INNATE IMMUNE RESPONSES ACTIVATED BY THE ADJUVANT POLY [DI (SODIUM CARBOXYLATOETHYLPHENOXY)PHOSPHAZENE](PCEP) IN PIGS
ABSTRACT Adjuvants are critical components of vaccines because they enhance antigen-specific immune responses to protect against disease. However, the mechanisms of action (MOA) of most adjuvants are not well understood and they particularly are under-investigated in large animal species including pigs and cattle. This knowledge gap may limit our ability to design effective vaccines for livestock. Understanding the mechanisms by which adjuvants mediate their effects could provide critical information on how innate immunity influences the development of adaptive immunity. Furthermore, knowledge on the MOA of adjuvants may inform vaccine the safety. In the present investigations, we studied the MOA of the experimental adjuvant polydi(sodium carboxylatoethylphenoxy)phosphazene (PCEP) in pigs. First, we administered PCEP by intradermal (i.d.) injection into pigs and assessed its impact on the expression of select immune response genes known as ‘adjuvant response genes’ over time. We observed that PCEP induced the expression of chemokine (C-C motif) ligand 2 (CCL-2), proinflammatory cytokine interleukin (IL)-6, IL-13 and macrophage scavenger receptor 1 (MSR1) genes at the site of injection. Next, we evaluated whether these gene expressions translate to protein transcription by accessing local and systemic production of cytokines after intradermal injection of PCEP into piglets and whether the cytokines produced induces recruitment of immune cells at the site of injection and in the draining lymph nodes. We observed that, at the site of injection, PCEP induced increased production of IL-1β and IL-13 cytokines, increased cellular infiltration of macrophages, T and B cells, and other leucocytes especially neutrophils as well as showing necrotic debris which might cause release of damage associated molecular patterns (DAMPs) and activate the inflammasome. In the draining lymph nodes, the cytokines IL-1β and IL-6 were elevated and there was increased leucocyte infiltration. No changes in cytokine levels were detected in the blood after PCEP injection indicating that the immunostimulatory effect of PCEP is local but not systemic. Because i.d. injection of PCEP induced signs of necrosis (cell death), we investigated whether reduction of the adjuvant dose reduced tissue damage without negatively impacting antigen-specific immune responses. We conducted two studies to address this issue. In the first study, we injected piglets i.d with varying doses of PCEP alone as follows: 500 μg, 100 μg, or 20 μg PCEP into piglets and evaluated the inflammatory responses. The four parameters evaluated were granuloma formation, lymphocytic infiltration, necrosis, and suppurative inflammation at the injection site and the draining lymph nodes over 14 days. When PCEP was injected alone, we observed that only 500 μg consistently induced significant necrosis and suppurative inflammation. However, the medium dose (100 μg) PCEP did induce significant skin granulomas and lymphocyte infiltration, where as the only significant response induced in the skin by the lower dose (20 μg) PCEP was lymphocyte infiltration. In the draining lymph nodes, only 500 ug PCEP significantly higher suppurative inflammation. No necrosis or granuloma was observed in the lymph nodes in all the doses. Thus, the high dose of adjuvant triggered the most significant pathological signs of tissue damage at both sites (skin and draining lymph nodes). In the second study, we co-injected i.d varying doses of PCEP 500 μg, 100 μg, 20 μg, or 4 μg co-formulated with inactivated swine inﬂuenza virus (SIV) H1N1 antigen and measured the four parameters of inflammatory response (granuloma formation, lymphocytic infiltration, necrosis, and suppurative inflammation) at days 20 and 41 after a single injection at each sites and also assayed SIV H1N1-specific antibody titers. We observed that the highest dose of the adjuvant PCEP (500 μg) induced significant inflammatory responses in 3 of the 4 parameters assessed at day 20, and by day 41, this high dose of adjuvant had caused significant tissue response in all the four parameters assessed. Interestingly, only 500 μg of PCEP induced significant granuloma formation and necrosis (the more severe lesions) at both time points. Of the lower doses, 100 μg and 20 μg of PCEP both induced significant lymphocytic infiltration compared to 4 μg of PCEP, SIV and PBS groups. The lowest dose, 4 μg of PCEP did not induce any significant inflammatory response in any of the four parameters assessed. When SIV H1N1-specific antibody titers were assessed in immunized animals, only 500 μg, 100 μg and 20 μg of PCEP induced significant antibody responses when compared to SIV H1N1, confirming that these doses of PCEP had adjuvant activity. In contrast, 4 μg of PCEP did not induce any significant SIV H1N1-specific antibody titers, indicating that this dose did not have adjuvant activity. These results suggest that induction of inflammatory responses at the site of injection is necessary for adjuvant activity. However, not all responses assessed may be required for adjuvant activity as induction of very severe inflammatory responses was not associated with any additional increase in antigen-specific antibody titres. Thus, the quality of the inflammatory response is important but severe inflammation was not beneficial to antigen-specific immune responses Together, our data indicate that in pigs, the adjuvant PCEP stimulated early inflammatory responses at the injection site, creating an immunocompetent environment that led to activation of innate immune response genes, production of cytokines and chemokines, and recruitment of various immune cells. These events contribute to the adjuvant activity of PCEP. We propose a MOA model whereby PCEP induces tissue damage at the site of injection and the subsequent release of damage associated molecular patterns (DAMPs) which may activate the inflammasome and contribute to increased immunogenicity of the co-administered antigens.
Innate immunity and adjuvants
Doctor of Philosophy (Ph.D.)
School of Public Health
Vaccinology and Immunotherapeutics