Cholera Toxin A1 Subunit Research Articles

Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice

The stimulation of gastric acid secretion from parietal cells involves both intracellular calcium and cAMP signaling. To understand the effect of increased cAMP on parietal cell function, we engineered transgenic mice expressing cholera toxin (Ctox), an irreversible stimulator of adenylate cyclase. The parietal cell-specific H(+),K(+)-ATPase beta-subunit promoter was used to drive expression of the cholera toxin A1 subunit (CtoxA1). Transgenic lines were established and tested for Ctox expression, acid content, plasma gastrin, tissue morphology, and cellular composition of the gastric mucosa. Four lines were generated, with Ctox-7 expressing approximately 50-fold higher Ctox than the other lines. Enhanced cAMP signaling in parietal cells was confirmed by observation of hyperphosphorylation of the protein kinase A-regulated proteins LASP-1 and CREB. Basal acid content was elevated and circulating gastrin was reduced in Ctox transgenic lines. Analysis of gastric morphology revealed a progressive cellular transformation in Ctox-7. Expanded patches of mucous neck cells were observed as early as 3 mo of age, and by 15 mo, extensive mucous cell metaplasia was observed in parallel with almost complete loss of parietal and chief cells. Detection of anti-parietal cell antibodies, inflammatory cell infiltrates, and increased expression of the Th1 cytokine IFN-gamma in Ctox-7 mice suggested that autoimmune destruction of the tissue caused atrophic gastritis. Thus constitutively high parietal cell cAMP results in high acid secretion and a compensatory reduction in circulating gastrin. High Ctox in parietal cells can also induce progressive changes in the cellular architecture of the gastric glands, corresponding to the development of anti-parietal cell antibodies and autoimmune gastritis.

Structural Basis for the Activation of Cholera Toxin by Human ARF6-GTP

The Vibrio cholerae bacterium causes devastating diarrhea when it infects the human intestine. The key event is adenosine diphosphate (ADP)-ribosylation of the human signaling protein GSalpha, catalyzed by the cholera toxin A1 subunit (CTA1). This reaction is allosterically activated by human ADP-ribosylation factors (ARFs), a family of essential and ubiquitous G proteins. Crystal structures of a CTA1:ARF6-GTP (guanosine triphosphate) complex reveal that binding of the human activator elicits dramatic changes in CTA1 loop regions that allow nicotinamide adenine dinucleotide (NAD+) to bind to the active site. The extensive toxin:ARF-GTP interface surface mimics ARF-GTP recognition of normal cellular protein partners, which suggests that the toxin has evolved to exploit promiscuous binding properties of ARFs.

Ganglioside GM1 binding toxins and human neuropathy-associated IgM antibodies differentially promote neuritogenesis in a PC12 assay

PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB). Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies. Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay. Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic. Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA. The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic. Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects. These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA. Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.

The CTA1-DD vaccine adjuvant binds to human B cells and potentiates their T cell stimulating ability

The present study demonstrates that the novel CTA1-DD-adjuvant, which combines the full enzymatic activity of the A1 subunit of cholera toxin (CT) with an immunoglobulin-binding domain of Staphylococcus aureus protein A (SpA), binds directly to human peripheral blood B lymphocytes of all classes and greatly augments B cell functions in vitro. These effects were reflected in strongly enhanced co-stimulation, resulting in augmented T cell responses to polyclonal-specific as well as Ag-specific activation in vitro. The CTA1-DD-adjuvant had pronounced effects on B cell functions with up-regulated expression of several important activation and co-stimulatory molecules, in particular CD86. Moreover, the adjuvant alone promoted cytokine and chemokine secretion by targeted B cells and in the presence of additional stimuli proliferative responses were augmented. These effects were dependent on the whole enzymatically active CTA1-DD molecule, since DD alone had no effects on the B cells. Collectively our data suggest that CTA1-DD acted via enhanced co-stimulation, which holds promise as to the use of CTA1-DD as a non-toxic adjuvant in future vaccines for human use.

Nasally administered cholera toxin A-subunit acts as a mucosal adjuvant

It is well established that cholera toxin (CT) produced by Vibrio cholerae acts as a potent mucosal adjuvant; however, the native form of this molecule causes severe diarrhea. Furthermore, both native CT and its B-subunit derivative bind to monosialogangliosides (GM1) in membrane raft micro-domains on neural tissues and are thus unsuitable for use in humans. In this study, we evaluated the adjuvanticity of the CT A-subunit (CT-A) administered with ovalbumin (OVA) by the nasal route. We found that nasal administration of OVA plus CT-A elicited both mucosal and systemic antibody (Ab) responses. Immunization of mice with OVA plus CT-A resulted in the induction of OVA-specific IgA Abs in saliva and nasal secretions. Furthermore, significant OVA-specific serum immunoglobulin (Ig) G and IgA Ab responses were induced. Antibody-forming cell (AFC) analysis confirmed the Ab titer findings by revealing significant numbers of OVA-specific IgA AFCs in submandibular glands. In addition, splenic lymphocytes restimulated with OVA in vitro exhibited significant proliferative responses. Thus, CT-A might be a candidate for an effective adjuvant for inducing antigen (Ag)-specific Ab responses in human systemic and mucosal compartments, such as the oral cavity.

A transgenic model of comorbid Tourette's syndrome and obsessive-compulsive disorder circuitry

The tic disorder Tourette's Syndrome (TS) and obsessive-compulsive disorder (OCD) are comorbid behavioral disorders, suggesting a shared but still unknown neuronal basis. To 'circuit-test' such behaviors, we previously engineered transgenic mice expressing a neuropotentiating protein (cholera toxin A1 subunit) within a cortical-limbic subset of dopamine D1-receptor expressing (D1+) neurons known to trigger glutamatergic excitation of orbitofrontal, sensorimotor, limbic and efferent striatal circuits thought to be hyperactive or affected in OCD and TS. These mice exhibited OCD-like behaviors including generalized behavioral perseveration and compulsion-like leaping and grooming-associated pulling and biting of skin and hair. We now report that these OCD-like mice, like humans, also exhibit comorbid TS-like behaviors, including juvenile-onset tics; increased tic number, complexity and flurries; increased tic severity in males; voluntary tic suppression; and tic responsiveness to a non-cataleptic TS+OCD drug therapy (clonidine, 0.01 mg kg(-1)). These data suggest that hormonal gender differences, apart from the influence of genetic or autoimmune etiologic factors, may be sufficient to aggravate tic severity in human TS males compared to TS females. These data also proffer a precise neuronal basis for TS+OCD, wherein tics and primary compulsions or obsessions are evoked by hyperactivity of various cortical-limbic projection neurons' glutamatergic output to efferent targets like the striatum. The 'Cortical-limbic Glutamatergic Neuron' (CGN) neuronal circuit model merges formerly opposed neurotransmitter models of TS and OCD, and is consistent with new clinical reports of increased cortical hyperactivity, striatal glutamate and striatal inhibitory D2 receptors, and reduced striatal responsiveness, in these disorders.

The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant

Intranasal (i.n.) immunization of BALB/c mice with chimeric murine rotavirus EDIM (epizootic diarrhea of infant mice) VP6 and attenuated E. coli heat-labile toxin (LT), LT(R192G), stimulated >99% protection against rotavirus shedding after EDIM challenge. Here, we evaluated other potential adjuvants with chimeric VP6 administered by two mucosal routes: i.n. and oral. Besides LT(R192G), the adjuvants examined included Adjumer ®, CpG oligodeoxynucleotides (CpG ODN), chimeric A1 subunit of cholera toxin (CTA1)-DD, and QS-21. All except QS-21 significantly ( P<0.05) increased VP6-specific serum IgG responses after i.n. immunization, but none significantly increased these responses when administered orally. The i.n. delivery of chimeric VP6 alone induced both rotavirus IgG1 and IgG2a whose relative titers suggested a skewed Th2-like response. Inclusion of Adjumer ® greatly increased Th2-like responses, while CpG ODN shifted the response to a less Th2-like response. The adjuvants CTA1-DD, LT(R192G), QS-21 had no significant effect on ratios of IgG1/IgG2a titers. Following EDIM challenge of mice immunized i.n. with chimeric VP6 and either LT(R192G), CTA1-DD, Adjumer ® or CpG ODN, shedding was reduced >99, 95, 80, 74, respectively, relative to that found in unimmunized mice ( P<0.05). QS-21 induced less protection (43%, not significant (N.S.)) while immunization with chimeric VP6 alone reduced shedding by only 16% (N.S.). Oral immunization with chimeric VP6 and all selected adjuvants except QS-21 was less effective than after i.n. immunization, with protection levels of 94 ( P<0.05), 71 ( P<0.05), 55, 35 and 28% for LT(R192G), QS-21, CpG ODN, CTA1-DD, and Adjumer ®, respectively, while immunization with chimeric VP6 alone gave no protection. Thus, different adjuvants induced different degrees of protection and oral immunization was generally less effective then the i.n. route.

Recombinant antigen-enterotoxin A2/B chimeric mucosal immunogens differentially enhance antibody responses and B7-dependent costimulation of CD4(+) T cells.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/B subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or LT-IIa with the saliva-binding region (SBR) from the streptococcal adhesin AgI/II. Intranasal immunization of BALB/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-SBR immunoglobulin A (IgA) and IgG Ab responses than SBR alone. Moreover, compared to SBR-LT-IIaA2/B, SBR-CTA2/B elicited significantly higher levels of plasma IgG1 and salivary IgA anti-SBR Ab responses. Ex vivo and in vitro experiments revealed that SBR-CTA2/B selectively up-regulated B7-2 expression on murine B cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, SBR-LT-IIaA2/B had little effect on B7-1 or B7-2 expression on B220(+), CD11b(+), or CD11c(+) cells. Analysis of the functional costimulatory activity of SBR-CTA2/B-treated B cells revealed a significant enhancement in anti-CD3-stimulated CD4(+) T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-B7-2 but not with anti-B7-1 or isotype control Abs. Thus, SBR-CTA2/B and SBR-LT-IIaA2/B exhibit distinct patterns of antibody responses associated with differential effects on B7-2 expression and subsequent costimulatory effects on CD4(+) T cells.

Identification of motifs in cholera toxin A1 polypeptide that are required for its interaction with human ADP-ribosylation factor 6 in a bacterial two-hybrid system.

The latent ADP-ribosyltransferase activity of cholera toxin (CT) that is activated after proteolytic nicking and reduction is associated with the CT A1 subunit (CTA1) polypeptide. This activity is stimulated in vitro by interaction with eukaryotic proteins termed ADP-ribosylation factors (ARFs). We analyzed this interaction in a modified bacterial two-hybrid system in which the T18 and T25 fragments of the catalytic domain of Bordetella pertussis adenylate cyclase were fused to CTA1 and human ARF6 polypeptides, respectively. Direct interaction between the CTA1 and ARF6 domains in these hybrid proteins reconstituted the adenylate cyclase activity and permitted cAMP-dependent signal transduction in an Escherichia coli reporter system. We constructed improved vectors and reporter strains for this system, and we isolated variants of CTA1 that showed greatly decreased ability to interact with ARF6. Amino acid substitutions in these CTA1 variants were widely separated in the primary sequence but were contiguous in the three-dimensional structure of CT. These residues, which begin to define the ARF interaction motif of CTA1, are partially buried in the crystal structure of CT holotoxin, suggesting that a change in the conformation of CTA1 enables it to bind to ARF. Variant CTA polypeptides containing these substitutions assembled into holotoxin as well as wild-type CTA, but the variant holotoxins showed greatly reduced enterotoxicity. These findings suggest functional interaction between CTA1 and ARF is required for maximal toxicity of CT in vivo.

Development and Validation of a Detection System for Wild-type Vibrio cholerae in Genetically Modified Cholera Vaccine

Orochol®, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intactctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol®.

Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells

The protective antigen (PA) component of anthrax toxin mediates delivery of either lethal factor (LF) or oedema factor into the cytosol of mammalian cells. The N-terminal domain of LF(1-254) (amino acids 1-254 of LF) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. In the present study, we fused the catalytic subunit of cholera toxin (CT-A) with LF(1-254) and showed that the fusion protein LF(1-254)-CT-A retains ADP-ribosylation activity in solution and increased intracellular cAMP levels in J774A.1 macrophage cells when added together with PA. A mutant fusion protein, in which arginine-7 of CT-A was replaced with lysine, did not show ADP-ribosylation activity in solution and failed to increase cAMP levels in macrophage cells. The data show that LF(1-254)-CT-A retains its catalytic activity in solution as well as when translocated into the cytosol of eukaryotic cells via an alternative pathway to the GM(1) receptor used by CT.

Determination of biological toxins using capillary electrokinetic chromatography with multiphoton-excited fluorescence.

We report a highly sensitive and rapid strategy for characterizing biological toxins based on capillary electrokinetic chromatography with multiphoton-excited fluorescence. In this approach, aflatoxins B1, B2, and G1 and the cholera toxin A-subunit are fractionated in approximately 80 s in a narrow-bore electrophoretic channel using the negatively charged pseudostationary phase, carboxymethyl-beta-cyclodextrin. The aflatoxins--highly mutagenic multiple-ringed heterocycles produced by Aspergillus fungi--are excited at the capillary outlet through the simultaneous absorption of two to three 750-nm photons to yield characteristic blue fluorescence; cholera toxin A-subunit, the catalytic domain of the bacterial protein toxin from Vibrio cholera, is excited through an unidentified multiphoton pathway that apparently includes photochemical transformation of an aromatic residue in the polypeptide. The anionic carboxymethyl-beta-cyclodextrin, used to chromatographically resolve the uncharged aflatoxins, enhances emission from these compounds without contributing substantially to the background. Detection limits for these toxins separated in 2.1-micron-i.d. capillaries range from 4.4 zmol (approximately 2700 molecules) for aflatoxin B2 to 3.4 amol for the cholera toxin A-subunit. Larger (16-micron-i.d.) separation capillaries provide concentration detection limits for aflatoxins in the 0.2-0.4 nM range, severalfold lower than achieved in 2.1-micron capillaries. These results represent an improvement of > 10(4) in mass detectability compared to previously published capillary separations of aflatoxins and demonstrate new possibilities for the analysis of proteins and peptides.

MHC Class I-Restricted Cytotoxic Lymphocyte Responses Induced by Enterotoxin-Based Mucosal Adjuvants

AbstractThe ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257–264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257–264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257–264. Both 51Cr release assays and IFN-γ enzyme-linked immunospot assays indicated that IFN-γ−/− and IL-12 p40−/− gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-γ and probably by a mechanism unrelated to cross-priming.

Intoxications medicamenteuses chez l'enfant au liban: elles sont frequentes et severes! etude transversale hospitaliere

The cholera toxin A-subunit (CTA) was genetically engineered at its amino end and tested for carriage of epitopes by fusion of the STa heat-stable enterotoxin analogue CAELCCNPAC. Efficient holotoxin formation by complementation in trans with cholera toxin B-subunit (CTB) indicated no decrease in affinity for CTB but evidence of reduced toxicity suggests steric interference by the decapeptide with the active site. The holotoxin was stable, able to bind to GM1 and was recognized by anti-STa and anti-CTA antibodies. The use of a full-length CTA might have been a key step for successful genetic fusions. Based on these findings, it seems worthwhile pursue the development of CTA for construction of recombinant mucosal immunoadjuvants.

A novel concept in mucosal adjuvanticity: the CTA1-DD adjuvant is a B cell-targeted fusion protein that incorporates the enzymatically active cholera toxin A1 subunit.

A promising novel concept in mucosal adjuvant research is demonstrated here. The adjuvant and toxic effects of the cholera toxin (CT) have been successfully separated in a gene fusion protein, CTA1-DD. This protein consists of the ADP-ribosylating A1 subunit of CT linked to a synthetic analogue of protein A. The CTA1-DD protein was found to exert comparable adjuvant activity to that of CT after systemic as well as mucosal immunizations with soluble protein antigens, such as KLH or ovalbumin (OVA). However, contrary to CT it was completely non-toxic. The CTA1-DD approach to the construction of a potential vaccine adjuvant is unique and highly promising. Conceptually, the CTA1-DD fusion protein demonstrates that: (i) contrary to CT the CTA1-DD is a highly targeted adjuvant, directed to B cells and possibly other antigen-presenting cells; (ii) it is possible to introduce ADP-ribosyltransferase activity into cells via an alternative pathway to the GM1 receptor pathway used by CTB; (iii) the adjuvant effect of CTA1-DD, and possibly also of CT, depend on the enzymatic activity; and (iv) one possible mechanism, shared by CT, that may explain the adjuvant effect of CTA1-DD is its ability to induce expression of the costimulatory molecule CD86 on B cells.

Thyroid-specific expression of cholera toxin A1 subunit causes thyroid hyperplasia and hyperthyroidism in transgenic mice.

Thyroid cell growth and function are regulated by hormones and growth factors binding to cell surface receptors that are coupled via G proteins, Gs and Gq, to the adenylyl cyclase and phospholipase C signal transduction systems, respectively. Activating mutations of the TSH receptor and G alpha s have been documented in subsets of thyroid neoplasms. To test the oncogenic potential of activated G alpha s in transgenic mice, we used the cholera toxin A1 subunit that constitutively activates G alpha s and used the rat thyroglobulin gene promoter for targeting this transgene (TGCT) to thyroid follicular cells. Three (M1392, F1358, and F1286) of six founders identified were able to transmit the transgene to their offspring and thyroid glands from these mice contained elevated levels of cAMP. Concentrations of serum thyroxine were elevated as early as 2 months of age (M 1392 and F 1286). F1358 mice were euthyroid until 8 months of age, at which time they developed hyperthyroidism. All three TGCT lines developed thyroid hyperplasia independent of their thyroxine levels. DNA image analysis of thyroid follicular cells from both the hyper and euthyroid mice showed that DNA index and "S+G2/M" phase were increased compared with normal, changes similar to that seen in poor prognosis human carcinomas. These data suggest that the G alpha s-adenylyl cyclase-cAMP pathway has an important role in thyroid hyperplasia and the transgenic mouse models reported herein will allow further examination of the role of this pathway in thyroid oncogenesis.

Thyroid-Specific Expression of Cholera Toxin A1 Subunit Causes Thyroid Hyperplasia and Hyperthyroidism in Transgenic Mice

Thyroid-Specific Expression of Cholera Toxin A1 Subunit Causes Thyroid Hyperplasia and Hyperthyroidism in Transgenic Mice

A genetically engineered non-toxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit

Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic. Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM. After i.v. injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo. Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT. In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity. The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.

A genetically engineered non-toxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit

A genetically engineered non-toxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit

Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit.

Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic. Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM. After i.v. injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo. Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT. In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity. The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.