clfA Antibody

What is ClfA and why is it significant in S. aureus research?

ClfA (Clumping Factor A) is a major virulence determinant expressed on the surface of Staphylococcus aureus. It functions as an MSCRAMM (microbial surface components recognizing adhesive matrix molecules) that mediates bacterial binding to fibrinogen, which contributes to S. aureus pathogenesis in multiple ways:

• Promotes bacterial adherence to host tissues and damaged endothelial surfaces

• Mediates clumping of S. aureus in blood plasma

• Contributes to evasion of host immune responses

• Plays critical roles in several experimental models of infection including septic arthritis, bacteremia, and endocarditis

ClfA is a 933 amino acid polypeptide that contains several domains: an N-terminal signal sequence (S) followed by three subdomains (N1, N2, and N3) that comprise the ligand-binding A region, a serine-aspartate repeat domain (R region), and C-terminal features required for cell wall anchoring such as the LPXTG motif . The N2-N3 domains (residues 221-559) are responsible for binding to the C-terminal region of the fibrinogen γ chain .

ClfA has emerged as a promising vaccine target because:

• It is highly conserved across virtually all clinical S. aureus strains

• It generates strong immune responses

• Genetic inactivation of clfA significantly reduces virulence in animal models

• Both active and passive immunization strategies targeting ClfA have shown protection in preclinical models

How do anti-ClfA antibodies exert their protective effects?

Anti-ClfA antibodies can provide protection through multiple mechanisms:

• Inhibition of fibrinogen binding: Functional anti-ClfA antibodies block the interaction between ClfA and fibrinogen, thereby preventing bacterial adherence to host tissues and inhibiting S. aureus clumping in plasma

• Displacement of bound bacteria: Some anti-ClfA antibodies can release bacteria that have already bound to fibrinogen, which may help clear existing infections. Studies show that functional antibodies can displace up to 35% of previously adherent S. aureus from fibrinogen-coated surfaces

• Opsonophagocytosis: Anti-ClfA antibodies can facilitate uptake and killing of S. aureus by neutrophils and other phagocytic cells

• Prevention of bacterial agglutination: Antibodies like MAb 11H10 prevent bacterial agglutination in human plasma, which may reduce bacterial load during infection

The protective effect has been demonstrated in multiple experimental models, including murine sepsis and septic arthritis models, where passive immunization with anti-ClfA antibodies significantly reduced mortality and disease severity .

What are the main approaches for generating anti-ClfA antibodies for research?

Researchers have employed several strategies to develop anti-ClfA antibodies:

• Hybridoma technology: This traditional approach involves immunizing mice with recombinant ClfA proteins (typically fragments containing the fibrinogen-binding domain, such as ClfA 221-559 or ClfA 40-531), followed by fusion of B cells with myeloma cells to create hybridomas that secrete monoclonal antibodies

  Example protocol:

  • Immunize mice using repetitive immunization at multiple sites (RIMMS) protocol with ClfA 221-559 and adjuvant every 2-3 days for 13 days

  • Collect lymph nodes and generate B-cell hybridomas after fusion with P3X myeloma partner

  • Screen hybridoma supernatants for anti-ClfA reactivity by ELISA and whole-cell S. aureus ELISA

  • Test purified antibodies for functional activity (fibrinogen binding inhibition, opsonophagocytic killing)

  • Clone the most active hybridomas by limiting dilution

• Recombinant antibody technology: Generating humanized or fully human antibodies by grafting human Fc regions to mouse variable regions or using transgenic mice expressing human immunoglobulin genes

• Polyclonal antibody production: Immunizing rabbits or other animals with recombinant ClfA proteins and collecting sera containing polyclonal anti-ClfA antibodies

The choice of ClfA protein construct is critical. Most researchers use recombinant fragments containing the fibrinogen-binding domain (N2-N3 domains, approximately residues 221-559, or somewhat extended versions like ClfA 40-531) for immunization .

What methods are used to assess the functionality of anti-ClfA antibodies?

Several assays have been developed to characterize anti-ClfA antibodies:

• Fibrinogen binding inhibition assay: This assay measures the ability of antibodies to prevent S. aureus binding to immobilized fibrinogen.

  Method Type	Description	Advantage
  ELISA-based	Anti-ClfA antibodies are pre-incubated with S. aureus or recombinant ClfA, then added to fibrinogen-coated wells	Simple, high-throughput
  Luminescence-based	Uses luminescent S. aureus strains for detection of bound bacteria	Higher sensitivity, can use live bacteria
  BIAcore analysis	Surface plasmon resonance to measure interference with ClfA-Fg interaction	Provides binding kinetics

  For the luminescence-based assay specifically developed for ClfA:

  • Coat microwells with human fibrinogen

  • Pre-incubate S. aureus with serial dilutions of anti-ClfA antibodies

  • Add the mixture to fibrinogen-coated wells

  • Wash to remove unbound bacteria

  • Detect bound bacteria using luminescence

  • Calculate percent inhibition compared to control

• Bacterial displacement assay: This assay measures the ability of antibodies to displace already bound S. aureus from fibrinogen-coated surfaces.

  • Allow S. aureus to bind to fibrinogen-coated surfaces

  • Add anti-ClfA antibodies at various concentrations

  • Measure the release of bound bacteria

  • Functional antibodies like MAb 12-9 can release up to 35% of adhered bacteria

• Opsonophagocytic killing (OPK) assay: This measures antibody-mediated uptake and killing by phagocytes.

  • Incubate S. aureus with anti-ClfA antibodies

  • Add human neutrophils or other phagocytes

  • Measure bacterial killing compared to controls

• Agglutination inhibition assay: This assesses the ability of antibodies to prevent S. aureus clumping in plasma.

  • Mix anti-ClfA antibody serial dilutions with human plasma

  • Add S. aureus suspension

  • Incubate at 37°C for 5 minutes

  • Evaluate visually for agglutination

  • Record the lowest antibody concentration that prevents agglutination

• Flow cytometry: Used to assess antibody binding to ClfA expressed on the surface of S. aureus strains

How do researchers determine the epitope specificity of anti-ClfA antibodies?

Understanding the epitope specificity of anti-ClfA antibodies is crucial for predicting their functional activity. Several approaches are used:

• Domain mapping: Testing antibody binding to different ClfA domain fragments (N1, N2, N3) to localize the epitope to a specific domain

• Binding competition experiments: Using instruments like the Octet BioLayer interferometer to identify interference or binding site overlap between different antibodies. This helps map relative epitope positions.

  Example findings:

  • MAb 12-9 (highly functional) recognizes an N3 subdomain epitope

  • MAb 15EC6 (non-functional) binds to an N2 domain epitope

  • MAb 23-3 recognizes an N3 domain epitope that interferes with MAb 12-9 binding

  • MAb 33-32 binds to an N2 domain site that does not overlap with other MAbs

  • MAb 32-105 requires both N2 and N3 domains for binding

• Competitive ELISA: Using soluble ClfA variants to compete for antibody binding to immobilized ClfA. This approach can reveal differences in antibody affinity for different ClfA variants

• Crystal structure analysis: For advanced epitope mapping, co-crystallization of the ClfA-antibody complex can provide precise epitope information at the atomic level

The location of the epitope often correlates with antibody functionality. Antibodies that recognize epitopes in or near the fibrinogen-binding site (particularly in the N3 domain) tend to have higher functional activity in blocking fibrinogen binding .

How should researchers evaluate strain coverage when developing anti-ClfA antibodies?

ClfA exhibits sequence variation across S. aureus strains (up to 14% variation in primary amino acid sequence), which can affect epitope composition and antibody recognition . To ensure broad strain coverage:

What are the critical experimental controls for anti-ClfA antibody studies?

For rigorous anti-ClfA antibody research, the following controls are essential:

• Antibody specificity controls:

  • Isotype-matched control antibodies (e.g., an irrelevant human IgG1 like anti-gp120 MAb R347)

  • Pre-immune sera when using polyclonal antibodies

  • Testing against clfA knockout strains to confirm specificity

• Functional assay controls:

  • Positive control: Validated functional anti-ClfA antibody (e.g., MAb 12-9)

  • Negative control: Non-functional anti-ClfA antibody (e.g., MAb 15EC6)

  • ClfA-deficient S. aureus strains to confirm assay specificity

  • ClfA-complemented strains to validate the role of ClfA

• In vivo experiment controls:

  • Wild-type vs. ΔclfA S. aureus strains

  • Complemented ΔclfA mutants carrying the clfA gene on a plasmid

  • Control antigens/antibodies unrelated to ClfA

  • Multiple animal models to validate findings

• Antibody validation controls:

  • Testing monoclonal antibody binding by ELISA, BIAcore, and flow cytometry

  • Confirming antibody functionality in multiple assays (binding, inhibition, displacement)

  • Using antibodies with known epitopes and functional properties

According to general antibody validation guidelines, researchers should also follow these best practices:

• Record detailed antibody information (source, catalog number, lot, dilution)

• Test new antibody lots before use in critical experiments

• Include appropriate positive and negative controls in each experiment

• Consider blinding for quantitative analyses

How do anti-ClfA antibodies perform in combination with other anti-S. aureus approaches?

Research indicates that combining anti-ClfA antibodies with other therapeutic approaches may provide enhanced protection:

• Combination with antibiotics: The humanized monoclonal antibody Aurexis (anti-ClfA) was tested in clinical trials in combination with antibiotic therapy for S. aureus bacteremia .

• Multiantigen vaccine approaches: ClfA is included in several multiantigen vaccine formulations currently in clinical development:

  • The SA3Ag vaccine includes a modified ClfA (rClfAm) that harbors a Y338A mutation to abolish fibrinogen-binding activity, which prevents potential interference with normal blood clotting

  • These multiantigen approaches aim to target multiple virulence mechanisms simultaneously

• Combination with other monoclonal antibodies: Studies have demonstrated enhanced protection when combining anti-ClfA antibodies with antibodies targeting other virulence factors:

  Example study: A combination of anti-alpha toxin (AT) MAb MEDI4893* and anti-ClfA MAb 11H10 provided improved protection against S. aureus bacteremia compared to either antibody alone:

  • Each MAb alone showed protection against some isolates

  • The MAb combination provided broader protection across multiple isolates

  • The combination provides multimechanistic protection by:

    • Neutralizing AT-mediated cytotoxicity

    • Blocking fibrinogen binding by ClfA

    • Preventing bacterial agglutination

    • Targeting bacteria for opsonophagocytic killing

This multimechanistic approach is increasingly viewed as necessary because previous attempts at passive or active immunization against S. aureus targeting single antigens have generally failed in clinical trials despite positive preclinical data .

How can researchers address the strain variation in ClfA when developing broadly protective antibodies?

The sequence variation in ClfA across S. aureus strains (up to 14%) presents challenges for developing broadly protective antibodies . Researchers can employ these strategies:

• Epitope mapping and targeting conserved regions: Identify conserved epitopes critical for ClfA function that are less likely to vary between strains.

  • Crystal structure analysis shows that most variable regions in ClfA are surface-exposed, while the fibrinogen-binding mechanism is largely conserved

  • Target antibody development to conserved regions within the functional domains

• Cocktail approaches: Develop antibody cocktails targeting multiple epitopes on ClfA to ensure broader strain coverage.

• Structure-guided antibody engineering: Use the crystal structure of the ClfA-fibrinogen interaction to design antibodies that specifically target the binding interface .

• Cross-reactivity screening: Screen candidate antibodies against a diverse panel of ClfA variants from clinical isolates.

  • Competitive ELISA can reveal differences in antibody affinity between ClfA variants

  • Flow cytometry can assess binding to native ClfA on diverse clinical isolates

• Combination with other targets: Combine anti-ClfA antibodies with antibodies targeting other conserved S. aureus antigens to ensure broader protection .

Research findings on cross-strain protection:

What are the key considerations when translating anti-ClfA antibodies from preclinical to clinical studies?

Translating anti-ClfA antibody research to clinical applications requires addressing several critical considerations:

• Humanization and safety: Convert murine antibodies to humanized or fully human versions to minimize immunogenicity.

  • Example: Aurexis, a humanized monoclonal antibody against ClfA, was developed for clinical trials

• Functional antibody assessment: Confirm that humanized antibodies retain the functional properties of the original murine antibodies.

  • Test fibrinogen binding inhibition, bacterial displacement, and opsonophagocytic activity

  • Compare EC50 values between original and humanized versions

• Dosing and pharmacokinetics: Determine appropriate dosing regimens based on antibody half-life and tissue distribution.

  • In preclinical models, single infusions of anti-ClfA antibodies have shown protection

• Timing of intervention: Consider the temporal aspects of ClfA's role in pathogenesis.

  • ClfA exhibits its primary effect early during infection as an adhesion factor

  • In animal models where S. aureus is delivered systemically (sepsis, peritoneal infections), the initial adhesion events requiring ClfA may be bypassed

  • This timing issue may affect the therapeutic window for anti-ClfA antibodies

• Clinical assay development: Develop assays to measure functional anti-ClfA responses in clinical trial participants.

  • The luminescence-based fibrinogen binding inhibition assay has been validated for measuring functional antibodies in human sera

  • Results show that despite having anti-ClfA antibodies, sera from unvaccinated individuals cannot prevent S. aureus binding to fibrinogen

  • In contrast, sera from vaccinated individuals with a ClfA-containing vaccine showed functional activity

• Model selection: Choose appropriate animal models that reflect the relevant pathophysiology.

  • Different models may yield different results based on the role of ClfA in that particular infection scenario

• Combination approaches: Consider combining anti-ClfA antibodies with other therapeutic agents or antibodies targeting different virulence factors .

How can researchers resolve contradictory findings about ClfA antibody efficacy in different experimental models?

Contradictory findings regarding anti-ClfA antibody efficacy can be addressed through:

• Understanding model differences: Recognize that different animal models may emphasize different aspects of S. aureus pathogenesis.

  • ClfA primarily functions as an adhesion factor early in infection

  • In models where bacteria are delivered systemically (bypassing initial adhesion events), anti-ClfA antibodies may show reduced efficacy

  • ClfA knockout strains showed reduced virulence in arthritis models and endocarditis models, but results may vary in other infection types

• Matching assays to mechanism: Use functional assays that specifically address the virulence mechanism of ClfA.

  • Opsonophagocytosis assays may not directly address ClfA's adhesion function

  • Fibrinogen binding inhibition assays more directly measure neutralization of ClfA's primary function

• Standardizing experimental conditions: Control variables that might affect results:

  • S. aureus strain selection (considering ClfA sequence variation)

  • Antibody dose and timing relative to infection

  • Route of administration

  • Animal genetic background

• Combining multiple readouts: Assess protection using multiple parameters:

  • Survival/mortality

  • Bacterial burden in tissues

  • Disease-specific pathology (e.g., arthritis severity)

  • Inflammatory markers

• Evaluating natural vs. vaccine-induced immunity: Studies show important differences:

  • Natural exposure to S. aureus generally fails to generate functional anti-ClfA antibodies

  • Even patients with chronic staphylococcal infections vaccinated with killed S. aureus had only marginally higher antibody levels than preexisting levels

  • In contrast, recombinant ClfA vaccines generate high titers of functional antibodies

  • This suggests that S. aureus may actively suppress host immune responses to surface virulence factors

• Examining strain-specific effects: Some contradictions may result from strain-specific differences in ClfA sequence or expression.

  • Different S. aureus isolates may show variable susceptibility to the same anti-ClfA antibody

  • Combining anti-ClfA with other approaches may provide more consistent protection across strains

How can modified ClfA antibodies be used to minimize potential adverse effects?

When developing anti-ClfA antibodies for therapeutic use, researchers need to consider potential adverse effects and how to mitigate them:

• Engineering non-interfering ClfA variants for vaccines: The SA3Ag vaccine contains a modified rClfA (rClfAm) with a Y338A mutation that abolishes fibrinogen-binding activity while maintaining immunogenicity.

  • This prevents the vaccine antigen from inhibiting normal platelet clumping and blood clotting

  • The mutation prevents interference with the binding of the platelet integrin αIIbβ3 receptor to the same C-terminal region of the fibrinogen γ chain

• Epitope-specific antibody design: Engineer antibodies that specifically block pathogenic interactions without interfering with physiological processes.

  • Target epitopes that specifically block S. aureus binding without affecting normal hemostasis

  • Rational antibody design based on the crystal structure of the ClfA-fibrinogen interaction

• Fragment-based approaches: Consider using antibody fragments (Fab, scFv) when full IgG might cause unwanted effects like complement activation or Fc receptor engagement in certain tissues.

• Site-specific delivery: Develop methods for delivering anti-ClfA antibodies specifically to infection sites to minimize systemic effects.

• Combination with anti-inflammatory agents: When using anti-ClfA antibodies that might temporarily increase free bacteria in circulation (through displacement from tissues), consider combining with anti-inflammatory agents to prevent sepsis exacerbation.

• Low-immunogenicity formats: Use fully human antibodies or deimmunized antibodies to minimize anti-drug antibody responses, especially for repeated or long-term use.

What are promising new methodologies for evaluating anti-ClfA antibody efficacy beyond traditional approaches?

Emerging methodologies that can advance anti-ClfA antibody research include:

• High-throughput functional screening: Developing miniaturized and automated versions of functional assays to screen large antibody libraries for fibrinogen binding inhibition and bacterial displacement.

• In vivo imaging: Using bioluminescent or fluorescent S. aureus strains to track infection progression and antibody effects in real-time in animal models.

  • This allows for temporal and spatial assessment of antibody efficacy

  • Can reveal tissue-specific effects that might be missed in endpoint analyses

• Single-cell analysis: Applying flow cytometry and mass cytometry to understand how anti-ClfA antibodies affect interactions between S. aureus and specific immune cell populations.

• Tissue-on-chip models: Using microfluidic systems with human cells to model interactions between S. aureus, host tissues, and anti-ClfA antibodies under physiologically relevant conditions.

  • Can model vascular flow and endothelial interactions where ClfA plays a crucial role

• Systems biology approaches: Integrating multi-omics data to understand how anti-ClfA antibodies affect global host responses to S. aureus infection.

• Advanced structural analyses: Using techniques like cryo-electron microscopy to visualize ClfA-antibody interactions in near-native conditions.

• Standardized human sample biobanks: Establishing collections of human samples with well-characterized S. aureus exposure history to evaluate naturally occurring anti-ClfA responses and correlate with protection.

• Multiplexed antibody analysis: Developing assays to simultaneously measure multiple parameters of antibody function:

  • Binding to multiple ClfA variants

  • Inhibition of fibrinogen binding

  • Displacement of bound bacteria

  • Opsonophagocytic activity

  • Complement activation