Native Cholera Toxin B Subunit Research Articles

Immunogenicity of Fusion Protein of Cholera Toxin B Subunit-Porphyromonas gingivalis 53-kDa Minor Fimbrial Protein Produced in Nicotiana benthamiana

Porphyromonas gingivalis induces destruction of periodontal tissues that surround and support the teeth, contributing to the development of periodontitis, which eventually results in tooth loss in adults. A 53-kDa protein of P. gingivalis is a major subunit variant protein of minor fimbriae (Mfa1), and is reported to be highly immunogenic and considered as a potential vaccine candidate. The gene encoding 53-kDa protein, was divided into three segments, and each DNA segment was fused to the gene coding for cholera toxin B subunit (CTB) to improve gut mucosal immune responses. The ctb-mfal fusion genes were expressed in the leaf tissues of Nicotiana benthamiana using agroinfiltration. Immunoblot analysis revealed that CTB-Mfa1 fusion proteins were produced in the agroinfiltrated leaves. The interaction of the plant-produced CTB-Mfa1 fusion proteins with GM1-ganglioside, which acts as the binding site for native CTB, was confirmed by GM1-ELISA. Mice immunized orally with the agroinfiltrated leaf powder containing the CTB-Mfa1 fusion proteins elicited serum IgG and fecal IgA antibodies to CTB and Mfa1. These results suggest that CTB-Mfa1 fusion proteins produced in plants can be used as an oral vaccine to control P. gingivalis infection-associated periodontitis.

N-glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential.

Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

A single point mutation within the coding sequence of cholera toxin B subunit will increase its expression yield.

Background: Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. Methods: ctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB. Results: SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies. Conclusion: Point mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield.

Transcytosis-Blocking Abs Elicited by an Oligomeric Immunogen Based on the Membrane Proximal Region of HIV-1 gp41 Target Non-Neutralizing Epitopes

CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

High‐yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts

The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G(M1)-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (approximately 100 mg for a dose of 500 microg antigen) and would permit encapsulation of freeze-dried material or pill formation.

Studies on the immunogenic potential of plant-expressed cholera toxin B subunit.

Nicotiana tabacum var. Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter. Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 microg recombinant CTB was injected intradermally into Balb/c (H2K(d)) mice. CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB. A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out. Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB. Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited. These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine. The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G(M1) ganglioside.

A novel method for the rational construction of well-defined immunogens: the use of oximation to conjugate cholera toxin B subunit to a peptide-polyoxime complex.

Cholera toxin B subunit (CTB), capable of binding to all mucous membranes in its pentameric form, is a potential carrier of mucosal vaccines. In our previous work we reported that the N-terminus of CTB, a threonine, could in principle undergo oxidation and oximation to form conjugates with a cascade of immunogenic peptides. In this study, we set up a model by chemically coupling CTB to a polyoxime that possessed five copies of influenza virus-derived peptides displayed in comblike form. The construct was reconstituted into pentameric form when eluted from a Superdex column after conjugation, and the pentameric nature of this CTB-viral peptide complex was confirmed by SDS-PAGE. GM(1)-ELISA assay showed that the binding properties of CTB-viral peptide complex were increased 4-5-fold over native CTB.

A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection

We have constructed and efficiently produced and purified a candidate vaccine against schistosomiasis consisting of a novel hybrid protein in which two dominant T- and B-cell epitopes from Schistosoma mansoni 28 kDa glutathione- S-transferase (Sm28GST) antigen (a.a 24–43 and 191–212) are fused to cholera toxin B subunit (CTB). Intranasal treatment of S. mansoni-infected mice with the hybrid protein, which similar to native CTB was assembled into receptor binding pentamers, significantly reduced total worm burden and liver egg counts due to the induction of Sm28GST-specific antibodies. Immunopathologic granuloma formation in the liver was also significantly suppressed and there was an almost complete suppression of delayed-type hypersensitivity reactions to both Sm28GST and to total soluble egg antigen in infected animals. The results suggest that this type of hybrid protein could be used as a combined anti-immunopathology and anti-infection vaccine against schistosomiasis.

Efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression/secretion system of Bacillus brevis and its mucosal immunoadjuvanticity

A gene encoding the mature Escherichia coli heat-labile enterotoxin B subunit (LTB) was introduced in a vector pNU212 and expressed at high levels in Bacillus brevis HPD31. The maximum amount of recombinant LTB (rLTB) secreted into the modified 5PY medium containing erythromycin was about 350 mg l −1 when cultivated at 30°C for 8 days. The rLTB purified directly from the culture supernatant by using d-galactose immobilized agarose was identical to the native LTB with respect to the molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the amino terminal amino acid sequence. Western blot analysis with antiserum to cholera toxin B subunit (CTB) indicated that rLTB had cross-reactivity to native CTB and its GM1 binding ability was almost the same as that of the CTB. The rLTB predominantly showed the pentameric form when non-boiled samples were applied to SDS–PAGE. When rLTB was administered intranasally to mice with diphtheria toxoid (D T), it resulted in the substantial stimulation of D T-specific serum IgG antibody, and in the induction of moderate levels of D T-specific mucosal IgA antibody responses in the nasal cavities and in the lung, suggesting that purified rLTB acts as a promising immunoadjuvant on mucosal immunizations.

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.

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.