• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.709-717
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• 2017

Mucosal tissues are the initial site through which most pathogens invade. As such, vaccines and adjuvants that modulate mucosal immune functions have emerged as important agents for disease prevention. Herein, we investigated the immunomodulatory mechanisms of the B subunit of Escherichia coli heat-labile enterotoxin type IIa ($LT-IIa-B_5$), a potent non-toxic mucosal adjuvant. Alternations in gene expression in response to $LT-IIa-B_5$ were identified using a genome-wide transcriptional microarray that focused on dendritic cells (DC), a type of cell that broadly orchestrates adaptive and innate immune responses. We found that $LT-IIa-B_5$ enhanced the homing capacity of DC into the lymph nodes and selectively regulated transcription of pro-inflammatory cytokines, chemokines, and cytokine receptors. These data are consistent with a model in which directional activation and differentiation of immune cells by $LT-IIa-B_5$ serve as a critical mechanism whereby this potent adjuvant amplifies mucosal immunity to co-administered antigens.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.490-497
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• 2004

Although the E. coli heat-labile enterotoxin B subunit (LTB) is known to be a potent mucosal adjuvant towards co-administrated unrelated antigens and immunoregulator in T-helper 1-type-mediated autoimmune diseases, a more efficient and useful LTB is still required for prospective vaccine adjuvants. To determine whether a novel chimeric LTB subunit would produce an enhanced mucosal adjuvant activity and immune response, a number of LTB subunits were genetically fused with chimeric proteins using the epitope genes of the envelope glycoprotein E2 (gp51-54) from the classical swine fever virus (CSFV). It was found that the total serum immunoglobulin (Ig) levels of BALB/c mice orally immunized with chimeric proteins containing an N-terminal linked LTB subunit (LE1, LE2, and LE3) were higher than those of mice immunized with LTB, E2 epitope, and chimeric proteins that contained a C-terminal linked LTB subunit. In particular, immunization with LE1 markedly increased both the total serum Ig and fecal IgA level compared to immunization with LTB or the E2 epitope. Accordingly, the current results demonstrated that the LTB subunit in a chimeric protein exhibited a strong mucosal adjuvant effect as a carrier molecule, while the chimeric protein containing the LTB subunit stimulated the mucosal immune system by mediating the induction of antigen-specific serum Ig and mucosal IgA. Consequently, an LE1-mediated mucosal response may contribute to the development of effective antidiarrhea vaccine adjuvants.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.283-291
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• 2010

Vaccine is one of the best known and most successful applications of immunological theory to human health and it protects human life through inducing the immune response in systemic compartment. However, when we consider the fact that mucosal epithelium is exposed to diverse foreign materials including viruses, bacteria, and food antigens and protects body from entry of unwanted materials using layer of tightly joined epithelial cells, establishing the immunological barrier on the lining of mucosal surfaces is believed to be an effective strategy to protect body from unwanted antigens. Unfortunately, however, oral mucosal site, which is considered as the best target to induce mucosal immune response due to application convenience, is prone to induce immune tolerance rather than immune stimulation. Since intestinal epithelium is tightly organized, a prerequisite for successful mucosal vaccination is delivery of antigen to mucosal immune induction site including a complex system of highly specialized cells such as M cells. Consequently, development of efficient mucosal adjuvant capable of introducing antigens to mucosal immune induction site and overcome oral tolerance is an important subject in oral vaccine development. In this review, various approaches on the development of oral mucosal adjuvants being suggested for effective oral mucosal immune induction.

• IMMUNE NETWORK
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• v.15 no.1
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• pp.9-15
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• 2015

Streptococcus pneumoniae (the pneumococcus) causes a major upper respiratory tract infection often leading to severe illness and death in the elderly. Thus, it is important to induce safe and effective mucosal immunity against this pathogen in order to prevent pnuemocaccal infection. However, this is a very difficult task to elicit protective mucosal IgA antibody responses in older individuals. A combind nasal adjuvant consisting of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligonucleotide (CpG ODN) successfully enhanced S. pneumoniae-specific mucosal immunity in aged mice. In particular, a pneumococcal surface protein A-based nasal vaccine given with pFL and CpG ODN induced complete protection from S. pneumoniae infection. These results show that nasal delivery of a combined DNA adjuvant offers an attractive potential for protection against the pneumococcus in the elderly.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.110-117
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• 2003

Background: The usefulness of DNA vaccine at priming step of heterologous prime-boost vaccination led to DNA vaccine closer to practical reality. DNA vaccine priming followed by recombinant viral vector boosting via systemic route induces optimal systemic immunity but no mucosal immunity. Mucosal vaccination of the reversed protocol (recombinant viral vector priming-DNA vaccine boosting), however, can induce both maximal mucosal and systemic immunity. Here, we tried to address the reason why the mucosal protocol of prime-boost vaccination differs from that of systemic vaccination. Methods: To address the importance of primary immunity induced at priming step, mice were primed with different doses of DNA vaccine or coadministration of DNA vaccine plus mucosal adjuvant, and immunity including serum IgG and mucosal IgA was then determined following boosting with recombinant viral vector. Next, to assess influence of humoral pre-existing immunity on boosting $CD8^+$ T cell-mediated immunity, $CD8^+$ T cell-mediated immunity in B cell-deficient (${\mu}K/O$) mice immunized with prime-boost regimens was evaluated by CTL assay and $IFN-{\gamma}$-producing cells. Results: Immunity primed with recombinant viral vector was effectively boosted with DNA vaccine even 60 days later. In particular, animals primed by increasing doses of DNA vaccine or incorporating an adjuvant at priming step and boosted by recombinant viral vector elicited comparable responses to recombinant viral vector primed-DNA vaccine boosted group. Humoral pre-existing immunity was also unlikely to interfere the boosting effect of $CD8^+$ T cell-mediated immunity by recombinant viral vector. Conclusion: This report provides the important point that optimally primed responses should be considered in mucosal immunization of heterologous prime-boost regimens for inducing the effective boosting at both mucosal and systemic sites.

• IMMUNE NETWORK
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• v.12 no.5
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• pp.165-175
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• 2012

Vaccination is one of the most effective methods available to prevent infectious diseases. Mucosa, which are exposed to heavy loads of commensal and pathogenic microorganisms, are one of the first areas where infections are established, and therefore have frontline status in immunity, making mucosa ideal sites for vaccine application. Moreover, vaccination through the mucosal immune system could induce effective systemic immune responses together with mucosal immunity in contrast to parenteral vaccination, which is a poor inducer of effective immunity at mucosal surfaces. Among mucosal vaccines, oral mucosal vaccines have the advantages of ease and low cost of vaccine administration. The oral mucosal immune system, however, is generally recognized as poorly immunogenic due to the frequent induction of tolerance against orally-introduced antigens. Consequently, a prerequisite for successful mucosal vaccination is that the orally introduced antigen should be transported across the mucosal surface into the mucosa-associated lymphoid tissue (MALT). In particular, M cells are responsible for antigen up-take into MALT, and the rapid and effective transcytotic activity of M cells makes them an attractive target for mucosal vaccine delivery, although simple transport of the antigen into M cells does not guarantee the induction of specific immune responses. Consequently, development of mucosal vaccine adjuvants based on an understanding of the biology of M cells has attracted much research interest. Here, we review the characteristics of the oral mucosal immune system and delineate strategies to design effective oral mucosal vaccines with an emphasis on mucosal vaccine adjuvants.

• Journal of Microbiology and Biotechnology
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• v.27 no.8
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• pp.1461-1471
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• 2017

Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli, we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2003.06a
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• pp.55-62
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• 2003

The mucosal immune system provides a first line of defense against invasion of infectious agents via inhalation, ingestion and sexual contact. For the induction of protective immunity at these invasion sites, one must consider the use of the CMIS, which interconnects inductive tissues, including PP and NALT, and effector tissues of the intestinal, respiratory and genitourinary tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant or use of mucosal antigen delivery vehicle has been shown to be essential. When vaccine antigen is administered via oral or nasal route, antigen-specific Th 1 and Th2 cells, cytotoxic T lymphocytes(CTLs) and IgA B cell responses are effectively induced by the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium(FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying DCs for the generation of antigen-specific T cells and IgA committed B cells. These antigen-specific lymphocytes are then home to the distant mucosal effector tissues for the induction of antigen-specific humoral(e.g., IgA) and cell-mediated (e.g., CTL and Th1) immune responses in order to form the first line of defense. Elucidation of the molecular/cellular characteristics of the immunological sequence of mucosal immune response beginning from the antigen sampling and processing/presentation by M cells and mucosal DCs followed by the effector phase with antigen-specific lymphocytes will greatly facilitate the design of a new generation of effective mucosal antigen-specific lymphocytes will greatly facilitate the design of a new generation of a new generation of effective mucosal adjuvants and of a vaccine deliver vehicle that maximizes the use of the CMIS.

• Korean Journal of Food Science and Technology
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• v.43 no.1
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• pp.72-76
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• 2011

The adjuvant effects of Korean mistletoe lectin-C (KML-C) were investigated following the oral administration of KML-C with ovalbumin (OVA) as an antigen. Mice were orally immunized with OVA alone or admixed with various doses of KML-C or cholera toxin (CT), and the titer of OVA-specific antibody in the serum and mucosal secretions were determined. OVA+KML-C-treated mice showed high titers of IgA specific to CT in mucosal secretions. The antibody titers in the serum of OVA+KML-C-treated mice were comparable to those in the serum of OVA+CT-treated mice. When mice were immunized with OVA+KML-C or with CT alone and subsequently injected with OVA on the footpads after the primary immunization, they showed a more significant increase in delayed-type hypersensitivity reactions than when they were administered CT alone. These results suggest that KML-C is a potent immunoadjuvant that enhances both humoral and cellular immunity by the mucosal immune system.

• IMMUNE NETWORK
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• v.15 no.1
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• pp.44-49
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• 2015

Interactions between microbes and epithelial cells in the gastrointestinal tract are closely associated with regulation of intestinal mucosal immune responses. Recent studies have highlighted the modulation of mucosal immunity by microbe-derived molecules such as ATP and short-chain fatty acids. In this study, we undertook to characterize the expression of the ATP-gated $P2X_7$ receptor ($P2X_7R$) on M cells and its role in gastrointestinal mucosal immune regulation because it was poorly characterized in Peyer's patches, although purinergic signaling via $P2X_7R$ and luminal ATP have been considered to play an important role in the gastrointestinal tract. Here, we present the first report on the expression of $P2X_7R$ on M cells and characterize the role of $P2X_7R$ in immune enhancement by ATP or LL-37.