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Enterotoxigenic E. coli (ETEC) are the major cause of traveler's diarrhoea and the CS3 fimbriae/fibrillae are expressed by most strains bearing the colonization factor CFA/II. The cstAH gene cluster determining CS3 biosynthesis has been previously cloned and sequenced and it has been shown that cstH encodes the major fimbrial subunit and cstA-G encode an assembly cassette. In the work described here we have sought to define the surface exposed domains on CS3 and to manipulate them so that CS3 can be used as a means of expressing foreign antigenic determinants on the bacterial surface. Using a panel of 21 monoclonal antibodies, which we have used in western blotting, immunofluorescence microscopy and colony blotting, together with computer predictions, we have identified three domains within CstH. Two of these sites were permissive for insertion and we have introduced, in-frame, either an epitope from the B subunit of LT (heat labile toxin) or the entire coding sequence of mature ST (heat stable toxin) to construct hybrid proteins. These proteins could be assembled into hybrid fimbriae which could be recognized by antibodies to both CS3 and the foreign epitope as shown by immunofluorescence microscopy and colony blotting. The immunogenicity of the constructs has been evaluated following both oral and intraperitoneal immunization of mice with the attenuated Salmonella typhimurium strain G30 harbouring the hybrid cst operons. Although plasmid stability is currently a problem, these experiments showed that antibodies to both the carrier and the foreign epitope were generated.

Synthetic lipopeptide analogues derived from the N-terminus of bacterial lipoprotein constitute potent B-lymphocyte and macrophage/monocyte activators in vitro. In vivo they act as immunoadjuvants in parenteral and oral immunization when administered in combination with antigens. When added to bacterial or viral vaccines, lipopeptides markedly enhance the vaccine effect. After the coupling of lipopeptides to haptens or non immunogenic low molecular mass antigens, a specific antibody response is induced often after only one application of the conjugate. The response can be further enhanced by introducing haplotype specific T helper cell epitopes into the conjugate. Lipopeptide antigen conjugates can also be applied as synthetic vaccines that give protection e.g. against foot-and-mouth-disease. The novel chemically well defined lipopeptides described here can be synthesized in gram amounts with high purity and reproducibility; they are non-toxic and can be stored for long time even at room temperature. For veterinary application, by replacing Freund's adjuvant, side reactions and inflammatory processes are avoided.

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different regions of a carrier protein. By using as carriers, two bacterial envelope proteins, the LamB and MalE proteins of E. coli K12, we were able to express the same epitope in different sites of the two proteins and in different compartments of the bacteria. This allowed us to analyze the influence of the localization in E. coli cells of a foreign B-cell epitope on the induction of specific antibody responses, and the role of the molecular environment on the immunological properties of foreign B- or T-cell epitopes, using either purified hybrid proteins or live recombinant bacteria. Several LamB and MalE hybrid proteins were expressed in the aroA attenuated strain of S. typhimurium, SL3261. Immunizations of mice with live recombinant bacteria by the intravenous route showed that it was possible to induce humoral responses against inserted foreign sequences. In order to improve the in vivo stability of the plasmids carrying the different contructions, and to increase the amounts of recombinant LamB and MalE hybrid proteins expressed in vivo, the LamB and malE genes were placed under the control of the anaerobically inducible pnirBpromoter control. The genetic factors susceptible of influencing the immune response to recombinant Salmonella in mice were also studied.

Infection of BALB/c mice with a standard and substantial number of Leishmania major parasites results in progressive disease, following the induction of a parasite-specific Th2 response. These mice have been designated as "susceptible" on this basis. We show that distinct types of immune response can be generated in "susceptible" BALB/c mice depending upon the number of parasites employed for infection, and that the pathophysiological consequences of such distinct responses are dramatically different. Infection with very low numbers of parasites results in the exclusive induction of a cell-mediated, Th1 response, and the generation of resistance to the standard and substantial challenge. Spleen cells from such resistant mice can confer resistance upon normal mice when transferred to them, but these spleen cells do not contain T cells expressing DTH or Th1 effector cells that produce IFN gamma on short term culture (48 hrs) with parasite antigen. The immune response in this case appears to result in the virtual elimination of parasites from the lymph node draining the site of infection and, by implication, from the infected mouse. We suggest that such elimination results in the absence of antigen stimulation and hence of effector T cells, and that "memory Th1 cells" are responsible for the capacity of spleen cells to confer resistance on normal mice. We predict such mice will not suffer parasitemia upon immune suppression, i.e. are not susceptible to reactivation disease. This is the "beneficial state". In contrast to this infection with a very low number of parasites infection with a low number usually results in one of two states: (i) The generation of a response with a very small Th2 component, production of a small amount of antibody, chronic parasitemia and hence chronic generation of parasite-specific effector Th1/Th2 cells, or (ii) The generation of a response with a greater Th2 component, the production of more antibody, the formation of a frank lesion, and the long term generation of a stable, mixed Th1/Th2 response. We refer to the latter state as borderline leishmaniasis in analogy with borderline leprosy. Parasites can be recovered from the draining lymph node in both these cases many months after infection. We therefore believe that mice infected with a low number of parasites, that harbour a chronic subclinical infection, will suffer reactivation disease upon immune suppression, and we consequently designate the state generated as potentially harmful. We consider mice with borderline disease to be in a harmful state. Mice immunised with high doses of parasite antigen produce in the long term Th2 responses, whereas those immunised with lower doses produce Th1 responses. Mice immunised to produce a Th2 response were subsequently infected with a very low number of parasites that is normally contained. The generation of a Th2 response results in the generation of a Th2 imprint, such that the response to the low dose infection is mo

Pseudomonas aeruginosa is a major pathogen in patients with cystic fibrosis (CF). In CF patients the opportunistic pathogen causes chronic pulmonary infections which are difficult to treat with antibiotics. Loss of lung function is the major cause of death in CF. Vaccination against P. aeruginosa is a possible way to prevent these infections and flagella antigens of P. aeruginosa seem to be promising vaccine candidates. In vitro and animal studies showed that flagella antigens were protective both as actively administered immunogens and in passive studies in compromised animals. Phase I studies using IMMUNO's flagella vaccines in healthy individuals revealed that, intramuscularly administered, these vaccine preparations were well tolerated, showed no adverse side effects and gave rise to high and longlasting antibody titers in the circulation of the individuals. Furthermore, immunisation with a flagella vaccine elicited specific anti-flagella antibodies not only systemically, but also in the secretory immune system of the airways. Consequently, a phase III multicenter vaccine trial using the Pseudomonas aeruginosa 5142/1210-Flagella Vaccine IMMUNO was initiated. The study design is placebo-controlled, randomized and double-blind, involving 400 CF patients without P. aeruginosa lung infection m 16 CF centers in Germany, France and Italy. The study will start in the fall of 1996 and will be carried out for 2 years.

DNA vaccination is an effective means of inducing both humoral and cell-mediated immunity in animal models of infectious disease. Presented here are data generated in two distinct disease models; one viral (influenza) and one bacterial (tuberculosis). Specifically, plasmid DNA encoding an influenza virus antigen (nucleoprotein; NP) and a Mycobacterium tuberculosis antigen (antigen 85; Ag85) were prepared and tested as DNA vaccines in mice. In both cases, high titer antibody responses and robust cell-mediated immune responses were induced against the respective antigens. With respect to the latter, lymphocyte proliferation, Th1-type cytokine secretion, and cytotoxic T lymphocyte responses were observed upon restimulation with antigen in vitro. Furthermore, protective efficacy in animal challenge models was demonstrated in both systems. The data support the hypothesis that DNA vaccination will prove to be a broadly applicable technique for inducing immunity against various infectious diseases.

When an antigen has passed the epithelial barrier of the skin or mucosal surfaces it has to be processed and presented by accessory cells to lymphocytes. These reactions take place in lymphoid organs, such as the regional lymph nodes, Peyer's patches and tonsils, but also in the spleen if the antigen entered the blood directly. The respective lymphocyte clone expands by proliferating, and primed lymphocytes of the B and T cell series emigrate from the lymphoid organs. The traffic of lymphocytes is regulated by the interaction of a series of adhesion molecules with endothelial cells and lymphocytes. Several earlier ideas, for instance one specific "homing receptor" for each organ and different receptors for B and T lymphocytes, or exclusive migratory routes for "memory" and "naive" lymphocytes, have had to be replaced by the concept of a much more complex, multistep, cascade-type reaction. Most migration routes show "preference" rather than "selectivity". The regulation of the entry of activated T and B lymphocytes into the parenchyma of non-lymphoid organs, e.g. the lamina propria of the gut, is not as well as understood as the entry into a lymph node. A further important aspect in lymphocyte traffic is the regulation of lymphocyte migration within the organs, including the interaction between lymphoid cells and the extracellular matrix. the basic mechanisms of lymphocyte migration have to be considered when the effects of vaccination procedures are interpreted.