Antibodies targeting the Crimean-Congo Hemorrhagic Fever Virus nucleoprotein protect via TRIM21

Crimean-Congo hemorrhagic fever (CCHF), caused by the Crimean-Congo hemorrhagic fever virus (CCHFV), is a significant global health threat. Spread primarily by Hyalomma genus ticks, CCHF is characterized by a non-specific febrile illness that can progress to a severe hemorrhagic disease with high mortality rates (5-70%). The geographic range of CCHFV is expanding due to factors such as climate change and global trade, increasing the risk to new populations. Currently, there are no widely approved vaccines or highly efficacious treatments, highlighting the urgent need for effective countermeasures. CCHFV is a negative-sense RNA virus with a tri-segmented genome (S, M, L). The S segment encodes the nucleoprotein (NP) and a non-structural protein (NSs), while the M segment encodes glycoproteins (Gn and Gc) and non-structural proteins. The L segment encodes the RNA-dependent RNA polymerase (RdRp). The NP plays multiple roles in viral replication, including genome encapsidation and apoptosis inhibition. While neutralizing antibodies against viral glycoproteins have been implicated in protective immunity, this study focuses on the role of antibodies targeting the NP.

Previous research has demonstrated the importance of neutralizing antibodies targeting viral glycoproteins in protective immunity against CCHFV. Studies have also shown that non-neutralizing antibodies can offer some protection. Recent work by the authors and others showed that both NP and NSs elicit protective antibodies, indicating that vaccines targeting NP could provide robust protection. Earlier studies by the authors showed that a replicating RNA vaccine expressing CCHFV NP conferred protection against lethal CCHFV challenge in mice, and this protection was B cell-dependent, but not T cell-dependent.

The study used several approaches to investigate the mechanisms of protection conferred by anti-NP antibodies. Passive transfer of serum from vaccinated mice to naive mice was performed to assess the protective efficacy of vaccine-elicited antibodies. Mice deficient in activating Fc receptors (FcγR-/-), the complement pathway (C3-/-), or NK cells were vaccinated to determine the involvement of these components in protection. TRIM21-/- mice were also vaccinated to evaluate the role of this cytoplasmic Fc receptor. An electroporated antibody-dependent neutralization assay (EDNA) was used to assess the ability of cytoplasmic anti-NP antibodies to inhibit CCHFV replication in vitro. Additionally, studies were conducted with mice depleted of CD4+ and CD8+ T-cells, and CD8-/- mice, to evaluate the role of T cells in protection. Immunological responses were measured using ELISA, ELISpot, and qRT-PCR. Viral loads were quantified using TCID50 and qRT-PCR. Histology was used to assess tissue pathology. Studies in cynomolgus macaques were also conducted to evaluate the generalizability of findings in mice. The study adhered to rigorous ethical standards and guidelines for animal care and human subject research.

Passive transfer of serum from replicating NP (repNP)-vaccinated mice conferred significant, albeit partial, protection to naive mice against lethal CCHFV challenge. This protection was not dependent on Fc receptors, complement, or NK cells. However, protection was completely absent in TRIM21-/- mice. Remarkably, repNP vaccination failed to protect TRIM21-/- mice, even though these mice mounted comparable antibody responses to wild-type mice. In contrast, mice depleted of CD4+ and CD8+ T cells, or CD8-/- mice, were fully protected after repNP vaccination, indicating that T cells are not essential for protection mediated by anti-NP antibodies. The EDNA showed that cytoplasmic anti-NP antibodies can inhibit CCHFV replication, and this inhibition was enhanced by TRIM21. Furthermore, serum from repNP-vaccinated cynomolgus macaques also inhibited CCHFV replication in vitro, and this inhibition was also enhanced by TRIM21. In summary, these data highlight the crucial role of TRIM21 in the protective mechanism mediated by anti-NP antibodies against CCHFV.

This study provides strong evidence that anti-NP antibodies elicited by the repNP vaccine protect against CCHFV through a TRIM21-dependent mechanism, independent of Fc-receptor mediated effector functions and T-cell responses. This is a novel finding for an enveloped virus, as TRIM21's role in antiviral immunity has been largely studied in the context of non-enveloped viruses. The mechanism by which antibody-bound NP interacts with cytoplasmic TRIM21 requires further investigation, but may involve endocytosis, release of NP into circulation, or disruption of endosomes during viral entry. While the study focuses on mouse models, the findings in cynomolgus macaques suggest that the TRIM21-mediated mechanism may be relevant across species, including humans. The results support the development of anti-NP antibodies as potential therapeutics for CCHF.

This research demonstrates the crucial role of TRIM21 in the protective efficacy of anti-NP antibodies against CCHFV. The findings highlight a novel mechanism of antiviral immunity and suggest that targeting the NP with vaccines or therapeutic antibodies may be a viable approach for combating CCHF. Future research could investigate the precise intracellular mechanisms of interaction between anti-NP antibodies, NP, and TRIM21. Moreover, further development and testing of anti-NP antibody-based therapeutics are warranted.

The study primarily utilized mouse models. Although results were corroborated in non-human primates, further studies in humans are needed to confirm the generalizability of findings. The mechanism by which anti-NP antibodies and NP access the cytoplasm to interact with TRIM21 remains to be fully elucidated. The partial protection observed in passive transfer studies versus complete protection with active vaccination may be attributed to differences in antibody levels or kinetics.