No abstract

The interaction of pathogenic microorganisms with host tissues, and the underlying genetic events which regulate these interactions, are difficult to analyse where no suitable animal model exists. The approach described here, for obtaining information on the genes involved in these interactions, employs an infection system based on the invasion of Henle cells by Salmonella typhi to select promoter-containing DNA sequences able to activate gene expression inside eukaryotic cells. Several DNA fragments exhibiting different promoter strengths and extent of selective activation within eukaryotic cells were identified. Three were selected and characterized according to the expression level of the reporter gene, the polynucleotide sequence, the transcription start, and the dependence upon regulatory proteins. All fragments gave much stronger expression of the reporter gene when the recombinant S. typhi carrier strains invaded cells compared with the expression measured in growth medium. One promoter-containing region exhibited sequence homology to sigma 54-dependent promoters, whereas another appears to be dependent on the stationary-phase RNA polymerase subunit sigma s. S. typhi containing the S1 subunit gene of pertussis toxin cloned under the control of these promoters, selectively expressed the S1 subunit following infection of different phagocytic and non-phagocytic cell lines of human or murine origin. Deletion and point mutant derivatives of two promoters enabled the identification of the main motif required for promoter activity. This method may be helpful for the analysis of pathogenesis in organisms previously difficult to study because of the lack of a convenient animal model, and could provide insights into the chronology and topology of gene expression during infection, including a possible genetic basis for tissue tropism.

Recombinant Salmonella strains expressing heterologous antigens can be delivered by oral route triggering the elicitation of efficient antigen-specific humoral, T helper and cytotoxic responses. The potential of attenuated Salmonella spp. to trigger anti-tumor immunity was evaluated for the first time by using g-galactosidase (g-gal) as a model tumor-associated antigen (TAA). g-gal was expressed in a Salmonella typhimurium aroA vaccine carrier strain either constitutively or under the control of a promoter activated upon infection. Oral immunization with both vaccine prototypes resulted in the elicitation of g-gal-specific humoral and cell-mediated immunity. Although both strains were able to trigger antigen-specific CTL, responses were more efficient when the expression was controlled by the promoter activated upon infection. The anti-tumor efficacy of the stimulated response was validated by challenging vaccinated animals with an aggressive fibrosarcoma transfected with g-gal, which operationally acts as a TAA. Both groups of vaccinated mice exhibited a significant reduction in tumor take and growth with respect to animals vaccinated with plasmidless carrier (p X 0.05). However, the overall efficiency was better in the group in which g-gal was controlled by the in vivo-activated promoter (85 % versus 54 %; p X 0.05).

Recombinant Salmonella strains expressing heterologous antigens can be delivered by oral route triggering the elicitation of efficient antigen-specific humoral, T helper and cytotoxic responses. The potential of attenuated Salmonella spp. to trigger anti-tumor immunity was evaluated for the first time by using beta-galactosidase (beta-gal) as a model tumor-associated antigen (TAA). Beta-gal was expressed in a Salmonella typhimurium aroA vaccine carrier strain either constitutively or under the control of a promoter activated upon infection. Oral immunization with both vaccine prototypes resulted in the elicitation of beta-gal-specific humoral and cell-mediated immunity. Although both strains were able to trigger antigen-specific CTL, responses were more efficient when the expression was controlled by the promoter activated upon infection. The anti-tumor efficacy of the stimulated response was validated by challenging vaccinated animals with an aggressive fibrosarcoma transfected with beta-gal, which operationally acts as a TAA. Both groups of vaccinated mice exhibited a significant reduction in tumor take and growth with respect to animals vaccinated with plasmidless carrier (p < 0.05). However, the overall efficiency was better in the group in which beta-gal was controlled by the in vivo-activated promoter (85% versus 54%; p < 0.05).

Hepatitis C virus isolates which disclosed a novel genotype 1-associated restriction pattern by restriction fragment length polymorphism analysis were characterized. Except for a mother and child pair, the patients were unrelated. Sequence analysis showed a G3A substitution leading to a new RsaI recognition site. Phylogenetic analysis revealed that these isolates constitute a novel genetic lineage within the main cluster of genotype 1 strains.Hepatitis C virus (HCV) is an enveloped virus which belongs to the genus Hepacivirus in the family Flaviviridae (15). It is the most important cause of posttransfusion non-A, non-B hepatitis worldwide. The genome of HCV consists of a single strand of positive RNA (Ϸ9.5 kb), which codes for at least 10 viral proteins and is flanked by 5Ј-and 3Ј-end noncoding regions.The 5Ј-end untranslated region (5ЈUTR) of HCV is the most highly conserved portion of the viral genome and has been used to develop sensitive assays for RNA detection as well as for genotyping. According to current recommendations, HCV isolates are classified into six major clades, 1 to 6, whose nucleotide and inferred amino acid sequences differ by 35% (15, 18). Clade assignment is achieved by sequencing and aligning the HCV core, E1, or NS5B sequences with those of the prototypical strains of each clade (15). Although exact and reliable, this method cannot be easily implemented in conventional diagnostic laboratories. Consequently, several other methods to determine HCV genotype have been developed, including HCV RNA amplification followed by either reamplification with genotype-specific primers in the core region (11), hybridization with type-specific probes in the 5ЈUTR (16), or digestion of PCR products with restriction endonucleases that recognize genotype-and even subtype-specific sequence polymorphisms in the 5ЈUTR of the HCV (restriction fragment length polymorphism [RFLP]) (3,9). Using the RFLP method described by Davidson et al. (3), a widely used technique in our region, we reported a high prevalence of genotype 1a/c in children and infants in Argentina (6). Although rapid and simple, RFLP turned out to be unsatisfactory for the identification of HCV genotypes in isolates for five of our cases. Four of them were children and represented 14% of our pediatric population under study. Our aim was to characterize these isolates and to evaluate their diversity by means of nucleic acid sequencing and subsequent phylogenetic analysis.Plasma samples from five patients with chronic HCV infection (four children and one adult) were analyzed. Viral RNA was reverse transcribed, and the 5ЈUTR was amplified as previously described (5), with Kwok and Higuchi's recommendations (8). Amplicons were digested with restriction enzymes, followed by 15% or 12% polyacrylamide gel electrophoresis to evaluate the HCV genotype or subtype, as described by Davidson et al. (3). In addition, fragments were purified and sequenced with the Big Dye terminator cycle sequencing kit, version 1.1, and the 3100 genetic analyzer (Applied Bios...