Behring Institute Mitteilungen最新文献

The lung infection with Pseudomonas aeruginosa is regarded as one of the major causes of health decline in patients with cystic fibrosis (CF). The CF host response to the persistent bacterial antigen load in the endobronchiolar lumen is characterized by a pronounced humoral response, local production of cytokines, influx of neutrophils into the lung and a protease-protease inhibitor imbalance predominantly sustained by released neutrophil elastase. CF is an autosomal recessive disease, and we could demonstrate for our local patient population that the age-dependent risk to become chronically colonized with P. aeruginosa can be differentiated by the disease-causing CFTR mutation genotype. The age-specific colonisation rates were significantly lower in pancreas sufficient than in pancreas insufficient patients. P. aeruginosa is occasionally detected in throat swabs already in infancy or early childhood in most patients although there is a lapse of several years amenable to preventive measures such as vaccination until onset of persistent colonization. The epidemiology of the infection with P. aeruginosa was investigated by quantitative macrorestriction fragment pattern analysis. The distribution and frequency of clones found in CF patients match that found in other clinical and environmental aquatic habitats, but the over-representation of specific clones at a CF clinic indicates a significant impact of nosocomial transmission for the prevalence of P. aeruginosa-positive patients at a particular center. Most patients remain colonized with the initially acquired P. aeruginosa clone. According to direct sputum analysis the majority of patients is carrying a single clonal variant at a concentration of 10(7)-10(9) CFU. Co-colonization with other species or other clones is infrequent. Independent of the underlying genotype, the CF lung habitat triggers a uniform, genetically fixed conversion of bacterial phenotype. Most CFP, aeruginosa strains become non-motile, mucoid, LPS-, pyocin- and phage-deficient, secrete less virulence determinants and shift the production of cytokines evoked in neutrophils. On the other hand, other properties such as antimicrobial susceptibility or adherence to bronchial mucins remain highly variable reflecting the capacity of P. aeruginosa to adapt to ongoing changes in the CF lung habitat.

Pseudomonas aeruginosa is an environmentally ubiquitous, extracellular opportunistic gram-negative bacteria that causes significant morbidity and mortality to a disproportionately high degree for infections with this bacteria compared with other gram-negative bacteria. Patients at particular risk of infection are those with compromised respiratory function, in intensive-care support and taking immunocompromising pharmaceutical agents. Once acquired, infection is difficult to eradicate with chemotherapy and attempts to vaccinate against infection have been of little success. Over the past five years, we have pursued the concept of mucosal immunisation against respiratory infection with P. aeruginosa. Initial studies in an acute animal model clearly demonstrated that mucosal immunisation with a killed whole bacterial cell preparation could induce protective immune responses in the lung. Subsequent studies have shown that the protective immune mechanisms were dependent on antigen specific CD4+ T cells, the activation of alveolar macrophages, the recruitment and activation of polymorphs, predominantly neutrophils, the controlled secretion of TNF-alpha, IL-1 and IFN gamma and the presence of antibody. We have hypothesised that the protective response is under the control of T cells. A pre-clinical human trial of an oral whole killed cell preparation has been completed with no adverse side effects. A limited open trial in patients with bronchiectasis has also been completed. Preliminary analysis of the results has demonstrated that after oral vaccination, specific lymphocyte responses were observed to P. aeruginosa.

Bacterial vaccine vectors have the potential to deliver a number of antigens from bacterial, protozoan and viral pathogens. To further develop the utility of bacterial vaccine vectors we are currently evaluating three model systems: 1. A Salmonella-ETEC Vaccine Vector; 2. A Salmonella-HIV Vaccine Vector, and 3. Novel Live Bacterial Nucleic Acid Vaccine Vectors. Through our studies, and those of others, significant progress has been made toward bacterial vaccine vector systems that effectively deliver subunit and nucleic acid vaccines to the organized lymphoid tissue of the intestine. The practical reality of these findings is discussed.

The induction of mucosal immune responses by oral delivery of vaccines is highly desirable. However vaccines to be used in this context will require protection from degradation in the gut and the use of specialised vehicles for their delivery and presentation. Using the biodegradable and biocompatible polymer, poly(lactide-co-glycolide), we have encapsulated bacterial and viral proteins, synthetic peptides and plasmid DNA in microparticles, and compared the immune responses resulting from their oral and parenteral administration to mice. The successful induction of specific systemic and mucosal humoral immune responses, as well as cell-mediated immune responses, demonstrates the potential of this polymer formulation as a vehicle for the oral delivery of vaccines.

Polynucleotide vaccines are a new approach to immunization that promises qualitative advances in vaccine technology. These vaccines mimic infection in that they result in expression of pathogen gene products in situ, which can elicit both cell-mediated immune responses and humoral responses. This approach has been applied primarily to vaccines against viral diseases, but may be significant for vaccines directed toward bacterial pathogens. Auragen has developed a generally applicable gene transfer technology and, for vaccine applications, has focused on particle-mediated gene transfer to epidermis. Results demonstrate that Accell polynucleotide vaccines induce immune responses toward human immunodefficiency virus (HIV) antigens, influenza A virus antigens, and hepatitis B virus (HBV) antigens in rodent,s swine and primates. Cellular immune responses toward these antigens have been demonstrated in rodents. In a swine influenza a challenge model Accell vaccination provides protection equivalent to that of a commercial killed-whole-virus vaccine. Vaccination of mice by this method toward a Chlamydia pneumoniae major outer-membrane protein elicits a species-specific antibody response.

A variety of gene delivery technologies can be used to express antigens within somatic tissues, resulting in systemic humoral and cellular immune responses. This observation has led to the development of polynucleotide vaccine preparations for stimulation of systemic immunity. Mucosal immune responses are functionally distinct from systemic immune responses, and are stimulated by antigen presentation within specialised mucosal-associated inductor tissues. We hypothesize that mucosal genetic vaccine will require gene transfer methods which target mucosal-associated inductor tissues such as the oropharyngeal Waldeyer's ring or intestinal Peyer's patches. We have tested this hypothesis by expressing a test antigen using a replication-defective recombinant Semliki Forest Virus (SFV) preparation. Mice treated with recombinant SFV via an intravascular or intratracheal route generated systemic immune responses against the test antigen. In contrast, intranasal inoculation resulted in the production of IgA within pulmonary fluids, one hallmark of a mucosal immune response. These results indicate that transfection of mucosal effector tissues may not be sufficient for the generation of a universal mucosal immune response. Furthermore, the results predict that techniques which target transfection or transduction to mucosal inductor tissues will enable the development of a new class of polynucleotide vaccines which exploit current concepts in mucosal immunology.

Peptide 10 (NATAEGRAINRRVE, residues 305-318 of mature protein F) is one of two linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa both of which have been shown to elicit whole cell-reactive antibodies and to afford protection in animal models against P. aeruginosa infection. Influenza A virus was chosen as a vector to present this epitope in a human-compatible vaccine. Various lengths of the peptide 10 epitope ranging from a 5-mer (GRAIN), 7-mer (AINRRVE), 8-mer (TAEGRAIN), 9-mer (GRAINRRVE), 11-mer (AEGRAINRRVE) to a 12-mer (TAEGRAINRRVE) were attempted to be presented into the antigenic B-site of the hemagglutinin (HA) of live recombinant influenza virus. Using PCR, DNA sequences encoding these various peptide 10 lengths were inserted into the HA gene of influenza A/WSN/33 virus. By using a reverse-genetics transfection system, RNA transcribed in vitro from these chimeric HA genes was reassorted into infectious virus. To date chimeric viruses have been rescued and purified containing the peptide 10 5-mer, 7-mer, 8-mer, and 11-mer. RT-PCR and sequencing have confirmed the presence of P. aeruginosa sequences in the HA RNA segment of each chimeric virus. Each of the four chimeric viruses produced to date was used to immunize mice to determine the ability of each chimeric virus to elicit antibodies reactive with whole cells of P. aeruginosa. The immunization protocol consisted of a series of three intranasal inoculations, followed by two intramuscular injections of the chimeric virus. The chimeric virus incorporating the 11-mer elicited IgG antibodies that reacted with various immunotype strains of P. aeruginosa in a whole cell ELISA at titers of 80 to 2,560, whereas the chimeric virus incorporating the 8-mer elicited whole cell-reactive IgG antibodies at titers of 320 to 2,560. These data suggest that these two chimeric viruses may have vaccine efficacy against P. aeruginosa infection. These studies may result in the development of a chimeric influenza virus-protein F vaccine which would prove to be suitable for use in children with cystic fibrosis for the prevention of pulmonary colonization of these children with P. aeruginosa.

Transepithelial transport of antigens and pathogens is the first step in the induction of a mucosal immune response. In the intestine, the delivery of antigens across the epithelial barrier to the underlying lymphoid tissue is accomplished by M cells, a specialized epithelial cell type that occurs only in the lymphoid follicle-associated epithelium. Selective and efficient transport of antigen by M cells is considered an essential requirement for effective mucosal vaccines. Therefore, particulate antigen formulations are currently being developed to take advantage of the capacity of M cells to endocytose particles. Based on pathogens that exploit the M cell as an invasion route into the body, live mucosal vaccines have been designed using genetically-engineered, attenuated strains of pathogens such as poliovirus and Salmonella. In an alternative approach, antigens are coupled to or encapsulated in particulate synthetic carriers. To enhance binding and uptake of such nonviable vectors, ligands are being attached which direct the vaccine particle to receptors on the M cell surface.

The E. coli hemolysin (HlyA) secretion apparatus represents a type I secretion system that is fully functional in Salmonella. The system which consists of the two specific membrane proteins HlyB and HlyD and the outer membrane protein TolC, recognizes on HlyA a C-terminally located signal sequence of about 60 amino acids. Fusion proteins to which this signal sequence is covalently linked at the C-terminus are also recognized by this secretion apparatus. The efficiency of secretion is dependent on the rate of folding of the reporter protein. Secretion-competent regions of a given reporter protein that is not secretable as entire protein can be screened by a recently constructed transposon TnhlyAs which allows the insertion of the secretion signal into any region of the reporter protein. The genetic information for antigens of any source ranging in size between 10 and 1000 amino acids can be easily inserted into a recently constructed secretion vector which will allow the secretion of the fused antigen(s) in attenuated Salmonella typhimurium strains and in other attenuated Enterobacteriaceae. By manipulation of the Hly secretion system the antigen can be either completely secreted into the environment, fixed on the outer membrane or arrested in the cytoplasm of the used carrier strain. By the use of appropriate attenuated Salmonella strains the antigen is delivered in isolated compartments or to the cytosolic compartment. The extracellular delivery of such antigens is also possible with the help of appropriate carrier strains. The immunological consequences of the different display of the processed antigen will be discussed in the paper by Hess et al in this volume. With a similar antigen delivery system the easy identification and molecular characterization of unknown antigens recognized by the immune system in an infection is also feasible.

Numerous experiments performed in humans and animals have revealed that stimulation of mucosal lymphoid inductive sites such as intestinal Peyer's patches results in parallel immune responses manifested by the appearance of S-IgA antibodies in the external secretions of remote glands. However, recent experiments suggest that inductive sites associated with the upper respiratory tract, rectum, and perhaps genital tract may also function as sources of lymphoid cells that populate, with some selectivity, certain remote mucosal effector sites. Furthermore, antigen-specific IgA antibodies can be induced in certain secretions (e.g., female genital tract) not only by immunization in the vicinity of corresponding mucosal tissues (e.g., vagina and rectum) but also by oral and especially intranasal immunization. The ineffectiveness of simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies of strategies for effective delivery systems that would (a) increase the antigen absorption, (b) prevent its degradation, and (c) skew the outcome of immunization to a desired goal (protective response to infectious diseases vs. tolerance; B vs. T cell responses; mucosal vs. systemic). The induction of immune responses at a desired mucosal site can be accentuated with the use of a suitable antigen-delivery system including relevant bacterial or viral vectors, edible transgenic plants expressing microbial antigens, incorporation of antigens in biodegradable microspheres or liposomes, and linkage or coadministration of antigens with cholera toxin B subunit. However, only a few antigen-delivery systems extensively used in animal experimentation have been evaluated for their efficacy in humans. The combination of various immunization routes and the use of suitable antigen-delivery systems may accomplish an important task-the induction of mucosal immune responses at a location relevant to the site of entry of a given pathogen.