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Oligomannose-coated liposomes (OMLs) comprised of dipalmitoylphosphatidylcholine, cholesterol and Man3-DPPE at a molar ratio of 1:1:0.1 and particle diameters of about 1000 nm can induce liposome-encased antigen-specific strong Th1 immunity. In this study, we evaluated the effect of particle sizes of OMLs on induction of Th1 immune responses in mice. Spleen cells obtained from mice immunized with antigen-encapsulating OMLs with 1000- and 800-nm diameters secreted remarkably high levels of IFN-γ upon in vitro stimulation. In addition, sera of mice that received these OMLs had significantly higher titers of antigen-specific IgG2a than those of IgG1, which are commonly associated with Th1 responses. In contrast, treatment with antigen-encapsulating OMLs with 400- and 200-nm diameters failed to induce IFN-γ secretion from spleen cells, although these OMLs did elicit elevation of antigen-specific IgGs. In addition, the titers of serum antigen-specific IgG2a were the same as those of IgG1 in mice that received 400-nm OMLs. Resident peritoneal mononuclear phagocytes (MNPs) treated with OMLs of diameter ≥ 600 nm secreted IL-12, which is essential for induction of Th1 immune responses, while those treated with OMLs of ≤ 400 nm failed to produce this cytokine. However, 400-nm OMLs did induce enhanced expression of MHC class II and costimulatory molecules on MNPs, similarly to OMLs of ≥ 600 nm. Taken together, these results strongly indicate that OMLs of diameter ≥ 600 nm are required to induce Th1 immune responses against OML-encased antigens, although OMLs of diameter ≤ 400 nm can activate MNPs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, we assessed the potential of liposomes coated with a neoglycolipid containing α1-3,α1-6-mannotriose residues (Man3-DPPE; Manα1-6(Manα1-3)Manitol-DPPE) for in vitro activation and maturation of human mononuclear phagocytes. In response to treatment with Man3-DPPE-coated liposomes (Man3-OMLs), PMA-stimulated human THP-1 cells showed enhanced expression of CD40, CD80 and HLA-DR and secreted significant levels of IL-12p40. Among various linkages of Man2-DPPE-coated liposomes, only liposomes coated with Manα1-6Manitol-DPPE (α1-6Man2-DPPE) induced these cellular responses similarly to Man3-OML treatment. Liposomes coated with Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manitol-DPPE (Man5-DPPE) failed to activate the cells. These results suggest that an unsubstituted α1-6Man branch bound to a mannitol unit at the reducing end in Man3-DPPE is required for in vitro activation of human mononuclear phagocytes. Man3-OML-induced IL-12p40 production was not inhibited by BAY11-7082, an inhibitor of the MyD88-dependent signaling network, suggesting that TLRs are not involved in activation of human mononuclear phagocytes by Man3-OMLs. Stimulation of inflammatory monocytes or monocyte-derived dendritic cells (moDCs) with Man3-OMLs also induced enhanced expression of co-stimulatory molecules, HLA-DR, and CCR7, and IL-12p40 production from both types of cells. In response to Man3-OML treatment, moDCs but not inflammatory monocytes produced bioactive IL-12p70, which was enhanced by CD40 ligation. Thus, Man3-OMLs can activate naïve human mononuclear phagocytes and lead human moDCs to a fully matured status in vitro to elicit CTLs and a Th1 response without addition of inflammatory cytokines or TLR agonists.

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii. N. caninum is thought to be a major cause of abortion in cattle worldwide. Given the current situation of drastic economic losses and a lack of efficient control strategies against such parasites, the challenge to develop potent vaccine candidates and technologies remains. We investigated the immune stimulating activity of N. caninum cyclophilin (NcCyp) with and without a formulation with oligomannose-coated-liposomes (OML) as the potential adjuvant. NcCyp-OML activated NF-κB in RAW 264.7 cells and triggered interleukin (IL)-12p40 production from murine peritoneal macrophages. In BALB/c mice, immunization with NcCyp-OML was associated with the production of specific antibodies (IgG1 and IgG2a). The specific antibody (IgG1) against naked NcCyp was also observed after the challenge infection, but it was significantly lower than those of NcCyp-OML. Moreover, significant cellular immune responses were induced, including spleen cell proliferation and interferon-gamma production. The immunization of mice with NcCyp-OML, and to lesser extent with naked NcCyp, induced significant protection against challenge with a lethal dose of N. caninum compared with the PBS control group. This protection was associated with a higher survival rate, slight changes in body weight, and lower clinical score of mice. In addition, the significant protective efficacy of NcCyp-OML was confirmed in another mouse strain, male C57BL/6 mice. The current study revealed the marked contribution of Toll-like receptor 2 (TLR2) to the protective immunity triggered by NcCyp-OML because higher numbers of TLR2 -/- mice succumbed to a lethal dose of N. caninum compared with C57BL/6 mice. Moreover, prominent spleen cell proliferation and IFN-γ production was induced in NcCyp-OML-immunized mice by a TLR2-dependent mechanism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Oligomannose-coated liposomes (OMLs), containing entrapped antigens, serve as effective antigen delivery vehicles and as a novel adjuvant to induce antigen-specific cellular immune responses. However, in vitro activation of antigen-presenting cells (APCs) by OMLs has not yet been demonstrated. In this paper, we found that OMLs can deliver the antigens and the stimulatory signals into inflammatory monocytes in vitro, leading to differentiation of the cells to mature APCs. When OMLs were co-cultured with peripheral blood mononuclear cells from C57BL/6 mice in the presence of mouse serum, OMLs were preferentially incorporated into both Ly6C high monocytes and Ly6C low monocytes, which are referred to as murine inflammatory and resident monocytes, respectively. The expression of CD11c, CD80, CD86, CCR7, and MHC class II on the Ly6C high monocytes was significantly enhanced during the 24 h after OML uptake, whereas upregulation of these molecules on the Ly6C low monocytes was limited. In addition, the antigenic peptide of OVA encased in OMLs was presented on MHC class I of only Ly6C high monocytes. Furthermore, OVA-encasing OML-ingesting monocytes can activate CD8 + T cells from OT-1 mice, suggesting that antigens encapsulated in OMLs were cross-presented in inflammatory monocytes. Adoptive transfer of the monocytes that engulf OVA-encasing OMLs led to induction of an antigen-specific Th1 immune response in mice. Taken together, mature APCs can be generated from inflammatory monocytes in peripheral blood by ex vivo treatment of the cells with OMLs without any additional stimuli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have previously demonstrated that administration of oligomannose-coated liposomes (OMLs), in which an antigen is encased, induce antigen-specific Th1 immune responses and CTLs. In the present study, we showed that TLR4 signaling is required for the induction of specific immune responses following OML administration. In C3H/HeJ mice, which express a dysfunctional TLR4, the antigen-specific Th1 immune response could not be elicited following intraperitoneal administration of OVA-encased OMLs (OML/OVA). However, OML uptake by peritoneal cells, the subsequent production of IL-12 and the upregulation of co-stimulatory molecules and MHC class II on the cells in response to OML uptake occurred in C3H/HeJ mice to the same extent as in wild type C3H/HeN mice. In addition, peritoneal phagocytic cells from TLR4(-/-) mice that ingest OML/OVA can activate CD4(+) T cells from OT-II mice. On the other hand, the number of OML-ingesting peritoneal cells that migrated into mesenteric lymph nodes in C3H/HeJ mice was significantly less than that in C3H/HeN mice. Therefore, the chemotactic capability of OML-ingesting peritoneal phagocytes to the draining lymph nodes rather than the activation and maturation of the cells in response to OML uptake is impaired by lack of TLR4 signaling, and disorder of the Th1 immune response elicited by OMLs in mice, which lack TLR4 signaling, is due to the impairment of cell migration following OML uptake., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: The design and development of an effective malaria vaccine against the pre-erythrocytic and erythrocytic-stages of infection present a great challenge., Methods: In the present study, protective efficacy of oligomannose-coated liposome (OML)-entrapped merozoite and sporozoite antigens against Plasmodium berghei challenge infection in BALB/c mice was evaluated., Results: Subcutaneous immunization with truncated merozoite surface protein 1 entrapped with OML (OML-PbMSP1) prolonged survival, but failed to protect the mice from erythrocytic-stage infection, despite the antigen-specific antibody responses induced by the immunization regimen. In contrast, immunization with circumsporozoite protein entrapped with OML (OML-PbCSP) elicited antigen-specific humoral and cellular responses, which correlated with substantial protection against sporozoite challenge infections., Conclusions: The current results represent the use of an oligomannose-coated liposome-based vaccine against pre-erythrocytic and erythrocytic stages malaria infection. This approach may offer a new vaccination strategy against malaria infection.

Toxoplasma gondii is an obligate intracellular parasite that can infect a variety of mammals and birds, causing toxoplasmosis. Several types of vaccines against T. gondii have been developed, but these have limitations in terms of their safety and inadequate efficacy. T. gondii profilin (TgPF) is a potential immunodominant antigen for a candidate vaccine. In this study, we encapsulated TgPF in oligomannose-coated liposomes (OMLs) to evaluate the immune response induced by this vaccine. C57BL/6 mice were immunized with TgPF-OML three times at 14-day intervals and challenged with T. gondii. TgPF-OML increased the survival of the mice and reduced the parasite burden in their brains after T. gondii infection. Immunization with TgPF-OML also induced TgPF-specific interferon-γ production and IgG antibodies in mice. Our results demonstrate that OML-encapsulated TgPF triggers strong humoral and cellular responses against T. gondii, and that TgPF-OML is a candidate vaccine that warrants further development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We evaluated the carbohydrate preferences of the C-type lectin receptors (CLRs) SIGNR1, SIGNR3, and Langerin as pathogen-uptake receptors based on uptake of liposomes consisting of cholesterol, DPPC, and various neoglycolipids at molar ratios of 10:10:1 and 10:7:4, respectively, using non-phagocytic CHO cells that express these receptors transiently. SIGNR1-expressing cells ingested liposomes coated with neoglycolipids with terminal mannose residues, such as Man2-, Man3-, and Man5-DPPE, and with a terminal N-acetylglucosamine. SIGNR1 mediated uptake of Man3-DPPE-coated liposomes most efficiently. Uptake of liposomes with lower neoglycolipid content by SIGNR3- or Langerin-expressing cells was slight or negligible, but uptake into these cells was detected for liposomes with higher neoglycolipid content. SIGNR1-expressing cells clearly ingested liposomes coated with Lewis X antigen, whereas SIGNR3- or Langerin-expressing cells barely ingested these liposomes, even at the higher neoglycolipid content. In contrast, SIGNR3 or Langerin, but not SIGNR1, mediated uptake of liposomes coated with blood group H antigen. These results indicate that CLRs with similar carbohydrate-recognition characteristics have distinct properties as pathogen-uptake receptors for carbohydrate-decorated particles.

Peritoneal macrophages (PEMs) preferentially and rapidly take up oligomannose-coated liposomes (OMLs) and subsequently mature to induce a Th-1 immune response following administration of OMLs into the peritoneal cavity. Here, we examine the contributions of complement component C3 and complement receptor type 3 (CR3) to carbohydrate-dependent uptake of OMLs by PEMs. Effective uptake of OMLs into PEMs in vitro was observed only in the presence of peritoneal fluid (PF), and OMLs incubated with PF were incorporated by PEMs in vitro in the absence of PF. These phenomena were inhibited by methyl-alpha-mannoside, N-acetylglucosamine or EDTA, but not by galactose. Pull-down analysis followed by peptide mass fingerprinting of PF-treated OMLs indicated that the OMLs were opsonized with complement fragment iC3b. In vivo uptake of OMLs by PEMs was inhibited by intraperitoneal injection of an antibody against CR3, a receptor for iC3b, and OML uptake by PEMs in the peritoneal cavity was not observed in C3-deficient mice. Thus, our results indicate that OMLs are opsonized with iC3b in a mannose-dependent manner in the peritoneal cavity and then incorporated into PEMs via CR3.

Highlights: [•] Cattle were immunized with M3-NcGRA7. [•] M3-NcGRA7 induced antigen-specific antibody and IFN-γ production from the PBMC. [•] M3-NcGRA7 vaccination reduced parasite load in the brain. [•] M3-NcGRA7 (50μg antigen) vaccination induced protective immune responses in cattle. [Copyright &y& Elsevier]