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| The Department of Immunology consists of two programs: 1) the Mucosal Immunology Program and 2) the Clinical Immunology Program. The Immunology Program is supported by the Korea Science and Engineering Foundation, the Ministry of Education of the Republic of Korea, the Bill and Melinda Gates Foundation, and the Swedish International Development Cooperation Agency (SIDA). |
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| Mucosal Immunology Program |
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Drs. Huan H. Nguyen (Molecular Immunology Section) and Nathalie Mielcareck (Neonatal Vaccinology Section) are analyzing with Mr. Sung-Moo Park (Mucosal Immunology Section) lymphoid cells during the course of experimental Salmonella infection.
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| Background |
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| As an alternative to vaccination by injection, mucosal vaccination offers obvious safety advantages, since it eliminates the risks of blood-borne infections from non sterile needles. Orally-administered vaccines are generally more readily accepted than injectable vaccines, and as the current global eradication of polio is demonstrating, they offer large logistical advantages for immunization programs. |
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| Mucosal cells, whether of the digestive, respiratory, or reproductive tracts, are constantly exposed to antigens of microbial, environmental, or food origin and require an effective defense system. The mucosal immune system in humans covers over 400 square meters of tissue and its cellular mass far exceeds the total mass of lymphoid cells in the bone marrow, thymus, spleen, and lymph nodes combined. Immune cells stimulated at one mucosal surface induce local as well as systemic protection, thus providing the potential for vaccines to be used for a broad spectrum of infectious diseases. |
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| Success with the trivalent attenuated oral Sabin polio vaccine triggered research to develop and license other mucosal vaccines. Oral vaccines against cholera and rotavirus are good examples of recently registered mucosal vaccines. The aerosolized administration of measles and influenza vaccines has also attracted interest. Another route of administration that the Program is exploring is transcutaneous immunization (TCI), which is the introduction of antigens to the host using topical application to intact skin. TCI is a simple, needle-free vaccine-delivery system, which may improve the safety and effectiveness of immunization programs. Studies show that TCI is able to induce primary and secondary humoral immune responses without signs of local or systemic toxicity. |
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| Goals |
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| The objectives of the Mucosal Immunology Program are to: |
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Facilitate the development of mucosal vaccines;
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Determine the role of mucosal dendritic cells in the regulation of immunity and inflammation induced by vaccination or by natural infection;
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Develop strategies for the improved delivery of vaccine antigens by selective targeting of resident cells involved in the uptake of antigens administered by various mucosal routes;
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Develop animal models of mucosal infection for pre-clinical testing of candidate vaccines.
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| Projects |
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| The Mucosal Immunology Program focuses on the development of strategies to effectively induce mucosal and systemic immune responses in the colonic mucosa (for Shigella and Salmonella) and in the airway mucosa (for influenza virus, Bordetella pertussis, and S. pneumoniae. We are exploring several mucosal delivery routes, including nasal, sublingual, oral, and rectal, as well as systemic delivery routes (e.g., intradermal, transcutaneous) to induce optimal and broad immune responses. These responses include secretory and systemic humoral responses and cytotoxic T cell responses to several antigens, including Shigella O antigens and protein antigens, influenza virus whole virions, and purified antigens (HA, neuraminidase, M2) in the colonic mucosa and in the respiratory tract. Animal models of influenza virus infection and shigellosis, using mice, guinea pigs and ferrets, have been established either in-house or through partnerships with outside collaborators to carry out preclinical efficacy studies. |
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| Our main projects to develop alternative vaccine delivery routes are the following: |
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| Sublingual immunization: |
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| In collaboration with Inserm in Nice, France and University of Gothenburg in Sweden, IVI scientists are exploring the capacity of the sublingual mucosa to serve as a site for inducing mucosal and systemic immune responses. Recent studies have indicated that the sublingual application of protein antigens, co-administered with a suitable adjuvant, induces systemic humoral and cellular immune responses, as well as vigorous secretory antibody and cytolytic T cell responses in the airway mucosa(see paper in J Immunol). We've also shown that sublingual immunization can lead to the dissemination of secretory antibody responses and cytolytic T cells to remote mucosal tissues, including the genital tract mucosa. |
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| Animal models of murine influenza infection have been established by IVI scientists. The studies found that the sublingual delivery of formalin-inactivated influenza virions or hemagglutinin (HA) antigen induced both systemic and mucosal IgG and IgA Ab responses and protected mice against an airway challenge with a lethal dose of live influenza virus. In addition and contrary to nasal vaccination, sublingual vaccination with live influenza A virus administered at different doses was not found to be lethal. Further, this vaccination strategy induced vigorous systemic and mucosal anti-viral responses. Work is in progress to assess the protective efficacy of this immunization strategy in terms of lung viral clearance and cross-protection against divergent subtypes of influenza viruses. |
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| The program has also been addressing important questions regarding the nature and the role of resident (sublingual mucosa) antigen-presenting cells (APC) as critical elements in the initiation of immune responses induced by this route. An interesting finding has been that the sublingual mucosa was enriched in cells co-expressing CD11b and CD11c and MHC class II molecules, in sharp contrast to other mucosal sites. These cells can uptake and process antigen in vitro. The program is studying the functional properties of these cells with regard to their capacity to activate various sub-populations of lymphocytes. This work could lead to the identification of cellular and/or molecular markers (e.g., cytokines, chemokines) that are predictive of the type of immune responses generated after sublingual administration of vaccines, and could facilitate the development of optimal formulations for this vaccination route. |
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| Transcutaneous immunization (TCI): |
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| It has been shown that transcutaneous immunization using a vaccine antigen combined with the mucosal adjuvant cholera toxin (CT) or its E. coli analog (heat-labile enterotoxin) induces both systemic and mucosal antibody responses(see paper in Vaccine). To further understand how TCI induces mucosal immunity, dendritic cells (DC) and Ag-specific CD4+ T cells were examined following transcutaneous immunization with a prototype vaccine antigen, tetanus toxoid (TT), co-administered with cholera toxin. Interestingly, the number of CD205+CD8a- DC increased in the spleen, skin-draining lymph nodes (LN) and mesenteric LN, but not in Peyer's patches after transcutaneous immunization. Furthermore, the number of TT-specific CD4+ T cells which expressed a4ß7 and aE integrins increased in the draining LN and mesenteric LN, but not in Peyer's patches. Consistent with these findings, the expression of IgA class switch recombination-related molecules, including AID, aCTs, and IµCa transcripts, was selectively increased in the mesenteric LN but not in skin-draining LN or in Peyer's patches after TCI. These results suggest the possibility that mesenteric lymph nodes, but not Peyer's patches or skin-draining lymph nodes, are indispensable for the induction of intestinal IgA responses following transcutaneous immunization. |
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| Rectal vaccination: |
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| The route of administration of a vaccine has a significant effect on the outcome of immune responses induced at the mucosal surfaces. Intrarectal vaccination of mice with a mixture of tetanus toxoid (TT) and cholera toxin adjuvant induced a significant Ag-specific IgA and IgG antibody response, measured in both mucosal and systemic compartments. To clarify the underlying mechanism of the adjuvant effect of cholera toxin, the time-dependent mobilization and maturation of mucosal dendritic cell (DC) subsets was examined. An immunohistochemical study demonstrated predominant numbers of CD11c+ MHC class II+ cells were accumulated in colonic patches and lamina propria of the large intestine and in draining lymph nodes after intrarectal vaccination with CT. In addition, a combination of vaccination routes¯with a primary subcutaneous dose and intrarectal booster¯enhanced large intestinal IgA Ab responses. These findings should contribute to the development of vaccine strategies against diseases caused by pathogens that invade the large intestine. |
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| Studies by IVI mucosal immunologists have demonstrated that rectal administration of a Shigella flexneri ribosome-based immunogen induced mucosal and systemic immune responses and could partially protect animals against live Shigella bacteria(see paper in Vaccine (in press)). The recent development of an animal model of Shigella-induced colitis will allow IVI scientists to evaluate candidate vaccines formulated for rectal administration. |
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| Study of influenza vaccine administered by different mucosal delivery routes: |
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| The Mucosal Immunology Program will also initiate a Phase I clinical trial in human volunteers of a licensed influenza vaccine administered by different routes to determine its safety and immunogenicity in relation to the route of administration. Special efforts will be devoted to the development of liquid and semi-liquid formulations (e.g., gel, spray) for adequate delivery of vaccine antigens to the target mucosa. We anticipate that these studies could lead to the development of dose-sparing vaccine strategies amenable to mass immunization against several enteric and respiratory pathogens, including influenza virus. |
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| Study of innate immunity following oral vaccination with attenuated Salmonella bacteria |
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| Toll-like receptors(TLRs) are known to mediate innate host responses, but the mechanisms involved in TLR-mediated adaptive immunity remain subject to debate. Provided that there is TLR signaling, dendritic cells mature in response to microbial infection in vitro; however, the role of such signaling in B cell activation of mucosal immunity in vivo is poorly understood. In collaboration with the Biodesign Institute at Arizona State University, we are exploring how innate immunity for induction of Ab responses at mucosal sites are controlled by TLR signaling following oral administration with recombinant attenuated Salmonella (S.) typhimurium vaccine (RASV) strains expressing pneumonia surface protein A (PspA). Interestingly, oral vaccination with RASV expressing PspA resulted in significantly higher levels of PspA-specific IgA Ab responses in the fecal extracts of MyD88-/- mice than in those of wild-type Balb/c mice, TLR4-/-, and TLR5-/- mice. Similarly, higher levels of PspA-specific IgA Ab-secreting cells were detected in the small and large intestines of MyD88-/- mice than of wild-type mice. Some current evidences indicating a Th2-dominant condition in the MyD88-/- mice following oral vaccination with RASV expressing PspA. These findings suggest that mucosal IgA responses are accelerated in the absence of MyD88 signaling following oral microbial challenge, perhaps due to the Th2 environment that predominates in these mice. |
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| Research on new adjuvants: |
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| Because mucosal vaccines are usually relatively less immunogenic than parenteral (injectable) vaccines, there is a need to develop adjuvants capable of enhancing the immune responses induced by these vaccines. A key focus of the Mucosal Immunology program is therefore to identify novel adjuvants and elucidate their mechanisms of action. In collaboration with several academic partner laboratories, IVI mucosal immunologists have characterized several pathways used by known adjuvants or novel ones to mediate enhancement of systemic and mucosal immune responses, when co-administered with relevant vaccine antigens(see paper in 1)Infec & Immunity 2)Trends Microbiol 3)Vaccine (in press)). Studies are ongoing to optimize adjuvant formulations for mucosal delivery by various routes and to characterize the mechanisms involved in the uptake of adjuvants and vaccines by specialized dendritic cells. |
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| Clinical Immunology Program |
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Ms. Sun Wha Moon testing blood samples for antibodies against Shigella flexneri, a leading cause of dysentery in developing countries.
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| Background |
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| The Clinical Immunology Program develops, standardizes and validates assays for measuring immune responses to vaccines in humans and establishes reference tests for the laboratory diagnosis of enteric and respiratory infections to support both disease burden studies and clinical trials conducted by IVI's Translational Research Division. Such assays can provide surrogate markers of vaccine efficacy and are thus essential for the approval of vaccines by regulatory agencies or to prove that the vaccine can be manufactured with the same immunological potency demonstrated during clinical trials. |
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| Dedicated laboratories for this program have been equipped and systems and assays have been established to measure cellular immune responses. These include: a flow cytometry assay to characterize B and T lymphocyte subsets, Elispot tests for detecting antibody-producing cells and cytokine-secreting cells in human blood, colorimetric assays for measuring lymphocyte proliferation, and assays for quantifying human cytokines and chemokines. A focus in all of our work is to develop tests that are simple and robust enough to be performed under field conditions. |
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| Goals |
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| The goals of the Clinical Immunology Program are to: |
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Develop standardized assays for measuring human humoral and cellular immune responses to vaccines;
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Establish a reference laboratory for the standardization and validation of assays for serotyping respiratory pathogens, including influenza virus, M. tuberculosis, and encapsulated bacteria;
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Develop new, cell-based, assays for measuring mucosal immune responses to candidate vaccines in young children and infants; and
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Develop and validate a method for measuring vaccine-induced immunological memory in human blood.
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| Projects |
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| Development of standardized sero-diagnostic assays to measure immune responses to enteric and respiratory vaccines: |
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| The Clinical Immunology Program is developing and standardizing assays for determining the magnitude, characteristics and duration of vaccine-specific humoral and cellular immune responses in clinical samples collected from actively infected patients and from vaccinees. IVI scientists have completed the development of quality-controlled, standardized sero-diagnostic assays for V. cholerae and S. pneumoniae, and are now developing similar tests for additional pathogens, including S. typhi, S. paratyphi and influenza. Towards this aim, the program is validating standardized ELISA for quantification of antibodies to S. typhi Vi polysaccharide, in collaboration with Dr. John Robbins' laboratory at the National Institutes of Health in the US. This standardized ELISA should also be useful in supporting future clinical trials of candidate typhoid vaccines.
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| A semi-automated vibriocidal assay against V. cholerae O1 strain was developed by IVI scientists, in close collaboration with Dr. Jan Holmgren's group at the University of Gothenburg in Sweden. The assay was validated for the following parameters: accuracy, precision, specificity and sensitivity. Robustness was documented under fluctuating conditions that mimicked conditions in the field. The assay has been used to follow cohorts of several hundred vaccinees in two separate Phase II clinical trials of a killed bivalent (O1/O139) whole-cell oral cholera vaccine, coordinated by IVI in India and in Vietnam. This assay has now been transferred to the National Institute of Cholera and Enteric Diseases (NICED) in Kolkata, India. In collaboration with the University of Gothenburg, IVI has also developed an assay - to be transferred to partner field laboratories - for the quantification of antibodies to the O139 strain of V. cholerae, which is included in the oral cholera vaccine undergoing a Phase III trial in Kolkata. |
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| Since peripheral blood is the most accessible lymphoid compartment in humans, IVI scientists will continue to develop and/or adapt new and existing assays to determine the magnitude, characteristics and duration of vaccine-specific humoral and cellular responses in blood samples collected from vaccine volunteers. We will continue to focus on developing simple, rapid and inexpensive procedures for processing blood samples and glandular secretions (e.g., nasal secretions, saliva, stools) from human subjects. Efforts will also focus on the miniaturization of ELISPOT tests for measuring T and B cell immune responses in small volumes of blood from pediatric cohorts. |
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| Development of methods to measure immunological memory: |
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| The Program will evaluate a new approach to measure immunological memory after vaccination. This assay, which combines immunomagnetic isolation of putative memory B cells with in situ ELISPOT assay after polyclonal stimulation, could provide an indication of the magnitude and duration of immune responses induced by vaccination. |
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