Gillian Douce

Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants.

Most vaccines are still delivered by injection. Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to contaminated syringes. However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant on effective delivery systems. Cholera toxin (CT) and Escherichia coli enterotoxin (LT) are powerful mucosal adjuvants when co-administered with soluble antigens. However, their use in humans is hampered by their extremely high toxicity. During the past few years, site-directed mutagenesis has permitted the generation of LT and CT mutants fully non toxic or with dramatically reduced toxicity, which still retain their strong adjuvanticity at the mucosal level. Among these mutants, are LTK63 (serine-to-lysine substitution at position 63 in the A subunit) and LTR72 (alanine-to-arginine substitution at position 72 in the A subunit). The first is fully non toxic, whereas the latter retains some residual enzymatic activity. Both of them are extremely active as mucosal adjuvants, being able to induce very high titers of antibodies specific for the antigen with which they are co-administered. Both mutants have now been tested as mucosal adjuvants in different animal species using a wide variety of antigens. Interestingly, mucosal delivery (nasal or oral) of antigens together with LTK63 or LTR72 mutants also conferred protection against challenge in appropriate animal models (e.g. tetanus, Helicobacter pylori, pertussis, pneumococci, influenza, etc). In conclusion, these LTK63 and LTR72 mutants are safe adjuvants to enhance the immunogenicity of vaccines at the mucosal level, and will be tested soon in humans.

Structure and mucosal adjuvanticity of cholera and Escherichia coli heat-labile enterotoxins

Escherichia coli heat-labile enterotoxin and cholera toxin are potent mucosal immunogens and adjuvants in animal models. Non-toxic mutants retaining adjuvant activity are useful tools to dissect the mechanism of mucosal adjuvanticity and promising candidates for development of human vaccines and immunotherapy. Clinical trials are expected to proceed in the near future.

Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of Clostridium difficile

Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.

Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.

Intranasal vaccination of mice against infection with Mycobacterium tuberculosis

The intranasal (i.n.) route of immunisation, has recently been of active interest in endeavours to improve the efficacy of vaccination against a number of respiratory infections. Here, we examined the outcome of tuberculous infection in BALB/c mice. I.n. application of the BCG-Pasteur strain was found to be highly protective against challenge infection with the pathogenic H37Rv strain given after a 4-week interval, reflected by the 100-fold reduction of CFUs in both lungs and spleens. Vaccination with the recombinant PstS-1 antigen and cholera toxin significantly protected against the challenge given 10 days later, but only marginally after 12 weeks. Histological examination showed, that i.n. vaccination abrogated the confluent infiltration of lungs with inflammatory cells, which surrounds the granulomas in H37Rv challenged control mice. In conclusion, the strong protection demonstrated by BCG suggests that the i.n. route of vaccine delivery deserves further attention toward improving vaccination against tuberculosis.

Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells

The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located prn gene were significantly more invasive than isogenic parental strains. This effect was most pronounced in the poorly invasive, semi-rough S. typhimurium strain LB5010. Escherichia coli K-12 strain HB101 harbouring the plasmid p41869D, which encodes the full-length prn gene under the control of the tac promoter on the broad-host-range plasmid pMMB66EH, was significantly more adhesive to HEp-2 and Chinese Hamster Ovary (CHO) cells growing in culture than E. coli HB101(pMMB66EH). However, the ability of E. coli to invade mammalian cells was not affected by P.69/pertactin expression. P.69/pertactin-mediated adhesiveness of HB101 to HEp-2 and CHO cells was not influenced by the viability of the bacterial cells. However, adherence was markedly reduced when assays were performed for less than 3 h, at 4 degrees C or in the presence of cycloheximide, suggesting the active participation of the eukaryotic cell in bacterial adhesion. Site-directed mutagenesis was used to mutate Asp to Glu in an Arg-Gly-Asp (RGD-->RGE) sequence present in mature P.69/pertactin and the mutated gene was cloned in the same broad-host-range vector (plasmid p41869E). This mutation had no detectable influence on the ability of P.69/pertactin to mediate adhesion of HB101 to HEp-2 or CHO cells. Plasmids p41869D and p41869E were introduced into the bvg-negative B. pertussis strain BP347. Expression of P.69RGD or P.69RGE did not enhance the adhesiveness of BP347 for epithelial (HEp-2 and CHO) cells.

Influence of codon usage on the immunogenicity of a DNA vaccine against tetanus

Two related DNA vaccine vector plasmids, harbouring either wild-type (pcDNA3/ntetC) or synthetic codon optimised (pcDNA3/stetC) DNA encoding fragment C (TetC) of tetanus toxin were constructed. COS-7 cells transformed with pcDNA3/stetC reproducibly expressed higher levels of TetC than similar cells transformed with pcDNA3/ntetC. BALB/c mice immunised intramuscularly with pcDNA3/stetC produced significantly higher levels of anti-TetC antibodies in their serum in the weeks following vaccination compared to mice immunised with pcDNA3/ntetC, even when differences in the CpG content between the two sequences were controlled for using non-expressing DNA.

Salmonella enterica Serovar Typhimurium surA Mutants Are Attenuated and Effective Live Oral Vaccines

ABSTRACT A previously described attenuated TnphoA mutant (BRD441) of Salmonella enterica serovar Typhimurium C5 (I. Miller, D. Maskell, C. Hormaeche, K. Johnson, D. Pickard, and G. Dougan, Infect. Immun. 57:2758–2763, 1989) was characterized, and the transposon was shown to be inserted in surA, a gene which encodes a peptidylprolyl-cis,trans-isomerase. A defined surA deletion mutation was introduced into S. enterica serovar Typhimurium C5 and the mutant strain, namedS. enterica serovar Typhimurium BRD1115, was extensively characterized both in vitro and in vivo. S. entericaserovar Typhimurium BRD1115 was found to be defective in the ability to adhere to and invade eukaryotic cells. Furthermore, S. enterica serovar Typhimurium BRD1115 was attenuated by at least 3 log units when administered orally or intravenously to BALB/c mice. Complementation of the mutation with a plasmid carrying the intactsurA gene almost completely restored the virulence of BRD1115. In addition, S. enterica serovar Typhimurium BRD1115 demonstrated potential as a vaccine candidate, since mice immunized with BRD1115 were protected against subsequent challenge withS. enterica serovar Typhimurium C5. S. entericaserovar Typhimurium BRD1115 also showed potential as a vehicle for the effective delivery of heterologous antigens, such as the nontoxic, protective fragment C domain of tetanus toxin, to the murine immune system.

LOV-based reporters for fluorescence imaging

Chromophore-binding domains from plant and bacterial photoreceptor proteins have recently gathered increasing attention as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (light, oxygen, or voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size, pH and thermal stability, utility under anaerobic conditions and their ability to generate reactive oxygen species. This review focuses on the potential applications of this emerging class of fluorescent reporters, discusses the advantages and limitations of LOV-based FPs, whilst offering insights regarding the further development of this technology for bioimaging and photodynamic therapy.