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.

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.

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.

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).

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.

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).

Genetic detoxification of bacterial toxins.

Several pathogens, such as Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Vibrio cholerae, enterotoxigenic Escherichia co1i (1), and even some emerging pathogens, such as Helicobacter pylori (2), produce potent toxins that are responsible for the pathology caused by the bacterium. In most cases the disease, and often even the infection, can be prevented by a vaccine that induces immunity against the toxin. In order to be used in vaccines, the dangerous toxins need to be depleted of their toxic activity in an effective and irreversible manner.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.