Assessing Risks of Plant-Based Pharmaceuticals: II. Non-Target Organism Exposure

Plant-based pharmaceuticals potentially offer a cleaner method of producing a protein for drug manufacturing than traditional methods because plants are free of mammalian infectious agents. However, in the open environment they have the potential for intra-and inter-species gene flow, protein exposure to the public and non-target organisms, and they also have the potential to contaminate livestock feed. This study used probabilistic approaches to quantify the non-target organism risks associated with three pharmaceutical proteins produced in field-grown maize. The risk assessment for plant-based pharmaceuticals was conducted for four receptor species used as surrogates for a wider range of species. Body weights and maize consumption rates for each species were modeled from currently available information and used to calculate the exposure based on expression levels of three proteins. The acute dietary exposure for the receptor species was a single-day event in which the total maize consumption came from the recombinant maize. The non-target organism risk assessment demonstrated that risks will vary between species and between proteins, based primarily on differences in toxic endpoint and consumption rates. It also shows the utility of probabilistic, quantitative risk assessment methodologies and the importance of assessing risks from plant-based pharmaceuticals on a case-by-case basis.     

表达大肠杆菌LT-B/ST融合抗原的减毒鼠伤寒沙门氏菌株的构建

编码LT-B/ST融合抗原的基因插入pYA248载体中,构建了重组质粒pXZL66。该重组质粒转入无毒鼠伤寒沙门氏菌SR-11,ΔCya,Δcrp,Δasd菌株X4072。此无抗药性的杂合菌株X4072(pXZL66)表达的LT-B/ST融合抗原具有LT和ST抗原性而没有生物毒性,可望成为预防ETEC腹泻和相应的沙门氏菌病双价口服活疫苗候选株。     

Assessing Risks of Plant-Based Pharmaceuticals: I. Human Dietary Exposure

Protein-based drugs are the fastest growing class of drugs for the treatment of disease in humans and other animals. However, the current method of producing proteins for pharmaceutical application is predicted to fall short because of population growth and demographic trends. This study characterized human dietary risks using quantitative risk assessment techniques for three pharmaceutical proteins produced in field-grown maize. The three proteins were aprotinin, gastric lipase, and Escherichia coli heat-labile enterotoxin B subunit (LT-B). The human dietary risks from the three proteins inadvertently occurring in food were evaluated using three different exposure scenarios so that potential risks could be compared. The three exposure scenarios ranged in conservatism to evaluate the range of risk between the proteins and scenarios. Risk quotients (RQs) were calculated for all three scenarios to integrate exposure and effect (toxicity). The risk assessments revealed that the most conservative scenario produced higher RQs than the other two scenarios. The dietary risks from scenario 1 for aprotinin were three orders of magnitude greater than for scenario 2, and four orders of magnitude greater than for scenario 3. This risk assessment revealed that dietary risks will vary dramatically and depend on factors such as the specific pharmaceutical protein, protein expression, and exposure scenarios. The assessment also reinforced the need for case-by-case assessments.     

Histidine-44 of the A subunit of Escherichia coli enterotoxin is involved in its enzymatic and biological activities

We examined the role in toxicity of histidine-44 of the A subunit of Escherichia coli enterotoxin, which is located in the active site cavity close to glutamic acid-112. Although amino acid substitution of histidine-44 usually renders a mutant toxin unstable to trypsin, one mutant, alanine-44 (His44Ala) was found to be stable. His44Ala did not show any agmatine:ADP-ribosyltransferase activity in the presence or absence of recombinant ADP-ribosylation factor. It showed no diarrheal or rabbit skin permeability activity and was a competitor in enterotoxin-ADP-ribosyltransferase assays containing recombinant ADP-ribosylation factor. These results suggest that like glutamic acid-112, histidine-44 plays an essential role in toxicity. A tentative model, which explains NAD⁺ catalysis and the transfer of the ADP-ribosyl moiety to a target amino acid, is proposed for histidine-44 and glutamic acid-112.     

大肠杆菌 LT B亚基基因表达载体对犬细小病毒VP2 DNA疫苗免疫应答的增强作用

【目的】通过融合基因表达载体和共免疫基因表达载体研究大肠杆菌不耐热肠毒素(LT)B亚基基因对犬细小病毒VP2DNA疫苗免疫应答的影响。【方法】提取大肠杆菌44815菌株基因组DNA,通过PCR方法从基因组DNA中扩增LTB基因,同时采用PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2的主要抗原表位基因(VP2-70,编码70个氨基酸)。将上述基因分别连接到含有人CD5信号肽序列的载体pcDNA-CD5sp上,分别构建成它们的分泌型真核表达载体,pcDNA-CD5sp-LTB和pcDNA-CD5sp-VP2-70。再利用酶切连接的方法构建LTB与VP2-70融合的真核表达载体pcDNACD5sp-LTB-VP2-70。然后用pcDNACD5sp-VP2-70(VP2-70组)、pcDNACD5sp-LTB-VP2-70(VP2-LTB融合组)、pcDNA-CD5sp-LTB/pcDNACD5sp-VP2-70(VP2-LTB共免疫组)和pcDNA3.1A(空载体对照组)分别免疫小鼠。免疫后用间接ELISA检测不同时间小鼠血清的抗体水平,用MTT方法检测小鼠免疫5周后脾脏淋巴细胞的增殖活性。【结果】经过测序表明本研究扩增的LTB和VP2基因序列和构建的相关表达载体结构正确。通过Western-blot检测证明构建的表达载体均能介导相应基因在真核细胞进行分泌表达。ELISA检测结果表明,3组实验组小鼠接受VP2DNA疫苗免疫后均能产生特异的体液免疫应答反应,特别是VP2-LTB基因融合组小鼠的抗体水平在第5周时高达1:5120,明显高于其它两组(P<0.01)。3组免疫小鼠抗体的亚型均表现IgG1抗体水平明显高于IgG2a抗体水平(P<0.01)。淋巴细胞增殖实验结果表明,在ConA的刺激下,3组免疫小鼠的淋巴细胞刺激指数均明显高于对照组(P<0.01),说明VP2DNA疫苗能够引起淋巴细胞的增殖。但3组免疫小鼠之间的刺激指数没有明显差异(P>0.05)。【结论】在小鼠体内,LTB基因表达载体可明显提高CPVVP2DNA疫苗的体液免疫应答水平。     

重组大肠杆菌不耐热肠毒素B亚单位基因表达系统构建

从E .coli 4 4 815株基因组DNA中扩增不耐热肠毒素B亚单位 (LTB)基因并分析了核苷酸序列 ,构建pET32a的LTB表达载体 ,在E .coliBL2 1DE3宿主菌中用不同浓度的IPTG诱导表达 ,采用SDS PAGE鉴定表达产物。克隆的LTB基因与报道的核苷酸和氨基酸序列同源性分别为 99.12 %～ 99.71%和 97.5 8%～ 99.19% ,pET32a LTB BL2 1DE3系统表达的rLTB量约占细菌总蛋白的 30 %。     

大肠杆菌热稳定肠毒素与霍乱毒素B亚单位融合蛋白免疫原性的评价

构建了霍乱毒素B亚单位(CTB)和大肠杆菌热稳定肠毒素(ST）的重组表达载体pMCST1、pMCST2。二的不同是,前为单拷贝ST,后为双拷贝ST。在大肠杆菌DH50α中融合蛋白高效表达。用这两种重组蛋白分别免疫小鼠,都诱导产生了高滴度的抗CTB和抗ST的血清。表明这两组融合蛋白具有良好的CTB和ST的免疫原性,为进一步构建抗CTB和抗ST的产毒性细菌腹泻疫苗打下了基础。     

Detection and purification of the free A subunit of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli

After removal of total B subunit and heat-labile enterotoxin (LT) from crude cell extracts of enterotoxigenic Escherichia coli (HB 101-EWD 299) by Bio-gel A 5 m column chromatography, the crude cell extract was shown to contain a free A subunit (A' subunit) that did not bind to the coligenoid of the B subunits. The A' subunit was found to be immunologically identical to the A subunit of holo-LT and was purified to show only one band in SDS-poly-acrylamide gel electrophoresis (PAGE). The mobility of the A' subunit was identical to that of the A subunit of holo-LT. The pI value of the A' subunit was also the same as that of the A subunit of holo-LT. These data suggest that in enterotoxigenic E. coli there is free A subunit which may be involved in formation of holo-LT, analogously to free B subunit (coligenoid), and that the free A subunit is physicochemically and immunologically identical to the A subunit of holo-LT.     

Effect of Substitution for Arginine Residues near Position 146 of the A Subunit of Escherichia coli Heat-Labile Enterotoxin on the Holotoxin Assembly

Escherichia coli heat-labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide-directed site-specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized d -galactose column and analyzed the purified mutant LTs by SDS-PAGE to examine the amount of A subunit associated with B-subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B-subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B-subunit oligomer. Subsequently, we measured the level of the intracellular B-subunit oligomer of these mutant strains. The measurement revealed that the amount of B-subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement.     

Role of verocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhoea

Abstract 273 Strains of Escherichia coli isolated from diarrhoeic and healthy control cattle and buffalo calves in Sri Lanka, were tested for Verocytotoxin (VT) and for heat-stable (ST) and heat-labile (LT) enterotoxins. VT and ST toxigenic E. coli were significantly associated with diarrhoea, accounting for 28% and 18% of diarrhoeic episodes, respectively. LT toxigenic E. coli were not significantly associated with diarrhoea.     

Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein

A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri-specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LT(B)) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5'-ST-LT(B)-3' DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLT(B) system was found to possess the capability of adhering to the mice gut, secreting GFP:STLT(B) product at 0.14 and 0.026 pgcell(-1) under induced and noninduced conditions, respectively. Further analysis of the GFP:STLT(B) product confirmed its ganglioside-binding ability, LT(B) antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLT(B) culture in mice elicited significant (P<0.01) serum IgG and mucosal IgA antibodies against the STLT(B) antigen. These immunized mice were subsequently challenged with ETEC and showed full protection against the fluid influx response in the gut. This is the first report of using L. reuteri as a vaccine carrier to induce complete immunologic protection against ETEC.     

A Functional Antigen in a Practical Crop: LT-B Producing Maize Protects Mice against Escherichia coli Heat Labile Enterotoxin (LT) and Cholera Toxin (CT)

We have produced a functional heat labile enterotoxin (LT-) B subunit of Escherichia coli in maize. LT-B is a multimeric protein that presents an ideal model for an edible vaccine, displaying stability in the gut and inducing mucosal and systemic immune responses. Transgenic maize was engineered to synthesize the LT-B polypeptides, which assembled into oligomeric structures with affinity for G_M1 gangliosides. We orally immunized BALB/c mice by feeding transgenic maize meal expressing LT-B or non-transgenic maize meal spiked with bacterial LT-B. Both treatments stimulated elevated IgA and IgG antibodies against LT-B and the closely related cholera toxin B subunit (CT-B) in serum, and elevated IgA in fecal pellets. The transgenic maize induced a higher anti-LT-B and anti-CT-B mucosal and serum IgA response compared to the equivalent amount of bacterial LT-B spiked into maize. Following challenge by oral administration of the diarrhea inducing toxins LT and CT, transgenic maize-fed mice displayed reduced fluid accumulation in the gut compared to non-immunized mice. Moreover, the gut to carcass ratio of immunized mice was not significantly different from the PBS (non-toxin) challenged control group. We concluded that maize-synthesized LT-B had features of the native bacterial LT-B such as molecular weight, G_M1 binding ability, and induction of serum and mucosal immunity. We have demonstrated that maize, a major food and feed ingredient, can be efficiently transformed to produce, accumulate, and store a fully assembled and functional candidate vaccine antigen.     

淋球菌porB和大肠杆菌ltB融合基因的构建、表达及其免疫活性

【目的】构建融合基因原核表达载体pET-30a/ltB-porB,并表达重组融合蛋白LTB-PorB,鼻饲途径免疫雌性BALB/c小鼠,分析重组融合蛋白的免疫活性,为研制抗淋病蛋白疫苗提供实验依据。【方法】构建大肠杆菌不耐热肠毒素B亚单位(LTB)与淋球菌外膜孔蛋白B(PorB)融合基因及LTB、PorB单基因pET-30a原核表达载体,在大肠杆菌BL21中表达重组蛋白;鼻饲途径免疫雌性BALB/c小鼠,检测体液免疫和细胞免疫水平。【结果】在大肠杆菌BL21中获得高效表达的重组蛋白;经鼻饲免疫小鼠后,重组融合蛋白LTB-PorB组生殖道黏膜产生的PorB特异性sIgA水平随免疫时间呈上升趋势,第42天A450值达0.66,明显高于对照组(P0.01),效价高达1∶1280;血清中产生的PorB特异性IgG第28天达最高,A450值为0.60,明显高于LTB和蛋白溶解液(Solution Buffer)对照组(P0.01),效价高达1:2560,但与PorB对照组血清IgG水平(A450:0.57)无明显差异(P0.05)。LTB-PorB组脾淋巴细胞刺激指数明显高于LTB和SolutionBuffer对照组(P0.05),但脾淋巴细胞诱生的IFN-γ水平与对照组无明显差异(P0.05)。【结论】重组融合蛋白LTB-PorB通过鼻饲途径免疫雌性BALB/c小鼠后,能诱导产生高水平的体液免疫和一定水平的细胞免疫。首次证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道粘膜免疫。     

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane

Abstract The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of ¹²⁵I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with ¹²⁵I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophorosis and gel filtration showed that the molecular mass was 25 kDa.     

A unique DNA sequence of human enterotoxigenic Escherichia coli enterotoxin encoded by chromosomal DNA

We detected Ent plasmids in 300 strains of human enterotoxigenic Escherichia coli, but one strain, E. coli 240-3, had neither a small nor a large plasmid and encoded the heat-labile enterotoxin (LTh(240-3)) gene on its chromosome. DNA sequences showed that LTh(240-3) differed by 12 and 14 base pairs from LT (LTh) and LT (LTp) from human H10407 and porcine EWD299 strains, respectively. In deduced precursor toxins, LTh(240-3), LTh and LTp differed from LTh, LTp and LTh(240-3) at nine, eight and eleven positions, respectively. These data suggest that although LTh(240-3) encoded in the chromosome is antigenically similar to LTh, it cannot be grouped with LTh due to differences in its DNA and amino acids sequences.     

大肠杆菌不耐热肠毒素B亚单位的克隆、表达及初步纯化

为研究大肠杆菌不耐热肠毒素B亚单位（LTB）的佐剂活性。从大肠杆菌中调出LTB的原始基因,将该基因克隆、构建pET21b—LTB表达载体、转化大肠杆菌B121（DE3）进行表达,并对表达产物进行初步纯化;经DNA测序、SDS—PAGE、ELISA检测,结果表明成功构建了能够稳定表达可溶性LTB的菌株,并获得初步纯化LTB的方法。为今后LTB的研究及应用奠定了基础。     

人产肠毒素大肠杆菌ST、LT—B肠毒素基因融合的研究

将人产肠毒素大肠杆菌(ETEC),编码耐热肠毒素(ST)的基因片段与编码不耐热肠毒素B亚基(LT-B)的基因进行融合,并在此基础上进行不同数目ST基因的串联,ELISA检测融合基因表达蛋白产物观察到ST与LT-B之间存在着相互影响。ST的检测滴度随基因串联个数增加而逐渐升高,而LT的ELISA滴度则减弱。说明了ST可以通过基因串联提高表达产物抗原活性,这为产肠毒素大肠杆菌多价疫苗的研制提供了重要的研究基础。     

Emission of carbonyl sulfide by Thiobacillus thioparus grown with thiocyanate in pure and mixed cultures

Abstract The biological activity of the heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was found to be predominantly associated with the periplasmic extract (about four-fold higher than the culture supernatant) of a recombinant E. coli (JM109) strain carrying the NAG-St toxin gene. Four molecular species of NAG-ST, two each from the periplasmic extract and culture supernatant of JM109, were purified. Amino acid sequence analysis of the four NAG-ST peptides isolated by HPLC revealed that they all differed from that of the mature 17-amino acid residue NAG-ST released by V. cholerae non-O1. The M _r-values of the peptides obtained from the periplasmic extract were 4331 and 2785, while those recovered from the culture supernatant were 3154 and 2785. It thus appears that V. cholerae NAG-ST is synthesized as larger molecules in the recombinant E. coli strain. The differences in sizes of the exported NAG-ST molecule could relate to difference in the enzyme cleavage system between E. coli and V. cholerea .     

Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.