Rabies Virus Vaccine：Revealing Potential Vaccine Targets
Introduction: Reasons For Studying The 3D Structure of Rabies Vaccine
Rabies is a vaccine-preventable zoonosis. Once clinical symptoms occur, rabies is almost 100 % fatal. Rabies vaccine was mainly inactivated by cell culture, and PEP/PrEP was confirmed to be effective. Adjuvant-free, intramuscular or intradermal simplified program is feasible. The rabies vaccine for human use is composed of inactivated viruses. Although the vaccine has a complete protective effect in the short term (6 months to 1 year after vaccination), it does not cause long-term or lifelong immunity. In the human population, the level of neutralizing antibodies induced by rabies vaccines usually decreases 1 to 5 years after vaccination. Scientific studies have shown that the deformation protein of rabies virus is one of the reasons why rabies vaccines cannot provide long-term protection. Rabies virus glycoprotein (RABV-G) is the only protein expressed on the surface of rabies virus, which means that it will be a target for neutralizing antibodies during infection.
Why Does the Deformed Rabies Glycoprotein Affect the Vaccine Efficacy?
In the process of virus invasion, RABV-G needs to switch back and forth between prefusion and postfusion conformations, and can dynamically balance different aggregation states such as trimers and monomers. This structural plasticity of folding-unfolding-refolding makes multiple key neutralizing epitopes in a sometimes non-existent exposure state: in some conformations, the epitopes are fully exposed and can be recognized by antibodies; in other conformations, the same epitope may be buried, distorted or significantly shifted in spatial position, thus escaping the binding of existing antibodies.
From an immunological point of view, human neutralizing antibodies are mostly lock-and-key binding with precise pairing of specific conformational epitopes. If the glycoprotein is often dissociated from a stable pre-fusion trimer into a monomer or transferred into a post-fusion conformation, the part of the antibody population induced by the vaccine that really targets the protective pre-fusion conformational epitope will be diluted; at the same time, the body will also produce a large number of antibodies against non-neutralizing or low-neutralizing conformations. Although these antibodies can bind to antigens, they cannot effectively block the entry of viruses. In the long run, this conformational heterogeneity not only reduces the quality of initial neutralization titers, but also affects the selection and maintenance of memory B cells, making immune memory more inclined to identify unstable or non-ideal conformations, which is manifested as a decrease in the persistence and protection breadth of vaccine immune response. Therefore, modern rabies vaccine design is increasingly inclined to structurally stabilized pre-fused trimer immunogens to lock the exposure state of key neutralizing epitopes and reduce the interference of conformational drift on protective immunity.
Fig.1 Schematic diagram of the glycoprotein domain of rabies virus.1
New Breakthrough: Construction of Rabies Glycoprotein 3D Image
The basic skeleton of 3D model: Typical "bullet-shaped" capsid: length of about~180nm, diameter of~75nm; the outer layer is a lipid envelope with~400 trimer G protein spikes; it is the M protein layer and spiral RNP (N-RNA as the core, accompanied by P, L). When modeling, the skeleton can be layered according to "coating→M layer→RNP spiral axis→one end flat and one end cone". Atomic/sub-nanometer resolution key components
Prefusion conformation of G protein (RABV-G) trimer: Ng. analyzed the structure of the pre-fused trimer RABV-G and its complexes with two protective antibodies, defined the neutralizing epitopes and reported the key mutations that can stabilize the pre-fused states such as H270P and H261L, which provided a direct template for the structural design of vaccines. For example, the pre-fused trimer was used as a spike grid with a density of about 350-450/particle, and the spikes in the flat end region were rare/missing.
L-P polymerase complex (3D architecture of transcription/replication machine):
Horwitz et al.gave the cryo-EM structure of RABVL-P complex, and analyzed the spatial organization of multi-enzyme functional domains such as cap/methylation/polyadenylation and the localization of P; when doing RNP axis details or animation, L-P can be attached to the trajectory of the N-RNA spiral groove and the multi-site anchoring of P can be maintained.
Note: The above two components are the atomic-level results that most directly affect 3D expression and immune design in recent years: G is responsible for the true morphology and epitope of the appearance "spine"; l-P to determine the relationship between the appearance and relative position of the internal replicase complex.
The Research Significance of 3D Model and the Difficulties That Need to Be Broken Through in the Future
The great significance of studying the structure of 3D rabies vaccine is to turn "visible shape" into "designable target". By analyzing the pre-fusion conformation of RABV-G trimer, the neutralizing epitopes can be accurately defined, and the immunogen is locked in the pre-fusion rather than the post-fusion state induced by low pH, thereby reducing the non-neutralizing antibody induced by false epitopes and improving the persistence of rabies vaccine. Secondly, the three-dimensional structure reveals the effect of spike density and arrangement on B cell cross-linking and complement deposition, which helps to optimize VLP/nanoparticle display. Moreover, the 3D model study of rabies vaccine will be expected to combine the L-P complex and the N-RNA nucleocapsid framework to support the rational screening of broad-spectrum neutralizing antibodies and small molecule inhibitors.
However, there are still some unsolved problems in the study of the 3D structure of rabies vaccine. For example, the intrinsic instability of the pre-fusion state, exogenous expression often loses trimers or transfers to the post-fusion state, which requires more techniques to determine the ideal environment for mutation, correct glycosylation, and membrane environment. In addition, although high-density display increases immunogenicity, it is easy to aggregate, enhance complement or affect rheology, which provides a technical test for industrial manufacturing vaccines. Industrial amplification needs to find the best point between particle size, density and stabilizer/adjuvant. In addition, the adjuvant of rabies vaccine also plays a significant role. The quantitative effects of aluminum salt/oil emulsion on conformational stability and immune bias (Th1/Th2, IgG subclasses) need to be systematically characterized. Researchers need to truly transform structural visualization into vaccine engineering, capture more images of the genus rabies virus and neutralizing antibodies, so as to hopefully provide more roles for revealing the common antibody targets of rabies virus.
In the future: the research on the three-dimensional structure of rabies vaccine will focus on the fine analysis of the in-situ conformation of G protein and the epitope of antibody. Combined with cryo-electron microscopy, hydrogen-deuterium exchange mass spectrometry and molecular dynamics, the conformational transformation path before and after fusion can be constructed, and the broad-spectrum neutralization and key mutation points can be locked, which provides a basis for the development of vaccines with long-lasting and stable efficacy. At the same time, the structural guidance was used to design stable trimers, epitope-focused immunogens and VLP display platforms, and the effects of glycosylation and lipid microenvironment on immunogenicity were evaluated to ensure the stability of vaccine efficacy. At the same time, AI assisted directed evolution and mRNA/glandular vector expression strategies to optimize vaccine thermal stability.
Creative Biolabs accelerates infectious disease research with specialized solutions for Rabies Virus (RABV) intervention. We offer a robust portfolio of high-purity RABV antigens, including recombinant glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and virus-like particles (VLPs), engineered for optimal bioactivity in diagnostics and basic research.
Our capabilities include:
- Vaccine Design & Engineering: Development of various vaccine platforms, including recombinant subunit vaccines, DNA/mRNA Vaccines, and virus-like particle (VLP) vaccines.
- Adjuvant Selection & Formulation: Optimization of adjuvant strategies to enhance the immunogenic response of RABV candidates.
- Preclinical Assessment: Comprehensive in vitro and in vivo testing, including immunogenicity evaluation, neutralizing antibody titer assays (FAVN/RFFIT), and safety profiling.
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Reference
- Fernando, Bastida-Gonzalez, et al. "Predicted 3D model of the rabies virus glycoprotein trimer." BioMed research international 2016.1 (2016): 1674580. CC BY 4.0. https://doi.org/10.1155/2016/1674580
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