HK1/2/3 Antibody

What are HK1, HK2, and HK3 antibodies and what is their origin?

H7.HK1, H7.HK2, and H7.HK3 are human monoclonal antibodies isolated from a 2013 H7N9 convalescent case in Hong Kong. These antibodies were obtained through single B cell RT-PCR after sorting IgG+ B cells that showed reactivity to H7-PE bait. They are part of a set of four H7-reactive monoclonal antibodies (including H7.HK4) recovered from peripheral blood mononuclear cells (PBMCs) of the convalescent donor . H7.HK1 and H7.HK2 are clonally related and target the hemagglutinin (HA) head domain (HA1), while H7.HK3 also targets HA1 but with different binding characteristics .

What viral targets do HK1/2/3 antibodies recognize?

These antibodies primarily target the hemagglutinin protein of influenza viruses. Specifically, H7.HK1 and H7.HK2 bind to a β14-centered surface on the HA globular head domain (HA1) and disrupt the 220-loop that makes hydrophobic contacts with sialic acid on an adjacent protomer. This mechanism effectively blocks viral entry by interfering with receptor binding . H7.HK3 also binds to the HA1 domain but displays different cross-reactivity patterns compared to H7.HK1 and H7.HK2, suggesting it targets a different epitope within HA1 .

How do HK1/2/3 antibodies compare to other virus-targeting antibodies?

The HK antibodies utilize a different neutralization mechanism compared to traditional anti-influenza antibodies. While many HA1-directed antibodies typically neutralize by directly interfering with the receptor binding site (RBS), H7.HK1 and H7.HK2 bind to a conserved lateral patch on the HA head, disrupting the interaction between the virus and host cell receptors through an alternative mechanism .

In comparative studies, H7.HK2 showed superior or comparable neutralization potency against both 2013 and 2017 H7N9 strains when compared to previous RBS-directed antibodies like L4A-14 and H7.167. H7.HK2 matched the performance of the non-RBS antibody 07-5F01 in neutralization assays .

How do the binding mechanisms of HK1/2 differ from traditional neutralizing antibodies?

H7.HK1 and H7.HK2 utilize a unique neutralization mechanism by binding to a conserved lateral patch on the HA head rather than the typical receptor binding site. Cryo-EM structural analyses revealed that these antibodies bind to a β14-centered surface and disrupt the 220-loop that makes hydrophobic contacts with sialic acid on an adjacent protomer . This distinctive binding mechanism:

• Targets a relatively conserved region of the HA head

• Disrupts the viral attachment process through an allosteric mechanism

• Maintains effectiveness against emerging strains that have undergone antigenic drift in the traditional antigenic sites A and B

This mechanism differs from traditional anti-influenza antibodies that directly compete with sialic acid binding at the receptor binding site, which is often subject to greater antigenic variation .

What explains the differential neutralization potency observed between HK1/2 and HK3/4?

The differential neutralization potency between these antibodies can be attributed to their distinct epitope targeting and binding mechanisms:

How do antigenic drift events affect the efficacy of HK1/2/3 antibodies?

A critical advantage of H7.HK1 and H7.HK2 antibodies is their maintained efficacy against antigenically drifted strains. While significant antigenic drift was documented in H7N9 strains from 2016-2017 compared to 2013 isolates, both H7.HK1 and H7.HK2 retained their binding and neutralization capacity against these later strains .

The binding curves of H7.HK1, H7.HK2, and H7.HK3 to both 2016 and 2017 HA1 proteins were fully retained, demonstrating remarkable conservation of their epitopes despite ongoing viral evolution. This stability is explained by structural data showing that the antigenic mutations during this timeframe occurred at the peripheries of their epitopes rather than in the core binding regions .

In contrast, many previously identified neutralizing antibodies against H7N9 lost activity against the 2016-2017 isolates, with only 3 out of 17 neutralizing monoclonal antibodies isolated from earlier cases maintaining activity against the newer strains .

What are the optimal techniques for isolating and characterizing HK1/2/3 antibodies?

Based on the successful isolation of H7.HK1, H7.HK2, and H7.HK3, the following methodological approach is recommended:

• Antigen preparation: Prepare soluble recombinant HA protein (e.g., based on A/Shanghai/2/2013 H7N9) for biotinylation, followed by streptavidin-PE conjugation to create an antigen bait .

• B cell isolation and sorting: Stain PBMCs from convalescent donors with the HA-PE bait and sort IgG+ B cells (defined as CD3-CD19+CD20+IgG+) that are HA-PE+ .

• Antibody recovery: Perform single B cell RT-PCR on the sorted B cells to recover antibody sequences .

• Expression and purification: Clone recovered antibody sequences into expression vectors and produce recombinant antibodies in appropriate expression systems.

• Characterization:

  • ELISA binding assays against various HA antigens to determine specificity and cross-reactivity

  • Pseudovirus neutralization assays using luciferase reporters

  • Live virus neutralization assays to confirm activity in more physiologically relevant systems

  • Structural analysis using cryo-EM to determine binding sites and mechanisms of action

What are the key differences between pseudovirus and live virus neutralization assays when evaluating HK antibodies?

The choice of neutralization assay significantly impacts the measured potency of HK antibodies:

• Sensitivity differences: Pseudovirus neutralization assays typically yield IC₅₀ values approximately 10-fold lower (indicating higher apparent potency) than those obtained using live replicating viruses. For instance, H7.HK1 and H7.HK2 showed IC₅₀ values of 20 ng/mL against pseudovirus but higher values against live virus .

• Assay characteristics:

  • Pseudovirus assays:

    • Use single-round infection with luciferase readout

    • More sensitive for initial screening of neutralizing mAbs

    • Less biohazardous, requiring lower containment levels

    • May not fully recapitulate all aspects of viral entry

  • Live virus assays:

    • Use replicating virus with cytopathic effect or plaque reduction endpoints

    • More physiologically relevant, incorporating all aspects of viral replication

    • Require higher biosafety containment

    • More stringent test of antibody function

• Application recommendations: Pseudovirus neutralization is recommended for initial screening of neutralizing antibodies due to its higher sensitivity, followed by confirmation with live replicating viruses for more definitive characterization .

How can HK antibodies be used in immunoprecipitation-mass spectrometry (IP-MS) approaches?

While not directly described for the H7.HK antibodies in the provided search results, the general approach for using antibodies in IP-MS studies can be adapted from result :

• Sample preparation: Coincubate sera containing antibodies of interest with antigen pools derived from relevant cell lysates (e.g., cells susceptible to viral infection) .

• Immunoprecipitation: Enrich antigen-antibody complexes using protein A/G beads .

• On-bead digestion: Perform in-solution on-bead trypsin digest of the complexes .

• Peptide labeling and analysis: Label peptide fragments and perform quantitative proteomics analysis using platforms like DEEP SEQ .

• Data analysis: Apply bioinformatics analysis to identify over-represented proteins in the experimental samples compared to controls .

This approach can be modified to use purified HK1/2/3 antibodies instead of sera, potentially providing insights into additional binding partners or conformational epitopes not identified through traditional structural studies.

How do HK antibodies compare with other neutralizing antibodies against different viruses?

The mechanism of action of H7.HK1 and H7.HK2 shares conceptual similarities with other virus-neutralizing antibodies:

• Comparison with anti-EBV antibodies: Like the anti-EBV gB neutralizing antibodies 3A3 and 3A5 that effectively neutralize EBV infection of both B and epithelial cells , H7.HK1 and H7.HK2 demonstrate broad neutralization against H7N9 strains. Both sets of antibodies target conserved regions essential for viral entry .

• Comparison with other influenza antibodies:

  • H7.HK1 and H7.HK2 target the HA head domain but bind to a conserved lateral patch rather than the typical receptor binding site (RBS)

  • Traditional RBS-directed antibodies (e.g., H7.167 and L4A-14) showed weaker neutralization against 2013 H7N9 than H7.HK2, and H7.167's activity was further reduced against the 2017 virus

  • The non-RBS antibody 07-5F01 showed comparable potency to H7.HK2 against both 2013 and 2017 H7N9 viruses

What synergistic effects have been observed when combining HK antibodies with other therapeutic agents?

While the search results don't specifically address synergistic effects for H7.HK antibodies with other therapeutic agents, there is evidence for beneficial combination effects:

• H7.HK4 augmentation: The HA2-directed mAb H7.HK4, despite lacking neutralizing activity on its own, moderately augmented mouse protection when used in combination with H7.HK2. This suggests that targeting different domains of the same viral protein can produce enhanced protective effects .

• Potential for combination approaches: By analogy with other antibody studies, the unique binding sites of HK antibodies suggest they could be valuable components in antibody cocktails targeting multiple epitopes simultaneously to prevent viral escape .

This concept is similar to approaches used with other viruses, such as the HK2 antisense oligonucleotide (HK2-ASO1) combined with oxidative phosphorylation inhibitor (DPI) and fatty acid oxidation inhibitor (perhexiline) to achieve synthetic lethality in certain cancer cells .

What are the prospects for developing universal influenza vaccines based on HK antibody epitopes?

The conserved lateral patch on HA targeted by H7.HK1 and H7.HK2 represents a promising target for developing broader influenza vaccines:

• Epitope conservation: Sequence analysis indicates that the lateral patch targeted by H7.HK1 and H7.HK2 is conserved among influenza subtypes, suggesting it could elicit broader protection than traditional vaccine approaches focused on highly variable regions .

• Resistance to antigenic drift: These antibodies retain binding and neutralization capacity to later H7N9 isolates from 2016–2017, demonstrating that their epitopes remain conserved despite ongoing viral evolution .

• Vaccine design strategies:

  • Structure-based immunogen design focusing on the β14-centered surface

  • Prime-boost strategies to direct immune responses toward this conserved epitope

  • Multivalent approaches combining this conserved epitope with other conserved targets

How might advanced structural biology techniques enhance our understanding of HK antibody mechanisms?

While cryo-EM has already provided valuable insights into the binding mechanisms of H7.HK1 and H7.HK2 , additional structural biology approaches could further enhance our understanding:

• Single-particle cryo-EM with improved resolution: Higher resolution structures could reveal additional molecular details of the antibody-antigen interface.

• X-ray crystallography: Complementary to cryo-EM, crystallography might reveal atomic-level details of specific interactions.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique could provide information about conformational changes in HA induced by antibody binding, potentially revealing allosteric mechanisms.

• Molecular dynamics simulations: Computational approaches could model the dynamic interactions between antibodies and their epitopes, providing insights into binding kinetics and energetics.

• Structure-guided mutagenesis: Systematic mutation of residues at the antibody-antigen interface could precisely map the contribution of specific interactions to binding and neutralization.