PER71 Antibody

What is EV71 and why are antibodies against it significant for research?

Enterovirus 71 (EV71) is a picornavirus that causes yearly outbreaks of hand, foot, and mouth disease, primarily in Southeast Asian countries including China and Malaysia . The clinical significance stems from its ability to cause severe neurological complications in infected children, including encephalitis that can be fatal or result in permanent brain damage . With no approved anti-EV71 therapeutic agents currently available, antibodies represent both valuable research tools and potential therapeutic candidates .

Understanding EV71 antibodies provides critical insights into human immune responses to the virus, informing vaccine development strategies and therapeutic approaches. These antibodies can also reveal the structural dynamics of virus-antibody interactions, offering mechanistic understanding of viral neutralization .

What are the structural characteristics of EV71 that influence antibody binding?

EV71 virions are nonenveloped particles with approximately 300 Å diameter. The capsid exhibits icosahedral, pseudo-T=3 symmetry composed of four viral proteins: VP1, VP2, VP3, and VP4 in each icosahedral asymmetric unit . The three external proteins (VP1, VP2, and VP3) display a jelly-roll fold common to many viruses, while VP4 is attached to the inner capsid face .

Two key particle forms exist with distinct antibody interactions:

• Mature infectious RNA-filled particles with VP1, VP2, VP3, and VP4

• Empty immature particles lacking genome that contain VP0 (precursor of VP4 and VP2)

These empty particles have approximately 5% larger diameter than mature virions, with the protomer formed by VP0, VP1, and VP3 rotated by 5.4° relative to the mature particle protomer with respect to the icosahedral symmetry axes . This structural difference creates unique epitopes that can generate antibodies with specific neutralizing properties.

How can researchers distinguish between different types of EV71 antibodies?

Researchers can categorize EV71 antibodies using several experimental approaches:

For distinguishing conformational vs. linear epitope recognition, researchers should use both ELISA with intact viral particles and western blot with denatured viral proteins . Neutralizing antibodies frequently recognize conformational epitopes that aren't detectable by western blot analysis, as demonstrated with antibodies E18 and E19 .

What techniques are most effective for isolating and characterizing EV71-specific antibodies?

Based on current research, the most productive approaches include:

For antibody isolation:

• Phage display libraries constructed from peripheral blood of EV71-infected donors have yielded numerous EV71-specific human antibodies . One study identified 27 EV71-specific human antibodies from eight infected donors, with four demonstrating neutralizing activity .

• Immunization with empty immature EV71 particles containing VP0 has successfully generated antibodies (like E18 and E19) with distinct neutralizing properties .

For antibody characterization:

• Neutralization assays using plaque reduction methods to evaluate potency

• Cryo-electron microscopy to determine binding sites and structural effects

• Deep sequencing of antibody heavy chains to analyze antibody conservation across patients

• Competitive ELISA to examine epitope specificity and antibody competition

The integration of these complementary techniques provides the most comprehensive characterization. For example, researchers identified that the heavy chains of EV71-specific antibodies were conserved among infected individuals but absent in controls, suggesting convergent evolution of human antibodies against EV71 .

How should researchers prepare Fab fragments for structural studies of EV71-antibody complexes?

Preparation of Fab fragments is essential for structural studies of EV71-antibody complexes, particularly for cryo-EM analyses. The methodological workflow includes:

• Isolate monoclonal antibodies from immunized animals or human donors

• Prepare Fab fragments using commercial kits (e.g., Pierce Fab Preparation Kit) according to manufacturer's instructions

• Verify Fab quality and binding activity using ELISA before complex formation

• Incubate Fab fragments with purified EV71 virions at appropriate ratios

• Prepare samples for cryo-EM imaging, ensuring sufficient distribution of particles

• Collect and process images for 3D reconstruction of the virus-Fab complex

For optimal results, researchers should ensure animal care and use complies with national welfare standards and guidelines (e.g., the Animals Act of 2006 in Malaysia) . The preparation of high-quality Fab fragments is critical for revealing the structural basis of neutralization mechanisms.

What mechanisms do neutralizing antibodies employ to inhibit EV71 infection?

Research has revealed several distinct neutralization mechanisms for EV71 antibodies:

Genome Release Induction:
The E18 antibody, generated by immunizing mice with empty immature EV71 particles, employs an unusual neutralization mechanism. When incubated with mature virus at temperatures between 4°C and 37°C, E18 induced a conformational change transforming infectious virions into A particles . This transformation results in genome release from the virion, effectively neutralizing the virus by rendering it non-infectious . Cryo-EM studies revealed that approximately 20% of EV71 particles incubated with E18 Fab had lost much or all of their RNA genome .

Conformational Locking:
Some antibodies bind to the virion and prevent the structural changes necessary for cell entry or uncoating. This "conformational locking" mechanism keeps the virus in a non-infectious state.

Epitope-Specific Neutralization:
Research has identified three major neutralizing epitopes on EV71:

• Plateau epitope (where a single residue change VP3 E81K can significantly reduce antibody binding)

• Fivefold vertex epitope

• Canyon epitope

Antibodies targeting the plateau epitope demonstrate particularly potent neutralization, with cryo-EM structures showing their binding footprints are conserved across most circulating EV71 strains .

How does the antibody repertoire evolve during EV71 infection?

Deep sequencing analysis of antibody heavy chains from EV71-infected donors has revealed fascinating insights into repertoire evolution:

• EV71-specific antibody heavy chains are remarkably conserved among infected individuals but absent in controls .

• This pattern suggests convergent evolution of human antibodies against EV71, with similar selection pressures driving antibody development across different patients .

One study sequenced B-cell transcriptomes from three EV71-infected patients at two time points (two days and twelve days after hospitalization) using next-generation sequencing technology . The abundance and distribution of EV71-specific antibody heavy chains showed conservation both in sequences and abundance patterns among infected individuals .

This conservation has significant implications for understanding human humoral immune responses against viral infection and provides important insights for diagnostics and therapeutics of EV71 .

What structural changes occur in EV71 virions when neutralizing antibodies bind?

Cryo-EM studies have revealed significant structural alterations when certain antibodies bind to EV71:

E18 Fab binding induces a dramatic conformational transformation of EV71 virions from the mature state to the A-particle state . This change was confirmed by comparing the cryo-EM electron density maps of:

• Heat-induced EV71 A particles

• EV71-E18 (full) complex

The correlation coefficient between these maps was 0.83, whereas the correlation between native virus capsid and the EV71-E18 complex was only 0.61 . This indicates that E18 Fab binding triggers the same structural transition as heat treatment, converting the virus to A particles.

This structural transformation exposes internal components of the virus and compromises the integrity of the RNA genome compartment, leading to RNA release and loss of infectivity .

How can EV71 antibodies be utilized for diagnostic purposes?

EV71 antibodies offer several diagnostic applications for research and clinical settings:

ELISA-Based Detection Systems:

• Indirect ELISA using heat-inactivated EV71-infected cell lysates can detect EV71-specific antibodies in patient samples

• Sandwich ELISA employing antibodies against VP1 (like R525) can capture viral particles, with detection by monoclonal antibodies such as E18 or E19

• Competitive ELISA can identify antibodies containing specific epitopes (e.g., E18 epitope) in mouse or human serum

Methodological Approach:
For researchers developing diagnostic assays, the following protocol has demonstrated effectiveness:

• Coat plates with antibody against VP1 (e.g., R525)

• Add PEG-precipitated EV71 (untreated or heat-inactivated)

• Detect bound particles using monoclonal antibodies (E18/E19) followed by HRP-conjugated secondary antibodies

• Develop with TMB substrate and measure absorbance at 450nm

This approach allows detection of EV71 in clinical samples with high sensitivity and specificity.

What are the therapeutic potentials and limitations of EV71 antibodies?

EV71 antibodies show significant therapeutic promise based on current research:

Therapeutic Potential:

• Neutralizing antibodies targeting the plateau epitope have demonstrated efficacy against lethal challenge in murine infection models

• Antibodies like E18 that induce genome release could be developed as anti-EV71 therapies

• Human monoclonal antibodies from infected donors offer naturally optimized therapeutic candidates

Key Limitations:

• Single amino acid mutations can significantly reduce antibody binding, as seen with the VP3 E81K mutation in the plateau epitope

• The diversity of circulating EV71 genotypes presents challenges for broad-spectrum coverage

• Some antibodies recognize conformational epitopes that may be difficult to reproduce in recombinant protein systems

Research-Based Recommendations:
For researchers pursuing antibody therapeutics, focus on antibodies with binding footprints conserved across the majority of circulating EV71 strains . Consider cocktail approaches combining antibodies targeting different epitopes to mitigate escape through mutation.

What critical knowledge gaps remain in understanding EV71 antibody responses?

Despite significant advances, several important questions remain unanswered:

• Epitope Identification: For many EV71-specific antibodies, precise epitope mapping remains challenging. Western blotting and peptide arrays have been attempted, but neutralizing antibodies often fail to react with denatured virions, suggesting complex conformational epitopes .

• Cross-Protection Mechanisms: How antibodies cross-protect against different EV71 genotypes needs further elucidation to inform broad-spectrum therapeutic development.

• Human Antibody Evolution: While conservation of antibody heavy chains has been observed, the mechanisms driving this convergent evolution require further investigation.

• Long-Term Protection: Durability of antibody responses and correlates of long-term protection against EV71 infection remain poorly understood.

• Antibody-Dependent Enhancement: Whether any antibodies might enhance infection under certain conditions needs careful evaluation.

Addressing these gaps will require integration of structural biology, deep sequencing of antibody repertoires, and functional characterization in relevant model systems.

How might new technological approaches advance EV71 antibody research?

Several emerging technologies offer transformative potential for EV71 antibody research:

Single B-Cell Sequencing:
This approach allows direct isolation and characterization of paired heavy and light chains from individual B cells of infected patients, providing more comprehensive understanding of the antibody repertoire than phage display or traditional hybridoma methods .

Cryo-Electron Tomography:
Building on successful cryo-EM studies of antibody-virus complexes , cryo-electron tomography could provide insights into antibody binding to heterogeneous virus populations or partially disrupted particles.

Deep Mutational Scanning:
Systematic analysis of how mutations in EV71 epitopes affect antibody binding could identify critical residues and predict escape variants, informing therapeutic antibody engineering.

Computational Epitope Prediction:
Integration of structural data with machine learning approaches could accelerate identification of vulnerable epitopes and design of optimized antibody therapeutics.

These technological advances promise to deepen our understanding of EV71-antibody interactions and accelerate development of effective diagnostics and therapeutics for EV71 infections.