Simple SummaryThe recent SARS-CoV-2 pandemic has underscored the impact of a single pathogen on public health and economic stability. Similarly, widespread disease outbreaks within agricultural animal populations are potentially devastating by producing food shortages and global starvation. Protecting agricultural animals from viral/bacterial disease outbreaks will depend on the development of countermeasures generating rapid innate immune protection. Accordingly, we have developed a rapidly deployable generalized immune stimulant activating innate immunity at mucosal sites. Past studies show that this platform, termedLTC(liposome-TLR complexes), can rapidly activate and recruit innate immune cells in rodents and companion animals. Moreover, prior rodent and cattle studies showed complete protection against lethal viral and bacterial challenge studies. In the studies described herein, we demonstrate that the LTC platform can be extended to demonstrate rapid activation of innate immune modulators from cell lines and immune cells from peripheral blood in three key agricultural animals: cattle, pigs, and poultry. Cell lines and immune cells from peripheral blood from all three of these species demonstrated significant increases in antibacterial and antiviral immune mediators when treated with LTC in vitro. These data provide justification for further testing of this platform in disease challenge models for these animals.AbstractGiven the rapid potential spread of agricultural pathogens, and the lack of vaccines for many, there is an important unmet need for strategies to induce rapid and non-specific immunity against these viral and bacterial threats. One approach to the problem is to generate non-specific immune responses at mucosal surfaces to rapidly protect from entry and replication of both viral and bacterial pathogens. Using complexes of charged nanoparticle liposomes with both antiviral and antibacterial toll-like receptor (TLR) nucleic acid ligands (termed liposome-TLR complexes orLTC), we have previously demonstrated considerable induction of innate immune responses in nasal and oropharyngeal tissues and protection from viral and bacterial pathogens in mixed challenge studies in rodents, cattle, and companion animals. Therefore, in the present study, we used in vitro assays to evaluate the ability of the LTC immune stimulant to activate key innate immune pathways, particularly interferon pathways, in cattle, swine, and poultry. We found that LTC complexes induced strong production of type I interferons (IFNα and IFNβ) in both macrophages and leukocyte cultures from all three species. In addition, the LTC complexes induced the production of additional key protective cytokines (IL-6, IFNγ, and TNFα) in macrophages and leukocytes in cattle and poultry. These findings indicate that the LTC mucosal immunotherapeutic has the capability to activate key innate immune defenses in three major agricultural species and potentially induce broad protective immunity against both viral and bacterial pathogens. Additional animal challenge studies are warranted to evaluate the protective potential of LTC immunotherapy in cattle, swine, and poultry.