Determining hyperimmune serum neutralizing effect on African Swine Fever Virus and serum enhancing effect on extracellular virion infectivity in adherent pig PBMC by flow cytometry

Authors: CANTER, JESSICA - Oak Ridge Institute For Science And Education (ORISE); APONTE, THERESA - Oak Ridge Institute For Science And Education (ORISE); Ramirez-Medina, Elizabeth; Pruitt, Sarah; NEILAN, JOHN - Us Deparment Of Homeland Security; Gladue, Douglas; Borca, Manuel; Zhu, James

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2022
Publication Date: 6/9/2022
Citation: Canter, J.A., Aponte, T., Ramirez Medina, E., Pruitt, S.E., Neilan, J., Gladue, D.P., Borca, M.V., Zhu, J.J. 2022. Determining hyperimmune serum neutralizing effect on African Swine Fever Virus and serum enhancing effect on extracellular virion infectivity in adherent pig PBMC by flow cytometry. Scientific Reports. https://doi.org/10.3390/v14061249.
DOI: https://doi.org/10.3390/v14061249

Interpretive Summary: African swine fever virus (ASFV) causes hemorrhagic fever with mortality rates up to 100% in domestic pigs, a global epidemy currently threating the global swine industry. There are no commercial vaccines for the disease yet. Although many vaccine candidates implemented with various techniques have been tested, only some live attenuated viruses were able to protect pigs from ASFV infection with a high degree of efficacy. The immune mechanisms involved in the protection from these experimental vaccines are not clear. ASFV antibodies can neutralize ASFV though incompletely. However, there is no standardized protocol for the assay. Traditional assays are tedious, time consuming, and highly technically variable. In this study, a new ASFV neutralization assay was developed for the test in the primary ASFV target cells to overcome the drawbacks. We found with this new assay that extracellular, but not intracellular, ASFV suspended in naïve sera were more infectious than those in the culture medium as reported for some enveloped viruses and both the intracellular and extracellular virions could not be completely neutralized by the hyperimmune sera. The findings indicate a novel mechanism for the virus to infect cells.

Technical Abstract: African swine fever virus (ASFV) causes hemorrhagic fever with mortality rates up to 100% in domestic pigs. Currently, there are no commercial vaccines for the disease. Although many vaccine candidates have been tested, only some live attenuated viruses were able to protect pigs from ASFV infection with a high degree of efficacy. The immune mechanisms involved in the protection from these experimental vaccines are not clear although humoral and cell-mediated immunities have been shown to be important for the protection. ASFV antibodies can neutralize ASFV though incompletely. However, there is no standardized protocol for the assay. In this study, a flow cytometry based ASFV neutralization assay was developed and tested in ex-vivo pig adherent PBMC using a virulent ASFV containing a green fluorescent protein gene inserted after a late expression promoter as a substrate for neutralization. Like previous studies, the percentage of infected macrophages was approximately five time higher than that of infected monocytes and nearly all infected cells displayed no staining with anti-CD16 antibodies. Hyperimmune sera from pigs immunized with a live attenuated experimental vaccine that fully protects against parental were used in the assay. This study describes the optimization of the ASFV neutralization assay in pig macrophages using a flow cytometry-based assay. The hyperimmune sera displayed incomplete ASFV neutralization with MOI-dependent neutralization efficacies. Extracellular, but not intracellular, virions suspended in naïve sera were more infectious than those in the culture medium as reported for some enveloped viruses, indicating a novel mechanism of ASFV to infect cells. Both the intracellular and extracellular virions could not be completely neutralized by the hyperimmune sera.