Development of protective immunity against African swine fever depends on host-environment interactions

African swine fever virus (ASFV) is a major threat for pig health and meat production in many countries. The development and commercialization of vaccine candidates are complicated by efficacy and safety concerns. Improved vaccine design requires further studies to identify factors that regulate immune responses to vaccines leading to protective immunity against a virulent challenge. In a previous study, we reported that infection with the moderately virulent ASFV field strain Estonia 2014 was less severe in specific pathogen-free (SPF) pigs than in conventional farm pigs, which differ in their gut microbiome and their basal immune activation status. As shown previously using intramuscular infection, SPF pigs were more resilient to oronasal infection with the ASFV Estonia 2014 strain compared to farm pigs, which showed increased fever and clinical signs. All SPF and farm pigs nevertheless survived the infection and remained viremic for approximately 4 months. When all animals had no detectable viremia, both groups were rechallenged with the virulent ASFV Armenia 2008 strain. SPF pigs were fully protected against disease and showed little or no viremia upon re-challenge. In contrast, farm pigs developed high viremia, high proinflammatory cytokine responses, severe clinical signs, and 40% (2 of 5 pigs) reached humane endpoints. Our findings suggest that limited prior immune exposure to other pathogens and/or the microbiome composition of SPF pigs promotes resilience to infection with a moderately virulent strain such as Estonia 2014, and importantly promotes the development of a strong protective immune response against a second challenge with a virulent ASFV strain. In conclusion, testing safety and efficacy of live attenuated vaccine candidates should take into account the specific hygiene conditions and the associated changes of general immune status of pigs in clinical trials.