Extracellular matrix from porcine small intestinal submucosa (SIS) as immune adjuvants

Abstract

Porcine small intestinal submucosa (SIS) of Cook Biotech is licensed and widely used for tissue remodeling in humans. SIS was shown to be highly effective as an adjuvant in model studies with prostate and ovarian cancer vaccines. However, SIS adjuvanticity relative to alum, another important human-licensed adjuvant, has not yet been delineated in terms of activation of innate immunity via inflammasomes and boosting of antibody responses to soluble proteins and hapten-protein conjugates. We used ovalbumin, and a hapten-protein conjugate, phthalate-keyhole limpet hemocyanin. The evaluation of SIS was conducted in BALB/c and C57BL/6 mice using both intraperitoneal and subcutaneous routes. Inflammatory responses were studied by microarray profiling of chemokines and cytokines and by qPCR of inflammasomes-related genes. Results showed that SIS affected cytokine and chemokines microenvironments such as up-regulation of IL-4 and CD30-ligand and activation of chemotactic factors LIX and KC (neutrophil chemotactic factors), MCP-1 (monocytes chemotactic factors), MIP 1-α (macrophage chemotactic factor) and lymphotactin, as well as, growth factors like M-CSF. SIS also promoted gene expression of Nod-like receptors (NLR) and associated downstream effectors. However, in contrast to alum, SIS had no effects on pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) or NLRP3, but it appeared to promote both Th1 and Th2 responses under different conditions. Lastly, it was as effective as alum in engendering a lasting and specific antibody response, primarily of IgG1 type.

Figure 1. Effects of porcine small intestinal submucosa (SIS) on chemokine and cytokine protein microenvironment in BALB/c peritoneal exudates.

BALB/c mice were injected i.p. with SIS preparations (SIS-H or SIS-M) without the antigens and peritoneal exudates harvested after 24 hr. Control groups were treated with PBS buffer. Peritoneal fluids were assayed to determine chemokine and cytokine expression as detailed in Materials and Methods. Data are expressed as the mean relative intensity relative to positive control of each chemokine or cytokine protein detected using pooled peritoneal fluids of 3 mice per group in duplicate. The result is an average of two separate experiments.

Figure 2. RT-PCR Microarray analysis of transcriptome profiles of inflammatory genes induced by vaccine adjuvants alone in mouse peritoneum.

Eighty-four genes were assessed and those genes up-regulated (A), or down regulated (B) with an average log2 ratio ≥1.5 were selected and plotted as a Venn diagram. Results for the alum-treated group refer to our previous study .

Figure 3. Effects of SIS adjuvants on anti-ovalbumin antibody response in C57BL/6 mice.

Mouse serum samples were collected on day 5 after (A) 2nd, and (B) 3rd immunizations with OVA plus adjuvants as described under Materials and Methods. Antibody responses were assessed using ELISA, (C) determination of IgG sub-classes of anti-OVA antibodies, induced with OVA containing adjuvants, in sera from the 3rd immunization. Commercial isotyping kits (Southern-Biotech) were used to perform ELISA in serum samples (starting at dilution 1∶1000). The results represent mean ± SD (n = 6 mice per group in two separate experiments). The significance in experimental groups relative to the group given antigen only (no adjuvant group) was calculated at the level of p≤0.05.

Figure 4. Evaluation of phthalate-KLH antibody response in BALB/c mice.

Mice were immunized with phthalate-KLH conjugate emulsified in different adjuvants. Serum samples were collected as described under Materials and Methods and diluted to 1∶1000 in 0.5% PBS/BSA. Anti-phthalate, anti-KLH, and anti-DNA antibody levels were determined using ELISA as described. The results represent the mean ± SD (n = 6 mice per group in two separate experiments). The significance in experimental groups was determined relative to the group given antigen only (no adjuvant group) at the level of p≤0.05.

Figure 5. Determination of IgG sub-classes of anti-phthalate antibodies induced with phthalate-KLH conjugates in different adjuvants.

This was done using commercial ELISA isotyping kits with serum samples (at dilution 1∶1000) collected after (A) intraperitoneal, or (B) subcutaneous immunization as described in Materials and Methods. Results represent mean ± SD (n = 6 mice per group in two separate experiments). The significance in experimental groups was determined relative to the group given antigen only (no adjuvant group) at the level of p≤0.05.