Jun Kunisawa

Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins.

In order to design an optimized liposome immunoadjuvant for inducing cell-mediated immune response against soluble proteinaceous antigens, we investigated the effect of liposomal surface charge on the immunoadjuvant action. Positively charged liposomes containing soluble antigens functioned as a more potent inducer of antigen-specific cytotoxic T lymphocyte responses and delayed type hypersensitivity response than negatively charged and neutral liposomes containing the same concentrations of antigens. To clarify the reason of the differential immune response, we examined the delivery of soluble proteins by the liposomes into the cytoplasm of macrophages, using fragment A of diphtheria toxin (DTA) as a marker. We found that positively charged liposomes encapsulating DTA are cytotoxic to macrophages, while empty positively charged liposomes, DTA in negatively charged and neutral liposomes are not. Consistent with this, only macrophages pulsed with OVA in positively charged liposomes could significantly stimulate OVA-specific, class I MHC-restricted T cell hybridoma. These results suggest that the positively charged liposomes can deliver proteinaceous antigens efficiently into the cytoplasm of the macrophages/antigen-presenting cells, where the antigens are processed to be presented by class I MHC molecules to induce the cell-mediated immune response. Possible development of the safe and effective vaccine is discussed.

Fusogenic liposomes efficiently deliver exogenous antigen through the cytoplasm into the MHC class I processing pathway

Exogenous soluble proteins enter the endosomal pathway by endocytosis and are presented in association with MHC class II rather than class I. In contrast, the delivery of exogenous protein antigens (Ag) into the cytosol generates MHC class I‐restricted cytotoxic T lymphocytes (CTL) responses. Although several immunization approaches, such as the utilization of liposomes, have induced the in vivo priming of MHC class I‐restricted CTL responses to protein Ag, it remains unclear whether this priming results from the direct delivery of protein Ag to the cytosol. Here we report that fusogenic liposomes (FL), which are prepared by fusing simple liposomes with Sendai virus particles, can deliver the encapsulated soluble protein directly into the cytosol of cells cultured concurrently and introduce it into the conventional MHC class I Ag presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FL but not in simple liposomes results in the potent priming of OVA‐specific CTL. Thus, FL function as an efficient tool for the delivery of CTL vaccines.

Sendai Virus Fusion Protein-Mediates Simultaneous Induction of MHC Class I/II-Dependent Mucosal and Systemic Immune Responses Via the Nasopharyngeal-Associated Lymphoreticular Tissue Immune System

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4+ Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.

Pharmacotherapy by intracellular delivery of drugs using fusogenic liposomes: application to vaccine development.

We prepared fusogenic liposomes by fusing conventional liposomes with an ultra-violet inactivated Sendai virus. Fusogenic liposomes can deliver encapsulated contents into the cytoplasm directly in a Sendai virus fusion-dependent manner. Based on the high delivery rates into the cytoplasm, we originally planned to apply the fusogenic liposomes to cancer chemotherapy and gene therapy. We have recently also examined the use of fusogenic liposomes as an antigen delivery vehicle. In terms of vaccine development, cytoplasmic delivery is crucial for the induction of the cytotoxic T lymphocyte (CTL) responses that play a pivotal role against infectious diseases and cancer. In this context, our recent studies suggested that fusogenic liposomes could deliver encapsulated antigens into the cytoplasm and induce MHC class I-restricted, antigen-specific CTL responses. In addition, fusogenic liposomes are also effective as a mucosal vaccine carrier. In this review, we present the feasibility of fusogenic liposomes as a versatile and effective antigen delivery system.

Transcription factor Spi-B–dependent and –independent pathways for the development of Peyer’s patch M cells

Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell–specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B–deficient mice showed reduced expression of most, but not all, other M-cell–specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B–deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B–deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.

Characterization of mucoadhesive microspheres for the induction of mucosal and systemic immune responses

In the present study, mucoadhesive polymer-dispersed microspheres (MS) were examined as a potential mucosal vaccine carrier. A major focus of the study was aimed at directly assessing the influence of antigen release and persistence in the mouse small intestine for the induction of mucosal and systemic immune responses. BALB/c mice were immunized with various forms of MS containing chicken egg ovalbumin (OVA) by administration into the duodenum. No detectable anti-OVA immune responses were observed following the administration of OVA alone or that of MS without mucoadhesive polymer (MS-0). MS-10 containing 10% mucoadhesive polymer rapidly released OVA and hardly induced anti-OVA antibody responses in either serum or fecal extracts. In contrast, MS-8 and MS-6 (with 8 and 6% mucoadhesive polymer) showed controlled release of OVA, which elicited strong OVA-specific IgG and IgA responses in serum and fecal extracts, respectively. Additionally, the strongest immune responses were induced in mice immunized with MS-8, which had both the optimal release-profile of OVA and the longest persistence in the small intestine. These findings indicate that antigen movement in the small intestine is an important factor and that appropriate microsphere forms with mucoadhesive polymers might be useful candidates as mucosal vaccine carriers.

Lack of antigen-specific immune responses in anti-IL-7 receptor α chain antibody-treated Peyer's patch-null mice following intestinal immunization with microencapsulated antigen

Peyers patches (PP) represent a well‐characterized inductive site in gut‐associated lymphoid tissue that actively acquires antigens from the intestinal lumen. It was reported that organized PP are not required for antigen‐specific IgA responses induced by oral immunization with soluble antigen mixed with the mucosal adjuvant, cholera toxin. However, the role of PP in the induction of mucosal and systemic immune responses remains to be clarified in the case of particulate antigen. Here, we created PP‐null mice by treating them with monoclonal anti‐IL‐7 receptor α chain (IL‐7Rα) antibody during gestation and then immunized with antigen‐encapsulated poly‐lactic acid (PLA) microspheres. Brisk OVA‐specific antibody responses were noted in serum and fecal extracts of normal mice following direct intestinal immunization with OVA in PBS (OVA‐PBS) as well as in PLA‐microspheres (OVA‐MS). Antibody production was similarly elevated in PP‐null mice immunized with OVA‐PBS via direct injection into the intestinal tract. In contrast, OVA‐specific antibody responses were dramatically decreased in both serum and fecal extracts collected from PP‐null mice immunized intestinally with OVA‐MS. These results were further supported by the number of OVA‐specific antibody‐forming cells detected in the spleen and intestinal lamina propria. PP deficiency also resulted in the reduction in OVA‐specific Th1/Th2 cell responses in the spleen and mesenteric lymph nodes of mice intestinally immunized with OVA‐MS. These results suggested that organized PP do, in fact, play a crucial role in the induction of antigen‐specific immune responses against ingested particulate antigen.