Viktorova, E. | Search bioRxiv

bioRxiv2025

Towards the development of a mucosal vectored vaccine against enterovirus D68

Enterovirus D68 (EVD68) is an emerging pathogen associated with neurological complications, including temporary and permanent paralysis, and lethality. Currently, no EVD68-specific vaccines or antivirals are available. Here, we used a Newcastle disease virus (NDV) vector to develop a live mucosal vaccine that produces EVD68 virus-like particles (VLPs) and evaluated its immunogenicity in a murine model. We made constructs producing VLPs of the prototype EVD68 strain Fermon and the USA/2018-23088 (Ohio-2018) strain from a recent outbreak in the USA. Our results demonstrate robust production and proper processing of EVD68 capsid proteins upon co-expression with the viral protease 3CD. Intranasally-immunized animals effectively seroconverted to NDV antigens, indicating successful replication of the vectors. Antibodies that could bind both the Fermon and Ohio-2018 virions were observed in the sera of mice immunized with either viral vector. At least some animals also demonstrated a strong respiratory mucosal IgA response against EVD68 capsid proteins. These data confirm the immunogenicity of EVD68 proteins expressed from a viral vector in the respiratory tract. Yet neither humoral nor mucosal antibodies were protective against EVD68 infection in cell culture. Analysis of cells infected with recombinant NDVs by electron microscopy indicated structural differences between bona fide EVD68 virions and VLPs, suggesting that the lack of neutralizing antibody response is likely due to antigenic disparity between the EVD68 virions and empty VLPs and/or limited stability of VLPs within the murine respiratory tract. These results have important implications for the development of VLP-based vaccines against enteroviruses.

microbiology↗

bioRxiv2022

A Proximity Biotinylation Assay with a Host Protein Bait Reveals Multiple Factors Modulating Enterovirus Replication

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, arguing that the GBF1 environment is linked to the viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified those previously associated with the enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins many of which normally localize in the nucleus constituted the largest group, underscoring the massive release of nuclear factors in the cytoplasm of infected cells and their involvement in the viral replication. Analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly increase the knowledge about the organization of enterovirus replication organelles and may provide new targets for anti-viral interventions.

microbiology↗