mut}} - \Delta G_{\text{wt}}$ ).
•Epitope Geometry Analysis: DSSP algorithm calculating secondary structure assignments and relative solvent accessible surface areas.
•Molecular Dynamics Simulations: CHARMM36m force field in OpenMM with TIP3P water box at 310 K and 1 bar pressure, monitoring root-mean-square deviations (RMSD).
•Cross-Neutralizing Epitope Matching: Structural alignment with known broadly neutralizing antibodies (bNAbs) from the Immune Epitope Database (IEDB).

KEY DATA SNAPSHOT

EPITOPES SCREENED320 PatchesStructural Surface Scans

TOP INVARIANT PATCHES4 SitesddG > 3.0 kcal/mol & RSA > 30%

MD SIMULATION TIME100 nsExplicit Solvent Stability

CROSS-STRAIN HIT RATE96.4%Predicted Structural Conservation

RESULTS

In silico screening identified four structurally constrained conformational epitope patches on the viral fusion glycoprotein stem that cannot mutate without destabilizing the trimer assembly (average delta-delta-G > 3.2 kcal/mol), maintaining 96.4% structural conservation across disparate viral lineages.

DISCUSSION

Epitopes located in the membrane-proximal external region (MPER) and fusion peptide base showed the highest thermodynamic fitness penalties for escape mutations.

Variable head domain loops tolerated extensive substitutions (ddG < 0.8 kcal/mol), explaining why receptor-binding domains evolve rapid immune evasion.

Targeting these four high-barrier structural patches provides an actionable blueprint for scaffold-based immunogen design.

LIMITATIONS

•In silico free energy estimations do not fully capture post-translational N-linked glycosylation steric shields without specialized glyco-modeling algorithms.
•Experimental wet-lab surface plasmon resonance (SPR) binding kinetics are required to validate antibody affinity constants.

IMPACT & APPLICATION

Structural Target PrioritizationDelivered a catalog of 4 structurally invariant neutralizing epitopes on polymorphic viral envelope proteins.

Open Screening ProtocolDeveloped automated Python scripts orchestrating AlphaFold structure parsing, FoldX mutagenesis, and solvent accessibility filtering.

DATA & REPRODUCIBILITY

Analytical code and specific target coordinates are currently held under institutional review and confidential protocol.

Epitope Screening PipelinePython scripts wrapping DSSP, FoldX, and OpenMM for structural epitope triage.

Immune Epitope Database (IEDB)Reference repository for validated monoclonal antibody epitopes.

REFERENCES

jumper2021Jumper, J., et al. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596(7873), 583–589.
schymkowitz2005Schymkowitz, J., et al. (2005). The FoldX web server: an online force field. Nucleic Acids Research, 33(suppl_2), W382–W388.