BOR6 Antibody

What is the B6R protein and why is it a significant target for antibody development?

B6R is a viral protein found in poxviruses including Monkeypox virus (MPXV) and Vaccinia virus (VACV). The protein consists of four sushi domains in its extracellular portion followed by a stalk region adjacent to its transmembrane domain . It represents a compelling antibody target because neutralizing antibodies directed against this protein can potentially inhibit viral infection. Recent research has demonstrated that antibodies targeting B6R can provide significant protection in animal models of poxvirus infection, highlighting its importance as a therapeutic target . Structurally, B6R contains distinct domains that offer multiple epitope targets for antibody binding, making it particularly valuable for developing targeted immunotherapeutics.

How is the structure of B6R protein related to antibody epitope binding?

The B6R protein has a complex structure that directly influences antibody binding. Advanced computational modeling using AlphaFold2 has revealed that the extracellular domain of B6R consists of a flexible N-terminal region (residues 1-19), followed by four sushi domains (residues 20-239), and a stalk region (residues 240-279) containing a connecting loop and partial alpha helix adjacent to the transmembrane domain .

Different antibodies target distinct regions of this structure. For example, research has shown that antibody B026 specifically binds to the stalk region (residues 240-279) of B6R, while antibody B019 recognizes domain 4 of the protein . This specificity was established through binding studies with truncated B6R protein variants, demonstrating that:

• B026 bound strongly to constructs containing the stalk region (clones 7, 9, 11, and 12)

• B026 showed no binding to fragments lacking this region (clones 3, 4, 5, 6, 8, and 10)

• B019 demonstrated strong binding to all truncated B6R proteins containing domain 4 (clones 1, 2, 6, 7, 8, 9, 10, and 11)

• B019 showed minimal or no binding to constructs lacking domain 4 (clones 3, 4, 5, and 12)

This understanding of structure-function relationships is crucial for developing highly specific antibodies and engineering improved variants for research and therapeutic applications.

What methods are most effective for producing research-grade B6R antibodies?

The most effective method for producing research-grade B6R antibodies involves recombinant technology using mammalian expression systems, particularly the Expi293F system. The detailed methodology includes:

• Gene cloning and vector construction: Following phagemid extraction, the heavy-chain and light-chain genes are amplified via PCR and cloned into antibody expression vectors encoding the constant regions of human IgG1 using enzymatic assembly techniques .

• Transfection and expression: The constructed plasmids containing heavy-chain and light-chain genes are mixed with polyethylenimine (PEI) and transfected into 293F cells, which are then incubated at 37°C with 7% CO₂ .

• Harvest and purification: Five days post-transfection, the culture supernatant containing antibodies is collected, centrifuged, and subjected to affinity purification using protein G (Solarbio, R8300). The antibodies are eluted with 100 mM Glycine-HCl (pH 3.0) and neutralized with 1 M Tris-HCl .

• Quality assessment: The purity of eluted proteins is assessed by 12% SDS-PAGE and Western blot, followed by buffer exchange into PBS using centrifugal filter units (Millipore, UFC8010) .

• Storage and analysis: The purified antibodies are aliquoted and stored at -80°C. Germline genes, germline divergence, framework regions, and CDR3 loop length can be analyzed using the IGBLAST program .

This recombinant approach offers significant advantages over traditional hybridoma methods, including greater reproducibility, consistency in glycosylation patterns, and the ability to engineer specific modifications to enhance antibody performance for research applications .

How can researchers accurately measure binding kinetics of antibodies to B6R protein?

Researchers can accurately measure binding kinetics of antibodies to B6R protein using Biolayer Interferometry (BLI), which provides real-time, label-free analysis of molecular interactions. The methodological approach includes:

For bispecific antibodies (bsAbs), while a 1:1 fitting model often provides better curve fitting than a bivalent analyte model, researchers should be cautious in interpretation as binding kinetics may be influenced by complex interactions .

Complementary approaches such as Surface Plasmon Resonance (SPR) can be used to validate BLI results, especially when dealing with antibodies that exhibit complex binding profiles or when precise kinetic constants are required for structure-activity relationship studies.

What are the optimal protocols for assessing B6R antibody neutralization efficacy in vitro?

Optimal protocols for assessing B6R antibody neutralization efficacy in vitro should include:

• ELISA-based binding assessment:

  • Coat 96-well plates with B6R antigen (1 μg/mL) in coating buffer overnight at 4°C

  • Block plates with 3% (w/v) BSA at 37°C for 1 hour

  • Apply serially diluted antibodies (3-fold dilutions recommended)

  • Detect binding using HRP-conjugated goat anti-human IgG (e.g., Santa Cruz, sc-2453)

  • Develop color reaction with TMB for 5 minutes and measure absorbance at 450/620 nm

• Functional neutralization assays:

  • Plaque reduction neutralization test (PRNT) to quantify the reduction in viral infectivity

  • Pre-incubate serially diluted antibodies with standardized viral inoculum

  • Add the mixture to susceptible cell monolayers and incubate for viral infection

  • Overlay with semi-solid medium to restrict viral spread to adjacent cells

  • Count plaques after appropriate incubation time to determine neutralization potency

• Combination testing:

  • To assess potential synergy between antibodies targeting different epitopes (like B026 targeting the stalk region and other antibodies targeting different domains), researchers should employ checkerboard dilution matrices

  • Calculate combination indices to determine whether antibody combinations exhibit synergistic, additive, or antagonistic effects

• Controls and standardization:

  • Include appropriate isotype control antibodies

  • Incorporate reference antibodies with established neutralization potency

  • Standardize viral input using PFU (plaque-forming units) quantification

  • Maintain consistent cell passage number and density across experiments

For comprehensive evaluation, researchers should compare EC50 values from binding assays with neutralization potency to establish structure-function relationships that can guide further antibody engineering efforts.

How can researchers optimize combinatorial antibody approaches for enhanced protection against poxviruses?

Researchers can optimize combinatorial antibody approaches for enhanced protection against poxviruses by implementing the following methodological strategies:

• Target selection based on structural and functional diversity:

  • Select antibodies that target non-overlapping epitopes on different viral proteins (such as combining B6R-targeting antibodies with those targeting M1R)

  • Prioritize combinations where constituent antibodies have demonstrated individual neutralizing activity

  • Consider antibodies that target conserved regions across multiple poxvirus species for broader protection

• Systematic in vitro evaluation:

  • Conduct neutralization assays with antibody combinations at varying ratios to identify optimal proportions

  • Measure viral load reduction in cell culture models using quantitative PCR or plaque assays

  • Assess if combinations can overcome potential viral escape mutants

• In vivo evaluation in animal models:

  • Test prophylactic efficacy by administering antibody combinations prior to viral challenge

  • Evaluate therapeutic efficacy by treating animals after established infection

  • Measure multiple endpoints including survival, weight loss, viral loads in target tissues, and disease-specific parameters

Recent research has demonstrated that combining antibodies targeting different viral proteins can substantially enhance protection against poxviruses compared to monotherapy. For example, the combination of B026 (targeting B6R) and A138 (targeting M1R) provided superior protection in a mouse model of VACV infection compared to either antibody alone . When challenged with 10⁹ PFU of virus:

• PBS control group: 0% survival by day 10

• A138 only: 83% survival with significant weight loss

• B026 only: 66% survival with significant weight loss

• B026 + A138 combination: Higher survival rate with only slight weight loss followed by recovery

Importantly, the viral load assessment demonstrated that the combination treatment resulted in extremely low or undetectable infectious viral levels in both lungs and spleen, even after high-dose challenge . This synergistic effect highlights the value of targeting multiple viral proteins simultaneously to enhance protective efficacy.

What strategies can resolve inconsistent B6R antibody binding results across different assay platforms?

Inconsistent B6R antibody binding results across different assay platforms can be resolved through:

• Protein conformation assessment:

  • The tertiary structure of B6R is critical for epitope presentation. Ensure proper folding by comparing binding to native and denatured forms of the protein

  • For antibodies targeting conformational epitopes (particularly those binding to the four sushi domains or the stalk region), validate proper protein folding using circular dichroism

  • Consider using multiple expression systems for B6R protein production to identify potential post-translational modification differences

• Buffer optimization:

  • Systematically test different buffer conditions (pH, ionic strength, detergent concentration) across platforms

  • For membrane-associated portions of B6R, include appropriate detergents or lipid nanodiscs to maintain native conformations

  • Evaluate the impact of calcium concentration, as the sushi domains in B6R may have calcium-dependent folding properties

• Cross-platform validation:

  • When discrepancies are observed between ELISA and BLI results, validate with orthogonal methods like SPR

  • For functional assays, compare cell-based neutralization with biochemical binding assays to identify potential co-factor requirements

  • Consider epitope mapping using hydrogen-deuterium exchange mass spectrometry to confirm consistent antibody-epitope interactions across platforms

• Reference standards implementation:

  • Establish internal reference standards with known binding characteristics

  • Include positive and negative control antibodies in each assay

  • Develop a qualification panel of B6R protein variants with known binding profiles to systematically identify platform-specific biases

When troubleshooting binding inconsistencies, researchers should particularly focus on the distinct domains of B6R, as antibodies targeting different domains may show differential sensitivity to experimental conditions. For example, antibodies targeting the stalk region (like B026) may show greater platform-dependent variability than those targeting the more stable sushi domains .

How should researchers design comprehensive epitope mapping studies for B6R antibodies?

Researchers should design comprehensive epitope mapping studies for B6R antibodies using a multi-method approach:

• Domain-level mapping using truncation mutants:

  • Generate a systematic series of truncated B6R protein constructs as demonstrated in recent research, where 12 different B6R fragments were created to span the various domains

  • Express these constructs with appropriate tags (e.g., 6×His-tag) for purification and immobilization

  • Test antibody binding against each construct using BLI or ELISA to determine domain-level specificity

  • This approach successfully identified that antibody B026 specifically targets the stalk region (residues 240-279) while B019 binds to domain 4

• Fine epitope mapping using alanine scanning mutagenesis:

  • After identifying the domain, create a panel of point mutants within that domain

  • Systematically replace individual amino acids with alanine (or with structurally divergent amino acids for alanine residues)

  • Evaluate the impact of each mutation on antibody binding

  • Identify critical binding residues where mutations significantly reduce binding affinity

• Structural characterization:

  • Attempt to solve crystal structures of B6R-antibody complexes (though this may be challenging as noted in the research where no crystals appeared for B6R-B019 or B6R-B026 Fab complexes)

  • When crystallography is unsuccessful, employ cryo-electron microscopy

  • Use hydrogen-deuterium exchange mass spectrometry to identify protected regions upon antibody binding

  • Validate using computational docking informed by experimental constraints

• Cross-competition binding studies:

  • Perform antibody cross-competition assays to group antibodies that target overlapping epitopes

  • Use a matrix approach where each antibody is tested against all others in the panel

  • This helps create an epitope map and identify antibodies that can be used in combination

The research demonstrated that this integrated approach successfully mapped B026 binding to the stalk region of B6R, establishing that constructs containing this region (clones 7, 9, 11, and 12) bound strongly to B026, while those lacking it showed no binding . Similarly, B019 was confirmed to recognize domain 4 through systematic testing of different B6R fragments .

What are the critical factors to consider when evaluating antibody-mediated protection in animal models of poxvirus infection?

When evaluating antibody-mediated protection in animal models of poxvirus infection, researchers should consider these critical factors:

• Experimental design considerations:

  • Challenge dose optimization: Titrate viral challenge doses to identify appropriate levels for distinguishing between partial and complete protection. Research has shown that at lower challenge doses (10⁷ PFU), even single antibodies may confer complete protection, necessitating higher doses (10⁹ PFU) to detect differences between treatment groups .

  • Route of administration: Match the antibody administration route to the expected clinical application (intravenous for systemic protection, intranasal for respiratory infections).

  • Timing of intervention: Evaluate both prophylactic (pre-exposure) and therapeutic (post-exposure) scenarios to determine optimal treatment windows .

• Comprehensive endpoint assessment:

  • Survival and clinical indicators: Monitor survival rates, body weight changes, and disease-specific symptoms .

  • Viral load quantification: Measure viral loads in multiple tissues (e.g., lungs and spleen) using plaque reduction neutralization test (PRNT) assays to determine if protection is associated with viral clearance or just symptom reduction .

  • Immunological parameters: Assess antibody titers, neutralizing activity in serum, and relevant inflammatory markers.

• Advanced evaluation criteria:

  • Antibody penetration: Evaluate the biodistribution of therapeutic antibodies to ensure they reach affected tissues.

  • Resistance development: Monitor for emergence of escape mutants, particularly in studies of combination antibody therapies.

  • Dose-response relationships: Establish dose-response curves for different antibodies and combinations to determine minimum effective doses.

• Translational considerations:

  • Species-specific differences: Consider how differences between animal models and humans might affect antibody efficacy.

  • Pharmacokinetic profiling: Determine antibody half-life and clearance rates in the model organism.

  • Immune status effects: Evaluate efficacy in both immunocompetent and immunocompromised animal models when possible.

Research with B6R antibodies has demonstrated that while single antibody administration (B026 or A138 alone) provided partial protection with 66-83% survival rates, the combination of B026 and A138 resulted in superior protection with minimal weight loss and rapid recovery even against high-dose challenges (10⁹ PFU) . This highlights the importance of combination strategies targeting multiple viral proteins simultaneously.

How can researchers assess the potential for viral escape from B6R antibody neutralization?

Researchers can assess the potential for viral escape from B6R antibody neutralization through a systematic approach:

• Serial passage under selective pressure:

  • Culture virus in the presence of sub-neutralizing concentrations of B6R antibodies

  • Gradually increase antibody concentration over multiple passages

  • Sequence the B6R gene from viral isolates that demonstrate resistance

  • Identify specific mutations associated with escape

• Structure-guided mutation analysis:

  • Using the AlphaFold2-predicted structure of B6R protein , identify amino acid residues likely to be critical for antibody binding

  • Generate viral mutants with specific substitutions at these positions using reverse genetics

  • Test neutralization efficacy against these engineered mutants

  • This approach is particularly valuable for antibodies like B026 that target the stalk region (residues 240-279) and B019 that target domain 4

• Combination resistance profiling:

  • Evaluate escape potential against individual antibodies compared to combinations

  • Assess whether viruses that escape neutralization by one antibody (e.g., B026) remain susceptible to another (e.g., A138)

  • Determine the frequency of dual-escape mutants emerging against combination therapy

• Deep mutational scanning:

  • Create libraries of B6R variants with comprehensive mutation coverage

  • Express these variants and test antibody binding

  • Identify mutations that reduce binding affinity

  • Correlate binding changes with functional escape in neutralization assays

• Natural variant analysis:

  • Compare B6R sequences across different poxvirus isolates and strains

  • Test neutralization efficacy against diverse viral isolates

  • Identify naturally occurring polymorphisms that affect neutralization sensitivity

Research has demonstrated that targeting multiple viral proteins simultaneously (such as combining antibodies against B6R and M1R) substantially reduces the likelihood of escape mutant emergence, as evidenced by enhanced protection in animal models . The combination of B026 (targeting B6R) and A138 (targeting M1R) provided superior protection compared to either antibody alone, supporting a dual-targeting strategy to minimize viral escape .

What longitudinal assays can effectively monitor the persistence of B6R antibodies in experimental models?

Effective longitudinal monitoring of B6R antibody persistence in experimental models requires:

• Quantitative ELISA protocols:

  • Develop a standardized ELISA method using purified B6R protein (1 μg/mL) coated onto 96-well plates

  • Establish a reference standard curve using purified antibody of known concentration

  • Process samples at multiple timepoints following antibody administration

  • Calculate absolute antibody concentrations based on standard curves

  • This approach allows precise quantification of antibody titers over time

• Functional persistence assessment:

  • Periodically collect serum samples from treated animals

  • Evaluate neutralization activity using plaque reduction neutralization tests (PRNT)

  • Compare functional activity decay with absolute concentration measurements

  • This reveals whether functional capacity diminishes at the same rate as concentration

• Tissue-specific antibody quantification:

  • Collect tissue samples from key sites of viral replication (e.g., lungs, spleen)

  • Extract and quantify antibody levels from tissue homogenates

  • Compare tissue penetration and persistence versus serum levels

  • This approach is critical for understanding the pharmacokinetics of antibody distribution

• Methodology validation:

  • Include appropriate positive and negative controls in each assay

  • Implement quality control samples with known antibody concentrations

  • Establish acceptance criteria for assay performance

  • Ensure consistent detection limits across timepoints

Research on antibody persistence in other viral systems has demonstrated that quantitative monitoring can provide valuable insights into treatment efficacy. For example, studies with Lyme disease showed that anti-IR6 antibody levels diminished sharply after successful antibiotic treatment (within 25 weeks), while levels of antibodies to other antigens remained relatively stable . This differential decline in antibody levels provided a useful biomarker for treatment success .

For B6R antibodies, longitudinal monitoring should include both quantitative concentration measurements and functional assessments to determine if structural changes (such as degradation or aggregation) affect neutralizing capacity over time.

What strategies can enhance the translational potential of B6R antibodies from animal models to clinical applications?

To enhance the translational potential of B6R antibodies from animal models to clinical applications, researchers should implement these strategies:

• Humanization and optimization of antibody sequences:

  • Convert murine or phage-derived antibodies to humanized versions to reduce immunogenicity

  • Use computational design to optimize complementarity-determining regions (CDRs) while maintaining epitope specificity

  • Apply germline-targeting approaches to minimize potential immunogenicity

  • For B6R antibodies isolated using phage display technology, analyze germline genes and divergence using tools like IGBLAST

• Production system refinement:

  • Transition from research-scale expression systems to GMP-compliant production

  • Implement the Expi293F expression system proven effective for research antibodies

  • Optimize production parameters including cell density, transfection efficiency, and purification protocols

  • Develop robust quality control metrics for lot-to-lot consistency

• Comprehensive preclinical assessment:

  • Evaluate efficacy across multiple animal models with varying degrees of disease severity

  • Establish detailed pharmacokinetic and pharmacodynamic profiles

  • Conduct thorough toxicology studies with attention to off-target effects

  • Build on successful prophylactic and therapeutic findings from mouse models

• Formulation development:

  • Optimize antibody stability through buffer formulation

  • Develop liquid and lyophilized formulations with appropriate shelf-life

  • Establish accelerated stability testing protocols

  • Consider delivery systems appropriate for emergency use scenarios

• Combination therapy development:

  • Leverage the enhanced protection demonstrated with antibody combinations (e.g., B026 targeting B6R and A138 targeting M1R)

  • Establish optimal ratios for antibody cocktails

  • Determine if fixed combinations or separate administration provides superior results

  • Evaluate combination with existing antiviral therapies for potential synergistic effects

The research demonstrating that combinations of antibodies targeting different viral proteins (B6R and M1R) provided superior protection against high-dose viral challenge compared to monotherapy suggests that multi-antibody approaches may offer the most promising path to clinical translation. The combination of B026 and A138 resulted in minimal weight loss and extremely low viral loads in both lungs and spleen even after challenge with 10⁹ PFU of virus , supporting a dual-targeting strategy for therapeutic development.

How can advanced computational approaches improve B6R antibody design and optimization?

Advanced computational approaches can significantly improve B6R antibody design and optimization through these methodological strategies:

• Structure-guided epitope prediction:

  • Leverage AlphaFold2-predicted structures of B6R protein to identify optimal epitope targets

  • Apply computational epitope mapping to predict regions with high antigenicity and accessibility

  • Identify conserved epitopes across multiple poxvirus variants to design broadly neutralizing antibodies

  • Model the four sushi domains and stalk region of B6R to identify structurally critical regions less likely to tolerate escape mutations

• In silico affinity maturation:

  • Simulate antibody-antigen interactions using molecular dynamics

  • Predict mutations that could enhance binding affinity through computational alanine scanning

  • Apply directed evolution algorithms to optimize complementarity-determining regions (CDRs)

  • Focus optimization efforts on specific domains, such as enhancing B026 binding to the stalk region (residues 240-279)

• Multivalent antibody design:

  • Computationally model bispecific antibodies targeting different epitopes within B6R

  • Design antibodies that simultaneously target B6R and other viral proteins (like M1R) to recapitulate the enhanced protection observed with combination therapy

  • Optimize linker length and flexibility for maximal dual-target engagement

  • Predict potential steric constraints in multivalent binding

• Machine learning applications:

  • Train neural networks on existing antibody-antigen binding data

  • Develop predictive models for antibody affinity and specificity

  • Apply deep learning to predict antibody developability properties

  • Use natural language processing to mine literature for insights on effective antibody designs

• Virtual screening approaches:

  • Create virtual libraries of antibody variants

  • Perform high-throughput computational screening against modeled B6R structures

  • Prioritize candidates based on predicted binding energy and other biophysical properties

  • Validate top computational candidates with experimental binding studies

Research has demonstrated the value of computational approaches in antibody development. For B6R antibodies, AlphaFold2 modeling successfully predicted the domain structure that informed the design of truncation constructs, enabling precise epitope mapping of antibodies like B026 and B019 . This structure-guided approach can be extended to design antibodies with enhanced specificity, improved neutralization potency, and reduced potential for escape mutation development.

What novel applications exist for B6R antibodies beyond direct neutralization of poxviruses?

B6R antibodies offer several novel applications beyond direct virus neutralization:

• Diagnostic development:

  • Create rapid lateral flow immunoassays for poxvirus detection using B6R antibodies

  • Develop multiplexed assays combining B6R and other viral protein antibodies for improved sensitivity

  • Design competition assays where patient samples compete with labeled B6R antibodies, allowing quantification of viral load

  • Engineer B6R antibody-based biosensors for continuous monitoring in high-risk settings

• Targeted drug delivery systems:

  • Conjugate B6R antibodies with antiviral payloads for targeted delivery to infected cells

  • Develop antibody-drug conjugates (ADCs) using B6R antibodies as the targeting moiety

  • Create immunotoxins that specifically eliminate cells expressing B6R protein on their surface

  • Design nanoparticle delivery systems coated with B6R antibodies for site-specific drug release

• Research tools for viral pathogenesis studies:

  • Use domain-specific antibodies (like B026 targeting the stalk region and B019 targeting domain 4) to probe the functional significance of different B6R regions

  • Develop fluorescently labeled B6R antibodies for live imaging of viral infection and spread

  • Create conditional inhibitors where B6R antibody fragments are linked to inducible effector domains

  • Engineer antibodies targeting distinct epitopes as tools to map conformational changes during infection

• Structural biology applications:

  • Employ B6R antibodies as crystallization chaperones to facilitate structural studies of challenging viral proteins

  • Use antibody-antigen complexes to stabilize specific conformations for cryo-electron microscopy

  • Apply antibody-mediated hydrogen-deuterium exchange mass spectrometry to probe dynamic structural features of viral proteins

• Combination immunotherapy platforms:

  • Design bispecific T-cell engagers (BiTEs) incorporating B6R-binding domains to redirect T-cell activity against infected cells

  • Develop chimeric antigen receptor (CAR) constructs using B6R-binding domains for adoptive cell therapy approaches

  • Create antibody-cytokine fusion proteins combining B6R targeting with immunomodulatory functions

The domain specificity of antibodies like B026 (targeting the stalk region) and B019 (targeting domain 4) provides unique opportunities to develop reagents that can distinguish between different functional states of the B6R protein . This specificity can be leveraged for applications ranging from high-resolution imaging to targeted therapeutic delivery.

How might emerging viral variants affect the efficacy of existing B6R antibodies?

Emerging viral variants could affect B6R antibody efficacy through several mechanisms that require systematic evaluation:

• Sequence variation assessment:

  • Monitor genetic diversity in the B6R gene across emerging poxvirus isolates

  • Compare sequences focusing on regions known to be targeted by antibodies like B026 (stalk region, residues 240-279) and B019 (domain 4)

  • Calculate conservation scores for specific epitopes

  • Predict impact of observed substitutions on antibody binding using computational modeling

• Cross-reactivity testing methodologies:

  • Express recombinant B6R proteins from multiple viral variants

  • Measure binding affinities of existing antibodies against variant proteins using BLI

  • Compare EC50 values in ELISA-based binding assays

  • Evaluate neutralization potency against diverse viral isolates

  • Create a heat map of neutralization efficiency across variant panels

• Structure-function correlation:

  • For variants showing reduced antibody susceptibility, map mutations onto the AlphaFold2-predicted B6R structure

  • Identify structural mechanisms of escape (direct epitope alteration vs. allosteric effects)

  • Engineer second-generation antibodies targeting highly conserved epitopes

  • Design combination strategies targeting multiple conserved epitopes simultaneously

• Antibody cocktail optimization:

  • Evaluate whether viral variants resistant to one antibody remain susceptible to others

  • Expand the combination approach beyond B026 and A138 to include additional antibodies targeting diverse epitopes

  • Determine minimum antibody combinations needed for broad variant coverage

  • Quantify the genetic barrier to resistance for different antibody combinations

• Surveillance and prediction frameworks:

  • Establish ongoing surveillance of B6R sequence evolution in circulating poxviruses

  • Develop predictive algorithms to identify potential emerging variants of concern

  • Create antibody sensitivity maps based on B6R sequence variations

  • Implement proactive antibody refinement programs for maintaining efficacy

The research demonstrating that combinations of antibodies targeting different viral proteins (B026 targeting B6R and A138 targeting M1R) provided superior protection supports the development of multi-target approaches to address viral variation. By targeting multiple distinct epitopes simultaneously, the impact of variation at any single site can be mitigated, enhancing the robustness of antibody-based interventions against emerging variants.