CPR5 Antibody

What is CCR5 and what are its primary biological functions?

CCR5 (CD195) is a seven transmembrane chemokine receptor that plays crucial roles in multiple physiological processes. It functions as a receptor for several inflammatory CC-chemokines including CCL3 (MIP-1-alpha), CCL4 (MIP-1-beta), and RANTES, transducing signals by increasing intracellular calcium levels . CCR5 participates in T-lymphocyte migration to infection sites by acting as a chemotactic receptor and may play a role in controlling granulocytic lineage proliferation or differentiation .

Most critically for infectious disease research, CCR5 serves as the major coreceptor for macrophage-tropic isolates of HIV-1, facilitating viral entry into CD4+ T lymphocytes upon transmission . The natural resistance to HIV infection observed in individuals with the CCR5-Δ32 genetic variant highlights its significance in disease dynamics .

What types of CCR5 antibodies are available for research purposes?

Researchers have access to several types of CCR5 antibodies for experimental applications:

• Polyclonal antibodies: Such as rabbit polyclonal antibodies that recognize specific regions of the CCR5 protein. For example, ab7346 targets a synthetic peptide within human CCR5's amino acids 1-50 .

• Monoclonal antibodies: Including clones like HM-CCR5 (7A4) that bind to specific domains such as the N-terminal extracellular domain of CCR5 .

• Therapeutic antibodies: Such as Leronlimab, which has been used in receptor occupancy studies and has potential clinical applications .

• Natural anti-CCR5 antibodies: Found in normal human IgG preparations (intravenous immunoglobulin or IVIG), these natural antibodies target CCR5 and may have immunoregulatory functions .

Each antibody type offers different advantages depending on the experimental goals, from high specificity (monoclonals) to broader epitope recognition (polyclonals).

How do I select the appropriate CCR5 antibody for my research application?

Selection should be based on several critical factors:

• Target species compatibility: Confirm the antibody recognizes your species of interest. For instance, HM-CCR5(7A4) binds mouse CCR5 with no cross-reactivity to human CCR5 .

• Application suitability: Verify the antibody has been validated for your specific application. For example, ab7346 is suitable for Western blot and immunohistochemistry-paraffin applications with human samples .

• Epitope specificity: Different experimental questions may require antibodies targeting different regions of CCR5. Some antibodies target the N-terminus, while others may target extracellular loops or intracellular domains.

• Functional properties: Determine whether you need a neutralizing or non-neutralizing antibody. Some anti-CCR5 antibodies block ligand binding and/or HIV entry, while others are primarily useful for detection .

Always carefully titrate antibodies for optimal performance in your specific assay of interest, as recommended by manufacturers .

What are the primary applications of CCR5 antibodies in HIV research?

CCR5 antibodies serve multiple critical functions in HIV research:

• Inhibition of viral entry: Anti-CCR5 antibodies can block HIV-1 infection of lymphocytes and monocytes/macrophages by preventing the virus from using CCR5 as a coreceptor for cell entry . This blocking effect is specific to R5-tropic HIV strains and does not inhibit X4-tropic HIV .

• Receptor occupancy (RO) analysis: Flow cytometric methods using anti-CCR5 antibodies like Leronlimab enable calculation of CCR5 receptor occupancy, which is a critical predictor of therapeutic efficacy for CCR5-targeting drugs .

• Monitoring CCR5+ cell populations: Antibodies allow researchers to track changes in CCR5-expressing cell populations during infection or treatment. For example, studies have shown that Leronlimab treatment leads to increased levels of CCR5+CD4+ T cells in peripheral blood .

• Development of passive immunotherapy approaches: Natural anti-CCR5 antibodies isolated from healthy donors may be suitable for developing novel passive immunotherapy regimens for specific clinical contexts in HIV infection .

How can CCR5 receptor occupancy (RO) be measured in experimental settings?

Two independent flow cytometric methods for calculating CCR5 receptor occupancy have been documented:

Method 1: Direct competition assay

• Uses the same anti-CCR5 antibody (e.g., Leronlimab) with different labels to determine occupied versus total receptors

• Provides sensitive measurements with low background on untreated CCR5+CD4+ T cells

• Can be applied to both blood and tissue-resident CD4+ T cells

Method 2: Indirect displacement assay

• Uses a competing anti-CCR5 antibody with a different epitope specificity

• Measures the displacement of the therapeutic antibody

• Correlates longitudinally with plasma concentrations in treated subjects

Both methods yield comparable CCR5 RO values and can detect occupancy that correlates with plasma concentrations of the anti-CCR5 agent. These approaches have demonstrated that weekly Leronlimab treatment can achieve complete CCR5 RO on peripheral blood CD4+ T cells in both macaques and humans .

What controls should be included when using CCR5 antibodies in flow cytometry?

When using CCR5 antibodies in flow cytometry experiments, researchers should include:

• Isotype controls: To establish background staining levels and identify non-specific binding

• CCR5-negative cell populations: As biological negative controls

• Blocking controls: To confirm specificity by pre-incubating with unlabeled antibodies or CCR5 ligands (RANTES, MIP-1α, MIP-1β)

• CCR5-Δ32 homozygous samples (when available): As genetic negative controls that naturally lack CCR5 expression

• Titration series: To determine optimal antibody concentration, as recommended for antibodies like HM-CCR5 (7A4)

These controls ensure accurate interpretation of results and help distinguish true CCR5 expression from technical artifacts.

How does the CCR5-Δ32 genetic variant affect CCR5 expression and function?

The CCR5-Δ32 variant represents a 32-base pair deletion in the CCR5 gene that has significant functional consequences:

• Molecular impact: The deletion occurs in the region encoding the second extracellular loop of the receptor, resulting in a frameshift mutation that leads to premature truncation of the CCR5 protein .

• Cellular expression: This truncated protein fails to reach the cell surface, effectively abrogating CCR5 availability and function .

• HIV resistance: Homozygous individuals (Δ32/Δ32) demonstrate remarkable resistance to R5-tropic HIV-1 infection, as the virus cannot use the absent coreceptor for cell entry .

• Evolutionary significance: This mutation has been maintained at relatively high frequencies in certain human populations, suggesting potential selective advantages. While initially hypothesized to provide protection against plague, more recent evidence suggests selection may have been driven by other infectious diseases such as smallpox .

• Research applications: Cells from CCR5-Δ32 homozygous individuals serve as important negative controls in CCR5 antibody validation and HIV entry studies .

What is the relationship between natural anti-CCR5 antibodies and immune function?

Natural anti-CCR5 antibodies have been identified in normal human serum and commercial IVIG preparations, revealing intriguing immunoregulatory properties:

• Origin: These natural autoantibodies are produced in the absence of deliberate immunization and are generated by positively selected autoreactive B cells .

• Functional effects:

  • They can inhibit the binding of RANTES to macrophages, demonstrating their interaction with the CCR5 receptor

  • They block infection with R5-tropic HIV strains but not X4-tropic HIV

  • They may participate in normal immune homeostasis and host defense mechanisms

• Potential therapeutic applications: Natural anti-CCR5 antibodies from healthy immunocompetent donors may be suitable for development of passive immunotherapy approaches for HIV infection .

• Relationship to other natural antibodies: IVIG preparations contain multiple natural antibodies targeting various immune molecules, including CD4, CD5, cytokine receptors, and adhesion motifs, suggesting coordinated roles in immune regulation .

This understanding of natural anti-CCR5 antibodies opens avenues for exploring their physiological significance and potential therapeutic applications.

How can researchers induce anti-CCR5 autoantibodies in animal models?

Researchers have successfully induced anti-CCR5 autoantibodies in macaque models using the following approach:

• Immunogen design: CCR5 peptide-conjugated virus-like particle (VLP) preparations have been used to break immune tolerance to self-CCR5 .

• Immunization protocol: Strategic vaccination schedules with appropriate adjuvants can generate high-avidity anti-CCR5 IgG autoantibody responses .

• Functional validation: In macaque studies, these induced autoantibodies could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV SF162P3 in vitro .

• Durability and boosting: While anti-CCR5 IgG titers naturally decline over time, levels can be effectively boosted through revaccination .

• Safety considerations: Long-term follow-up (over 3 years) of immunized macaques showed no adverse health effects and no detectable decline in CCR5-expressing T cell populations, suggesting this approach may be safe .

This methodology provides a potential framework for inducing protective anti-CCR5 antibodies that could be translated to human therapeutic applications.

What are the recommended protocols for validating CCR5 antibody specificity?

A comprehensive validation approach should include:

• Multiple detection methods: Verify specificity using independent techniques such as Western blot, flow cytometry, and immunohistochemistry .

• Genetic controls: Test antibody binding to cells from CCR5-Δ32 homozygous individuals (which lack CCR5 expression) to confirm specificity .

• Competitive binding assays: Demonstrate that binding can be blocked by CCR5-specific ligands such as RANTES, MIP-1α, and MIP-1β, or by other validated anti-CCR5 antibodies targeting the same epitope .

• Cross-reactivity assessment: Test against related chemokine receptors (e.g., CXCR4, CCR2) to ensure specificity within the chemokine receptor family .

• Functional validation: Confirm that the antibody can inhibit known CCR5-dependent processes, such as chemokine-induced calcium flux or HIV entry for neutralizing antibodies .

These validation steps ensure that experimental observations can be confidently attributed to CCR5-specific effects.

How should CCR5 antibodies be titrated for optimal performance in different applications?

Proper titration is essential for obtaining reliable results with CCR5 antibodies:

• Flow cytometry titration:

  • Start with the manufacturer's recommended concentration (e.g., ≤1.0 μg per test for HM-CCR5 antibody)

  • Prepare a serial dilution series (typically 2-fold dilutions)

  • Stain a constant number of cells (10^5 to 10^8 cells/test) with each dilution

  • Identify the concentration that provides optimal signal-to-noise ratio

• Western blot titration:

  • Test dilutions ranging from 1:500 to 1:5000

  • For ab7346, 1:1000 dilution has been validated for Western blot applications

• Immunohistochemistry titration:

  • Start with 2.5 μg/ml for paraffin-embedded tissue sections (as validated for ab7346)

  • Adjust concentration based on tissue type and fixation method

The optimal antibody concentration will provide maximum specific signal with minimal background staining. Always include appropriate controls at each concentration tested.

What factors can interfere with CCR5 antibody binding and how can they be mitigated?

Several factors can affect CCR5 antibody binding:

• Receptor internalization: CCR5 can be internalized following ligand binding or activation. Perform experiments in the absence of chemokines or after allowing for receptor recycling to the surface .

• Post-translational modifications: Variations in glycosylation or other modifications may affect epitope recognition. Consider using multiple antibodies targeting different epitopes .

• Fixation and permeabilization effects: Some fixatives can alter CCR5 conformation or epitope accessibility. Optimize fixation protocols or use live-cell staining when possible .

• Competitive inhibition by endogenous ligands: Pre-existing bound chemokines may block antibody access. Washing steps with acidic buffers can sometimes dissociate bound ligands .

• Receptor occupancy by therapeutic agents: In samples from treated subjects, pre-existing bound drugs may prevent antibody binding. This can be addressed by using antibodies targeting different epitopes or by employing specialized receptor occupancy assays .

By addressing these potential issues, researchers can enhance the reliability and reproducibility of CCR5 antibody-based experiments.

What are the emerging applications of CCR5 antibodies beyond HIV research?

CCR5 antibodies are increasingly being explored for applications in:

• Inflammatory disease research: Given CCR5's role in inflammatory conditions, antibodies targeting this receptor can help elucidate pathological mechanisms and potentially lead to new therapeutic approaches .

• Cancer immunotherapy: Emerging evidence suggests CCR5 plays roles in cancer progression, making it a potential target for intervention .

• Neuroinflammatory disorders: CCR5 is involved in neuroinflammatory processes, and antibodies could help understand these mechanisms and develop treatments.

• Transplantation medicine: CCR5 participates in transplant rejection processes, and antibodies may provide insights for improving graft survival.

• Autoimmune disease studies: The role of CCR5 in immune cell trafficking makes it relevant to autoimmune pathologies, where antibodies could serve as both research tools and potential therapeutics.

These expanding applications highlight the growing importance of CCR5 antibodies across multiple fields of biomedical research.

What technological advances are improving CCR5 antibody development and applications?

Several technological advancements are enhancing CCR5 antibody research:

• Improved receptor occupancy assays: New flow cytometric methods provide more sensitive and accurate measurements of CCR5 occupancy by therapeutic antibodies, with low background and the ability to monitor both blood and tissue-resident cells .

• Single-cell analysis approaches: Integration of CCR5 antibody staining with single-cell RNA sequencing allows researchers to correlate CCR5 protein expression with transcriptional profiles.

• Advanced imaging techniques: Super-resolution microscopy combined with fluorescently-labeled CCR5 antibodies enables detailed visualization of receptor distribution and trafficking.

• Humanized mouse models: These models facilitate in vivo testing of human-specific anti-CCR5 antibodies that wouldn't bind to mouse CCR5.

• Antibody engineering: Technologies for developing bispecific antibodies or antibody fragments with enhanced tissue penetration or modified effector functions are expanding the potential applications of anti-CCR5 therapeutics.

These advances are collectively accelerating both basic research on CCR5 biology and the development of CCR5-targeted therapeutic approaches.