Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia

Abstract

Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4 + lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4 + CD25 − FoxP3 − LAP + T cells (but not of CD4 + CD25 + FoxP3 + T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4 + CD25 − LAP + Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4 + CD25 + T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.

Green giant—a tiny chloroplast genome with mighty power to produce high‐value proteins: history and phylogeny

“…Upon oral delivery, PDs are protected in the stomach from digestion by enzymes because human or animal enzymes do not break beta linkage of plant cell wall polymers. However, when intact plant cells reach the gut, enzymes released by commensal microbes in the gut digest plant cell wall, releasing PDs into the gut lumen (Kumar et al ., 2020). When tags are fused to PDs, they cross gut epithelium and reach the circulatory or immune system (Figure 2).…”

Preclinical development of plant‐based oral immune modulatory therapy for haemophilia B

“…CTB functions as a transmucosal carrier, while plant cell wall provides bioencapsulation. The enzymatic activity provided by commensal bacteria causes the CTB fusion protein to be released in small intestine (Kumar et al., 2020 ; Xiao et al., 2016 ). Pentameric CTB fusion proteins then bind to the GM1 receptor that is abundantly present on the surface of gut epithelial cells and on dendritic cells.…”

“…Treg induction, including LAP + Treg, occurs in the lamina propria of the small intestine (Kumar et al, 2020). While the capacity for microbial degradation of plant cell wall is greater in the large intestine, our recent microbiome analysis shows that bacterial species producing enzyme that hydrolyse several cell wall components are present in the small intestine, in particular in sections closer to the stomach such as the duodenum (Kumar et al, 2020). Berglund et al (2017) recently established key regulatory criteria for FDA approval of orally delivered plant cells expressing therapeutic proteins including detailed product characterization, product safety, integrity, dose, potency, stability, moisture content and bioburden.…”

“…Therefore, new algorithms were recently developed using >130 sequenced chloroplast genomes to optimize expression of human genes in chloroplasts (Kwon et al, 2016, Kwon et al, 2018Park et al, 2020;Daniell et al, 2020). Bioencapsulation in plant cells protected by cell wall also prevents degradation from harsh degrading enzymes after oral administration until the drugs are acted upon by enzymes produced by specific commensals in the mammalian gut (Kumar et al 2020). When the target antigen is tagged with carrier proteins such as CTB (cholera toxin subunit B), efficient transmucosal uptake and delivery to the immune or circulatory system is accomplished (Daniell et al, 2019;Kwon and Daniell, 2016;Xiao et al, 2016).…”

“…This approach takes advantage of the small intestine’s natural ability to promote tolerance to food antigen. Antigen processing by the immune system of the small intestine induces regulatory T cells (T reg ) that can systemically suppress immune responses (Bertolini et al., 2021 ; Kuhn and Weiner, 2016 ; Kumar et al., 2020 ; Wang et al., 2015 ).…”