Use of Recombinant Virus Replicon Particles for Vaccination against Mycobacterium ulcerans Disease

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans.     

Evolutionary stability of the R1 retrotransposable element in the genus Drosophila

R1 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts into a specific sequence of insect 28S ribosomal RNA genes. We have previously shown that this element has been maintained through vertical transmission in the melanogaster species subgroup of Drosophila. To address whether R1 elements have been vertically transmitted for longer periods of evolutionary time, the analysis has been extended to 11 other species from four species groups of the genus Drosophila (melanogaster, obscura, testecea, and repleta). All sequenced elements appeared functional on the basis of the preservation of their open-reading frames and consistently higher rate of substitution at synonymous sites relative to replacement sites. The phylogenetic relationships of the R1 elements from all species analyzed were congruent with the species phylogenies, suggesting that the R1 elements have been vertically transmitted since the inception of the Drosophila genus, an estimated 50-70 Mya. The stable maintenance of R1 through the germ line appears to be the major mechanism for the widespread distribution of these elements in Drosophila. In two species, D. neotestecea of the testecea group and D. takahashii of the melanogaster group, a second family of R1 elements was also present that differed in sequence by 46% and 31%, respectively, from the family that was congruent with the species phylogeny. These second families may represent occasional horizontal transfers or, alternatively, they could reflect the ability of R1 elements to diverge into new families within a species and evolve independently.      

Immunoreactivity of six monoclonal antibodies directed against 1,25-dihydroxyvitamin-D3 receptors in human skin

Using confocal laser scanning microscopy, we tested the suitability of five monoclonal mouse antibodies (IVA7E7, IVB12G12, IVG9C11, VD2F12, and VIIID8C12) that had been raised against different domains of the porcine intestinal 1,25-dihydroxyvitamin-D₃ receptor (VDR), for the immunohistological detection of VDR in human skin. The VDR immunoreactivity of these antibodies was compared with the well-characterized VDR-staining pattern of the mouse monoclonal antibody 9A7 raised against chick intestinal VDR. All six antibodies revealed strong nuclear and qualitatively similar immunoreactivity in all cell layers of the viable epidermis. Our data demonstrate that the five mouse monoclonal antibodies are suitable for immunohistochemical detection of VDR in frozen sections. These antibodies show comparable staining patterns in human skin even though they had been raised against different functional domains of the 1,25-dihydroxyvitamin-D₃ receptor.     

Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain

Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.     

Effect of light and oxygen and adaptation to changing light conditions in a photosynthetic mutant in which the LHII complex of Rhv. sulfidophilum was heterologously expressed in a strain of Rb. capsulatus whose puc operon was deleted

In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII⁻ mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation. Received: 20 November 2001 / Accepted: 31 December 2001