HIV-1 gp41, HRP

What is the structure and function of HIV-1 gp41?

HIV-1 gp41 is a transmembrane glycoprotein that forms part of the envelope spike along with gp120. While gp120 initiates virus entry by binding to host receptors, gp41 mediates fusion between viral and host membranes . Structurally, gp41 contains several domains including the fusion peptide, heptad repeat regions (HR1 and HR2), membrane-proximal external region (MPER), transmembrane domain, and cytoplasmic domain . The two heptad repeat regions (HR1 and HR2) play crucial roles in the conformational changes necessary for membrane fusion. During fusion, gp41 transitions from a native prefusion state through a prehairpin intermediate to a post-fusion six-helix bundle structure, bringing viral and cellular membranes into proximity for fusion .

What applications are HRP Anti-HIV1 gp41 antibodies suitable for?

HRP-conjugated Anti-HIV1 gp41 antibodies, such as the goat polyclonal antibody ab68622, are primarily suitable for Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . These antibodies have been validated to react specifically with HIV1 gp41 in viral and infected tissue samples . The horseradish peroxidase (HRP) conjugation provides a direct detection method, eliminating the need for secondary antibodies in immunoassays. This makes these antibodies particularly useful for detecting HIV-1 gp41 expression in various experimental contexts, including viral preparation quality control, infected cell cultures, and assessment of recombinant gp41 protein expression .

How can researchers assess antibody responses against gp41 in HIV-1-infected patients?

Researchers can assess antibody responses against gp41 using soluble fusion proteins containing different segments of the gp41 ectodomain. One effective approach is using glutathione S-transferase (GST) fusion proteins encompassing C-terminal fragments of varying lengths (e.g., 30, 64, 100, 142, or 172 amino acids) of gp41 ectodomain . These proteins can be used in ELISA to test patient plasma samples for antibody reactivity. Additionally, overlapping peptides covering the gp41 sequence can help map linear epitopes .

The assessment methodology should include:

• Generating soluble recombinant gp41 fragments

• Validating antigenic integrity with known monoclonal antibodies (such as 2F5 and 4E10)

• Testing patient sera against equimolar amounts of different gp41 fragments

• Using appropriate negative controls (e.g., GST protein alone)

• Correlating antibody responses with neutralizing activity through neutralization assays

Why is it challenging to express gp41 in soluble form?

Expression of soluble gp41 is challenging due to several factors:

• Extreme hydrophobicity of gp41, particularly in the fusion peptide, transmembrane domain, and parts of the cytoplasmic domain

• Tendency of gp41 to form aggregates through non-specific hydrophobic interactions

• Formation of intramolecular interactions between HR1 and HR2 regions that can lead to misfolding

• Difficulty in properly folding when expressed in bacterial systems, resulting in accumulation in inclusion bodies

To overcome these challenges, researchers have developed strategies including:

• Fusion to solubility-enhancing partners like GST

• Introduction of mutations that disrupt intramolecular HR1-HR2 interactions while preserving intermolecular HR1-HR1 interactions

• Attachment of trimerization domains like foldon

• Careful denaturation, refolding, and renaturation protocols

What strategies can be employed to design soluble near full-length HIV-1 gp41 trimers?

Designing soluble near full-length HIV-1 gp41 trimers requires multifaceted approaches to overcome inherent challenges:

• Strategic Mutations: Introduce mutations in HR1 and HR2 regions that specifically disrupt intramolecular HR1-HR2 interactions while preserving intermolecular HR1-HR1 interactions. This reduces non-specific aggregation and improves solubility .

• Trimerization Domain Fusion: Attaching a 27-amino acid foldon domain (derived from phage T4 fibritin) at the C-terminus stabilizes the trimeric structure. The foldon helps channel gp41 into trimers during the folding process .

• Controlled Refolding: A slow refolding process is critical for proper trimerization. This typically involves:

  • Complete denaturation in high concentrations of denaturants like urea or guanidine hydrochloride

  • Gradual removal of denaturant through dialysis or dilution

  • Refolding in the presence of stabilizing agents

• Fusion to Carrier Proteins: Fusion to carrier proteins such as GST or T4 small outer capsid protein (Soc) can enhance solubility and facilitate display on phage nanoparticles for structural studies or immunogen development .

These techniques have successfully led to the design of soluble gp41 trimers containing both the fusion peptide and the cytoplasmic domain, which are stabilized in a prehairpin-like conformation .

How does antibody reactivity against different regions of gp41 correlate with neutralization capacity?

Research has revealed significant correlations between antibody reactivity patterns and neutralization capacity:

• MPER Reactivity and Broad Neutralization: Patients with stronger antibody responses against the membrane-proximal external region (MPER) of gp41, which contains epitopes for broadly reactive neutralizing antibodies 2F5 and 4E10, exhibit broader and more potent neutralizing activity .

• GST-gp41-30 Reactivity: Patients with high antibody reactivity against GST-gp41-30 (containing the MPER) show statistically significant stronger neutralizing activity (p < 0.01) against primary HIV-1 isolates including difficult-to-neutralize strains like HIV-1 AD8 .

• Variation in Neutralization Breadth: Some patients develop antibodies that can neutralize a wide range of viral isolates across multiple clades, while others show limited neutralization capacity despite having antibodies against gp41 .

The table below summarizes the relationship between gp41 antibody reactivity and neutralization capacity observed in one study:

This correlation supports the hypothesis that antibodies targeting the MPER region, particularly those with specificity similar to 2F5 and 4E10, contribute significantly to broad neutralization capacity .

What methods can be used to confirm the structural conformation of recombinant gp41 trimers?

Confirming the structural conformation of recombinant gp41 trimers requires multiple complementary approaches:

• Binding to Conformation-Specific Antibodies:

  • Testing binding to hexa-helical bundle-specific antibodies (e.g., NC-1 mAb)

  • Assessing binding of HR2 peptides to exposed HR1 grooves (which occurs in the prehairpin conformation but not in the post-fusion six-helix bundle)

• Neutralizing Antibody Binding Assays:

  • Evaluating binding to broadly neutralizing antibodies like 2F5 and 4E10 that recognize specific conformational epitopes

  • Inhibition of virus neutralization by these antibodies can indicate proper folding of the target epitopes

• Biophysical Characterization:

  • Size exclusion chromatography to confirm trimeric state

  • Circular dichroism spectroscopy to assess secondary structure content

  • Analytical ultracentrifugation to determine oligomeric state and homogeneity

• Functional Assays:

  • Cell fusion inhibition assays to test whether the recombinant gp41 can interfere with viral entry

  • Binding to cellular receptors or co-receptors in a manner consistent with the proposed structural state

• Structural Analysis:

  • Negative-stain electron microscopy to visualize trimeric structure

  • X-ray crystallography or cryo-EM for high-resolution structural determination if possible

A combination of these methods provides comprehensive validation of the structural conformation of recombinant gp41 trimers.

How can researchers overcome antigen variability when developing gp41-based diagnostics or vaccines?

Overcoming gp41 antigen variability for diagnostic or vaccine development requires several strategic approaches:

• Consensus Sequence Utilization: Use of M group consensus envelope sequences (e.g., MCON6) for generating gp41 antigens can enhance recognition by antisera from patients infected with diverse HIV-1 clades .

• Conserved Epitope Targeting: Focus on highly conserved regions of gp41, particularly:

  • The membrane-proximal external region (MPER) containing 2F5 and 4E10 epitopes

  • The immunodominant loop between HR1 and HR2 (cluster I)

  • The region between HR2 and the 2F5 epitope (cluster II)

• Multi-Clade Antigen Cocktails: Development of antigen panels representing major HIV-1 clades to ensure broad coverage of viral diversity.

• Structure-Based Design: Engineering gp41 constructs that present conserved neutralizing epitopes in their native conformation while minimizing exposure of variable, non-neutralizing epitopes .

• Stabilization in Specific Conformational States: Stabilizing gp41 in pre-hairpin intermediates that expose conserved epitopes targeted by broadly neutralizing antibodies rather than in the post-fusion six-helix bundle .

The use of soluble gp41 fragments of varying lengths (e.g., GST-gp41-30, -64, -100) can help identify which regions elicit the most cross-reactive antibodies among diverse patient populations .

What are the optimal protocols for expressing and purifying soluble gp41 constructs?

Based on successful approaches in the literature, the optimal protocol for expressing and purifying soluble gp41 constructs involves:

• Expression System Selection:

  • E. coli BL21(DE3) for GST fusion proteins

  • The use of specialized E. coli strains designed for expression of disulfide-bonded or membrane proteins may improve yield

• Expression Construct Design:

  • Fusion to solubility-enhancing partners (GST, MBP, or SUMO)

  • Addition of a C-terminal foldon trimerization domain if trimeric structure is desired

  • Introduction of strategic mutations to reduce hydrophobicity and aggregation

  • Inclusion of a polyhistidine tag for purification

• Expression Conditions:

  • Induction at lower temperatures (16-25°C) to slow protein synthesis and improve folding

  • Use of reduced IPTG concentration (0.1-0.5 mM) for induction

  • Extended expression time (overnight) at lower temperatures

• Extraction and Solubilization:

  • Isolation of inclusion bodies through detergent washing

  • Complete denaturation using 6-8 M urea or 6 M guanidine hydrochloride

• Refolding Strategy:

  • Stepwise dialysis to gradually remove denaturant

  • Inclusion of redox couples (reduced/oxidized glutathione) to facilitate disulfide bond formation

  • Addition of glycerol or arginine to prevent aggregation during refolding

• Purification Steps:

  • Initial capture via affinity chromatography (glutathione for GST fusions or Ni-NTA for His-tagged proteins)

  • Secondary purification via ion exchange chromatography

  • Final polishing by size exclusion chromatography to remove aggregates and ensure homogeneity

• Quality Control:

  • Verification of antigenic integrity by binding to conformation-specific antibodies

  • Assessment of oligomeric state by native PAGE or analytical size exclusion chromatography

What factors should be considered when designing ELISA assays to detect anti-gp41 antibodies?

When designing ELISA assays to detect anti-gp41 antibodies, researchers should consider several critical factors:

• Antigen Selection and Preparation:

  • Use of well-characterized recombinant gp41 fragments of different lengths to distinguish antibody responses against different domains

  • Equimolar coating of antigens to enable direct comparison of antibody responses

  • Inclusion of appropriate controls (e.g., GST alone for GST-fusion proteins)

• Assay Optimization:

  • Determination of optimal antigen coating concentration through titration

  • Selection of appropriate blocking reagents to minimize background

  • Optimization of antibody dilution ranges to ensure detection within the linear range

• Patient Sample Considerations:

  • Use of paired plasma/serum samples for longitudinal studies

  • Standardization of sample collection and processing procedures

  • Consideration of antiretroviral therapy status, as this can affect antibody responses

• Data Interpretation:

  • Establishment of cutoff values based on negative controls

  • Normalization strategies for comparing results across different plates or experiments

  • Statistical approaches for analyzing variations in antibody responses among patient groups

• Conformational Considerations:

  • Recognition that some epitopes may be conformational rather than linear

  • Assessment of whether the immobilized antigen retains native-like conformation

  • Use of properly folded proteins rather than just peptides to detect antibodies against conformational epitopes

• Cross-Reactivity Assessment:

  • Testing for potential cross-reactivity with other proteins or with different HIV clades

  • Inclusion of competing antigens to assess antibody specificity

How can researchers evaluate neutralization breadth and potency of anti-gp41 antibodies?

Evaluating neutralization breadth and potency of anti-gp41 antibodies requires systematic approaches:

• Virus Panel Selection:

  • Include diverse HIV-1 isolates representing multiple clades

  • Incorporate both laboratory-adapted strains and primary isolates

  • Include viruses with varying neutralization sensitivities (Tier 1, 2, and 3)

  • Representative panel might include:

    • Easily neutralizable "Tier 1" viruses (e.g., MN, SF162.LS)

    • "Tier 2" viruses representing typical circulating strains of moderate neutralization resistance

    • Difficult-to-neutralize strains (e.g., AD8)

• Neutralization Assay Selection:

  • Pseudovirus-based single-round infection assays using TZM-bl cells

  • PBMC-based assays with primary isolates

  • Cell-cell fusion inhibition assays to assess antibody function

• Controls and Standards:

  • Include well-characterized broadly neutralizing antibodies (e.g., 2F5, 4E10) as positive controls

  • Use non-HIV envelope glycoprotein (e.g., VSV-G) pseudotyped viruses as specificity controls

  • Include serum/plasma from HIV-negative individuals as negative controls

• Breadth Assessment:

  • Calculate the percentage of viruses neutralized above a defined threshold

  • Group results by viral clades to assess cross-clade neutralization

• Potency Evaluation:

  • Determine IC50 values (antibody concentration giving 50% neutralization)

  • Calculate geometric mean titers across virus panels

  • Prepare titration curves to visualize neutralization potency

• Statistical Analysis:

  • Correlate neutralization data with antibody binding to specific gp41 domains

  • Perform significance testing to identify associations between binding patterns and neutralization capacity

What approaches can be used to map epitopes recognized by anti-gp41 antibodies?

Epitope mapping of anti-gp41 antibodies can be accomplished through several complementary techniques:

• Peptide-Based Methods:

  • Overlapping peptide ELISA: Using a series of overlapping peptides spanning the gp41 sequence

  • Peptide competition assays: Testing if specific peptides can block antibody binding to full-length gp41

  • Phage display peptide libraries: Identifying mimotopes that bind to the antibody of interest

• Protein Fragment Analysis:

  • Testing binding to truncated gp41 constructs of varying lengths (e.g., GST-gp41-30, -64, -100)

  • Comparing binding patterns to deduce the location of epitopes

  • Using domain-swapped chimeric proteins to localize epitope regions

• Mutagenesis Approaches:

  • Alanine-scanning mutagenesis: Systematically replacing amino acids with alanine

  • Site-directed mutagenesis of predicted contact residues

  • Testing mutant proteins for altered antibody binding

• Structural Biology Techniques:

  • X-ray crystallography of antibody-antigen complexes

  • Cryo-electron microscopy of antibody-trimer complexes

  • Hydrogen-deuterium exchange mass spectrometry to identify protected regions upon antibody binding

• Computational Methods:

  • Epitope prediction algorithms

  • Molecular modeling and docking studies

  • Analysis of sequence conservation across HIV-1 clades to identify likely epitopes

• Competition Assays:

  • Testing if the antibody of interest competes with known mAbs (like 2F5 or 4E10) for binding to gp41

  • Using this approach to determine if the epitope overlaps with known neutralizing epitopes

A systematic combination of these approaches provides comprehensive epitope mapping and can reveal whether antibodies target conformational epitopes or linear sequences, which is critical for understanding their neutralization mechanism.

How can understanding gp41-specific antibody responses inform next-generation HIV vaccine design?

Understanding gp41-specific antibody responses offers several strategic insights for next-generation HIV vaccine design:

• MPER-Focused Immunogen Design:

  • The correlation between strong antibody responses against MPER (membrane-proximal external region) and broader neutralization suggests that vaccines should be designed to specifically elicit MPER-targeted antibodies

  • Presenting MPER in its native conformation while eliminating immunodominant non-neutralizing epitopes

  • Potential approaches include stabilized gp41 trimers or nanoparticle presentation of MPER epitopes

• Prehairpin Intermediate Targeting:

  • Vaccines designed to elicit antibodies against the transient prehairpin intermediate conformation of gp41 that forms during viral entry

  • Stabilization of gp41 in this conformation through strategic mutations and trimerization domains

  • This approach could generate antibodies that interrupt the fusion process

• Sequential Immunization Strategies:

  • Using a series of immunogens that gradually guide B cell maturation toward broadly neutralizing antibody production

  • Initial priming with engineered minimalist constructs followed by boosting with more native-like structures

• Lessons from Natural Infection:

  • Analysis of the tremendous variation in antibody responses against different gp41 regions among individual patients can inform personalized vaccine approaches

  • Understanding why some patients develop broadly neutralizing responses while others do not

• Adjuvant and Delivery Optimization:

  • Development of adjuvants that specifically enhance antibody responses against hydrophobic or membrane-proximal regions

  • Exploration of display platforms that present gp41 in native-like trimeric configuration, such as phage nanoparticles

• Cross-Clade Coverage:

  • Use of consensus sequences or conserved epitope focusing to develop vaccines with broad coverage against diverse HIV-1 clades

  • Development of multivalent vaccines containing gp41 components from different clades

What are the challenges in correlating in vitro antibody binding with in vivo neutralization efficacy?

Several challenges exist in correlating in vitro antibody binding with in vivo neutralization efficacy:

• Conformational Disparities:

  • Recombinant gp41 proteins used in binding assays may not perfectly mimic the conformation of gp41 on viral particles

  • Native gp41 exists in a dynamic equilibrium between different conformational states during the fusion process

• Context-Dependent Epitope Accessibility:

  • Some epitopes accessible in recombinant proteins may be occluded in the context of the viral envelope trimer

  • MPER accessibility may differ between binding assays and the dynamic environment of viral fusion

• Antibody Characteristics Beyond Binding:

  • Neutralization efficacy depends not only on binding affinity but also on:

    • Angle of approach to the viral envelope

    • IgG subclass and Fc-mediated functions

    • Ability to interfere with conformational changes during fusion

• Assay Variability Factors:

  • Different neutralization assay formats (PBMC-based vs. TZM-bl) can yield varying results

  • Cell line differences in receptor/co-receptor expression levels affect neutralization sensitivity

  • Variations in virus preparation methods influence envelope conformation and stability

• In Vivo Complexities:

  • Factors present in vivo but absent in vitro:

    • Complement activation

    • Antibody-dependent cellular cytotoxicity (ADCC)

    • Mucosal environment effects on antibody function

    • Tissue-specific differences in virus neutralization requirements

• Temporal Aspects of Neutralization:

  • The kinetics of antibody binding versus the rapid kinetics of the fusion process

  • Time-dependent conformational changes in gp41 during fusion that affect epitope accessibility

Understanding these challenges is crucial for designing improved in vitro assays that better predict in vivo efficacy and for developing more effective HIV-1 immunogens.

How can researchers address aggregation issues when working with recombinant gp41 proteins?

Recombinant gp41 proteins are prone to aggregation due to their hydrophobicity. Researchers can address this challenge through several approaches:

• Protein Engineering Strategies:

  • Introduction of strategic mutations that disrupt intramolecular HR1-HR2 interactions while preserving intermolecular HR1-HR1 interactions

  • Replacement of highly hydrophobic residues in non-essential regions

  • Addition of solubility-enhancing tags such as GST, MBP, or SUMO

  • Incorporation of trimerization domains like foldon to promote proper oligomerization

• Optimized Expression Conditions:

  • Lower temperature expression (16-20°C) to slow protein synthesis rate

  • Reduced inducer concentration to prevent overwhelming the folding machinery

  • Co-expression with chaperones to assist proper folding

• Improved Solubilization and Refolding:

  • Complete denaturation with strong denaturants (8M urea or 6M guanidine hydrochloride)

  • Gradual, step-wise refolding through dialysis with decreasing denaturant concentration

  • Inclusion of stabilizing agents during refolding:

    • 0.4-1.0 M L-arginine to prevent aggregation

    • 5-10% glycerol to stabilize native conformations

    • Redox couples (reduced/oxidized glutathione) for proper disulfide formation

• Buffer Optimization:

  • Screening of various buffer compositions, pH values, and ionic strengths

  • Addition of non-ionic detergents at concentrations below CMC

  • Use of amino acid additives like proline or arginine that disrupt protein-protein interactions

• Handling and Storage Protocols:

  • Maintaining protein at moderate concentrations (0.5-1 mg/ml) to prevent concentration-dependent aggregation

  • Flash-freezing aliquots to prevent freeze-thaw induced aggregation

  • Addition of cryoprotectants for long-term storage

• Purification Considerations:

  • Final size exclusion chromatography step to remove aggregates

  • On-column refolding techniques to prevent aggregation during concentration steps

  • Use of specialized matrices designed for hydrophobic proteins

Implementation of these strategies has successfully allowed researchers to generate soluble gp41 constructs containing both the fusion peptide and cytoplasmic domain .

What controls are essential when validating anti-gp41 antibody specificity?

Validating anti-gp41 antibody specificity requires rigorous controls:

• Positive Controls:

  • Well-characterized anti-gp41 monoclonal antibodies (e.g., 2F5, 4E10, 98-6) with known epitope specificity

  • Polyclonal HIV-Ig preparation from HIV-1-positive patients

  • Recombinant gp41 protein of verified sequence and conformation

• Negative Controls:

  • Isotype-matched irrelevant antibodies

  • Serum/plasma from HIV-negative individuals

  • For fusion proteins, the tag-only protein (e.g., GST alone for GST-gp41 fusions)

  • Pre-immune serum when assessing newly generated antibodies

• Specificity Validation:

  • Competition assays with soluble gp41 or specific peptides

  • Testing against a panel of unrelated viral proteins

  • Demonstration of reactivity with HIV-1 infected cells but not uninfected cells

• Cross-Reactivity Assessment:

  • Testing against gp41 proteins from different HIV-1 clades

  • Evaluation using gp41 mutants with altered key epitopes

  • Testing against related proteins from HIV-2 or SIV to assess specificity

• Functional Validation:

  • For neutralizing antibodies, testing neutralization of HIV-1 but not control viruses (e.g., VSV-G pseudotyped viruses)

  • For non-neutralizing antibodies, testing appropriate effector functions

• Technical Controls:

  • Secondary antibody-only controls to assess background

  • Blocking optimization to minimize non-specific binding

  • Dilution series to establish dose-dependency of binding

Inclusion of these controls ensures that observed results truly reflect specific anti-gp41 activity rather than non-specific binding or technical artifacts.