Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system.

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

The affinity of cholera toxin for Ni2+ ion.

Cholera toxin (CT) was shown to bind to immobilized Ni2+ ion. The affinity of CT for the complex required the presence of the Ni2+ ion, since CT was unable to bind in its absence. Binding was mediated by the B-subunit (CTB) as both CT and CTB bound to the resin, but not the A-subunit (CTA). Binding was reversible in the presence of imidazole and suggested that the affinity of CT for the Ni2+ ion was mediated by His residues. The heat-labile enterotoxin of Escherichia coli (LT), which is closely related to CT, was unable to bind to the Ni2+ ion. Comparison of amino acid sequences revealed the presence of three His residues in CT (positions 13, 57 and 94), but only one in LT (position 57). To confirm that the residues at positions 13 and 94 of CTB were responsible for the binding, they were changed to residues found in LTB. Changing His13-->Arg completely abrogated the ability of CTB to bind to Ni2+ ion. In contrast, the mutation of His 94-->Asn reduced, but did not abrogate, the ability of CTB to bind to Ni2+ ion. Based on calculated interatomic distances, it is unlikely that His13 and His94 are part of the same complex. There appear to be two separate binding sites, with the principal site involving His13 and a much weaker site involving His94. This latter site can only participate in binding if the complex involving His13 has formed.

Genetically engineered vaccines: an overview.

Despite the early success demonstrated with the hepatitis B vaccine, no other recombinant engineered vaccine has been approved for use in humans. It is unlikely that a recombinant vaccine will be developed to replace an existing licensed human vaccine with a proven record of safety and efficacy. This is due to the economic reality of making vaccines for human use. Genetically engineered subunit vaccines are more costly to manufacture than conventional vaccines, since the antigen must be purified to a higher standard than was demanded of older, conventional vaccines. Each vaccine must also be subjected to extensive testing and review by the FDA, as it would be considered a new product. This is costly to a company in terms of both time and money and is unnecessary if a licensed product is already on the market. Although recombinant subunit vaccines hold great promise, they do present some potential limitations. In addition to being less reactogenic, recombinant subunit vaccines have a tendency to be less immunogenic than their conventional counterparts. This can be attributed to these vaccines being held to a higher degree of purity than was traditionally done for an earlier generation of licensed subunit vaccines. Ironically, the contaminants often found in conventional subunit vaccines may have aided in the inflammatory process, which is essential for initiating a vigorous immune response. This potential problem may be overcome by employing one of the many new types of adjuvants that are becoming available for use in humans. Recombinant subunit vaccines may also suffer from being too well-defined, because they are composed of a single antigen. In contrast, conventional vaccines contain trace amounts of other antigens that may aid in conferring an immunity to infectious agents that is more solid than could be provided by a monovalent vaccine. This problem can be minimized, where necessary, by creating recombinant vaccines that are composed of multiple antigens from the same pathogen. These issues are less of a concern with a live attenuated vaccine, since these vaccines are less costly, require fewer steps to manufacture, and elicit long-lived immunity after only a single dose. Unfortunately, live vaccines carry a higher risk of vaccine-induced complications in recipients that make their use in highly developed, litiginous countries unlikely. In lesser developed countries, where the prevalence of disease and the need for effective vaccines outweighs the risk associated with their administration, live vaccines may play an important role in human health. This review has attempted to make the reader aware of some of the current approaches and issues that are associated with the development of these vaccines. Genetically engineered vaccines hold great promise for the future, but the potential of these vaccines to improve human and animal health has yet to be fully realized.

Strong mucosal adjuvanticity of cholera toxin within lipid particles of a new multiple emulsion delivery system for oral immunization.

Cholera toxin (CT) is an effective mucosal adjuvant but causes significant intestinal secretion which limits its usefulness. In the present study we developed a new multiple emulsion (ME) delivery system into which antigen and CT could be incorporated and asked whether CT would retain its mucosal adjuvanticity when sequestered within emulsion particles. ME were selectively taken up into Peyer's patches, and those containing antigen plus CT generated intestinal secretory IgA and serum IgG antibody responses in mice comparable quantitatively and qualitatively to those occurring after oral immunization with soluble antigen plus CT. The ME particles containing CT did not cause intestinal secretion. The adjuvanticity of CT within ME was due to the CT present in the inner aqueous phase of the ME and was lost if CT binding was blocked by pre-incubation with GM1 ganglioside. Proteins incorporated in ME were protected from external acid, protease, and bile. We conclude that CT sequestered in ME, although unable to bind to the epithelium and thus stimulate intestinal secretion, still retains its mucosal adjuvanticity. Thus, the ability of CT to bind to enterocytes is not obligatory for its mucosal adjuvanticity.

A Francisella tularensis DNA clone complements Escherichia coli defective for the production of Era, an essential Ras-like GTP-binding protein.

We cloned the era gene of Francisella tularensis from a plasmid library by heterologous genetic complementation of an Escherichia coli mutant conditionally defective for the production of Era, an essential protein for cell growth. Nucleotide sequence analysis indicated that, in F. tularensis, era constitutes a single gene operon. ORFs aspC and mdh encoding aspartate aminotransferase and malate dehydrogenase, respectively, flank era in F. tularensis. Although classified as Gram-, the flanking regions and the relative location of era in F. tularensis are distinctly different from those of typical Gram- and Gram+ bacteria. Computer analysis of bacterial Era protein sequences identified conserved domains in addition to the common G domains of most GTP-binding proteins.

Mapping of protective and cross-reactive domains of the type A neurotoxin of Clostridium botulinum.

The purpose of this study was to identify the location of domains within the serotype A neurotoxin of Clostridium botulinum (BoNT/A) that conferred protection against botulism. The BoNT/A gene was subcloned into a series of 10 overlapping fragments that were expressed in Escherichia coli. The expressed proteins were partially purified and used to immunize mice. The resulting antisera were screened by immunoblotting analysis for the presence of BoNT/A-specific antibody. All fragments, except one, elicited antibody that recognized BoNT/A in an immunoblot. Serological screening identified several fragment-specific cross-reactive epitopes that were shared by heterologous serotypes of BoNT. Most of these epitopes immunoreactive by enzyme-linked immunosorbent assay, but not by immunoblot. Only two fragments were shown to confer protection against BoNT/A intoxication. Both of these proteins were derived from segments of the heavy chain and encoded amino acid residues H455-661 and H1150-1289 of BoNT/A.

Analysis of the DnaK molecular chaperone system of Francisella tularensis.

We have cloned the Francisella tularensis (Ft) grpE-dnaK-dnaJ heat-shock genes which are organized in that order. These genes allow heterologous genetic complementation of each respective mutant strain of Escherichia coli (Ec) for bacteriophage lambda growth. The nucleotide sequences of the Ft grpE-dnaK-dnaJ genes and the deduced amino-acid sequences share significant homologies with their respective Ec counterparts. The Ft DnaK and DnaJ proteins cross-react with polyclonal antibodies raised against the respective Ec proteins. The grpE-dnaK-dnaJ genes of Ft are organized in a fashion that is more characteristic of Gram+ bacteria.

Morphologic and functional alterations of mucosal T cells by cholera toxin and its B subunit.

Despite the mucosal immunogenicity and adjuvanticity in vivo of cholera toxin (CT), both CT and CT B subunit are strong inhibitors of T cell activation in vitro. This study asked whether such T cell inhibition is relevant to the mucosal effects of CT in vivo. The activation of T cells pulsed in vitro for only 15 to 120 min with CT or CT B subunit, respectively, was inhibited, consistent with the expected short exposure times in vivo. Although both CD8+ and CD4+ T cells were inhibited in vitro, CD8+ T cells bound more toxin and were inhibited to a greater degree than were CD4+ T cells. Intestinal gavage of mice with 10 micrograms CT did not alter the overall composition of Peyer's Patch, mesenteric lymph node, or spleen but did cause a marked depletion of intraepithelial lymphocytes, mainly CD8+ T cells, and of lymphocytes in the dome epithelium over Peyer's Patch. To determine whether such inhibition of T cells was functionally relevant in vivo, T cells from mice fed keyhole limpet hemocyanin (KLH) were adoptively transferred into naive recipients, who were then parenterally immunized. T cells from mice fed KLH alone inhibited both the systemic IgG and secretory IgA anti-KLH response, but T cells from mice fed KLH plus CT did not, indicating that mucosally applied CT was able to abrogate the induction of this suppressor T cell. We conclude that one of the mechanisms of CT's mucosal effects in vivo is the inhibition of certain mucosal T cell functions and alteration of the regulatory T cell environment in gut-associated lymphoid tissue.

Reduction in oral immunogenicity of cholera toxin B subunit by N-terminal peptide addition.

The mucosal adjuvanticity of cholera toxin and the potential of the B subunit of cholera toxin (CtxB) to serve as an oral vaccine carrier have prompted interest in the coupling of immunogenic peptides to this protein. The purpose of this study was to determine how such fusions affect the function of CtxB. Oligonucleotides were genetically fused to the 5' terminus of the ctxB gene to encode additional amino acids of 8, 12, and 24 residues in length. None of these additions affected the ability of CtxB to oligomerize, as determined by nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Circular dichroism revealed no difference in conformation between the modified B subunits, regardless of the length of the addition. However, when compared with native CtxB, additions to the N terminus induced a consistent change in the net conformation of the protein. By using a competitive enzyme immunoassay, the affinity of the modified B subunits for GM1 ganglioside was shown to gradually decrease with increasing length of the N-terminal addition. A similar pattern was observed for the ability of the chimeras to inhibit proliferation of concanavalin A-stimulated spleen cells in vitro, which is a previously described functional property of CtxB that is dependent on its binding to cells. Lastly, the oral immunogenicity of these chimeras was found to be less than that of native CtxB. These results indicate that large fusions to the N terminus of CtxB can significantly affect its biological properties and could reduce its value as a mechanism for effective mucosal immunization.

Comparative effectiveness of the cholera toxin B subunit and alkaline phosphatase as carriers for oral vaccines.

The purpose of this study was to determine whether the B subunit of cholera toxin (CtxB) has adjuvant activity over and above serving as a carrier protein for orally administered vaccines. An oligonucleotide that encodes an antigenic determinant (GtfB.1) from the glucosyltransferase B gene (gtfB) of Streptococcus mutans was genetically fused to the 5' terminus of either the CtxB gene (ctxB) or the Escherichia coli alkaline phosphatase gene (phoA). The resulting chimeric proteins were expressed in a phoA mutant strain of E. coli and then purified. The antigenicities of the proteins were confirmed by immunoblotting analysis using antisera specific for GtfB, CtxB, or PhoA. An equimolar amount of peptide on each carrier was administered by gastric intubation to mice three times at 10-day intervals. Antibody titers to the peptide, CtxB, and PhoA (in the serum, intestine, vagina, saliva, and bronchus) were determined by enzyme immunoassay. Antibody to the peptide was detected only in the sera of mice immunized with the peptide fused to CtxB. No antipeptide antibody was detected in mice immunized with the peptide fused to PhoA. The lack of detectable levels of antipeptide antibody in intestinal lavage fluid was attributed to dilution of the sample beyond the sensitivity of the assay. This was confirmed by cultivation of Peyer's patch and mesenteric lymph node tissue from mice orally immunized with the GtfB.1::CtxB chimera. Using this method, antipeptide antibody was detected in the culture fluid. We conclude that CtxB possesses unique properties that allow it to act as more than a simple carrier protein.

The mucosal immune system: from fundamental concepts to vaccine development.

Recent studies in experimental animals and humans have shown that the mucosal immune system, which is characterized by secretory IgA (S-IgA) antibodies as the major humoral defence factor, contains specialized lymphoid tissues where antigens are encountered from the environment, are taken up and induce B- and T-cell responses. This event is followed by an exodus of specific lymphocytes, which home to various effector sites such as the lamina propria regions and glands. These responses are regulated by T cells and cytokines and lead to plasma cell differentiation and subsequent production of S-IgA antibodies in external secretions. This knowledge has led to practical approaches for vaccine construction and delivery into mucosal inductive sites in an effort to elicit host protection at mucosal surfaces where the infection actually occurs.

Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide.

An antigenic 15-amino-acid peptide sequence (gtfB.1) from the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans GS-5 was identified previously from the genetic fusion of this sequence to the B subunit of cholera toxin. The resulting chimeric protein was used to raise antiserum in rabbits. This antiserum was shown to recognize the native glucosyltransferase enzyme and to inhibit its activity. The antiserum inhibited the synthesis of water-soluble glucan by approximately 40% and the synthesis of water-insoluble glucan by greater than 90%. The antiserum was shown to partially inhibit fructosyltransferase activity as well. The ability of this antipeptide antiserum to inhibit several enzymes from S. mutans suggests that these enzymes share an epitope related to enzymatic activity.

Genetic approaches to the study of oral microflora: a review.

As the study of oral microorganisms intensified almost 2 decades ago, the application of genetic techniques resulted in important contributions to the understanding of this clinically and ecologically important group of bacteria. The isolation and characterization of mutants of cariogenic streptococci helped to focus attention on traits that were important in colonization and virulence. Such classic genetic approaches gave way to molecular genetic techniques, including recombinant DNA methodology in the late 1970s. Gene cloning systems and methods to move DNA into cells have been developed for oral streptococci. Many streptococcal genes thought to be important in colonization and virulence have since been cloned and their nucleotide sequence determined. Mutant strains have been constructed using defective copies of cloned genes in order to create specific genetic lesions on the bacterial chromosome. By testing such mutants in animal models, a picture of the cellular and molecular basis of dental caries is beginning to emerge. These modern genetic methodologies also are being employed to develop novel and efficacious cell-free or whole cell vaccines against this infection. Genetic approaches and analyses are now being used to dissect microorganisms important in periodontal disease as well. Such systems should be able to exploit advances made in genetically manipulating related anaerobes, such as the intestinal Bacteroides. Gene cloning techniques in oral anaerobes, Actinomyces and Actinobacillus, are already beginning to pay dividends in helping understand gene structure and expression. Additional effort is needed to develop facile systems for genetic manipulation of these important groups of microorganisms.

Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein.

A synthetic peptide, encoding amino acid residues 345 to 359 of the glucosyltransferase B enzyme of Streptococcus mutans GS-5, was genetically fused to the N-terminal end of the B-subunit gene of cholera toxin. The protein was overexpressed in Escherichia coli and retained the antigenicity associated with cholera toxin B subunit (CTB) as well as that associated with glucosyltransferase B. The addition of 15 amino acids to the N-terminal end of CTB did not appear to affect the gross structure of the protein significantly. The chimeric protein monomers assembled into a functional oligomer which exhibited only minor conformational differences from native CTB as measured by circular dichroism. The chimera bound to GM1 ganglioside and thus retained the biological activity of CTB. These results demonstrate that genetic fusion of small peptides to the N terminus of CTB has only a minimal effect on the structure and function of the protein. Furthermore, the chimera was shown to be immunogenic when fed to mice. This work has important implications in the construction of CTB chimeras for use as oral vaccines.

Plasmid vectors for constructing translational fusions to the B subunit of cholera toxin.

A family of plasmid cloning vectors has been developed for creating translational fusions to the ctxB gene encoding the B subunit of cholera toxin (CTB) in Escherichia coli. These vectors permit insertion of transcriptionally and translationally competent gene sequences upstream from ctxB. To test the utility of the system, a portion of the glucosyltransferase B (GTF) gene (gtfB) from the cariogenic bacterium Streptococcus mutans GS-5 (Bratthall serotype c), encoding the N-terminal one-third of the protein, was inserted into each vector. E. coli lysates containing the constructs were partially purified by passage over a GM1 ganglioside affinity column. Western blotting analysis of the column retentate from one of the lysates revealed the presence of a novel 58-kDa protein which cross-reacted with antisera to GTF and CTB. These vectors are of general use for making other translational fusions to ctxB. The high binding affinity of CTB can be exploited in purifying large polypeptides fused to this relatively small protein. Moreover, these vectors can be used to create neoantigens with altered immunogenicity for use in polypeptide-based vaccines.

An enzyme immunoassay for the detection of staphylococcal protein A in affinity-purified products.

Rabbit antiserum, specific for protein A from Staphylococcus aureus, was conjugated to alkaline phosphatase and used in a double antibody solid-phase enzyme immunoassay. The assay was developed to monitor eluate from a large-scale protein A-Sepharose affinity column used to purify monoclonal antibodies for human clinical trials. The assay detected soluble protein A in the presence of immunoglobulin at concentrations as low as 4 ng/ml. Analysis of the product purified by affinity chromatography revealed the presence of protein A at ng/ml concentrations. The assay developed here can provide a reliable and convenient method for detecting soluble protein A.