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M Pizza

Publications and source records attributed to M Pizza.

At least 37 records · Page 2Linked to original sources
PubMed1999

Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants.

Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i. n. immunizations were consistently higher than the equivalent p.o. -delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response.

Adjuvants, Immunologic↗
PubMed1998

KDEL receptor (Erd2p)-mediated retrograde transport of the cholera toxin A subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p.

A cholera toxin mutant (CTX-K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTX-A-K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-gamma-S inhibits the internalization as well as Golgi-ER transport of CTX-A-K63. The appearance of CTX-A-K63 in the Golgi induces a marked dispersion of Erd2p and p53 but not of the Golgi marker giantin. Erd2p is translocated under these conditions most likely to the intermediate compartment as indicated by an increased colocalization of Erd2p with mSEC13, a member of the mammalian coat protein II complex. IgGs as well as Fab fragments directed against Erd2p, beta-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A-K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63-induced translocation of Erd2p and p53.

Adenosine Diphosphate Ribose↗
PubMed1998

Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

Adjuvants, Immunologic↗
PubMed1998

Effects of the insertion of a nonapeptide from murine IL-1beta on the immunogenicity of carrier proteins delivered by live attenuated Salmonella.

A nonapeptide from IL-1beta has been reported to be an immunostimulant and adjuvant. To investigate the possibility of enhancing the immunogenicity of recombinant antigens delivered by live-attenuated Salmonella strains, we inserted an oligonucleotide coding for the nonapeptide from murine IL-1beta into the genes of three model proteins: LamB, MalE, and flagellin. The hybrid proteins were expressed and delivered in vivo by Salmonella aroA strains, and serum antibody responses were analyzed. The results showed that the nonapeptide induced an increase in the immune response against Salmonella-delivered flagellin, measured on day 28 post-immunization. However, the adjuvant effect was lost by day 42. In no case was an adjuvant effect detected for Salmonella-delivered LamB or MalE. Thus, by comparing the immune responses raised by purified MalE with and without the peptide, we investigated whether the insertion of the peptide affected the immunogenicity of the protein itself. Also in this case, a modest adjuvant effect was shown only after primary immunization and when very low doses of antigen were used. In conclusion, the immunomodulatory properties of the IL-1beta peptide can also be detected when it is delivered in vivo by Salmonella; however, the effect is modest and antigen-dependent.

Adjuvants, Immunologic↗
PubMed1998

Molecular basis of vaccination.

Vaccines represent the most cost-effective means to prevent infectious diseases. Most of the vaccines which are currently available were developed long before the era of molecular biology and biotechnology. They were obtained following empirical approaches leading to the inactivation or to the attenuation of microorganisms, without any knowledge neither of the mechanisms of pathogenesis of the disease they were expected to protect from, nor of the immune responses elicited by the infectious agents or by the vaccine itself. The past two decades have seen an impressive progress in the field of immunology and molecular biology, which have allowed a better understanding of the interactions occurring between microbes and their hosts. This basic knowledge has represented an impetus towards the generation of better vaccines and the development of new vaccines. In this monograph we briefly summarize some of the most important biotechnological approaches that are currently followed in the development of new vaccines, and provide details on an approach to vaccine development: the genetic detoxification of bacterial toxins. Such an approach has been particularly successful in the rational design of a new vaccine against pertussis, which has been shown to be extremely efficacious and safe. It has been applied to the construction of powerful mucosal adjuvants, for administration of vaccines at mucosal surfaces.

Adjuvants, Immunologic↗
PubMed1998

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.

Adjuvants, Immunologic↗
PubMed1998

A recombinant live attenuated strain of Vibrio cholerae induces immunity against tetanus toxin and Bordetella pertussis tracheal colonization factor.

An attenuated strain of Vibrio cholerae was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor from Bordetella pertussis (Tcf). In vitro, high levels of protein were obtained when the Escherichia coli nirB promoter was used and the bacteria were grown with low aeration. Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type B. pertussis. These data are in agreement with the putative role of Tcf in Bordetella tracheal colonization. In conclusion, we have demonstrated that V. cholerae may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved.

Administration, Intranasal↗
PubMed1998

Adjuvant effect of non-toxic mutants of E. coli heat-labile enterotoxin following intranasal, oral and intravaginal immunization.

Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) are known to be very effective mucosal adjuvants, but their toxicity limits their use in humans. We genetically detoxified LT by substituting single residues in the active site of the enzymatic A subunit and obtained mutant molecules that retain mucosal adjuvant activity but are devoid of toxicity. These mutant LT molecules induce mucosal and systemic responses to antigens delivered intranasally, orally and intravaginally in mice. Furthermore, mucosal immunization with these molecules confers protection against systemic challenge with tetanus toxin (TT) and mucosal challenge with Helicobacter pylori.

Adjuvants, Immunologic↗
PubMed1997

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.

Adjuvants, Immunologic↗
PubMed1997

Protease susceptibility and toxicity of heat-labile enterotoxins with a mutation in the active site or in the protease-sensitive loop.

To generate nontoxic derivatives of Escherichia coli heat-labile enterotoxin (LT), site-directed mutagenesis has been used to change either the amino acid residues located in the catalytic site (M. Pizza, M. Domenighini, W. Hol, V. Giannelli, M. R. Fontana, M. M. Giuliani, C. Magagnoli, S. Peppoloni, R. Manetti, and R. Rappuoli, Mol. Microbiol. 14:51-60, 1994) or those located in the proteolytically sensitive loop that joins the A1 and A2 moieties of the A subunit (C. C. R. Grant, R. J. Messer, and W. J. Cieplack, Infect. Immun. 62:4270-4278, 1994; B. L. Dickinson and J. D. Clements, Infect. Immun. 63:1617-1623, 1995). In this work, we compared the in vitro and in vivo toxic properties and the resistance to protease digestion of the prototype molecules obtained by both approaches (LT-K63 and LT-R192G, respectively). As expected, LT-K63 was normally processed by proteases, while LT-R192G showed increased resistance to trypsin in vitro and was digested by trypsin only under denaturing conditions (3.5 M urea) or by intestinal proteases. No toxicity was detected with the LT-K63 mutant, even when 40 micrograms and 1 mg were used in the in vitro and in vivo assays, respectively. In marked contrast, LT-R192G showed only a modest (10-fold) reduction in toxicity in Y1 cells with a delay in the appearance of the toxic activity and had toxicity comparable to that of wild-type LT in the rabbit ileal loop assay. We conclude that mutagenesis of the active site generates molecules that are fully devoid of toxicity, while mutagenesis of the A1-A2 loop generates molecules that are resistant to trypsin in vitro but still susceptible to proteolytic activation by proteases other than trypsin, and therefore they may still be toxic in tissue culture and in vivo.

Animals↗
PubMed1997

Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection.

Chronic infection of the gastroduodenal mucosae by the gram-negative spiral bacterium Helicobacter pylori is responsible for chronic active gastritis, peptic ulcers, and gastric cancers such as adenocarcinoma and low-grade gastric B-cell lymphoma. The success of eradication by antibiotic therapy is being rapidly hampered by the increasing occurrence of antibiotic-resistant strains. An attractive alternative approach to combat this infection is represented by the therapeutic use of vaccines. In the present work, we have exploited the mouse model of persistent infection by mouse-adapted H. pylori strains that we have developed to assess the feasibility of the therapeutic use of vaccines against infection. We report that an otherwise chronic H. pylori infection in mice can be successfully eradicated by intragastric vaccination with H. pylori antigens such as recombinant VacA and CagA, which were administered together with a genetically detoxified mutant of the heat-labile enterotoxin of Escherichia coli (referred to as LTK63), in which the serine in position 63 was replaced by a lysine. Moreover, we show that therapeutic vaccination confers efficacious protection against reinfection. These results represent strong evidence of the feasibility of therapeutic use of VacA- or CagA-based vaccine formulations against H. pylori infection in an animal model and give substantial preclinical support to the application of this kind of approach in human clinical trials.

Animals↗
PubMed1997

Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin.

Genetically modified derivatives of cholera toxin (CT), harboring a single amino acid substitution in and around the NAD binding cleft of the A subunit, were isolated following site-directed mutagenesis of the ctxA gene. Two mutants of CT, designated CTS106 (with a proline-to-serine change at position 106) and CTK63 (with a serine-to-lysine change at position 63), were found to have substantially reduced ADP-ribosyltransferase activity and toxicity; CTK63 was completely nontoxic in all assays, whereas CTS106 was 10(4) times less toxic than wild-type CT. The mucosal adjuvanticity and immunogenicity of derivatives of CT were assessed by intranasal immunization of mice, with either ovalbumin or fragment C of tetanus toxin as a bystander antigen. Mice immunized with wild-type CT produced both local (immunoglobulin A in mucosal washes) and systemic immune responses to both CT and bystander antigens. CTS106 showed good local and systemic responses to bystander proteins and to itself. Interestingly, mice immunized with the nontoxic derivative of CT, CTK63, generated weak immune responses to the bystander antigens which were similar to those achieved when CT B subunit was used as an adjuvant. In parallel experiments, an equivalent nontoxic mutant of the Escherichia coli heat-labile enterotoxin, LTK63 (with a serine-to-lysine change at position 63), was tested (9). In contrast to CTK63, LTK63 was found to be more immunogenic and a better intranasal adjuvant than recombinant heat-labile enterotoxin B subunit or CTK63. This information, together with data on immunoglobulin subclass responses, suggests that although highly homologous, CT and heat-labile enterotoxin should not be considered biologically identical in terms of their ability to act as intranasal adjuvants.

Adjuvants, Immunologic↗
PubMed1996

Novel molecular biology approaches to acellular vaccines.

Bacterial toxins are commonly detoxified by chemical treatment in order to use them in human vaccines. We have used site-directed mutagenesis of toxin genes to obtain bacteria that produce naturally nontoxic mutants of bacterial toxins, such as pertussis toxin (PT), cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT). Genetically detoxified PT showed a superior safety and immunogenicity in animal models, phase I and phase II clinical trials, and a superior protective efficacy in the early and late stage of a phase III efficacy trial, proving in a definitive and extensive way that genetic detoxification of bacterial toxins can, and should, replace chemical treatment. The results obtained with genetically inactivated LT and CT indicate that genetic detoxification of bacterial toxins can be used not only to produce vaccines for systemic immunization that are superior to the ones produced by conventional technologies, but suggest that these type of molecules may be the prototype molecules for the design and construction of innovative vaccines with a totally new design, such as mucosally delivered preventive and therapeutic vaccines.

Animals↗
PubMed1996

The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide.

The intranasal route has been shown to be effective for immunization. However, immunization via this route may require the use of potent and safe adjuvant. The construction of non-toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines. In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co-immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope. LTK63 was more effective at potentiating the in vivo induction of peptide-specific and measles virus-specific CTL responses than was administration of the peptide in saline. A concentration of 10 micrograms/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide-specific and measles virus-specific CTL responses. These findings highlight the potential of the non-toxic mutant of LT as a safe mucosal adjuvant for use in humans.

Adjuvants, Immunologic↗
PubMed1996

Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908.

Salmonella typhi vaccine strain CVD 908 can deliver heterologous antigens to the host immune system following mucosal immunization. Stable expression of foreign proteins in Salmonella cells often requires antigen-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antigens to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis toxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxyl terminus of fragment C were expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal antibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these recombinant plasmids. All fusion proteins elicited serum antibody responses to fragment C following intranasal immunization, whereas oral inoculation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less effective than S1 fused to the carboxyl terminus in generating anti-fragment C antibodies. CVD 908 expressing truncated S1 fused to the carboxyl terminus of fragment C elicited neutralizing serum pertussis antitoxin following intranasal immunization of mice.

Animals↗
PubMed1996

Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin.

Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex. A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis. Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys). We have found that mutations in the A subunit may have profound effects on the ability to form the AB5 structure and on the stability and trypsin sensitivity of the purified proteins. Unstable mutants, during long-term storage at 4 degrees C, showed a decrease in the amount of the assembled protein in solution and a parallel appearance of soluble monomeric B subunit. This finding suggests that the stability of the B pentamer is influenced by the A subunit which is associated with it. Among the seven nontoxic mutants tested, LT-K63 was found to be efficient in AB5 production, extremely stable during storage, resistant to proteolytic attack, and very immunogenic. In conclusion, LT-K63 is a good candidate for the development of antidiarrheal vaccines and mucosal adjuvants.

Endopeptidases↗
PubMed1996

Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant.

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.

Animals↗