Recombinant Cocal virus Glycoprotein (G)

What is the molecular structure of Cocal virus glycoprotein (G)?

Cocal virus glycoprotein (G) is a transmembrane glycoprotein that consists of 512 amino acids with two potential N-linked glycosylation sites. When expressed in mammalian cells, it produces a glycoprotein with a molecular weight of approximately 71,000 daltons. The protein is not palmitylated but possesses the ability to induce cell fusion under acidic pH conditions. Phylogenetic analysis confirms that Cocal G represents a distinct lineage within the Vesicular Stomatitis Virus Indiana (VSV-IND) serotype while maintaining significant sequence homology .

At the amino acid level, Cocal G shares 71.5% identity with VSV-G Indiana strain, explaining some of their similar functional properties while maintaining distinct serological characteristics . The protein structure follows the typical vesiculovirus glycoprotein organization with defined domains responsible for receptor binding, fusion, and trimerization.

How does Cocal G compare phylogenetically to other vesiculovirus glycoproteins?

Phylogenetic analysis of amino acid sequence differences among vesiculovirus G proteins has revealed that Cocal virus represents a distinct evolutionary lineage within the VSV Indiana serotype . Despite this distinction, Cocal G maintains significant structural homology with other vesiculovirus G proteins, particularly with VSV-G Indiana strain (71.5% amino acid identity) .

This evolutionary relationship explains why some broadly neutralizing antibodies like 8G5F11 can cross-react with multiple vesiculovirus G proteins including those from VSV Indiana, Cocal, and Maraba viruses, while other antibodies like IE9F9 exhibit strain-specific binding only to VSV Indiana G . The phylogenetic position of Cocal G makes it particularly valuable for research scenarios where alternative pseudotyping options to VSV-G are required.

What are the optimal systems for recombinant expression of Cocal virus glycoprotein?

For efficient expression of recombinant Cocal virus glycoprotein, mammalian cell expression systems have proven most effective. The glycoprotein has been successfully expressed by cloning a cDNA copy of the Cocal G mRNA into mammalian expression vectors. When producing Cocal-pseudotyped lentiviral vectors, human codon-optimized versions of the Cocal G open reading frame have demonstrated superior expression efficiency, requiring less plasmid DNA (3 μg) compared to VSV-G (6 μg) or RD114/TR (9 μg) envelope plasmids during transient transfection protocols .

For laboratory-scale production, standard transient transfection methods in HEK293T cells using calcium phosphate or lipid-based transfection reagents provide sufficient expression levels. The expressed protein undergoes proper post-translational modifications including glycosylation in mammalian systems, which is crucial for its functionality.

What methodological approaches should be used to confirm successful expression of recombinant Cocal G?

Multiple complementary techniques should be employed to verify successful expression of recombinant Cocal G:

• Western Blot Analysis: Using antibodies that recognize vesiculovirus G proteins, such as the cross-reactive monoclonal antibody 8G5F11, which binds to G proteins from VSV (Indiana, New Jersey, and Alagoas strains), Cocal, and Maraba viruses . For intracellular domain detection, antibodies like P5D4 can be used.

• Flow Cytometry: Surface expression can be measured by flow cytometry using external epitope-targeting antibodies. This approach allows quantification of expression levels and comparison between different G protein variants.

• Functional Assays: Confirming the ability of expressed Cocal G to pseudotype lentiviral vectors and mediate cell entry serves as a functional verification. Transduction efficiency with EGFP reporter genes can provide a quantitative measure of functional expression.

• pH-dependent Cell Fusion Assay: Since recombinant Cocal G induces cell fusion at acidic pH, fusion assays can be employed as additional functional verification .

How does Cocal G compare to VSV-G for lentiviral vector pseudotyping in terms of titer and stability?

Cocal-pseudotyped lentiviral vectors demonstrate performance characteristics comparable to VSV-G pseudotypes in several critical aspects:

Cocal-pseudotyped lentiviral vectors can be produced at titers as high as VSV-G pseudotypes while requiring less plasmid DNA during transfection, likely due to the human codon-optimization of the Cocal G expression construct . Like VSV-G, Cocal-pseudotyped vectors demonstrate excellent stability allowing for efficient concentration by centrifugation, an important consideration for applications requiring high-titer vector preparations.

How does human serum affect Cocal G-pseudotyped lentiviral vectors compared to other pseudotypes?

A significant advantage of Cocal G-pseudotyped lentiviral vectors is their enhanced resistance to inactivation by human serum compared to VSV-G pseudotypes, making them potentially more suitable for in vivo applications:

Human serum inactivation studies examining vectors pseudotyped with Cocal G, VSV-G, and RD114/TR revealed that:

• Cocal-pseudotyped vectors showed significantly higher resistance to human serum neutralization in 7 out of 10 human donors tested compared to VSV-G pseudotypes (P < 0.01 for 5 donors, P < 0.05 for 2 donors) .

• The level of serum inactivation varied significantly between human individuals for both VSV-G and Cocal (P < 0.0001), while RD114/TR showed more consistent resistance across individuals (P = 0.12) .

• In contrast, canine serum potently inactivated both Cocal and VSV-G pseudotypes, with RD114/TR showing significantly higher resistance in all five dogs tested (P < 0.01) .

This enhanced resistance to human serum inactivation represents a key advantage for Cocal G in human gene therapy applications, particularly for in vivo delivery approaches where serum neutralization presents a significant barrier.

What is the cell and tissue tropism of Cocal G-pseudotyped vectors compared to VSV-G and other pseudotypes?

Notably, Cocal G-pseudotyped vectors demonstrated particularly enhanced performance in non-human primate cells, with 3.2-fold higher transduction efficiency in pigtailed macaque CD34+ cells compared to VSV-G pseudotypes and 6.7-fold higher efficiency compared to RD114/TR pseudotypes . This makes Cocal G especially valuable for preclinical studies in non-human primate models.

How efficient is Cocal G at transducing hematopoietic stem cells compared to other pseudotypes?

Cocal G-pseudotyped lentiviral vectors have demonstrated excellent capacity for transducing hematopoietic stem cells (HSCs), particularly in clinically relevant models:

• Human CD34+ Cells: Cocal-pseudotyped vectors efficiently transduce human CD34+ cells at levels comparable to or higher than VSV-G pseudotypes .

• Non-human Primate CD34+ Cells: In pigtailed macaque CD34+ cells, Cocal-pseudotyped vectors achieved 3.2-fold higher transduction efficiency than VSV-G and 6.7-fold higher efficiency than RD114/TR at an MOI of 5 .

• Canine CD34+ Cells: Transduction efficiency was similar between Cocal and VSV-G pseudotypes, while both were approximately 4.3-fold more efficient than RD114/TR pseudotypes .

• Long-term Repopulating Cells: In competitive repopulation studies in a clinically relevant non-human primate model, Cocal-pseudotyped vectors demonstrated efficient transduction of long-term repopulating cells, indicating their suitability for HSC gene therapy applications .

These findings collectively suggest that Cocal G represents an excellent alternative pseudotype for HSC gene therapy applications, particularly for preclinical studies in non-human primate models.

Which antibodies recognize Cocal virus glycoprotein and what are their binding epitopes?

Several antibodies have been characterized for their ability to recognize Cocal virus glycoprotein, with distinct binding epitopes and cross-reactivity patterns:

• 8G5F11 (Cross-reactive): This commercially available monoclonal antibody binds to and neutralizes G proteins from three strains of VSV (Indiana, New Jersey, and Alagoas) as well as Cocal and Maraba viruses . The epitope for 8G5F11 has been mapped to amino acid residues 257-259 (DKD) on VSV-G Indiana, a region that undergoes conformational changes during the G protein's transition to its post-fusion structure. The antibody likely neutralizes vesiculovirus G proteins by inhibiting these conformational changes .

• IE9F9 (VSV-G Indiana-specific): This monoclonal antibody binds to and neutralizes only VSV-G Indiana but not other vesiculovirus G proteins including Cocal G . Its epitope has been mapped near the receptor binding site (involving amino acid residues 352-353 and 356-358), and it competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSV-G Indiana, explaining its mechanism of neutralization .

• VSV-Poly (Polyclonal): This antibody can detect multiple vesiculovirus G proteins including Cocal G in their extracellular domains .

• P5D4 (Intracellular domain): This antibody recognizes the intracellular domains of both VSV-G and Cocal G proteins .

Understanding these antibody interactions is crucial for developing detection methods, neutralization assays, and for understanding the antigenic relationships between different vesiculovirus G proteins.

How can epitope mapping of Cocal G be performed to characterize antibody binding sites?

Epitope mapping of Cocal G can be accomplished using several complementary approaches as demonstrated in the literature:

• Chimeric G Protein Construction: Creating chimeras between Cocal G and other vesiculovirus G proteins (particularly VSV-G Indiana) has been successfully used to narrow down antibody binding regions. By systematically swapping domains between G proteins with different antibody binding properties, researchers identified the amino acid region 137-369 as containing key epitopes for antibodies like 8G5F11 and IE9F9 .

• Site-Directed Mutagenesis: After identifying candidate regions through chimeric approaches, site-directed mutagenesis targeting specific amino acids can pinpoint exact binding determinants. For instance, mutations of amino acids 257-259 (DKD) in VSV-G Indiana dramatically reduced 8G5F11 binding, while introducing these residues into Cocal G enabled binding .

• Flow Cytometry-Based Binding Assays: Expressing mutant G proteins in mammalian cells and quantifying antibody binding by flow cytometry provides a rapid method to assess the impact of specific mutations on antibody recognition .

• Western Blot Analysis: Complementing flow cytometry with western blot analysis can confirm binding patterns under denaturing conditions, helping distinguish conformational from linear epitopes .

• Neutralization Assays: Testing the ability of antibodies to neutralize lentiviral vectors pseudotyped with mutant G proteins helps correlate specific amino acid changes with functional neutralization .

These methodologies have successfully identified key determinants for antibody binding and neutralization, such as the DKD motif (amino acids 257-259) for 8G5F11 binding and the LSR and AA regions (amino acids 356-358 and 352-353, respectively) for IE9F9 binding .

What advantages does Cocal G offer for gene therapy applications compared to VSV-G?

Cocal G offers several distinct advantages for gene therapy applications compared to the widely used VSV-G:

• Enhanced Resistance to Human Serum Inactivation: Cocal-pseudotyped vectors demonstrate significantly higher resistance to neutralization by human serum in the majority of individuals tested . This is particularly valuable for in vivo gene therapy applications where serum neutralization can severely limit vector efficacy.

• Improved Transduction of Non-human Primate Cells: Cocal G shows enhanced transduction efficiency in multiple non-human primate cell types, particularly in CD34+ hematopoietic stem cells, where it achieved 3.2-fold higher efficiency than VSV-G . This makes it particularly valuable for preclinical testing in non-human primate models.

• Lower Plasmid Requirements: Due to human codon optimization, Cocal G requires approximately half the amount of plasmid DNA (3 μg vs. 6 μg) during vector production compared to VSV-G, potentially improving production economics .

• Antigenic Distinctness: Cocal G is serologically distinct from VSV-G Indiana , providing an alternative pseudotype option for patients who may have pre-existing immunity to VSV-G or for sequential gene therapy treatments to avoid immune responses against the vector envelope.

• Broad Tropism: Like VSV-G, Cocal G maintains a broad cell tropism, making it suitable for diverse target cell populations .

These advantages collectively position Cocal G as a valuable alternative to VSV-G for various gene therapy applications, particularly those targeting hematopoietic stem cells and applications requiring in vivo administration.

How can Cocal G be engineered to enhance specific properties for research applications?

Several engineering approaches can be employed to enhance Cocal G properties for specific research applications:

• Codon Optimization: Human codon optimization has already been demonstrated to enhance expression efficiency, requiring less plasmid DNA during vector production . Further codon optimization for specific expression systems or target species could further improve performance.

• Targeted Mutagenesis: Based on epitope mapping studies, specific mutations in Cocal G could be introduced to alter antibody binding properties. For example, modifying the DKD motif (amino acids 257-259) could alter interactions with neutralizing antibodies like 8G5F11 .

• Domain Swapping: Creating chimeric proteins between Cocal G and other vesiculovirus G proteins could generate novel variants with combined advantageous properties. This approach has already been used successfully for epitope mapping and could be extended to functional engineering .

• Glycosylation Site Modification: Since Cocal G contains two potential N-linked glycosylation sites , modifying these sites could alter protein stability, immunogenicity, or receptor binding properties.

• Fusion with Targeting Ligands: Adding tissue-specific targeting ligands to Cocal G could enhance specificity for particular cell types while maintaining its favorable properties like serum resistance.

• pH-dependent Fusion Optimization: Since Cocal G induces cell fusion at acidic pH , engineering the pH threshold for fusion activity could enhance transduction efficiency in specific cellular environments.

These engineering approaches could be combined to create custom Cocal G variants optimized for specific research or therapeutic applications, building on the already favorable natural properties of this glycoprotein.

What are common challenges in expressing functional Cocal G and how can they be addressed?

Researchers working with recombinant Cocal virus glycoprotein may encounter several challenges that can be addressed with specific methodological approaches:

• Protein Misfolding: As a complex transmembrane glycoprotein, Cocal G may experience misfolding issues during recombinant expression.

  • Solution: Use mammalian expression systems rather than bacterial systems to ensure proper post-translational modifications and folding. Including molecular chaperones or expressing at lower temperatures (30-32°C) may improve folding efficiency.

• Cytotoxicity: Overexpression of viral envelope proteins can be toxic to producer cells.

  • Solution: Utilize inducible expression systems or optimize transfection conditions to balance expression levels and cytotoxicity. Adding sodium butyrate (1-5 mM) during production can enhance expression while managing toxicity.

• Protein Degradation: Rapid turnover of expressed protein can reduce yields.

  • Solution: Add protease inhibitors during purification and consider adding stabilizing agents. Optimizing codon usage for the expression system can also improve protein stability.

• Verification of Functionality: Ensuring the expressed protein maintains fusion activity.

  • Solution: Employ pH-dependent cell fusion assays to confirm functionality. Using protamine sulfate (8 μg/ml) during transduction assays has been shown to enhance transduction approximately sevenfold .

• Serum Effects During Testing: Serum components can interfere with functional assays.

  • Solution: Carefully control serum conditions during functional testing, and consider using serum-free media for critical comparisons between different G protein variants.

How can researchers optimize transfection protocols for producing high-titer Cocal G-pseudotyped lentiviral vectors?

Based on research findings, the following optimized protocol can be used to generate high-titer Cocal G-pseudotyped lentiviral vectors:

• Plasmid Ratios: Use 3 μg of human codon-optimized Cocal G envelope plasmid in standard transient transfection protocols, compared to 6 μg for VSV-G or 9 μg for RD114/TR envelopes . Optimize the ratio of envelope plasmid to packaging plasmids for specific vector backbones.

• Producer Cell Line: HEK293T cells are the standard for lentiviral vector production. Maintain cells at low passage number and 70-80% confluence at the time of transfection for optimal results.

• Transfection Method: Calcium phosphate precipitation provides cost-effective transfection for large-scale production, while lipid-based transfection reagents may offer more consistent results for smaller scales.

• Media Conditions: Harvest vector-containing supernatant at 48-72 hours post-transfection. Consider adding sodium butyrate (1-10 mM) 8-16 hours post-transfection to enhance expression.

• Concentration Methods: Cocal G-pseudotyped vectors, like VSV-G pseudotypes, are stable enough for concentration by ultracentrifugation (typically 50,000-100,000g for 2 hours) or tangential flow filtration.

• Enhancing Transduction: Include protamine sulfate at 8 μg/ml during transduction to enhance efficiency approximately sevenfold . Alternatively, polybrene (8 μg/ml) can be used as a transduction enhancer.

• Storage Conditions: Store produced vectors at -80°C in small aliquots to avoid freeze-thaw cycles. For short-term storage (1-2 weeks), 4°C is acceptable with minimal loss of titer.

• Quality Control: Verify vector titer using appropriate target cells and include both Cocal G and VSV-G pseudotyped vectors as controls for comparison of relative efficacy.

By following these optimized protocols, researchers can reliably produce high-titer Cocal G-pseudotyped lentiviral vectors suitable for diverse experimental and potential clinical applications.