Abstract
African swine fever (ASF) is a lethal disease of swine caused by the ASF virus (ASFV), for which no licensed vaccine is currently available in non-endemic countries. Here, replication-competent adenovirus-vectored ASFV multi-antigen constructs were shown to express ASFV antigens in primary swine cells. Pigs immunized with a cocktail of these constructs, with or without Quil-A adjuvant, tolerated the formulation well. The constructs elicited robust ASFV-specific IgG responses (p < 0.0001), which were boosted upon reimmunization (p < 0.05). Following challenge with the virulent ASFV Georgia 2007/1 strain using a natural transmission model, five of six pigs vaccinated without Quil-A survived, while all pigs receiving adjuvanted constructs succumbed to ASF. Survivors cleared the virus, exhibited only mild clinical signs, gained weight, and remained healthy for the remainder of the study. Histopathological analysis revealed an absence of ASFV-associated lesions in survivors, whereas severe lesions were observed in pigs vaccinated with adjuvanted constructs and in negative controls. Although neutralizing antibodies were undetectable, granzyme B-producing CD8α⁺ T cell responses were observed in survivors, indicating a likely correlation for cellular immunity in protection. These findings highlight the protective potential of ASFV antigen expression constructs and inform subunit vaccine design.
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All data supporting the findings of this study are available from the corresponding author upon request.
References
Zhu, G. et al. Structural basis of RNA polymerase complexes in African swine fever virus. Nat. Commun. 16, 501 (2025).
Spinard, E. et al. A re-evaluation of African swine fever genotypes based on p72 sequences reveals the existence of only six distinct p72 groups. Viruses 15, 2246 (2023).
Njau, E. P. et al. The first genotype II African swine fever virus isolated in Africa provides insight into the current Eurasian pandemic. Sci. Rep. 11, 13081 (2021).
Coelho, I. M. P., Paiva, M. T., Da Costa, A. J. A. & Nicolino, R. R. African Swine Fever: Spread and seasonal patterns worldwide. Prev. Vet. Med. 235, 106401 (2025).
OIE. African Swine Fever (ASF)—Situation Reports https://www.woah.org/en/disease/african-swine-fever/ (2025).
Dixon, L. K. Advances in African swine fever virus molecular biology and host interactions contributing to new tools for control. J. Virol. 99, e00932–00924 (2025).
Ruedas-Torres, I., Thi To Nga, B. & Salguero, F. J. Pathogenicity and virulence of African swine fever virus. Virulence 15, 2375550 (2024).
Guinat, C. et al. Transmission routes of African swine fever virus to domestic pigs: current knowledge and future research directions. Vet. Rec. 178, 262–267 (2016).
Montgomery, R. E. On a form of swine fever occurring in British East Africa (Kenya Colony). J. Comp. Pathol. 34, 242 (1921).
de Carvalho Ferreira, H. C. et al. African swine fever virus excretion patterns in persistently infected animals: a quantitative approach. Vet. Microbiol. 160, 327–340 (2012).
Chandana, M. S. et al. Recent progress and major gaps in the vaccine development for African swine fever. Braz. J. Microbiol. 55, 997–1010 (2024).
Montgomery, R. E. Immunization with Attenuated Virus—Annual Reports of the Chief Veterinary Research Officer, Agricultural Department, Kenya (Agricultural Department,1921–1927).
Sanford, B. et al. Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus. Virus Res. 213, 165–171 (2016).
Sang, H. et al. Progress toward development of effective and safe African swine fever virus vaccines. Front. Vet. Sci. 7, 84 (2020).
Rock, D. L. Thoughts on African swine fever vaccines. Viruses 13, 943 (2021).
Diep, N. V. et al. Genotype II live-attenuated ASFV vaccine strains unable to completely protect pigs against the emerging recombinant ASFV genotype I/II strain in Vietnam. Vaccines (Basel) 12, 1114 (2024).
Nefedeva, M., Titov, I., Tsybanov, S. & Malogolovkin, A. Recombination shapes African swine fever virus serotype-specific locus evolution. Sci. Rep. 10, 18474 (2020).
Zhao, D. et al. Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs. Nat. Commun. 14, 3096 (2023).
van den Born, E. et al. African swine fever virus vaccine strain Asfv-G-∆ I177l reverts to virulence and negatively affects reproductive performance. npj Vaccines 10, 46 (2025).
Zhang, Y. et al. ASFV subunit vaccines: strategies and prospects for future development. Microb. Pathog. 197, 107063 (2024).
Escribano, J. M., Galindo, I. & Alonso, C. Antibody-mediated neutralization of African swine fever virus: myths and facts. Virus Res. 173, 101–109 (2013).
Yang, X. et al. The antibodies against the A137R protein drive antibody-dependent enhancement of African swine fever virus infection in porcine alveolar macrophages. Emerg. Microbes Infect. 13, 2377599 (2024).
Sunwoo, S. Y. et al. DNA–protein vaccination strategy does not protect from challenge with African swine fever virus Armenia 2007 strain. Vaccines (Basel) 7, 12 (2019).
Schafer, A. et al. Adaptive cellular immunity against African swine fever virus infections. Pathogens 11, 274 (2022).
Takamatsu, H. H. et al. Cellular immunity in ASFV responses. Virus Res. 173, 110–121 (2013).
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. J. Gen. Virol. 86, 2445–2450 (2005).
Lokhandwala, S. et al. Adenovirus-vectored African swine fever virus antigen cocktails are immunogenic but not protective against intranasal challenge with Georgia 2007/1 isolate. Vet. Microbiol. 235, 10–20 (2019).
Zajac, M. D. et al. Immunization of pigs with replication-incompetent adenovirus-vectored African swine fever virus multi-antigens induced humoral immune responses but no protection following contact challenge. Front. Vet. Sci. 10, 1208275 (2023).
Portugal, R. et al. Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge. J. Virol. 98, e0062224 (2024).
Goatley, L. C. et al. A pool of eight virally vectored African swine fever antigens protect pigs against fatal disease. Vaccines (Basel) 8, 234 (2020).
Peng, B. et al. Replicating rather than nonreplicating adenovirus–human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies. J. Virol. 79, 10200–10209 (2005).
Li, Y. et al. Immunization with recombinant Sao protein confers protection against Streptococcus suis infection. Clin. Vaccin. Immunol. 14, 937–943 (2007).
Singh, M. & O’Hagan, D. T. Recent advances in veterinary vaccine adjuvants. Int. J. Parasitol. 33, 469–478 (2003).
Trapani, J. A. Granzymes: a family of lymphocyte granule serine proteases. Genome Biol. 2, Reviews3014 (2001).
Jawalagatti, V., Kirthika, P., Park, J. Y., Hewawaduge, C. & Lee, J. H. Highly feasible immunoprotective multicistronic SARS-CoV-2 vaccine candidate blending novel eukaryotic expression and Salmonella bactofection. J. Adv. Res. 36, 211–222 (2022).
Kim, J. H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6, e18556 (2011).
Alexander, J. et al. Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin. PLoS ONE 7, e31177 (2012).
Elkashif, A., Alhashimi, M., Sayedahmed, E. E., Sambhara, S. & Mittal, S. K. Adenoviral vector-based platforms for developing effective vaccines to combat respiratory viral infections. Clin. Transl. Immunol. 10, e1345 (2021).
Mudrick, H. E. et al. Comparison of replicating and nonreplicating vaccines against SARS-CoV-2. Sci. Adv. 8, eabm8563 (2022).
Shaimardanova, A. A. et al. Production and application of multicistronic constructs for various human disease therapies. Pharmaceutics 11, https://doi.org/10.3390/pharmaceutics11110580 (2019).
Blome, S., Gabriel, C. & Beer, M. Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation. Vaccine 32, 3879–3882 (2014).
Howey, E. B., O’Donnell, V., de Carvalho Ferreira, H. C., Borca, M. V. & Arzt, J. Pathogenesis of highly virulent African swine fever virus in domestic pigs exposed via intraoropharyngeal, intranasopharyngeal, and intramuscular inoculation, and by direct contact with infected pigs. Virus Res 178, 328–339 (2013).
Guinat, C. et al. Dynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission. Vet. Res. 45, 93 (2014).
Bosch-Camós, L. et al. Cross-protection against African swine fever virus upon intranasal vaccination is associated with an adaptive-innate immune crosstalk. PLoS Pathog. 18, e1010931 (2022).
Arias, M. et al. Approaches and perspectives for development of African swine fever virus. Vaccines 5, 35 (2017).
Bosch-Camós, L., López, E. & Rodriguez, F. African swine fever vaccines: a promising work still in progress. Porcine Health Manag. 6, 17 (2020).
Neilan, J. G. et al. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection. Virology 319, 337–342 (2004).
Salguero, F. J. Comparative pathology and pathogenesis of African swine fever infection in swine. Front. Vet. Sci. 7, 282 (2020).
Galindo-Cardiel, I. et al. Standardization of pathological investigations in the framework of experimental ASFV infections. Virus Res. 173, 180–190 (2013).
Lokhandwala, S. et al. Induction of robust immune responses in swine by using a cocktail of adenovirus-vectored African swine fever virus antigens. Clin. Vaccin. Immunol. 23, 888–900 (2016).
Friedrichs, V., Streitz, M., Beer, M., Blome, S. & Schäfer, A. Maternal immunity and African swine fever virus: understanding the limits of passive protection. Front. Immunol. 16, 1593820 (2025).
Schlafer, D. H., Mcvicar, J. W. & Mebus, C. A. African swine fever convalescent sows—subsequent pregnancy and the effect of colostral antibody on challenge inoculation of their pigs. Am. J. Vet. Res. 45, 1361–1366 (1984).
Onisk, D. V. et al. Passively transferred African swine fever virus antibodies protect swine against lethal infection. Virology 198, 350–354 (1994).
Wardley, R. C., Norley, S. G., Wilkinson, P. J. & Williams, S. The role of antibody in protection against African swine fever virus. Vet. Immunol. Immunopathol. 9, 201–212 (1985).
Attreed, S. E. et al. A highly effective African swine fever virus vaccine elicits a memory T cell response in vaccinated swine. Pathogens 11, 1438 (2022).
Zajac, M. D. et al. Adenovirus-vectored African swine fever virus pp220 induces robust antibody, IFN-gamma, and CTL responses in pigs. Front. Vet. Sci. 9, 921481 (2022).
Vanselow, B. A., Abetz, I. & Trenfield, K. A bovine ephemeral fever vaccine incorporating adjuvant Quil A: a comparative study using adjuvants Quil A, aluminium hydroxide gel and dextran sulphate. Vet. Rec. 117, 37–43 (1985).
Crawley, A., Raymond, C. & Wilkie, B. N. Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines. Vet. Immunol. Immunopathol. 91, 141–154 (2003).
Crawley, A. & Wilkie, B. N. Porcine Ig isotypes: function and molecular characteristics. Vaccine 21, 2911–2922 (2003).
Liu, W. et al. A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine. Emerg. Microbes Infect. 12, 2233643 (2023).
Lokhandwala, S. et al. Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine. PLoS ONE 12, e0177007 (2017).
Su, Q., Sena-Esteves, M. & Gao, G. Purification of the recombinant adenovirus by cesium chloride gradient centrifugation. Cold Spring Harb. Protoc. 2019, (2019). https://doi.org/10.1101/pdb.prot095547.
Singleton, H., Graham, S. P., Bodman-Smith, K. B., Frossard, J.-P. & Steinbach, F. Establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with PRRSV-1. Front. Microbiol. 7, https://doi.org/10.3389/fmicb.2016.00832 (2016).
Goatley, L. C., Nash, R. & Netherton, C. L. Primary macrophage culture from porcine blood and lungs. Methods Mol. Biol. 2503, 63–72 (2022).
Ho, C. S. et al. Nomenclature for factors of the SLA system, update 2008. Tissue Antigens 73, 307–315 (2009).
Hammer, S. E. et al. Importance of the major histocompatibility complex (swine leukocyte antigen) in swine health and biomedical research. Annu Rev. Anim. Biosci. 8, 171–198 (2020).
Ho, C. S. et al. Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations. Anim. Genet. 40, 468–478 (2009).
Ho, C. S. et al. Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations. Anim. Genet. 41, 428–432 (2010).
Gao, C. et al. Swine leukocyte antigen diversity in Canadian specific pathogen-free Yorkshire and Landrace pigs. Front. Immunol. 8, 282 (2017).
Hammer, S. E. et al. Comparative analysis of swine leukocyte antigen gene diversity in European farmed pigs. Anim. Genet. 52, 523–531 (2021).
Techakriengkrai, N., Nedumpun, T., Golde, W. T. & Suradhat, S. Diversity of the swine leukocyte antigen Class I and II in commercial pig populations. Front. Vet. Sci. 8, 637682 (2021).
Hammer, S. E. et al. in 6th European Veterinary and Immunology Workshop (EVIW) (ed. Rutten, V.) The European Veterinary Immunology Group (EVIG).
Sørensen, M. R. et al. Sequence-based genotyping of expressed swine leukocyte antigen Class I alleles by next-generation sequencing reveal novel swine leukocyte antigen Class I haplotypes and alleles in Belgian, Danish, and Kenyan fattening pigs and Göttingen Minipigs. Front. Immunol. 8, https://doi.org/10.3389/fimmu.2017.00701 (2017).
Luetkemeier, E. S., Malhi, R. S., Beever, J. E. & Schook, L. B. Diversification of porcine MHC class II genes: evidence for selective advantage. Immunogenetics 61, 119–129 (2009).
Le, M. T. et al. Comprehensive and high-resolution typing of swine leukocyte antigen DQA from genomic DNA and determination of 25 new SLA class II haplotypes. Tissue Antigens 80, 528–535 (2012).
Maccari, G. et al. IPD-MHC 2.0: an improved inter-species database for the study of the major histocompatibility complex. Nucleic Acids Res. 45, D860–D864 (2017).
Acknowledgements
We thank the Kansas State University Comparative Medicine Group for their help in animal sourcing, clinical care, and all the proper general animal welfare. We are also grateful to Carly Cole, Aubrie Rich, Riley Cox, Heidi Anderson, Gabriele Leighow, Katie Hoch, Ian Stoll, Emma McClure, and Katelin Megee for their invaluable help in the animal health check, sample collection, and PBMC isolation. In addition, we are grateful to Sandra Groiss (Department of Biological Sciences and Pathobiology, University of Veterinary Medicine, Vienna, Austria) for her technical assistance in SLA typing. We thank the individuals at the KSVDL histology laboratory for histological processing of tissues.
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W.M. designed and oversaw the development and characterization of the experimental vaccine. The vaccine was generated by R.K. and T.K. Animal immunization, animal monitoring, sample collection, and challenge experiments were done by R.K., T.K., M.Z., H.S., E.H., D.K., J.M., S.O., and W.M. Evaluation of antibody responses, clinical outcomes, viremia, and neutralization assays was conducted by R.K., T.K., M.Z., and E.H. Generation and characterization of Granzyme B mAbs was conducted by N.S., L.B., K.M. and M.Z. Evaluation of T cell responses was conducted by M.Z., B.A., and J.M. Necropsy was conducted by J.T., S.A., R.K., T.K., M.Z., H.S., E.H., D.K., J.M., and J.A.R. Gross and histopathology were performed by R.K. and J.T. SLA typing was conducted by S.H. R.K., T.K., S.H., M.Z., and W.M. were involved in data analysis and result interpretation. R.K., T.K., J.T., S.H., and W.M. wrote the manuscript.
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Kumar, R., Kim, T., Zajac, M.D. et al. Live-vectored antigen cocktail confers protection against African swine fever virus (ASFV) Georgia 2007/1 challenge. npj Vaccines (2026). https://doi.org/10.1038/s41541-026-01399-8
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DOI: https://doi.org/10.1038/s41541-026-01399-8
