VACWR150 Antibody

What is the VACWR150 protein and why is it a significant antibody target?

VACWR150 refers to the Vaccinia virus A27 protein, a structural component of the viral envelope that is conserved across the Orthopoxvirus genus. This protein has multiple critical functions: it binds to cell surface heparan sulfate, anchors A26 protein into mature virions, and plays an essential role in the egress of mature virus from infected cells . As a surface-exposed protein with critical functions, A27 serves as an excellent neutralizing target for antibody development, making it valuable for both basic research and potential therapeutic applications .

What is the molecular structure of the A27 protein that antibodies target?

The A27 protein forms a unique structural assembly with important functional implications. Crystallographic studies at 2.2 Å resolution reveal that A27 uses its N-terminal region interface (NTR) to form a trimeric assembly as the basic unit. This trimer contains two parallel α-helices and one unusual antiparallel α-helix. In a serpentine pattern, two trimers stack together to form a hexamer using the C-terminal region interface (CTR) . This complex oligomeric structure is critical for understanding antibody binding sites and developing antibodies that can effectively target functional domains.

How does A27 protein interact with other viral components?

The A27 protein forms disulfide-linked protein complexes with the A26 protein, which modulates the membrane fusion activity of mature virions upon cell entry . Additionally, A27 protein is anchored to the mature virion membrane through interaction with the A17 transmembrane protein. This network of interactions makes A27 a central player in viral structure and function, providing multiple potential mechanisms through which anti-A27 antibodies might exert their effects .

What are the primary research applications of anti-VACWR150 antibodies?

Anti-VACWR150 antibodies serve several important research functions:

• Neutralization studies: Evaluating the capacity of antibodies to prevent viral infection in cell culture systems, which can be measured through neutralization assays

• Structural analysis: Investigating protein-protein interactions essential for viral assembly

• Immunological research: Studying host immune responses to orthopoxvirus infections

• Development of antiviral therapeutics: Validating A27 as a target for inhibiting viral replication

• Diagnostic applications: Detecting vaccinia or related orthopoxviruses in research samples

What experimental methods are most effective for evaluating anti-VACWR150 antibody specificity?

For rigorous evaluation of antibody specificity:

• ELISA (Enzyme-Linked Immunosorbent Assay): Provides quantitative measurement of antibody binding to purified recombinant A27 protein

• Western Blotting: Confirms antibody recognition of denatured A27 protein from viral lysates

• Immunofluorescence microscopy: Visualizes A27 localization in infected cells

• Virus neutralization assays: Assesses functional blocking of viral infectivity

• Competition binding assays: Determines epitope specificity using characterized antibodies

Researchers should use multiple complementary methods to comprehensively validate antibody specificity, particularly when studying closely related orthopoxviruses .

How does the oligomeric structure of A27 affect antibody binding and neutralization potential?

The complex oligomeric structure of A27 creates unique challenges and opportunities for antibody development. Since A27 forms concentration-dependent oligomers (from dimers to hexamers) in solution , antibodies targeting different epitopes may have variable accessibility depending on the oligomerization state.

Key considerations include:

• Antibodies targeting the NTR or CTR interfaces might disrupt protein-protein interactions critical for A27 function

• Certain epitopes may be masked in higher-order oligomers but exposed in monomers or dimers

• Antibodies targeting conserved regions involved in oligomerization could have broader neutralizing potential across orthopoxviruses

Research indicates that mutations in either the NTR or CTR of A27 impair wild-type dimer/trimer formation, resulting in defects in virus egress and altered membrane fusion activity . Antibodies that interfere with these interfaces might therefore demonstrate potent antiviral effects.

How can researchers design antibody combinations to prevent viral escape mechanisms?

Drawing from research on other viral antibody therapies, combination approaches targeting multiple non-overlapping epitopes on A27 or different viral proteins could prevent viral escape. Studies with SARS-CoV-2 antibodies have shown that:

• While single antibody treatments rapidly select for escape variants both in vitro and in vivo, combinations of two non-competing antibodies significantly delay or prevent escape

• Three non-competing antibodies provide even greater protection against escape variant emergence

For VACWR150 antibody research, investigators should:

• Map epitopes for multiple anti-A27 antibodies to identify non-competing pairs

• Test antibody combinations in serial passage experiments to evaluate escape potential

• Consider combining A27 antibodies with antibodies targeting other viral proteins (such as A26)

What methodologies are most effective for studying neutralization mechanisms of anti-VACWR150 antibodies?

Advanced methodologies for studying neutralization mechanisms include:

• Cryo-electron microscopy: Visualizes antibody binding to intact virions and conformational changes in the A27 protein

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps epitopes and conformational changes upon antibody binding

• Bio-layer interferometry (BLI) or surface plasmon resonance (SPR): Quantifies binding kinetics and affinity of antibodies to recombinant A27

• Plaque reduction neutralization tests (PRNT): Measures functional neutralization capacity

• Cell fusion inhibition assays: Assesses the ability of antibodies to block A27-mediated membrane fusion events

These methods can illuminate whether antibodies neutralize by blocking attachment to host cells, preventing viral fusion, interfering with A27-A26 complex formation, or disrupting viral egress .

How do mutations in A27 affect antibody recognition and neutralization potential?

Mutations in key residues of A27 can significantly impact antibody recognition and neutralization. Research has shown that:

• Mutations at the NTR (Leu47, Leu51, and Leu54) and CTR (Ile68, Asn75, and Leu82) affect A27 self-assembly

• These mutations not only impair viral functions but could also potentially affect antibody binding

• Antibodies targeting highly conserved regions of A27 may have broader spectrum activity against related orthopoxviruses

For researchers developing therapeutic antibodies, targeting epitopes with lower mutation tolerance would be advantageous to minimize escape variant development, similar to strategies employed for other viruses like SARS-CoV-2 .

What controls should be included when using anti-VACWR150 antibodies in experimental procedures?

Rigorous experimental design requires appropriate controls:

• Positive controls:

  • Verified recombinant A27 protein (full-length or specific domains)

  • Vaccinia virus-infected cell lysates with confirmed A27 expression

• Negative controls:

  • Uninfected cell lysates

  • Lysates from cells infected with A27-deletion mutant viruses

  • Isotype-matched irrelevant antibodies

• Specificity controls:

  • Pre-absorption of antibody with recombinant A27 protein

  • Testing against related orthopoxvirus proteins to assess cross-reactivity

  • Competitive binding assays with characterized anti-A27 antibodies

How can researchers optimize immunoprecipitation protocols using anti-VACWR150 antibodies?

For successful immunoprecipitation of A27 and associated proteins:

• Lysis conditions: Use buffers that preserve protein-protein interactions while effectively solubilizing membrane-associated A27 (e.g., RIPA buffer with 1% NP-40 or 0.5% Triton X-100)

• Cross-linking considerations:

  • For capturing transient interactions, consider mild cross-linking (1-2% formaldehyde)

  • For studying A27-A26 disulfide-linked complexes, use non-reducing conditions

• Antibody coupling:

  • Covalently couple antibodies to protein A/G beads to prevent antibody contamination in eluates

  • For co-immunoprecipitation studies, use oriented coupling methods that preserve antibody binding domains

• Elution strategies:

  • pH gradient elution for gentle recovery of protein complexes

  • Competitive elution with excess recombinant A27 peptide for higher specificity

How can researchers utilize anti-VACWR150 antibodies to investigate the role of A27 in orthopoxvirus entry and fusion?

Understanding A27's role in viral entry and fusion requires sophisticated experimental approaches:

• Binding interference assays: Use antibodies that target the heparan sulfate binding domain of A27 to block viral attachment to host cells

• Synchronized infection systems:

  • Pre-bind virus to cells at 4°C

  • Add antibodies at different time points during temperature shift to 37°C

  • Monitor fusion events by fluorescence microscopy or reporter systems

• Split fusion reporter assays:

  • Express viral fusion machinery and target cell receptors in separate cell populations

  • Mix cells with or without antibody treatment

  • Measure fusion events through complementation of split reporters

• Site-directed mutagenesis studies:

  • Generate A27 variants with mutations in key functional domains

  • Test antibody binding and neutralization against each variant

  • Map functionally critical epitopes through correlation of binding and neutralization data

What are the technical challenges in developing therapeutic monoclonal antibodies targeting VACWR150?

Developing therapeutic antibodies against A27 presents several challenges:

• Epitope accessibility: Since A27 forms oligomeric structures on the virion surface, determining which epitopes remain accessible in the native state is critical

• Cross-reactivity considerations:

  • Ensuring specificity for orthopoxvirus A27 without binding to human proteins

  • Balancing broad cross-reactivity against related orthopoxviruses with specificity

• Antibody engineering requirements:

  • Modifications to prevent antibody-dependent enhancement (ADE) of infection (e.g., N297A modification as used in some SARS-CoV-2 antibodies)

  • Optimizing half-life through Fc engineering for prolonged protection

• Combination approaches:

  • Identifying synergistic antibody pairs targeting different epitopes

  • Developing antibody cocktails that minimize escape risk, similar to successful approaches used against SARS-CoV-2 and Epstein-Barr virus

Recent advances in antibody therapies for other viral pathogens provide valuable insights for researchers working on anti-VACWR150 therapeutic development .

How might novel antibody engineering approaches enhance anti-VACWR150 antibody functionality?

Recent advances in antibody engineering offer new possibilities for enhancing anti-VACWR150 antibodies:

• pH-dependent binding antibodies:

  • Engineering antibodies with "sweeping" capabilities that bind antigen at neutral pH but release it in the acidic endosome

  • This approach increases antigen clearance from circulation by enabling FcRn-mediated recycling of the antibody while directing the antigen to lysosomal degradation

• Bispecific antibodies:

  • Targeting A27 with one binding arm and another viral protein (e.g., A26) with the second arm

  • Developing bispecifics that target A27 and host immune effector cells to enhance viral clearance

• Intracellular antibodies (intrabodies):

  • Expressing antibody fragments inside cells to interfere with A27 function during viral assembly

  • Using cell-penetrating antibodies to deliver neutralizing activity to the intracellular compartment

These innovative approaches could overcome limitations of conventional antibodies and provide new research tools .

How can structural information about A27 guide rational antibody design and engineering?

The detailed structural information available for A27 protein provides opportunities for structure-guided antibody engineering:

• Computational epitope mapping:

  • Identifying conserved, functionally critical, and accessible epitopes on A27

  • Predicting antibody binding modes through molecular docking simulations

• Structure-based antibody optimization:

  • Using the crystal structure to guide affinity maturation by identifying key interaction residues

  • Engineering antibodies that specifically target oligomerization interfaces to disrupt viral function

• Novel binding modalities:

  • Designing single-domain antibodies or alternative scaffolds that can access cryptic epitopes in the A27 structure

  • Developing antibodies that specifically recognize certain oligomeric states of A27

This rational approach has proven successful in developing antibodies against other viral targets, including the recent development of antibodies that can neutralize all SARS-CoV-2 variants .