Developing Human Intestinal in vitro Organ Culture and an in vitro M-Cell Model as Platforms for Investigation of Novel Oral Vaccines

In Taipei Medical University Shuang Ho Hospital, we are developing human intestinal in vitro organ culture (IVOC) to maintain human intestinal biopsies viable outside human bodies for 3-8 hours. This model has evolved from non-polarized IVOC to polarized IVOC (pIVOC), providing an apical exposure to simulate an in vivo enteric infection route. Because human tissue samples are irreplaceable for studying the genuine human host responses, IVOC provides a valuable platform that is suitable for studying human mucosal immune responses after early interactions with enteric pathogens (or foods, vaccines, and drugs). Additionally, an in vitro M cell model has been developed using Transwells to culture polarized Caco-2 cell monolayers for 14 days and subsequently co-culture with basolateral Raji B lymphocytes for another 4-6 days. This model can transform intestinal Caco-2 cells into M cells which are characterized morphologically having sparse, irregular microvilli on their apical surface, with a basolateral cytoplasmic pocket harboring immune cells, and functionally having transcytotic capacity of particles to the invaginated immune cells. Since intestinal M cells, the specialized antigen-sampling cells of the mucosal immune system, are exploited by Salmonella as a route of invasion and play a key role in activation of host immunity, the in vitro M cell model is feasible for further studies in the interactions between oral vaccines and human M cells/B cells. In my preliminary study, a big-scale gene screen was conducted in Cambridge using 1,440 transposon mutants of Salmonella Typhimurium wild-type SL1344 to infect HeLa cells in vitro and Transposon Directed Insertion-site Sequencing (TraDIS) was subsequently applied for detection of the attenuated transposon mutants in the conditioned output pools. Two unreported Salmonella genes wzxE and speG were discovered to be responsible for bacterial invasion and intracellular replication in human epithelial cells, respectively. The wzxE mutant can adhere to but can not significantly invade HeLa cells, and the speG mutant can invade but can not significantly replicate inside various human cells. Therefore, these two attenuated mutants are potential candidates as oral vaccine vectors. Taken together, this study aims to develop the pIVOC and in vitro M cell models as platforms for testing oral vaccines and candidate vaccine vectors like the attenuated S. Typhimurium mutants identified in our preliminary studies. Since two licensed rotavirus oral vaccines Rotarix and RotaTeq are available with known immunogens, it is valuable to investigate these two vaccines in the proposed models for assessing host immunity using the relevant parameters such as rotavirus-specific IgG and IgA, IL-15 and TLR-3 expression on intestinal lymphocytes, and T-cell cytokines (e.g. IL-2, IL-6, IL-10, IFN-γ). Furthermore, recombinant oral vaccines will be constructed using attenuated Salmonella Typhimurium mutants which carry foreign DNA to express rotavirus immunogens such as VP4 and VP7. These recombinant oral vaccines will be examined in both models to determine whether they can trigger host immunity similarly to two rotavirus vaccines. If successful, the human intestinal IVOC and in vitro M-cell models can be feasible platforms to assess novel oral vaccines before in vivo animal studies and clinical trials are practicable.