F6'H2 Antibody

What is the RIID F6-H2 antibody cocktail and what is its composition?

RIID F6-H2 is a Sudan virus (SUDV)-specific antibody cocktail comprising two human chimeric monoclonal antibodies (mAbs) targeting different epitopes of the SUDV glycoprotein (GP). The cocktail consists of 16F6, a base-binding mAb, and X10H2, a glycan cap-binding mAb. This combination was down-selected from a panel of SUDV GP-specific human chimeric mAbs based on their neutralizing activity, epitope specificity, and in vivo protective efficacy .

How was the F6'H2 antibody cocktail developed and selected?

The development process involved generating a panel of SUDV GP-specific human chimeric mAbs using both plant and mammalian expression systems. Researchers conducted comprehensive head-to-head in vitro and in vivo evaluations to select the optimal antibody combination. The selection criteria focused on identifying neutralizing antibodies targeting non-competing epitopes of SUDV GP, with neutralizing potency and protective efficacy being key parameters. After initial screening and testing in mouse models, the combination of 16F6 and X10H2 was down-selected for evaluation in a macaque model of SUDV infection, where it demonstrated significant protective efficacy .

What experimental systems were used to produce the F6'H2 components?

Both plant and mammalian expression systems were employed to produce the SUDV GP-specific human chimeric mAbs. Interestingly, no significant differences were observed between plant and mammalian-produced mAbs in any of the in vitro or in vivo evaluations, suggesting that both expression platforms can yield functionally equivalent antibodies for therapeutic applications .

What epitopes on the Sudan virus glycoprotein are targeted by the components of F6'H2?

The two components of RIID F6-H2 target different structural regions of the SUDV glycoprotein:

• 16F6: Binds at the base of the GP trimer, targeting an epitope consisting of residues located within both GP1 and GP2. The binding site was previously determined by X-ray crystallography .

• X10H2: Targets an epitope located within the glycan cap of SUDV GP. Viral escape variant studies identified a critical glutamine (Q) to lysine (K) mutation in the glycan cap region that conferred resistance to X10H2 neutralization, confirming its binding specificity .

How were the binding footprints of F6'H2 components determined?

Multiple complementary techniques were employed to determine the binding footprints:

• X-ray crystallography: The 16F6 epitope was previously mapped using this technique, showing binding at the base of the GP trimer .

• Viral escape variant selection: X10H2's binding site was identified by serially passaging recombinant VSV expressing SUDV GP (rVSV-SUDV GP) in the presence of X10H2 until resistance emerged. Sequencing of resistant clones revealed a critical Q→K mutation in the glycan cap .

• Cleaved GP neutralization assays: Consistent with other glycan cap binding antibodies, X10H2 was unable to neutralize cleaved SUDV GP, further confirming its glycan cap binding specificity .

• SPOT membrane analysis: For mAbs that recognize linear epitopes (such as 17F6, another mAb studied but not included in the final cocktail), researchers employed SPOT membranes coated with 13-amino acid-long linear peptides of SUDV GP to identify binding sequences .

What analytical methods were used to characterize antibody binding affinities?

Binding affinities of the mAbs to recombinant GPs were determined by Bio-Layer Interferometry (BLI) using the Octet Red96 system. The specific protocol involved:

• Loading antibodies onto anti-human Fc (AHC) biosensors at 3 μg/mL in kinetic buffer

• Association of recombinant SUDV-Bon GP, SUDV-Gulu GP, EBOV-Kik GP, or BDBV GP across threefold serial dilutions

• Binding kinetics determination using a 1:1 binding model, although the researchers noted this interaction is likely more complex due to bivalency of mAbs and trivalent configuration of recombinant GPs .

How were competition groups for SUDV-binding antibodies determined?

Competition groups were determined using Bio-Layer Interferometry (BLI) with the following methodology:

• Histidine-labeled recombinant SUDV-Bon GP was loaded onto Anti-Penta-HIS biosensors at 25 μg/mL in kinetic buffer

• A "binning mAb" was allowed to associate at 50 μg/mL to near saturation

• A second mAb at 50 μg/mL was then introduced in the presence or absence of the binning mAb

• Area Under the Curve (AUC) measurements quantified binding competition

• Antibodies were classified as:

  • Competitive: <30% binding

  • Partially competitive: 30-70% binding

  • Noncompetitive: ≥70% binding in both directions

The results were confirmed using competitive ELISA, where chimeric mAbs were incubated with SUDV GP-coated plates prior to adding murine versions of each mAb .

What competition groups were identified for Sudan virus glycoprotein-binding mAbs?

Two main competition groups were identified through BLI analysis:

• mAbs competing with 16F6 (base-binding): 16F6 and X10B1

• mAbs competing with X10H2 (glycan cap-binding): X10H2, X10B6, and X10F3

The competition between some mAbs was directional, correlating with antibody affinity. For example, 16F6 and X10B1 significantly inhibited binding of one another, though somewhat directionally with residual 16F6 binding observed in the presence of X10B1 .

How does the neutralizing activity of different SUDV mAbs compare?

The base-binding mAbs (16F6 and X10B1) were more potent neutralizers and demonstrated greater protective efficacy than glycan cap-binding (X10H2, X10F3, X10B6) or mucin-like domain-binding mAbs (17F6). Neutralizing activity was assessed using a microneutralization assay where antibodies were incubated with SUDV-Bon and then exposed to Vero E6 cells at an MOI of 0.5 pfu/cell. Infected cells were quantified by fluorescence microscopy and automated image analysis using Operetta high-content device and Harmony software .

What was the protective efficacy of RIID F6-H2 in non-human primates?

RIID F6-H2 demonstrated significant protective efficacy in rhesus macaques infected with SUDV. The key findings include:

• Survival rate: 100% of treated macaques survived, compared to 25% of control animals

• Treatment regimen: 50 mg/kg administered on days 4 and 6 post-infection

• Viral clearance: Following treatment on day 4, viral titers decreased rapidly in all experimental macaques and remained below detection limits through the end of the study

• PCR negativity: All treated macaques were PCR negative by day 8 post-exposure, just 2 days after the final antibody treatment

• Antibody response: SUDV GP-specific IgG titers increased rapidly following antibody delivery and remained elevated through day 28

What biomarkers can be used to assess F6'H2 treatment success?

Several biomarkers were monitored to assess treatment efficacy in the non-human primate model:

• Liver function markers: Control animals showed elevated ALT, AST, and ALP levels, while treated animals maintained normal levels

• Hematological parameters: Control animals developed thrombocytopenia, which was absent in treated animals

• Kidney function markers: Elevated BUN, GGT, and creatinine were observed in control animals that succumbed to infection

• SUDV GP-specific IgG antibody titers: Rapid increase following antibody treatment, remaining elevated through day 28

• Viral load: Measured by plaque assay and PCR, showing rapid clearance in treated animals

How does the timing of F6'H2 administration impact treatment outcomes?

The timing of antibody administration was a critical factor in the non-human primate studies. RIID F6-H2 was administered to macaques on days 4 and 6 post-infection, which is significant because by day 4, animals were already PCR-positive for SUDV. This represents a clinically relevant "trigger-to-treat" milestone, as treatment was initiated after confirmation of infection. This administration schedule resulted in 100% protection, demonstrating that the antibody cocktail is effective as a post-exposure therapy even when treatment is delayed until after viral replication is established .

How does F6'H2 compare to other ebolavirus antibody cocktails?

While RIID F6-H2 is specifically designed for SUDV, its design principles and epitope targeting show similarities to other ebolavirus antibody cocktails like ZMapp (developed for Ebola virus):

• Epitope targeting: Both RIID F6-H2 and ZMapp target similar structural regions on their respective viral glycoproteins:

  • Base-binding antibodies: 16F6 in RIID F6-H2 is similar to 2G4 and 4G7 in ZMapp

  • Glycan cap-binding antibodies: X10H2 in RIID F6-H2 is similar to 13C6 in ZMapp

• Cross-reactivity: Unlike some newer pan-ebolavirus antibodies, RIID F6-H2 components do not recognize glycoproteins from other ebolaviruses like EBOV or BDBV

• Development strategy: Both cocktails were down-selected from a relatively small panel of mAbs derived from animals vaccinated with viral GP (SUDV GP for RIID F6-H2, EBOV GP for ZMapp)

What is the proposed mechanism of action for the base-binding and glycan cap-binding components?

The two components of RIID F6-H2 likely neutralize SUDV through different mechanisms:

• Base-binding mAb (16F6):

  • May "lock" GP in a prefusion conformation, impeding structural rearrangements required for membrane fusion

  • Could block cathepsin cleavage of GP, similar to mechanisms reported for KZ52 and 2G4 antibodies against EBOV

  • Targets residues located within both GP1 and GP2

• Glycan cap-binding mAb (X10H2):

  • Targets an epitope within the glycan cap

  • Cannot neutralize cleaved SUDV GP (after cathepsin processing), indicating its mechanism depends on binding to pre-cleaved GP

  • The mechanism may involve blocking receptor binding or interfering with viral attachment

What structural features of ebolavirus glycoproteins make them vulnerable to antibody therapies?

The similarities in epitope specificity between ZMapp (for EBOV) and RIID F6-H2 (for SUDV) suggest that structural features of GP vulnerability to antibody therapies may be conserved across ebolavirus species. Specifically:

• The base region of GP, which contains elements of both GP1 and GP2, represents a conserved vulnerability site across ebolaviruses

• The glycan cap region, despite sequence variation between viral species, contains structurally similar epitopes that can be targeted by neutralizing antibodies

• These conserved vulnerability sites likely reflect functional constraints on GP structure that are necessary for viral entry

What are the recommended protocols for assessing F6'H2 neutralization activity?

Based on the methods described in the research, a standardized microneutralization assay protocol would include:

• Antibody preparation: Dilute antibodies to desired concentrations in appropriate culture media

• Virus-antibody incubation: Mix diluted antibodies with SUDV (strain Boniface recommended) for 1 hour

• Cell infection: Expose Vero E6 cells to the antibody/virus inoculum at MOI of 0.5 pfu/cell for 1 hour

• Post-infection care: Remove inoculum and add fresh culture medium

• Fixation and staining (48h post-infection):

  • Fix cells with formalin

  • Block with 1% BSA

  • Incubate with SUDV GP-specific detection antibody (e.g., mAb 3C10)

  • Apply secondary antibody (e.g., goat anti-mouse IgG conjugated to Alexa 488)

  • Counterstain with Hoechst stain

• Quantification: Use fluorescence microscopy with automated image analysis (e.g., Operetta high-content device with Harmony software)

How should viral escape variants be generated and characterized for antibody resistance studies?

To generate and characterize viral escape variants, researchers can follow this methodology:

• Serial passage: Culture recombinant VSV expressing SUDV GP (rVSV-SUDV GP) in the presence of the antibody of interest, gradually increasing antibody concentration if needed

• Resistance monitoring: Continue passages until resistance to antibody neutralization is observed (typically 3-4 passages)

• Plaque purification: Isolate individual viral clones from the resistant population

• Sequencing: Sequence the GP gene from multiple clones to identify mutations that confer resistance

• Validation: Confirm that identified mutations indeed cause resistance using:

  • Neutralization assays with the mutant virus

  • Testing antibody binding to recombinant mutant GP

• Structural mapping: Map the resistance mutations onto the GP structure to define the antibody epitope

What considerations are important when designing antibody cocktails for viral hemorrhagic fevers?

Based on the findings from RIID F6-H2 development, key considerations for designing effective antibody cocktails include:

• Epitope diversity: Include antibodies targeting non-competing epitopes to prevent viral escape and maximize neutralization potential

• Functional complementarity: Combine antibodies with different mechanisms of action (e.g., preventing receptor binding, blocking membrane fusion)

• Potency: Prioritize antibodies with strong neutralizing activity in vitro

• In vivo efficacy: Validate protective efficacy in relevant animal models before finalizing cocktail composition

• Structural considerations: Target conserved, functionally important regions of viral glycoproteins

• Administration timing: Design cocktails that remain effective when administered post-infection/exposure, as this represents the most likely clinical scenario

• Expression system compatibility: Ensure selected antibodies can be reliably produced in the chosen expression system (plant or mammalian) without loss of functionality