Behring Institute Mitteilungen最新文献

The protozoan parasite Toxoplasma gondii comprises three clonal lineages that are associated with the clinical outcome in infected individuals. Whereas group C strains are mainly found in animals, group A and B strains are associated with human disease (Howe and Sibley, 1995). An increased level of transcripts of the tachyzoite-specifically expressed gene SAG1 could be identified in group A T. gondii strains compared to group B strains. Since SAG1-mediated host-cell invasion seems to be important for parasite replication, the observed higher replication rate in group A T. gondii strains might explain the association with clinically overt symptoms at the acute stage in patients who are infected with this group of parasite strains. The presence of external stress factors, such as interferon-gamma (IFN-gamma)-mediated nitric oxide (NO) formation has been identified to stabilize the cyst stage, most likely by activation of promoter(s) which drive the expression of genes encoding bradyzoite-specific antigens. Reactivation of chronic toxoplasmosis thus might occur in the absence of external stress factors, as has been observed in AIDS patients with decreases levels of IFN-gamma. Since group B T. gondii strains might form more cysts in infected individuals due to an increased potential to convert into bradyzoites, reactivation with resulting toxoplasmic encephalitis could be a more common event in those AIDS patients who were infected with persistent cysts of this group of parasite strains.

African trypanosomes can escape destruction by the immune system of their mammalian host by antigenic variation of the trypanosome surface coat. This coat is mainly composed of a single protein species, the Variant Surface Glycoprotein or VSG. The genes for VSGs are expressed in a polycistronic telomeric expression site together with at least eight expression site-associated genes (ESAGs). Trypanosomes may switch coat either by replacing the VSG gene in the active expression site by a different one, or by activating another expression site with concomitant silencing of the previously active one. Here we review our present knowledge of antigenic variation in Trypanosome brucei. We focus on four questions: How do trypanosomes switch from one VSG gene expression site to another one? What is the role of the novel base J in silencing expression sites? What is the functional significance of the antigenic variation of the heterodimeric transferrin receptor encoded by two ESAG genes? Why do trypanosomes have multiple expression sites at all?

Plasmodium vivax is the most widely distributed human malaria with an estimate of 35 million cases per year. The deduced amino acid sequence comparisons of the Merozoite Surface Protein 1 (MSP1) from several plasmodial species, including that of P. vivax (PvMSP1), revealed the existence of highly conserved blocks and polymorphic blocks. We had previously shown that sequences within conserved blocks from the N-terminal region of the PvMSP1 were poorly immunogenic in natural human infections. These results suggest that these regions code for important and unknown structural and/or functional features and thus they could potentially be tested as a sub-unit PvMSP1 vaccine. In the present study, a battery of monoclonal antibodies (Mabs) was produced against the N-terminal region of the PvMSP1 in an attempt to determine whether these N-terminal ICBs contained all the epitopes exposed on the native molecule. The results suggest that the most terminal ICB2 and ICB3 blocks are not exposed on the surface of the PvMSP1 native molecule and clearly eliminate the possibility of considering the N-terminal domains as unique components of a sub-unit PvMSP1 vaccine candidate.

The wide occurrence of molecular rearrangements associated with expression of specific members within multigene families led us to investigate whether this also happens during antigenic variation of malaria parasites. We have investigated here the Pf60 multigene family restriction patterns of four distinct variants of the Plasmodium falciparum Palo Alto line propagated in Saimiri monkeys. The O and its cloned Oc variant both express the O serotype, while the R variant (derived from O parasites) and the Vc variant (derived from Oc parasites) express distinct serotypes. We show that a specific modification of the restriction pattern could be associated with antigenic switching in this line. The DNA of the variants which expressed the O serotype (O and Oc) had a specific 5.5 kb Hind III/Eco RI restriction fragment which was absent from the R or Vc parasite DNA, whereas both R and Vc DNA presented a 3.5 kb Hind III/Eco RI restriction fragment, which was absent from the O and Oc parasites. These results indicate that both expression and silencing of the O serotype were associated with specific restriction patterns, suggesting that some molecular rearrangement or some modification of the DNA might control expression of the variant surface antigen in malaria parasites.

Flagellar murine MAbs (53B and 29) to strain a-type 170018 (a0,a3,a4, 45 kDa) and a human a-type MAb were studied in ELISA, Immunoblot, colony blot, agglutination and motility assays to evaluate the degree of cross-reactivity within the dominant a0 epitope. No specific effect of possible subtypes (a1, a2, a3, a4) was observed. An association of cross-reactivity and molecular weight was observed for 53B. A broader cross-reactivity as seen with MAbs 29 and A522 including high molecular weight flagellins (49-52 kDa), and particularly in the motility assay, may predict protective potential. Moderate reactivity with strain 5939 (a0 a3) was only seen with A522 MAb. These data indicate the presence of several cross-reactive sites associated with the common a0 antigen.

The hepatitis B virus nucleocapsid antigen (HBcAg) was investigated as a carrier moiety for circumsporozoite protein (CS) repeat B cell epitopes of the rodent malaria agent Plasmodium yoelii. A vector expressing a hybrid gene coding for the dominant CS repeat epitope (QGPGAP)4 was constructed and transformed into avirulent Salmonella typhimurium. The resulting hybrid HBcAg-CS polyproteins were purified from recombinant Salmonella typhimurium. They purified as particles and displayed HBc as well as P. yoelii CS antigenicity. To investigate immunogenicity and protective efficacy, BALB/c mice were immunized with the hybrid HBcAg-CS particles. Immunization resulted in high titered antinative CS serum IgG antibody litres. BALB/c mice immunized with hybrid HBcAgCS particles were between 90-100% protected against subsequent P. yoelli challenge. Protective immunity persisted for a minimum of three months. These data confirm the previous suggestion (Schödel et al., 1994), that hybrid HBcAg particles could become a useful component of future human malaria vaccines.

Despite a complex sputum bacteriology, the progressive decline in pulmonary function that is the hallmark of the genetic disease cystic fibrosis (CF) is attributable to a single infecting pathogen, mucoid Pseudomonas aeruginosa. Therefore, active and passive immunotherapies that target this particular variant of the bacterium should be of value in attenuating infection and interfering with the decline in pulmonary function. The major surface antigen of mucoid P. aeruginosa is referred to as either mucoid exopolysaccharide (MEP) or alginate, a random polymer of D-mannuronic and L-guluronic acid residues linked beta 1-4. During chronic infection CF patients make antibodies to MEP that fail to mediate opsonic killing of bacteria in vitro. These antibodies can be elicited by vaccination in 35-40% of plasma donors given a preparation of MEP comprised of only the highest molecular-weight polymers; inclusion in human vaccines of smaller polymers normally produced by the bacterium fails to elicit opsonic antibodies, just like in infected CF patients. Opsonic, but not non-opsonic, antibodies to MEP protect animals against chronic endobronchial infection. CF patients do produce opsonic antibodies to mucoid P. aeruginosa that are in a planktonic or suspended state, but these antibodies are not directed at the MEP antigen and they fail to kill P. aeruginosa growing in a biofilm. This is the state that the bacteria grow in the lung. Therefore immunoglobulin G preparations with opsonic antibodies to MEP could provide CF patients with antibodies that they normally do not produce during chronic lung infection and may improve their clinical course.

Mucosal surfaces of the respiratory, digestive and urogenital tracts are covered by a specialized epithelium which constitutes an efficient physical barrier against environmental pathogens. These surfaces differ greatly in their cellular organisation and in antigen sampling. In stratified epithelia, professional antigen-presenting cells, the dendritic cells or Langerhans cells, are intimately associated with the epithelial barrier and take up samples of foreign material from the external environment which they transport to local or distant organized lymphoid tissues. In simple epithelia highly specialised cells, the so-called M cells, sample foreign material and microorganisms and deliver them by transepithelial transport from the lumen to the underlying organized lymphoid tissue (MALT). The interaction of lymphocytes with the follicle-associated epithelium (FAE) is responsible for the loss of digestive functions and the acquisition of transepithelial transport activity. The three way interaction of epithelium, lymphoid cells, and microorganisms seen in the FAE which controls the formation of MALT provides a dramatic demonstration of the phenotypic plasticity of the intestinal epithelium and probably of all simple epithelia. We have shown that all mucosal surfaces, covered by stratified or simple epithelia are able to sample and transport live recombinant bacterial vaccines, which elicit systemic and local immune responses against the carrier and the foreign antigen. In gut and nasal-associated lymphoid tissue, Salmonella are taken up by dendritic cells which form a dense cellular network in the dome regions of MALT. Targeting bacterial vaccine candidates to dendritic or M cells is likely to facilitate their sampling by epithelial tissues and to contribute to strong mucosal and systemic immune responses.

New vaccination approaches and new delivery systems have been subject of intensive research activities recently. Controlled release vaccine delivery systems depend on the microencapsulation of antigens into biodegradable polymers, yielding small spherical polymeric particles, in the size range of 1-100 microns. By manipulating the micromorphology of the microparticles and degradation properties of the polymer either continuous or pulsatile release patterns can be adjusted. As biodegradable polymers mainly copolymers of lactic- and glycolic acid have been utilized, since these materials are known to be biocompatible and non-toxic. Apart from modulation of antigen release, an improvement of the adjuvant effect and an increase of in vitro (shelf-life) and in vivo stability of the antigen are issues of general interest with respect to parenteral vaccine delivery systems. Using different microparticles that release antigens in a pulsatile pattern at predetermined timepoints one hopes to induce protective immunity by a single administration of the vaccine delivery system. Using tetanus toxoid (TT) as a model antigen we have examined the stability during preparation, in vitro release and storage of TT microparticles. TT is a complex protein mixture sensitive to changes in pH conditions (pH < 5) and to thermal stress. TT microparticles can be prepared by a W/O/W double emulsion technique with satisfactory encapsulation efficiencies in good yields. In accordance with other investigators we observe an adjuvant effect of TT microspheres in mice upon sc administration leading to a long-lasting antibody response. In challenge experiments we could demonstrate a protective effect. The issue of an ideal release pattern remains open, since a boosting of the antibody titers during the bioerosion of the TT microspheres was not observed, possibly due to desactivation of TT in the degrading microspheres.