The Neisseria gonorrhoeae lpxLII gene encodes for a late-functioning lauroyl acyl transferase, and a null mutation within the gene has a significant effect on the induction of acute inflammatory responses.

LPS is a fundamental constituent of the outer membrane of all Gram-negative bacteria, and the lipid A domain plays a central role in the induction of inflammatory responses. We identified genes of the Neisseria gonorrhoeae lipid A biosynthetic pathway by searching the complete gonococcal genome sequence with sequences of known enzymes from other species. The lpxLII gene was disrupted by an insertion-deletion in an attenuated aroA mutant of the gonococcal strain MS11. Lipopolysaccharide (LPS) and lipid A analysis demonstrated that the lpxLII mutant had synthesized an altered LPS molecule lacking a single lauric fatty acid residue in the GlcN II of the lipid A backbone. LPS of the lpxLII mutant had a markedly reduced ability to induce the proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human macrophages and IL-8 from polymorphonuclear cells. This study demonstrates that the lpxLII gene in gonococci encodes for a late-functioning lauroyl acyl transferase that adds a lauric acid at position 2' in the lipid A backbone. The presence of lauric acid at such a position appears to be crucial for the induction of full inflammatory responses by N. gonorrhoeae LPS.

Expression and immunogenicity of an Echinococcus granulosus fatty acid-binding protein in live attenuated Salmonella vaccine strains.

Fatty acid-binding proteins (FABPs) are candidate molecules for vaccines against several parasitic platyhelminths. A FABP from the cestode Echinococcus granulosus (EgDf1) was expressed in Salmonella vaccine strains as a C-terminal fusion to fragment C of tetanus toxin (TetC) by using expression vector pTECH. The fusion protein was equally expressed in several attenuated vaccine strains derived from bacteria with different genetic backgrounds and different attenuating mutations. Single-dose immunization experiments with the aroA Salmonella typhimurium strain SL3261 carrying the pTECH-EgDf1 construct were conducted with mice, using both the intravenous and the oral routes. Surprisingly, the antibody response to EgDf1 and the antigen-specific cytokine production in spleen cells were stronger in mice immunized orally. Furthermore, immune mouse sera strongly reacted with fixed sections of the worm's larval stage. Analysis of the isotype distribution of the specific anti-EgDf1 antibodies showed similar production of immunoglobulin G1 (IgG1) and IgG2a together with specific IgA antibodies. In addition, stimulation of spleen cells from mice immunized with the different constructs with either Salmonella lysate, TetC, or EgDf1 showed that, together with Th1-related cytokines (gamma interferon and interleukin 2 [IL-2]), significant levels of a Th2 cytokine (IL-5) were produced specifically, indicating a Th2 component to the response to the Salmonella carrier and to the recombinant antigens. Salmonellae expressing the TetC-rEgDfl fusion are currently under evaluation as potential vaccines against E. granulosus.

Correlates of protection induced by live Aro- Salmonella typhimurium vaccines in the murine typhoid model.

Live attenuated salmonella vaccines generally confer better protection than killed vaccines. The immune responses in BALB/c mice elicited by immunization with a live attenuated Aro Salmonella typhimurium vaccine given orally, intravenously or subcutaneously were compared with those elicited by killed whole-cell vaccines (acetone or heat-treated) given subcutaneously. Live vaccines given by all routes elicited higher interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) responses in spleen cells against an alkali-treated whole-cell salmonella lysate than did killed vaccines. Live and killed vaccines elicited high total antibody levels to smooth lipopolysaccharide (LPS) (enzyme-linked immunosorbent assay), but all live vaccine regimes elicited higher IgG2a, suggesting a Th1 response. Oral and intravenous vaccination with live organisms elicited IgA against smooth LPS which subcutaneous vaccination with live or killed salmonellae failed to evoke. Western blots using rough whole-cell lysates showed that all vaccines elicited a varied anti-protein response; however, all groups immunized with live organisms recognized three unidentified bands of MW 52,000, 46,000 and 18,000 which were consistently absent in groups immunized with killed organisms. The results indicate that immunization with live aroA salmonellae elicited a Th1 type of response, including bystander T-cell help to LPS, and a response to proteins not seen in mice that received killed vaccines.

Protection against oral challenge three months after i.v. immunization of BALB/c mice with live Aro Salmonella typhimurium and Salmonella enteritidis vaccines is serotype (species)-dependent and only partially determined by the main LPS O antigen.

The role of the main LPS O antigen in the specificity of protection as mediated by systemic mechanisms following immunization with live attenuated Aro Salmonella vaccines was studied in mice. Innately Salmonella-susceptible (Itys) BALB/c mice were immunized intravenously with a single dose of either Salmonella typhimurium SL3261 aroA (LPS O4,5,12) or Salmonella enteritidis Se795aroA (LPS O1,9,12), and challenged orally 2-3 months later with either S. typhimurium C5 or S. enteritidis Thirsk. Nearly isogenic transductants of the two challenge strains expressing either their own LPS or that of the other serotype (S. typhimurium C5 O4 or O9, and S. enteritidis Thirsk O9 or O4) were also used. Both vaccines conferred similar high protection against the virulent strain of the homologous serotype expressing its own LPS. There was no protection against the heterologous serotype expressing its own LPS. However, when vaccinated mice were challenged with either the same serotype as the vaccine but expressing the heterologous LPS, or with the heterologous serotype expressing the LPS of the vaccine, protection was always lower than protection against the fully homologous serotype. Anti-smooth LPS antibodies showed higher titres against the homologous LPS, but with significant crossreactivity with the heterologous LPS. Antibodies to O-rough S. typhimurium and S. enteritidis LPS were present following immunization with either of the two vaccine strains. The LPS alone cannot fully account for the specificity of protection in this model; other (protein) antigens may be responsible. It remains to be seen whether there is a T-cell mediated component to the specificity of protection conferred by live Salmonella vaccines.

A serum-sensitive, sialyltransferase-deficient mutant of Neisseria gonorrhoeae defective in conversion to serum resistance by CMP-NANA or blood cell extracts.

A stable, sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62 totally defective in CMP-NANA-dependent lipopolysaccharide (LPS) sialylation was isolated by insertion mutagenesis with transposon Tn 1545-delta 3 and screened for unlabelled colonies following incubation with CMP-14C-NANA. In contrast to the parental strain which became serum resistant on incubation with CMP-NANA or blood cell extracts, the mutant, JB1, remained serum sensitive. French press extracts of strain F62 catalysed LPS sialylation, but corresponding extracts of the mutant were inactive. Five LPS components were detected by SDS-PAGE in the parental strain. Five components of the same Mr were also found in the mutant. Three identical components were detected by Western blotting using MAb 3F11, which recognizes the Gal beta 1-4GlcNAc groups in the conserved LPS components of F62 which can be sialylated. The mutant, JB1, is therefore deficient in the sialyltransferase that is essential for both LPS sialylation and conversion of serum-sensitive gonococci to serum resistance by either CMP-NANA or blood cell extracts. No evidence was obtained for an LPS sialylation pathway by blood cell extracts that is independent of CMP-NANA.

Construction, expression, and immunogenicity of multiple tandem copies of the Schistosoma mansoni peptide 115-131 of the P28 glutathione S-transferase expressed as C-terminal fusions to tetanus toxin fragment C in a live aro-attenuated vaccine strain of Salmonella.

Genetic fusions have been constructed between the highly immunogenic but atoxic fragment C of tetanus toxin and a guest peptide, aa115-131, from the protective 28-kDa glutathione S-transferase Ag of Schistosoma mansoni. Fusions have been assembled to express one, two, four, and eight tandem copies of the peptide. The recombinant vectors have been electroporated into the nonvirulent aroA strain of Salmonella typhimurium SL3261. The fusion proteins are soluble and stably expressed in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice have been immunized i.v. with a single dose of the live recombinant salmonellae. The strains are stable in mice and elicit Ab responses directed against fragment C, as determined by enzyme-linked immunosorbent assays. Ab responses were also detected against the guest peptide. The Ab responses improved dramatically toward the aa115-131 peptide with increasing copy number, with the octameric "repitope" fusion displaying the greatest potency. This approach may represent a general strategy for eliciting immune responses against peptides in live bacterial vaccines.

Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella.

A vector has been constructed to allow genetic fusions of guest antigens via a hinge domain to the C terminus of the highly immunogenic C fragment of tetanus toxin. A fusion has been constructed with the gene encoding the protective 28-kDa glutathione S-transferase (EC 2.5.1.18) from Schistosoma mansoni. The recombinant vector has been electroporated into the nonvirulent Salmonella typhimurium aroA live vaccine strain SL3261. The corresponding chimeric protein is stably expressed in a soluble form in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice immunized intravenously with a single dose of the live recombinant bacteria elicit antibodies to both fragment C and glutathione S-transferase as detected by enzyme-linked immunosorbent assays. Furthermore, all of the mice were solidly protected when challenged with lethal doses of either tetanus toxin or the virulent Salmonella typhimurium strain C5. Mice have also elicited antibodies to fragment C and glutathione S-transferase after oral immunization. It may be that a live trivalent vaccine against typhoid, tetanus, and schistosomiasis is feasible.

Toxicity of lipopolysaccharide and of soluble extracts of Salmonella typhimurium in mice immunized with a live attenuated aroA salmonella vaccine.

Mice immunized intravenously 10 days earlier (but not those immunized 2 months earlier) with an attenuated Salmonella typhimurium SL3261 aroA live vaccine and tested for delayed-type hypersensitivity by injection of crude Salmonella extracts in the footpad can die within 24 to 48 h of an unexplained allergic reaction. The lethal reaction could be prevented by prior administration of anti-tumor necrosis factor alpha serum. Injection of lipopolysaccharide (LPS) (either purified phenol-water-extracted [Westphal] LPS or protein-rich trichloracetic acid-extracted [Boivin] LPS) was also lethal for mice immunized 10 days before. An LPS-rich crude Salmonella extract was more toxic than one which contained less LPS, suggesting that LPS may have been involved in the lethal reactions to crude antigens. Mild alkaline hydrolysis removes O-linked acyl groups from lipid A and eliminates many toxic effects of LPS; however, both Boivin LPS and Westphal LPS remained toxic for immunized mice after alkaline hydrolysis. In contrast, alkaline hydrolysis of crude whole Salmonella extracts (which caused marked protein degradation) reduced the lethal toxicity of the extracts, especially for an LPS-rich preparation. Mice immunized orally with the live vaccine did not show hypersensitivity to either LPS or crude extracts. The results suggest that the lethal reaction to crude Salmonella antigens in mice immunized 10 days earlier is complex, that tumor necrosis factor alpha is involved, and that allergic reactions to crude antigens (but not to LPS alone) can be reduced by mild alkaline hydrolysis.

Neisseria gonorrhoeae strain MS11 harbouring a mutation in gene aroA is attenuated and immunogenic.

An aroA mutant of gonococcal strain MS11 was constructed (JKD298) and compared with the wild-type (JKD288). The requirement of JKD298 for aromatic compounds, typical of an aroA mutant, was demonstrated using defined media. Other than the expected auxotrophy, no further differences could be demonstrated between the parent and the aroA mutant. SDS-PAGE analysis of protein and lipopolysaccharide (LPS) profiles showed no differences between the strains. Bactericidal assays using human and guinea-pig normal sera showed that both strains were serum sensitive and were similarly converted to serum resistance by in vitro sialylation using CMP-NANA. Infectivity experiments in guinea-pig subcutaneous chambers showed considerably reduced virulence of the aroA JKD298, which could only infect chambers at very high doses. Established infections by either strain elicited a strong PMN response in which similar proportions of each strain were seen intracellularly. Infections by JKD298 provoked a strong antibody response as detected by ELISA using whole sonicated gonococci. This is the first demonstration of attenuation of Neisseria gonorrhoeae by introduction of a defined mutation in a metabolic gene.

Delayed (footpad) hypersensitivity and Arthus reactivity using protein-rich antigens and LPS in mice immunized with live attenuated aroA Salmonella vaccines.

Footpad reactions to protein-rich salmonella extracts and lipopolysaccharide (LPS) were studied in BALB/c mice 2 and 8 months after immunization with the Salmonella typhimurium SL3261 aroA live vaccine. T-cell depletion in vivo and adoptive serum transfer showed that protein-rich antigens induced T-cell dependent delayed hypersensitivity reactions, whereas LPS only elicited Arthus reactions. The footpad reactions to crude protein extracts were not always T-cell mediated, but depended on the nature and the dose of the antigen. Selective depletion of CD4+ T cells alone had a greater effect than depletion of CD8+ T cells alone, but neither was as marked as simultaneous depletion of both CD4+ and CD8+ T cells, which abolished the delayed type hypersensitivity (DTH) response. Crude protein-rich extracts subjected to alkaline hydrolysis (which removes some ester-linked fatty acids and causes disaggregation of LPS resulting in decreased toxicity while conserving O-specificity) still gave positive T-cell dependent reactions, but with reduced T-cell independent reactivity. Purified phenol-water LPS (2.5 micrograms) produced Arthus reactivity which could be confused with DTH. LPS induced positive reactions which still occurred in T-cell depleted mice and were transferable by immune serum. Arthus reactions did not occur when using alkali-treated LPS, which showed reduced complement fixation in vitro when using serum from immunized mice. The results indicate that footpad testing using salmonella antigens containing LPS elicit DTH but can also produce toxic reactions, some of which are T-cell independent and not necessarily a true measure of DTH. Arthus reactivity to LPS can be confused with DTH. Alkaline hydrolysis of the antigens can eliminate non-specific reactogenicity while retaining the ability of the (protein-rich) antigen to elicit a true T-cell dependent footpad response, which requires the participation of both CD4+ and CD8+ T cells.

Serum TNF alpha inhibitor in mouse typhoid.

Administration of anti-TNF alpha antiserum enhanced a sublethal infection with salmonellae of moderate virulence (Salmonella typhimurium M525) in innately susceptible (Ity(s)) BALB/c mice, indicating that TNF alpha is important in the early response which suppresses bacterial growth in the reticuloendothelial system (RES). However, only transient low levels of TNF alpha were detectable on day 3 in sera from some, but not all, sublethally infected mice. Conversely, on day 4 of the same infection, clear TNF alpha inhibitory activity was detected in some sera. Neither TNF alpha or any inhibitory activity were detected in sera of lethally infected BALB/c mice undergoing an acute, overwhelming Salmonella infection. In contrast, TNF alpha inhibitory activity, but not TNF alpha, was detected in sera of mice showing a cachectic syndrome induced by persistent high bacterial numbers following intravenous inoculation of a very high dose (2 x 10(7)) of the attenuated aro- S. typhimurium SL3261 strain.

Alterations of the LPS determine virulence of Neisseria gonorrhoeae in guinea-pig subcutaneous chambers.

The virulence of lipopolysaccharide (LPS) variants of Neisseria gonorrhoeae strain Gc40 was studied in vivo using the guinea-pig subcutaneous chamber model. Survival of variants D1, D2, D4 and D5 was assessed by viable counts made on chamber fluid at various times after inoculation. Chemotactic effect was measured by counts of white cells in the chambers. Differential cell counts and assessments of the location of the gonococci were made on Giemsa-stained smears of chamber fluid. Sensitivity of the variants to normal guinea-pig serum was determined by in vitro bactericidal assays. D1 and D5 had relatively high Mr LPS which was shed in the medium, were serum resistant, produced intense infections and were mainly extracellular. Large number of damaged white cells were present. D2 and D4, had low Mr LPS which was poorly shed in the medium, were serum sensitive and produced low grade infections. D2 was the least infective and was seen mainly inside neutrophils. Collectively the data indicates that the type of LPS on the gonococcal surface and possibly the amount of shed LPS strongly influence the fate of gonococci in vivo, in an environment in which antibodies, complement and phagocytic cells are freely available. This may be decisive at some stages of the human infection.

The surface structure seen on gonococci after treatment with CMP-NANA is due to sialylation of surface lipopolysaccharide previously described as a 'capsule'.

Phenotypic serum resistance of gonococci in urethral exudates is due to sialylation of lipopolysaccharide (LPS) by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). A surface structure was visible on gonococci [strain BS4 (agar)] that had been stained with ruthenium red after incubation with CMP-NANA. This structure was not visible after neuraminidase treatment, which released sialic acid but not LPS. The LPS profiles of strain BS4 (agar) had another in vivo-selected strain Gc40 (variant D1), were similar. A monoclonal antibody (mAb) which recognises epitope C on the LPS of 'capsulated' gonococci was shown by immunoblotting to react with several LPS components, including one of about 4.5 kDa which contains the probable site of sialylation by CMP-NANA. The reactions with the mAb were not affected by growing the strains with CMP-NANA nor by neuraminidase treatment of the sialylated LPS. The mAb also gold-labelled the surface of both strains before and after treatment with CMP-NANA. These data indicate that sialylation by CMP-NANA and staining with ruthenium red renders more visible the surface LPS which, sometimes in the past, has been seen as a 'capsule'.

Neisseria gonorrhoeae LPS variation, serum resistance and its induction by cytidine 5'-monophospho-N-acetylneuraminic acid.

Inherent serum resistance and the effect of the serum resistance inducing factor cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were studied on Neisseria gonorrhoeae with different lipopolysaccharides (LPS). Strain M01 and LPS variants of strain Gc40 (variants D1, D2, D4 and D5) were examined after incubation in the presence or absence of CMP-NANA by bactericidal assays using normal human or immune sera and by SDS-PAGE followed by silver staining or Western blotting. The blots were probed with monoclonal antibody CC1, specific to epitope C of the LPS. Variants D1 and D5 were inherently serum resistant, variants D2 and D4 and strain M01 were susceptible. CMP-NANA induced marked changes in the LPS of all gonococci. However, only some gonococci were converted to serum resistance. Gonococci which were converted to serum resistance had LPS with components of relatively large molecular mass, expressing epitope C. Variants which did not convert to serum resistance had LPS with low molecular mass components only, without epitope C. Conversion to serum resistance increased the size of the large LPS components without affecting the expression of epitope C. The results indicate that conversion to serum resistance by CMP-NANA is not a general occurrence but depends on the quality of the LPS.

Yersinia enterocolitica aroA mutants as carriers of the B subunit of the Escherichia coli heat-labile enterotoxin to the murine immune system.

Plasmid p5F which directs the expression of the Escherichia coli heat-labile enterotoxin B subunit (LT-B) from the ptac promoter was introduced into the attenuated Yersinia enterocolitica O:8 aroA mutant strain YAM.1. YAM.1 (p5F) expressed high levels of cell-associated and secreted LT-B in a stable fashion when grown on normal laboratory medium. The strain was used as a live oral vaccine in BALB/c mice and vaccinated mice developed high levels of gut-associated and systemic antibodies to both LT-B and the lipopolysaccharide (LPS) of the vaccine strain. Anti-LT-B and anti-LPS responses in the sera were predominantly of the IgG class whereas gut-associated antibodies were predominantly IgA. ELISPOT assays carried out on selected tissues prepared from vaccinated mice showed significant numbers of cells synthesising IgG and IgA antibodies to LT-B. These results show that Y. enterocolitica aroA mutants can be used effectively as carriers of heterologous antigens to the murine immune system.

Immunization of guinea pigs with epitope C-rich lipopolysaccharide from Neisseria gonorrhoea; in vivo selection of gonococcal variants.

The immunogenic potential of lipopolysaccharide (LPS) of a variant of Neisseria gonorrhoeae strain Gc 40 selected by growth in vivo (vivo variant) was investigated in guinea pigs. LPS extracts obtained from the water (WLPS) and the phenol (PLPS) phases of a hot phenol-water extraction were compared for their antigenic capacity and protective effect against infection in subcutaneous chambers. Immunization with PLPS induced significant levels of anti-LPS and anti-epitope C antibodies but WLPS was not antigenic. Two days after challenge, all guinea pigs immunized with WLPS had infections similar to those seen in unimmunized control animals while most animals immunized with PLPS and challenged with either 10(3) or 10(5) gonococci per ml showed low numbers of or no viable gonococci in their chambers. Five days after challenge, however, the same animals had chamber infections with high viable counts similar to controls. Gonococci reisolated from three such animals had physically and antigenically altered lipopolysaccharide and showed patterns of serum sensitivity to pre-challenge chamber fluid from immunized animals which were different from those of the parent vivo variant used for immunization and challenge. The results demonstrate that selection of LPS variants occurs in vivo. This could constitute an immune evasion mechanism.

Antibodies to the C epitope of Neisseria gonorrhoeae are present in patients with gonorrhoea and absent in normal sera.

We have previously described a surface oligosaccharide antigen (epitope C) present in fresh isolates of Neisseria gonorrhoeae and in variants grown in subcutaneous chambers, but poorly formed by variants repeatedly subcultured in vitro. We have now investigated the presence of antibodies to epitope C in sera from normal individuals and from patients with gonorrhoea. Sera were analysed by Western blotting and ELISA, and compared with a pool of sera from normal individuals with no known history of gonorrhoea. Antigenic extracts and monoclonal antibody to the C epitope were used for competition and inhibition studies. Only the sera from patients contained antibodies to epitope C. Antibodies to several other gonococcal antigens were found in sera from patients, and also in normal sera. Collectively, the results indicate that epitope C is expressed in humans, that patients with gonorrhoea develop antibodies to it, and that such antibodies are absent in sera of normal individuals.

Gonococcal variants selected by growth in vivo or in vitro have antigenically different LPS.

Lipopolysaccharide (LPS) was extracted from two variants of strain gc40 of Neisseria gonorrhoeae obtained by repeated subculture in vitro or by growth in vivo in a subcutaneous chamber. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by silver stain analysis revealed that both variants had three main LPS components, but the large size components were predominant in gonococci selected in vivo and the smallest size in those selected in vitro. Western blotting, ELISA and ELISA inhibition using monoclonal and polyclonal antibodies showed that the two variants had antigenically different LPS and that serum sensitivity may be due to the antigenic specificity of the large components. These results indicate that during infection clones of gonococci are selected with LPS of antigenic and physicochemical composition different from those seen after repeated subcultures.

An attenuated aroA Salmonella typhimurium vaccine elicits humoral and cellular immunity to cloned beta-galactosidase in mice.

beta-Galactosidase (GZ) is an intracellular protein that is frequently used to express cloned antigens as fusion proteins in Escherichia coli. Salmonella typhimurium strain SL3261, an attenuated aroA vaccine strain, was used as a carrier for the plasmid pXY411, which directs the expression of GZ in salmonellae (which do not normally produce this protein). The resulting strain--SL3261(pXY411)--expressed GZ as an intracellular antigen. The plasmid was stable in vitro and in vivo and did not significantly alter the behavior of strain SL3261 in mice. Animals intravenously vaccinated with this construct developed circulating antibodies to GZ, as measured by ELISA, and delayed hypersensitivity to the antigen injected in the footpad. These results indicate that attenuated salmonellae may be expected to elicit both humoral and cellular responses to intracellular cloned antigens.

Definition of a virulence-related antigen of Neisseria gonorrhoeae with monoclonal antibodies and lectins.

Variants of one strain of Neisseria gonorrhoeae, grown in vivo or in vitro, that have been previously shown to differ in infectivity, serum resistance, and capsule production were compared with use of monoclonal antibodies and lectins. Monoclonal antibodies to virulent gonococci recognized an antigenic site of the lipopolysaccharide (LPS) produced in large amounts by gonococci grown in vivo but present only in a small proportion of in vitro-grown gonococci. This antigen (C-LPS) was found in all 85 different gonococcal isolates studied but not among nonpathogenic neisseriae. It was shared by group B and C meningococci but not by groups A and D. Enzyme-linked immunosorbent assay and Western blot analysis showed that N-acetylglucosamine and N-acetylgalactosamine form part of the epitope. The C-LPS antigen was shown by immunofluorescence to be present on the surface of the gonococci and also free as slime. This antigen appears to confer resistance to killing by normal sera.