A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Abstract Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) at doses as low as 0.8 ng·ml⁻¹, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertusis , containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for PT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.     

F型肉毒毒素的制备及类毒素免疫原性初探

将F型肉毒梭菌经适宜条件产毒培养后,以硫酸铵盐析和酸沉两种不同工艺制备的F型肉毒毒素,用分段脱毒法脱毒制备类毒素,分别免疫豚鼠、马匹后测定免疫血清抗体效价。结果显示,两种工艺制备的毒素其类毒素都具有较好的免疫原性。     

Comparative analysis of antigenic strength and in vivo serum antibodies concentration of tetanus toxoid vaccine adsorbed in Pakistan

Clostridium tetani produce tetanospasmin, a potent exotoxin; that causes tetanus or lockjaw disease. Scientists developed an anti-tetanus toxoid to protect the body from the spasm's neurotoxic effect. In Pakistan recently, 478 cases of neonatal tetanus were reported. The study was carried out at The National Control Laboratory for Biologicals Islamabad, aiming to decipher the effectiveness of the most frequently used tetanus toxoid vaccine adsorbed in Pakistan in comparison to standard reference vaccine having earlier known consistent values. The vaccines included domestic public sector, domestic private sector, imported private sector I, and imported private sector II. The triplicate experiments on purebred Swiss albino mice were performed by immunizing with Tetanus toxoid and then tested parallel with standard reference vaccine. Various analytical tests were performed on the test organism that included flocculation test/identity test, antibody quantification using enzyme-linked immunosorbent assay (ELISA), potency test, abnormal toxicity test, osmolality, pH test, liquid sub-visible particle test, and sterility test. Results of all the vaccines were compared in comparison with the standard reference vaccine. Absorbances of test vaccines were recorded at the lowest dilution by ELISA. The domestic private sector, imported private sector I, imported private sector II and standard reference vaccine were flocculated at mean dilution (Mean: 0.24, 95% CI: 0.1903–0.2897), and the domestic public sector was flocculated at mean dilution (Mean: 0.23, 95% CI: 0.2052–0.2548). All the products were found within the normal ranges where it was concluded that the maximum average titer of 2.81 was observed at dilution 10^?1.6, indicating that these vaccines were adequate/suitable for the prevention of tetanus.     

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vβ type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21–32, 93–107 and 202–217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.     

肉毒A、B、E、F型抗毒素血浆的试生产与检定

按照《中国生物制品规程》(2000版)的规定,在肉毒A、B、E、F型抗毒素血浆试生产前,制定了肉毒A、B、E、F型类毒素马匹免疫计划及相应抗毒素血浆效价测定方法,免疫结果显示：肉毒A、B、E、F型类毒素免疫马匹成功率分别为75%、75%、60%、100%。     

Inhibition of mouse SP2/0 myeloma cell growth by the B7-H4 protein vaccine

B7-H4 is a member of B7 family of co-inhibitory molecules and B7-H4 protein is found to be overexpressed in many human cancers and which is usually associated with poor survival. In this study, we developed a therapeutic vaccine made from a fusion protein composed of a tetanus toxoid (TT) T-helper cell epitope and human B7-H4IgV domain (TT-rhB7-H4IgV). We investigated the anti-tumor effect of the TT-rhB7-H4IgV vaccine in BALB/c mice and SP2/0 myeloma growth was significantly suppressed in mice. The TT-rhB7-H4IgV vaccine induced high-titer specific antibodies in mice. Further, the antibodies induced by TT-rhB7-H4IgV vaccine were capable of depleting SP2/0 cells through complement-dependent cytotoxicity (CDC) in vitro. On the other hand, the poor cellular immune response was irrelevant to the therapeutic efficacy. These results indicate that the recombinant TT-rhB7-H4IgV vaccine might be a useful candidate of immunotherapy for the treatment of some tumors associated with abnormal expression of B7-H4. [BMB Reports 2014; 47(7): 399-404]     

Development of a carbohydrate binding assay for the B-oligomer of pertussis toxin and toxoid

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and, in its detoxified form PTd, is an important component of pertussis vaccines. The in vivo histamine sensitization test (HIST) is currently used for the safety testing of these vaccines. However, an alternative test is needed because of large assay variability and ethical concerns with regard to animal usage. PTx has two functionally distinct domains: the enzymatic A-protomer and the B-oligomer that facilitates host-cell binding and entry of PTx into the cell. The development of a quantitative PTx binding assay using glycoproteins or defined oligosaccharides is reported. PTx was found to bind preferentially to multiantennary N-glycans, with the highest binding toward the fully sialylated structures. In contrast, PTd lost the ability of PTx to bind to sialylated multiantennary structures but retained some capacity to bind to neutral multiantennary structures. The developed assay was shown to be specific, sensitive, and robust and could be used for investigating the mechanisms of PTx detoxification and for monitoring PTx binding activity in vaccine formulations. This assay could also be used to complement a PTx-enzymatic assay, developed recently, and together they may form the basis of a potential alternative in vitro assay to replace the in vivo HIST.     

C型肉毒毒素的制备及类毒素保护力初探

将C型肉毒梭菌经适宜条件的产毒培养后纯化,并进行相关鉴定。制备的C型肉毒毒素用分段脱毒法脱毒,并进行类毒素保护力的初步研究。以不同蛋白含量C型肉毒类毒素免疫小鼠后攻毒,结果显示,蛋白含量为0.625μg的类毒素免疫2针或蛋白含量为1.25μg的类毒素免疫1针均可保护50LD50的C型肉毒毒素攻击。蛋白含量为5μg的C型肉毒类毒素与福氏不完全佐剂配制的抗原免疫小鼠3次所得抗血清的保护力(Anti LD50/ml)为4.3×104。说明用该纯化工艺制备的C型肉毒类毒素具有很好的免疫原性,作为抗原成分用于C型肉毒疫苗和C型肉毒抗毒素的研究和生产具有较好的应用潜力。     

Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the “3R” principles of replacing, reducing and refining animal tests, the “binding and cleavage” (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.     

Optimal conditions for the toxoiding of pertussis toxin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl

Abstract The optimal conditions for toxoiding a pertussis toxin (PT) preparation with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl (EDAC) were determined. The prime factor affecting the toxoiding of PT was the EDAC to protein ratio. A ratio of 40–80 : 1 EDAC to protein by weight was optimal for abolishing the acute toxicity, histamine-sensitising and leucocytosis-promoting activities associated with PT, whilst maintaining the antigenicity of the vaccine antigens. An EDAC-toxoid also manifested no late histamine-sensitising activity. Duration of exposure to EDAC, temperature and pH value of the reaction were found not be be critical for toxoiding. The data indicated that the use of EDAC for toxoiding PT in a B. pertussis extract is a simple and reproducible procedure and should be considered as a method for the production of acellular pertussis vaccines.