Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens.

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.

Positive-negative selection for homologous recombination in Arabidopsis.

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.

Mucosal and systemic immune responses in humans after primary and booster immunizations with orally administered invasive and noninvasive live attenuated bacteria.

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.

European multi-centre validation study of NucliSens Extractor in combination with HCV Amplicor 2.0 assay for HCV-NAT screening of plasma pools.

The NucliSens Extractor in combination with the 2.0 version of the Roche Cobas HCV Amplicor test has been validated by five European blood screening laboratories in a multi-centre study. For testing the performance characteristics of this HCV-NAT method, the European Pharmacopoeia validation guidelines were followed. The CLB VQC reference reagents were used for testing robustness and sensitivity. After a technical improvement in the extraction stations, the NucliSens Extractor appeared to be contamination-free as was proved by testing negative controls alternating with samples containing a high HCV-RNA concentration. The Pelicheck HCV-RNA genotype 1 dilution panel was tested 74 times in the five laboratories and an overall 95% detection limit of 80 genome equivalents (geq)/ml was found. In one laboratory the Pelicheck panel was tested in 25 runs and here a 95% detection limit of 32 geq/ml was achieved. In this laboratory the Pelispy HCV-RNA run control samples of 140 geq/ml were consistently picked up in all extractor stations. In addition the laboratories have tested a WHO HCV-RNA genotype 1 standard dilution series 39 times and a Pelicheck HCV-RNA genotype 3 reference panel in 32 test runs. The limiting dilution analysis enabled us to compare the detection efficiency of the NucliSens-Amplicor method for the genoype 1 and genotype 3 isolates and to calibrate the reference reagents against each other. The combined Nuclisens-Amplicor method was found to detect the genotype 3 isolate in the Pelicheck HCV-RNA panels with 2-3 fold lower efficiency than the genotype 1 standard (assuming that the historical calibration of the genotype 3 against the genotype 1 standard is correct). In this study of a single method 1 IU of the WHO HCV-RNA standard was found to be equivalent to 5.1 geq of the VQC HCV-RNA standard (95% confidence intervals 3.1-9.1 geq). To avoid confusion with the use of the CLB VQC reagents we accept the NIBSC collaborative study in which calibration by a variety of methods showed that the Pelispy 380 geq/ml run control is equivalent to 100 IU/ml of the WHO standard. This multi-centre validation study demonstrates that the 95% detection limit of the NucliSens HCV Amplicor method lies far below the detection limits required by the international regulatory bodies.

Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease.

The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.

Versatile cosmid vectors for facilitated analysis and subcloning of the insert.

A series of cosmid vectors, termed pSSVI215, pSSVI216-1, pSSVI216-2, pSSVI217, and pSSVI218, were constructed in order to facilitate the downstream processing of large inserts. Each vector has dual cos sites as well as a kanamycin resistance (KmR) gene flanked by recognition sites for the very rare cutter I-SceI meganuclease as well as symmetrical NotI and SwaI sites (SCEKAN cassette). Several unique cloning sites, including BamHI, are present on one side of the cassette between the I-SceI and NotI/SwaI sites. The various cosmids differ from each other by one or more of the following features: origin of replication (ori), size, host range, and conjugal transfer capability. Inserts combined with the SCEKAN cassette can be isolated on a NotI or SwaI fragment from any of these vectors and easily subcloned into the vector of choice by selecting for the adjacent KmR gene which can later be removed by I-SceI restriction and self-ligation. In addition, the SCEKAN cassette can be conveniently excised from plasmid pSSVI214 such that any plasmid can easily be fitted with the present system. The subcloning strategy afforded by the new vectors was successfully applied to an approximately 37-kb fragment from the V. cholerae O139 genome carrying the rfb locus which encodes the O-serotype specificity of this organism.

Construction and characterization of a potential live oral carrier-based vaccine against Vibrio cholerae O139.

The rfb region from Vibrio cholerae O139 strain MO45 was cloned from cosmid gene banks established in Escherichia coli HB101, using an immunoblot assay for screening of the correct clones. Immunoblot analysis of lipopolysaccharide (LPS) preparations revealed the presence of two types of positive clones: (i) those expressing only a short core-linked O polysaccharide (SOPS) and (ii) those also expressing a highly polymerized capsular polysaccharide (CPS) not bound to the E. coli K-12 LPS core. In addition, the latter clones appear to contain a locus which may encode a putative regulator of SOPS and CPS chain length. Further characterization in E. coli showed that CPS constitutes a barrier against large particles such as the bacteriophage Ffm but not against bacteriophage lambda or P1. In addition, a portion of the K-12 LPS core may not be substituted with SOPS. Loci associated with the two clonal types were transferred into V. cholerae CH19, an rfbAB deletion mutant of CVD103-HgR deficient in the production of the homologous Inaba O polysaccharide. This resulted in the stable expression of SOPS, alone or together with CPS, that was indistinguishable from that of wild-type V. cholerae O139. Strains CH25 and CH26, which correspond to CH19 bearing the V. cholerae O139 rfb region integrated into the chromosome, were found to be genetically stable and essentially identical to the parent CVD103-HgR with respect to physiological properties such as cell motility, mercury resistance, toxicity, and production of the cholera toxin B subunit. Rabbits immunized with CH25 elicited high titers of anti-O139 SOPS- and CPS-specific serum antibodies. These strains possess characteristics desirable in candidate live oral vaccines against V. cholerae O139.

Further molecular characterization and stability of the live oral attenuated cholera vaccine strain CVD103-HgR.

Vibrio cholerae CVD103-HgR, the first live attenuated vaccine licensed for human use produced by recombinant DNA technology, was genetically compared to its parent strains 569B and CVD103. The genetic stability for both lyophilized vaccine in final container form and for viable organisms shed from vaccinees was determined. Results obtained lead us to conclude: (i) the genetic composition of the examined genes in CVD103-HgR is identical to that of the parent strains except for the alterations induced; (ii) the level of mercury resistance depends on the orientation of the mer operon within hlyA, with the highest level being observed for the orientation found in CVD103-HgR; (iii) no DNA sequences from plasmids used in construction remain in the genome; (iv) the strain is genetically stable; and (v) both CVD103-HgR and its parent strains contain defective lysogenic prophages. We have further confirmed that a certain amount of restriction fragment length polymorphism (RFLP) exists around the chromosomal ctx locus within V. cholerae strains of the classical biotype (detectable on chromosomal DNA restricted by either HindIII or EcoRI, but not PstI).

Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae.

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103-HgR. Expression studies showed that the production of complete S. sonnei O-polysaccharide (O-PS)-bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host-encoded Inaba O-PS. Conversely, high amounts of LPS carrying S. sonnei O-PS are produced in monovalent Inaba-deficient derivatives, even in those strains which do not co-express the compatible R1 LPS core. Thus, the non-enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O-PS provided that competition with the host O-PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103-HgR. Both monovalent and bivalent strains stimulated high serum-antibody titres specific for their respective O-serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O-serotypes is discussed.

Development of Shigella sonnei live oral vaccines based on defined rfbInaba deletion mutants of Vibrio cholerae expressing the Shigella serotype D O polysaccharide.

Previous experimentation has highlighted a number of difficulties in the development of carrier-based bivalent vaccines (J.-F. Viret and D. Favre, Biologicals 22:361-372, 1994) In an attempt to obviate these carrier strains. Toward this aim, a series of defined rfbInaba deletion (delta rfbInaba) mutants of the cholera vaccine strain V. cholerae CVD103-HgR (O1 Inaba serotype) and derivative bearing the chromosomally integrated locus encoding the S. sonnei O-PS were constructed and characterized. The various mutations disrupt genes thought to be involved in either the synthesis of perosamine, the synthesis of 3-deoxy-L-glycero tetronic acid, or the O-PS transport functions together with synthesis of the perosamine synthetase. Some deletions were obtained only in strains expressing the heterologous lipopolysaccharide (LPS). Viable delta rfbInaba deletions in CVD103-HgR profoundly altered some of its phenotypic properties. The same deletions present in CVD103-HgR derivatives expressing the heterologous LPS affected their phenotypes only to a lesser extent. Only in strains in which perosamine synthesis was specifically abolished could high amounts of core-bound S. sonnei O-PS be synthesized. Two such strains (CH21, which expresses both the R1 core and the S. sonnei O-PS, and CH22, which expresses only the latter antigenic determinant) were further analyzed and were found to be indistinguishable from CVD103-HgR with regard to lack of enterotoxin activity, choleragenoid production, mercury resistance, pilin production, and, for CH22, motility. Mice immunized with CH22 produced high titers of S. sonnei O-PS-specific antibodies.

Activity of the yeast FLP recombinase in Arabidopsis.

The coding sequence for FLP recombinase, originally from the 2 mu plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations.

Polyclonal preparations of anti-tetanus toxoid antibodies derived from a combinatorial library confer protection.

We have compared the in vivo therapeutic potential of anti-tetanus toxin (TT) human Fab antibodies derived from a combinatorial phage display library to established polyclonal and monoclonal reagents. The oligoclonality and fine specificity distribution of the synthetic anti-TT Fab preparations was comparable to the antibody spectrum present in the donor serum and the affinities determined for the synthetic phage-bound Fab (Phab) and soluble Fab were in the same range as their monoclonal and polyclonal counterparts. On a weight basis, the protective capacity of the new oligoclonal preparations in vivo (16.4 IU/100 micrograms Fab) was comparable to those of the best combinations of hybridoma derived human monoclonal antibodies, and far better than those exhibited by the polyclonal serum antibodies of the donor (0.29 IU/100 micrograms IgG) or by a standard commercial human tetanus immunoglobulin preparation. These data suggest that recombinant antibodies may become a safe and effective alternative to human plasma-derived immunoglobulins for passive immunization.

Bivalent vaccines against bacterial enteropathogens: construction of live attenuated vaccine strains with two O-serotype specificities.

A considerable interest exists worldwide in the development of live attenuated oral vaccines against diarrhoeal diseases. In addition to vaccination against the corresponding pathogens, such vaccine strains can be used as carriers for the expression of protective antigens from other organisms. The antigenic repertoire of a given vaccine strain may thereby be extended, potentially leading to a bivalent vaccine. The lipopolysaccharide is known to be a major antigenic surface component of bacterial enteric pathogens. The feasibility of the development of combined vaccines based on live attenuated carriers expressing two O-serotype specificities is illustrated here by the development of candidate live oral vaccines against Shigella sonnei using Salmonella typhi and Vibrio cholerae as carriers. Various factors that may limit the potential of such hybrid strains as bivalent vaccines are discussed.

Transcriptional photoregulation of cell-type-preferred expression of maize rbcS-m3: 3' and 5' sequences are involved.

In the C4 plant maize, members of the rbcS gene family, encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, are not expressed in mesophyll cells (MC) but are expressed strongly in the adjacent bundle sheath cells (BSC). Expression of genes in an in situ transient expression assay indicates that the photostimulated expression seen in BSC during the first 24 h that leaves of dark-grown seedlings are illuminated requires rbcS-m3 sequences lying between -211 bp and +434 bp of the transcription start site. Photoregulated partial suppression of rbcS-m3 expression in MC, on the other hand, requires gene sequences that lie between -907 bp and -445 bp together with sequences that lie between +720 and +957 bp within the 3' transcribed region of the gene. Suppression in MC occurs during the second 24-h period that dark-grown seedlings have been illuminated, but not during the first 24 h. The 3' +720- to +957-bp region is also effective in lowering MC expression when it is relocated to a position > 2 kbp upstream of the transcription start site. Thus, suppression of rbcS-m3 expression in MC has, at the least, a substantial transcriptional component. As reported earlier, a converse pattern of suppression in BSC and stimulation of expression in MC is seen in the control of cab-m1 in maize leaves.

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains.

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype-specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei O polysaccharide is covalently bound to LPS cores of both the K-12 and R1 types, but neither to Salmonella (Ra-type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1-type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfasonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by R1-type LPS. Co-expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of rfaR1 DNA are required for the expression of complete phase-I-like S. sonnei LPS in E. coli K-12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae. S. sonnei O antigen expressed in a V. cholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.

The cab-m7 gene: a light-inducible, mesophyll-specific gene of maize.

Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclear cab multigene family encoding the chlorophyll a- and b-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3' probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designated cab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. This cab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts of cab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of the cab-m7 gene resemble those found in the 5'-flanking regions of some other plant genes.

Characterization of the Shigella serotype D (S. sonnei) O polysaccharide and the enterobacterial R1 lipopolysaccharide core by use of mouse monoclonal antibodies.

In the course of developing a live vaccine, we generated three murine monoclonal antibodies (MAb) specific for Shigella sonnei. The specificities of these MAb were determined by enzyme-linked immunosorbent assay and immunoblot analyses with whole cells or purified lipopolysaccharides (LPSs) as antigens. Two of them are specific for the Shigella serotype D O-polysaccharide determinant, whereas one specifically binds to the core hexose region of R1-type LPSs. With these MAb, it was possible to analyze clinical isolates and a hybrid Salmonella typhi strain for their expression of the corresponding LPS moieties. In addition to their use in the screening of candidate vaccine strains, the new MAb provide a powerful tool for epidemiological and phylogenetic studies of natural enterobacterial populations.