Untreated rabies infection is almost 100 percent fatal, killing between 50000 and 60000 people a year, many of whom are children. Rabies vaccine for human use consists of inactivated virus, which has completed protective effect only in the short term (6 months to one year after vaccination) and cannot induce lifelong immunity. In humans, vaccine-induced neutralizing antibody levels usually decrease in 1 to 5 years after vaccination, so frequent vaccinations are needed to maintain neutralizing antibody titers and provide protection against rabies infection. In most low-income countries where rabies death cases are high, it is difficult to afford regular vaccination and treatment after exposure to the virus. Therefore, the research and development of long-acting rabies vaccine is urgent and critical.
Rabies virus glycoprotein (RABV-G) is the only protein exposed to the virus surface and is the target of vaccine-induced neutralizing antibodies. On the surface of the virus, the structure of RABV-G is heterogeneous, with only a part being a recognizable trimer. The structural heterogeneity may affect the production of neutralizing antibodies against the fourth epitope and may lead to a shorter time of immune response after vaccination.
In a recent study, a team led by the La Jolla Institute of Immunology and the Pasteur Institute reported the structure of a trimeric wild-type RVA122 that binds to human antibody RABV-G, which can be used to guide improved vaccine design and identify therapeutic drug targets. The related research results are published on Science Advances under the title “Structure of the rabies virus glycoprotein trimer bound to a prefusion-specific neutralizing antibody”.
Scientists cannot accurately answer why rabies vaccines do not provide long-term protection, but the heterogeneity of RABV-G structure is a key issue. Like a Swiss Army knife, the sequence of RABV-G can be unfolded and flipped upwards as needed. The glycoprotein can move back and forth between pre-fusion and post-fusion forms, or it may disintegrate and change from trimer structure to monomer structure.
This change seems to provide an invisibility cloak for rabies virus, because human antibodies usually recognize only a single site on a protein, but when the protein hides or moves, the antibodies cannot accurately recognize them, and thus are unable to produce an effective immune response.
In this study, the researchers combined the glycoprotein RABV-G with the monoclonal antibody RVA122 to identify the sites in the structure of the virus that are vulnerable to antibody attack. RVA122 is the type of antibody needed for rabies immunization and can effectively neutralize rabies virus. The combination with RVA122 increased the proportion of RABV-G trimer visible through cryogenic electron microscopy by more than 30 times, made high-resolution reconstruction possible, and locked RABV-G in the pre-fusion conformation, because RVA122 may neutralize rabies virus by inhibiting the conformational transition of RABV-G to fusion.
Using a cryogenic electron microscope, the researchers captured a high-resolution image of the structure of RABV-G, which highlights the natural morphology of two key parts of the viral structure, the fusion peptide. When the RABV-G trimer is not anchored to the micelle or cell membrane, the fusion peptides are either disordered or interact with each other. These results suggest that in addition to driving the fusion of virus and cell membrane after endosomal acidification, the fusion peptide also affects the extracellular domain binding and trimerization of glycoprotein.
In order to determine whether the fusion peptide also affects the conformation and stability of full-length RABV-G, the researchers expressed the fusion peptide mutant as a full-length glycoprotein, including transmembrane domain and cytoplasmic tail, and analyzed it by flow cytometry and immunofluorescence. All full-length versions of RABV-G mutants were well expressed and easily reached the cell surface, but the full-length proteins in the pre-fusion conformation of W121A mutants were significantly reduced. The results show that for full-length glycoproteins, W121 stabilizes the pre-fusion conformation by interacting with the membrane and prevents glycoproteins from transforming into alternative conformations. The interaction between W121 and viral membrane may also stabilize RABV-G on the surface of virions.
RABV-G is not only an important component of vaccine, but also a target for antibody therapy and potential antiviral drugs, but its structural heterogeneity may lead to suboptimal antibody response. Pre-fusion trimer RABV-G will be an ideal vaccine immunogen, but the presentation of this form is challenging because of its instability. This study reported the structure of the pre-fusion trimer RABV-G, demonstrated the importance of the fusion peptide for successful trimerization and conformational stability, and visualized effective and widely neutralized antibody epitopes, which will guide and improve the development of rabies vaccine and post-exposure treatment.
Through this study, scientists have a clearer understanding of the structure of rabies virus, which facilitates the research for the design and development of long-acting rabies vaccine and vaccine covering the entire genus of rabies virus. According to the researchers, the next step is to capture more images of rabies virus, its relatives and neutralizing antibodies. Scientists are trying to find several of these viral structures to reveal common antibody targets for rabies viruses.