Recombinant African swine fever virus Virus attachment protein p12 (Ken-110)

What is the origin and classification of ASFV p12 (Ken-110)?

The p12 (Ken-110) protein is derived from African swine fever virus isolate Pig/Kenya/KEN-50/1950. ASFV belongs to the Asfarviridae family, which is endemic to sub-Saharan Africa. This specific p12 variant has been identified and cataloged in the UniProt database with the identifier P0C9Y1 . The virus naturally exists in a sylvatic cycle involving soft ticks of the Ornithodoros genus and wild pigs, including bushpigs and warthogs . The Ken-110 variant specifically refers to the attachment protein derived from this Kenyan isolate, which has been recombinantly expressed in E. coli systems for research purposes.

How does ASFV p12 (Ken-110) differ from other p12 variants?

The p12 (Ken-110) variant (aa 1-62) differs from other variants such as the Warthog/Namibia/Wart80/1980 isolate (aa 1-61) primarily in amino acid length and sequence variations that reflect their different geographical origins . These differences, though minor in terms of length (62 amino acids versus 61 amino acids), may contribute to functional variations in host specificity, virulence, and immunogenicity. The Ken-110 variant has been assigned the UniProt ID P0C9Y1, while the Warthog-derived variant has been assigned P0C9Y4 , indicating distinct protein records in biological databases.

What is the functional role of p12 in ASFV infection?

The p12 protein functions as a virus attachment protein, playing a crucial role in the early stages of viral infection. It facilitates the virus's ability to recognize and bind to receptors on susceptible host cells, particularly in swine species. While specific binding mechanisms of p12 (Ken-110) have not been fully characterized in the provided search results, viral attachment proteins generally initiate the infection process by mediating virus-host cell interactions that ultimately lead to virus internalization. The protein's relatively small size (62 amino acids for Ken-110) suggests it may function as part of a larger attachment complex rather than independently .

What expression systems are commonly used for producing recombinant ASFV p12?

E. coli is the predominant expression system used for recombinant production of ASFV p12 proteins, including the Ken-110 variant . While the search results don't provide specific expression protocols for p12, similar ASFV proteins like p32 and p54 have been successfully expressed using Semliki Forest virus (SFV) vector systems in baby hamster kidney (BHK-21) cells . For p12 specifically, bacterial expression in E. coli represents a cost-effective method that yields sufficient quantities of protein for research applications, though post-translational modifications may differ from those in the native viral context.

What structural characteristics of p12 (Ken-110) contribute to its function in viral attachment?

While the search results don't provide specific structural data for p12 (Ken-110), its classification as a virus attachment protein suggests it likely contains domains involved in receptor recognition. The relatively short sequence (62 amino acids) indicates a compact structure that may function alongside other viral proteins in the attachment complex. Researchers investigating this protein would need to employ techniques such as X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy to determine its three-dimensional structure and identify the specific amino acid residues involved in host cell receptor binding. Computational modeling based on the known UniProt sequence (P0C9Y1) may provide initial insights into structure-function relationships .

How do the immunogenic properties of p12 (Ken-110) compare with other ASFV antigens?

Based on research with other ASFV antigens such as p32 and p54, recombinant viral proteins can elicit robust humoral and cellular immune responses when properly delivered . While the search results don't provide specific immunogenicity data for p12 (Ken-110), similar attachment proteins often contain B-cell and T-cell epitopes that can stimulate adaptive immunity. In studies with p32 and p54, Semliki Forest virus replicon particles (SFV-RPs) expressing these proteins induced immune responses in BALB/c mice without significant adverse effects on their growth or health status . Similar approaches could be applied to evaluate the immunogenic properties of p12, potentially in combination with other ASFV antigens for synergistic immune stimulation.

What is the role of p12 (Ken-110) in ASFV genotype diversity and virulence determination?

ASFV exists in at least 24 known genotypes with variable virulence profiles . While the search results don't explicitly connect p12 variants to specific virulence phenotypes, attachment proteins like p12 can influence host range and tissue tropism, potentially contributing to virulence differences between strains. The p12 (Ken-110) variant comes from a historical Kenyan isolate (KEN-50/1950), which may exhibit different virulence characteristics compared to currently circulating strains. Researchers investigating the relationship between p12 sequence variation and virulence would need to conduct comparative studies across multiple ASFV isolates, potentially using recombinant viruses with chimeric or mutated p12 sequences to assess the protein's specific contribution to pathogenesis.

How can researchers optimize expression and purification of functionally active p12 (Ken-110)?

While specific protocols for p12 (Ken-110) are not detailed in the search results, researchers can adapt methods used for similar ASFV proteins. For bacterial expression, considerations include codon optimization for E. coli, selection of appropriate fusion tags (e.g., His-tag, GST) to facilitate purification, and optimization of induction conditions to maximize yield while maintaining protein solubility. For mammalian expression, viral vector systems like the SFV replicon particles used for p32 and p54 could be adapted . Purification strategies should account for the relatively small size of p12 (62 amino acids) and potential challenges in maintaining its native conformation. Functional activity assessments could include binding assays with susceptible cell lines and conformational analysis using circular dichroism or other spectroscopic methods.

What are the recommended protocols for assessing p12 (Ken-110) interactions with host cell receptors?

To investigate p12-receptor interactions, researchers should consider a multi-faceted approach:

• Binding assays: Utilizing purified recombinant p12 (Ken-110) labeled with fluorescent dyes or biotin to identify binding to susceptible cell types. Flow cytometry can quantify binding kinetics and saturation.

• Receptor identification: Employing techniques such as co-immunoprecipitation followed by mass spectrometry, virus overlay protein binding assay (VOPBA), or yeast two-hybrid screening to identify cellular proteins that interact with p12.

• Inhibition studies: Using anti-p12 antibodies, soluble receptor mimics, or peptide competitors to block viral attachment and quantify the specific contribution of p12 to the attachment process.

• Mutagenesis: Creating alanine-scanning or targeted mutants of p12 to identify critical residues involved in receptor binding, followed by functional binding assays to quantify the impact of these mutations.

Each approach should include appropriate controls and multiple biological replicates to ensure reproducibility of results.

How can researchers effectively compare immunogenicity of different p12 variants in vaccine development?

Based on immunogenicity studies with other ASFV proteins , researchers should implement:

• Animal model selection: While BALB/c mice have been used for initial immunogenicity assessments , pig models are essential for translational relevance due to species-specific immune responses to ASFV antigens.

• Vaccine platform comparison: Test p12 (Ken-110) in multiple delivery systems, including:

  • Viral vectors (adenovirus, modified vaccinia Ankara)

  • Replicon particles (as demonstrated with SFV-RPs for p32 and p54)

  • DNA vaccines

  • Protein subunit formulations with various adjuvants

• Immune response assessment: Measure both humoral and cellular immunity:

  • Antibody responses (ELISA, virus neutralization assays)

  • T-cell responses (ELISpot, intracellular cytokine staining)

  • Cytokine profiles (multiplex assays)

• Comparative analysis: Include side-by-side testing of p12 variants from different ASFV isolates to identify those with optimal immunogenicity profiles.

• Challenge studies: Ultimately, protective efficacy must be assessed through challenge with virulent ASFV in susceptible swine, measuring parameters such as viremia, clinical scores, and survival rates.

What techniques are most effective for studying p12 expression during different stages of ASFV infection?

To characterize p12 expression dynamics during infection:

• Quantitative RT-PCR: Design primers specific for p12 (Ken-110) mRNA to monitor transcript levels at different time points post-infection.

• Western blotting: Develop specific antibodies against p12 to track protein expression in infected cells, with careful sample collection at multiple time points.

• Immunofluorescence microscopy: Use anti-p12 antibodies for spatial localization of the protein within infected cells, potentially with co-staining for cellular compartment markers.

• Reporter systems: Generate recombinant ASFV with p12 fused to fluorescent proteins to monitor expression in real-time during infection.

• Proteomic analysis: Apply mass spectrometry-based approaches to quantify p12 abundance relative to other viral proteins throughout the infection cycle.

These approaches should be implemented in relevant cell types including porcine macrophages, which are primary targets for ASFV infection.

How can recombinant p12 (Ken-110) be utilized in serological diagnostic assays for ASFV?

Recombinant p12 protein can serve as a valuable antigen in ELISA-based serological assays for ASFV detection. While the search results don't specifically address p12-based diagnostics, the methodology would be similar to approaches using other ASFV proteins:

• ELISA development: Coat plates with purified recombinant p12 (Ken-110) to capture anti-p12 antibodies from test samples.

• Validation parameters to establish:

  • Analytical sensitivity and specificity

  • Diagnostic sensitivity and specificity using well-characterized serum panels

  • Cross-reactivity assessment with antibodies against classical swine fever and other porcine pathogens

  • Reproducibility across different laboratories

• Applications:

  • Surveillance in endemic regions

  • Confirmation of infection in clinically suspect cases

  • Monitoring of immune responses following vaccination

  • Epidemiological studies

The relatively conserved nature of p12 across some ASFV isolates could make it valuable for broad detection, though validation against multiple genotypes would be essential.

What are the key considerations when designing experiments to assess p12's role in ASFV virulence?

To investigate p12's contribution to virulence:

• Gene manipulation approaches:

  • CRISPR/Cas9 or homologous recombination to generate p12 deletion mutants

  • Complementation studies with different p12 variants

  • Site-directed mutagenesis targeting specific functional domains

• In vitro assessments:

  • Growth kinetics in different cell types

  • Cell-to-cell spread efficiency

  • Cytopathic effect quantification

  • Macrophage response modulation

• In vivo studies:

  • Clinical presentation monitoring using standardized scoring systems

  • Viral load quantification in tissues

  • Immune response characterization

  • Pathological assessment

• Comparative analysis:

  • Side-by-side testing of wild-type virus versus p12-modified variants

  • Comparison across different host genetic backgrounds

Researchers must account for the potential compensatory effects of other viral proteins and conduct experiments in both laboratory-adapted cell lines and primary porcine cells.

What are the promising avenues for combining p12 (Ken-110) with other ASFV antigens in multivalent vaccine development?

Based on immunogenicity studies with other ASFV proteins like p32 and p54 , future research should explore:

• Antigen combinations: Test p12 (Ken-110) in combination with other ASFV proteins that have shown immunogenic potential, such as:

  • p32 and p54, which have demonstrated robust immune responses in SFV-RP delivery systems

  • p72 (major capsid protein)

  • CD2v (hemadsorption protein)

  • Additional structural or non-structural proteins with conserved epitopes

• Delivery platforms for multivalent expression:

  • Viral vectors expressing multiple antigens

  • DNA vaccines with polycistronic constructs

  • Protein cocktails with optimized adjuvant formulations

  • Virus-like particles incorporating multiple ASFV proteins

• Epitope optimization:

  • Identification of protective B-cell and T-cell epitopes within p12

  • Creation of chimeric antigens containing multiple protective epitopes from different ASFV proteins

  • Modification of epitopes to enhance presentation by relevant swine MHC alleles

• Immune correlates of protection:

  • Systematic assessment of antibody and T-cell responses to identify correlates of protection

  • Longitudinal studies to determine duration of immunity with different antigen combinations

This multivalent approach may overcome the limitations of single-antigen vaccines and address the challenge of ASFV genetic diversity.

How might structural biology approaches advance our understanding of p12 (Ken-110) function?

Advanced structural biology techniques would significantly enhance our understanding of p12 function:

• High-resolution structure determination:

  • X-ray crystallography of purified p12

  • Cryo-electron microscopy of p12 in complex with potential receptors

  • NMR spectroscopy for dynamic structural elements

• Structure-function analysis:

  • Mapping of receptor-binding domains

  • Identification of conformational epitopes recognized by neutralizing antibodies

  • Comparison with attachment proteins from other viral families

• Molecular dynamics simulations:

  • Modeling of p12 interactions with cell membrane components

  • Prediction of conformational changes during receptor binding

  • Virtual screening for potential inhibitors of p12-receptor interactions

• Structural comparisons across isolates:

  • Analysis of structural conservation and variation among p12 proteins from different ASFV genotypes

  • Correlation of structural features with virulence phenotypes

These approaches would provide fundamental insights into viral entry mechanisms and identify potential targets for therapeutic intervention.

What comparative analysis methods can assess functional differences between ASFV p12 variants?

The following table outlines methodological approaches for comparing different p12 variants:

Researchers should implement these methods in a systematic manner, including the Ken-110 variant alongside other p12 proteins from different ASFV genotypes to establish functional correlates.

How do different expression systems affect the yield and functionality of recombinant p12 (Ken-110)?

Based on recombinant protein expression principles and data from similar viral proteins:

• Expression system comparison:

  • E. coli: Typically yields 5-15 mg/L of culture, with potential for inclusion body formation requiring refolding

  • Yeast: Moderate yields (2-8 mg/L) with improved folding but potential hyperglycosylation

  • Insect cells: Lower yields (1-5 mg/L) but better post-translational modifications

  • Mammalian cells: Lowest yields (<1 mg/L) but most authentic modification pattern

• Critical factors affecting functionality:

  • Proper disulfide bond formation

  • Absence of contaminating bacterial endotoxins

  • Correct folding and oligomeric state

  • Stability during purification and storage

• Optimization strategies:

  • Codon optimization for the expression host

  • Fusion partners to enhance solubility (SUMO, thioredoxin)

  • Directed evolution to select for variants with improved expression

  • High-throughput screening of culture conditions

Researchers should conduct side-by-side functional comparisons of p12 expressed in different systems to identify the optimal approach for their specific application.