Self-assembling nanoparticle engineered from the ferritinophagy complex as a rabies virus vaccine candidate

Rabies, caused by the rabies virus (RABV), remains a significant public health concern, particularly in developing nations. Current rabies vaccines, while effective, often require multiple doses and may suffer from limitations in stability and immunogenicity. Ferritin-based nanoparticles are attractive vaccine platforms due to their inherent advantages, including the ability to present antigenic repetitive arrays analogous to viral surfaces, leading to high safety and biocompatibility. However, challenges such as particle heterogeneity, inefficient antigen presentation, and immunogenicity related to bulky protein adaptors have hampered their development. This study addresses these issues by designing a universal ferritin-based delivery platform inspired by the natural ferritinophagy complex. The complex involves the interaction between the human ferritin heavy chain (FTH1) and nuclear receptor coactivator 4 (NCOA4). Understanding the structural basis of this interaction is crucial for designing an efficient peptide adapter for the ferritin nanoparticle, which will ensure stable and high-affinity antigen presentation. The researchers chose rabies virus glycoprotein domain III (GDIII) as a model antigen to evaluate the efficacy and safety of this novel platform. The aim was to engineer a self-assembling rabies vaccine that would overcome limitations of existing approaches and provide a highly effective and safe vaccination strategy.

Extensive research has explored the use of ferritin nanoparticles as vaccine platforms. Several ferritin-based vaccine candidates have shown promise in preclinical and even early clinical trials. The advantages of using ferritin are multiple: high safety and biocompatibility, rapid production, broad applicability, excellent stability, and the capacity to elicit enduring and robust immune responses. However, many existing ferritin-based vaccine approaches face challenges including particle heterogeneity resulting from chemical conjugation of antigens, improper antigen folding caused by antigen-ferritin gene fusion, and unintended immune responses against protein adaptors used in Tag/Catcher systems. These limitations necessitate the development of novel strategies for efficient and safe antigen delivery using ferritin nanoparticles. The researchers aimed to improve on these prior approaches by focusing on the naturally occurring interaction between ferritin and its receptor during ferritinophagy for improved antigen presentation and vaccine efficacy. This natural interaction provides a template for designing a high-affinity peptide adapter with reduced immunogenicity compared to previous adapter systems.

The study employed a multi-faceted approach. Firstly, the researchers determined the atomic structure of the FTH1/NCOA4 complex at high resolution using cryo-EM, providing structural insights into their interaction. Based on this structure, they computationally designed a 16-amino acid peptide adapter (Fagy-tag) that exhibits significantly enhanced binding affinity to FTH1 compared to the wild-type NCOA4 peptide. The binding affinity of various peptides was assessed using ELISA and SPR. Next, they constructed a self-assembling rabies virus vaccine candidate by noncovalently conjugating the Fagy-tagged GDIII antigen to the ferritin nanoparticle. The homogeneity, stability, and antigen presentation efficiency of the resulting nanoparticle vaccine were characterized using DLS, negative-stain EM, and AUC. The immunogenicity of this vaccine was then compared to alternative rabies subunit vaccines (GDIII, GDIII-Fc, and full-length G) in a mouse model. Immunogenicity was assessed by measuring IgG antibody titers, neutralizing antibody titers, and analyzing T cell responses (Th1/Th2 balance, IFN-γ, IL-4, Granzyme B production) using ELISA and flow cytometry. Finally, in vivo protective efficacy was evaluated using an intracerebral RABV challenge model in mice, assessing survival rates, weight changes, and viral loads in brain tissue via qRT-PCR and DFA.

Cryo-EM revealed the high-resolution structure of the FTH1/NCOA4 complex, providing the basis for the design of the Fagy-tag. The Fagy-tag demonstrated significantly enhanced binding affinity to FTH1 compared to the wild-type NCOA4 peptide. The self-assembling GDIII-Ferritin nanoparticle vaccine showed superior homogeneity, stability, and antigenic effectiveness compared to other subunit vaccines. The vaccine elicited potent, rapid, and durable humoral immune responses in mice, with significantly higher titers of GDIII-specific IgG and neutralizing antibodies. Furthermore, the vaccine induced a Th1-biased CD4+ T-cell response, contributing to its strong protective efficacy. Remarkably, a single dose of the GDIII-Ferritin nanoparticle vaccine (60 µg) provided 100% protection against a lethal intracerebral RABV challenge in mice, exceeding the protection afforded by the standard rabies vaccine at higher dosages and multiple immunization rounds. Even a two-dose regimen of 30 µg/dose provided full protection. Sustained protective neutralizing antibody levels were observed for over 9 months post-vaccination. Analysis of brain tissue from challenged mice confirmed the complete elimination of the virus in mice vaccinated with the GDIII-Ferritin nanoparticle vaccine.

This study successfully addressed the limitations of previous ferritin-based vaccine platforms. The Fagy-tag, derived from the natural ferritinophagy complex, provides a highly efficient and specific system for antigen conjugation to ferritin nanoparticles, resulting in a homogeneous and stable vaccine. The superior immunogenicity of the GDIII-Ferritin nanoparticle vaccine, particularly its ability to elicit high titers of neutralizing antibodies and a protective Th1 response, suggests that the multivalent presentation of the antigen on the nanoparticle surface plays a critical role in enhancing immune responses. The single-dose efficacy observed in the mouse model demonstrates a significant improvement over existing rabies vaccines and highlights the potential for this platform to simplify vaccination strategies. The versatility of the Fagy-tag system, allowing the conjugation of multiple antigens, opens possibilities for developing multi-component vaccines against various pathogens. The results strongly suggest that this approach has the potential to significantly improve the efficacy and ease of administration of rabies vaccines and to be adapted to other viral or bacterial vaccines.

This study presents a novel self-assembling rabies vaccine candidate based on a ferritin nanoparticle platform utilizing a rationally designed Fagy-tag. The vaccine exhibits superior homogeneity, stability, and immunogenicity compared to existing approaches, demonstrating 100% protection against a lethal rabies virus challenge in mice after a single dose. The versatility and efficiency of the Fagy-tag system suggest broad applicability for developing vaccines against a wide range of pathogens. Future studies should focus on clinical trials to evaluate the safety and efficacy of this vaccine in humans and further explore its multi-antigen presentation capabilities for combination vaccines.

The study was conducted in a mouse model, and the results may not be directly translatable to humans. The use of Freund's adjuvant in the mouse model could influence the immune response, and the efficacy of the vaccine without adjuvant warrants further investigation. The long-term safety and durability of the immune response require further evaluation in larger animal models and clinical trials. While the Fagy-tag system demonstrates high efficiency, a thorough comparison with other conjugation methods (e.g., gene fusion, other Tag/Catcher systems) is warranted for a complete assessment of its advantages.