Cloning and sequencing of the glucosyl transferase-encoding gene from converting bacteriophage X (SFX) of Shigella flexneri.

Shigella flexneri type Y strains (-;3,4) are converted to type X (-;7,8) by bacteriophage X (SFX) that causes glucosylation of the O-antigenic polysaccharide chain. The gene (gtr) encoding glucosyl transferase from bacteriophage X has been cloned and sequenced. The protein encoded by gtr consists of 416 amino acids with a M(r) of 47,369. The cloned gtr product was able to convert a S. flexneri strain type Y (SFL 124, a live attenuated candidate vaccine strain) to type X. The importance of the hybrid strain in vaccine development is discussed.

Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV.

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI-BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.

Molecular characterization of the genes involved in O-antigen modification, attachment, integration and excision in Shigella flexneri bacteriophage SfV.

Bacteriophage SfV is a temperate phage of Shigella flexneri responsible for converting serotype Y (3,4) to serotype 5a (V; 3,4) through its glucosyl transferase gene. The glucosyl transferase (gtr) gene of SfV has been cloned and shown to partially convert S. flexneri serotype Y to serotype 5a. In this study, we found that the serotype-converting region of SfV was approximately 2.5 kb in length containing three continuous ORFs. The recombinant strain carrying the three complete ORFs expressed the type V and group antigen 3,4, both indistinguishable from that of S. flexneri 5a wild-type strain. The interruption of orf5 or orf6 gave partial conversion in the S. flexneri recombinant strain indicated by the incomplete replacement of group antigen 3,4. The region adjacent to the serotype-conversion genes was found to be identical to the attP-int-xis region of phage P22. Altogether, an approximately 2.2-kb sequence covering a portion of the serotype-conversion (approximately 500 nt)-attP-int-xis regions of SfV was remarkably similar to that of P22.

The pathology of Shigella flexneri infection in rhesus monkeys: an endoscopic and histopathological study of colonic lesions.

Twenty-two Rhesus monkeys were orally fed 1 x 10(11) live virulent Shigella flexneri of either serotypes 1b, 2a, 4a or Y. On the basis of colonoscopic findings they were classified into: group A - normal endoscopic picture (10 monkeys), and group B - pathological endoscopic picture (12 monkeys). Pathological findings, distributed over the entire colon, were seen as either red patches (+/- erosions) or diffuse lesions, i.e. fragile red mucosa, mucosal bleeding and broad edemas. Histopathological examination of concomitant biopsies showed an acute inflammation restricted to the mucosa in 8/12 of group B as compared to 2/10 of group A. The Shigellae were most commonly demonstrated in the surface epithelium and more rarely in the deep layer of the lamina propria. Immunohistochemical staining, using monoclonal antibodies directed against Shigella flexneri O-antigenic polysaccharide, showed a high correlation with histopathological findings. Clinically all 10 monkeys in group A remained healthy, whereas 7/12 (all displaying histopathological signs of acute inflammation) in group B developed dysenteric symptoms. Colonoscopy should be combined with histopathological and immunohistochemical examinations of biopsies to study the pathological events taking place in the colon tissue during the course of a Shigella infection and will be of great value to assess the protective efficacy of S. flexneri vaccine candidates.

Immune responses in Vietnamese children after a single dose of the auxotrophic, live Shigella flexneri Y vaccine strain SFL124.

The live, auxotrophic Shigella flexneri vaccine strain SFL124 was given in a single dose of 10(7), 10(8) or 10(9) colony forming units (cfu), respectively, to each of three groups of 10 Vietnamese children aged 9-14 years. The vaccine was well tolerated by all the children without any severe side effects such as diarrhoea or fever being observed. Mild symptoms were reported by five children. Only five children were found by culture to excrete SFL124 but, by PCR, 28 of 30 children were found to excrete the vaccine strain for up to 5 days (mean 2.8 days) with insignificant differences among the groups. Local mucosal immune responses and antibody secreting cell (ASC) responses to S. flexneri lipopolysaccharide (LPS) and invasion plasmid-coded antigens (Ipa) were elicited in the children in a dose-dependent manner. Doses of 10(9) cfu induced most prominent responses, followed by those of 10(8) and 10(7) cfu. The sIgA responses were the highest whereas the ASC were modest. High titres of serum antibodies to Shigella LPS and Ipa were found in all the children before ingestion of the vaccine which elicited increases in serum antibody titres in only a few of them. The immune response patterns seen indicate a booster rather than a primary response and may be a consequence of the endemic nature of shigellosis in Vietnam.

Immunogenicity of the Shigella flexneri serotype Y (SFL 124) vaccine strain expressing cloned glucosyl transferase gene of converting bacteriophage SfX.

The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O-antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild-type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O-antigen immunity in the host defense against shigellosis.

Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers.

The live, auxotrophic dependent Shigella flexneri Y vaccine strain SFL124 with a deleted aroD gene was tested in 30 healthy adult male Vietnamese volunteers. A single dose of 2 x 10(9) live bacteria was given orally to 15 volunteers, whereas 15 received three doses every other day. None of the volunteers reacted with fever or diarrhoea and SFL124 was excreted by all for a mean of 2.8 (single dose) and 2.6 (three doses) days. A total of 27 of 30 (90%) and 26 of 30 (87%) responded with significantly (0.001 < p < 0.01) increased antibody-secreting cell (ASC) numbers against Shigella flexneri Y lipopolysaccharide (LPS) and invasion plasmid-coded antigens (Ipa). A faecal IgA antibody response to LPS and Ipa was seen in 20 of the 30 (67%) volunteers against both antigens. Serum antibody responses were seen in 23 of 30 (77%) against the LPS and in 17 of the 30 against Ipa. The three-dose schedule elicited only somewhat stronger immune responses than the single-dose schedule. A booster dose of 2 x 10(9) live bacteria was given to half of the volunteers in each group after 6 months, the other half received the same dose after 12 months. Following the booster at 6 or 12 months (i) the excretion of SFL124 was significantly shorter (p < 0.05) than after primary vaccination; (ii) the anti-S. flexneri LPS and anti-Ipa faecal sIgA titres were significantly higher (p < 0.05 to p < 0.01) than after primary vaccination; (iii) the anti-LPS and anti-Ipa ASC responses were significantly lower (p < 0.05) and of shorter duration than after primary vaccination, and (iv) the serum anti-LPS and anti-Ipa responses were significantly elevated (p < 0.05) and similar to those seen after primary vaccination. The results indicate that SFL124 is a safe, live vaccine strain with a negligible reactogenicity in adults living in a Shigella endemic area. SFL124 induces specific immune responses against LPS and Ipa with a mucosal memory lasting for at least 1 year.