ompP2 Antibody

What is OmpP2 and why is it an important target for antibody development?

OmpP2 (Outer Membrane Protein P2) is a porin family protein found in the outer membrane of various gram-negative bacteria, including Haemophilus influenzae, Glaesserella parasuis (formerly Haemophilus parasuis), and other clinically significant pathogens. It serves as an important target for antibody development because:

• It plays multifunctional roles in bacterial pathogenicity

• It can stimulate proinflammatory cytokine responses in host cells

• It contains both variable and conserved epitopes that can be targeted by antibodies

• It has been identified as a potential vaccine candidate for preventing infections

Research has demonstrated that OmpP2 from H. parasuis can upregulate mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, and IL-8 in porcine alveolar macrophages in a dose-dependent manner, suggesting its role in inflammatory responses during infection .

How does OmpP2 structure vary across different bacterial species and strains?

OmpP2 exhibits significant structural variation across different bacterial species and even between strains of the same species. This variation manifests primarily in:

• Sequence variations in specific regions of the protein

• Different antigenic determinants exposed on the bacterial surface

• Variable immunogenicity of different OmpP2 variants

For instance, comparative analysis of Haemophilus parasuis OmpP2 sequences has revealed that while the gene is relatively stable (with 97% nucleotide homology and 92.5% amino acid homology between strains), there are distinct variable regions concentrated in three base sequences: positions 40-65, 110-156, and 180-202 . These variable regions may affect antibody recognition and binding efficacy.

What are the optimal methods for producing monoclonal antibodies against OmpP2?

The production of high-quality monoclonal antibodies against OmpP2 requires careful consideration of several methodological factors:

Recommended Protocol Based on Research Literature:

• Antigen preparation:

  • Recombinant OmpP2 expression in E. coli

  • Purification to >90% purity using affinity chromatography

  • Conjugation to carrier proteins (e.g., keyhole limpet hemocyanin) for increased immunogenicity

• Immunization schedule:

  • Initial subcutaneous injection with 100-125 μg antigen emulsified in Freund's complete adjuvant

  • 2-3 booster injections at 2-week intervals using 50 μg antigen in incomplete Freund's adjuvant

  • Final intravenous boost (20 μg in PBS) 3 days before spleen harvest

• Hybridoma production:

  • Fusion of splenocytes with SP2/0 myeloma cells at 3:1 ratio using polyethylene glycol 1450

  • Selection and subcloning of positive hybridomas three times by limited dilution

  • Screening by indirect ELISA using purified OmpP2

• Antibody characterization:

  • Determination of isotype (IgG subclass)

  • Specificity verification by ELISA and Western blotting

  • Epitope mapping

This approach has yielded successful results in multiple studies, producing monoclonal antibodies with high titers (up to 1:2,048,000) and specific binding characteristics .

What techniques are most effective for mapping B-cell epitopes on OmpP2?

Effective mapping of B-cell epitopes on OmpP2 involves multiple complementary approaches:

• Overlapping peptide arrays:

  • Synthesize overlapping 15-20mer peptides spanning the entire OmpP2 sequence

  • Screen using ELISA to identify reactive peptides

  • Refine using shorter peptides to pinpoint specific epitopes

• Phage display libraries:

  • Construction of random peptide libraries displayed on phage surfaces

  • Selection of peptides binding to anti-OmpP2 antibodies

  • Sequencing of positive clones to identify mimotopes

• Mutagenesis-based approaches:

  • Site-directed mutagenesis of specific residues

  • Expression of mutant OmpP2 proteins

  • Assessment of antibody binding to identify critical residues

• Computational prediction combined with experimental validation:

  • Prediction of B-cell epitopes using algorithms based on hydrophilicity, accessibility, and flexibility

  • Experimental validation using synthetic peptides

One study successfully identified nine epitopes on OmpP2, including genotype-common (GC) epitopes (Pt7/Pt7a, Pt9a, Pt17, and Pt19) and genotype-specific (GS) epitopes (Pt5, Pt11/Pt11a for genotype I; Pt5-II and Pt11a-II for genotype II) .

How can researchers effectively assess the opsonophagocytic activity of anti-OmpP2 antibodies?

Opsonophagocytosis is a critical function of antibodies that enhances bacterial clearance. To assess the opsonophagocytic activity of anti-OmpP2 antibodies, researchers should employ the following methods:

• Flow cytometry-based opsonophagocytosis assay:

  • Label bacteria with fluorescein isothiocyanate (FITC)

  • Incubate labeled bacteria with test antibodies and complement

  • Add human polymorphonuclear leukocytes (PMNs)

  • Measure fluorescence of PMNs by flow cytometry

  • Define positive opsonophagocytosis as PMNs becoming fluorescent due to association with FITC-labeled bacteria

• Microscopy-based evaluation:

  • Fluorescence microscopy to visually confirm phagocytosis

  • Electron microscopy for detailed visualization of bacterial internalization

• Killing assays to assess functional consequences:

  • Quantify surviving bacteria after incubation with antibodies, complement, and phagocytes

  • Compare colony-forming units (CFUs) between test and control conditions

• Controls to include:

  • Bacteria + PMNs (no antibody)

  • Bacteria + PMNs + complement (no antibody)

  • Bacteria + PMNs + heat-inactivated antibody + complement

  • Bacteria + PMNs + antibody (no complement)

Research has shown that opsonophagocytosis is dependent on both antibodies and complement, and antibodies directed to variable parts of OmpP2 have different opsonophagocytic capabilities .

What factors influence the protective efficacy of anti-OmpP2 antibodies against bacterial infections?

The protective efficacy of anti-OmpP2 antibodies is influenced by multiple factors:

These factors should be carefully considered when designing vaccines or immunotherapeutic strategies targeting OmpP2.

How can researchers address the strain specificity limitations of OmpP2 antibodies for broader protection?

Addressing strain specificity limitations of OmpP2 antibodies requires strategic approaches:

• Identification of conserved epitopes:

  • Comprehensive sequence analysis across multiple strains

  • Structural modeling to identify conserved surface-exposed regions

  • Focused antibody development against these conserved regions

• Multi-epitope vaccine design:

  • Inclusion of epitopes from multiple variant regions

  • Construction of chimeric OmpP2 proteins containing critical epitopes from different strains

  • Expression of these multi-epitope constructs as immunogens

• Adjuvant optimization:

  • Testing different adjuvant formulations to enhance breadth of antibody response

  • Targeting specific immune pathways that promote broad protection

• Combining with other antigens:

  • Including other conserved outer membrane proteins

  • Formulating multivalent vaccines targeting multiple bacterial structures

• Mucosal immunization strategies:

  • Evidence suggests mucosal immunization induces antibodies that recognize surface-exposed epitopes on multiple strains

  • Optimization of mucosal delivery systems and adjuvants

Despite these approaches, research indicates that OmpP2-dependent opsonophagocytosis remains strictly strain-specific, highlighting the challenge of developing broadly protective antibodies .

What mechanisms explain the different immunogenic properties of OmpP2 serotypes, particularly O1 versus O2 in Klebsiella pneumoniae?

The differential immunogenic properties of OmpP2 serotypes, especially between O1 and O2 in Klebsiella pneumoniae, involve several mechanisms:

• B-cell repertoire differences:

  • Significantly lower frequency of O2-specific memory B cells compared to O1-specific in both IgM and IgG repertoires

  • Antibody responses to O2 are disproportionately lower than O1 or O1/O2 cross-reactive responses

• Structural determinants:

  • O2 antigens may have reduced structural complexity or altered presentation of immunodominant epitopes

  • Differences in polysaccharide composition affecting recognition by pattern recognition receptors

• Immune stealth mechanisms:

  • O2 serotypes show increased prevalence in drug-resistant strains despite being more serum-sensitive

  • This immune stealth advantage allows O2 strains to evade antibody-mediated clearance

• Clinical relevance:

  • O2 serotype strains have increased prevalence in extended-spectrum β-lactamase (ESBL) and carbapenem-resistant Enterobacteriaceae (CRE) multidrug-resistant groups

  • This prevalence suggests selective pressure favoring O2 strains in antibiotic-rich environments

Research has documented that human plasma samples show dominantly higher IgG responses to O1 LPS compared to O2, and analysis of memory B cell repertoires confirmed significantly lower frequencies of O2-specific memory B cells .

How does the proinflammatory response induced by OmpP2 affect pathogenesis and vaccine development?

The proinflammatory response induced by OmpP2 has complex implications for both pathogenesis and vaccine development:

Pathogenesis implications:

• OmpP2 can upregulate mRNA expression of multiple proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8) in macrophages

• OmpP2 induces more prolonged cytokine responses than lipooligosaccharide (LOS)

• This sustained inflammatory response may contribute to tissue damage during infection

• The inflammatory response may differ between bacterial species and strains based on OmpP2 variability

Vaccine development considerations:

• Balancing immunity vs. inflammation:

  • Need to generate protective immunity without excessive inflammatory responses

  • Epitope selection to maximize protection while minimizing inflammatory damage

• Adjuvant selection:

  • Careful selection of adjuvants that complement rather than exacerbate OmpP2-induced inflammation

  • Potential for using modified OmpP2 variants with reduced inflammatory properties

• Monitoring inflammatory markers:

  • Inclusion of cytokine profiling in vaccine safety assessments

  • Development of correlates of protection that include both antibody titers and inflammatory parameters

• Route of administration:

  • Different routes may affect the balance between protective immunity and harmful inflammation

  • Mucosal delivery might enhance protective responses while limiting systemic inflammation

Research suggests that OmpP2's proinflammatory properties play an important role in the pathogenesis of bacterial infections, making this a critical consideration for vaccine development strategies .

What approaches can resolve contradictory results between in vitro binding assays and functional protection studies with OmpP2 antibodies?

Researchers frequently encounter discrepancies between binding assays (like ELISA) and functional protection studies. To resolve these contradictions:

• Comprehensive antibody characterization:

  • Determine if antibodies recognize conformational vs. linear epitopes

  • Evaluate binding to whole bacteria vs. purified OmpP2

  • Assess binding under different conditions (pH, salt concentration)

• Surface accessibility analysis:

  • Use flow cytometry with intact bacteria to confirm that epitopes are accessible

  • Compare results between binding to purified protein and whole cells

  • Construction of OmpP2-deficient mutants as negative controls

• Isotype and subclass analysis:

  • Determine if antibodies belong to isotypes capable of mediating protection

  • Consider converting antibodies to different isotypes to test functional effects

• Complement activation assessment:

  • Measure complement activation by different antibodies

  • Research shows non-opsonophagocytic MAbs may be unable to activate complement

• Multiple protection assays:

  • Employ various functional assays beyond opsonophagocytosis

  • Include serum bactericidal assays, neutrophil activation assays, and in vivo models

Research has shown that antibodies cross-reacting in ELISA with non-related strains may not promote opsonophagocytosis of those strains, highlighting the importance of functional studies beyond binding assays .

What are the current technological limitations in developing therapeutic OmpP2 antibodies for multidrug-resistant bacterial infections?

Development of therapeutic OmpP2 antibodies faces several technological challenges:

• Strain variation and specificity issues:

  • OmpP2 proteins show significant variability between strains

  • Strictly strain-specific opsonophagocytic activity limits broad application

  • Need for rapid strain typing before antibody administration

• Penetration into infection sites:

  • Limited antibody penetration into biofilms

  • Challenges in reaching bacteria in abscesses or intracellular locations

  • Need for delivery systems to enhance antibody distribution

• Combination therapy requirements:

  • Monotherapies may be insufficient for multidrug-resistant infections

  • Optimization of antibody-antibiotic combinations

  • Determining optimal timing of combination treatments

• Manufacturing and stability:

  • Ensuring consistent glycosylation patterns affecting antibody functions

  • Maintaining stability during storage and administration

  • Developing formulations suitable for different administration routes

• Immune escape mechanisms:

  • Bacterial downregulation of target antigens

  • Selection pressure leading to epitope mutations

  • Bacterial mechanisms that interfere with opsonophagocytosis

Recent research has demonstrated that antibodies can provide synergistic protection when combined with standard-of-care antibiotics like meropenem, confirming the importance of immune assistance in antibiotic therapy .

How might genetic engineering and synthetic biology advance OmpP2 antibody development?

Genetic engineering and synthetic biology offer promising approaches to enhance OmpP2 antibody development:

• Engineered antibody formats:

  • Bispecific antibodies targeting OmpP2 and other bacterial antigens simultaneously

  • Antibody-antibiotic conjugates for targeted delivery

  • Single-chain variable fragments (scFvs) with enhanced tissue penetration

• Rational epitope design:

  • Computational design of immunogens displaying conserved, protective epitopes

  • Scaffold proteins presenting multiple OmpP2 epitopes in optimal orientation

  • Structure-based engineering of hyper-stable epitopes

• Expression system optimization:

  • Development of improved bacterial or mammalian expression systems

  • Cell-free protein synthesis for rapid production

  • Engineering strains for high-yield production of conformationally correct antigens

• Novel antibody discovery platforms:

  • Phage display libraries enriched for anti-bacterial antibodies

  • Yeast display systems for affinity maturation

  • Synthetic antibody libraries designed for bacterial surface recognition

• Gene delivery approaches:

  • In vivo expression of antibody genes using viral vectors

  • DNA vaccines encoding both OmpP2 antigens and neutralizing antibodies

  • Graphene oxide nanoparticles to enhance gene transfer, as demonstrated in one study

Research has shown that graphene oxide nanoparticles can aid transformation processes, which might be leveraged for genetic engineering approaches in antibody development .

What novel insights can proteomics approaches provide regarding OmpP2-host protein interactions for therapeutic targeting?

Proteomics approaches offer significant potential for understanding OmpP2-host interactions:

• Interaction partner identification:

  • TurboID proximity labeling systems have identified host proteins interacting with OmpP2

  • One study identified 240 unique proteins from immortalized porcine alveolar macrophage cells that could interact with G. parasuis OmpP2

  • Among these, membrane proteins CAV1, ARF6, and PPP2R1A were found to be involved in bacterial recognition and phagocytosis

• Temporal dynamics analysis:

  • Proteomics can reveal how host-pathogen protein interactions change over time

  • Understanding the sequence of interactions can identify optimal intervention points

• Post-translational modification studies:

  • Characterization of how post-translational modifications affect OmpP2-host interactions

  • Identification of modifications that might be targeted therapeutically

• Comparative interactomics:

  • Comparing interaction networks between virulent and avirulent strains

  • Identifying strain-specific interactions that correlate with disease severity

• Therapeutic target prioritization:

  • Ranking interaction partners based on druggability and functional importance

  • Focusing on host proteins involved in bacterial recognition for therapeutic intervention

This approach has already provided valuable insights, identifying three novel host membrane proteins (CAV1, ARF6, and PPP2R1A) involved in the recognition and phagocytosis of G. parasuis by alveolar macrophages .

How can OmpP2 antibody research inform broader strategies against bacterial biofilms?

OmpP2 antibody research provides valuable insights for combating bacterial biofilms:

• Targeting newly released bacteria:

  • Bacteria newly released from biofilms may have a unique, vulnerable phenotype

  • Research has shown transient up-regulated expression of major porins (including OmpP2) in newly released bacteria

  • This increased expression correlates with increased membrane permeability and vulnerability to antibiotics

• Combination therapy approaches:

  • Anti-OmpP2 antibodies could be combined with antibiofilm agents

  • Timing such combinations to target both established biofilms and dispersed bacteria

• Preventive strategies:

  • OmpP2 antibodies might prevent initial bacterial attachment

  • Coating medical devices with antibodies could reduce biofilm formation

• Enhanced biofilm penetration:

  • Developing antibody fragments with improved biofilm penetration

  • Conjugating antibodies with enzymes that degrade biofilm matrix components

• Leveraging biofilm-specific expression patterns:

  • Targeting OmpP2 epitopes specifically exposed in biofilm environments

  • Developing antibodies against biofilm-associated OmpP2 conformations