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M Pizza

Publications and source records attributed to M Pizza.

At least 55 records · Page 3Linked to original sources
PubMed1996

The reaction of bacterial toxins with formaldehyde and its use for antigen stabilization.

Since the discovery of diphtheria toxin inactivation in the early 1920s, formaldehyde has been used to inactivate bacterial toxins and viruses used as vaccine antigens. More recently, formaldehyde was used to inactivate pertussis toxin (PT), a component of the newly developed diphtheria-tetanus-acellular pertussis (DTaP) vaccine. This application however illustrated the complexity of the reaction. To eliminate the need for inactivation, the mutant PT-9K/129G was developed. This toxin analogue is irreversibly devoid of toxicity and is a more immunogenic antigen than chemically detoxified PT. Native antigens however proved less stable than detoxified antigens upon storage or heating. We investigated the use of low concentrations of formaldehyde as a stabilizing agent for PT-9K/129G. Under the conditions selected, its antigenic characteristics were retained. Enhanced immunogenicity compared to detoxified preparations was demonstrated in clinical trials in infants where DTaP vaccines containing formalin-stabilized PT-9K/129G were compared to other DTaP vaccines containing detoxified wild type PT. Additional studies with filamentous haemagglutinin (FHA), another component of acellular pertussis vaccines, showed how high formaldehyde concentrations could depress the presentation of epitopes to T-cells by limiting the antigen processing. In conclusion, mild formaldehyde treatment can be applied to stabilize vaccine antigens while retaining optimum antigenic activity.

Antigens, Bacterial↗
PubMed1995

The Arg7Lys mutant of heat-labile enterotoxin exhibits great flexibility of active site loop 47-56 of the A subunit.

The heat-labile enterotoxin from Escherichia coli (LT) is a member of the cholera toxin family. These and other members of the larger class of AB5 bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including Gs alpha. The A subunit is responsible for this covalent modification, while the B pentamer is involved in receptor recognition. We report here the crystal structure of an inactive single-site mutant of LT in which arginine 7 of the A subunit has been replaced by a lysine residue. The final model contains 103 residues for each of the five B subunits, 175 residues for the A1 subunit, and 41 residues for the A2 subunit. In this Arg7Lys structure the active site cleft within the A subunit is wider by approximately 1 A than is seen in the wild-type LT. Furthermore, a loop near the active site consisting of residues 47-56 is disordered in the Arg7Lys structure, even though the new lysine residue at position 7 assumes a position which virtually coincides with that of Arg7 in the wild-type structure. The displacement of residues 47-56 as seen in the mutant structure is proposed to be necessary for allowing NAD access to the active site of the wild-type LT. On the basis of the differences observed between the wild-type and Arg7Lys structures, we propose a model for a coordinated sequence of conformational changes required for full activation of LT upon reduction of disulfide bridge 187-199 and cleavage of the peptide loop between the two cysteines in the A subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Toxins↗
PubMed1995

Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.

A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants.

Adjuvants, Immunologic↗
PubMed1995

Mucosal and systemic immunogenicity of a recombinant, non-ADP-ribosylating pertussis toxin: effects of formaldehyde treatment.

The effect of formaldehyde treatment on the mucosal and systemic immunogenicity of the genetically detoxified pertussis toxin (PT-9K/129G) was investigated. Groups of BALB/c were immunized intranasally (i.n.) or subcutaneously (s.c.) with untreated, lightly formaldehyde treated (LFT) or heavily formaldehyde treated (HFT) recombinant pertussis toxin (PT) mutant, PT-9K/129G. Intranasal immunization with native PT-9K/ 129G induced significant levels of anti-toxin antibodies in serum and IgA anti-toxin responses in nasal and lung lavages of these mice. Similar local and systemic responses were observed following intransal immunization with LFT toxin. However, i.n. immunization with HFT toxin failed to induce a local IgA response and elicited a much diminished anti-toxin response in the serum. In contrast, the total antibody response following s.c. immunization was not significantly affected. In addition, i.n. immunization with native PT-9K/129G induced low but detectable levels of toxin neutralizing antibodies in the serum. These results show that native PT-9K/129G protein acts as a mucosal immunogen in mice and that this activity is greatly diminished by HFT of the protein.

Administration, Intranasal↗
PubMed1995

A mutant pertussis toxin molecule that lacks ADP-ribosyltransferase activity, PT-9K/129G, is an effective mucosal adjuvant for intranasally delivered proteins.

We examined the capacity of a genetically detoxified derivative of pertussis toxin (PTX), PT-9K/129G, to act as a mucosal adjuvant for an intranasally (i.n.) administered tetanus vaccine. Groups of mice were immunized i.n. with the nontoxic C-terminal 50-kDa portion of tetanus toxin (fragment C [Frg C]) either alone or mixed with PT-9K/129G, PTX, or cholera toxin (CT) or were immunized subcutaneously (s.c.) with an equivalent amount of Frg C adsorbed to alhydrogel. In response to a single immunization, mice receiving Frg C plus PT-9K/129G or CT i.n. and parenterally immunized mice developed high-titer (> 20,000) anti-Frg C antibodies, whereas mice immunized i.n. with Frg C plus PTX or with Frg C alone seroconverted only after being boosted. The serum anti-Frg C response was dominated by immunoglobulin G1 (IgG1) in mice immunized with Frg C plus PT-9K/129G, with Frg C plus PTX, or s.c. In contrast, IgG1, IgG2a, and IgG2b contributed almost equally to the Frg C response when CT was the adjuvant. Anti-Frg C IgE was detected only in the sera of mice immunized i.n. with Frg C plus PTX and immunized s.c. with Frg C plus alhydrogel. High levels of IgA antibodies were present in nasal lavage fluid from mice immunized i.n. with Frg C plus PT-9K/129G, PTX, or CT but not in that from mice given Frg C alone i.n. or parenterally. The mucosal adjuvanticity of PT-9K/129G was manifested in inbred as well as outbred mice. A single i.n. dose of Frg C plus either PT-9K/129G or PTX (with high specific activity) was sufficient to protect all immunized mice from tetanus toxin challenge, in contrast to the case for mice that received Frg C alone i.n. We conclude that the pertussis toxin analog PT-9K/129G, which is devoid of ADP-ribosyltransferase activity, is a potent mucosal adjuvant for vaccines delivered via the respiratory tract.

Adjuvants, Immunologic↗
PubMed1995

Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit.

Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated. Six of them encoded proteins that were fully assembled in the AB5 structure and were nontoxic; these proteins were CT-D53 (Val-53-->Asp), CT-K63 (Ser-63-->Lys), CT-K97 (Val-97-->Lys), CT-K104 (Tyr-104-->Lys), CT-S106 (Pro-106-->Ser), and the double mutant CT-D53/K63 (Val-53-->Asp, Ser-63-->Lys). Two of the mutations encoded proteins that were assembled into the AB5 structure but were still toxic; these proteins were CT-H54 (Arg-54-->His) and CT-N107 (His-107-->Asn). Finally, one of the mutant proteins, CT-E114 (Ser-114-->Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized. The CT-K63 mutant, which was the most efficient in assembly of the AB5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits. This molecule may be useful for the construction of improved vaccines against cholera.

Animals↗
PubMed1995

Genetic detoxification of bacterial toxins: a new approach to vaccine development.

Chemically detoxified bacterial toxins (toxoids) have been successfully used as vaccines for the prevention of many bacterial infectious diseases. Today, nontoxic derivatives of bacterial toxins can be obtained by mutagenesis of the toxin genes. These genetically inactivated toxins are superior to the classical toxoids both in safety and in immunogenicity and therefore they should replace the old toxoids in the existing vaccines. In addition, they represent a novel class of immunogens with unique properties, some of which may be used for innovative approaches to vaccination.

Antigen Presentation↗
PubMed1995

Acellular pertussis vaccine composed of genetically inactivated pertussis toxin.

Whooping cough, an acute respiratory disease affecting over sixty million infants, can be prevented by vaccination. The vaccine currently used, composed of killed bacterial cells, however, has been associated with many side effects. An improved vaccine against the disease should contain pertussis toxin (PT), a major virulent factor of Bordetella pertussis (B. pertussis). In order to be included in the vaccine, PT needs to be detoxified and the chemical methods used so far are not completely satisfactory, since they give a product with reduced immunogenicity and possible residual toxicity. To avoid this problem, we have used recombinant DNA technologies to clone the PT gene, express it in bacteria, map the B and T cell epitopes of the molecule and identify the amino acids that are important for the enzymatic activity and toxicity. Based on this information, the gene coding for PT was mutated to produce an inactive protein. This genetically modified PT was non toxic, highly immunogenic and able to protect mice from intracerebral challenge with virulent B. pertussis. The mutant was included as a main component of an acellular pertussis vaccine which has been shown in numerous clinical trials to be more safe and immunogenic than the old cellular vaccine.

Animals↗
PubMed1994

A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

Amino Acid Sequence↗
PubMed1994

Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins.

Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophilic residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins.

ADP Ribose Transferases↗
PubMed1994

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.

Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.

Amino Acid Sequence↗
PubMed1992

Progress towards the development of new vaccines against whooping cough.

Acellular vaccines against whooping cough are in the final stage of clinical testing and are likely to become available for mass immunization in the near future. Over a dozen vaccines of similar composition have been developed by vaccine companies and research laboratories; all of them contain a detoxified form of pertussis toxin (PT) that may be present alone or combined with one or more other non-toxic proteins, such as filamentous haemagglutinin (FHA), pertactin (69 kDa), and the agglutinogens (AGG). Most of the vaccines contain a PT that has been inactivated by chemical treatment, a process that reduces the immunogenicity of the molecule and may not completely eliminate the risk of reversion to toxicity. To avoid these problems, we have constructed by genetic manipulation a mutant of Bordetella pertussis that produces a non-toxic form of PT. This molecule (PT-9K/129G) contains two amino acid substitutions in the S1 subunit (Arg9-->Lys and Glu129-->Gly) which abolish the enzymatic activity of the S1 subunit and all the toxic properties of PT, without changing the immunological properties of the wild-type toxin. Following extensive preclinical studies, which have shown that PT-9K/129G is safe and more antigenic than the toxin treated with chemical agents, this molecule was tested for safety and immunogenicity in adult volunteers, 18-month-old children and 2-month-old infants. The molecule has been tested alone, combined with FHA and pertactin and also combined with diphtheria and tetanus toxoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Development and clinical testing of an acellular pertussis vaccine containing genetically detoxified pertussis toxin.

In 1924 Ramon described the inactivation of diphtheria toxin by formaldehyde treatment. This method allowed the introduction of mass vaccination against diphtheria and tetanus and opened the way to the inactivation of viruses by chemical treatment. In this review we describe the use of genetic manipulations for the inactivation of pertussis toxin. The toxin inactivated by this new method is an antigen superior to those obtained by chemical treatment and has been used to develop a new vaccine against whooping cough.

Animals↗
PubMed1992

Recombinant acellular pertussis vaccine--from the laboratory to the clinic: improving the quality of the immune response.

Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.

Amino Acid Sequence↗
PubMed1992

Cellular pertussis vaccine containing a Bordetella pertussis strain that produces a nontoxic pertussis toxin molecule.

Bordetella pertussis 165-9K/129G, which produces a nontoxic form of pertussis toxin (PT), was used to prepare a whole-cell diphtheria-tetanus-pertussis (DTP) vaccine. The in vivo potency and the serological response induced by this vaccine were comparable to those of the conventional DTP vaccine which contains active PT. The toxic activities induced by PT such as leukocytosis, histamine sensitivity, and potentiation of anaphylactic reactions, which are present in the conventional DTP vaccine, were absent in the new vaccine. These results suggest that the introduction of a whole-cell vaccine containing B. pertussis 165-9K/129G would induce the same immunity as the conventional vaccine and would avoid the administration of a harmful toxin to children.

Animals↗