ASFV P72

What is the ASFV P72 protein and why is it significant in ASFV research?

P72 is a major capsid structural protein of African Swine Fever Virus that exhibits strong antigenicity, typically eliciting high antibody titers in ASFV-infected pigs . It is considered one of the immunodominant proteins of ASFV, making it a critical target for both diagnostics and vaccine development . The significance of P72 lies in its essential structural role in the virus and its strong immunological properties that make it valuable for both detecting infection and potentially protecting against it. P72 bears T-cell antigenic epitopes which can trigger T cells to secrete pro-inflammatory cytokines once presented by major histocompatibility complexes . This protein is highly conserved across ASFV isolates, which makes it ideal for broad spectrum detection and potential cross-protection strategies.

How does the P72 protein contribute to ASFV immunology?

The P72 protein plays multiple roles in ASFV immunology:

• Antibody production: P72 elicits strong humoral immune responses, generating high antibody titers in infected animals, making it useful for serodiagnosis .

• T-cell responses: P72 contains T-cell epitopes that can trigger cellular immunity. Studies have shown that peripheral blood mononuclear cells (PBMCs) from pigs recovered from ASFV infection produced IFN-γ upon stimulation with the virus, and specifically with P72 peptides .

• Cytokine modulation: P72 influences the production of various cytokines, including pro-inflammatory cytokines such as IFN-γ, IL-1β, IL-6, and IL-12, which orchestrate immune responses .

Research has demonstrated that P72 can stimulate splenocytes to secrete IFN-γ, which is an important pro-inflammatory cytokine in ASFV infection . This highlights the protein's role not just as an antigenic target but as an active modulator of host immune responses.

How do P72 structural mutations impact immunological responses and what are the implications for vaccine development?

Research on P72 mutants, particularly p72Δ377–428 (which contains a deletion of amino acids 377-428), has revealed significant impacts on immunological responses:

Structural changes: Swiss-Model analysis predicts that this deletion converts the native P72 trimer from a compacted tertiary configuration to a loose configuration . This structural alteration appears to fundamentally change how the protein interacts with the immune system.

Immunological effects of p72Δ377–428 compared to wild-type P72:

• Enhanced antibody production: p72Δ377–428 induced stronger antibody production in mice on days 42 and 56 post-immunization

• Reduced pro-inflammatory cytokine production: The mutant protein reduced IFN-γ production in splenocytes from immunized mice and treated swine macrophages

• Decreased IL-1β, IL-6, and IL-12 production: Lower levels of these pro-inflammatory cytokines were observed with the mutant protein

• Mechanistic pathway alterations: p72Δ377–428 reduced pro-inflammatory cytokine gene induction by inhibiting AKT phosphorylation and HIF1α expression

These findings suggest that strategic modifications to P72 could potentially produce vaccine candidates that enhance protective antibody responses while reducing the risk of cytokine storms caused by excessive pro-inflammatory cytokine release. This is particularly important for ASFV, as severe disease is often associated with dysregulated immune responses.

What signaling pathways does P72 interact with and how might these be targeted in therapeutic interventions?

P72 interacts with several important immune signaling pathways:

• AKT/HIF1α Pathway: Research indicates that p72 activates the AKT/HIF1α signaling pathway, which regulates the production of pro-inflammatory cytokines . The mutant p72Δ377–428 reduces the induction of pro-inflammatory cytokines by decreasing HIF1α expression through inhibition of AKT phosphorylation .

• T-cell receptor signaling: P72 contains T-cell antigenic epitopes that, when presented by MHC molecules, trigger T-cell activation and subsequent cytokine secretion .

• Inflammatory cascades: P72 influences the production of multiple pro-inflammatory cytokines, including IFN-γ, IL-1β, IL-6, and IL-12, suggesting interaction with various inflammatory pathways .

Potential therapeutic targets based on these interactions include:

• AKT inhibitors: Could potentially reduce excessive inflammation during ASFV infection

• HIF1α modulators: May help control cytokine storms associated with severe disease

• Tailored immunomodulatory approaches: Designing interventions that maintain protective immunity while preventing pathological inflammation

Future research using experimental approaches such as phosphor-proteomics, pathway inhibition, and gene silencing would be valuable to further validate these targets and develop effective therapeutic strategies .

What are the optimal methods for expressing and purifying recombinant P72 protein for immunological studies?

Based on the research data, the following methodological approach is recommended for optimal expression and purification of recombinant P72:

Expression System Selection:

• E. coli has been successfully used for P72 expression, particularly for the p72Δ377–428 mutant which showed stable and high expression

Expression Protocol:

• Culture bacteria in LB selective medium with appropriate antibiotic (e.g., 50 μg/mL kanamycin)

• Dilute overnight culture in fresh medium at 1:100 ratio

• Induce protein expression when cell density (OD600nm) reaches 0.6 using IPTG

Optimization Parameters to Consider:

• IPTG concentration: Test range from 0.1 to 2.0 mM

• Temperature: Evaluate expression at 20, 25, 32, 37, or 40°C

• Duration: Test induction periods of 2, 3, 4, 5, or 6 hours

Protein Extraction and Purification:

• Harvest cells by centrifugation and wash with PBS

• Resuspend in appropriate lysis buffer

• Lyse cells via sonication (200 cycles of two working seconds and four resting seconds on ice)

• Separate supernatant and pellet (inclusion bodies) by centrifugation

• Analyze yield using 10% SDS-PAGE gel

For researchers working with P72 variants, it's essential to evaluate each parameter individually to determine optimal expression conditions for the specific construct. The research indicates that structural modifications like those in p72Δ377–428 can significantly impact expression efficiency and stability.

What are the current most sensitive detection methods for P72 antibodies and how do they compare?

Several detection methods for P72 antibodies have been developed and compared in research settings:

Chemiluminescent Immunoassay (CLIA):

• A recently developed dual-antigen sandwich automated chemiluminescent immunoassay (DAgS-aCLIA) showed excellent performance for P72 antibody detection

• This method uses P72 protein conjugated to magnetic particles (MPs) and acridinium ester (AE)

• Quantitative range: 0.21 to 212.0 ng/mL or 0.21 to 424.0 ng/mL depending on the specific protocol

• Shows excellent diagnostic accuracy with AUC > 0.9

Comparison with ELISA Methods:
The following table summarizes the comparative performance between CLIA and different commercial ELISA kits:

Coincidence Rate Analysis:

The research indicates that while ELISA methods remain valuable and widely accessible, the newer CLIA methodology offers superior sensitivity and a wider detection range, making it particularly valuable for research applications requiring precise quantification of antibody responses.

What are the most effective PCR protocols for detecting the ASFV p72 gene in research samples?

Real-time PCR assays targeting the p72 gene have been developed for highly sensitive and specific detection of ASFV. The following methodological considerations should be noted:

Recent PCR Development:
A newly developed multiplex real-time PCR assay has shown improved performance compared to the widely used Zsak assay. This assay was designed after analyzing 1,012 p72 sequences from GenBank and 23 additional p72 sequences to ensure broader coverage of ASFV strains/isolates .

Key Features of the Advanced PCR Protocol:

• Targets the conserved p72 gene region

• Incorporates porcine beta-actin (ACTB) as an endogenous internal control to reduce false-negative detections

• Uses a multiplex approach combining a new p72 assay with a modified Zsak assay

• Achieves broader strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available

Performance Specifications:

• Limit of Detection (LOD): 6 plasmid copies or 0.1–1 TCID₅₀/ml of ASFV isolates per reaction

• Specificity: 100%, with only targeted ASFV isolates and positive clinical samples being detected

• Superior performance compared to the standard Zsak assay when tested with 26 ASFV isolates from different countries

The inclusion of an endogenous internal control (ACTB) is particularly valuable for research applications as it helps verify sample quality and extraction efficiency, reducing the risk of false-negative results due to PCR inhibition or nucleic acid degradation.

How can multi-epitope approaches to P72 be designed for improved diagnostic or vaccine applications?

Multi-epitope approaches to P72 (MeP72) represent an advanced strategy for designing more effective diagnostic tools and vaccine candidates. Based on the research data:

Design Process for Multi-epitope P72:

• Epitope prediction: Use computational tools to identify dominant linear epitopes within the P72 sequence

• Screening: Select epitopes based on their predicted antigenicity, conservation across strains, and accessibility

• Construction: Engineer a recombinant protein containing multiple selected epitopes, potentially with appropriate linkers between epitopes

Advantages of Multi-epitope Approaches:

• Enhanced sensitivity: By incorporating multiple epitopes, the construct can detect antibodies targeting different regions of P72

• Improved specificity: Carefully selected epitopes can reduce cross-reactivity with other pathogens

• Broader protection: As a vaccine candidate, multi-epitope constructs may elicit immune responses against multiple crucial epitopes simultaneously

Research Applications:

• Diagnostic development: Multi-epitope constructs can serve as antigens in various assay formats (ELISA, CLIA, lateral flow)

• Vaccine research: May induce more balanced immune responses compared to whole protein

• Epitope mapping studies: Help identify which regions of P72 are immunologically dominant in different host species or infection stages

Based on epitope prediction results, researchers can screen and select dominant linear epitopes for constructing multi-epitope proteins . This approach may be particularly valuable when certain regions of P72 elicit stronger protective immune responses than others, allowing for focused targeting of these critical epitopes.

What are the prospects for using P72 mutants like p72Δ377–428 in ASFV vaccine development?

The p72Δ377–428 mutant shows promising attributes for vaccine development based on several key findings:

• Enhanced antibody production: p72Δ377–428 elicited stronger humoral immunity in mice compared to wild-type p72

• Reduced pro-inflammatory cytokine production: The mutant reduced levels of IFN-γ, IL-1β, IL-6, and IL-12, potentially attenuating the risk of cytokine storms that can cause severe disease

• Recognition of ASFV antisera: p72Δ377–428 could recognize ASFV-infected swine sera, confirming its diagnostic potential

• Structural modifications: The deletion creates a distinct structure compared to wild-type p72, which may contribute to its altered immunological properties

These characteristics suggest that p72Δ377–428 could serve as a novel candidate for ASFV vaccines by providing strong antibody-mediated protection while reducing the risk of harmful inflammatory responses. Future research should focus on:

• In vivo protection studies in pigs to evaluate efficacy against ASFV challenge

• Combination with other ASFV immunogens for potentially synergistic protection

• Optimization of delivery platforms to enhance immune responses

• Investigation of cellular immune responses beyond antibody production

• Long-term immunity studies to assess duration of protection

The ability of p72Δ377–428 to modulate immune responses through the AKT/HIF1α pathway also opens avenues for better understanding the immunopathology of ASFV infection and developing strategies to control it .