Adverse reactions after injection of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine are not due only to pertussis organisms or pertussis components in the vaccine.

Reactions to adsorbed diphtheria-pertussis-tetanus (DPT) vaccine have mostly been attributed to the pertussis organisms or pertussis components in the vaccine. Nevertheless reactions may also be due to other factors such as sensitization induced by aluminium adjuvants and impurities present in crude toxoids that cannot be removed by purification of toxoids after formalinization. Aluminium compounds such as aluminium phosphate and aluminium hydroxide are the most commonly used adjuvants with vaccines for human use. Due to the increasing concern about the toxicity of aluminium, other adjuvants like calcium phosphate may be evaluated as an alternative to aluminium adjuvants. To minimize reactions after immunization with DPT vaccine due to impurities in the toxoids, the use of toxoided purified toxins is suggested.     

The exotoxins of Corynebacterium ulcerans.

The exotoxins produced by ten strains of C. ulcerans (two human, six bovine and two equine) have been studied. On the criteria of toxin-antitoxin neutralisation and immunoprecipitation tests using highly specific diphtheria and C. ovis antitoxins with crude toxic filtrates, (NH4)2SO4 concentrates, and partially purified chromatographic preparations of these, together with the presence or absence of inhibition of the action of staphylococcal beta-haemolysin, and the reaction produced when injected intradermally into rabbits, two toxins could be identified, namely diphtheria toxin and C. ovis toxin. There was no evidence for the production of a third toxin specific for C. ulcerans. Five strains produced both diphtheria and C. ovis toxins. In four diphtheria toxin predominated, but in the fifth C. ovis toxin predominated. Two strains produced only diphtheria toxin and two only C. ovis toxin, though there was good but not complete evidence that a third strain (Revell) also fell into this latter group. Considerable variation occurred in the concentration of each toxin and, where both were present, in the proportion of each.     

Potential of Klebsiella pneumoniae cytotoxin toxoid as vaccine against klebsiellosis in rabbits and mice

The protective efficacy of toxoids prepared from crude cytotoxin (polymyxin-B extract, PBE) and purified Klebsiella cytotoxins (KCTs) was studied in rabbits and mice. The toxoids of KCT-I and PBE were found to be protective against homologous as well as heterologous Klebsiella challenges, while toxoids of KCT-II and KCT-III afforded protection in mice against homologous Klebsiella infections. KCT-I and PBE toxoids also induced good humoral anti-Klebsiella response in rabbits with ELISA titres ranging from 20480 to 81480. Immunized female rabbits passed protective anti-Klebsiella immunoglobulins to their offsprings through colostra. Baby rabbits fed on colostrum of immunized rabbits withstood lethal Klebsiella infection up to 1 month of age, but not on the 50th day. Baby rabbits having an anti-Klebsiella IgG titre > or =1280 were fully protected against lethal dose of Klebsiella. The study revealed a significant protective efficacy of KCT-I and PBE toxoids against klebsiellosis in mice and rabbits.     

A surface antigen influenza vaccine. 2. Pyrogenicity and antigenicity.

Conventional influenza vaccine containing whole virus particles purified on a zonal centrifuge is pyrogenic and can cause systemic and local adverse side effects. An improved vaccine was therefore prepared which contained only the surface antigens of the virus adsorbed to aluminium hydroxide. The antigenicity of this vaccine was compared with conventional vaccine in chickens. Both vaccines induced similar titres of serum haemagglutination-inhibition and neuraminidase inhibition antibody. The dose response curves, however, were different. The surface antigens at vaccine strength without aluminium hydroxide were of negligible pyrogenicity in rabbits.     

Potential protective immunogenicity of recombinant Clostridium perfringens α-β2-β1 fusion toxin in mice, sows and cows

Clostridial toxins are main pathogenic virulence of Clostridium perfringens that have been associated with a wide range of diseases in both humans and domestic animals. Genetically engineered toxoids have been shown to function as potential vaccine candidates in the prevention of Clostridium derived infectious diseases. In this study, we have developed recombinant α-toxin (CPA), β2/β1-fusion toxin (CPB2B1) and α/β2/β1 trivalent fusion-toxin (CPAB2B1) as vaccine candidates that may be used to vaccinate against C. perfringens α, β1 and β2-toxins. Mice immunized with these recombinant toxoids demonstrated a strong protective immunological response when administered a lethal dose of C. perfringens type C culture filtrate with high titers of neutralizing antibodies to the toxins in the sera, as well as the intestinal mucosal s-IgA level. Specific neutralizing antibodies to the toxins were also detected in the sera and colostrum of sows and cows vaccinated with the toxoids. Furthermore, the CPA and CPB2B1 recombinant toxoid cocktail was capable of stimulating relatively higher levels of immune responses compared to that of CPA, CPB2B1 and CPAB2B1 alone. The CPAB2B1 trivalent fusion toxoid also displayed increased immunogenicity relative to CPA and CPB2B1 alone. These results suggest that recombinant toxoids are potential vaccine candidates against Clostridial toxins; the use of mixed cocktails and/or multivalent recombinant toxoids against different types of toxins may be an effective approach in the prevention of diseases caused by toxins produced by C. perfringens.     

Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge

Shiga toxin type 1 (Stx1) and type 2 (Stx2) are produced by Escherichia coli O157:H7 and are responsible for the life-threatening sequela, the hemolytic uremic syndrome. Whether antisera to Stx1 or Stx2 are cross-neutralizing remains controversial, so we constructed genetic toxoids of Stx1 and Stx2 and evaluated them as vaccines. Antisera from mice immunized with a single toxoid type recognized and neutralized the homologous toxin but not the heterologous toxin. Furthermore, only mice immunized with Stx1 and Stx2 toxoids were protected against a lethal challenge of both toxins. We conclude that Stx1 and Stx2 are distinct antigens for mice.     

Determination of trichothecenes in cereals

A simple method is described of identification and determination of deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS) and T-2 toxin in cereals. Chloroform-ethanol extracts were purified in columns filled with active charcoal, aluminium oxide and celite and were analysed by the method of thin-layer chromatography. The results were evaluated exposing fluorescent trichothecene derivatives to ultraviolet light, after they had been obtained on a chromatographic plate with aluminium chloride and sulphuric acid. The detectability of the method for various toxins was: DON--37, NIV--100, DAS--50, and T-2 toxin--100 mcg/kg.     

Toxicity assessment of Clostridium difficile toxins in rodent models and protection of vaccination

Clostridium difficile is the leading cause of hospital-acquired diarrhea, also known as C. difficile associated diarrhea. The two major toxins, toxin A and toxin B are produced by most C. difficile bacteria, but some strains, such as BI/NAP1/027 isolates, produce a third toxin called binary toxin. The precise biological role of binary toxin is not clear but it has been shown to be a cytotoxin for Vero cells. We evaluated the toxicity of these toxins in mice and hamsters and found that binary toxin causes death in both animals similar to toxins A and B. Furthermore, immunization of mice with mutant toxoids of all three toxins provided protection upon challenge with native toxins. These results support the concept that binary toxin contributes to the pathogenicity of C. difficile and provide a method for monitoring the toxicity of binary toxin components in vaccines.     

Rapid method for purification of Clostridium botulinuh type C neurotoxin by High Performance Liquid Chromatography (HPLC )

The culture supernatant of Clostridium botulinum type C, concentrated by addition of RNA, acid precipitation and subsequent protamine treatment was used as starting material for rapid purification of L toxin (mol. wt. ca. 500K) and M toxin (mol. wt. ca. 350K) of C1 neurotoxin by ion-exchange chromatography on a Mono S column by fast performance liquid chromatography (FPLC). L and M toxins were highly purified further by gel permeation chromatography through a TSK G3000SW column at pH 6.0 by high performance liquid chromatography (HPLC). Purified S toxin (mol. wt. ca. 150K, Cl neurotoxin without a nontoxic component) was then obtained from L toxin rapidly by gel permeation chromatography at pH 7.3 through a TSK G3000SW column by HPLC. Purified S toxin was also obtained rapidly from M and L toxins by ion-exchange chromatography on a Mono Q column at pH 8.0 using an FPLC system. The purified preparations of L, M and S toxins gave single bands on conventional polyacrylamide gel electrophoresis, and had specific activities of 2.8, 6.7, and 14–21 × 10⁷ LD₅₀/mg N, respectively, in mice. On immunoelectrophoresis, purified S toxin gave a single arc against anti-crude toxin serum. The yield of toxicity as L and M toxins was 73.1% (32.5% as L toxin and 40.6% as M toxin) from the protamine-treated concentrated culture supernatant. The recovery of toxicity as S toxin from purified L or M toxin was almost 100% (97.6–100% of L toxin and 97.5% of M toxin). These procedures provide a rapid method for purifying L and M toxins, which have stable toxicities. The method will also be very useful for rapid preparation of the toxic component (S toxin) of C1 neurotoxin, which is unstable, in small amounts from the L and M toxins just before its use in experiments.     

Bacterial toxins: friends or foes?

Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus alpha-toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we discuss the information available on its synthesis and structure, mode of action, and contribution to virulence. We also review the role certain toxins have played in unraveling signal pathways in eukaryotic cells and summarize the beneficial uses of toxins and toxoids. Our intent is to illustrate the importance of the analysis of bacterial toxins to both basic and applied sciences.     

Antibody response of pregnant women to two different absorbed tetanus toxoids

The antibody response in pregnant women vaccinated with either of two different adsorbed tetanus toxoids has been studied. One vaccine (A), prepared by toxoiding purified tetanus toxin followed by its adsorption onto calcium phosphate, exhibited a low titre expressed as international immunizing units, 69 IIU/0.5 ml. The other vaccine (B), prepared by purifying formalinized crude tetanus toxin and adsorbing it onto aluminium phosphate showed a high titre, 212 IIU/0.5 ml. No significant differences between titres of circulating antibodies were obtained after the first injection of either vaccine, but titres after the second injection were much higher for vaccine A as compared with those obtained using vaccine B. The results showed that the immune response in human beings is not correlated to titres expressed in IIU. These results confirm that other methods should be adopted for evaluating the potency of vaccines. A simplified technique based on the comparison of circulating antitoxin levels after vaccination of mice has recently been proposed.     

Evidence of a role for permeability factors in the pathogenesis of salmonellosis.

Two clinical isolates of Salmonella typhimurium were shown to produce two skin permeability factors. One factor was heat stable and rapid in onset while the other was heat labile and elicited maximal induration by 18 to 24 hr. The rapid, erythematous permeability factor (PF) response could not be prevented by antisera to cholera toxin or Salmonella antisomatic serum, but it could be simulated by high concentrations of lipopolysaccharide from S. typhimurium. The appearance of the delayed PF reaction was indistinguishable from that of purified cholera toxin. Histological comparisons of rabbit skin injected with Salmonella-delayed PF and cholera toxin revealed that both toxins resulted in gross edema and infiltration of polymorphonuclear leukocytes after 18 hr. The Salmonella-delayed PF was shown to be resistant to a variety of enzymes, sensitive to extremes in pH, and had an isoelectric point of pH 4.8. Unlike Salmonella lipopolysaccharide skin activity, the Salmonella-delayed PF was destroyed at 100 C and was neutralized by monospecific cholera antitoxin. The Salmonella-delayed PF, which shares antigenic determinants with cholera toxin, appears to be elaborated by living S. typhimurium cells in the rabbit ligated intestine, since rabbits immunized with procholeragenoid were protected against fluid loss from live cell challenge. Finally, production of the rapid PF is a stable genetic trait, while delayed PF production is apparently an unstable characteristic among the salmonellae.     

Structural perturbation of diphtheria toxoid upon adsorption to aluminium hydroxide adjuvant

Aluminium-containing adjuvants are often used to enhance the potency of vaccines. In the present work we studied whether adsorption of diphtheria toxoid to colloidal aluminium hydroxide induces conformational changes of the antigen. Diphtheria toxoid has a high affinity for the aluminium hydroxide particles based on a high adsorption degree, adsorption rate and adsorptive capacity. The conformation and stability of diphtheria toxoid in solution and adsorbed to aluminium hydroxide adjuvant were characterized using five physicochemical techniques: intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, infrared spectroscopy and differential scanning calorimetry. Diphtheria toxoid adsorbed to aluminium hydroxide resulted in a minimal shift of the tryptophan fluorescence spectrum, whereas a large increase in the emission of the Bis-ANS probe was observed, indicating that hydrophobic sites of the protein became accessible due to adsorption. In addition, circular dichroism and infrared spectroscopy revealed that adsorption to aluminium hydroxide caused an increase of β-sheet content and a decrease of α-helix content in diphtheria toxoid. Differential scanning calorimetry demonstrated a major decrease in the enthalpy of denaturation upon adsorption. In conclusion, the adsorption of diphtheria toxoid to aluminium hydroxide adjuvant leads to substantial conformational changes in the antigen. Since physicochemical methods can be used to monitor these conformational changes, these analytical methods might offer a tool in regulatory required vaccine quality control by demonstrating consistency in production.     

A phase 1, placebo-controlled,randomized study of the safety,tolerability, and immunogenicity of a Clostridium difficile vaccine administered with or without aluminum hydroxide in healthy adults

IntroductionClostridium difficile is a significant cause of morbidity and mortality in hospitals, nursing homes, and long-term care facilities. The bacteria can produce 3 toxins, of which the C. difficile toxin A and C. difficile toxin B are the principal virulence factors for C. difficile-associated disease.MethodsA phase 1, first-in-human, placebo-controlled, dose-escalation study was performed to assess the safety and immunogenicity of an investigational vaccine candidate consisting of genetically and chemically detoxified, purified toxins A and B. The toxoids, either alone or in combination with aluminum hydroxide (Al(OH)₃), were administered to healthy adults 50–85 years of age at antigen dose levels of 50, 100, or 200 μg in a 3-dose regimen administered at 0, 1, and 6 months.ResultsOverall, the C. difficile vaccine formulations and doses administered were generally well tolerated. Local reactions and systemic events were predominantly mild to moderate, were more common in the 50–64-year age cohort, and comprised mostly injection site pain, headache, and fatigue.In subjects who received the vaccine formulations, both the toxin A- and toxin B-specific neutralizing antibody geometric mean concentrations increased substantially at 1 month after Dose 2 and after Dose 3 compared to baseline. In the 50–64-year age cohort, geometric mean fold rises (GMFRs) in toxin A-specific neutralizing antibodies from baseline at Month 7 ranged from 59.19 to 149.23 in the vaccine groups compared to 2.47 in the control group. For toxin-B specific neutralizing antibodies, the GMFRs from baseline at Month 7 ranged from 116.67 to 2503.75 in the vaccine groups compared to 2.48 in the control group. In the 65–85-year age cohort, GMFRs in toxin A-specific neutralizing antibodies from baseline at Month 7 ranged from 42.73 to 254.77 in the vaccine groups compared to 2.03 in the control group. For toxin-B specific neutralizing antibodies, the GMFRs from baseline at Month 7 ranged from 136.12 to 4922.80 in the vaccine groups compared to 1.58 in the control group. Potent antitoxin neutralizing responses were still evident in immunized subjects in both age groups at Month 12. Although there was no clear dose-level response pattern, the data suggest that both the antitoxin A- and B-specific neutralizing responses were trending higher in the toxoid-only groups compared to the toxoid + Al(OH)₃ groups. Furthermore, the magnitude of the immune response was similar in the 2 age cohorts.ConclusionThe vaccine formulations studied in this phase 1 study were immunogenic and well tolerated. The results presented support further development of the C. difficile vaccine candidate in a larger population of subjects to determine the optimal dose and immunization schedule.Clinical trial registryNCT01706367.     

Experiments on monkeys with cholera toxin partially purified and detoxified with formol and glycine

Cholera toxoid partially purified and detoxified with formol and glycine and inoculated in monkeys proved safe. Histological examination revealed no changes in the skin of the animals treated. Blood samples taken from monkeys immunized with 2 doses of toxoid 12 days after the second inoculation revealed appreciable levels of antitoxin in the animals that had received two 100-μg doses of toxoid. The monkeys vaccinated with toxoid, when inoculated with challenge doses of cholera toxin, had the capacity to neutralize the toxin. Detoxification with formol and glycine may be the first step towards the preparation of a highly purified, innocuous, and antigenic cholera toxoid.     

Formaldehyde treatment increases the immunogenicity and decreases the toxicity of Haemophilus ducreyi cytolethal distending toxin

Haemophilus ducreyi cytolethal distending toxin (HdCDT) is a tripartite AB toxin, which causes DNA damage in affected cells. We investigated the effects of formaldehyde on the chemical, biological, and immunological properties of the HdCDT complex, which was purified by immobilizing the glutathione S-transferase (GST)-CdtB fusion protein, followed by binding of the CdtA and CdtC recombinant proteins. The HdCDT was treated with increasing concentrations of formaldehyde in the presence of lysine. The treatment of HdCDT at 1 and 0.1 mg protein/ml with 320 and 80 mM of formaldehyde, respectively, resulted in the complete abrogation of cytotoxic activity, loss of DNase activity, and loss of binding capacity to HeLa cells. The toxoid showed protein bands of 75-150 kDa in SDS-PAGE, composed of the three cross-linked CDT components detected by immunoblotting. Three doses of 10 microg protein/mouse of the formaldehyde-treated HdCDT elicited toxin-neutralizing antibodies at titers about 200 times higher than those elicited by the native toxin. The described methodology may be applied to produce immunogenic toxoids from other CDTs, which might be used as candidate components in vaccines against CDT-producing bacteria, including H. ducreyi.     

Interference of outer membrane protein PalA with protective immunity against Actinobacillus pleuropneumoniae infections in vaccinated pigs

The role of antibodies to the outer membrane protein PalA of Actinobacillus pleuropneumoniae in protective immunity was studied in pigs vaccinated with purified PalA alone and PalA in combination with toxoids of the RTX toxins ApxI and ApxII using an established challenge model with the virulent serotype 1 of A. pleuropneumoniae. Pigs that developed antibody titers against PalA after immunization were more significantly affected by challenge with A. pleuropneumoniae serotype 1. Following challenge, pigs that were immunized with PalA showed more severe respiratory symptoms, had a higher mortality rate and died faster. They also displayed much more severe lung lesions after necropsy than animals not immunized with PalA. Pigs that were immunized with toxoids of the two cytotoxins ApxI and ApxII were protected against challenge with A. pleuropneumoniae. In contrast, the protective efficacy of the ApxI and ApxII vaccine was completely lost when it was supplemented with PalA. Hence, antibodies induced against the outer membrane protein PalA of A. pleuropneumoniae aggravated the consequences of infection and counteracted the protective effect of anti-ApxI and anti-ApxII antibodies. Due to the high similarity between protein analogues of PalA from various bacteria of the Pasteurellaceae family such as P6 of Haemophilus influenzae or 16kDa Omp of Pasteurella multocida, this deleterious effect of PalA in vaccination should be taken into consideration in the development of vaccines against infections with other Pasteurellaceae.     

精制锡克氏毒素人群使用效果观察及三种方法测定白喉抗毒素比较

我们发现精制白喉毒素十分稳定,用这种毒素制备的锡克氏毒素,其中和白喉抗毒素的能力恒定,符合英国药典(1980)规定,並显著地低于原制锡克氏毒素的中和力,又因其中无“类毒素”成分,故其ST的敏感性大为提高。用这种精制锡克氏毒素进行人群观察结果表明,锡克氏阳性反应及阴性反应与标准动物法测得的对应人血白喉抗毒素单位符合率分别为96.67%及90%,且无过敏反应发生。其假阳性反应与混合反应皆明显地低于用陈旧原毒素制备的锡克氏毒素。我们结果还证明,iHA试验的敏感性低于ST和动物测定,iHA试验与动物测定结果的符合率为68.83%,与ST的符合率为70.47%。     

The in vitro displacement of adsorbed model antigens from aluminium-containing adjuvants by interstitial proteins.

Vaccines prepared by adsorbing an antigen onto an aluminium-containing adjuvant are usually administered by intramuscular or subcutaneous injection. The vaccine then comes in contact with interstitial fluid which contains proteins. In vitro displacement studies were performed to determine whether antigens, which are adsorbed to aluminium-containing adjuvants, can be displaced by interstitial proteins. It was found that when previously adsorbed model antigens such as lysozyme or myoglobin were exposed to interstitial proteins such as albumin or fibrinogen that extensive displacement occurred. A factorial study of the displacement of myoglobin from aluminium hydroxide adjuvant by albumin was performed. The displacement occurred rapidly with the majority of the displacement occurring in less than 15 min. Whether the concentration of the adsorbed myoglobin was above or below the adsorptive capacity of the aluminium hydroxide adjuvant affected the amount which could be displaced. Less myoglobin was displaced when the concentration was below the adsorptive capacity. The age of the model vaccine (1, 2 or 7 days) prior to exposure to the interstitial protein did not influence the amount of myoglobin that was displaced. The affinity of model antigens and interstitial proteins for aluminium hydroxide or aluminium phosphate adjuvant was characterized by the adsorption coefficient in the Langmuir equation. In every case studied, the protein having the larger adsorption coefficient was able to displace the protein with the smaller adsorption coefficient.     

Immunogenicity of IRIV- versus alum-adjuvanted diphtheria and tetanus toxoid vaccines in influenza primed mice

The immunogenicity and protectivity of two different toxoid vaccines were compared in mice. In one formulation, toxoids (diphtheria or tetanus) were adsorbed to alumoxid, whereas in the other formulation the toxoids were crosslinked to immunopotentiating reconstituted influenza virosomes (IRIVs). A preimmunization with influenza antigens is necessary for a good anti-toxoid antibody response when the IRIV formulation was administered. After two immunizations with the IRIV- or alum-based vaccines, the IRIV-based formulation induced a higher humoral immune response than toxoids adsorbed to alum. Using an in vitro test, diphtheria toxin neutralizing antibodies were tested. Di-IRIV induced a significantly (p = 0.002) higher titer of diphtheria toxin neutralizing antibodies than Di-alum. Tetanus challenge experiments showed, that the IRIV-based tetanus vaccine induced a threefold higher titer of protective antibodies than the tetanus toxoid adsorbed to alum. Therefore, the IRIV-based formulations appeared to be superior to the alum-based vaccines in terms of immunogenicity and protectivity.