In vivo functional assessment of recombinant adeno-associated viruses carrying genes of protectively significant antigens of the African swine fever virus

Relevance. African swine fever (ASF) is a viral hemorrhagic disease with exceptionally high mortality in members of the family Suidae, with serious economic consequences associated with production losses, trade restrictions and eradication programs. To date, no effective commercial vaccine against ASF has been developed. Of particular interest in the design of candidate vaccines are viral vectors, in particular the adenoassociated virus of the 2nd serotype (AAV2), which has successfully proven itself as a gene therapy agent. We previously reported the ability of rAAV2 to effectively deliver ASF virus genes B646L, E183L, CP530R, CP204L into porcine cells in vitro.

The aim of the study was to evaluate the in vivo functionality of adenoassociated viruses of the 2nd serotype carrying genes of protectively significant antigens of the African swine fever virus.

Methods. By cloning pairwise combined genes B646L-CP530R, E183L-CP204L into the pAAV-MCS vector, bicistronic constructs with the self-cleaving P2A peptide were created. Assembly of rAAV2 was accomplished by calcium phosphate transfection of AAV293 cells. After iodixanol density gradient purification, rAAV2 was administered to pigs at a dose of 3 × 10¹¹ viral particles and humoral and cellular immunity was assessed for 180 days. The dynamics of antibody genesis were assessed by indirect ELISA, and immunophenotyping of peripheral blood T-lymphocytes was assessed by flow cytometry.

Results. It was found that the developed bicistronic constructs based on rAAV2 are safe and easily tolerated by animals and cause the induction of both humoral and cellular immune responses: the formation of virus-specific antibodies was observed, which persisted until the end of the experiment, as well as increased expression of CD8+ and CD4+ lymphocytes. The AAV platform we propose is a promising tool for creating a vaccine, however, a comprehensive characterization of rAAV2 can only be compiled after assessing its protective effect.

1. Makarov V.V. African swine fever. Russian veterinary journal. 2018; 6: 15–19 (in Russian). https://doi.org/10.32416/article_5c050abbcf8d70.94861250

2. Brown V.R., Bevins S.N. A Review of African Swine Fever and the Potential for Introduction into the United States and the Possibility of Subsequent Establishment in Feral Swine and Native Ticks. Frontiers in Veterinary Science. 2018; 5: 11. https://doi.org/10.3389/fvets.2018.00011

3. Netherton C.L. et al. Identification and Immunogenicity of African Swine Fever Virus Antigens. Frontiers in Immunology. 2019; 10: 1318. https://doi.org/10.3389/fimmu.2019.01318

4. Gaudreault N.N., Richt J.A. Subunit Vaccine Approaches for African Swine Fever Virus. Vaccines. 2019; 7(2): 56. https://doi.org/10.3390/vaccines7020056

5. Kolbasov D. African swine fever: creation of vaccine is urgent Animal. Husbandry of Russia. 2020; 7: 29–33 (in Russian). https://doi.org/10.25701/ZZR.2020.48.46.008

6. Chathuranga K., Lee J.-S. African Swine Fever Virus (ASFV): Immunity and Vaccine Development. Vaccines. 2023; 11(2): 199. https://doi.org/10.3390/vaccines11020199

7. Zhang H., Zhao S., Zhang H., Qin Z., Shan H., Cai X. Vaccines for African swine fever: an update. Frontiers in Microbiology. 2023; 14: 1139494. https://doi.org/10.3389/fmicb.2023.1139494

8. Ravilov R.Kh. et al. Viral Vector Vaccines Against ASF: Problems and Prospectives. Frontiers in Veterinary. Sciences. 2022; 9: 830244. https://doi.org/10.3389/fvets.2022.830244

9. Wang D., Tai P.W.L., Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery. 2019; 18(5): 358–378. https://doi.org/10.1038/s41573-019-0012-9

10. Pillay S. et al. An essential receptor for adeno-associated virus infection. Nature. 2016; 530(7588): 108–112. https://doi.org/10.1038/nature16465

11. Deyle D.R., Russell D.W. Adeno-associated virus vector integration. Current. Opinion in Molecular. Therapeutics. 2009; 11(4): 442–447.

12. Efimova M.A., Galeeva A.G., Khamidullina A.I., Ravilov R.Kh. Analysis of immunodominant African swine fever virus peptides for candidate vaccine design. Agrarian science. 2023; 3: 40–45 (in Russian). https://doi.org/10.32634/0869-8155-2023-368-3-40-45

13. Ravilov R. et al. Efficient delivery of the immunodominant genes of African swine fever virus by adeno-associated virus serotype 2. Veterinary World. 2023; 16(12): 2425–2430. https://doi.org/10.14202/vetworld.2023.2425-2430

14. Nieto K., Salvetti A. AAV vectors vaccines against infectious diseases. Frontiers in Immunology. 2014; 5: 5. https://doi.org/10.3389/fimmu.2014.00005

15. Zhou X. et al. Comparison of mucosal immune responses to African swine fever virus antigens intranasally delivered with two different viral vectors. Research in Veterinary Science. 2022; 150: 204–212. https://doi.org/10.1016/j.rvsc.2022.06.025

16. Mingozzi F., High K.A. Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape. Annual Review in Virology. 2017; 4(1): 511–534. https://doi.org/10.1146/annurev-virology-101416-041936

17. Goatley L.C. et al. Cellular and Humoral Immune Responses after Immunisation with Low Virulent African Swine Fever Virus in the Large White Inbred Babraham Line and Outbred Domestic Pigs. Viruses. 2022; 14(7): 1487. https://doi.org/10.3390/v14071487

18. Silva E.B. et al. The Presence of Virus Neutralizing Antibodies Is Highly Associated with Protection against Virulent Challenge in Domestic Pigs Immunized with ASFV live Attenuated Vaccine Candidates. Pathogens. 2022; 11(11): 1311. https://doi.org/10.3390/pathogens11111311

19. Oura C.A.L., Denyer M.S., Takamatsu H., Parkhouse R.M.E. In vivo depletion of CD8 + T lymphocytes abrogates protective immunity to African swine fever virus. Journal of General Virology. 2005; 86(9): 2445–2450. https://doi.org/10.1099/vir.0.81038-0

20. Attreed S.E. et al. A Highly Effective African Swine Fever Virus Vaccine Elicits a Memory T Cell Response in Vaccinated Swine. Pathogens. 2022; 11(12): 1438. https://doi.org/10.3390/pathogens11121438