Recombinant African swine fever virus Inner membrane protein p54 (War-136)

What is p54 (War-136) protein and what is its significance in ASFV research?

The p54 protein, also known as War-136 in the isolate Warthog/Namibia/Wart80/1980, is an envelope protein encoded by the E183L gene of African swine fever virus (ASFV). It serves as a structural component of the viral particle and plays a crucial role in virus attachment and entry into host cells. The protein is highly significant in ASFV research due to its immunogenic properties, making it valuable for both diagnostic assay development and vaccine research. ASFV belongs to the Asfarviridae family and is endemic to sub-Saharan Africa, where it naturally circulates in a cycle involving ticks and wild pigs, including warthogs and bushpigs .

What are the molecular characteristics of recombinant p54 protein?

Recombinant p54 protein can be successfully expressed in bacterial systems, most commonly as a maltose binding protein (MBP)-tagged fusion protein in Escherichia coli. When expressed with the MBP tag, the fusion protein has an approximate molecular weight of 65 kDa as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The protein can be purified using affinity chromatography methods such as MBP Trap columns, and its identity can be verified through Western blot analysis using anti-p54 antibodies. The recombinant protein maintains immunogenic properties similar to the native viral protein, making it suitable for various research applications .

How does p54 contribute to ASFV pathogenesis and immune response?

The p54 protein is recognized as one of the most antigenic proteins of ASFV. During infection, it triggers a significant humoral immune response in pigs, resulting in the production of high levels of anti-p54 antibodies. These antibodies appear as early as 10 days post-infection and persist in the blood of infected animals for several weeks. This strong immunogenicity makes p54 a key target for immune recognition during natural ASFV infection. While the exact mechanism of p54's contribution to ASFV pathogenesis is still being investigated, its position as an envelope protein suggests its involvement in virus entry and host cell interactions. Research indicates that antibodies against p54 may play a role in neutralizing viral particles, although complete protection likely requires immune responses against multiple viral antigens .

What are the primary research applications for recombinant p54 protein?

Recombinant p54 protein has several key research applications:

• Diagnostic tool development: The protein serves as an antigen in various serological assays for ASFV antibody detection, including ELISA and Western blot.

• Vaccine research: As an immunogenic viral component, p54 is incorporated into experimental vaccine formulations, often in combination with other ASFV antigens.

• Immunological studies: Recombinant p54 is used to investigate host immune responses to ASFV infection, including antibody dynamics and cellular immunity.

• Structural biology: The protein enables studies of ASFV envelope architecture and virus-host interactions.

• Monoclonal antibody production: Purified recombinant p54 serves as an immunogen for generating specific antibodies for research and diagnostic applications .

How can researchers express and purify recombinant p54 protein?

To express and purify recombinant p54 protein, researchers can follow this methodological approach:

• Gene cloning: Amplify the E183L gene (excluding the predicted transmembrane domain) from ASFV isolates by PCR and clone it into an expression vector with an affinity tag, such as the pMAL-c5X vector for MBP fusion expression.

• Transformation and expression: Transform the recombinant plasmid into a suitable E. coli strain such as ER2523. Culture transformed bacteria at optimal conditions (typically 37°C) until reaching appropriate density, then induce protein expression using IPTG (isopropyl-β-D-1-thiogalactoside).

• Cell lysis: Harvest bacterial cells by centrifugation and lyse them using sonication or commercial lysis buffers to release the intracellular recombinant protein.

• Affinity purification: Purify the fusion protein using affinity chromatography specific to the tag (e.g., MBP Trap column for MBP-tagged proteins), following the manufacturer's protocols.

• Protein verification: Confirm protein identity and purity by SDS-PAGE analysis and Western blotting using anti-p54 or anti-tag antibodies.

• Additional purification: If necessary, perform further purification steps such as size exclusion chromatography or ion exchange chromatography to achieve higher purity.

• Quality control: Assess protein functionality through immunogenicity testing or binding assays .

How is recombinant p54 utilized in ASFV diagnostic assay development?

Recombinant p54 protein serves as a critical component in developing serological diagnostic assays for ASFV detection. The protein's high immunogenicity makes it an excellent target antigen for antibody detection in infected animals. Researchers have successfully developed various ELISA formats using p54, including indirect, competitive, and blocking ELISAs. In competitive ELISA (cELISA) development, the recombinant p54 protein is typically immobilized on microplates and used to detect competition between test serum antibodies and specific monoclonal antibodies. Previous studies have demonstrated that p54-based ELISAs achieve approximately 98% sensitivity and 97% specificity compared to OIE-recommended ELISAs. Additionally, p54 serves as a protein of choice for confirmation of ASFV antibody detection by Western blot, enhancing diagnostic reliability through multiple testing methodologies .

What are the performance characteristics of p54-based competitive ELISA for ASFV detection?

A competitive ELISA based on the p54 protein and using the monoclonal antibody designated as 2A7 has demonstrated excellent diagnostic performance. In a validation study using 365 pig serum samples (178 negative and 187 positive), the assay underwent receiver-operating characteristic (ROC) analysis to determine optimal cut-off values. The analysis revealed an area under the curve (AUC) of 0.982 (95% confidence interval: 96.9% to 99.4%), indicating excellent discriminatory ability. When using a percent inhibition threshold of 20%, the assay achieved a sensitivity of 92.5% and a specificity of 98.9%. Furthermore, this cELISA showed excellent agreement when compared to commercially available blocking ELISA methods (kappa value = 0.912) and demonstrated no cross-reactivity with other swine pathogens. These performance characteristics confirm that p54-based cELISA offers a reliable, rapid, and convenient approach for ASFV serodiagnosis .

What advantages do monoclonal antibodies against p54 offer for ASFV diagnostics?

Monoclonal antibodies (mAbs) against p54 provide several advantages for ASFV diagnostic applications:

• High specificity: These mAbs recognize specific epitopes on the p54 protein, reducing false positive results from cross-reactivity with other proteins.

• Consistency: Unlike polyclonal antibodies, mAbs offer batch-to-batch consistency, ensuring reproducible assay performance over time.

• Versatility: Anti-p54 mAbs can be used in multiple diagnostic formats, including ELISA, immunofluorescence assays, and Western blots.

• Competitive assay development: These mAbs enable the creation of competitive ELISAs, which often show improved specificity compared to indirect formats.

• Early detection: When targeting highly immunogenic proteins like p54, these assays can detect antibodies that appear early in infection (around 10 days post-infection).

Research has characterized several anti-p54 mAbs (2A7, 2D9, 4G5, 3F2, and 3D1), all belonging to the IgG1 isotype with kappa light chains. These mAbs effectively detect both recombinant p54 and ASFV-infected cells in immunofluorescence assays, demonstrating their utility across multiple diagnostic platforms .

How is p54 being utilized in ASFV vaccine research?

The p54 protein has emerged as a key component in ASFV vaccine development strategies, particularly in multi-antigen approaches. Researchers have incorporated the E183L gene encoding p54 into various vaccine platforms, with adenovirus-vectored vaccines showing promising results. In these constructs, replication-incompetent type-2 adenoviruses are engineered to carry ASFV antigens, including p54 alongside other immunogenic viral proteins like p30 (CP204L), CD2v (EP402R), p72 (B646L), and p72 chaperone (B602L). These adenovirus-vectored vaccines are designed to express ASFV antigens in vivo, stimulating both humoral and cellular immune responses without the risks associated with attenuated live virus vaccines. Vaccine cocktails containing multiple ASFV antigens, including p54, have demonstrated the ability to robustly elicit both systemic and mucosal immune responses in animal models, suggesting their potential efficacy against ASFV infection .

What vaccination regimens incorporating p54 have shown the most promise against ASFV?

Vaccination regimens that combine multiple delivery routes with multi-antigen formulations including p54 have demonstrated the most promising results. A notable approach involves simultaneous intramuscular (IM) and intranasal (IN) administration of adenovirus-vectored ASFV antigens. This combined IM/IN approach effectively stimulates both systemic immunity through intramuscular delivery and mucosal immunity via the intranasal route, creating a more comprehensive immune response against the virus. In experimental studies, such vaccine cocktails containing p54 alongside other ASFV antigens have shown highly effective protection against circulating ASFV strains in farmed pigs. These multi-antigen formulations were well-tolerated in vaccinated animals with no significant interference observed between the different antigens. The dual-route administration strategy appears to enhance protective efficacy compared to single-route approaches, suggesting that this combined delivery method warrants further evaluation for providing safe and effective protection against ASFV infection and transmission .

What are the challenges in using recombinant p54 in ASFV vaccine development?

Despite its promising attributes, several challenges exist in utilizing recombinant p54 for ASFV vaccine development:

• Incomplete protection: While p54 is highly immunogenic, antibodies against p54 alone do not confer complete protection against ASFV infection, necessitating multi-antigen approaches.

• Strain variation: Genetic differences between ASFV isolates may affect the cross-protection potential of p54-based vaccines against diverse field strains.

• Immune correlates: The specific immune correlates of protection against ASFV remain incompletely defined, complicating vaccine design and evaluation.

• Expression system limitations: Recombinant p54 produced in bacterial systems may lack post-translational modifications present in the native viral protein, potentially affecting immunogenicity.

• Delivery challenges: Optimal delivery methods and adjuvant formulations for recombinant protein vaccines must be determined to maximize immune response.

• Durability of immunity: Ensuring long-lasting protection through recombinant protein vaccination requires careful consideration of dosing schedules and adjuvant selection.

• Scalability: Production of consistent, high-quality recombinant p54 at scales necessary for widespread vaccination presents technical challenges .

What techniques are available for characterizing monoclonal antibodies against p54?

Researchers can employ several sophisticated techniques to characterize monoclonal antibodies (mAbs) against p54:

• Isotype determination: Commercial isotyping kits can identify antibody isotypes and light chain types, as exemplified in the characterization of five anti-p54 mAbs (2A7, 2D9, 4G5, 3F2, and 3D1), all found to be IgG1 with kappa light chains.

• Western blot analysis: This technique confirms the specificity of mAbs by demonstrating their ability to recognize the recombinant p54 protein at the expected molecular weight (approximately 65 kDa for MBP-tagged p54).

• Immunofluorescence assay (IFA): IFA using cells transfected with the p54 gene or ASFV-infected cells verifies the ability of mAbs to recognize both recombinant and native forms of the protein in cellular contexts.

• Epitope mapping: Techniques such as peptide array analysis or phage display can identify the specific epitopes recognized by each mAb, providing insights into antibody-antigen interactions.

• Affinity determination: Surface plasmon resonance (SPR) or bio-layer interferometry can measure binding affinities and kinetics of mAbs to p54.

• Cross-reactivity testing: Assessing mAb reactivity against various ASFV isolates and related proteins evaluates their specificity and potential for broad application.

• Functional analyses: Virus neutralization assays and antibody-dependent cellular cytotoxicity (ADCC) tests can determine the functional properties of the mAbs in virus inhibition .

How can researchers optimize recombinant p54 expression for structural studies?

Optimizing recombinant p54 expression for structural studies requires specialized approaches:

• Expression construct design: Remove transmembrane domains and flexible regions that may interfere with structural analysis. Consider generating truncated constructs focusing on stable domains.

• Expression host selection: While E. coli is commonly used, eukaryotic expression systems like insect cells (using baculovirus) may provide better folding and post-translational modifications for structural integrity.

• Fusion tag optimization: Beyond MBP, consider tags specifically designed for structural biology, such as SUMO or thioredoxin, which can enhance solubility while being removable with specific proteases.

• Expression condition screening: Systematically test various induction temperatures (typically lowering to 16-18°C), IPTG concentrations, and induction durations to maximize soluble protein yield.

• Solubility enhancement: Include additives such as glycerol, mild detergents, or specific ions in the buffer to maintain protein stability and prevent aggregation.

• Purification strategy refinement: Implement multi-step purification combining affinity chromatography with size exclusion chromatography and ion exchange to achieve the homogeneity required for structural studies.

• Protein quality assessment: Utilize techniques like dynamic light scattering, thermal shift assays, and negative stain electron microscopy to evaluate sample homogeneity before proceeding to structural studies .

How can researchers address low yields of soluble recombinant p54 protein?

Researchers encountering low yields of soluble recombinant p54 can implement several strategies:

• Optimize expression temperature: Lowering the expression temperature to 16-20°C can slow protein synthesis, allowing more time for proper folding and increasing solubility.

• Adjust induction parameters: Reduce IPTG concentration (0.1-0.5 mM) and extend induction time to promote slower, more efficient protein expression.

• Modify fusion tags: The maltose-binding protein (MBP) tag has proven effective for p54 expression, but alternative solubility-enhancing tags like SUMO, thioredoxin, or NusA can be tested if yields remain low.

• Co-express chaperones: Co-expressing molecular chaperones (e.g., GroEL/GroES, DnaK/DnaJ) can assist with protein folding and increase soluble yield.

• Optimize lysis conditions: Include additives in lysis buffers such as non-ionic detergents (0.1-0.5% Triton X-100), glycerol (5-10%), or higher salt concentrations (300-500 mM NaCl) to improve protein extraction and stability.

• Evaluate E. coli strains: Test expression in specialized strains designed for difficult proteins, such as Rosetta (for rare codons) or SHuffle (for disulfide bond formation).

• Consider refolding approaches: If inclusion bodies are unavoidable, develop a renaturation protocol using step-wise dialysis or on-column refolding techniques .

What strategies can improve specificity in p54-based diagnostic assays?

To enhance specificity in p54-based diagnostic assays, researchers can implement the following approaches:

• Monoclonal antibody selection: Carefully screen and select monoclonal antibodies based on their specificity and affinity for p54. The 2A7 monoclonal antibody, for example, has demonstrated excellent performance in competitive ELISA formats.

• Assay format optimization: Competitive or blocking ELISA formats often provide higher specificity than indirect ELISAs. These formats showed 98.9% specificity in published studies compared to conventional methods.

• Cut-off value determination: Apply rigorous statistical methods like ROC analysis to establish optimal cut-off values. Studies identified a 20% inhibition threshold as ideal for p54-based competitive ELISA, balancing sensitivity (92.5%) and specificity (98.9%).

• Cross-reactivity elimination: Pre-absorb reagents with potential cross-reactive proteins or include blocking agents in assay buffers to reduce non-specific binding.

• Sample preparation refinement: Optimize sample dilution and pre-treatment steps to minimize matrix effects that could impact assay specificity.

• Validation with diverse panels: Test the assay against a comprehensive panel of confirmed positive and negative samples, including challenging samples from animals with similar clinical presentations.

• Dual-antigen approaches: Consider combining p54 with other ASFV antigens like p30 or p72 in multiplex or sequential testing strategies to further increase diagnostic confidence .

What are promising new approaches for utilizing p54 in ASFV research?

Several innovative approaches for utilizing p54 in ASFV research show particular promise:

• Structure-function relationship studies: Determining the three-dimensional structure of p54 using X-ray crystallography or cryo-electron microscopy could reveal important insights into its role in viral entry and assembly.

• Epitope-focused vaccine design: Identifying specific protective epitopes within p54 could enable more targeted vaccine approaches, potentially improving efficacy while reducing the protein components needed.

• CRISPR-based diagnostics: Coupling p54-specific antibodies with CRISPR-Cas systems could enable ultra-sensitive point-of-care diagnostic platforms with simplified readouts.

• Nanoparticle delivery systems: Incorporating p54 into nanoparticle formulations may enhance its immunogenicity and stability, potentially improving vaccine efficacy.

• Synthetic biology approaches: Creating simplified synthetic versions of p54 that focus on key epitopes while eliminating regions that might contribute to adverse effects could lead to improved vaccine candidates.

• Combination with adjuvant discovery: Screening novel adjuvant formulations specifically optimized for p54-based vaccines could enhance immune responses and durability of protection.

• Single-domain antibody (nanobody) development: Generating camelid-derived nanobodies against p54 could provide new tools for diagnostic and therapeutic applications with advantages in stability and tissue penetration .

How might advancements in protein engineering enhance p54-based vaccines and diagnostics?

Protein engineering advancements offer several promising avenues to enhance p54-based applications:

• Epitope optimization: Computational prediction and experimental validation can identify immunodominant epitopes within p54, allowing for the creation of optimized constructs that present these epitopes in their most immunogenic form.

• Stability engineering: Introducing stabilizing mutations or disulfide bonds can enhance the thermal and storage stability of recombinant p54, extending shelf life for vaccines and diagnostic reagents.

• Surface display technologies: Engineering p54 or its epitopes for display on virus-like particles or nanoparticles can dramatically increase immunogenicity by mimicking the repetitive antigen display of viruses.

• Fusion protein strategies: Creating fusion proteins linking p54 with immune-stimulating molecules (like flagellin or cytokines) or other ASFV antigens can enhance immune responses and simplify vaccine production.

• Glycoengineering: Modifying or introducing glycosylation sites in recombinant p54 expressed in eukaryotic systems can potentially improve protein folding, stability, and immunogenicity.

• Scaffold protein approaches: Grafting key epitopes from p54 onto stable protein scaffolds could improve epitope presentation and immune recognition.

• Directed evolution: Applying directed evolution techniques to generate p54 variants with enhanced properties such as increased expression, solubility, or immunogenicity could yield improved reagents for both vaccines and diagnostics .

How do different expression systems for recombinant p54 compare in research applications?

This comparative analysis demonstrates that while E. coli expression systems with MBP fusion tags have been successfully employed for p54 expression in diagnostic applications, researchers should consider alternative expression platforms based on their specific research goals and requirements .

What are the performance characteristics of different diagnostic assays utilizing p54?

This data demonstrates that p54-based competitive ELISA using the 2A7 monoclonal antibody offers an excellent balance of sensitivity and specificity for ASFV diagnosis, while each method presents distinct advantages for specific research or diagnostic applications .

How do various vaccination strategies incorporating p54 compare in protective efficacy?