# Multiple directed mutagenesis reduces enzymatic activity and antibody recognition of the African Swine Fever Virus E2 ubiquitin-conjugating protein (ASFV-pI215L)

## TL;DR

Scientists found that changing specific parts of a key protein in African Swine Fever Virus reduces its activity and immune recognition, offering a path to develop a better vaccine.

## Contribution

The study identifies specific mutations that impair both enzymatic activity and antibody recognition of the ASFV-pI215L protein.

## Key findings

- Single- and multiple-residue substitutions in R11-E15 and D130-S134 reduce E2 ubiquitin-conjugating activity of ASFV-pI215L.
- Mutations between P61 and F69 residues reduce or abolish IgG and IgM recognition and E2 activity.
- Targeted mutagenesis can create a live attenuated vaccine that differentiates infected from vaccinated animals.

## Abstract

African Swine Fever virus (ASFV) causes a contagious and fatal disease in domestic pigs and Eurasian wild boars, representing a serious threat to the global pig industry, since no antivirals are available and vaccine use is currently restricted to Vietnam. Notably, ASFV encodes for an E2 ubiquitin-conjugating enzyme (ASFV-pI215L) which is essential for viral replication and evasion from immune interferon type I responses, suggesting that its functional impairment could lead to a live attenuated vaccine. In this study, we showed that ASFV-pI215L is highly conserved among 222 ASFV isolates, including the emerging ones, emphasizing its value as a target for vaccine design. Furthermore, our mutagenic studies revealed that single- and multiple-residue substitutions comprising the R11-E15 and D130-S134 residues reduced ASFV-pI215L E2 ubiquitin-conjugating activity. In parallel, a strong immunodominant B-cell epitope was mapped and mutated between P61 and F69 resides, reducing or abolishing both IgG and IgM recognition, and ASFV-pI215L E2 activity. In sum, this study highlights that rational targeted mutagenesis can reduce E2 ubiquitin-conjugating activity and immune recognition of ASFV-pI215L, providing a strategy to develop an attenuated vaccine able to differentiate infected from vaccinated animals.