Browsing by Subject "Inflammatory Bowel Disease"

Inflammatory bowel disease (IBD) is a chronic disorder characterized by persistent inflammation in the gastrointestinal tract. Current therapies for IBD, such as anti-inflammatory or immunosuppressant drugs and anti-cytokine biologics, only temporarily alleviate symptoms and vary widely in effectiveness among patients. Consequently, there exists a critical unmet need for a long-lasting and broadly effective IBD treatment. Natural antibodies against the small molecule epitope phosphorylcholine (PC) are an important component of innate immunity with diverse functions including the clearance of bacterial and autologous targets in a non-inflammatory manner. The cells that produce these antibodies, B1a cells, however, have been shown to be reduced in patients with IBD, with this decrease being associated with a more advanced disease state. It has also been demonstrated that the adoptive transfer of B1a cells in a murine model of IBD results in the increased production of anti-PC antibodies and lessens disease severity. Furthermore, active immunotherapies are an alternative to monoclonal antibody biologics and a promising approach for generating a long-lasting IBD therapy because they exploit the ability of a patient’s own immune system to produce antibodies against a therapeutic target. Building on these concepts, we strove to develop an active immunotherapy consisting of PC as an epitope displayed on self-assembling peptide nanofibers to produce a therapeutic anti-PC antibody response for the treatment of IBD. The first part of this dissertation (Chapter 3) describes the process of designing, determining the therapeutic efficacy of, and gaining mechanistic insights into a nanofiber-based anti-PC active immunotherapy. We began by developing conjugation strategies for attaching PC to Q11 self-assembling peptides to render PC immunogenic. In an effort to find a balance between immunogenicity, stability, and ease of synthesis, we compared phosphoramidite and phosphodiester linkages for PC and concluded that the phosphodiester linkage was critical for PC epitope integrity. We then investigated how altering the multivalency of PC on Q11 nanofibers could further augment the anti-PC antibody response by synthesizing two nanofiber and PC conjugates with either 1 or 1-4 PC copies per Q11 peptide termed PC-Q11 and PCM-Q11, respectively. Intraperitoneal (i.p.) immunization with PCM-Q11 was found to induce a significantly greater anti-PC antibody response than i.p. immunization with PC-Q11. Additionally, PCM-Q11 was more selectively taken up by and able to activate natural antibody-producing B1a cells compared to all other B cells than PC-Q11 or Q11 alone. Further, control over the immune phenotype elicited was achieved via the inclusion of a T-cell epitope and/or CpG adjuvant, with the addition of both greatly augmenting the immune response elicited. We then evaluated the efficacy of immunizations with PCM-Q11/T-cell epitope with or without CpG in several different dextran sodium sulfate (DSS)-induced murine colitis models. We first investigated the ability of these immunizations to prevent severe disease when administered prior to the induction of a 30-day chronic DSS colitis model. Interestingly, immunization with both formulations was protective, significantly improving weight loss, disease severity indices, and colon lengths over unimmunized controls. This efficacy was repeated in a 1 cycle (10-day) DSS colitis model in both male and female mice and not attributed to CpG administration alone. Immunizations against PC also lowered bacterial spread to the spleen due to colon damage, with this effect being more pronounced in female mice. Additionally, we determined the efficacy of PCM-Q11 immunizations in a therapeutic setting where mice received one cycle of DSS colitis followed by three immunizations and then one more cycle of DSS colitis. This showed that immunizations with PCM-Q11 have therapeutic efficacy by significantly improving weight loss, disease activity indices, colon lengths, and bacterial spread to the spleen in this model. Furthermore, we have conducted several other studies to gain mechanistic insight into the observed efficacy of PCM-Q11 immunizations. We found that anti-PC immunization decreases microbiome diversity. We were also able to use flow cytometry to detect IgG and IgM antibodies in serum from mice immunized with PCM-Q11 that bind apoptotic colon epithelial cells in vitro. Additionally, we have shown through passive transfer of PCM-Q11 immunized sera that induced anti-PC antibodies offer some protection against severe DSS-induced colitis. Moreover, through both histological examination of colon damage and immunofluorescence imaging of tight junction proteins, we determined that PCM-Q11 immunizations were not acting by improving barrier function in the colon. Finally, we observed reduced efficacy of PCM-Q11 immunizations in an Il10-/- murine model of colitis. Collectively, this data demonstrates that immunization with PCM-Q11 was both preventative and therapeutic in multiple DSS-induced models of colitis in mice, with considerable efficacy attributed to the induced anti-PC antibody response. In the second part of this dissertation (Chapter 4), peptide nanofibers were modified for use as oral vaccines. While oral delivery offers direct access for eliciting immune responses within the gastrointestinal tract, it poses substantial obstacles for vaccines to overcome including acidic and proteolytic environments, thick mucus barriers, and a limited window for absorption. We therefore focused on two main design improvements to peptide nanofibers: synthesis with protease-resistant D-amino acids and incorporation of muco-penetrative peptide sequences rich in proline, alanine, and serine (PAS). We showed that D-amino acid Q11 was not degraded in simulated gastrointestinal environments in contrast to its L-amino acid counterpart. Additionally, we determined that PASylation enhanced muco-penetration in vitro and accelerated nanofiber transport through the GI tract in vivo. Ultimately, however, we found that oral immunization with PASylated L-amino acid nanofibers with cholera toxin B subunit mucosal adjuvant was the optimal formulation for the generation of both local and systemic immune responses. The primary areas affected in IBD are the distal small intestines and the colon, so the induction of therapeutic antibodies in these tissues is paramount. Thus, we sought to translate the above design principles to the small molecule epitope, PC, to enable oral administration. Oral immunization with PASylated anti-PC formulations was able to generate both local and systemic anti-PC immune responses. We then illustrated that oral vaccination with PASylated PC-bearing nanofibers was effective in both therapeutic and prophylactic models of DSS-induced colitis, observing comparable reductions in disease severity to i.p. anti-PC immunizations, thus, increasing the translatability of our therapy by offering a needle-free formulation. Overall, this data indicates that PASylated supramolecular peptide nanofibers are a promising platform for oral immunization. This dissertation outlines an encouraging first example of an active immunotherapy engaging natural antibody responses against phosphorylcholine as a durable therapy for IBD. More broadly, the strategies developed offer a potentially versatile approach for engaging natural antibody therapies and oral nanofiber peptide vaccines towards a variety of inflammatory and infectious diseases.