Efficient delivery of the immunodominant genes of African swine fever virus by adeno-associated virus serotype 2

Research (Published online: 06-12-2023)

5. Efficient delivery of the immunodominant genes of African swine fever virus by adeno-associated virus serotype 2

Rustam Ravilov, Antonina Galeeva, Gennadiy Frolov, Marina Efimova, Elena Zakirova, Albert Rizvanov, Almaz Hisamutdinov, Lenar Garipov, and Danil Mingaleev

Veterinary World, 16(12): 2425-2430

Rustam Ravilov: Kazan State Academy of Veterinary Medicine named after N.E. Bauman, Kazan, Russia.

Antonina Galeeva: Kazan State Academy of Veterinary Medicine named after N.E. Bauman, Kazan, Russia; Federal Center for Toxicological, Radiation and Biological Safety, Kazan, Russia; Laboratory of Regenerative Veterinary Medicine, Kazan (Volga region) Federal University, Institute of Fundamental Medicine and Biology, Kazan, Russia.

Gennadiy Frolov: Kazan State Academy of Veterinary Medicine named after N.E. Bauman, Kazan, Russia.

Marina Efimova: Kazan State Academy of Veterinary Medicine named after N.E. Bauman, Kazan, Russia; Federal Center for Toxicological, Radiation and Biological Safety, Kazan, Russia.

Elena Zakirova: Laboratory of Regenerative Veterinary Medicine, Kazan (Volga region) Federal University, Institute of Fundamental Medicine and Biology, Kazan, Russia.

Albert Rizvanov: Laboratory of Regenerative Veterinary Medicine, Kazan (Volga region) Federal University, Institute of Fundamental Medicine and Biology, Kazan, Russia.

Almaz Hisamutdinov: Main Directorate of Veterinary Medicine, Ministry of the Tatarstan Republic, Kazan, Russia.

Lenar Garipov: Ministry of Agriculture and Food of Tatarstan Republic, Kazan, Russia.

Danil Mingaleev: Kazan State Academy of Veterinary Medicine named after N.E. Bauman, Kazan, Russia; Federal Center for Toxicological, Radiation and Biological Safety, Kazan, Russia.

Background and Aim: Adeno-associated virus serotype 2 (AAV2) represents a promising basis for developing a virus-vector vaccine against African swine fever (ASF). This study aimed to create genetic constructs based on AAV2 to deliver the immunodominant genes of ASF virus (ASFV) and to evaluate their functionality in vitro. The efficiency and specificity of transgene expression, as well as their non-toxicity in cells of target animals, were evaluated.

Materials and Methods: Bioinformatics analysis methods were used to identify the immunodominant genes of ASFV. The target genes B646L, E183L, CP204L, and CP530R were identified and subsequently cloned into the pAAV-MCS vector. Assembly of recombinant AAV2 (rAAV2) was performed by cotransfection of AAV293 cells with the following plasmids: pAAV-MCS with the gene of interest, envelope, and packaging. Quantitative polymerase chain reaction was used to determine the AAV2 titer. The functionality of the constructs was evaluated in HEK293 and SPEV cells by determining the presence of mature proteins in the cell lysate and the expression levels of messenger RNA. The specificity of the target proteins in cell lysates was confirmed by Western blotting.

Results: The proposed AAV2 assembly protocol makes it possible to achieve a concentration of mature viral particles of at least 280 billion/mL of virus-containing material. The rAAV2 could effectively transduce host SPEV cells. The expression of both cistrons was detectable during the transduction of cells; therefore, the combined expression of immunogens in the cells of target animals should be possible using this method.

Conclusion: This study demonstrated the potential of using genetic constructs based on AAV2 for the delivery of ASFV genes in vitro.

Keywords: adeno-associated virus, African swine fever virus, gene delivery, virally vectored vaccine.