Wild-type LT Research Articles

Merkel cell polyomavirus and non-small cell lung cancer

Sir, We congratulate Hashida et al (2013) on an interesting report that Merkel cell polyomavirus (MCV) is present in ∼18% of non-small cell lung carcinoma (NSCLC). An important clarification is needed; however, we have not yet discovered a naturally occurring mutation in the MCV large T retinoblastoma-binding motif (LFCDE). We engineered the sequence that the authors refer to as Appendix206 ({type:entrez-nucleotide,attrs:{text:JN038578,term_id:354683949,term_text:JN038578}}JN038578) to have a lysine substitution mutation (LFCDK) to serve as a negative control for the wild-type appendix-derived LT deposited as {type:entrez-nucleotide,attrs:{text:JN038579,term_id:354683952,term_text:JN038579}}JN038579, which possesses a wild-type LXCXE motif. Post-submission editing at NCBI obfuscated the description of {type:entrez-nucleotide,attrs:{text:JN038578,term_id:354683949,term_text:JN038578}}JN038578, leading to the confusion described in this paper. Thus, all but one of the MCV sequences Hashida et al (2013) report are consistent with wild-type virus and do not have the tumour-specific mutations we described that eliminate MCV LT helicase activity (Shuda et al, 2008). The authors do describe one virus (AC43) with a terminally truncated LT consistent with a tumour-derived mutation. Together with its high viral copy number, this case represents a particularly intriguing tumor that deserves careful follow-up, such as Southern blotting for clonality and histopathological characterization with reliable biomarkers for NSCLC to investigate a potential MCV contribution to its tumorigenesis.

Safety and immunogenicity of bacterial and tobacco plant cell line derived recombinant native and mutant Escherichia coli heat-labile toxin in chickens

The safety and immunogenicity of the mammalian mucosal adjuvants, Escherichia coli wild-type heat-labile holotoxin (LT) and E. coli mutant LT (LTA-K63/LTB), were examined in 1-day-old chicks and 10-day-old to 21-day-old broilers. Biologically active, E. coli recombinant wild-type LT and recombinant LTA-K63/LTB produced in a transgenic Nicotiana tabacum (NT-1) tobacco cell line (SLT102) were tested for safety and antigenicity following various routes of administration. Safety was assessed by clinical signs, body weight gain, gross organ pathology and wet organ weight, and histopathology. Antigenicity was assessed using LT-B-specific serum IgG enzyme-linked immunosorbent assay. Parenteral administration of E. coli recombinant wild-type LT did not have any discernible effect on bird health and was well tolerated at levels up to 400 µg per dose. Recombinant, SLT102-derived mutant LT derived from SLT102 cells retained in vitro ganglioside binding and was safe and antigenic following repeated mucosal administration to birds. The highest systemic LT-B-specific IgG titres were detected in birds that received three on-feed doses of SLT102-derived mutant LT. Among the various SLT102-derived mutant LT preparations tested, whole, wet cells or whole cell lysates were the most antigenic. These results demonstrate for the first time that E. coli-derived recombinant, wild-type LT holotoxin is well tolerated following multiple administrations to young birds at body weight doses previously reported to be enteropathogenic and toxic in mammalian species. Moreover, these data also demonstrate the feasibility of using recombinant wild-type and mutant LT produced in transgenic NT-1 tobacco cells as safe and potent vaccine adjuvants in poultry.

LT(K63/R72), a New Mutant of Escherichia coli Heat-labile Enterotoxin, Exhibits Characteristics More Similar to LT(K63) than LT(R72)

LT(K63), a non-toxic mutant and LT(R72), a low toxic mutant of E. coli heat-labile enterotoxin are frequently used mucosal adjuvants. In many cases, the adjuvanticity of LT(K63) is lower than that of LT(R72), but LT(K63), which induces a mixed Th1/Th2 response, exhibits a higher level of protection than LT(R72) which induces a polarized Th2-type response. To utilize the advantages of both adjuvants, a double-mutation LT(K63/R72) was generated and purified. The characterization results showed that there was no significant difference in production rate and immunogenicity between wild type LT and LT mutants. The results also showed that the toxicity and the trypsin sensitivity of LT(K63/R72) are between that of LT(K63) and LT(R72). Using HPLC, when samples in an OHpak SB-800 column were eluted by denatural buffer (TEAN containing 10 mg/ml SDS), we found the stability of LT(K63/R72) was higher than that of LT(R72) and lower than that of LT(K63). Through further analyzes, we found that LT(K63/R72) exhibits characteristics more closely related to LT(K63) than LT(R72).

LT(K63/R72), a New Mutant of <italic>Escherichia coli</italic> Heat-labile Enterotoxin, Exhibits Characteristics More Similar to LT(K63) than LT(R72)

Abstract LT(K63), a non-toxic mutant and LT(R72), a low toxic mutant of E. coli heat-labile enterotoxin are frequently used mucosal adjuvants. In many cases, the adjuvanticity of LT(K63) is lower than that of LT (R72), but LT(K63), which induces a mixed Th1/Th2 response, exhibits a higher level of protection than LT (R72) which induces a polarized Th2-type response. To utilize the advantages of both adjuvants, a doublemutation LT(K63/R72) was generated and purified. The characterization results showed that there was no significant difference in production rate and immunogenicity between wild type LT and LT mutants. The results also showed that the toxicity and the trypsin sensitivity of LT(K63/R72) are between that of LT(K63) and LT(R72). Using HPLC, when samples in an OHpak SB-800 column were eluted by denatural buffer (TEAN containing 10 mg/ml SDS), we found the stability of LT(K63/R72) was higher than that of LT(R72) and lower than that of LT(K63). Through further analyzes, we found that LT(K63/R72) exhibits characteristics more closely related to LT(K63) than LT(R72).

Modulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives

Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood. This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells. Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells. LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells. LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants. LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran. Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments. This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules.

Mutant Escherichia coli heat-labile enterotoxin [LT(R192G)] enhances protective humoral and cellular immune responses to orally administered inactivated influenza vaccine

Influenza vaccines capable of inducing both systemic and mucosal antibody responses are highly desirable. Optimal induction of mucosal IgA is accomplished by mucosal delivery of vaccine. Mucosal adjuvants may improve the immunogenicity and efficacy of vaccines delivered by this route. Here, we compare the adjuvant activities of a mutant of heat-labile enterotoxin from Escherichia coli [LT(R192G)] with those of the wildtype LT (wtLT) for oral vaccination with inactivated influenza vaccine in BALB/c mice. Compared with administration of oral influenza vaccine alone, co-administration of vaccine with LT(R192G) provided enhanced protection from infection in the upper and lower respiratory tract equivalent to and at similar doses as that obtained with wtLT. Likewise, LT(R192G) augmented virus-specific IgG and IgA responses in serum, lung and nasal washes and the numbers of virus-specific antibody-forming cells in spleen, lung and Peyer’s patches in a manner comparable to wtLT. Virus-specific splenic CD4 + cells from mice administered oral vaccine with either adjuvant produced a mixed Th1- and Th2-type cytokine response pattern. Taken together, these results indicate that LT(R192G), like wtLT, is a potent adjuvant for oral vaccination of mice with influenza vaccine.

Evaluation of the adjuvant effect of Escherichia coli heat-labile enterotoxin mutant (LTK63) on the systemic immune responses to intranasally co-administered measles virus nucleoprotein. Part I: antibody responses.

The adjuvanticity and immunogenicity of the heat-labile enterotoxin (LT) of Escherichia coli and of its non-toxic mutant, LTK63, was evaluated after intranasal administration of CBA mice with recombinant measles virus nucleoprotein (rMVNP) with or without LT or LTK63. Both LT and LTK63 were shown to be highly immunogenic with higher responses observed 4 weeks after the booster immunization. Although the nucleoprotein was immunogenic on its own, mice immunized with the nucleoprotein plus wild type LT produced significantly high antibody responses (p< 0.01). Mice that received the rMVNP with LTK63 also generated strong antibody responses to rMVNP. These antibodies were also significantly higher than those of rMVNP alone (p< 0.05). No significant differences were observed between groups of mice immunized intranasally with rMVNP plus LT or LTK63 (p> 0.05). Data on IgG antibody isotype profiles showed that IgG 1 and IgG 2a were predominant in mice immunized with rMVNP + LT or LTK63 whereas IgG 1 predominated when rMVNP was given on its own implying that LT and LTK63 induce both Th1 and Th2-type immune responses. These results highlight the great potential of this non-toxic mutant of LT as a safe vaccine adjuvant.

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.

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.

Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides

The adjuvanticitity of heat-labile enterotoxin (LT) of Escherichia coli and its non-toxic mutant LTK63 was assessed and compared for intranasal immunization of synthetic peptides. Mice immunized intranasally with LT, or its mutant LTK63, generated strong systemic proliferative and cytotoxic T-cell responses to co-administered synthetic peptides. The wild LT toxin promoted higher peptide-specific proliferative and cytotoxic T-cell responses than the LTK63 mutant. Moreover, the wild-type LT toxin was shown to promote peptide-specific memory CTL responses which were detectable 1 year after intranasal priming. Both LT and LTK63 molecules were shown to be immunogenic, with serum antibody subclasses being predominantly IgG1 and to a lesser extent IgG2a. These findings demonstrate that cellular immune responses to small synthetic peptide antigens administered by the intranasal route can be potentiated with the use of mucosal adjuvants. Moreover, the ability of LT and LTK63 to promote both CD4+ and CD8+ T-cell responses will have relevance to the design and production of future mucosal vaccines.

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.

Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal influenza vaccine

The effectiveness and safety of known mutants of Escherichia coli heat-labile enterotoxin (LT) as an adjuvant for nasal influenza vaccine were examined. Six mutants, called LT7K (Arg to Lys), LT61F (Ser to Phe), LT112K (Glu to Lys), LT118E (Gly to Glu), LT146E (Arg to Glu) and LT192G (Arg to Gly) were constructed by the replacement of one amino acid at one position of the A1 subunit to another using site-directed mutagenesis. All mutants were confirmed to be less toxic than wild-type LT when analyzed using Y-1 adrenal cells in vitro. When influenza vaccine was administered intranasally with LT7K and LT192G, BALB c mice developed high levels of serum and local antibodies to the HA molecules. The adjuvant activity of these mutant LTs corresponded to that of wild-type LT when 1 μg of these mutant LTs (or wild-type LT) was coadministered with the vaccine. From the point of view of safety, LT7K was considered to be the most potent mucosal adjuvant and was examined in more detail. The adjuvant activity of the mutant was lowered more rapidly with a decrease in dose than was that of wild-type LT. The low level of adjuvant activity of a relatively small amount of LT7K was heightened by adding LTB to the mutant LT. These results suggest that LT7K supplemented with LTB can be used as a less toxic, effective adjuvant for nasal influenza vaccine.

Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.

Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion. At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold. KDEL-mutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction. KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.