Recombinant African swine fever virus Virus attachment protein p12 (War-108)

What is the structure and function of p12 (War-108) protein?

The p12 (War-108) protein is a truncated form of the p12 protein encoded by the O16R gene of ASFV genotype I. It is localized to the inner envelope of ASFV virions and plays a critical role in viral attachment to host cells. The protein exists in different forms: a 17 kDa mature form found in virions and 10-12 kDa immature forms present in infected cells.

The full amino acid sequence of this 61-amino acid protein is:
MALDGSSGGGSNVETLLIVAIIVVIMAIMLYYFWWMPRQQKKCSKAEECTCNNGSCSLKTS

Functionally, p12 mediates ASFV binding to susceptible cells, particularly porcine macrophages, through specific receptor interactions. This attachment protein is highly conserved across ASFV genotypes, indicating its evolutionary importance in the viral lifecycle. The protein's role in host cell receptor binding makes it a significant target for both diagnostic applications and therapeutic interventions in ASFV research.

How is recombinant p12 (War-108) protein produced for research purposes?

Recombinant p12 (War-108) can be expressed through multiple expression systems, with bacterial and insect cell systems being the most common. The methodological approaches include:

• Bacterial Expression: The protein can be expressed in Escherichia coli with an N-terminal histidine tag to facilitate purification. This approach typically yields protein in a lyophilized powder form with greater than 90% purity as determined by SDS-PAGE .

• Insect Cell Expression: Baculovirus expression systems using Sf9 cells represent another production method, yielding approximately 50 mg/L with purity exceeding 90% when purified using aqueous phase partition techniques.

The recombinant protein production workflow typically involves:

For optimal handling of the purified protein, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL with the addition of 5-50% glycerol is recommended before storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided to maintain protein integrity.

What experimental applications can p12 (War-108) be used for in ASFV research?

The recombinant p12 (War-108) protein serves multiple experimental purposes in ASFV research:

• Viral Attachment Studies: The protein can be used to investigate the mechanisms of ASFV attachment to host cells. In vitro assays demonstrate that recombinant p12 blocks viral attachment and infectivity in a dose-dependent manner, with pre-treatment of macrophages resulting in 80-90% reduction in ASFV titers at concentrations of 10 μg/mL.

• Receptor Identification: The protein serves as a tool for identifying and characterizing cellular receptors that interact with ASFV during the initial stages of infection.

• Antibody Production: Recombinant p12 can be used to generate specific antibodies for immunological studies and diagnostic test development.

• Structural Analysis: The protein enables investigation of structural characteristics through techniques like SDS-PAGE and immunoaffinity assays, which have helped identify the 17 kDa mature protein in virions versus the 10-12 kDa cellular forms.

• Vaccine Development: Although immunization with recombinant p12 alone has proven insufficient for protective immunity, the protein remains valuable for subunit vaccine research, particularly in multi-antigen approaches combining p12 with other ASFV proteins like p30 or p54.

Researchers typically employ SDS-PAGE for initial characterization of the recombinant protein, confirming its molecular weight and purity before proceeding to more specialized applications .

How does p12 (War-108) contribute to ASFV neutralization mechanisms?

The neutralization mechanisms involving p12 (War-108) are complex and involve both antibody-mediated processes and direct protein interactions. Research indicates that neutralizing antibodies targeting different ASFV proteins, including p12, can inhibit both virus attachment and internalization . This dual inhibitory effect suggests a multifaceted role for p12 in the viral entry process.

The methodological approach to studying p12's role in neutralization typically involves:

• Neutralization Assays: Incubating ASFV with antibodies against p12 before infection to measure reduction in viral titers.

• Competitive Binding Studies: Using recombinant p12 to block viral receptors on target cells, demonstrating a dose-dependent inhibition with IC50 measurements.

• Time-of-Addition Experiments: Adding recombinant p12 at different time points during infection to determine when the protein exerts its inhibitory effect.

The literature suggests that while p12 antibodies contribute to neutralization, they alone aren't sufficient for complete protection. This indicates that effective neutralization likely requires targeting multiple viral proteins simultaneously to disrupt the attachment-internalization cascade .

What are the challenges in developing p12-based vaccines against ASFV?

Despite p12's critical role in viral attachment, developing effective p12-based vaccines faces several significant challenges:

• Limited Immunogenicity: Immunization with recombinant p12 alone fails to induce sufficient protective immunity in pigs, suggesting inadequate stimulation of relevant immune responses.

• Conformational Requirements: The native conformation of p12 in virions (17 kDa) differs from recombinant forms, potentially affecting epitope presentation and antibody recognition.

• Viral Escape Mechanisms: Though p12 is highly conserved, ASFV's complex structure with over 150 proteins provides multiple redundant entry mechanisms that may bypass p12 neutralization.

• Delivery System Limitations: Current delivery platforms may not effectively present p12 to the immune system in a manner that elicits robust protective responses.

Research approaches to address these challenges include:

Future directions suggest that crystallography studies to map receptor-binding epitopes could provide critical insights for rational vaccine design targeting p12.

How does p12 (War-108) interact with specific cellular receptors during ASFV infection?

The molecular interactions between p12 (War-108) and host cell receptors remain incompletely characterized, representing an active area of investigation. Current understanding suggests that p12 mediates ASFV binding to susceptible cells, particularly porcine macrophages, through specific receptor interactions.

Methodologies to investigate these interactions include:

• Receptor Identification Techniques:

  • Virus overlay protein binding assays (VOPBA)

  • Co-immunoprecipitation with crosslinking

  • Surface plasmon resonance for binding kinetics

  • Yeast two-hybrid screening for protein-protein interactions

• Receptor Blocking Studies:

  • Pre-treatment of cells with recombinant p12

  • Competition with synthetic peptides derived from p12 sequence

  • Antibody blocking of potential receptors

• Structural Biology Approaches:

  • Cryo-electron microscopy of p12-receptor complexes

  • X-ray crystallography (though this remains a research need)

The highly conserved nature of p12 across ASFV genotypes suggests evolutionary pressure to maintain specific receptor interactions, but the identity of these receptors and their distribution across tissues may influence viral tropism and host range. Further research is required to map the specific binding domains within p12 and identify corresponding cellular receptor components.

What methodologies are most effective for evaluating p12 inhibition of viral attachment?

Evaluating p12 inhibition of viral attachment requires robust methodologies that can quantitatively assess the protein's effect on the initial virus-host interaction. Based on the available literature, the following approaches have demonstrated effectiveness:

• Pre-treatment Inhibition Assays: This methodology involves pre-treating susceptible cells (typically porcine macrophages) with varying concentrations of recombinant p12 before viral challenge. Studies have shown that this approach can reduce ASFV titers by 80-90% at concentrations of 10 μg/mL, establishing a clear dose-response relationship.

• Competitive Binding Assays: These assays measure the ability of recombinant p12 to compete with whole virus for receptor binding sites, typically using labeled virus or labeled p12 to quantify displacement.

• Fluorescence-Based Attachment Assays: Utilizing fluorescently-labeled virus particles to visualize and quantify attachment in the presence or absence of recombinant p12.

• Time-Course Experiments: Sequential addition of p12 and virus at different time intervals to determine the temporal window during which p12 exerts its inhibitory effect.

• Temperature-Shift Experiments: Conducting binding assays at 4°C (which permits attachment but prevents internalization) followed by temperature shift to 37°C to distinguish effects on attachment versus entry.

Research protocols often incorporate multiple complementary approaches to comprehensively characterize inhibition. For example, combining flow cytometry to quantify virus binding with confocal microscopy to visualize attachment patterns provides both quantitative and qualitative insights into p12's inhibitory mechanisms.

How can researchers optimize multi-antigen vaccine strategies incorporating p12 (War-108)?

Given that p12 alone fails to induce protective immunity, optimizing multi-antigen vaccine strategies represents a promising research direction. The methodological framework for this optimization includes:

• Antigen Selection and Combination:

  • Rational selection of complementary ASFV proteins (e.g., p12 with p30 or p54)

  • Testing various stoichiometric ratios of antigens

  • Evaluating both physical mixtures and fusion proteins

• Expression System Optimization:

  • Comparing bacterial, insect, and mammalian expression systems

  • Assessing post-translational modifications impact on immunogenicity

  • Optimizing codon usage for enhanced expression

• Adjuvant Selection:

  • Screening adjuvants that preferentially stimulate cell-mediated immunity

  • Testing oil-in-water emulsions, toll-like receptor agonists, and cytokine-based adjuvants

  • Evaluating mucosal adjuvants for oral or intranasal delivery

• Delivery Platform Development:

  • Viral vector systems (e.g., adenovirus, modified vaccinia Ankara)

  • DNA vaccine approaches with optimized promoters

  • Nanoparticle-based delivery for enhanced antigen presentation

• Immunological Assessment:

  • Comprehensive antibody profiling (neutralizing titers, isotype distribution)

  • T-cell response characterization (CD4+/CD8+ activation, cytokine profiles)

  • Challenge studies in relevant animal models with clinical and virological endpoints

Research indicates that fusion constructs combining p12 with other ASFV antigens may enhance immunogenicity through improved epitope presentation and processing. Additionally, prime-boost strategies using different delivery platforms for initial and booster immunizations have shown promise in preliminary studies for other viral diseases and warrant investigation for ASFV.

What are the optimal storage and handling conditions for recombinant p12 (War-108)?

Proper storage and handling of recombinant p12 (War-108) is crucial for maintaining its structural integrity and biological activity. Based on the literature, the following protocol is recommended:

• Storage Temperature:

  • Store lyophilized protein at -20°C or -80°C upon receipt

  • For working aliquots, storage at 4°C is acceptable for up to one week

• Reconstitution Procedure:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being recommended)

• Aliquoting Strategy:

  • Divide reconstituted protein into single-use aliquots

  • Use low-binding microcentrifuge tubes to minimize protein loss

  • Label clearly with concentration, date, and number of freeze-thaw cycles

• Stability Considerations:

  • Avoid repeated freeze-thaw cycles as these significantly reduce activity

  • Monitor potential degradation using SDS-PAGE before experimental use

  • For proteins with His-tags, periodic verification of tag integrity may be necessary

• Buffer Compatibility:

  • The protein is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • Buffer exchanges should maintain similar pH and ionic strength

  • Compatibility testing is recommended before mixing with experimental buffers

These recommendations are based on manufacturer guidance and research protocols that have successfully maintained protein activity. For applications requiring extended storage, lyophilized formats generally offer superior stability compared to reconstituted solutions.

How can researchers verify the functional activity of recombinant p12 (War-108)?

Verifying the functional activity of recombinant p12 (War-108) is essential before proceeding with experimental applications. Multiple complementary approaches can be employed:

• Binding Assays:

  • Cell-based binding assays using susceptible porcine macrophages

  • Solid-phase receptor binding assays if specific receptors are available

  • ELISA-based binding to potential receptor molecules or antibodies

• Inhibition Assays:

  • Pre-treatment of macrophages with recombinant p12 followed by ASFV challenge

  • Measurement of reduction in viral titers (80-90% reduction at 10 μg/mL indicates functional activity)

  • Dose-response analysis to calculate IC50 values

• Structural Verification:

  • Circular dichroism spectroscopy to assess secondary structure

  • Limited proteolysis to evaluate proper folding

  • Size-exclusion chromatography to detect aggregation

• Immunological Methods:

  • Western blotting with conformation-specific antibodies

  • Immunoprecipitation with known binding partners

  • Dot blot analysis for epitope accessibility

• Biophysical Characterization:

  • Thermal shift assays to determine stability

  • Surface plasmon resonance for binding kinetics

  • Dynamic light scattering to assess homogeneity

A systematic approach combining multiple methods provides the most comprehensive assessment of functional activity. For example, a researcher might first confirm structural integrity via SDS-PAGE and Western blotting, then proceed to binding assays, and finally validate functional activity through inhibition assays using live virus under appropriate biosafety conditions.

What biosafety considerations apply when working with recombinant ASFV proteins?

While recombinant p12 (War-108) doesn't contain infectious viral material, research with this protein still requires appropriate biosafety considerations:

While the recombinant protein itself poses minimal risk, maintaining strict biosafety practices prevents cross-contamination with live virus studies and establishes good laboratory habits for researchers who may later work with infectious materials .

What analytical techniques best characterize p12 (War-108) structural properties?

Comprehensive characterization of p12 (War-108) structural properties requires multiple analytical techniques that provide complementary information:

• Primary Structure Analysis:

  • Mass spectrometry for accurate molecular weight determination

  • Amino acid analysis to confirm composition

  • N-terminal sequencing to verify the start of the protein

  • Peptide mapping with LC-MS/MS for sequence coverage

• Secondary and Tertiary Structure Determination:

  • Circular dichroism (CD) spectroscopy for secondary structure content (α-helix, β-sheet)

  • Fourier-transform infrared spectroscopy (FTIR) for complementary secondary structure information

  • Nuclear magnetic resonance (NMR) for solution structure (challenging for membrane-associated proteins)

  • X-ray crystallography for high-resolution structure (currently a research need for p12)

• Aggregation and Homogeneity Assessment:

  • Size-exclusion chromatography (SEC)

  • Dynamic light scattering (DLS)

  • Analytical ultracentrifugation (AUC)

  • Native PAGE for oligomeric state determination

• Thermal and Chemical Stability:

  • Differential scanning calorimetry (DSC)

  • Thermal shift assays with fluorescent dyes

  • Chemical denaturation monitoring with spectroscopic techniques

  • Stability studies under varying pH and ionic strength conditions

• Membrane Interaction Studies (relevant for this viral envelope protein):

  • Lipid binding assays

  • Model membrane systems (liposomes, nanodiscs)

  • Atomic force microscopy for membrane insertion visualization

How can researchers design experiments to study p12's role in cross-protection against various ASFV genotypes?

Given that p12 is highly conserved across ASFV genotypes, investigating its potential for cross-protection represents an important research direction. A systematic experimental approach includes:

• Sequence Analysis and Epitope Mapping:

  • Comparative sequence analysis of p12 across all known ASFV genotypes

  • In silico prediction of B-cell and T-cell epitopes

  • Experimental validation of conserved epitopes using peptide arrays

  • Structural modeling to identify surface-exposed conserved regions

• Cross-Reactivity Assessment:

  • Production of recombinant p12 variants from diverse ASFV genotypes

  • Generation of polyclonal and monoclonal antibodies against reference p12

  • Cross-reactivity testing via ELISA, Western blot, and neutralization assays

  • Epitope-specific antibody binding studies

• In Vitro Protection Studies:

  • Attachment inhibition assays using p12 against viruses of different genotypes

  • Neutralization assays with anti-p12 antibodies against diverse viral isolates

  • Correlation of neutralization efficiency with sequence variation

• Immunization Studies:

  • Development of p12-based vaccine candidates focusing on conserved regions

  • Single-genotype versus multi-genotype p12 formulations

  • Prime-boost strategies with heterologous p12 variants

  • Challenge studies with viruses of different genotypes

• Immunological Analysis:

  • T-cell epitope conservation assessment through IFN-γ ELISPOT

  • Cross-reactive antibody profiling through competitive binding assays

  • Memory B-cell analysis for recognition of heterologous p12 variants

  • Correlation of immune parameters with protection status

Experimental design should incorporate statistical planning with adequate sample sizes and appropriate controls. A factorial design allowing for assessment of both genotype-specific and cross-protective responses would provide the most comprehensive data. Additionally, researchers should consider using pseudotyped viral systems for initial screening when possible, reserving live virus challenges for validation of promising approaches under appropriate biosafety conditions .