Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals.

Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (Ags), the precise requirements for in vivo MAIT cell responses to infection remain unclear. Using major histocompatibility complex-related protein 1 (MR1) tetramers, the MAIT cell response was investigated in a model of bacterial lung infection employing riboflavin gene-competent and -deficient bacteria. MAIT cells were rapidly enriched in the lungs of C57BL/6 mice infected with Salmonella Typhimurium, comprising up to 50% of αß-T cells after 1 week. MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long-term accumulation in the lungs, draining lymph nodes and spleen. Lung MAIT cells from infected mice displayed an activated/memory phenotype, and most expressed the transcription factor retinoic acid-related orphan receptor γt. T-bet expression increased following infection. The majority produced interleukin-17 while smaller subsets produced interferon-γ or tumor necrosis factor, detected directly ex vivo. Thus the activation and expansion of MAIT cells coupled with their pro-inflammatory cytokine production occurred in response to Ags derived from microbial riboflavin synthesis and was augmented by co-stimulatory signals.

Control of gonococcal pilin-encoding gene expression in Escherichia coli.

The pilE gene from Neisseria gonorrhoeae, unlike other type-4 pilin-encoding genes, is well expressed in Escherichia coli. Two putative promoters have been implicated in the transcription of this gene. Besides the -24/-12 promoter used to transcribe type-4 pilin-encoding genes in most species, the consensus sequence for a conventional promoter is also present. The two promoters overlap and would have almost identical transcription start points (tsp). Transcription from a -24/-12 promoter should be abolished in an E. coli rpoN mutant. A recombinant plasmid carrying pilE could not be transformed into such a mutant, apparently because the synthesis of the N-terminal hydrophobic domain of pilin is lethal to the rpoN mutant. This suggests that pilE is expressed at a higher level in an rpoN mutant than it is in a wild-type (wt) strain of E. coli. This suggestion was confirmed by constructing fusions between the pilE promoter region and a promoter-less cat gene. We suggest that the conventional promoter is primarily responsible for the transcription of pilE, but that the binding of the RpoN sigma factor partially represses transcription of this gene in wt strains. In an rpoN mutant, the repression is removed and transcription occurs at a level that is lethal to the mutant host.

Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains.

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimurium chromosome which directed the expression of tetanus toxin fragment C and the Treponema pallidum lipoprotein. The expression of both antigens was detected by Western blotting.

Nucleic acid vaccines: tasks and tactics.

There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.

Developmental expression of a Fasciola hepatica sequence homologous to ABC transporters.

Polymerase chain reaction and cDNA library screening approaches were employed to identify a putative member of the highly conserved family of ATP-binding cassette transport proteins from Fasciola hepatica. At the predicted protein level, the F. hepatica sequence identified in the present study shares 43% and 36% identity with the Schistosoma mansoni SMDR2 and human MDR1 ATP-binding cassette transport sequences, respectively. Northern blot and reverse transcriptase-PCR analyses have demonstrated that expression of the F. hepatica ABC-transporter homologue is confined to immature parasites. The biochemical basis for the stage-specific expression of the ATP-binding cassette transporter homologue within F. hepatica remains to be determined.

Use of the polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid.

The polymerase chain reaction was used to detect Treponema pallidum in specimens of cerebrospinal fluid (CSF), as a means of diagnosing syphilis. Segments of the TmpA and 4D genes were amplified to provide an estimated threshold sensitivity of approximately 65 organisms in 0.5 ml. A spectrum of pathogens known to cause meningitis, and several non-pathogenic treponemes were unreactive. Treponema pertenue, and only one of 30 control specimens of CSF were positive. In contrast, 10 of 19 CSFs from patients being evaluated for latent or tertiary syphilis were positive, as were 7 of 28 specimens from HIV-positive patients.

Cloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development.

Corynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA- background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA- C. pseudotuberculosis was 10-12-fold lower than that in the recA+ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA- background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.

The sacB gene cannot be used as a counter-selectable marker in Pasteurella multocida.

The use of the Bacillus subtilis sacB gene as a counter-selectable marker was assessed in serogroup A and B strains of Pasteurella multocida. Expression ofsacB failed to render any of the strains sensitive to sucrose, indicating that the sacB gene can not be used as a positive selection system in P. multocida.

Kinetics of the mucosal antibody secreting cell response and evidence of specific lymphocyte migration to the lung after oral immunisation with attenuated S. enterica var. typhimurium.

The kinetic of mucosal secretory responses elicited by the vaccine vector Salmonella enterica var. typhimurium (S. typhimurium) was examined by enzyme linked immunospot (ELISPOT) and compared with serum responses. Mice immunised orally with BRD509, the aroA, aroD mutant of virulent S. typhimurium SL1344 expressing the C Fragment of tetanus toxin (TT), simultaneously developed an IgA antibody secreting cells (ASC) response in the gastrointestinal lamina propria, the spleen and the lung, against both S. typhimurium lipopolysaccharide (LPS) and TT. The magnitude of the ASC response was greatest in the gut, was boosted by a secondary immunisation at day 25, and the kinetic of the response did not correlate with the appearance of serum antibodies. This study suggests that S. typhimurium can engage the common mucosal immune system to effect mucosal secretory responses at distal sites, however, the magnitude of the responses is both greatest in the gut and antigen-specific. The ASC origin of the serum antibodies specific for S. typhimurium and antigens expressed by the bacterium is yet to be elucidated.

Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica.

The aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, from Pasteurella haemolytica biotype A, serotype 1 was cloned by complementation of the aroA mutation in Escherichia coli strain AB2829 after electroporation with a DNA library constructed in pUC18. The cloned P. haemolytica aroA gene was inactivated by insertion of a kanamycin resistance gene and reintroduced by allelic exchange into the chromosome of the parental P. haemolytica using PbluescriptII SK+. The P. haemolytica aroA mutant was highly attenuated in a mouse septicaemic model. Mice immunized intraperitoneally with two doses of live P. haemolytica aroA mutant were protected against a lethal parental strain challenge.

A comparison of DNA vaccines expressing the 45W, 18k and 16k host-protective antigens of Taenia ovis in mice and sheep.

The immunogenicity of DNA vaccines encoding three different Taenia ovis host-protective antigens was compared in mice and sheep. DNA vaccines encoding the 45W, 18k and 16k antigens of T. ovis were constructed. The ability of DNA vaccines encoding the 45W and 18k genes to express antigen was confirmed by Western blotting of transfected Cos-7 cells. BALB/c mice were vaccinated intramuscularly with 45W, 18k or 16k DNA vaccines and the humoral immune response analysed by ELISA. DNA vaccines expressing 45W, 18k or 16k antigen were immunogenic in mice and generated significant titres of antigen-specific antibody. Intramuscular vaccination of outbred sheep with the T. ovis DNA vaccines generated significantly lower titres of 45W-specific antibody and failed to generate 18k or 16k-specific antibody. The findings of this study show that each of the three T. ovis host-protective antigens are amenable to delivery via DNA vaccines, and that the parameters governing the efficacy of DNA vaccines in sheep require further investigation.

Crude saponins improve the immune response to an oral plant-made measles vaccine.

Millions of people live in areas where infectious diseases, such as measles, are endemic and resources are scarce. Heat-stable vaccines that are delivered orally will greatly enhance vaccination programs in these areas. A stumbling block in the development of oral vaccines is the availability of safe and effective mucosal adjuvants, especially for use with subunit vaccines. The experiments presented here examine the ability of CTB/CT, LT(R192G) and crude Quillaja saponin extracts to stimulate MV-specific immune responses in mice, following oral immunisation with plant-made measles virus hemagglutinin (MV-H) protein. LT(R192G) and crude saponin extracts both functioned as potent mucosal adjuvants when ad-mixed with plant-made MV-H protein, and were more effective than CTB/CT. MV-H protein supplemented with saponin extract induced the strongest MV-specific responses, in the greatest number of mice. Crude saponins are routinely used by the food and beverage industry at concentrations greater than those required for adjuvanticity, and as such, they have a better safety profile than bacterial enterotoxins. This study demonstrates their potential as adjuvants for use with oral plant-made vaccines.

Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination.

Plant-made oral vaccines have the potential to overcome many of the limitations of traditional vaccines. Here we report on progress towards a lettuce-made measles vaccine. Lettuce is a palatable species which exhibits rapid growth in contained hydroponic systems and produces negligible quantities of toxins. Measles virus hemagglutinin (MV-H) protein was successfully expressed in transgenic lettuce and found to be immunogenic in mice. Lettuce extracts containing MV-H protein induced MV neutralising antibodies following intraperitoneal injection and intranasal inoculation of mice. Using a sequential prime-boost strategy in which mice were vaccinated with MV-H DNA followed by an orally delivered freeze-dried MV-H lettuce formulation a 10-fold increased in MV-specific IgG titers was observed relative to mice vaccinated with control lettuce formulations (p=0.05). MV-H protein was stable in freeze-dried lettuce for up to 13 months at room temperature, and survived at least a week at temperatures as high as 50 degrees C. This research represents a significant step towards the development of measles vaccine formulation that is effective, temperature-stable, easy to administer in a resource-poor setting and amenable to large scale manufacture.

Antibody responses and epitope specificities to the Taenia solium cysticercosis vaccines TSOL18 and TSOL45-1A.

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. This study examined the antibody responses in pigs immunized with the TSOL18 and TSOL45-1A recombinant vaccines against T. solium cysticercosis. Immunization with these proteins induced specific, complement-fixing antibodies against the recombinant antigens that are believed to be associated with vaccine-induced protection against T. solium infection. Sera from immunized pigs were used to define the linear B-cell epitopes of TSOL18 and TSOL45-1A. Prominent reactivity was revealed to one linear epitope on TSOL18 and two linear epitopes on TSOL45-1A. These, and oncosphere antigens from other taeniid cestodes, contain a protein sequence motif suggesting that they may show a tertiary structure similar to the fibronectin type III domain (FnIII). Comparison of the location of linear antigenic epitopes in TSOL18 and TSOL45-1A within the proposed FnIII structure to those within related cestode vaccine antigens reveals conservation in the positioning of the epitopes between oncosphere antigens from different taeniid species.

Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated.

Live Salmonella as vaccines and carriers of foreign antigenic determinants.

Salmonella species can be rationally attenuated by introducing non-reverting defined mutations into the genome to produce live vaccine strains. Several genes have been identified which when mutated, will attenuate Salmonellae. In particular, salmonella strains harbouring mutations in genes involved in the pre-chorismate biosynthetic pathway make excellent oral vaccines evoking strong humoral, local and cellular immune responses in the host. Because of the spectrum of immune responses induced by live vaccine strains they have the potential to be used for delivery of heterologous antigens to the mammalian immune system. A number of antigens from other bacteria, viruses and parasites have been expressed in live salmonella vaccine strains. Such hybrid strains have the potential to be used as multivalent vaccines against a number of infectious diseases.

Cross protective immunity conferred by a marker-free aroA mutant of Pasteurella multocida.

The aroA gene from Pasteurella multocida serotype A:1 (X-73) was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The cloned aroA gene was inactivated by deletion of a 300 bp internal sequence and reintroduced by homologous recombination into the chromosome of X-73 and P-1059 (serotype A:3) using a Pasteurella-E. coli shuttle vector pPBA1100. By subjecting the transformed cells to repeated subculturing in the presence of antibiotic selection coupled with auxotrophic enrichment, marker-free aroA mutants of X-73 and of P-1059 were isolated and designated PMP1 and PMP3, respectively. PMP1 and PMP3 were highly attenuated and capable of conferring complete protection against subsequent lethal challenge infection in a mouse model. Moreover, PMP3-immunized mice were protected against heterologous challenge infection with serotype A:1 or A:4.

Response of syphilitic rabbits to reinfection with homologous and heterologous Treponema pallidum strains.

Rabbits infected intradermally with 10(3) Treponema pallidum (Melbourne 1) cells were examined for their susceptibility to reinfection with 10(2) T. pallidum cells (homologous or heterologous strains) at various intervals after the initial infection. At 2.5 weeks after infection, the rabbits were extremely sensitive to reinfection and developed syphilitic lesions significantly faster (i.e., shorter latent periods) than control rabbits that had not received the initial infection. This phenomenon may represent a state of immunosuppression or hypersensitivity in the infected rabbits. Whatever its etiology (at present unknown), it was a transient state since at 5 weeks after infection the rabbits were no longer different from control rabbits in their susceptibility to reinfection. They showed neither immunity (i.e., longer latent periods) nor immunosuppression or hypersensitivity (shorter latent periods) upon reinfection. At 6.5 weeks after infection, two of the three experimental rabbits were fully immune (no lesions upon reinfection), whereas the other rabbit exhibited immunosuppression or hypersensitivity upon reinfection. At 7.5 and 10 weeks after infection, all of the experimental rabbits were immune to reinfection. We conclude that syphilitic rabbits show a biphasic response to reinfection, consisting of an early phase of enhanced sensitivity to T. pallidum and a later phase of immunity to T. pallidum.

Studies of immunity and bacterial invasiveness in mice given a recombinant salmonella vector encoding murine interleukin-6.

Interleukin-6 (IL-6) was expressed in Salmonella typhimurium in an attempt to increase the mucosal immune response against the bacterium. Murine IL-6 was PCR amplified from cDNA, cloned, sequenced, and found to be functionally active when expressed in S. typhimurium BRD509, the (delta)aroA (delta)aroD vaccine strain. Expression of murine IL-6 did not appear to adversely affect the growth of salmonellae, as the construct was retained in the absence of antibiotic selection and the growth rate was unaffected compared with that of the parent strain in vitro. However, IL-6 expression led to a significant reduction in bacterial invasiveness in vitro and in vivo. Splenocytes and small intestinal lamina propria lymphocytes were isolated from mice orally immunized with BRD509 expressing IL-6 (pKK233-2/IL-6), and the number of antibody-secreting cells was determined by the ELISPOT technique. No differences were observed between mice immunized with BRD509(pKK.233-2/IL-6) and those immunized with BRD509(pKK233-2) with respect to the antibody subclass-specific responses elicited despite the markedly reduced invasiveness of the former. Serum antibody responses were also examined by a kinetic enzyme-linked immunosorbent assay (ELISA), and equivalent levels of antibody response were detected in mice given BRD509(pKK233-2/IL-6) and those given BRD509(pKK233-2). The humoral immune response against bacterial lipopolysaccharides was also examined in transgenic IL-6-deficient mice given oral inocula of BRD509. Equivalent numbers of antibody-secreting cells (ELISPOTs) were observed in the spleens and laminae propriae of both IL-6-deficient (-/-) mice and control (+/+) mice harboring an intact IL-6 gene, whereas small, yet significant differences in the serum immunoglobulin A ELISA titers were observed. These data suggest that the immunoglobulin A response against Salmonella lipopolysaccharides is largely IL-6 independent.