Recombinant Irkut virus Glycoprotein G (G)

What is Irkut virus and where has it been isolated?

Irkut virus (IRKV) is a member of the Lyssavirus genus within the Rhabdoviridae family. It has been isolated from bats in different geographic locations, including China and Russia. A notable isolate, IRKV-THChina12, was identified from the brain tissue of an adult male Murina leucogaster bat in Jilin Province, China. This isolate shares high sequence identity (>98%) with IRKV isolate Ozernoe, which was obtained from a human case of rabies following a bat bite in Russia in 2007 . Another human case was documented in Russia, demonstrating the zoonotic potential of this virus. IRKV has been confirmed to have high pathogenicity in experimental mice, with inoculated suckling mice developing neurological signs between days 7-9 post-inoculation in initial passages, with the incubation period reducing to 3-4 days in subsequent passages .

How is Irkut virus genetically related to other lyssaviruses?

Irkut virus belongs to phylogroup I of lyssaviruses, which includes the classical rabies virus (RABV). Phylogenetic analysis based on full nucleoprotein (N) sequences clearly demonstrated that IRKV-THChina12 clustered with other IRKV isolates . The N gene nucleotide identities provide unequivocal separation of all lyssavirus species with an identity threshold of 82% .

IRKV-THChina12 shares 92.4%/98.9% (nucleotide) and 92.2%/98.8% (amino acid) identity with the N protein of previously identified IRKV isolates. In comparison, the identity with other lyssaviruses ranges from 70.0%–79.2% (nucleotide) and 70.2%–79.1% (amino acid) . This genetic relationship has significant implications for vaccine development, as current rabies vaccines provide sufficient protection against phylogroup I lyssaviruses but limited protection against more divergent lyssaviruses .

What is the genomic organization of Irkut virus?

The genome of IRKV-THChina12 is 11,980 nucleotides in length with a G+C content of 44.72%, similar to the G+C content of other IRKV isolates . The genomic organization follows the typical pattern of lyssaviruses with the following structure:

• 70-nt 3′ untranslated region (UTR)

• 1356-nt nucleoprotein (N)

• 92-nt N-P UTR

• 897-nt phosphoprotein (P)

• 83-nt P-M UTR

• 609-nt matrix protein (M)

• 214-nt M-G UTR

• 1575-nt glycoprotein (G)

• 569-nt G-L UTR

• 6384-nt large protein (L)

• 131-nt 5′ UTR

The coding sequences (CDS) and UTR sequences of IRKV-THChina12 are located at the same positions as in other IRKV isolates, with no variation in length. Intragenotypic comparison of the five structural proteins shows different percent identity orders between IRKV isolates, which doesn't follow the general order (N>L>M>G>P) observed in many lyssaviruses .

What are the key structural features of Irkut virus glycoprotein G?

The glycoprotein G of IRKV has several distinct structural features compared to RABV. Analysis of the deduced amino acid sequences revealed that while RABV contains N-linked glycosylation sites at positions 37, 158, 247, and 319, IRKV only has two such sites at positions 247 and 319 .

Recent structural studies of lyssavirus glycoproteins have revealed that the 'fusion-loop substitution' represents a universal strategy for preparing the G-ecto proteins of lyssaviruses . This approach has facilitated the crystallization of glycoproteins and determination of their structures in both pre- and post-fusion states.

How is recombinant Irkut virus glycoprotein G produced?

The production of recombinant IRKV glycoprotein G typically involves molecular cloning of the glycoprotein gene into an appropriate expression vector followed by expression in a suitable host system. One validated approach is the use of adenoviral expression systems. For instance, researchers have generated recombinant human adenovirus type 5 (rHAd5)-THChina12-G using an E1- and E3-deleted cytomegalovirus (CMV) adenoviral expression system (RAPAd) .

The detailed procedure involves:

• Insertion of the IRKV glycoprotein gene into multiple cloning sites of a shuttle vector (e.g., pacCMVK-NpA)

• Co-transfection of HEK-293 cells with the shuttle vector and a backbone vector (e.g., pacAd5 9.2–100, which is devoid of the left inverted terminal repeat and packaging signal) using a transfection reagent like Lipofectamine

• Passaging the transfected cells until a typical cytopathic effect is observed

• Verification of glycoprotein expression using appropriate antibodies, such as Light Diagnostics rabies FITC-globulin conjugate

This methodology produces recombinant viral vectors expressing IRKV glycoprotein G that can be used for various research applications, including immunogenicity studies and vaccine development.

What expression systems are suitable for producing soluble Irkut virus glycoprotein ectodomain?

For structural and functional studies, producing soluble forms of the glycoprotein ectodomain (G-ecto) is often preferable. Research has shown that 'fusion-loop substitution' represents a universal strategy for preparing the G-ecto proteins of lyssaviruses with favorable solution behaviors when analyzed by gel filtration .

While the search results don't provide specific details for IRKV G-ecto production, the methods used for other lyssavirus glycoproteins likely apply. These typically involve:

• Cloning the ectodomain sequence (excluding the transmembrane and cytoplasmic domains) into an expression vector with an appropriate secretion signal

• Introduction of fusion-loop substitutions to improve protein stability and solubility

• Expression in mammalian cells (e.g., HEK293) or insect cells

• Purification using affinity chromatography followed by size-exclusion chromatography

The successful crystallization of lyssavirus glycoproteins at specific pH conditions (e.g., IKOV-G-ecto at pH 8.3) suggests that buffer conditions are critical for maintaining the native conformation of these proteins .

How can the antigenic properties of Irkut virus glycoprotein G be analyzed?

The antigenic properties of IRKV glycoprotein G can be analyzed through various immunological and molecular techniques:

• Serological cross-reactivity assays: These involve testing the reactivity of antibodies raised against RABV or other lyssaviruses against IRKV glycoprotein, and vice versa. This approach assesses the extent of antigenic similarity and potential cross-protection .

• Antigenic site swapping: This technique involves swapping defined antigenic sites between different lyssavirus glycoproteins (e.g., between phylogroup-I and -II glycoproteins) and assessing the impact on antibody recognition and neutralization. This approach has been used to identify immunodominant antigenic sites and their contribution to virus neutralization .

• Virus neutralization assays: These assays measure the ability of antibodies to neutralize virus infectivity. They can be performed using live viruses or pseudotype viruses (PTVs) expressing IRKV glycoprotein. The results, expressed as virus-neutralizing antibody (VNA) titers, provide quantitative data on the protective potential of antibodies against IRKV .

• In vivo protection studies: These involve challenging immunized animals (e.g., hamsters) with IRKV and assessing survival rates. This approach provides direct evidence of protective immunity against virus infection .

The following table shows an example of VNA titers and protection rates from a study comparing different immunization strategies against IRKV and RABV:

Note: Different groups represent different immunization strategies .

What approaches are used for structural characterization of Irkut virus glycoprotein G?

Structural characterization of lyssavirus glycoproteins, including IRKV glycoprotein G, typically employs several complementary techniques:

• X-ray crystallography: This technique has been successfully used to determine the three-dimensional structure of lyssavirus glycoproteins in both pre- and post-fusion states. For example, IKOV-G-ecto protein was crystallized at pH 8.3 and its structure was determined at 2.9-Å resolution . For IRKV, similar approaches could be applied.

• Cryo-electron microscopy (cryo-EM): This technique can provide structural information at near-atomic resolution without the need for crystallization.

• Molecular modeling and sequence analysis: Comparative analysis of glycoprotein sequences from different lyssaviruses helps identify conserved and variable regions, including antigenic sites and potential N-linked glycosylation sites .

• Protein engineering: Fusion-loop substitution has been shown to be a universal strategy for preparing stable lyssavirus G-ecto proteins suitable for structural studies . This approach likely applies to IRKV glycoprotein as well.

• pH-dependent conformational changes: Lyssavirus glycoproteins undergo conformational changes in response to pH that are critical for the fusion process. Characterizing these changes provides insights into the mechanism of virus entry. Proteins can be crystallized at different pH values to capture different conformational states .

Understanding the structural details of IRKV glycoprotein G is essential for rational vaccine design and development of therapeutic antibodies.

How do antigenic sites in Irkut virus glycoprotein G compare to those in rabies virus?

Detailed comparison of the deduced amino acid sequences revealed significant differences in several key antigenic sites between IRKV and RABV glycoproteins. Specifically:

• Antigenic site I (amino acids 226 to 231): Different in IRKV compared to RABV

• Antigenic site II (amino acids 34 to 42 and 198 to 200): Different in IRKV compared to RABV

• Antigenic site III (amino acids 330 to 338): Different in IRKV compared to RABV

• Antigenic site G1 (amino acids 242 to 243): Different in IRKV compared to RABV

• Antigenic site IV (amino acid 251): Fully conserved

• Antigenic site G5 (amino acids 261 to 264): Fully conserved

• Antigenic site VI (amino acid 264): Fully conserved

• B-cell epitope (amino acids 14 to 19): Fully conserved

These differences in antigenic sites likely contribute to the limited cross-neutralization observed between IRKV and RABV. Studies involving antigenic site swapping between phylogroup-I and -II glycoproteins have shown that site II (particularly amino acids 34–42 and 198–200) appears to be immunodominant . Pseudotype viruses presenting a phylogroup-I glycoprotein containing phylogroup-II antigenic site II were efficiently neutralized by antibodies raised against phylogroup-II PTV, while those with site IV swaps were poorly neutralized .

What is known about the pre- and post-fusion states of lyssavirus glycoproteins?

While the search results don't provide specific information about the pre- and post-fusion states of IRKV glycoprotein, they do mention recent structural studies of other lyssavirus glycoproteins that are likely applicable:

Researchers have successfully crystallized the IKOV-G-ecto protein at pH 8.3 and determined its structure at 2.9-Å resolution. In the crystallographic asymmetric unit, six IKOV-G molecules, which are of essentially the same structure, were observed to assemble into two IKOV-G trimers . The individual domains and secondary structural elements of IKOV-G showed similar topological structure to RABV-G.

Additionally, structures of both the pre-fusion state of IKOV-G and the post-fusion state of MOKV-G have been determined . These structures provide valuable insights into the conformational changes that lyssavirus glycoproteins undergo during the fusion process, which is critical for virus entry into host cells.

The fusion-loop substitution strategy has proven to be a universal approach for preparing stable G-ecto proteins of lyssaviruses suitable for structural studies . This approach could be applied to IRKV glycoprotein to determine its structure in different conformational states.

Why is understanding cross-neutralization between lyssaviruses important?

Understanding cross-neutralization between lyssaviruses is crucial for several reasons:

• Vaccine efficacy assessment: Current rabies vaccines confer protection against all reported phylogroup I lyssaviruses, including RABV, but provide little or no protection against more divergent lyssaviruses . Evaluating cross-neutralization helps determine whether existing vaccines can protect against emerging lyssaviruses like IRKV.

• Epitope identification: Cross-neutralization studies help identify protective epitopes that could be targeted for vaccine development. For instance, antigenic site II appears to be immunodominant based on site-swapping experiments .

• Risk assessment: By understanding the extent of cross-neutralization, researchers can assess the risk posed by newly identified lyssaviruses to public health.

• Therapeutic antibody development: Knowledge of cross-neutralization patterns guides the development of broadly neutralizing antibodies that could protect against multiple lyssaviruses.

Research has shown demonstrable intra- but limited inter-phylogroup cross-neutralization among lyssaviruses . For IRKV specifically, rabies biologics available in the United States elicited only partial protection, highlighting the need for further investigation into epitopes that dictate a neutralizing response against divergent lyssaviruses .

How effective are current rabies biologics against Irkut virus infection?

In experimental studies, hamsters immunized with human rabies vaccine and human rabies immune globulin (HRIG) showed complete protection (10/10 survival) when challenged with RABV BD06, but only limited protection (2/10 survival) when challenged with IRKV-THChina12 . This significant difference in protection highlights the need for the development and evaluation of new biologics specifically targeting IRKV.

Interestingly, combining HRIG with experimental vaccines based on recombinant adenoviruses expressing IRKV glycoprotein (rHAd5-THChina12-G) improved protection against IRKV challenge (6/10 survival) . Even better protection (8/10 survival) was achieved when HRIG was combined with interferon-α2a (IFN-α2a) . These results suggest potential strategies for improving protection against IRKV infection.

What approaches are being explored for developing vaccines against Irkut virus?

Several approaches are being explored for developing vaccines against IRKV, with a focus on the viral glycoprotein G as the primary immunogen:

• Recombinant viral vector vaccines: Researchers have generated recombinant human adenovirus type 5 (rHAd5) expressing IRKV glycoprotein (rHAd5-THChina12-G). This approach leverages the adenovirus's ability to efficiently deliver the glycoprotein gene to host cells, resulting in robust expression and immune responses .

• Glycoprotein subunit vaccines: While not explicitly mentioned in the search results for IRKV, this approach typically involves the production of purified glycoprotein or its immunogenic domains for direct immunization.

• Chimeric/modified glycoprotein vaccines: This approach involves creating chimeric glycoproteins by swapping antigenic sites between different lyssavirus glycoproteins or introducing specific modifications to enhance immunogenicity and cross-protection .

• Combination therapies: Studies have explored combining vaccines with immunomodulators like interferon-α2a (IFN-α2a) or passive immunotherapy with human rabies immune globulin (HRIG) to enhance protection .

The development of these vaccine candidates is guided by detailed understanding of the antigenic sites within the glycoprotein and their role in eliciting neutralizing antibodies.

What protection levels do experimental vaccines provide against Irkut virus?

Experimental vaccines have shown varying levels of protection against IRKV challenge in animal models. The following data from hamster challenge studies illustrate the protective efficacy of different vaccination strategies:

• Traditional rabies vaccine: Hamsters immunized with human rabies vaccine and HRIG showed only 20% survival (2/10) when challenged with IRKV-THChina12, despite showing 100% survival (10/10) against RABV BD06 challenge .

• Recombinant adenovirus expressing IRKV glycoprotein: When HRIG was combined with rHAd5-THChina12-G (expressing IRKV glycoprotein), protection against IRKV-THChina12 challenge improved to 60% survival (6/10) .

• Recombinant adenovirus expressing RABV glycoprotein: HRIG combined with rHAd5-BD06-G (expressing RABV glycoprotein) provided 40% protection (4/10) against IRKV-THChina12 challenge .

• Interferon-α2a: Surprisingly, IFN-α2a alone provided 40% protection (4/10) against both RABV BD06 and IRKV-THChina12 .

• HRIG + Interferon-α2a: The combination of HRIG and IFN-α2a showed the best protection against IRKV-THChina12, with 80% survival (8/10) .

These results demonstrate that experimental recombinant vaccines containing IRKV glycoproteins induced more reliable protection against IRKV than against RABV infection. The data also suggest that combination therapies may offer the best approach for protecting against IRKV infection.

How does antigenic site swapping influence vaccine design for lyssaviruses?

Antigenic site swapping is a powerful approach for understanding the immunological relevance of specific regions within the glycoprotein and for designing improved vaccines. Research has shown that:

• Identification of immunodominant sites: Pseudotype viruses (PTVs) presenting a phylogroup-I glycoprotein containing phylogroup-II antigenic sites were differentially neutralized by antibodies raised against phylogroup-II PTV. Site II (IIb, aa 34–42 and IIa, aa 198–200)-swapped PTVs were efficiently neutralized, while site IV-swapped PTV was poorly neutralized . This indicates that site II is immunodominant.

• Enhanced cross-protection: By swapping immunodominant antigenic sites between different lyssavirus glycoproteins, it may be possible to create chimeric glycoproteins that elicit broader neutralizing antibody responses, potentially providing protection against multiple lyssavirus species.

• Rational vaccine design: Understanding which antigenic sites are responsible for protective immunity guides the rational design of vaccines. For instance, vaccines could be designed to focus the immune response on conserved antigenic sites that elicit cross-neutralizing antibodies.

• Functional constraints: Live lyssaviruses containing antigenic site-swapped glycoproteins have demonstrated that specific residues within the lyssavirus glycoprotein dictate functionality and enable differential neutralizing antibody responses . These functional constraints must be considered when designing modified glycoproteins for vaccines.

This approach could be particularly valuable for developing vaccines against IRKV, as it could help identify the most promising antigenic sites to target for inducing protective immunity.

What are the potential approaches for improving protection against Irkut virus infection?

Based on the research findings, several approaches show promise for improving protection against IRKV infection:

• Develop IRKV-specific vaccines: Experimental recombinant vaccines containing IRKV glycoproteins induced more reliable protection against IRKV than against RABV infection . This suggests that IRKV-specific vaccines may be necessary for optimal protection.

• Combination therapies: The combination of HRIG and IFN-α2a provided the best protection (80% survival) against IRKV-THChina12 challenge in hamsters . This approach leverages both passive immunotherapy and immunomodulation.

• Chimeric glycoproteins: Creating chimeric glycoproteins that incorporate protective epitopes from multiple lyssavirus species could potentially elicit broader protection against various lyssaviruses, including IRKV .

• Focus on immunodominant antigenic sites: Research has identified antigenic site II (amino acids 34–42 and 198–200) as immunodominant . Vaccines designed to focus the immune response on this site might elicit stronger neutralizing antibody responses.

• Improved adjuvants: While not specifically mentioned in the search results, the use of advanced adjuvants could enhance the immunogenicity of IRKV glycoprotein-based vaccines.

• Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) protocols: Development of specific PrEP and PEP protocols for IRKV is required to ensure sufficient protection in regions where this virus is present .

The development and evaluation of these approaches should be guided by a comprehensive understanding of the antigenic properties of IRKV glycoprotein and its interactions with the immune system.

How should virus neutralization assay results for Irkut virus be interpreted?

Virus neutralization assays measure the ability of antibodies to neutralize virus infectivity and are typically expressed as virus-neutralizing antibody (VNA) titers. When interpreting these results for IRKV:

• Correlation with protection: In general, higher VNA titers correlate with better protection. For instance, hamsters with a mean VNA titer of 3.50 ± 0.95 IU/ml showed 60% protection against IRKV-THChina12, while those with a titer of 1.10 ± 0.42 IU/ml showed only 20% protection .

• Cross-neutralization: VNA titers against IRKV should be compared with those against RABV to assess cross-neutralization. Limited cross-neutralization suggests the need for IRKV-specific vaccines.

• Antigenic distance: The interpretation should consider the antigenic distance between RABV and IRKV, as determined by in vitro tests . Greater antigenic distance generally correlates with reduced cross-neutralization.

• Threshold for protection: While specific thresholds for IRKV protection aren't defined in the search results, the World Health Organization considers VNA titers ≥0.5 IU/ml as indicative of adequate immunization against RABV. For IRKV, higher titers may be necessary for protection.

• Variability factors: Results may vary based on the virus strain, cell line, and assay methodology used. These factors should be considered when comparing results across different studies.

Researchers should interpret neutralization results in conjunction with in vivo protection studies whenever possible, as neutralizing activity in vitro doesn't always perfectly predict protection in vivo.

What factors influence the structural stability of recombinant Irkut virus glycoprotein G?

While the search results don't provide specific information on the structural stability of recombinant IRKV glycoprotein G, insights from studies on other lyssavirus glycoproteins suggest several factors to consider:

• pH sensitivity: Lyssavirus glycoproteins undergo conformational changes in response to pH, which are critical for the fusion process. IKOV-G-ecto protein was successfully crystallized at pH 8.3 , suggesting that neutral to slightly alkaline conditions may favor the pre-fusion conformation.

• Fusion-loop modifications: The 'fusion-loop substitution' strategy has been shown to be universal for preparing stable G-ecto proteins of lyssaviruses with favorable solution behaviors . This approach likely applies to IRKV glycoprotein as well.

• Glycosylation: IRKV glycoprotein contains N-linked glycosylation sites at positions 247 and 319 . Proper glycosylation is often critical for protein folding, stability, and function.

• Expression system: The choice of expression system (bacterial, insect, or mammalian cells) can significantly impact glycoprotein folding and post-translational modifications, affecting stability.

• Disulfide bonds: Lyssavirus glycoproteins contain multiple disulfide bonds that are essential for maintaining their tertiary structure. Ensuring proper disulfide bond formation during recombinant expression is crucial for stability.

• Buffer composition: The composition of the buffer (salt concentration, presence of stabilizing agents, etc.) can significantly influence glycoprotein stability, especially during purification and storage.

Understanding these factors is essential for producing stable recombinant IRKV glycoprotein G for structural studies, immunological assays, and vaccine development.

How can researchers troubleshoot issues in glycoprotein expression and purification?

While the search results don't specifically address troubleshooting for IRKV glycoprotein expression and purification, general principles for lyssavirus glycoproteins include:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Try different promoters or expression vectors

  • Consider using secretion signals optimized for the host system

  • Test different cell lines or expression conditions (temperature, induction time, etc.)

• Protein misfolding:

  • Express the protein in mammalian cells to ensure proper glycosylation and disulfide bond formation

  • Include molecular chaperones to assist protein folding

  • Try expressing only the ectodomain (G-ecto) without the transmembrane and cytoplasmic domains

  • Apply the 'fusion-loop substitution' strategy that has proven successful for other lyssavirus glycoproteins

• Aggregation during purification:

  • Optimize buffer conditions (pH, salt concentration, addition of stabilizers)

  • Include mild detergents for full-length glycoprotein purification

  • Use size-exclusion chromatography as a final purification step to remove aggregates

  • Consider adding glycerol or other stabilizing agents to the storage buffer

• Degradation:

  • Include protease inhibitors during purification

  • Optimize storage conditions (temperature, buffer composition)

  • Consider flash-freezing aliquots for long-term storage

• Verifying proper folding:

  • Assess binding to conformational antibodies

  • Perform functional assays (e.g., receptor binding if the receptor is known)

  • Use circular dichroism or other spectroscopic techniques to assess secondary structure

These troubleshooting approaches should be adapted to the specific challenges encountered during IRKV glycoprotein expression and purification.

What control measures should be implemented when working with Irkut virus?

Working with IRKV requires strict biosafety measures due to its pathogenicity. While specific biosafety guidelines for IRKV aren't detailed in the search results, general principles for working with lyssaviruses include:

• Biosafety level (BSL): Live IRKV should be handled in BSL-3 or BSL-4 facilities, depending on local regulations and risk assessment. IRKV has demonstrated high pathogenicity in experimental mice .

• Personal protective equipment (PPE): Appropriate PPE, including laboratory coats, gloves, and eye protection, is essential. For aerosol-generating procedures, respiratory protection may be required.

• Vaccination: Personnel should receive pre-exposure rabies vaccination, although it's important to note that current rabies vaccines provide only partial protection against IRKV .

• Alternatives to live virus: When possible, use safer alternatives such as:

  • Pseudotype viruses expressing IRKV glycoprotein

  • Recombinant glycoprotein for immunological studies

  • Molecular techniques that don't require live virus

• Animal experiments: Special precautions are needed for animal experiments involving IRKV, as demonstrated in studies where suckling mice developed neurological signs following inoculation .

• Decontamination procedures: Effective disinfection protocols should be established for equipment, surfaces, and waste materials.

• Incident response plan: A clear plan should be in place for dealing with potential exposures, including post-exposure prophylaxis options.

These control measures should be implemented in accordance with institutional and national biosafety guidelines, with regular risk assessments to ensure they remain appropriate for the work being conducted.