1: Scand J Immunol 2000 Aug;52(2):138-47Characterization of murine dendritic cells derived from adherent blood mononuclear cells in vitro.

Agger R, Petersen MS, Toldbod HE, Holtz S, Dagnaes-Hansen F, Johnsen BW, Bolund L, Hokland M.

Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark. Agger@microbiology.au.dk

The therapeutic potential of dendritic cells loaded with tumour antigens for the induction of effective immune responses against cancer is currently being tested in numerous clinical trials. In most cases, the dendritic cells are generated in vitro from peripheral blood monocytes. Many aspects of dendritic cell-based vaccination have not yet been examined in detail, and homologous mouse model systems may prove very valuable for optimizing clinical procedures. In the murine system, however, dendritic cells are usually isolated from either lymphoid tissues or bone marrow cultures. To date, murine monocyte-derived dendritic cells have been described only sporadically. Here, we describe a culture system for the generation of murine dendritic cells from adherent peripheral blood mononuclear cells by culturing in the presence of granulocyte-macrophage colony stimulating factor and interleukin-4. After 7 days of culture the nonadherent cells were harvested from the cultures. Most of these cells exhibited well-accepted characteristics of mature dendritic cells (e.g. veiled appearance, high expression of major histocompatibility complex class II and CD86) and stimulated vigorous proliferation of allogeneic T cells in a primary mixed leucocyte reaction following stimulation with bacterial lipopolysaccharide. Interestingly, staining the cells for expression of the putative antigen-uptake receptor DEC-205 revealed a distinct bimodal distribution.

Free as elsevierJ Immunol Methods 2000 May 26;239(1-2):95-107A two-step culture method starting with early growth factors permits enhanced production of functional dendritic cells from murine splenocytes.

Berthier R, Martinon-Ego C, Laharie AM, Marche PN.

Laboratoire Immunochimie, CEA-G, DBMS/ICH, INSERM U238, Universit inverted question marke Joseph Fourier, 17, rue des Martyrs, 38054 Cedex 9, Grenoble, France. berthier@dsvgre.cea.fr

Dendritic cells (DC) are professional antigen presenting cells (APC) able to activate naive T cells and initiate the immune response. They are present in most tissues at very low concentrations and are difficult to isolate. DC can be obtained in larger numbers by their propagation from progenitors present in blood, bone marrow and spleen. However, biochemical studies and biological analysis of DC functions require very large numbers of these cells. In this paper, we described a two-step culture system using unfractionated splenocytes from BALB/c mice as a source of DC progenitors. The proliferative capacity of the progenitors is amplified in the first step of the culture (day 0-6) using different combinations of early acting cytokines combined or not with granulocyte-macrophage CSF (GM-CSF). The second step of the culture starts at day 6 with the removal of early growth factors in order to allow the differentiation and final maturation of DC during 2-3 weeks of culture with flt-3 ligand (flt-3L) and GM-CSF. The addition of Stem Cell Factor (SCF) or IL-6 to the standard combination of flt-3L+/-GM-CSF produces a large increase in the proliferation of GM and DC progenitors (28 times and 11 times respectively) in the first step of the culture. This proliferative wave of DC progenitors is followed by the production of a high percentage of immature and mature DC in flt-3L+GM-CSF stimulated cultures. The best combination of early cytokines in terms of proliferative activity and subsequent level of DC production was flt-3L+IL-6+GM-CSF, which permitted the generation of 1 to 2x10(9) DC from one single spleen. Using this growth factor cocktail, a mixture of immature (2/3) and mature (1/3) DC was produced until day 14 of culture, and levels of MHC class II and costimulatory molecules (CD40, B7.2) increased between 2 and 4 weeks of incubation, or within 2 days when stimulated by IL-4 or LPS. The splenic DC produced after 2 weeks of culture are fully functional, exhibiting a high capacity of endocytosis when immature, a strong stimulatory reactivity in mixed leukocyte reaction and consistently producing high levels of bioactive IL-12 p70 after CD 40 ligation in the presence of LPS between 13 and 43 days of culture.

Microbiol Immunol 2002;46(1):23-8Immunogenicity of a recombinant MVA and a DNA vaccine for Japanese encephalitis virus in swine.

Nam JH, Cha SL, Cho HW.

Department of Virology, Korean National Institute of Health, Eunpyung-Gu, Seoul.

We previously reported that mice immunized with recombinant modified vaccinia virus Ankara (MVA) encoding Japanese encephalitis virus (JEV) prM and E genes were completely protected against JEV challenge (Nam, J.H., Wyatt, L.S., Chae, S.L., Cho, H.W., Park, Y.K., Moss, B. Vaccine 1999,17: 261-268). In this study, we examined the immunogenicity in swine of this recombinant MVA (vJH9) or a DNA vaccine (pcJH-1) expressing the same JEV genes. Although the booster effect in mice with a combination of vJH9, pcJH-1 and inactivated JEV commercial vaccine was not apparent by measuring JEV antibodies, the recombinant MVA vaccine (vJH9) and the DNA vaccine (pcJH-l) efficiently produced neutralizing antibodies in swine and 2 doses of each showed a booster effect in mice and swine. Therefore, both vJH9 and pcJH-1 are good candidates for a second generation JEV vaccine.

Crit Rev Biochem Mol Biol 2002;37(1):29-54

DNA vaccine against malaria: a long way to go.

Tuteja R.

International Centre for Genetic Engineering and Biotechnology, New Delhi. renu@icgeb.res.in

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle--antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.

Curr Mol Med 2001 May;1(2):217-43

DNA vaccines.

Reyes-Sandoval A, Ertl H C.

The Wistar Institute, Philadelphia, PA 19104, USA.

Within the last decade bacterial plasmids encoding foreign antigens have revolutionized vaccine design. Although

no DNA vaccine has yet been approved for routine human or veterinary use, the potential of this vaccine modality has been demonstrated in experimental animal models. Plasmid DNA vaccination has shown efficacy against viral, bacterial and parasitic infections, modulated the effects of autoimmune and allergic diseases and induced control over cancer progression. With a better understanding of the basic immune mechanisms that govern induction of protective or curative immune responses, plasmid DNA vaccines and their mode of delivery are continuously being optimized. Because of the simplicity and versatility of these vaccines, various routes and modes of delivery are possible to engage the desired immune responses. These may be T or B effector cell responses able to eliminate infectious agents or transformed cells. DNA vaccines may also induce an immunoregulatory/modulatory or immunosuppressive (tolerizing) response that interferes with the differentiation, expansion or effector functions of B and T cells. In this sense a DNA vaccine may be thought of as a 'negative' vaccine. Pre-clinical and initial small-scale clinical trials have shown DNA vaccines in either of these modes to be safe and well tolerated. Although DNA vaccines induce significant immune responses in small animal trials their efficacy in humans has so far been less promising thus necessitating additional optimizations of this novel vaccine approach.

Immunol Lett 2002 Apr 1;81(1):13-24

A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-gamma and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor.

Kumar S, Villinger F, Oakley M, Aguiar JC, Jones TR, Hedstrom RC, Gowda K, Chute J, Stowers A, Kaslow DC, Thomas EK, Tine J, Klinman D, Hoffman SL, Weiss WW.

Malaria Program, Naval Medical Research Center, 20910, Silver Spring, MD, USA

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein1 (pMSP1(42)) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP1(42) after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP1(42) produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1(19) (yMSP1(19)). Immunization also induced antigen specific T cell responses as measured by interferon-gamma production, and by classical CTL chromium release assays. In addition, immunization with pMSP1(42) primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1(19). We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1(42.) We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP1(42) induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1(19). The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

Nurse Pract 2002 Jan;27(1):53-9; quiz 60-1Advances DNA vaccines.

Simmerman JM.

US Centers for Disease Control and Prevention, National Center for Infectious Diseases, International Emerging Infections Program, Bangkok, Thailand.

Extraordinary advances in biotechnology make DNA vaccines the most promising area of vaccinology. This article reviews the public health impact of vaccines in the 20th century, summarizes immunologic concepts, and updates the status of DNA vaccine development and its impact on clinical practice.

Naturwissenschaften 2001 Dec;88(12):504-13DNA vaccines.

Gregersen JP.

Chiron Behring GmbH, Postfach 1630, 35006 Marburg, Germany. jens-peter_gregersen@chiron-behring.com

Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

Mol Immunol 2002 Mar;38(11):803-16GPI-anchoring of GM-CSF results in active membrane-bound and partially shed cytokine.

Poloso NJ, Nagarajan S, Mejia-Oneta JM, Selvaraj P.

Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, 1639 Pierce Drive, Woodruff Memorial Building, Room 7309, 30322, Atlanta, GA, USA

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the generation and activation of dendritic cells (DCs), the most potent of antigen presenting cells (APCs). Tumors secreting GM-CSF have been shown to induce strong anti-tumor immune responses. In this report, we have constructed a glycosylphosphatidyl-inositol (GPI) anchored form of GM-CSF (GPI-GM-CSF). This protein subsequently was found expressed on the cell membrane and sensitive to phosphatidyl-inositol-specific phospholipase C (PIPLC), confirming that it is GPI-anchored. However, GM-CSF was also found in the culture supernatant of cells expressing GPI-GM-CSF. Inhibition studies using brefeldin A and para-formaldehyde fixation revealed that GM-CSF found in the supernatant was not secreted, but due to shedding or proteolytic cleavage. Accumulation of GM-CSF in the media from isolated membranes was time and temperature-dependent. The released portion represented 10-15% of all membrane-bound GM-CSF after 72h under culture conditions. GPI-GM-CSF retained functional activity to induce bone marrow cell proliferation and administration of GPI-GM-CSF expressing membranes induced the generation of DCs in vivo. These results demonstrate that GPI-anchored GM-CSF retains all functional activity of native GM-CSF while gaining the ability to attach to cell membranes. The ability of GPI-GM-CSF to be expressed on membranes and be partially released, can possibly lead to formation of a cytokine gradient, while retaining ability to target associated

membrane antigens to DCs. This novel form of GM-CSF may have wide range of clinical applicability.

J Immunol Methods 2002 Mar 1;261(1-2):49-63Generation of functional and mature dendritic cells from cord blood and bone marrow CD34(+) cells by two-step culture combined with calcium ionophore treatment.

Liu A, Takahashi M, Narita M, Zheng Z, Kanazawa N, Abe T, Nikkuni K, Furukawa T, Toba K, Fuse I, Aizawa Y.

First Department of Internal Medicine, Faculty of Medicine, Niigata University, Niigata, Japan

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34(+) hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34(+) progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34(+) hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208plus minus66 (meanplus minusSD, n=13), or 94plus minus29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34(+) cells at the time of starting culture. Out of the total nucleated cells, 23plus minus10.4% of cells in CB cell culture and 25plus minus5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a(+) cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34(+) cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a(+) DC generated from CD34(+) cells, and the phenotypes and functions of these CD1a(+) DC could be enhanced efficiently by treatment with a calcium ionophore agent.

Transplant Proc 2001 Nov-Dec;33(7-8):3814-5

Dendritic cells, generated in vitro, are immunocompetent and very useful in the induction of specific cytotoxic T lymphocyte activity.

Hasel T, Yoshimura R, Wada S, Chargui J.

Immunology 2001 Oct;104(2):175-84Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties.

Carrasco CP, Rigden RC, Schaffner R, Gerber H, Neuhaus V, Inumaru S, Takamatsu H, Bertoni G, McCullough KC, Summerfield A.

Institute of Virology and Immunoprophylaxis, Mittelhausern, Switzerland.

Despite the central role that dendritic cells (DC) play in immune regulation and antigen presentation, little is known about porcine DC. In this study, two sources of DC were employed. Bone marrow haematopoietic cell-derived DC (BM-DC) were generated using granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of tumour necrosis factor-alpha (TNF-alpha). Monocyte-derived DC (Momicron-DC) were generated with GM-CSF and interleukin-4 (IL-4). In both systems, non-adherent cells developed with dendritic morphology, expressing high levels of major histocompatibility complex (MHC) class II. The presence of TNF-alpha increased the BM-DC yield, and enhanced T-cell stimulatory capacity. Both BM-DC and Momicron-DC expressed the pan-myeloid marker SWC3, as well as CD1 and CD80/86, but were also CD14+ and CD16+. The CD16 molecule was functional, acting as a low-affinity Fc receptor. In contrast, the CD14 on DC appeared to differ functionally from monocyte CD14: attempts to block CD14, in terms of lipopolysaccharide (LPS)-induced procoagulant activity (PCA), failed. The use of TNF-alpha or LPS for DC maturation induced up-regulation of MHC class II and/or CD80/86, but also CD14. Allogeneic mixed leucocyte reactions and staphylococcal enterotoxin B antigen presentation assays demonstrated that these DC possessed potent T-cell stimulatory capacity. No T helper cell polarization was noted. Both the BM-DC and the Momicron-DC induced a strong interferon-gamma and IL-4 response. Taken together, porcine DC generated in vitro possess certain characteristics relating them to DC from other species including humans, but the continued presence of CD14 and CD16 on mature and immature porcine DC was a notable difference.

J Immunol 2002 Feb 15;168(4):1704-9Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals.

de Jong EC, Vieira PL, Kalinski P, Schuitemaker JH, Tanaka Y, Wierenga EA, Yazdanbakhsh M, Kapsenberg ML.

Department of Cell Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. E.C.deJong@AMC.UvA.NL

Upon microbial infection, specific Th1 or Th2 responses develop depending on the type of microbe. Here, we demonstrate that different microbial compounds polarize the maturation of human myeloid dendritic cells (DCs) into stably committed Th1 cell-promoting (DC1) or Th2 cell-promoting (DC2) effector DCs that polarize Th cells via different mechanisms. Protein extract derived from the helminth Schistosoma mansoni induced the development of DC2s that promote the development of Th2 cells via the enhanced expression of OX40 ligand. Likewise, toxin from the extracellular bacterium Vibrio cholerae induced development of DC2s as well, however, via an OX40 ligand-independent, still unknown mechanism. In contrast, toxin from the intracellular bacterium Bordetella pertussis induced the development of DC1s with enhanced IL-12 production, which promotes a Th1 cell development. Poly(I:C) (dsRNA, mimic for virus) induced the development of extremely potent Th1-inducing DC1, surprisingly, without an enhanced IL-12 production. The obtained DC1s and DC2s are genuine effector cells that stably express Th cell-polarizing factors and are unresponsive to further modulation. The data suggest that the molecular basis of Th1/Th2 polarization via DCs is unexpectedly diverse and is adapted to the nature of the microbial compounds.

J Immunol 2002 Jan 15;168(2):537-40Cutting edge: Th2 response induction by dendritic cells: a role for CD40.

MacDonald AS, Straw AD, Dalton NM, Pearce EJ.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

We investigated the influence of dendritic cell (DC) CD40 expression on Th2 and Th1 development by in vivo transfer of Ag-pulsed bone marrow-derived DC generated from wild-type (WT) or CD40(-/-) mice. Contrary to

expectation, CD40(-/-) DC primed with Ag that inherently induce a Th2 response (soluble egg Ag from Schistosoma mansoni) failed to induce a Th2 response or any compensatory Th1 response, whereas CD40(-/-)DC primed with Ag that inherently induce a Th1 response (Propionibacterium acnes) generated a competent Th1 response. Thus, DC expression of CD40 is a prerequisite for initiation of Th2, but not Th1, responses by these Ag. Consistent with this, CD154(-/-) mice, unlike WT mice, failed to mount a Th2 response when directly injected with schistosome eggs but mounted a normal Th1 response after challenge with P. acnes. CD40-CD154 interaction can therefore play a major role in Th2 response induction.

Parasite Immunol 2001 Dec;23(12):627-32Defect of protective immunity to Schistosoma mansoni infection in Mongolian gerbils involves limited recruitment of dendritic cells in the vaccinated skin.

Sato H, Kamiya H.

Department of Parasitology, Hirosaki University School of Medicine, Hirosaki, Japan.

In Mongolian gerbils, Meriones unguiculatus, the attenuated Schistosoma mansoni vaccine, is known to induce marginal or no resistance to a homologous infection. To clarify the base of defective acquisition of the resistance, we have focused on the induction phase of protective immunity to S. mansoni, i.e. cellular responses in the skin and skin-draining lymph nodes (SLN). Percutaneous exposure to normal or ultraviolet (18mJ/cm2)-attenuated cercariae induced comparable increases in SLN leucocyte counts, in contrast to other attenuated schistosome vaccine models in rodents where attenuated parasites induce more notable increases in SLN leucocyte counts than normal ones. Using serial sections, it was demonstrated that greater numbers of attenuated larvae remained for a longer period in the exposed skin than normal ones. Correlated with cellular responses in the SLN, attenuated and normal schistosomes elicited a comparable degree of response of epidermal Langerhans' cells/putative dermal dendritic cells that were visualized by immunohistochemistry using a monoclonal antibody to a gerbil major histocompatibility complex class II molecule (HUSM-M.g.30). It is speculated that in Mongolian gerbils limited recruitment of dendritic cells around attenuated S. mansoni larvae, at least partially, contribute to defective induction of protective immunity by the attenuated vaccine.

J Immunol 2001 Aug 15;167(4):1982-8CD8- dendritic cell activation status plays an integral role in influencing Th2 response development.

MacDonald AS, Straw AD, Bauman B, Pearce EJ.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Whether dendritic cells (DC) play a passive or active role in Th2 response induction is poorly understood. In this study, we show that CD8- DC pulsed with Th2-polarizing Ag (soluble egg Ag (SEA)) from Schistosoma mansoni potently stimulate Th2 responses in vivo and in vitro while failing to undergo a conventional maturation process. Thus, in contrast to DC pulsed with the Th1 response inducing Ag Propionebacterium acnes, SEA-exposed DC exhibit a phenotype that is most similar to that of immature DC, failing to up-regulate expression of CD40, CD54, CD80, CD86, or OX40L; producing no detectable IL-4, IL-10, or IL-12; and displaying only a minor increase in MHC class II expression. Importantly, in vitro derived DC exposed to SEA were phenotypically similar to CD8- DC isolated from active S. mansoni infection. By discriminating between different types of pathogen and responding appropriately, CD8- DC play a major role in the decision process to mount either a Th1 or Th2 response.

J Exp Med 2001 May 21;193(10):1135-47Role of the parasite-derived prostaglandin D2 in the inhibition of epidermal Langerhans cell migration during schistosomiasis infection.

Angeli V, Faveeuw C, Roye O, Fontaine J, Teissier E, Capron A, Wolowczuk I, Capron M, Trottein F.

Centre d'Immunologie et de Biologie Parasitaire, Institut National de la Sante et de la Recherche Medicale (INSERM) U547, Lille, France.

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-alpha, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-alpha-triggered migration of LCs through the adenylate cyclase-coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

: Immunol Today 1999 Dec;20(12):561-7T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal.

Kalinski P, Hilkens CM, Wierenga EA, Kapsenberg ML.

Parasite Immunol 1998 Jul;20(7):337-43Accelerated influx of dendritic cells into the lymph nodes draining skin sites exposed to attenuated cercariae of Schistosoma mansoni in guinea-pigs.

Sato H, Kamiya H.

Department of Parasitology, Hirosaki University School of Medicine, Japan.

We compared temporal changes in the cell composition of the skin-draining lymph nodes (SLN) in guinea-pigs exposed percutaneously to normal or attenuated cercariae of Schistosoma mansoni. Different populations were analysed by flow cytometry of double-stained cells by monoclonal antibodies to the major histocompatibility complex class II molecule and lymph node cells of guinea-pigs. Exposure to S. mansoni caused a marked increase in the proportion of B cells and dendritic cells (DC) on day 2, reaching a peak number on day 4. These changes were comparable in both infected and vaccinated animals exposed to normal or attenuated parasites, respectively. Total number of DC, however, showed different kinetics; in infected animals, the number peaked on day 2 and then gradually declined, whereas it reached a higher peak on day 4 in vaccinated animal. Daily injection of bromo-deoxyuridine after exposure to the parasite reduced the total number of DC in the SLN on day 4. A reduction in DC counts in the contralateral side SLN was also evident in vaccinated animals. Our results indicate that a significant number of newly formed DC are recruited to the skin by 4th day of vaccination, followed by increased efflux to the SLN. It is possible that retention of attenuated S. mansoni in the skin may cause accelerated recruitment of newly formed DC from the bone marrow, and facilitate transport and processing of antigens highly expressed on attenuated parasites.

Hybridoma 1997 Dec;16(6):529-36Production of murine monoclonal antibodies to guinea pig leukocytes and immunohistochemistry of guinea pig skin exposed to Schistosoma mansoni.

Sato H, Inaba T, Kamiya H.

Department of Parasitology, Hirosaki University School of Medicine, Japan.

Using histochemical ATPase-staining of the guinea pig epidermal sheet, we have demonstrated remarkable accumulations of ATPase-positive cells after exposure to attenuated Schistosoma mansoni cercariae. To characterize further the cells accumulating in the skin after exposure to S. mansoni, we produced a panel of monoclonal antibodies (MAb) to guinea pig leukocytes. These were immunohistochemically classified into 15 types and included MAb to the major histocompatibility complex (MHC) Class I and Class II molecules, shared antigens of all lymph node cells or between lymph node cells and Langerhans' cells (LC), T cells and macrophages (M phi), and M phi including the large Tingible body M phi in the secondary follicle. Varied MAb to M phi, including commercially available MAb (MR-1), were negative with ATPase- and MHC Class II-positive cells accumulated in the skin exposed to S. mansoni. Three MAb (HUSM-30 and 46, and commercially available MSgp2) detected an identical staining profile of accumulated cells with epidermal LC, but two MAb (HUSM-12 and 42) positively stained accumulated cells but not resident LC. These results indicate that the cells accumulated in the guinea pig skin within a few days after exposure to attenuated cercariae of S. mansoni are closest to LC, not to Mo, and may be blood-borne LC/dendritic cells.

Immunology 1995 Feb;84(2):233-40Role of epidermal Langerhans' cells in the induction of protective immunity to Schistosoma mansoni in guinea-pigs.

Sato H, Kamiya H.

Department of Parasitology, Hirosaki University School of Medicine, Japan.

Percutaneous exposure of guinea-pigs to attenuated or normal larvae of Schistosoma mansoni induced proliferative T-cell responses in the skin-draining lymph nodes (SLN). The responses elicited by attenuated larvae were stronger and more prolonged [2-12 days post-infection (p.i.)] than those by normal larvae (3-8 days p.i.). The former were coincident with greater and more sustained increases in numbers of SLN dendritic cells. During this event, epidermal Langerhans' cells (LC) showed marked changes in their distribution and morphology. Resident LC were similarly exhausted by either attenuated or normal larvae between 12 hr and 1 day p.i., but thereafter more blood-borne LC were recruited around the former, since reaggregation of LC around these persisted larvae was more frequent and intensive, and enhanced replenishment of epidermal LC was achieved by 8 days p.i. When the skin depleted of epidermal LC by short-wavelength ultraviolet (UVC) irradiation was exposed to attenuated larvae, consequent T-cell responses were delayed. Excision of the whole exposed skin on day 4 p.i. also reduced T-cell responses to marginal levels. These results indicate that during the afferent phase of immunity to S. mansoni, efficient T-cell responses in the SLN need an active involvement of not only resident LC but also blood-borne LC as immunostimulatory cells.

J Parasitol 1998 Aug;84(4):764-70

Overproduction of SM28GST in a baculovirus expression vector and its use to evaluate the in vivo immune responses of mice vaccinated against Schistosoma mansoni with naked DNA encoding the SM28GST gene.

Kayes SG, Shaneyfelt RC, Monteiro C, O'Brien JJ.

Department of Structural and Cellular Biology, College of Medicine, University of South Alabama, Mobile 36688-0002, USA.

To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.

Mol Biochem Parasitol 1998 Oct 30;96(1-2):15-25Characterization of Sm20.8, a member of a family of schistosome tegumental antigens.

Mohamed MM, Shalaby KA, LoVerde PT, Karim AM.

Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt.

Two cDNA clones each encoding a 20.8-kDa protein (Sm20.8) were identified from the human blood fluke Schistosoma mansoni sporocyst and adult worm cDNA expression libraries by antibodies derived from rabbits vaccinated with irradiated cercariae and purified over an NP-40 extract of 3h schistosomula. Each identified cDNA has an open reading frame encoding a protein of 181 amino acids and shows homology (29-30%) with Sm21.7, Sm22.6, and Sj22.6, previously identified as belonging to a family of soluble schistosome tegumental antigens. An EF-hand calcium-binding motif is found in Sm20.8 protein in two different positions. However, neither motif binds 45calcium (45Ca) Recombinant Sm20.8 showed immunoreactivity with sera from infected humans and rabbits vaccinated with irradiated cercariae. Polyclonal rabbit sera against the Sm20.8 recognized the native protein in an extract of infected snail (sporocyst), cercariae, 3 hour schistosomules (3 h NP-40) and an adult worm preparation but not in uninfected snail tissue or eggs. Further demonstration that Sm20.8 was expressed in the different developmental stages of the parasite was by RT-PCR. Confocal microscopy demonstrates that Sm20.8 localizes to the tegument of adult worms and 3 h np-40. The IgG fraction specific to Sm20.8 mediated complement killing of schistosomules in vitro by 34%. Vaccination of mice with naked DNA containing the Sm20.8 gene and subsequently challenged with cercariae showed 30% reduction in worm burden compared to controls.

Vaccine 1999 Mar 17;17(11-12):1338-54Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model.

Hota-Mitchell S, Clarke MW, Podesta RB, Dekaban GA.

The John P. Robarts Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Canada.

Schistosomiasis is a parasitic disease affecting over 200 million people every year in tropical regions of the world. Drug treatment and other existing control measures are costly and have failed to eliminate the incidence of infection, morbidity and mortality due to Schistosoma mansoni infection. Vaccination of susceptible individuals using recombinant vaccines encoding key S. mansoni antigens may be the most effective and least expensive means of controlling schistosomiasis. A candidate vaccine antigen is p80, the large subunit of the S. mansoni protease, calpain. In our vaccine studies, we have employed both the wild-type p80 and a mutant p80 (mut p80) in which an active site amino acid was genetically altered to create a less proteolytically-active enzyme. Two vaccine delivery approaches were implemented using p80 or mut p80 as vaccine antigen: recombinant vaccinia virus (RVV) inoculation and DNA immunization via the Accell gene gun (GG) delivery system. RVV's expressing p80 and mut p80 were generated and tested for recombinant protein expression in vitro. These RVV's were tested for protective capacity in mouse challenge studies. Neither subcutaneous nor intranasal vaccinations with RVV-p80 or RVV-mut p80 were capable of significantly reducing the mean worm burdens of vaccinated mice. A GG-RVV combination immunization regime using WRG-vectors encoding p80 and mut p80 for GG priming and the RVV's for boosting prior to S. mansoni challenge infection was performed and no significant protection was obtained over two repeated studies. However, duplicate challenge studies involving GG immunization of mice with WRG-vectors encoding p80 or mut p80 revealed that 3 inoculations of mice with WRG-full5' mut p80 (containing the full 5' untranslated region of mut p80) provided 60% protection which was statistically significant (p < 0.05). These preliminary in vivo studies demonstrate the potential for further study of the protection afforded by gene gun-delivered WRG-full5' mut p80 into subsequently-challenged mice. Such studies may pave the way to effective vaccination of humans using WRG DNA vectors expressing a schistosomal calcium-activated neutral protease.

J Infect Dis 1999 Aug;180(2):454-63Control of schistosomiasis pathology by combination of Sm28GST DNA immunization and praziquantel treatment.

Dupre, Herv M, Schacht AM, Capron A, Riveau G.

Telethon Institute for Gene Therapy, Science Park DIBIT-HSR, 20132 Milan, Italy.

Today the control of schistosomiasis infection relies only on the use of praziquantel (PZQ) chemotherapy. However, PZQ treatment cannot prevent reinfection and progressive development of the pathology. We assessed in a mouse model the efficiency of a combined therapy, based on the combination of PZQ chemotherapy with Schistosoma mansoni 28-kDa glutathion S-transferase (Sm28GST) DNA vaccination, designed to limit the pathology. Following this combined therapy, the long-term survival of the mice was significantly enhanced in comparison with the survival of mice either vaccinated only or treated with PZQ only. In addition, the development of the pathology observed in the control groups was almost completely prevented in the vaccinated-PZQ-treated mice and was associated with a dramatic reduction of egg deposition in the tissues. We showed that PZQ treatment induced the unmasking of the native GST enzyme at the surface of the worms, thus permitting its neutralization by the antibodies raised by DNA immunization. This study provides insights into the synergistic mechanisms involved in an immunointervention strategy associated with chemotherapy for the control of a chronic infection and its associated pathology.

Vaccine 2000 Apr 14;18(20):2102-9

Immunogenicity of plasmid DNA encoding the 62 kDa fragment of Schistosoma japonicum myosin.

Zhang YY, Taylor MG, Gregoriadis G, McCrossan MV, Bickle QD.

Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, UK.

The recombinant Schistosoma mansoni 62 kDa myosin fragment, rIrV-5, is highly protective in experimental animals, however, vaccination of mice and rats with the recombinant Schistosoma japonicum homologue, rSj62, did not induce significant resistance against S. japonicum infection. To explore alternative ways of presenting this antigen, we further constructed a plasmid (VRSj62) which encodes Sj62 using the VR1020 vector and tested it in vaccination experiments. Four immunisations with 10 microg VRSj62 DNA alone were sufficient to induce high and progressively increasing levels of IgG antibodies against rSj62 with increasing numbers of injections in CBA/Ca mice (IgG titre > or =1:25000), and three injections with 50 microg VRSj62 DNA alone induced significant IgG responses in C57Bl/6 mice (IgG titre, 1:1600). However, vaccination with plasmid DNA entrapped in cationic liposomes or together with pUC19 DNA as a source of CpG motifs, both of which have been reported to enhance immune responses, did not enhance specific antibody production. In spite of the stimulation of specific antibodies against rSj62 with the naked DNA construct no resistance to challenge was demonstrated.

Vaccine 2001 May 14;19(25-26):3606 Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection.

Dupre L, Kremer L, Wolowczuk I, Riveau G, Capron A, Locht C.

Laboratoire des Relations Hotes-Parasite et Strategies Vaccinales, INSERM U 167, Institut Pasteur de Lille, F-59019 Cedex, Lille, France.

In vivo delivery of DNA encoding antigens is a simple tool to induce immune responses against pathogens. This approach to vaccination also offers the possibility to codeliver plasmids encoding immunomodulatory molecules in order to drive immune responses towards optimal protective effects. In the murine model of Schistosoma mansoni infection, vaccination inducing a Th1 profile has been shown to be protective. In this study, we used a plasmid encoding the Th1-promoting cytokine IL-18, since we observed that percutaneous infection of Balb/c mice strongly induced the production of IL-18 mRNA in the skin. Intradermal injection of the IL-18-encoding plasmid prior to infection did not interfere with parasite migration through the skin although it led to a local and transient cellular infiltration. When the IL-18-encoding plasmid was codelivered with a S. mansoni glutathione S-transferase (Sm28GST)-encoding plasmid, a 30-fold increase of antigen-specific IFN-gamma secretion by spleen cells was observed in comparison to spleen cells from mice that had received only the Sm28GST-encoding plasmid. This immunostimulatory effect was related to a significant protective effect (28% reduction in egg laying and 23% reduction in worm burden) which was attributed to a cooperative effect between both plasmids. Therefore, this study shows that codelivery of an IL-18-encoding plasmid with an antigen-encoding plasmid can stimulate specific cellular responses and induce protective effects against S. mansoni infection.

Vaccine 2001 Nov 12;20(3-4):359-69Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice.

Da'dara AA, Skelly PJ, Wang MM, Harn DA.

Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA.

Schistosomes are helminth parasites infecting at least 200 million people worldwide. In this study, we evaluated the feasibility of using a nucleic acid vaccine to induce protective immune responses to the Schistosoma mansoni integral membrane protein Sm23. C57BL/6 mice were immunized by intramuscular injection in three separate vaccination trials. ELISA and Western Blot analyses indicated that mice immunized with a DNA plasmid construct encoding Sm23 (Sm23-pcDNA) generated specific IgG for Sm23, while sera from mice immunized with the control pcDNA plasmid did not. The vaccine elicited IgG(2a), and IgG(1) antibody isotypes. We also tested the adjuvant activity of IL-12 and IL-4 on humoral responses to Sm23. Co-immunization with plasmid encoding IL-12 did not affect the level of anti-Sm23 IgG(2a), but did reduce the IgG(1) level. In contrast, co-injection with a plasmid encoding IL-4 significantly reduced the level of anti-Sm23 IgG(2a), while the level of IgG(1) was largely unchanged. Importantly, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection (21-44%, P<0.001-0.02). Co-administration of plasmids encoding either IL-12 or IL-4 did not significantly enhance this protective effect.

Vaccine 2001 Nov 12;20(3-4):439-47DNA vaccination with asparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni: anti-fecundity effect induced in mice.

Chlichlia K, Bahgat M, Ruppel A, Schirrmacher V.

Department of Tropical Hygiene, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. katerina.chlichlia@urz.uni-heidelberg.de

DNA-based vaccine technology was used to induce an immune response in mice against a schistosome cysteine proteinase, asparaginyl endopeptidase (Sm32). The cDNA coding for Sm32 was cloned in a mammalian expression vector under control of the CMV promoter/enhancer and expressed for the first time in transfected mammalian cells as well as in mice immunized with the Sm32-encoding DNA construct. These mice developed antibodies which recognized the native protein not only in homogenates of Schistosoma mansoni worms but also in the gut on cryostat sections of the parasites. This DNA vaccine led to an anti-fecundity effect: female worms of a challenge infection produced 37% less eggs than those growing in naive mice. The results suggest that Sm32 may be a candidate antigen for the generation of an anti-pathology vaccine against schistosomes.

Int J Med Microbiol 2002 Feb;291(6-7):577-82Delivery of protein antigens and DNA by attenuated intracellular bacteria.

Gentschev I, Dietrich G, Spreng S, Pilgrim S, Stritzker J, Kolb-Maurer A, Goebel W.

Department of Microbiology, University of Wuerzburg, Germany. gentsch@biozentrum.uni-wuerzburg.de

On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors. The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the alpha-hemolysin secretion system (type I) of Escherichia coli. The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen-presenting cells (APC) like macrophages and dendritic cells in vitro and can be also used in animal models.

J Med Virol 2002 Mar;66(3):320-8

Co-delivery of GM-CSF gene enhances the immune responses of hepatitis C viral core protein-expressing DNA vaccine: Role of dendritic cells.

Ou-Yang P, Hwang LH, Tao MH, Chiang BL, Chen DS.

Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan.

Hepatitis C virus (HCV) infection has become a critical public health problem worldwide. In Taiwan, it has been estimated that more than 300,000 people, 2% of the general population, have HCV infection. It has been well documented that direct delivery of gene intramuscularly can generate both humoral and cellular immunity, which more closely simulates the conditions of infection. In this study, female Balb/c mice immunized with HCV core plasmid DNA with or without adjuvant GM-CSF cytokine gene could induce both cellular immune response and HCV core-specific antibody titers after injection. Furthermore, the mice immunized with HCV core plus GM-CSF genes showed higher antibody titer and cytotoxic T cell activity compared to those of mice immunized with HCV core gene only (P < 0.05). To explore the effect of GM-CSF gene, the mice were immunized with reporter gene and cytokine gene plasmid. Increased levels of reporter protein and infiltrating cells around muscle tissue were noted. Moreover, the protein could be detected in inguinal node 24 hr after injection, especially in mice immunized with HCV/core plasmid plus GM-CSF gene. It was also observed that reporter protein expressing CD11c(+) dendritic cells could be seen in the inguinal node. These data suggest that the GM-CSF gene did enhance HCV core specific immune response when co-immunized with HCV core DNA plasmid. Although more studies are needed, dendritic cells that appeared around the naked DNA injection area and that local lymph nodes might play a critical role in the immune response induced by naked DNA immunization. Copyright 2002 Wiley-Liss, Inc.

: Immunol Lett 2002 Feb 1;80(2):89-96Oral DNA vaccination: antigen uptake and presentation by dendritic cells elicits protective immunity.

Cochlovius B, Stassar MJ, Schreurs MW, Benner A, Adema GJ.

Division of Diagnostics and Experimental Therapy, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

Melanoma differentiation antigens, such as glycoprotein 100 (gp100), have been shown to induce both cellular and humoral immune responses against melanoma in mouse and man. They are therefore considered as potential targets for melanoma immunotherapy. In this study, we have used the attenuated auxotrophic mutant strain SL7207 of Salmonella typhimurium as vehicle for a human gp100 (hgp100) DNA vaccine against melanoma. In vitro studies indicate that Salmonella/pCMV-hgp100 is efficiently scavenged by dendritic cells, resulting in the expression of the hgp100 transcription unit in the DC. In addition, oral administration of Salmonella/pCMV-hgp100 results in the expression of hgp100 RNA and protein by cells exhibiting DC-morphology in mesenteric lymph nodes as soon as 3 days after vaccination. Analysis of the efficacy of the Salmonella/pCMV-hgp100 vaccine in the B16/hgp100 model demonstrated the induction of strong anti-hgp100 CTL responses and protective immunity in 70% of the vaccinated mice, but not in control mice. Based on these data, we consider S. typhimurium as a useful vehicle for the design of recombinant DNA based anti-cancer vaccines.

Dev Biol (Basel) 2000;104:159-64The cellular basis of immune induction at mucosal surfaces by DNA vaccination.

Barnfield C, Brew R, Tilling R, Rae A, Wheeler C, Klavinskis LS.

Peter Gorer Department of Immunobiology, Guy's Hospital, GKT, London, UK.

The nasal mucosa provides a simple, non-invasive route to deliver DNA encoding the gene of interest to stimulate mucosal and systemic immune responses. However, unlike the intradermal or intramuscular routes for plasmid DNA (pDNA) delivery, immune responsiveness to antigen exposure at the respiratory mucosa is tightly regulated, consistent with the balance between active immunity and non-responsiveness to pathogenic or inert environmental antigens. We have characterised the antigen presenting cell types, their distribution and activation status following nasal vaccination with pDNA-cytofectin complexes encoding model antigens. We demonstrate that nasal immunisation is associated with expression of the encoded protein in a small population of dendritic cells and macrophages at the site of pDNA delivery, in the draining lymph nodes (LN) and in the spleen. Antigen expression

by nasal dendritic cells was associated with up-regulation of surface MHC class II and CD86 expression and functional activation of T-lymphocytes. The results highlight the potential of intranasal vaccination with pDNA, provided the activation / costimulatory requirements for an active immune response are achieved.

Immunol Lett 2001 Sep 3;78(2):103-11Dendritic cell discoveries provide new insight into the cellular immunobiology of DNA vaccines.

Coombes BK, Mahony JB.

Department of Medical Sciences, Father Sean O'Sullivan Research Centre, St. Joseph's Hospital, McMaster University, 50 Charlton Avenue East, Hamilton, Ontario, Canada L8N 4A6. coombebk@mcmaster.ca

The evolution of increasingly virulent human pathogens, together with the rapid onset of antimicrobial resistance has created a need for new vaccination strategies. Nucleic acid vaccines, based on recombinant DNA technology are a promising new vaccine formulation capable of eliciting both humoral and cellular immune responses. This technology has been experimentally validated in animal models of pathogen challenge and tumor protection following administration of a DNA vaccine and has led to extensive research into the mechanisms of protective immunity. We focus here on the cellular and molecular mechanisms leading to cell-mediated immune responses to DNA vaccines and discuss these mechanisms in light of recent advances in the field of dendritic cell immunobiology. In particular, the potential involvement of: (i) the CpG pattern-recognition receptor, toll-like receptor-9; (ii) the dendritic cell-specific surface adhesion molecule, DC-SIGN; and (iii) the molecular interactions between CD40 and CD154 in the evolution of protective cell-mediated immunity to DNA vaccines are discussed. An improved understanding of the precise mechanisms leading to protective cellular immunity following DNA vaccination may help in the design of novel DNA constructs containing immunostimulatory features that target one or more of these mechanisms, with the aim of increasing the immunogenic potential and protective efficacy of DNA vaccines.